Ultrasound Imaging of Anatomy and Milk Ejection in the
Human Lactating Breast
By
Donna T Ramsay (PostGrad Dip Sci)
This thesis is presented for the degree of
Doctor of Philosophy
of The University of Western Australia
Biochemistry and Molecular Biology
The University of Western Australia
35 Stirling Highway
Crawley WA 6009
Australia
2004
Preface
The work in this thesis was supervised by Professor Peter Hartmann,
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The
University of Western Australia. I was supported financially by a scholarship provided by
Medela AG (January 2001 – June 2004).
Chapters 4 and 5 have been published in peer-reviewed journals (1 and 2). In
addition parts of this thesis have been published in a scientific review (3), and presented
at scientific conferences (4, 5, 6, and 7) as follows:
1. Ramsay, D.T., Kent, J.C., Owens, R.A. and Hartmann, P.E. (2004). Ultrasound
imaging of milk ejection in the breast of lactating women. Pediatrics 113(2):361-367.
2. Kent, J.C., Ramsay, D.T., Doherty, D., Larsson, M. and Hartmann, P.E. (2003).
Response of breasts to different stimulation patterns of an electric breast pump.
Journal of Human Lactation 19(2):179-186.
3. Hartmann, P.E., Cregan, M.D., Ramsay, D.T., Simmer, K. and Kent, J.C. (2003).
Physiology of lactation in preterm mothers: Initiation and Maintenance. Pediatric
Annals 32-35.
i
4. Ramsay, D.T., Kent, J.C. and Hartmann, P.E. Breast anatomy redefined by
Ultrasound in the lactating breast. Conference proceedings of Australian Society for
Ultrasound in Medicine. Perth, Australia. September, 2003
5. Ramsay, D.T., Kent, J.C. and Hartmann, P.E. (2002). Ultrasound imaging of the
anatomy of the human lactating breast. Conference proceedings of the Perinatal
Society of Australia and New Zealand. Christchurch, New Zealand. March 2002.
6. Ramsay, D.T., Kent, J.C. and Hartmann, P.E. (2001). Ultrasound imaging of milk
ejection in the human lactating breast. 32nd Annual Conference for the Society for
Reproductive Biology. CSIRO Publishing. http://www.publish.csiro.au.
7. Kent, J.C, Ramsay, D.T. and Hartmann, P.E. (2001). Stimulation of the human milk
ejection reflex by an electric breast pump. 32nd Annual Conference for the Society for
Reproductive Biology. CSIRO Publishing. http://www.publish.csiro.au.
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Acknowledgments
The completion of this PhD would not have been possible without the help and
support of colleagues, family and friends and it is here that I would like to acknowledge
their support.
During this PhD my supervisor Professor Peter Hartmann has not only provided
me with the benefit of his vast knowledge and years of experience in the field of lactation
but inspired me to extract more from my research than I initially thought possible. He has
also been extremely tolerant and understanding of the rather tumultuous personal journey
that I have experienced during the course of my PhD.
I would like to gratefully acknowledge the generous financial support of Medela
AG (Michael Larsson) for my research. I have especially appreciated the interest shown
by the company for research into the basic physiology of lactation.
Almost all of the work presented in this research required the participation of
volunteer mothers and babies in the studies and without these mothers this work would
not have been possible. A large proportion of these mothers were recruited by the West
Australian Branch of the Australian Breastfeeding Association. To the mothers and
babies I have especially enjoyed the reciprocal sharing of information, experiences and
the laughter associated with breastfeeding and motherhood.
I would like to thank Mrs. Robyn Hartmann very much for her contribution of an
artists’ interpretation of my ultrasound findings of the anatomy of the breast. Her ability
to perceive a 2-dimensional image and convert it to a 3-dimensional diagram was not an
easy task.
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I would like thank Dr Jacqueline Kent for her assistance in operating the breast
pump and collating the data for the stimulation study (Chapter 5) and for calculating both
24-hour milk productions and storage capacities for the all of the mothers. Also, my
thanks to Dr Leon Mitoulas, Dr Jacqueline Kent and Mr Ching Tat Lai for their help in
the operation of the breast pump and collection of samples for the expression study
(Chapter 6).
I am very grateful to Dr Dorota Doherty for the statistical advice provided for
Chapters 5 and 6. I also thank her for her enthusiasm and interest in these studies.
I give special thanks to Dr Alfred and Pearl Cowie for their enthusiastic
welcoming of me into their home and their interest in my research. I am deeply indebted
to Dr Alfred Cowie for his kind gift of Sir Astley Cooper’s original text to Professor
Peter Hartmann as it has provided us with a far greater understanding of the anatomy of
the breast.
The members of the Hartmann Lab, I thank you all for your contribution towards
making my PhD life both interesting and enjoyable. Special thanks to Dr Jacqueline Kent
for her willingness to share her expertise, support, patience and kindness throughout the
course of my research. I am grateful to Dr Leon Mitoulas for his freely sharing his
knowledge and entertaining many enthusiastic discussions about milk ejection and also
for his good humour and patience. Thank you Mr. Ching Tat Lai for your invaluable IT
assistance that has enabled me to complete this thesis. Thank you also Mr. Mark Cregan,
Mrs. Catherine Fetherson, Ms Jenni Henderson and Miss Tracey Williams for your
interest and participation in discussions on any topic. People outside the lab that I would
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like to thank include the office staff (Ms Jenny Gillett, Ms Jude Moyle and Mr. Greg
Allen) and Perth Imaging who have allowed me the flexibility of being able to carry out
research and present it internationally while still working in the clinical field. And of
course support and motivation given by close friends (Lisa, Michelle, Kate and
Emmerick, Sally and Phil, and Lorraine and Rob) was essential to remaining somewhat
balanced during this PhD.
Lastly and mostly I would like to acknowledge the two very special people that I
could not have completed this research without. Firstly my partner Tim Geddes who has
displayed much patience and understanding, has freely offered his support and
encouragement and has simply believed I could complete a PhD. But most of all I thank
him for making me laugh at times I have only wanted to cry. Secondly thanks to my
beautiful 6 year old daughter Gabrielle who has traveled the PhD journey since she was
conceived and who shares as much fascination of breasts and breastfeeding as her
mother.
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Table of Contents
Preface………………………………………………………………………………i Acknowledgements………………………………………………………………..iii Table of Contents………………………………………………………………....vi List of Tables………………………………………………………………………xii List of Figures……………………………………………………………………..xv Abstract……………………………………………………………………………xvii Chapter 1 Literature Review 1.1 Introduction…………………………………………………………………1
1.2 The development of the non-lactating Breast………………………… ........4
1.2.1 Fetal development……………………………………………..4 1.2.2 Neonatal and pre-pubertal development………………………6 1.2.3 Puberty………………………………………………………..6 1.2.4 Menstrual cycle………………………………………………..7 1.2.5 Post menopausal involution…………………………………..7
1.3 Gross anatomy of the mature non-lactating breast………………………….8
1.3.1 Arterial supply………………………………………………..10 1.3.2 Venous drainage…………………………… ...........................11 1.3.3 Innervation……………………………………………………12 1.3.4 Lymphatic drainage…………………………………………..14
1.4 The development of the lactating Breast……………………………………15
1.4.1 Pregnancy……………………………………………………..15 1.4.2 Lactogenesis I………………………………………………... 17 1.4.3 Lactogenesis II………………………………………………..19
1.4.4 Post lactation involution…………………………………… ...20
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1.5 The gross anatomy of the lactating breast…………………………………..21
1.5.1 Arterial and venous changes during lactation…………………23 1.5.2 Sensitivity of the lactating breast……………………………...24 1.5.3 Lymphatics of the lactating breast…………………………….25
1.6 Ultrasound imaging of the breast……………………………………………25
1.6.1 Ultrasound technique for imaging the breast………………….25 1.6.2 Ultrasound appearance of the non-lactating breast……………27 1.6.3 Ultrasound appearance of the lactating breast……………… ..28
1.7 Milk ejection reflex ………………………………………………………..29 1.8 ..... Oxytocin........................................................................................................32
1.8.1 Release of oxytocin during suckling………………………….34 1.8.2 Stimuli for oxytocin release……….......................................... 37
1.8.2.1 Ferguson reflex…………………………………………….37 1.8.2.2 Touch………………………………………………............37 1.8.2.3 Temperature………………………………………………..38 1.8.2.4 Olfactory…………………………………………………...38
1.9 Oxytocin receptor in the breast……………………………………………..39
1.9.1 Other oxytocin receptor locations…………………………….40 1.9.1.1 Effects of oxytocin…………………………………............41 1.9.1.2 Effect of oxytocin on myoepithelial cells………………….41 1.9.1.3 Effect of oxytocin on the mammary gland
during suckling.....................................................................42 1.9.1.4 Effect of oxytocin on the mammary gland
during expression .................................................................45 1.9.1.5 Endocrine effects of oxytocin…………………………… ..47 1.9.1.6 Physiological effects of oxytocin……………………….....47 1.9.1.7 Behavioural effects………………………………………...49 1.9.1.8 Effect of exogenous oxytocin…………………………… ..50 1.9.1.9 Other effects of oxytocin…………………………………..51
1.10 Inhibition of milk ejection………………………………………………… .52
1.10.1 Factors that inhibit milk ejection…………………………… ..53 1.10.2 Stress……………………………………………………….....53 1.10.3 Alcohol……………………………………………………......54 1.10.4 Opiates……………………………………………………… ..55 1.10.5 Relaxin………………………………………………………..56
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1.10.6 Breast Surgery…………………………………………...........54 1.11 Aims……………………………………………………………………..55
Chapter 2 Materials and Methods 2.1 Materials……………………………………………………………………. 57
2.1.1 Participants………………………………………………….. 57 2.1.2 Physics and principles of ultrasound ........................................
2.1.2.1 Ultrasound controls .............................................................. 2.1.2.2 Artifacts................................................................................ 2.1.2.3 Basic principles of breast ultrasound ...................................
2.1.3 Ultrasound equipment……………………………………….58 2.1.4 Validation of ultrasound equipment…………………………58 2.1.5 Ultrasound gel……………………………………………….59 2.1.6 Condoms…………………………………………………….59 2.1.7 Electric breast pump .................................................................
2.2 Methods .........................................................................................................62 2.2.1 Ultrasound imaging of breast milk………………………….. .62 2.2.2 Ultrasound imaging of the breast tissues……………………. .62 2.2.3 Ultrasound imaging of the milk ducts………………………...70 2.2.4 Ultrasound imaging of resting milk ducts………………….....71 2.2.5 Ultrasound imaging of milk ducts during breastfeeding……. .72
2.2.6 Ultrasound imaging of milk ducts during stimulation or expression of the breast using an electric breast pump.............72 2.2.7 Measurement of 24-hour milk production………………….. ..73 2.2.8 Calculation of degree of fullness and storage capacity…….....73 2.2.9 Measurement of milk intake during a breastfeed……………..75
2.2.10 Statistical analysis………………………………………….....75
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Chapter 3 Anatomy of the Lactating Human Breast Redefined with Ultrasound
Imaging
3.1 Introduction……………………………………………………………........87
3.2 Materials…………………………………………………………………….90
3.2.1 Participants………………………………………………...90 3.2.2 Ultrasound equipment………………………………… ......90
3.3 Methods……………………………………………………………………..90
3.3.1 Measurement of 24-hour milk production……………… ...90 3.3.2 Calculation of storage capacity…………………………… 90 3.3.3 Ultrasound imaging of the anatomy of the breast………… 91 3.3.4 Statistical analysis………………………………………… 91
3.4 Results………………………………………………………………………91
3.4.1 Participant characteristics…………………………….........91 3.4.2 Milk duct system………………………………………….. 92 3.4.3 Breast tissues……………………………………………….93
3.5 Discussion………………………………………………………………….. 97 Chapter 4 Ultrasound imaging of milk ejection in the breast of lactating women 4.1 Introduction……………………………………………………………........103 4.2 Materials…………………………………………………………………… 105
4.2.1 Participants…………………………………………….......105 4.2.2 Ultrasound equipment……………………………………. .105
4.3 Methods……………………………………………………………………..105
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4.3.1 Measurement of 24-hour milk production……………… ...105 4.3.2 Measurement of milk intake ……………………………....105 4.3.3 Ultrasound imaging of breast milk…………………….......106 4.3.4 Ultrasound monitoring of resting milk ducts…………… ...106 4.3.5 Ultrasound imaging during the entire breastfeed…………. 106 4.3.6 Statistical analysis………………………………………… 106
4.4 Results............................................................................................................107
4.4.1 Ultrasound imaging of breast milk…………………….......107 4.4.2 Ultrasound monitoring of resting milk ducts…………… ...108 4.4.3 Ultrasound imaging of the entire breastfeed………… ........110
4.5 Discussion…………………………………………………………..............115 Chapter 5 Response of the breast to different stimulation patterns of an electric breast pump 5.1 Introduction……………………………………………………………........120 5.2 Materials ……………………………………………………………… .......121
5.2.1 Participants………………………………………………...121 5.2.2 Ultrasound equipment ……………………………… .........122 5.2.3 Electric breast pump………………………………….........122
5.3 Methods..........................................................................................................122
5.3.1 Measurement of 24-hour milk production……………….. .122 5.3.2 Calculation of degree of fullness and storage capacity
of the breast………………………………………………..122 5.3.3 Protocol…………………………………………………... .123
5.3.3.1 Prestudy protocol…………………………………….....123 5.3.3.2 Study protocol………………………………………... ..123
5.3.4 Statistical analysis………………………………………... .125
5.4 Results……………………………………………………………………....127
5.4.1 Participant characteristics……………………………….....127
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5.4.2 Milk ejection……………………………………………... .127 5.4.3 Milk removal……………………………………………....129 5.4.4 Changes in milk ducts…………………………………… ..130 5.4.5 Mothers perceptions……………………………………... ..131
5.5 Discussion………………………………………………………….. ............134 Chapter 6 The use of ultrasound to characterize milk ejection in women using an electric breast pump 6.1 Introduction……………………………………………………………........138 6.2 Materials ……………………………………………………………… .......139
6.2.1 Participants…………………………………………….......139 6.2.2 Ultrasound equipment………………………………… ......139 6.2.3 Breast pump and vacuum patterns……………………. ......140
6.3 Methods………………………………………………………………..........140
6.3.1 Measurement of 24-hour milk production…………….. .....140 6.3.2 Calculation of degree of fullness and storage capacity... .....140 6.3.3 Ultrasound imaging of milk ducts in the non-expressed.
Breast....................................................................................140 6.3.4 Expression parameters…………………………………. ....141 6.3.5 Milk sampling during the expression period…………........141 6.3.6 Statistical analysis……………………………………… ....142
6.3 Results…………………………………………………………………........142
6.3.1 Participant characteristics……………………………….....142 6.3.2 Expression characteristics……………………………... .....143 6.3.3 Vacuum characteristics………………………………… ....144
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6.3.4 Ultrasound monitoring of milk duct diameter for the initial milk ejection………………………………… ...144
6.3.5 Ultrasound monitoring of milk duct diameter for the entire expression period……………………………. ......146
6.4 Discussion………………………………………………………………......147 Chapter 7 Clinical Implications of Ultrasound Imaging of the Anatomy of the Lactating Human Breast and Milk Ejection……………………........152
References………………………………………………………………….. ...... 160
List of Figures ............................................................................................ 160 Figure 1.1 The gross anatomy of the female breast
(adapted from Johnston, 1949). .........................................................10 Figure 1.2 The innervation of the human breast.
(Adapted from Vorherr, 1974)...........................................................14
Figure 1.3 The mean (±SD) increase in breast volume (ml) during pregnancy (n=8; Adapted from Cox et al., 1999 ...........................17 Figure 1.4 The output (mmol/24h) of lactose in the urine (mean ± SD) during pregnancy (n=8; Adapted from Cox et al., 1999)............................................18 Figure 1.5 (a) Ultrasound scanning technique for systematic transverse and sagittal scanning of the breast. (b) Ultrasound scanning technique for radial scanning of the breast.......................................................................................26 Figure 2.1 The vacuum profile of the Classic pumping pattern (47 cycles/minute)
for Chapter 6. .....................................................................................71 Figure 2.2 The vacuum profile of the 3-Phase pumping pattern
consisting of A: stimulation phase (pre-milk ejection, 120 cycles/minute) and B: expression phase A (0-2 minutes post-milk ejection, 20 cycles/minute) and C: expression phase B (2-10 minutes post-milk ejection, range 55-78 cycles/minute). This pattern was tested in Chapter 6 ..............71
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Figure 2.3 Ultrasound image of tissues of the lactating breast. The skin (SK)
is shown as an echogenic (bright) line at the top of the image. The subcutaneous fat (S) is less echogenic and situated below the skin. The intraglandular fat (I) is of similar echogenicity to the subcutaneous fat. The glandular tissue is echogenic (G) while the milk duct (←D) appears as a hypeoechoic (low echo) tubular structure. The retromammary fat (R) is a thin hypoechoic band along the chest wall......................................................................................73
Figure 2.4 (a) The right breast divided into sectors according to
the face of the clock. (b) The arrows show the four images documented along the 3 o’clock axis. The number of images was dependent on the size of the breast, the axis measured and the extent of the glandular tissue. ...............................74
Figure 2.5 A sagittal section of the breast showing the intervals at which measurements were made of the depths of breast tissues along the 12 o’clock axis........................................................75
Figure 2.6 (a) Sagittal section of the breast demonstrating the
breast structures (b) Diagram demonstrating the measurements of depths of the subcutaneous fat (S) and retromammary fat (R) at a 30 mm radius from the base of the nipple (c) Diagram demonstrating multiple measurements of glandular tissue at a 30 mm radius from the base of the nipple (d) diagram showing the measurement (X) which represents the total of the glandular tissue and intraglandular fat measured at a 30 mm radius from the base of the nipple. ........................................76
Figure 2.7 (a) Ultrasound image of milk duct in the lactating
breast. The duct is traced from deep in the breast to the nipple (N). The walls are echogenic (↑) and the lumen hypoechoic (→). Note the echogenic flecks within the duct that are consistent with the appearance of fat globules in the breast milk. A small branch is noted ( ) (b) The milk duct is outlined..............................................................81
Figure 3.1 Artists impression of the lobes of the breast. The ducts
were injected with coloured wax prior to dissection (from Cooper, 1840). .........................................................................89
Figure 3.2 Ultrasound image of milk duct in the lactating breast.
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The duct is traced from the nipple (N) to the periphery of the breast. The walls are echogenic (↑) and the lumen hypoechoic (*). The first branch of this duct (→) is imaged almost directly under the nipple............................................93
Figure 3.3 The distribution of different tissues in the right breast
of 2 different lactating women. Cumulative totals of each tissue were calculated by summing all of the measurements made in the breast at 0, 3 ,6 ,9 and 12 cm distances from the base of the nipple. (a) mother 1 had a total tissue sum of 1465 mm and a 24-hour milk production of 299 g . (b) mother 9 had a total tissue sum of 790 mm and a 24-hour milk production of 176 g. ...........................................96
Figure 3.4 A drawing of the gross anatomy of the lactating breast
based on ultrasound observations made of the milk duct system and distribution of different tissues within the breast................................................................................98
Figure 4.1 An ultrasound image of two main milk ducts in the lactating breast. The ducts in the nipple (N) are narrow. The more superficial duct (→) displays a constant diameter for approximately 20 mm from the nipple. The deeper duct branches almost immediately before entering the nipple. Note the glandular tissue (G, bright echoes) directly below the nipple......................................109 Figure 4.2 Monitoring of milk ducts for 5 minute. A relatively
large (4.2mm) duct ( ! and ∀) and small (1.9mm) duct (% and 3) monitored for 5 minutes at least 7 days apart are shown.......................................................................110
Figure 4.3 Multiple milk ejections. 4 milk ejections are detected in
this mother during an 11.5 min breastfeed. The maximum diameter for each milk ejection was consistent. The duration of each duct dilation was between 90 and 120 seconds. The infant finished feeding as the duct diameter reached a maximum (↓). .....................................................112
Figure 4.4 Milk intake of the infant in relation to the number of milk
ejections detected during a breastfeed. ..............................................113
Figure 4.5 Variation of the duration of milk duct dilation. Single milk ejection (∀). One duct dilation lasted 231seconds
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during this mothers’ breastfeed and her infant consumed 140g of milk. Multiple milk ejections (%). Three duct dilations of 45, 64 and 89 seconds were observed during this mothers’ breastfeed of 4 minutes and 49 seconds and her infant consumed 100g of milk. ....................114
Figure 5.1 Mothers’ ratings of the frequencies (A) and strengths
(B) of the stimulation patterns on a scale of 1 (slow, soft) to 10 (fast, strong). Bars indicated the range of responses, boxes indicate the first and third quartiles, and the dashed line indicates the median...........................................133
Figure 6.1 Changes in milk duct diameter (times 10) in 10 second periods (x) and changes in the volume of expressed milk in 30 second periods (+) over a full 10 minute expression period using the 3-Phase pattern...................................................................................147
List of Tables Table 1.1 Location of oxytocin receptors in tissues
as shown by oxytocin binding (Adapted from Gimpl and Fahrenholz, 2001)....................................40 Table 1.2 Actions of oxytocin on behaviours of
different species (Adapted from Gimpl and Fahrenholz, 2001)....................................50
Table 1.3 Additional effects of oxytocin on different
Species ...............................................................................................51 Table 2.1 Maternal and infant characteristics of mothers
studied in Chapters 3 (mothers 1-22), 4 (mothers 1-17 and 22-28) and 6 (mothers 1-28)................................61
Table 2.2 Maternal and infant characteristics of mothers
studied in Chapter 6. ..........................................................................62
Table 2.3 B mode seeting for ACUSON XP10 ultrasound machine.................68 Table 2.4 Characteristics of the stimulation patterns* for
Chapter 5............................................................................................70
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Table 3.1 The distribution (depths) of the tissues in the
breast (mm) .......................................................................................94 Table 5.1 Time for milk ejection, vacuums chosen and
amount of milk removed for each stimulation pattern*.....................129
Table 5.2 Comparison of perceived frequency and strength of each pattern with the infant’s frequency and strength (% of responses)...................................................................132
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Abstract
In women, as in other mammals, the ability to successfully lactate depends on
both complete functional development of the mammary gland and the stimulation of the
milk ejection reflex to enable the suckling young to remove stored milk. Prior to my
studies, Sir Astley Cooper’s carried out the most comprehensive investigation of the
gross anatomy of the lactating human breast in 1840. I have used ultrasound to image the
anatomy of the breasts of fully breastfeeding women (1-6 months, n=22) with particular
emphasis on the distribution of the main milk ducts, glandular and adipose tissue.
Scanning of the milk duct system demonstrated that the anatomy in the region of the
areola and nipple is different to that depicted in standard anatomical textbooks. The main
milk ducts were small (diameter, left: 1.9 ± 0.6 mm; right: 2.1 ± 0.7 mm), superficial
(depth, left: 4.50 ± 1.98 mm; right: 4.74 ± 1.59 mm) and branched close to the nipple
(within 8.20 ± 6.27 mm, left; 7.00 ± 3.98 mm, right) (mean ± SD). The lactiferous
sinuses (described in current textbooks) were not observed and the number of main ducts
detected at the base of the nipple was less than the quoted 15-20 (9.4, range 4-18).
Quantitative descriptions of the morphology of either the lactating or, indeed, the
non-lactating breasts have not been attempted using ultrasound. I developed a systematic
approach to ultrasound imaging of the breast that provided a semi-quantitative
description of the distribution of glandular and adipose tissues within the lactating breast.
xvii
Approximately two thirds of the breast was comprised of glandular tissue. Intraglandular
fat was identified as hypoechoic transects within the hyperechoic glandular tissue. Over
65% of the glandular tissue together with 50% of the intraglandular fat and 25% of the
subcutaneous fat was located within a 30 mm radius of the base of the nipple. The
absence of lactiferous sinuses and the arrangement of tissue within a 30 mm radius of the
nipple suggested that the current conceptualisation of sucking dynamics of the infant
requires revision.
Successful milk removal depends on the stimulation of the milk ejection reflex
and currently subjective assessments of milk ejection such as the mother’s sensations and
an alteration in the infants sucking and swallowing are used clinically to confirm milk
ejection whereas in research two stressful invasive procedures; changes blood oxytocin
and intra-ductal pressure have been used. I have developed a non-invasive ultrasound
technique to detect milk ejection in women. In the absence of stimulation no significant
changes in milk duct diameter (CV = 1.4% to 8.3%) were observed. During a breastfeed
(or breast expression) a milk duct in the contralateral breast was monitored with
ultrasound. The milk duct expanded by 1.15 ± 0.22 mm and milk flowed towards the
nipple at the time the mother first sensed milk ejection. Milk was not removed from the
monitored breast and within 90 seconds the duct diameter decreased as milk flowed back
into the glandular tissue. The mean duration of a milk ejection was 86 ± 51 seconds (n =
146). Serial measurements of duct diameter during breastfeeds detected multiple milk
ejections (2.5 ± 1.5, n=62) in 76% of breastfeeds. Although few mothers were able to
xviii
sense second and subsequent milk ejections, the number of milk ejections during a
breastfeed was related to the milk intake of the infant.
Although electric breast pumps are widely used by lactating women, there is little
information on the physiology of breast expression. The milk ejection response to
different stimulation vacuum patterns was investigated in fully breastfeeding women (1-6
months, n= 28). The time to milk ejection was reduced with increased time since the last
breastfeed and with increased degree of fullness of the breast. The increase in milk duct
cross sectional area was similar to that observed at a breastfeed indicating that the
stimulation patterns did not compromised milk ejection. The milk ejection response in 11
fully breastfeeding women (1-6 months) during a 10-minute expression period was
measured using 2 markedly different expression patterns (Classic: 47 cycles/minute; 3-
Phases: stimulation phase 120 cycles/minute, expression phase 20 cycles/minute for 2
minutes then 55-78 cycles/minute). Milk was collected at 30-second intervals over the
expression period. Multiple milk ejections were observed by ultrasound and were
associated with acute increases in milk flow. The volume of milk removed at the initial
milk ejection was similar for both patterns and represented between 40-50% the total
volume expressed. The number of milk ejections was similar for each pattern (Classic:
3.27 ± 2.05; 3-Phase: 3.72 ± 1.19) and to those observed during a breastfeed. There was
no relationship between multiple milk ejections and volume of milk removed. Since, the
Classic pattern was consistent throughout the 10 minutes of expression, the stimulus for
multiple milk ejections remains unknown.
xix
These studies have been the first to use ultrasound to both extensively study the
anatomy of the lactating breast and the milk ejection, and provide a new opportunity for
future objective investigations of the physiology of human lactation and clinical problems
associated with breastfeeding.
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Chapter 1
Literature Review
Breast Anatomy and Milk Ejection in Women
1.1 Introduction
The benefits of breastmilk for both the term and preterm infants are well
documented in the literature. Breastmilk provides optimal nutrition for the growth and
development of the infant as well as enhancing the infants immune system and providing
protection from many diseases such as necrotizing eterocolitis, diarrhoea, otitis media,
respiratory tract infections, asthma and diabetes. Furthermore there is evidence to suggest
that breastfed infants have higher intelligence scores, less risk of obesity and increased
visual acuity. However, the benefits of breastfeeding are not limited to the infant. Women
who have breastfed their babies have a reduced risk of osteoporosis, pre-menopausal
breast cancer and ovarian cancer as well as improved iron status. In addition
breastfeeding enhances maternal nurturing behaviour, feelings of self-competency and
social interaction (Prentice, 1997; American Academy of Pediatrics, 1997).
Human milk is the natural diet for infants and provides many benefits that cannot
be replicated in substitute food. This has led the WHO to recommend that infants should
be exclusively breastfed for the first 6 months of life. Although a large proportion of
1
women (83%) in Australia choose to breastfeed by 3 months post partum only 54% of
women are fully breastfeeding. These rates fall further to 32% by 6 months post partum
(Australian Bureau of Statistics, 2000).
Successful lactation is dependent upon the ability of the breast to synthesise,
secrete and release milk (milk ejection) for removal by the infant and for increasing
numbers of women by the use of a breast pump. Unlike other organs of the body the
breast does not reach its full functional capacity until reaching maturity during pregnancy
and childbirth. Thus breast development occurs in distinct phases: fetal, neonatal/pre
pubertal and post pubertal. Development of the breast can then proceed through a number
of lactation cycles (pregnancy, lactogenesis I, lactogenesis II, involution) then finally to
post menopausal involution. These developmental phases are influenced by key
hormones and growth factors (Hovey, Trott and Vonderhaar, 2002). It is during
pregnancy and lactation that the most extensive growth of the secretory tissue of the
breast occurs. During pregnancy the mammary epithelial cells lining the milk ducts
undergo extensive proliferation to form the secretory parenchyma with the formation of
alveoli lined with mammary epithelial cells that differentiate into lactocytes and become
capable of producing some milk components (lactogenesis I). Although milk secretion is
suppressed until after parturition (McManaman and Neville, 2003) where upon
lactogenesis II is triggered by the withdrawal of progesterone (Kulski, Smith and
Hartmann, 1977) and the breast, in its fully differentiated state synthesises and secretes
copious quantities of breastmilk. During weaning, the alveoli regress and the breast
returns back to a resting state. There has been limited investigation into the anatomy of
the lactating human breast. The most extensive work was performed by Sir Astley
2
Cooper on lactating cadavers in 1840. The means by which Cooper acquired the lactating
cadavers in the 1800’s was at best dubious and currently the availability of lactating
cadavers for dissection is very limited and fraught with legal difficulties. Therefore
Coopers work remains the benchmark for anatomical texts. Cooper injected the ductal
systems of the breast with either coloured wax or size and demonstrated that the ducts
draining the alveoli of the breast merge into larger ducts and finally into one large duct
before opening onto the nipple. This large duct had a dilated portion termed the
lactiferous sinus, which he thought acted as a reservoir for milk in a similar manner to the
milk cisterns of ruminants.
Nevertheless a significant proportion of the milk produced by the lactocytes is
stored in the alveolar portion of the breast rather than the ductal system. The process by
which milk is forced into the larger ducts to become available for removal by the infant is
termed the milk ejection reflex. Stimulation of the teat/nipple causes the release of
oxytocin from the neurohypophysis into the bloodstream. Oxytocin causes the
contraction of myoepithelial cells around the milk filled alveoli thereby forcing the milk
into the milk ducts. Historically it was well recognised that milk ejection (milk ‘let
down’) occurred during the period of infant suckling and that it was required for
successful breastfeeding. Milk ejection was also observed to occur occasionally between
breastfeeds.
Considerable investigation of the milk ejection reflex has been carried out in
animals by measuring the increase in intra-ductal pressure that occurs when milk is
forced out of the alveoli and by taking frequent blood samples to detect the release of
oxytocin. However, the application of these methods to women is difficult as both
3
investigations are invasive and the stress associated with these proceedures may impair
milk ejection resulting in decreased milk yield (Newton and Newton, 1948). As a result
there are relatively few studies of milk ejection in women.
Since complete development of the breast is fundamental to the synthesis of
breast milk it is important to have a thorough understanding of the anatomy of the breast.
Furthermore milk ejection is essential for the release of milk from the breast to both
satisfy the infant and ensure continued milk production, therefore, knowledge of the
response of the breast to milk ejection is fundamental to understanding normal lactation.
1.2 The development of the non-lactating breast
1.2.1 Fetal development
The human breast begins to develop at 6 weeks of gestation when a thickened
ectodermal ridge situated along the anterior body wall from the groin to the axilla of the
embryo becomes apparent (milk line). Eventual regression of this ridge occurs except for
the pectoral region (2-6th rib) to form the mammary gland. Supernumerary glands may
develop any where along the ectodermal ridges and may either mature into mammary
glands or remain as accessory nipples in 2-6% of women (Vorherr, 1974).
During the 7th and 8th week of gestation the mammary parenchyma invades the
stroma and a raised portion called the mammary disc appears. Epithelial buds begin to
form between 10 and 12 weeks and parenchymal branching during the 13th-20th week.
Between the 12th and 16th weeks of gestation the smooth musculature of areola and nipple
are formed and at approximately 20 weeks gestation 15-25 (Gould, 1983; Hovey, Trott
and Vonderhaar, 2002) solid cords form in the subcutaneous tissue. Branching continues
4
and canalization of the cords occurs by apoptosis of the central epithelial cells to become
the primary milk ducts by 32 weeks gestation (Hovey, Trott and Vonderhaar, 2002).
Mesenchymal cells differentiate into erythroblasts and primitive blood vessels at 7
weeks gestation. Subsequently small capillaries appear between 9 and 10 weeks gestation
and by 12 to 13 weeks gestation a concentric vascular network has formed in the
mammary gland. Development of the vascular system is complete by week 16 and blood
begins circulating to the skin, secretory, adipose and connective tissues of the mammary
gland.
In the last 8 weeks of gestation there is little lobulo-alveolar development
however the periductal stroma increases in density (Naccarato et al., 2000). Also at 32
weeks of gestation the ducts open onto an area which develops into the nipple (Tobon
and Salazar, 1974). The ectoderm around the nipple becomes pigmented and free of hair
and Montgomery glands develop in this region. The adipose tissue of the mammary gland
is assumed to be formed by connective tissue that has lost its capacity to form fibres and
is considered essential as a medium for further growth of the parenchyma of the
mammary gland (Vorherr, 1974).
Shortly after birth colostrum (“witches milk”) can be expressed from the infant’s
mammary glands. This is thought to be due to the pro-lactation hormones present in the
fetal circulation at birth. Regression of the mammary gland usually occurs by 4 weeks
postpartum and coincides with a decrease in the secretion of prolactin from the anterior
pituitary gland of the infant (Vorherr, 1974).
5
1.2.2 Neonatal and pre pubertal development
The newborn breast consists only of rudimentary ducts that have small club-like
ends that regress soon after birth. Prior to puberty the growth of the breast is isometric
but has not been well documented. At puberty (8-12 years) allometric growth of both the
epithelium and stroma occurs. In ruminants allometric growth begins before puberty
(Hovey, Trott and Vonderhaar, 2002) and impaired mammary development and
subsequent lactation performance has been observed in ruminants that have a dietary
intake in excess of their energy requirements (Sejrsen and Purup, 1997).
1.2.3 Puberty
At puberty the human breast increases in size mainly due increased deposition of
adipose tissue within the gland (Russo and Russo, 1987). Epithelial proliferation also
begins with elongation and branching of the ductal system to form a more extensive
ductal network. Although the hormonal regulation of mammary growth during this period
is poorly understood in women it has been associated with increased levels of oestrogen,
prolactin, lutenising hormone, follicle stimulating hormone and growth hormone (Lee et
al., 1976; Thorner et al., 1977; Rose et al., 1991; Ankarberg-Lindgren et al., 2001).
However, care must be taken in extrapolating the findings in animals to women because
the human mammary gland undergoes much greater growth and development at puberty
than in other mammals.
6
1.2.4 Menstrual cycle
The menstrual cycle in women generally ranges from 25-30 days with ovulation
occurring at day 10-16 of the cycle. During the luteal phase (days 3-15) the lobules and
alveoli develop with open lumens, basal clear cells become prominent and the stroma
becomes loose and edematous (Vogel et al., 1981; Longacre and Bartow, 1986). Mitotic
activity is at its greatest during the luteal phase (Anderson, Ferguson and Raab, 1982;
Longacre and Bartow, 1986; Potten et al., 1988). Whereas, during the follicular phase
(days 16-26) small lobules with few alveoli and dense cellular stroma are apparent along
with low mitotic activity. From day 27 to menstruation these changes regress typified by
epithelial degeneration and there is an increase in the proportion of dense stroma.
However the degeneration of the epithelial growth is not complete (Vogel et al., 1981;
Longacre and Bartow, 1986) and some of the follicular growth remains prior to the next
cycle. With advancing age there is a concomitant decrease in mitotic activity such that at
about 35 years of age there is a plateau in breast development (Vorherr, 1974).
1.2.5 Post menopausal involution
After menopause, declining ovarian function is associated with diminishing levels
of oestrogen and progesterone and involution of the breast. This involution is typified by
the reduction and eventual atrophy of the glandular tissue of the breast resulting in the
ductules in the lobules decreasing by approximately a third (Tavassoli, 1992). Dilation of
the ductules (cystic atrophy) may be present in some breasts. As the glandular tissue
decreases there is a concomitant increase in adipose tissue (Bannister, 1995) usually
beginning at the periphery of the breast and progressing towards the nipple (Vorherr,
7
1974). This is clearly apparent on mammograms (Laya et al, 1995; Sterns and Zee, 2000).
A decrease in elasticity of the supporting connective tissues of the breast is also apparent
(Hutson, Cowen and Bird 1985). However, there is wide variation between women in the
proportion of glandular and adipose tissue in the breast at this time (Bannister, 1995).
1.3 Gross anatomy of the mature non-lactating breast
The current descriptions of the anatomy of the breast have changed little in the
past 150 years and are based on Coopers dissections of lactating breasts in 1840. The
breast is composed of glandular (secretory) and adipose (fatty) tissue and is supported by
a loose framework of fibrous connective tissue called Cooper’s ligaments. Apart
from the external number, position and appearance of the mammary glands the internal
structure (glandular tissue and supporting tissues) is similar in practically all species
(Cowie 1974). In women the glandular tissue is composed of 15-20 lobes that are
comprised of lobules containing 10-100 alveoli that are approximately 0.12mm in
diameter (Hartmann, 1991; Lawerence, 1994). Up until recently it was believed that each
lobe existed as a single entity, however, a study that created three dimensional
reconstructions of the entire ductal system (16 lobes) of a mastectomized breast of a 69
year old woman demonstrated connections between lobes on two occasions. These
connections were located more than 40 mm from the nipple (Ohtake et al., 2001). The
amount of tissue associated with each lobe is variable and may show 20-30 fold
differences (Moffatt and Going, 1996).
The ratio of glandular to adipose tissue in the breasts of 30-80 year old women
was found to be on average 1:1 in Malaysian, Australian and German women (Heggie,
8
The ratio of glandular to adipose tissue in the breasts of 30-80 year old women
was found to be on average 1:1 in Malaysian, Australian and German women (Heggie,
1996; Klein et al., 2002; Jamal et al., 2004) when measured by mammography.
Furthermore the proportion of glandular tissue decreased with advancing age (Soares et
al., 2002; Jamal et al., 2004) and increasing breast size (Cruz-Korchin, 2002). Although
quantitative measurements of the amount of tissue in the breast have not been made, one
study has correlated mammographic images with the ultrasound parenchymal pattern of
the non-lactating breast (Kaizer et al., 1988). The alveoli of the breast are drained by
small ducts and these ducts coalesce into larger ducts (2mm) that eventually converge
into one main milk duct, that dilates slightly to form the lactiferous sinus (2-4.5mm)
before narrowing as it passes through the nipple and opens onto the nipple surface
(Figure 1.1). The nipple pores are 0.4-0.7mm in diameter (Fawcett, 1986) and are
surrounded by circular muscle fibres (Vorherr, 1974; Bannister et al., 1995). There is
disagreement over the number of main ducts in the human breast with some authors
stating that there are 6-10 (Hicken, 1937, 1940; Love, 1990; Lanfranchi, 2000), whereas,
standard anatomy textbooks state that there are 15-20 (Bannister et al., 1995). The
authors that quote fewer than 15-20 ducts appear to base their estimates on observation
rather than methodical study. Whereas those that describe 15-20 ducts are probably
referring to either Coopers (1840) work or possibly more recent histological sections of
the nipple (Tavassoli, 1992). Histological sections of the nipple allow the number of
openings in the nipple to be counted but do not allow one to determine if all of the ducts
are connected to functional lobes within the breast. Galactography is a procedure
whereby the duct is cannulated through the nipple and radio-opaque contrast is injected in
9
order to outline the ductal system on subsequent radiographs (Horatio, Slawson, and
Johnson, 2001). Hicken (1937) used galactography and found that the ducts were
relatively superficial in the breast. The main ducts of the breast are depicted as being
regularly spaced around the nipple (Bannister ,1995; Moffatt and Going, 1996) despite
observations that the lobes are difficult to separate on dissection (Bannister, 1995). In
addition one study of the breast of a 19-year old cadaver showed that her 10 main ducts
branched within 4 to 28 mm of the nipple (Moffatt and Going, 1996).
Nipple pore
Lactiferous sinus
Milk ducts
Alveoli
Figure 1.1 The gross anatomy of the female breast (adapted from Johnston, 1949)
1.3.1 Arterial supply
Descriptions of the blood supply to the breast have been based on the classic
dissections of lactating cadavers by Cooper (1840). Methods used to investigate the
10
vasculature of the mammary gland in cadavers include injection of either coloured wax or
mercury into the vessels (Cooper, 1840), surgical dissection (Anson, Wright and Wolfer,
1939) and injection of a suspension of fine lead and radiography of the blood supply in
one non-lactating woman (Salmon, 1939). The breast is supplied mainly by the anterior
and posterior medial branches of the internal mammary artery (60%) and the lateral
mammary branch of the lateral thoracic artery (30%) (Vorherr, 1974; Cunningham 1977;
Doughty et al., 1996).
The limited number of studies performed since Cooper (1840) have highlighted
the variation in blood supply to the breast between women. The anterior perforating
branch at the second intercostal space has been considered to be the main source of blood
due to its large size (Maliniak, 1934). Other studies show that two
main anterior perforating branches supply the breast (Anson, Wright and Wolfer, 1939)
rather than four branches quoted in textbooks (Vorherr, 1974; Bannister et al., 1995).
Although the lateral thoracic artery is considered to supply up to a third of the blood to
the breast, recent studies have shown that the lateral thoracic artery is infact absent in as
many as a third of women (Doughty et al., 1996). Smaller sources of arterial blood also
include the posterior intercostal arteries and the pectoral branch of the thoracoacromial
artery (Freeman et al., 1981; Bannister et al., 1995). There is also a wide variation in the
proportion of blood supplied by each artery between women (Doughty et al., 1996) and
little evidence of symmetry between breasts (Anson, Wright and Wolfer, 1939, Morehead
1982). Furthermore the course of the arteries does not appear to be associated with the
ductal system of the breast (Cooper 1840).
11
1.3.2 Venous drainage
The venous drainage of the breast is divided into the deep and superficial systems
(Cooper, 1840) joined by short connecting veins. Both systems drain into the internal
thoracic, axillary and cephalic veins. The deep veins are assumed to follow the
corresponding mammary arteries (Anson, Wright and Wolfer, 1939; Cunningham, 1977),
while the superficial plexus consists of subareola veins that arise radially from the nipple
and drain into the periareolar vein which circles the nipple and connects the superficial
and deep plexus. Symmetry of the superficial venous plexus is, as with the arterial
supply, not apparent (Isard and Ostrum, 1974; Cunningham, 1977).
1.3.3 Innervation
Cooper (1840) showed that the 2nd to 6th intercostal nerves supply the breast and
that they have a relatively superficial course within the gland. Eckhard (1850) reported
that the intercostal nerves divide into superficial and deep branches (Figure 1.2). The
deep branches supply the nipple and glandular tissue while the superficial branches
supply the nipple and areola. The distribution and course of the nerves innervating the
nipple and areola are complex and frequently variable. However the nipple and areola are
always supplied by the anterior and lateral cutaneous branches of the 3rd to 5th intercostal
nerves (Craig and Sykes, 1970; Schlenz, et al., 2000). The anterior branches of the 3rd to
5th intercostal nerves lie along the ducts to the nipple (Craig and Sykes, 1970). However,
Sarhadi et al. (1996) found that the anterior branch of the 2nd intercostal nerve also
contributed to the innervation of the nipple and areola. The lateral supply of the nipple
12
and areola is less variable than the medial supply. The lateral supply is provided by the
4th lateral cutaneous nerve (Farina, 1980; Schlenz, et al., 2000) and it most often takes a
sub-glandular course within the pectoral fascia to the posterior aspect of the nipple (Craig
and Sykes, 1970; Gonzalez et al., 1993; Schlenz, et al, 2000). Less commonly it takes a
superficial course (Cooper, 1840; Farina, 1980; Sarhadie et al., 1996). Detailed
descriptions of the course of the anterior cutaneous branches are scant and conflicting. A
deep course is descried by Craig and Sykes (1970) whereas Sarhadi et al. (1996) and
Schlenz et al. (2000) describe a superficial course.
Nerves have been demonstrated in association with the major duct system but
none have been identified near the smaller ducts (Linzell, 1971). The areola and nipple
are sparsely innervated with all neural elements concentrated at the base of the nipple and
only few at the side of the nipple, and practically none in the areola (Montagna and
MacPherson, 1974). These nerves are sensory nerves and together with the lack of
evidence of motor innervation of either the lactocytes or myoepithelial cells suggests that
both the synthesis and secretion of milk is independent of neural stimulation. However,
there is motor innervation of the smooth muscle of the areola and nipple (Courtiss and
Goldwyn, 1976) and the arteries of the breast (Cowie, 1974)
13
Supraclavicular nerves
Medial branches thoracic intercostal
Lateral branches thoracic intercostal
Nerves supplying the areola and nipple
Figure 1.2 The innervation of the human breast. (Adapted from Vorherr, 1974)
1.3.4 Lymphatic drainage
The drainage of lymph from the breast has been extensively investigated because
of its importance in the spread of breast carcinoma. The lymph is drained by two main
pathways; to the axillary nodes (Turner-Warwick, 1959) and to the internal mammary
nodes (Hultborn et al., 1955; Turner-Warwick, 1955; Vendrell-Torné, Setoain-Quinquer
and Domenech-Torne, 1972). The axillary nodes have been reported to receive more than
75% of the lymph from both the medial and lateral portions of the breast (Turner-
Warwick 1959; Borgstein, Meijer and Pijpers, 2000), whereas, the internal mammary
nodes receive lymph from the deep portion of the breast (Aukland and Reed, 1993).
14
However, there is a wide variation in the drainage of lymph from the breast and less
common pathways have been demonstrated. Lymph may occasionally pass through either
the interpectoral nodes (Bannister, 1995) or lymph nodes in the breast parenchyma (Tanis
et al., 2001). Sometimes direct drainage of lymph occurs to the supraclavicular nodes
(Tanis et al., 2001) and infrequently lymph may pass retrosternally into the contralateral
internal mammary nodes. In addition lymph has been shown to drain into the posterior
(Turner-Warwick, 1959) and anterior intercostal nodes (Tanis et al., 2001).
1.4 The development of the lactating breast
1.4.1 Pregnancy
At 3-4 weeks gestation ductal branching and lobular formation escalates
exceeding normal premenstrual changes and often the increased breast tenderness
associated with these changes is the first indicator of pregnancy. During the first half of
pregnancy extension and branching of the ductal system occurs along with intensified
lobular-alveolar growth (mammogenesis). Growth of the mammary gland is influenced
by oestrogen, progesterone, prolactin, growth hormone, epidermal growth factor,
fibroblast growth factor and insulin-like growth factor (Oka et al., 1991; Kelly et al.,
2002) and more recently parathyroid hormone related protein (Wysolmerski et al., 1995).
It appears that the glandular tissue grows by invading the fat lobules hence decreasing the
amount of adipose tissue relative to glandular tissue in the breast (Vorherr, 1974). This
may be assisted by the hormone relaxin which has been shown to be essential for
mammary growth by causing both an increase the amount of adipose tissue in the
mammary gland and dilation of the mammary microvessels in rats (Bani, 1997). Prior to
15
16 weeks gestation the alveoli and ducts have a two layer epithelial lining. After 16
weeks gestation the superficial layer disappears in the alveoli and this single layer
persists for the rest of pregnancy and for the duration of lactation (Vorherr, 1974). There
is some secretory development (colostrum) by mid-pregnancy with colostrum beginning
to dilate both the alveolar and ductal lumina. In the last trimester there is a further
increase in lobular size associated with hypertrophy of the cells to twice their resting size
and further accumulation of secretion in the lumen of the alveoli. These changes usually
lead to a marked increase in breast size during pregnancy. However, breast growth
during pregnancy varies greatly between women ranging from either little or no increase
to a considerable increase in size that can occur either rapidly during the first trimester or
more gradually over the entire pregnancy (Figure 1.3; Cox et al. 1999). While the major
increase in breast size is usually completed by week 22 of pregnancy, it is clear that for
some women significant breast growth occurs during the last trimester of pregnancy
(Figure 1.3). The rate of growth of the mother’s breast during pregnancy was correlated
with the increase in the concentration of human placental lactogen in the mother’s blood
suggesting that this hormone may stimulate breast growth in women as it does in some
other mammals (Cox et al., 1999). The effect of nutrition on the development of the
mammary gland during pregnancy has not been extensively studied, however, a recent
study of rats has shown that the restriction of energy intake by 40% in the first part of
pregnancy resulted in increased mammary cell proliferation (46%) and suppressed
apoptosis resulting in a 14% increase in milk yield (Kim and Park, 2004). In women no
such studies have been performed, however, one study has associated the reduced
16
maternal energy intake associated with morning sickness in the first trimester with
increased placental weight at birth (Huxley, 2000).
Change in Breast Volume
(ml)
Figure 1.3 The mean (±SD) increase in breast volume (ml) during pregnancy (n=8; Adapted from Cox et al., 1999)
1.4.2 Lactogenesis I
Lactogenesis I is defined as the stage of breast development during pregnancy
when mammary epithelial cells differentiate into lactocytes and are able to secrete milk-
specific components (Kulski and Hartmann, 1981). Lactogenesis I can be confirmed by
identifying milk-specific components such as lactose, casein, and α-lactalbumin in blood,
urine or colostrum. Milk specific components move from the colostrum to the blood
through the paracellular pathway (Arthur et al., 1991a). Lactose in the blood is unable to
be metabolised in the body and is consequently excreted in the urine (Carleton and
Roberts, 1959). Therefore increased excretion of lactose in the urine can confirm
lactogenesis I in pregnant women (Arthur et al., 1991). Lactogenesis I identified by
Stage of gestation (weeks)
0 4010 20 30
0
-100
100 200 300 400
Conception Birth
# #
#
# ##
17
increased excretion of lactose in urine normally occurs by 22 weeks of gestation and
coincides with the observation of secretion in the lobules (Russo and Russo, 1987).
However, lactogenesis I may occur between 10 and 22 weeks gestation indicating a large
variation between women (Figure 1.4).
Although there is little information about hormonal regulation of breast growth
and development in women during pregnancy changes in circulating hormones are now
well established for women during lactogenesis I (Hartmann, 1991). Extrapolation from
animal studies suggests the growth of the milk ducts is due to oestrogen, growth hormone
and corticosteroids, while proliferation of the alveoli requires progesterone and prolactin.
The high concentration of progesterone in maternal blood is also thought to inhibit the
lactocytes from secreting milk during pregnancy (Cowie et al., 1980).
0 40 10 20 30
Conception Birth
0
1
2
3
Urinary Lactose mmol/24h
# #
#
##
Stage of gestation
(weeks)
Figure 1.4 The output (mmol/24h) of lactose in the urine (mean ± SD) during pregnancy (n=8;
Adapted from Cox et al., 1999)
18
1.4.3 Lactogenesis II
Lactogenesis II is defined as the onset of copious milk secretion. For many years
lactogenesis II was identified by a sudden or gradual increase in the feeling of fullness of
the breasts (milk ‘coming in’) (Cadogan, 1748; Rendle-Short and Rendle-Short, 1966)
which occurs between 24 and 102 hours postpartum (mean 59–64 hours) (Kulski et al.,
1981; Arthur et al., 1989) However, lactogenesis II can be confirmed by measuring either
changes in milk (colostrum) composition or an increase in milk production and these
changes often occur before the sensation of the milk ‘coming in’.
A sharp drop in progesterone in the maternal blood initiates Lactogenesis II after
birth (Kulski, Smith and Hartmann, 1977). However, adequate levels of prolactin, insulin
and adrenal corticosteroids are also necessary to facilitate the initiation of lactation
(Forsyth, 1983). Milk production, determined by test weighing, normally increases
between 30-40 hours post-partum (Arthur et al., 1989b) to 39–169 g/day in term women
(Saint et al., 1984; Arthur et al., 1989b; Glasier and McNeilly, 1990). The increase in
milk production is also accompanied by marked changes in milk composition such as
increased lipid content and concentrations of casein, lactose, calcium, phosphate, citrate
and potassium and decreased concentrations of protein, sodium and chloride (Harzer et
al., 1986; Arthur, 1989b; Neville et al., 1991; Kunz and Lönnerdal, 1992). These changes
stablize within the 5 days postpartum (Arthur et al., 1989b). By day 6 post partum term
mothers who breastfeed on demand have a milk production of 440 to 1220 g/day (Saint et
al., 1984; Casey et al., 1985; Arthur et al., 1989b; Hartmann, Sherriff and Kent, 1995;
Sherriff and Hartmann 1995; Kent, 1999).
19
Factors involved in the delay of lactogenesis II include premature delivery, frequency of
breastfeeding (De Carvalho et al., 1983) and expression (Hopkinson, Schanler and Garza,
1988), type of delivery (Evans et al, 2003; Chapman and Perez-Escamilla, 1999), obesity
(Chapman and Perez-Escamilla, 1999), and Type 1 diabetes (Arthur, Smith and
Hartmann, 1989). Other factors such as maternal medication and anaesthetic drugs that
may affect initiation have not yet been investigated. However, it is recognised that the
early commencement of breastfeeding (within an hour of birth) for normal term mothers
and frequent breastfeeding thereafter provides the optimum stimulation for the initiation
of successful lactation. In fact, Neville and Morton (2001) observed that delayed removal
of colostrum is more likely to result in an unsuccessful lactation.
The repeated application of the suckling stimulus has been reported to initiate
lactation in non-pregnant women from non-industrialized societies and women in
industrialized societes who have adopted babies. Although there is little research into this
phenomenon reports do suggest that the capacity of the women to respond to this
stimulus is variable (Cowie, Forsyth and Hart, 1980; Auerbach, 2001)
The transition of the breast through lactogenesis I and II results in the
development of a fully functional breast with a high metabolic capacity capable of
synthesising up to 500 mL/24hour of breast milk.
1.4.4 Post lactation involution
When suckling is discontinued involution of the mammary gland occurs. The milk
accumulates in the ducts causing atrophy of the lactocytes. If cessation of breastfeeding is
abrupt engorgement of the breast is more intense than a gradual weaning that decreases
20
milk production slowly. The secretions in the ducts and alveoli are absorbed and the
alveoli gradually collapse and undergo phagocytosis and resorption (Vorherr, 1974). Also
there is an increase in the adipose and periareolar connective tissue. During the involution
process changes occur in milk composition with increased concentrations of sodium,
potassium and protein and decreased concentrations of lactose. As a result the
composition of the involution secretion is not dissimilar to that of colostrum (Hartmann
and Kulski, 1978). The breast does not however, return back to its pre-pregnancy state as
some of the alveoli formed during pregnancy do not involute but rather return to a resting
state and the breast is again influenced by the hormonal changes of the menstrual cycle.
Secretions may remain in the ducts for at least 2 months (Hartmann and Kulski, 1978;
Fawcett, 1986).
1.5 Gross anatomy of the lactating breast
During lactation the alveoli of the breast are lined with lactocytes that synthesise
breast milk and little or no difference has been demonstrated in the structure of these cells
between species (Cowie, 1974). Although the structure of the mammary parenchyma is
remarkably similar in all mammals including women, the arrangement of the ducts and
the size of the storage spaces vary from species to species. For example, goats and cows
have large cisterns that may store as much as 20% of the available milk and have only
one duct from the cistern to the surface of the teat. In contrast the rabbit and bitch
(Cooper, 1840) are similar to women in that they do not have cisterns and have multiple
ducts leading to the nipple. In general in animals with no cisterns almost all of the milk is
21
stored in the alveolar portion of the gland and very little in the ductal system (Cowie
1974).
Standard descriptions of the human mammary gland are based on Coopers (1840)
magnificent dissections of the breasts of lactating cadavers (section 1.2). Although,
imaging modalities have become more sophisticated in the last 20 years research has
focused extensively on the abnormal non-lactating breast and little attention has been
given to the anatomy of the lactating breast. Mammography of the lactating breast is
limited due to increase in glandular tissue and the secretion of breast milk (Tobon and
Salazar, 1975) causing an increase in radio-density which makes the images of the breast
difficult to interpret (Madjar, 2000). Galactography (the injection of radio-opaque
contrast media into the duct orifice at the nipple and subsequent radiography) has
illustrated only a portion of the ductal system and few studies have examined lactating
women. Some studies using galactography have described the milk ducts to be
significantly larger in the lactating breast than those of the non-lactating breast (Leborne
1943; Cardeonsa, Doudna and Eklund, 1994). However, Cardenosa and Eklund (1996)
have used galactography in a very small number of lactating breasts and suggest that the
ducts do not enlarge during lactation. These studies have not measured duct diameter nor
can they provide accurate information on distribution of the ducts as radiography of the
breast requires substantial compression be applied to the breast (Gold, Bassett and
Kimme-Smith, 1986, Kopans, 1989).
To date both Computed Tomography (CT) and Magnetic Resonance Imaging
(MRI) have had little to offer in elucidating mammary anatomy particularly that of the
lactating breast. However, a recent report using MRI illustrated a duct after injection of
22
the duct with contrast (Kanemaki et al., 2004), and an attempt has been made to quantify
fatty and glandular tissue volumes in the non-lactating breast (Lee et al., 1997). MRI has
been used successfully in pregnant and lactating goats to observe mammary growth and
to calculate milk volume of the udder (Fowler et al., 1990a; Fowler et al., 1990b).These
findings suggest that this modality may offer some new insights into the anatomy of the
breast in future.
Although ultrasound imaging has been used extensively in the breast to detect
abnormalities (mainly malignancy) it has not been used systematically to investigate the
anatomy of the breast during lactation. A more detailed discussion of the ultrasound
appearances of the breast is provided in a subsequent section (sections 1.6.2 and 1.6.3).
1.5.1 Arterial and venous changes during lactation
During pregnancy blood flow to the breast doubles by 24 weeks and then remains
constant during lactation (Vorherr, 1974; Thoresen and Wesche, 1988). Whereas breast
skin temperature increases by 1oC by week 9 of pregnancy and then remains stable until 2
days postpartum when the temperature increases again (Burd et al., 1977). As with the
non-lactating breast Aljazaf (2002) has shown that there is a wide variation between
women in the proportion of blood supplied by each artery and there is little evidence of
symmetry between breasts. Along with an increase in blood flow, the superficial veins of
the breast also become more prominent during pregnancy and lactation.
During breastfeeding Doppler ultrasound of the lateral thoracic artery of 4 women
has shown that blood flow decreases by 40-50% just prior to milk ejection and then
increases in the following 1-2 minutes. These blood flow changes are also observed after
23
intravenous injections of oxytocin (Janbu et al., 1985). Furthermore a significant decrease
in blood flow at milk ejection also has been observed in other species (Petersen, 1942;
Pearl, Downey and Lepper, 1973; Davis et al., 1995; Eriksson, Lundeberg and Uvnas-
Moberg, 1996).
1.4.2 Sensitivity of the lactating breast
Investigation of the innervation and sensitivity of the breast has predominantly
focused on women that have undergone breast surgery such as reduction mammoplasty.
Only one study investigated the sensitivity of the breast during lactation. This study
found that women displayed a marked increase in areola and nipple sensitivity within 24
hours post-partum (Robinson and Short, 1977) although this tends to decrease in the
following days. In addition Kent et al., (unpublished observations) has found that women
in established lactation (1-6 months) displayed limited sensory discrimination of the skin
of the breast, areola and nipple using the 2-point discrimination method. Lack of
sensitivity of the epidermis of the nipple has been noted previously (Vorherr, 1974).
Clinical evidence supports the limited distribution of nerve fibres in the glandular tissue
in that while women may recognise the overall fullness of a distended breast and pain
associated with some abnormalities they are often unable to accurately localise either
sensation (Cowie, Forsyth and Hart, 1980). Indeed, many women experience influenza-
like symptoms associated with mastitis before they are aware of tenderness in their
breasts.
24
1.5.2 Lymphatics in the lactating breast
Cooper (1840) dissected and injected the lymphatic vessels of the lactating breast
and concluded correctly that fluid in the lymph vessels flowed away from the breast.
Prior to this it was proposed by the Italian anatomist Gasparo Aselli in 1622 that these
vessels carried chyle to the breast for formation of milk. Apart from Cooper (1840) there
has been no investigation of the lymphatic drainage of the lactating breast despite its
importance in clinical conditions such as engorgement and mastitis.
1.6 Ultrasound imaging of the breast
1.6.1 Ultrasound technique for imaging the breast Ultrasound imaging of the breast requires a high-resolution linear transducer in
the frequency range of 7-12 MHz to resolve fine structures within the breast (Smith,
2001). Although higher frequencies provide better image resolution the frequency must
be adequate to penetrate the breast parenchyma to the pectoral muscles to ensure the
entire breast is imaged. The time compensation curve (compensates for the normal
attenuation of the sound waves in the tissue) ranges between a gentle slope for
predominately fatty breasts to a steep slope for dense breasts. One or two focal zones are
used to improve resolution of the image, by narrowing the ultrasound beam, at selected
depths of insonation. The power setting should be high enough to ensure adequate
visualisation of all of the tissues of the breast from the skin to the pectoral muscle (Smith,
2001).
25
The ultrasound technique used to scan the breast for the detection of abnormalities
requires the woman to lay supine and roll away from the side that is to be examined
(approximately 30-45 degrees). She is given support in the form of a foam pad and raises
the arm of the side that is to be scanned above her head. In this position the breast is
flattened against the chest wall ensuring a complete survey of the breast. Systematic
scans can be performed using two methods. The first employs systematic transverse
(horizontal) and sagittal (vertical) scanning of the entire breast (Figure 1.5a). The second
method involves making radial scans around the nipple then moving gradually out
towards the periphery of the breast (Figure 1.5b). Radial scanning is preferred by some
examiners as it follows the more natural course of the ductal system and lobes, but it is
difficult and time consuming. Therefore, the first method is more commonly used to
locate an abnormality and selective radial scans are performed to detect either intra-ductal
changes or tumour extension along a duct (Madjar, 2000, Lanfranchi, 2000).
(a) (b)
Figure 1.5 (a) Ultrasound scanning technique for systematic transverse and sagittal scanning of the
breast. (b) Ultrasound scanning technique for radial scanning of the breast.
26
1.6.2 Ultrasound appearances of the non-lactating breast
The skin of the breast appears as a two thin echogenic lines that vary in thickness
between 1.0 and 3.0 mm (Mendelson, 1998; Smith, 2001). The areola may appear as a
thicker skin layer compared to the rest of the breast whereas the nipple is demonstrated as
a well-defined oval area of medium echogenicity. Due the uneven and fibrous nature of
the nipple distortion of the ultrasound beam may occur resulting in posterior acoustic
shadowing making visualisation of the parenchyma behind the nipple poor (Chersevani et
al., 1995; Smith, 2001). Either the application of extra gel and pressure or angling around
the nipple will ensure satisfactory documentation of this area (Smith, 2001; Baker, Scott
Soo and Rosen, 2001). The superficial fascia of the breast is occasionally seen as another
thin echogenic line below the skin (Mendelson, 1998).
The subcutaneous fat appears as a hypoechoic (low echo) layer of tissue beneath
the skin lines. Cooper’s ligaments (fibrous bands) run between the superficial and deep
fascia of the breast providing a framework for the parenchyma and are represented as
echogenic (bright white) bands running obliquely from the posterior of the breast to the
skin. The curved and fibrous nature of the ligaments may reflect the beam causing
posterior shadowing. Changing the transducer pressure and angle will reduce or eliminate
this artefact (Mendelson, 1998; Baker, Scott Soo and Rosen, 2001).
The breast tissue is comprised of glandular and adipose tissue. The glandular
tissue appears echogenic on ultrasound and may extend into the axilla while the fat is less
echogenic. There is a wide range of ultrasonic patterns of the breast depending on the
amount of fat interspersed throughout the glandular tissue. Each lobe or segment of the
breast is unable to be recognised as distinct entities due to their intertwined nature
27
(Kopans, 1989) however the pattern of glandular tissue is observed more clearly by
ultrasound than by mammography (Kaizer et al., 1988). The retromammary fat appears as
a hypoechoic layer above the pectoral muscles, which display a typical fibrillar pattern.
Blood vessels, nerves and lymphatics comprise the remainder of the breast.
Nerves and normal lymphatics are not visualised on ultrasound, however when the
lymphatics are dilated due to either inflammation or carcinoma invasion they become
visible as very thin anechoic/hypoechoic lines running parallel and perpendicular to the
skin in the subcutaneous tissues (Chersevani et al., 1995). Lymph nodes are demonstrated
in the breast and axilla as well defined oval masses with an echogenic hilum and
hypoechoic cortex (Yang et al., 1995).The arteries and veins of the breast can be
visualised and assessed with colour Doppler ultrasound. In addition veins are
occasionally imaged as anechoic tubular structures that compress with gentle pressure.
1.6.3 Ultrasound appearances of the lactating breast
During pregnancy and lactation the glandular tissue undergoes extensive
proliferation and the ultrasonic appearance of this tissue has been described as increasing
in volume and echogenicity along with thinning of the subcutaneous fat layer
(Mendelson, 1998; Staren and O’Neill, 1998; Sohn, Hamper and Blohmer, 1999). The
main milk ducts can be visualised within the glandular tissue as either hypoechoic or
echo free branching tubular structures that increase in size towards the nipple (Rizzatto et
al., 1992; Rizzatto and Chersevani, 1994; Smith, 2001). The diameter of the main ducts
in the non-lactating breast measured by ultrasound vary but generally range between 1
and 3 mm and ducts above 3mm are considered ectatic (Chung and Chun, 1994; Venta et
28
al., 1994, Rizzatto and Chersevani, 1994; Baker, Scott Soo and Rosen, 2001). Accounts
of the ultrasonic appearance of ducts in the lactating breast describe the ducts to be
dilated (Mendelson, 1998; Staren and O’Neill, 1998; Sohn, Hamper and Blohmer, 1999).
These descriptions appear to be subjective rather than evidence based as they do not state
actual duct diameter measurements. Only one study has traced the ductal systems of
lactating women albeit intermittently using ultrasound (Chung and Chun, 1994).
Unfortunately the aim the study was to visualise the smallest anatomical units of the
ductal system (terminal duct lobular unit) and therefore the study did not provide any
measurements or extensive descriptions of ductal anatomy.
Studies of the anatomy of the lactating breast been extremely limited particularly
with regard to the ductal system. Similarly there has been little investigation into the into
the physiology of the ductal system during lactation even though it is well documented
that intra-ductal pressure increases in response to oxytocin at milk ejection.
1.7 Milk ejection reflex
Although historically it was recognised that a process occurred during suckling
that caused the milk to flow the mechanism of the ‘draught’ as it was referred to (Cooper,
1840; Waller, 1937) was not fully understood. Milk secretion and milk release was
thought to occur simultaneously. In 1911 Ott and Scott discovered that an injection of an
extract from the neurohypopysis into a lactating goat caused in milk flow and Gaines
(1915) concluded that milk secretion and milk removal where two distinct processes.
Then for many years it was assumed that milk ejection was a neural reflex until 1941
when Ely and Petersen established that it was infact a neuroendocrine reflex in cows.
29
Subsequently in 1948, Newton and Newton’s study supported Ely and Petersen’s theory
of the mechanism of milk ejection in women.
Breast milk synthesis occurs within the alveoli of the breast and the greater
portion of the milk is stored here until required by the suckling young. In ruminants a
considerable proportion of the milk passes into a cistern between suckling or milking.
Sheep and goats store a larger fraction of the total milk volume in their gland cistern than
cows (Bruckmaier and Blum, 1992). In dairy cows up to 20% of the total milk volume
can be removed prior to milk ejection and this fraction of milk decreases as lactation
advances (Knight, Hirst and Dewhurst, 1994; Pfeilsticker, Bruckmaier and Blum, 1996).
The critical nature of milk ejection is widely acknowledged in both women and
animals (Cowie, 1974; Newton, 1992; Knight, Hirst and Dewhurst, 1994; Scott Young et
al., 1996; Nishimori et al., 1996) with complete elimination of milk ejection resulting in
lactation failure (Scott Young et al., 1996). Furthermore recent evidence suggests that
oxytocin is not essential for normal pregnancy and birth of the young in both mice (Scott
Young et al., 1996) and women (Volz, Heinrich and Volz-Koster, 2002). However, milk
ejection is required for successful lactation and therefore oxytocin is almost certainly
fundamental to the survival of the mammalian species.
The milk ejection reflex is a classical neuroendocrine reflex where the neural
component constitutes the afferent arm and the hormonal component the efferent arm of
the reflex. When the nipple is stimulated, afferent nervous impulses from the areola and
nipple travel via the spinothalamic tracts to stimulate the release of oxytocin from the
neurohypophysis. Oxytocin is transported in the blood stream to the mammary gland
where it interacts with receptors on the myoepithelial cells surrounding the milk filled
30
alveoli. Activation of these receptors causes the myoepithelial cells to contract forcing
the milk from the alveoli into the milk ducts to the nipple for removal by the suckling
infant.
Although suckling is considered the primary stimulus for the milk ejection reflex
in both women (Luther et al., 1974) and animals (Stormorken and Vanschoubroek, 1960;
Akers and Lefcourt, 1982; Bar-Peled et al., 1995) oxytocin is also released in response to
many other stimuli. As with other species (Cowie, Forsyth and Hart, 1980) the milk
ejection reflex often becomes conditioned in women to factors such as the sight of the
infant, the infant’s cry and other routines (Newton and Newton, 1950; McNeilly and
McNeilly, 1983). Spontaneous milk ejections between breastfeeds are also common
(McNeilly et al., 1978; Cobo, 1993).
Symptoms and sensations of milk ejection are varied and differ greatly between
women. During the first few days’ postpartum uterine contractions are associated with
the release of oxytocin due to infant suckling. Also the dripping of milk from the nipple
as a result of milk ejection may be observed in some women before and during
breastfeeding (Newton and Newton, 1950). Sensations such as pins and needles, pain,
pressure, thirst and nausea may be experienced by women at milk ejection (Isbister,
1956), however, it is estimated that up to 30% of women do not recognise milk ejection
at all (Kent et al., unpublished observations) which is in contrast an earlier study in which
women focused on the sensation of milk ejection reported 95% of women were conscious
of milk ejection. They also suggested a positive association between the intensity of the
sensation and the amount of milk in the breast (Isbister, 1956). In addition, the intensity
of the sensations appear to wane with advancing lactation. In contrast women may
31
experience sensations of milk ejection years after weaning suggesting some residual
functional mypoepithelial cells are retained after the involution of the lactating breast.
Currently objective measurements of milk ejection include the detection of
oxytocin in the maternal plasma (Fabian et al., 1969) and the measurement of intra-ductal
pressure (Cobo et al., 1967) both of which are invasive. Alternatively ultrasound has been
used to detect an increase in cisternal area at milk ejection in sheep, goats and cows
(Bruckmaier and Blum, 1992; Bruckmaier et al., 1997; Ayadi et al., 2003). Clinically it is
common to observe the slowing of infant sucking and increased swallowing during a
breastfeed as an indication that the infant is receiving milk (Bowen-Jones, Thompson and
Drewett, 1982; Woolridge 1986; Righard and Alade, 1992) signifying that milk ejection
has occurred.
1.8 Oxytocin
Oxytocin is comprised of nine amino acids and has a molecular weight of 1007
Da. The synthesis of oxytocin occurs as a 30 kD prehormone in the supraoptic and
paraventricular nucleus of the hypothalamus. (Brownstein et al., 1980; Zimmerman and
Defendini, 1977). Cleavage of the prohormone occurs and then it is converted into
secretory granules and transported down the magnocellular neurons to the
neurohypophysis. The magnocellular neuronal bodies have diameters of 25-30
micrometers and short dendrites that may be joined by gap junctions with other oxytocin
neurons. Varacosities are present on some of the dendrites suggesting they may release
oxytocin locally within the hypothalamus. In the neurohypophysis it undergoes further
cleavage into oxytocin and the oxytocin-carrier peptide neurophysin. The human
32
neurohypophysis is able to store large amounts of oxytocin far in excess of that required
to cause milk ejection (Lincoln and Paisley, 1982). Vasopressin is a similar molecule to
oxytocin which is also synthesised in the hypothalamus (Zimmerman and Defendini,
1977) and released from the neurohypophysis in smaller amounts and has approximately
5-6 fold less milk ejection activity than oxytocin.
Stimulation of the sensory nerves in the nipple and areola transmit impulses via
the spinothalamic tract to the brain stem. The nerves synapse at the hypothalamus and
travel down the hypothalamo-neurohypophysial tract to the posterior lobe causing the
release secretory granules containing the oxytocin-neurophysin complex (stabilising
peptide) by exocytosis into the circulation (Belin et al., 1984; Fuchs et al., 1984; Higuchi
et al., 1985; Dyball and Leng, 1986; Crowley and Armstrong, 1992) and other areas of
the brain including other hypothalamic nuclei, the striatum and the brainstem (Sofroniew,
1983; Tribollet, Bubois-Dauphin and Dreifuss, 1992; Uvnas-Moberg and Eriksson,
1996). The synthesis of oxytocin in hypothalamus and its transport to the
neurohypophysis is rapid and occurs independently of the stimulation of the milk ejection
reflex.
The release of oxytocin also causes the release of neurogenic peptides such as
vasoactive intestinal polypeptide and calcitonin gene-related peptide that have been
shown to relax vascular smooth muscle cells. This may assist in the relaxation smooth
muscle around the large milk ducts in some species (Uvnas-Moberg and Eriksson, 1996).
Although smooth muscle around the larger ducts has been observed in rodents
(Richardson, 1949) it has not been demonstrated in women. Therefore, the release of
oxytocin at milk ejection in women is more likely to be involved in the relaxation of the
33
smooth muscle of the nipple (Vorherr, 1974; Guyton, 1986) decreasing resistance to milk
flow during suckling.
Oxytocin released from the neurohypophysis increases plasma levels, however,
oxytocin released from the paraventricular nucelus increases oxytocin levels in both the
blood and cerbrospinal fluid (Harris, Jones and Robinson, 1981). Oxytocin in the
bloodstream does not cross the blood-brain barrier readily nor does the amount of
oxytocin released from the neurohypophysis into the bloodstream affect the level of
oxytocin in the cerebrospinal fluid (Harris, Jones and Robinson, 1981; Gimpl and
Fahrenholtz, 2001). The half-life of plasma oxytocin is species dependent with ewes (40-
50 seconds), rats (1 minute 40 seconds) (Folley and Knaggs, 1965) and sows (30-44
seconds) (Ellendorff et al., 1982) having shorter half-lives than rabbits (3 minutes 20
seconds) and women (up to 4 minutes) (Folley and Knaggs, 1965). The half-life of
oxytocin in the cerbrospinal fluid oxytocin (28 minutes) is considerably longer (Jones and
Robinson, 1982; Meyer, et al., 1987). Circadian rhythms of oxytocin in the cerbrospinal
fluid have been demonstrated in humans and monkeys (Amico et al., 1983) but not in
other species, however, a circadian rhythm for oxytocin in the blood has not been
observed (Amico, Finn and Haldar, 1988)
1.8.1 Release of oxytocin during suckling
The basal levels of oxytocin are generally higher in women who exclusively
breastfeed their infants compared to those who breastfeed and use supplements of
formula. In breastfeeding women the basal levels of oxytocin are higher at 4 days post
34
partum than at 4 months lactation and then remain constant for up to12 months lactation
(Uvnas-Moberg et al., 1990a). This trend is comparable to basal levels of oxytocin
measured during expression with a mechanical breast pump during early (10-90 days),
mid (90-190 days) and late (180-365 days) lactation (Leake et al., 1983). However,
concentrations of the basal level of oxytocin do not appear to have an affect on the milk
intake of the infant (Uvnas-Moberg et al., 1990a).
Oxytocin is released in a pulsatile fashion in response to suckling (Wagner and
Fuchs, 1968; Fox and Knaggs, 1969, Lucas Drewett and Mitchell, 1980; McNeilly et al.,
1983; Yokoyama et al., 1994) and is able to be detected in the maternal blood (Coch et
al., 1968; Fielitz et al., 1970). Due to the very short half-life of oxytocin, the pulses may
last as little as a minute to three and a half minutes (Drewett, Bowen-Jones and Dogterm,
1982; McNeilly et al., 1983; Uvnas-Moberg et al., 1990a) therefore frequent blood
sampling is necessary for accurate detection (McNeilly and McNeilly, 1983; Udea et al.,
1994). In addition to suckling, spontaneous rises in oxytocin levels have been detected in
the 3 to 10 minutes prior to breastfeeding (McNeilly and McNeilly, 1983) and are more
frequent in early lactation (4 days post partum) (Uvnas-Moberg et al., 1990a).
Although the release of oxytocin in response to suckling does not appear to
decrease during the first 6 months of lactation (Johnson and Amico, 1986) the peak
oxytocin value is greater in early lactation than late lactation when measured during
breast expression (Leake et al., 1983). Recently it has been found that the mode of
delivery may influence the release of oxytocin. Women who deliver by emergency
caesarean section show a lower number of pulses of oxytocin during breastfeeding
compared to those that have a normal vaginal delivery (Nissen et al., 1996).
35
Measurements of intra-oral pressure of the infant at the beginning of a breastfeed
(1-2 minutes) typically show a rapid sucking rate with a relatively high vacuum
compared to the remainder of the feed when the sucking rate is slower and less vacuum is
applied by the infant. The high sucking rate at the beginning of the feed is believed to
stimulate the breast in order to initiate milk ejection whereas the slower sucking rate is
associated with the infant removing milk from the breast (Luther et al., 1974; Drewett
and Woolridge, 1979; Bowen-Jones, Thompson and Drewett, 1982; Woolridge, 1986;
Righard and Alade, 1992). Similarly pre-stimulation patterns at the beginning of milking
of dairy cows are commonly employed to initiate oxytocin release and promote efficient
milk removal (Bruckmaier and Blum, 1996) and recently Weiss and associates (2003)
demonstrated that increased intensity of stimulation of the teat during milking resulted in
increased oxytocin release. In contrast, however, a particular breed of ewe has a
significantly delayed release of oxytocin in response to milking both with and without
pre-stimulation resulting in a longer milking time (Bruckmaier et al., 1997).
Currently no relationship has been found between the amount of oxytocin
released during a breastfeed to either, milk volume, prolactin release or parity of the
mother (McNeilly et al., 1983). However, the number of pulses of oxytocin detected
during a breastfeed at Day 2 of lactation has been positively related to the amount of milk
consumed by the infant (Nissen et al., 1998).
36
1.8.2 Stimuli for oxytocin release
Many stimuli other than stimulation of the nipple have been shown to stimulate
the release of oxytocin. Stimulation of the reproductive tract (Ferguson Reflex), touch
and temperature have been shown to illicit the release of oxytocin.
1.8.2.1 Ferguson Reflex
Oxytocin is released as a result of the distention and relaxation of the vagina
causing uterine contractions (Ferguson, 1941). This neurohormonal reflex is termed the
Ferguson reflex. The Ferguson reflex is not stimulated during early pregnancy but is
demonstrated late in pregnancy and is thought to play a major role in parturition.
Furthermore, a greater amount of oxytocin is released during genital tract distention than
suckling in dairy cows (Bruckmaier, Schams and Blum, 1992). Indeed in the past, vaginal
stimulation in traditional societies was used to stimulate milk ejection and enhance
milking (Cowie, Forsyth and Hart, 1980).
1.8.2.2 Touch
Newborn infants massage the breast with their hands and an increase in this
activity is associated with the release of oxytocin in the mother (Matthiesen et al.,
2001).This activity is reduced in mothers who receive analgesia (intramuscular pethidine,
mepivacaine via pudendal block and bupivacaine via epidural) during labour (Nissen, et
al., 1997; Ransjo-Arvidson, et al., 2001). This may be likened to the tap reflex in animals
where the young bunt the udders with their heads to release maternal oxytocin prior to
suckling (Cowie, 1980). Similarly hand massage of the lactating breast by a trained nurse
37
causes an increase in oxytocin levels (Yokoyama et al., 1994). In addition massage
therapy releases oxytocin in humans (Uvnas-Moberg, 1998), as does ventral stroking,
touch and light pressure with animals (Agren et al., 1995; Uvnas-Moberg et al., 1993).
Indeed repeated ventral stroking has been demonstrated to have long-term effects
attributed to oxytocin (Lund et al., 2002) compared to dorsal stroking that has an effect of
much shorter duration (Lund, et al., 1999). Furthermore massage is associated with
decreased cortisol levels, anxiety (Field et al., 1992) and blood pressure (Kurosawa et al.,
1995).
1.8.2.3 Temperature
There is evidence that increased temperature triggers oxytocin release in rats
(Argen et al., 1995). In cows washing of the udder with warm water (40 C) elicits an
antidiruetic response that is associated with oxytocin release and milk ejection but only in
cows that were conditioned to this practice (Stormorken and Vanschoubroek, 1960).
However, despite evidence of thermal stimulation of milk ejection in animals there is no
evidence to support this in women (Sala et al., 1974b).
o
1.8.2.4 Olfactory
Oxytocin may be released in response to olfactory cues. In two studies of a
similar design, rats that were given oxytocin had decreased tail temperatures (reduced
energy loss through thermoregulation) and increased hind paw withdrawal latency to
heat. Both of these responses were also present in control-treated rats in the same cage
whereas control rats in other cages did not respond. When olfaction was blocked using
38
zinc sulphate, the oxytocin effects were not produced in the control-treated rats in the
same cage suggesting the olfactation plays a role in the dissemination of oxytocin effects
(Agren, et al., 1997; Agren, Uvnas-Moberg and Lundeberg, 1997).
1.9 Oxytocin receptor in the breast
The human oxytocin receptor is comprised of 389 amino acids and belongs to G-
protein coupled receptor family (Inoue et al., 1994). In addition it is coupled to the
inositol phospholipid-protein kinase C system. During labor the oxytocin receptor
undergoes a massive up-regulation in the myometrium (Fuchs et al., 1995; Kimura et al.,
1996; Tahara et al., 2000) and a 5-fold increase in the decidua (Takemura et al., 1994) but
not the mammary gland. Although a gradual increase in oxytocin receptors has been
demonstrated during pregnancy peaking at lactation in the rat mammary gland (Soloff et
al., 1979; Breton, Di Scala-Guenot and Zingg, 2001) there is little evidence of an increase
in the human lactating breast (Kimura et al., 1998). Furthermore there appears to be more
oxytocin receptors present in the ductal/glandular epithelium than the myoepithelial cells
in women (Kimura et al., 1998) which is in contrast to earlier reports showing oxytocin
receptors only on the myoepithelial cells of the human breast (Bussolati et al., 1996) and
the rat mammary gland (Adan et al., 1995). In light of the low numbers of oxytocin
receptors on the myoepithelial cells in women it has been speculated that the function of
the receptors is to produce prostaglandins (Kimura et al., 1998) such as prostaglandin F2α,
which could increase the intra-ductal pressure at milk ejection (Cobo et al., 1974).
However, it is possible that the increased oxytocin receptors in the glandular tissue may
simply reflect the myoepithelial cell distribution along the ducts.
39
1.9.1 Other oxytocin receptor locations
Oxytocin receptors have been identified in many organs in both humans and
animals (Table 1.1) further emphasising the extensive affects of this hormone on
physiological processes other than milk ejection.
Table 1.1 Location of oxytocin receptors in tissues as shown by oxytocin binding (Adapted from Gimpl and Fahrenholz, 2001) Tissue Species Female reproductive system
Myometrium Human, cow, sheep, rat, cow, rabbit, guinea pig, pig Endometrium Human, rat, cow, sheep, rabbit, pig Amnion Human, cow, rabbit Chorion Human Decidua Human, rat, guinea pig, rabbit Ovary Human, marmoset monkey, cow, guinea pig, brushtail
possum, Corpus luteum Human, cow, sheep Male reproductive system
Testis Human, macaquet monkey Epididymis Human, wallaby, pig Prostate gland Human, macaquet monkey, wallaby, rat, guinea pig, dog Mammary gland Human, marmoset monkey, cow, rat, cow, pig,
Central nervous system
(olfactory and limbic system, coritcal areas, basal ganglia, thalamus, hypothalamus, brain stem, pituitary gland)
Human, rat
Kidney Rat, rabbit, brushtail possum
Heart Rat Vascular endothelium Human Thymus Human, rat, Adiposites Rat Pancreas Rat Adrenal gland Cow Osteoblasts Human
Breast cancer cell lines Human
40
1.9.1.1 Effects of oxytocin
The role of oxytocin in the milk ejection reflex is critical for the success of
lactation however, due to the many locations of oxytocin receptors, oxytocin has many
physiological affects that are believed to be conducive to lactation and maternal
behaviour.
1.9.1.2 Effect of oxytocin on myoepithelial cells
An irregular network of stellate shaped myoepithelial cells surrounds the milk
filled alveoli of the lactating breast. These myoepithelial cells extend onto the milk ducts
becoming more spindle shaped and parallel to the longitudinal axis of the duct
(Richardson, 1949; Linzell, 1955). The myoepithelial cell has a receptor that binds to
oxytocin (Soloff et al., 1975; van Leuwen et al., 1985). The binding of oxytocin
stimulates myoepithelial cell contraction, which in turn expels milk from the alveoli into
the larger milk ducts making it available to be removed by either the suckling infant or by
breast expression. Contraction of the myoepithelial cells of the milk ducts assists in the
propulsion of milk by causing widening and shortening of the duct.
There is a rich network of capillaries in close proximity to the myoepithelial cells
surrounding the alveoli therefore Linzell (1955) assumed that oxytocin would reach all
myoepithelial cells at approximately the same time and cause contraction of most if not
all of the alveoli. More recent studies suggest otherwise. It has been observed that not all
myoepithelial cells contract in response to oxytocin (Moore et al., 1987). De Nuccio and
41
Grosvenor (1971) found that myoepithelial cells surrounding distended alveoli were more
sensitive to oxytocin in the rat mammary gland. Furthermore there is evidence to suggest
that the regulation of myoepithelial cell contraction may depend, in part, upon the
secretory activity of the lactocytes (Moore et al., 1987; Sopel, 1995).
1.9.1.3 Effect of oxytocin on the mammary gland during suckling
The ejection of milk from the alveoli into the ductal system of the breast causes
an increase in intra-ductal pressure (Folley, 1956). The method of measuring intra-ductal
pressure was first developed in animals (Cross and Harris, 1952) and then later adapted
for women (Sica-Bianco, 1959; Cobo et al., 1967). The technique involves the insertion
of one or more polyethylene canulas into a milk duct through the nipple pore and
connecting the other end of the cannula to a pressure transducer. Analgesia may be given
to mother to reduce stress associated with the procedure (Sala et al., 1974a; Sala et al.,
1974b; Luther et al., 1974). Unfortunately, when more than one duct was cannulated (up
to 3) only the duct exhibiting the greatest change in pressure was analysed (Sala et al.,
1974a). The differences in pressures between ducts were attributed to the different
amounts of milk contained in each ductal system (Sandholm, 1968; Sala et al., 1974a). A
range of 4-10 increases in pressure (5-25 mmHg) was observed during a 10-minute
breastfeed by Cobo et al. (1967). Similarly Sala et al. (1974a) measured as many as 16
increases in pressure during a breastfeed with an average of 2 increases per minute.
Unfortunately no information was provided on the milk intake of the infants in these
studies. Alternatively a less invasive technique can be used that involves placing perspex
cylinder over the breast and holding it in place with a suction cup. The system is then
42
filled with water and a tube connecting the cylinder to a pressure transducer records
changes in mammary pressure. Although this technique gives similar results to intra-
ductal cannulation the values are a tenth of that measured with intra-ductal cannulation
(Sandholm, 1968).
. Increases in intra-ductal pressure measured during a breastfeed can be replicated
by rapid serial intravenous injections of oxytocin (Cobo et al., 1967) and are therefore
taken to be representative of milk ejection. The threshold dose of oxytocin during the first
weeks of pregnancy is up to 100 mU compared to non-pregnant women that do not
respond to doses up to 1000 mU. At approximately 18-20 weeks the dose drops
dramatically to 1 mU and there is little change in the last half of pregnancy (Sala, 1964).
This change in mammary sensitivity to oxytocin approximates the onset of lactogenesis I
(Cox et al., 1999). During lactation the dose required to produce milk ejection ranges
from as little as 0.65 mU up to 9.5 mU (Sala and Althabe, 1965; Cobo et al., 1967; Luther
et al., 1974; Sala et al., 1974a) with no change in the response to oxytocin in late lactation
(Wiederman and Stone, 1962; Cobo et al., 1967)
The most effective stimulus of milk ejection has found to be suckling (Luther et
al., 1974; Sala et al., 1974b) and milk ejection normally occurs within 60 seconds of the
start of a breastfeed. Mechanical stimulation of the nipple (Cobo et al., 1965; 1967) and
the application of negative pressure have also been shown to illicit a milk ejection
response. However, thermal stimulation (37oC), positive pressure and tactile stimulation
of the areola are ineffective in producing milk ejection. (Sala et al., 1974b)
The first pressure increase during breastfeeding is often the highest although this
is not statistically significant when compared to subsequent increases (Sala et al., 1974a).
43
The variation in amplitude may be due to the redistribution of milk through the ductal
system resulting in differing pressure losses with milk flow. Intra-ductal pressure
responses have not been shown to be related to suckling pressure (-59 to -135 mmHg) or
frequency (64-122 sucks/minute), days of lactation, infant weight, and time since the last
breastfeed in normal term infants (Luther et al., 1974). Multiparae women have
demonstrated larger increases in intra-ductal pressure compared to that of primiparae
women. This difference is assumed to be due to the increased likelihood of primiparae
women to react to stress (Sandholm, 1968).
Increases in intra-ductal pressure have been observed when a mother either sees
or hears her infant cry (Caldeyro-Barcia, 1969; Cobo et al., 1967) and prior to
breastfeeding (Sandholm, 1968) consistent with increases in maternal plasma oxytocin
levels. The frequency of spontaneous milk ejections (those between breastfeeds) tends to
increase from 1 to 9 weeks of lactation (Cobo 1993) and it has been postulated that
spontaneous milk ejections are more likely to occur when the breast is full of milk
(McNeilly and McNeilly, 1978). In support of this theory Cobo (1993) found a higher
frequency of spontaneous milk ejections in fully breastfeeding women compared to partly
breastfeeding women. Furthermore, an increase in intramammary pressure has been
shown to decrease mammary blood flow in the lactating goat (Pearl, Downey and Lepper,
1973). Therefore, it is conceivable that the milk ejection response may be stimulated as a
signal to empty the breast to ensure continued milk synthesis and avoid pathologies
associated with milk stasis such as blocked ducts, mastitis and engorgement.
44
1.9.1.4 Effect of oxytocin on the mammary gland during breast expression
Pre-stimulation of the teats of cows for approximately 30-60 seconds at the
beginning of machine milking results in an increased milk flow rate and reduced milking
times but does not affect total milk yield (Mayer et al., 1984; Bruckmaier, Rothenanger
and Blum, 1995; Bruckmaier and Blum, 1996). This effect is consistent for high and low
yielding cows (Bruckmaier and Blum, 1998) irrespective of breed (Bruckmaier,
Rothenanger and Blum, 1995).
During milking adequate stimulation must be continuously applied to maintain
oxytocin levels in order to remove the maximum amount of milk. However the residual
milk in the udder constitutes on average 10% of the total milk volume and this can be
almost but not completely removed by supraphysiological doses of oxytocin (Koshi and
Petersen, 1955). For sheep, however, pre-stimulation does not affect the milk flow pattern
because of the comparatively large fraction of milk contained in the cistern (Bruckmaier
et al., 1994) and the timing of oxytocin release is variable depending on breed
(Bruckmaier et al., 1997)
Few studies have investigated milk ejection during expression in women and none
have studied the effects of a pre-stimulation pattern incorporated into a breast pump. An
early study measured changes in intra-ductal pressure in women expressing milk with an
electric breast pump and determined that they were similar to those demonstrated during
a breastfeed (Sandholm, 1968).
Alekseev et al., 1998 have investigated milk ejection during expression with an
experimental pump that applied areola compression by measuring intra-ductal pressure
and measuring milk flow in a 150 mL graduated bottle recording volumes at 30-second
45
intervals. They found that both intra-ductal pressure and milk flow changed in pulsatile
manner at 1-2 minute intervals and the highest rates of milk flow were associated with
the greatest rises in intra-ductal pressure. Furthermore, when the tactile component was
switched off there was up to a 2 fold increase in the latency to milk ejection and up to a 2
fold reduction in milk flow rate indicating that the compression of the breast enhanced
milk removal at milk ejection. In a further study using only measurement of milk flow
(Alekseev, Omel'yanyuk and Talalaeva, 2000) 2 to 7 peaks in milk flow were
demonstrated during an expression period and the milk flow response was characteristic
for each woman. In addition women that had not breastfeed their infants in the first few
days post-partum had a significantly longer latency time until milk flow. However, it is
unclear whether this was due to either compromised milk production or difficulty in
observing small increases in milk flow as the bottle was unable to be removed from the
pump rather than an actual delay of the milk ejection reflex.
The release of oxytocin and prolactin in response to different types of breast
pumps was investigated by Zinaman et al. (1992). He found no difference in the oxytocin
response and some variability in prolactin response. However, the protocol for this study
involved drawing continuous blood samples over periods of 10 minutes giving a
measurement of the average level of oxytocin during this time. Frequent blood sampling
is necessary to detect the pulsatile release of oxytocin, therefore, any differences in the
milk ejection response to the breast pumps with regard to milk ejection and hence milk
volume may not be detected using 10 minute blood collection protocol.
46
1.9.1.5 Endocrine effects of oxytocin
During suckling insulin and glucagon are released in rats and this is sometimes
accompanied by an increase in concentration of blood glucose (Eriksson, Linden and
Uvnas-Moberg, 1987), therefore, the effect of glucagon tends to dominate the glucose
response. Administration of an oxytocin antagonist eliminates these changes
(Bjorkstrand, Eriksson and Uvnas-Moberg 1996). Since there is an increased number of
insulin receptors in the mammary gland compared to other maternal energy stores (Flint,
1982) the release of insulin during suckling may enhance the transfer of glucose to the
mammary gland for milk synthesis.
During lactation a heightened efficiency of the digestive system and anabolic
metabolism is apparent. In many species including women suckling is associated with
increased levels of gastrin and cholecystokinin whereas somatostatin levels show a slight
increase or decrease (Linden et al., 1987; Algers et al., 1991; Uvnas-Moberg, 1994;
Eriksson et al., 1994; Bjorkstrand, Eriksson and Uvnas-Moberg, 1996). Furthermore,
these changes are abolished by vagotomy (Algers et al., 1991) and blocked by oxytocin
antagonists. Acute doses of oxytocin will increase concentrations of cholecystokinin
(Bjorkstrand et al., 1996) and growth hormone in the blood of rats (Bjorkstrand, Hulting
and Uvnas-Moberg, 1997).
1.9.1.6 Physiological effects of oxytocin
Hyperphagia is considered to be an adaptation of lactation to ensure adequate
nourishment for the production of milk. It is well documented that lactating rats increase
their food intake in response to the sucking stimulus (Fleming, 1976) and if the milk
47
ejection reflex is interrupted in the midbrain of lactating rats they will reduce their food
intake to the same level as non-lactating rats (Hansen and Kohler, 1984; Hansen and
Ferreira, 1986). Subsequently oxytocin administered by the intracerbroventricular route
to non-lactating rats causes an increase in their food consumption (Bjorkstrand and
Uvnas-Moberg, 1996). One study, however, has shown a short-term decrease in food
intake in response to oxytocin (Arletti, Benelli and Bertolini, 1989). This response would
be expected to disappear with longer observation periods (Bjorkstrand and Uvnas-
Moberg, 1996). Furthermore, a recent study suggests that oxytocin may increase weight
gain with a restricted food intake (Uvnas-Moberg, Alster and Petersson, 1996; Uvnas-
Moberg et al., 1998). This result is likely to be due to the increased release of insulin
which enhances digestion by slowing the passage of food through the digestive system.
Repeated exposure to suckling in lactating rats results in decreased blood pressure
compared to non-lactating rats (Uvnas-Moberg, 1996; Uvnas-Moberg and Eriksson,
1996). Similarly repeated doses of oxytocin given to non-lactating rats induces a long-
term decrease in blood pressure (Petersson et al., 1996) as does postnatal ventral stroking
(Holst, Uvnas-Moberg and Petersson, 2002). Similar findings have been demonstrated in
lactating women who show a significant decrease in cortisol levels and blood pressure
during and after a breastfeed (Nissen et al., 1996). In addition when breastfeeding and
bottle-feeding women are subjected to physical stress, breastfeeding women show a
smaller rise in cortisol than bottle-feeding women (Altemus et al., 1995). Women with
panic disorder have experienced a reduction in symptoms during lactation (Klein,
Skrobola and Garfinkel, 1995). These may be adaptive changes that occur during
lactation to enhance maternal behaviour.
48
The surface temperature of the mammary gland increases in response to oxytocin.
This effect is due to vasodilation of the superficial blood vessels of the skin (Eriksson et
al., 1996) and is likely to assist in the transfer of heat to the young during suckling.
Oxytocin when administered to non-lactating rats has a sedative effect (Uvnas-
Moberg et al., 1994). Also pain thresholds related to heat and mechanical stimuli are
increased. This effect is subsequently eliminated by administration of an oxytocin
antagonist (Agren et al., 1995).
1.9.1.7 Behavioural effects
Oxytocin has been shown to stimulate the maternal behaviour including bonding
in lactating animals (Kendrick, Keverne and Baldwin, 1987; Keverne and Kendrick,
1992) and decreased reactions to stress (Hansen and Ferreira, 1986). There is also
evidence to suggest that oxytocin may have antidepressant effects (Windle et al., 1997).
Furthermore, oxytocin has been shown to mediate the antidepressant effects of selective
serotonin reuptake inhibitors in rats (Uvnas-Moberg et al., 1999). Blood pressure and
cortisol levels decrease in women during breastfeeding (Amico, Johnston and Vagnucci,
1994; Nissen et al., 1996). In addition high levels of basal oxytocin have been associated
with reduced anxiety and higher tolerance to monotonous tasks in lactating women at 4
days post partum (Uvnas-Moberg et al., 1990b). These traits are more pronounced in
women that had experienced a vaginal delivery as opposed to a cesarean delivery. It is
also of note that an increased number of pulses of oxytocin are related to increased social
interaction and desirability (Nissen et al., 1998). Therefore, oxytocin is considered to
have a role in maternal behaviour and temperament. Thus it can also be concluded from
49
these physiological responses that breastfeeding in women is likely to be a social rather
than solitary activity.
Oxytocin has also been shown to have to have effects on many other types of
behaviour and these are detailed in the Table 1.2.
Table 1.2 Actions of oxytocin on behaviours of different species. ↑ denotes an increase in the
behaviour and ↓ a decrease in the behaviour. NE represents no effect. (Adapted from Gimpl and Fahrenholz, 2001)
Behaviours Species:
Rat Mouse Prairie vole Sheep Human
Maternal behaviour ↑ ↑ ? ↑ ? Female sexual behaviour ↑ NE ↑ or ↓ ↓ ↑ Male sexual behaviour ↑ or ↓ NE ↓ ? ↑ Female affiliative behaviour ? NE ↑ ↑ ? Male affiliative behaviour ↑ NE ↑ or NE ? ? (Auto)grooming ↑ ↑ ↑ ? ? Social memory ↑ ↑ ↑ ↑ ? Male aggression ? ↑ ↑ or NE ? ? Female aggression ↑ or ↓ ? ↓ ? ? Nociception ↓ ↓ ? ? ↓ Anxiety ↓ ↓ ? ? ? Feeding ↓ ? ? ? ? Memory and learning ↓ ↑ or ↓ ? ? ↓ Tolerance to opiates ↓ ↓ ? ? ?
1.9.1.8 Effect of exogenous oxytocin
Early studies determined that the minimum dose of intranasal oxytocin required to
produce milk ejection (measured by an increase in intra-ductal pressure) was
approximately 2 IU (Wiederman and Stone 1962; Sandholm, 1968). However, one dose
produced repeated increases in intra-ductal pressure for 10 to 20 minutes. With increasing
doses the duration of the effect increased (Sandholm, 1968). In animals intravenous doses
50
of oxytocin prior to milking has increased milk yield. This is presumed to be due to either
complete removal of milk and hence the feedback inhibitor of lactation resulting either in
a decrease in the involution of lactocytes (Nostrand et al., 1991; Ballou et al., 1993) or
that the milk ejection reflex is enhanced by supraphysiological does of oxytocin (Heap et
al., 1986; Knight, 1994). A recent Cochrane review of the literature found that sublingual
and buccal doses of oxytocin (10-100 IU) increased in milk production in women.
However, they encouraged appropriate support and information for the breastfeeding
mother prior to considering oxytocin to augment lactation (Renfrew, Lang and
Woolridge, 2002). Caution is likely to be wise in view of a recent study finding that
chronic oxytocin treatment of diary cows reduced milk ejection measured as a decrease in
the amount of milk removed during milking with a spontaneous milk ejection
(Bruckmaier, 2003).
1.9.1.9 Other effects of oxytocin
Oxytocin has a wide range of effects consistent with its numerous receptor locations
in the body. The known effects of oxytocin are detailed in Table 1.3.
Table 1.3 Additional effects of oxytocin on different species
Effects
Species
Increased milk production Cow, goat, sheep Analgesia Human, rat, mouse, dog Cardiovascular effects Human, rat, frog, snake Thymus and T-cell differentiation Human, rat Peripheral dilation/constriction Human, rat Natriuresis Rat, rabbit Thermoregulation Rat
51
1.10 Inhibition of milk ejection
Either complete or incomplete inhibition of milk ejection results in the
accumulation of residual milk in the mammary gland resulting eventually in diminished
milk synthesis (Wilde and Knight, 1990). This response can be explained by the action of
an autocrine feedback mechanism involving a peptide in milk called Feedback Inhibitor
of Lactation (FIL). Evidence for the effects of FIL has been found in various species
including cows and goats etc (Wilde et al., 1995). FIL has been shown to rapidly reduce
milk secretion (Wilde et al., 1995; Peaker and Wilde, 1996) as well as cause diminished
cell differentiation in both cell culture (Wilde, Blatchford and Peaker, 1991) and lactating
animals (Wilde, Calvert and Peaker, 1988; Li et al., 1999). The degree of inhibition of
milk synthesis is related to the concentration of FIL in the milk (Wilde et al., 1995). In
addition, Daly et al. (1993) has shown that as the breast is emptied of milk the rate of
milk synthesis increases providing evidence for local control of milk synthesis in women.
Unfortunately there are few studies that have investigated the role of long-term either
complete or incomplete inhibition of milk ejection in lactation failure in women.
However, a recent study of Sahiwal Fresian cattle showed 30% of cows suffer a severe
reduction in milk yield associated with lactation failure within 8 weeks postpartum. This
was associated with high residual milk volumes of more than 25% of the total milk yield.
The decrease in milk production was attributed to a failure of milk ejection as all of the
cows studied had similar mammary anatomy and storage characteristics. In addition those
in lactation failure had a history of lactation failure in previous lactations suggesting a
possible genetic influence (Murugaiyah et al., 2001).
52
1.10.1 Factors that inhibit milk ejection
Certain factors have been shown to reduce or eliminate milk ejection by either
inhibiting the central release of oxytocin or by restricting the amount of oxytocin
reaching the mammary gland.
1.10.1.1 Stress
The effects of stress on milk ejection in animals are well documented (Ely and
Petersen, 1941). For example, the milking of dairy cows in unfamiliar surroundings
inhibits the release of oxytocin and increases levels of cortisol resulting in the decreased
milk yield (Varner et al., 1983). Maternal stress appears to interfere with the release of
oxytocin (Lincoln and Plaisley, 1982; Crowley and Armstrong, 1992; Dewey, 2001)
where as adrenaline inhibits milk ejection peripherally (Cross 1953). A reduction in the
amount of oxytocin reaching the myoepithelial cells may be due to vascular contraction.
In 1948 Newton and Newton showed that 3 different types of stressors (cold, pain
and electric shocks) applied to one woman reduced the amount of milk transferred during
a breastfeed by 59% of that transferred with no stress. When pictocin was injected prior
to the stressors milk intake of the infant was similar to that on the days that no stress was
applied. Therefore, Newton and Newton concluded that milk secretion was inhibited by
stress due to inhibition of milk ejection. Similarly in another study 22 women were
randomly exposed to control, mental stress and noise stress during breastfeeding.
Although no difference was found in either milk intake or prolactin concentrations the
frequency of oxytocin pulses were significantly lower in those women exposed to mental
(43%) and noise stress (52%) (Udea et al., 1994). Unfortunately it was assumed that the
53
infants in the control group normally received the same amount of milk as the infants in
the treatment groups.
Mothers of pre-term infants experience a large amount of stress associated with
the hospitalization and the expression of milk for their compromised infants. The effect
of this stress was shown in a study that gave mothers of pre-term infants relaxation
exercises to practice once a day, particularly prior to milk expression for one week. These
mothers demonstrated a significant increase in the amount of milk expressed for one
expression compared to women that did not use the relaxation exercises (Feher et al.,
1989).
To date the studies investigating the effect of stress on milk ejection have used
severe stressors that are not normally encountered in an everyday setting. Historically the
milk ejection reflex appears to be quite resilient as women successfully breastfeed their
infants during times of major conflict. In contrast subtle stresses that undermine a
mothers’ confidence such as conflicting advice and the stress of the hospital environment,
particularly for mothers of a premature infants, have been associated with poor milk
production (Morton, 2003; McGuire and Wood, 2003).
1.9.1.1 Alcohol
An early study observed that small amounts of alcohol increased the likelihood of
milk ejection while using a breast pump (Newton and Newton, 1948). However, in a later
study alcohol was shown to inhibit milk ejection, measured by intra-ductal pressure, in a
dose dependent manner. Doses of alcohol resulting in a blood level less than 0.1%, did
not affect the increases in intra-ductal pressure but in some cases uterine contractions
54
were eliminated suggesting that the myoepithelial cells in the breast are more sensitive to
oxytocin than the myometrium (Cobo, 1973). A 3 g/kg maternal dose of alcohol has been
shown to significantly reduce both the amount of milk expressed in the short-term
(Mennella, 1998) and the amount of milk consumed by the breastfeeding infant
(Mennella, 2001). In response to maternal alcohol intake infants were found to consume
less milk in the first 4 hours after maternal alcohol exposure and then exhibited a
compensatory increase in milk consumption in the following 8-12 hours that was
associated with an increased feeding frequency (Mennella, 2001). Alcohol has been
shown to inhibit oxytocin release during suckling in the lactating rat (Subramanian, 1999)
and doses of 50 mL of ethyl alcohol eliminate the oxytocin rise caused by breast
stimulation in non-lactating women (Coiro et al., 1992). Furthermore, studies in rats
suggest that impaired lactation as a result of chronic alcohol administration is due to a
decrease in oxytocin release rather than a reduced release of prolactin during suckling
(Heil and Subramanian, 2000). In this instance one would expect that poor milk removal
would result in diminished milk production (See section 1.9).
1.9.1.2 Opiates
Opioids such as morphine have been shown to inhibit the release of oxytocin
(milk ejection) in rats (Bicknell and Leng, 1982; Rayner et al., 1995). The administration
of naloxone an opioid antagonist reverses the effects of opioids by restoring the central
release of oxytocin in both rats (Douglas et al., 1998) and sows (Lawrence et al., 1992).
Little is known about the effects of opioids in lactating women.
55
1.9.1.4 Relaxin
Relaxin has important physiological affects on the pregnant mother that prepare
her for birth. Exogenous relaxin given to rodents during lactation has been shown to
inhibit the milk ejection reflex by affecting the central release of oxytocin (Summerlee et
al., 1984; O’Byrne et al., 1986). Furthermore, during suckling even though the basal
oxytocin levels increase the pulsatile release of oxytocin is obliterated (Summerlee,
O’Byrne and Poterski, 1998). In the sow relaxin is released during suckling and is
thought to reverse the oxytocin induced myoepithelial cell contraction thus resulting in
the very short period of milk availability (10-20 seconds) at milk ejection (Afele, et al.,
1979).
1.9.1.5 Breast surgery
Mammoplasty reduction surgery has been associated with impaired lactation.
Although extensive studies have not been performed regarding the success of lactation
post-mammoplasty a lower proportion of women are able to successfully breastfeed for
as long as women that have not undergone surgery. Partial breastfeeding is also
considered possible in a number of women post surgery (Marshall, Callan and Nicholson,
1994). The inability to successfully lactate has been attributed to disruption of the nerves
innervating the nipple, which subsequently impairs the milk ejection reflex. However, it
is possible for milk ejection to be conditioned and this could compensate for the
56
disruption of the neural pathway therefore the alternate possibility that the cutting of
some of the main ducts in the breast during surgery could be a major contributor to the
inhibition of milk flow must be considered. In addition the proportion of glandular tissue
removed at surgery may also affect milk production (Marshall, Callan and Nicholson,
1994).
Information regarding successful breastfeeding after breast augmentation is scant.
Two studies have shown delayed initiation of lactogenesis II and insufficient lactation in
more than half of the women studied (Hughes and Owen, 1993; Hurst, 1996). Disruption
of nerve fibres, pressure of the implant upon glandular and ductal structures of the breast
and insufficient glandular tissue prior to surgery have all been suggested as possible
factors contributing to insufficient milk production in these women.
57
Aims
The success of lactation depends in part upon both the full development of the
synthetic tissue of the breast and facilitation of milk ejection. Recent technological
advances in ultrasound imaging has enabled the resolution of small structures such as the
milk ducts of the breast and the aim of this thesis was to use ultrasound to investigate the
anatomy and ductal physiology in the normal lactating human breast (1-6 months
lactation). My studies have had two main objectives.
The main objective of the first part of the study was to quantify the gross anatomy
of the normal lactating breast in women.
Specifically my investigations aimed to:
1) measure and describe characteristics of the main portion of the ductal
system of the breast such as duct diameter, branching of the ducts and
their location in the breast.
2) develop a method using ultrasound imaging to describe and semi-
quantify the distribution of the two major tissues (glandular and
adipose tissue) of the breast.
3) determine if the glandular tissue was related to either milk production
or calculated storage capacities of the breast.
The main objective of the second part of the study was to determine if ultrasound
imaging could detect changes in milk duct diameter as a consequence of milk ejection
58
and whether this technique could be used to investigate the physiology of milk ejection in
breastfeeding and expressing women with normal lactations (1-6 months).
Specifically my investigations aimed to:
4) develop a non-invasive technique using ultrasound imaging to identify
milk ejection by showing changes in milk duct diameter.
5) determine if changes in duct diameter occurred in the absence of
stimulation of the nipple (resting state).
6) determine if there was a relationship between changes in duct diameter
and milk production, milk intake of the infant, amount of milk
expressed and degree of fullness of the breast during both
breastfeeding and breast expression.
59
60
Chapter 2
Materials and Methods
2.1 Materials
2.1.1 Participants
Exclusively breastfeeding mothers of healthy term infants (1-6 months of age)
were recruited from either the West Australian Branch of the Australian Breastfeeding
Association, private health care centres or were contacts of women that participated in the
studies. All mothers supplied written, informed consent to participate in the studies,
which were approved by the Human Research Ethics Committee, The University of
Western Australia. Prior to each study, the procedure was explained to the mother and a
written explanation was provided. At each visit mothers were asked to confirm
exclusivity of breastfeeding and that throughout the study periods breastfeeding
behaviour was not altered. Maternal and infant characteristics for the mothers that
participated in the studies in Chapter 3, 4 and 5 are summarised in Table 2.1. Mothers 1-
22 were studied in Chapter 3, mothers 1-17 and 22-28 were studied in Chapter 4 and
mothers 1-28 were studied in Chapter 5. The characteristics for the mothers that
participated in the study in Chapter 6 are summarised in Table 2.2.
Mother
Mothers age
Parity
Infants age
24-hr milk production (g)
Average
Breastfeed(g)
Storage Capacity
Frequency of breastfeeds 24-hr
(years) (weeks) Left Right Left Right Left Right Left Right 1 38 1 23 236 299 53 56 184 223 5 5 2 36 1 20 419 430 106 113 212 232 4 4 3 23 1 22 340 553 78 98 150 174 5 6 4 26 1 17 446 556 80 98 192 177 6 6 5 26 3 22 196 176 25 23 64 56 7 6 6 25 2 12 455 488 90 113 245 192 5 5 7 32 1 22 426 408 93 74 236 223 4 5 8 28 1 20 239 159 62 34 157 86 4 5 9 32 1 18 566 309 83 43 307 187 6 6 10 26 2 12 199 338 67 108 113 182 4 3 11 33 4 12 417 318 91 115 137 199 4 4 12 _ 2 13 401 410 59 53 102 121 7 8 13 26 2 16 377 436 111 119 209 195 4 4 14 30 1 23 371 586 75 121 135 202 5 5 15 28 2 7 321 368 57 48 195 142 7 7 16 32 2 7 383 598 64 98 132 355 5 5 17 22 1 23 420 472 46 49 149 171 8 9 18 30 1 19 418 341 61 44 155 136 7 8 19 26 3 22 497 358 116 99 180 154 5 4 20 32 2 6 502 379 54 43 199 161 7 7 21 32 1 15 531 570 110 91 193 197 4 4 22 37 2 26 297 319 84 84 167 118 4 4 23 26 2 17 353 397 53 62 95 143 7 7 24 36 3 19 387 407 91 97 147 147 5 5 25 37 4 26 257 417 46 76 159 134 6 6 26 38 1 15 215 337 32 58 131 160 5 5 27 33 1 22 454 518 52 53 193 201 8 9 28 36 2 11 399 332 100 78 189 184 5 5
Table 2.1 Maternal and infant characteristics of mothers studied in Chapters 3 (mothers 1-22), 4 (mothers 1-17 and 22-28) and 6 (mothers 1-28)
61
Mother Mothers Age
Parity Infant age
24-hr milk (g)
production
Mean feed volume
volume (g)
Frequency
feed/ 24 hr
(years) (weeks) Left Right Left Right Left Right 1 34 1 24 454 518 56 55 9 9 2 28 2 23 262 275 59 81 6 4 3 33 2 26 260 615 57 122 5 7 4 31 2 23 300 454 53 66 6 8 5 32 2 16 386 322 66 60 6 5 6 35 2 8 348 474 75 89 5 4 7 29 2 13 177 383 26 44 7 8 8 33 2 12 383 598 64 98 5 5 9 35 1 7 309 270 31 27 9 8 10 28 3 15 241 496 58 91 5 5 11 33 1 15 531 570 137 116 4 4
Table 2.2 Maternal and infant characteristics of mothers studied in Chapter 6.
62
2.1.2 Physics and Principles of Ultrasound
The sound waves used in medical ultrasound lie in the frequency range of 1-15
MHz. Ultrasound transducers emit sound waves and as they pass through tissue they are
attenuated by absorption, reflection and scattering of the wave. The echoes that are
reflected back to the transducer are assigned a shade of grey which is amplitude
dependent and these echoes build up an image that is displayed on a monitor. This is
termed B-mode or Brightness mode ultrasound.
Ultrasound transducers are comprised of piezoelectric elements such as lead
zirconate titanate. The elements of the transducer are excited by a short burst of electric
current that causes it to vibrate at its resonance frequency. The frequency of the
transducer is determined by the thickness of the element and the thinner the element the
higher the frequency of the transducer. For example a 0.4mm element will produce sound
waves of 5.0 MHz and a 0.2mm thick element will produce sound waves of 10.0 MHz
frequency (Kremkau, 1998). A backing material placed behind the elements damps the
vibration of the element very soon after the excitation by the electrical pulse thereby
stopping backward propagation of the sound wave into the transducer. Impedance-
matching layers in front of the elements improve the ability of the transducer to transmit
sound waves into soft tissue as well as detect very weak echoes.
The elements of the transducer emit a sound pulse containing energy at the
resonance frequency however this pulse also includes a range of frequencies above and
below the resonance frequency and this is termed the frequency bandwidth. Shorter pulse
63
durations result in a wider frequency bandwidth. Transducers with a wide bandwidth are
able to be operated at more than one frequency (Kremkau, 1998).
For sound waves to be transmitted through tissue an aqueous gel must be used as
a coupling medium. This is because the thin layer of air between the transducer and the
skin has low impedance and will reflect most of the ultrasound (Kremkau, 1998).
The sensitivity or resolution of the ultrasound system is defined as the ability of
the system to resolve two closely spaced reflectors. Axial or depth resolution is the ability
of the system to resolve two relectors along the axis of the ultrasound beam. Axial
resolution increases as frequency increases however as a result penetration of the
ultrasound beam decreases (Kremkau, 1998). In addition, a shorter the pulse duration will
result in better axial resolution. The axial resolution of a 10.0 MHz transducer (two-cycle
pulse) is approximately 0.15mm and will penetrate to a depth of 6cm (Kremkau, 1998).
Lateral resolution refers to the ability to resolve two reflectors perpendicular to the axis
of the ultrasound beam. Lateral resolution is affected by the width of the ultrasound beam
in that the narrower the beam width the better the lateral resolution. Since an unfocussed
ultrasound beam naturally diverges focusing the beam will improve lateral resolution. In
addition, higher frequency beams have less beam divergence. The resolution of the image
is also dependent on the ability of the monitor to display the echoes (Kremkau, 1998).
The sensitivity of the ultrasound system can be tested using an ultrasound phantom.
Measurements of axial and lateral resolution can be made by measuring the shortest
distance between 2 structures within the phantom both along and perpendicular to the
axis of the ultrasound beam.
64
Several types of array transducers (multi-element) are available. Typically these
transducers are comprised of an array of many elements and can be linear (sequential),
phased (electronically sectored) and annular (circular). Each of the elements in the array
is excited individually and the received echoes are detected and amplified individually.
Arrays are focused electronically and often multiple focal points are available. Real-time
scanning is achieved by steering the beam through the tissue rapidly and automatically so
that many frames can be presented in a second of time (typically 30 frames/second).
Array transducers can also be focused in during the transmission and receiving of echoes.
2.1.2.1 Ultrasound controls
The quality of the ultrasound image can be influenced by the adjustment of a
number of controls. The input power can be set and it is considered that power levels
should be set at the level required to produce satisfactory images. Attenuation of the
ultrasound beam as it passes through soft tissue is compensated for by the time-gain
compensation curve enables one to amplify echoes that are received from a greater depth.
The gain control however changes the brightness of the whole image by increasing or
decreasing the ratio of the amplified output to the electrical power input. The dynamic
range essentially affects the contrast of the image by changing the ratio of the largest
power to the smallest power of the systems capabilities.
2.1.2.3 Artifacts
During ultrasound imaging artifacts may occur. An artifact is considered to be any
structure in the image that is not indicative of the actual structure scanned (Kremkau,
65
1998). Ultrasound systems work on the assumption that sound travels in a straight line
and that received echoes originate along the axis of the beam. Therefore it is possible that
the sound beam maybe refracted by a tissue boundary and cause received echoes to be
wrongly positioned on the display. Ultrasound criteria for the identification of breast
cysts include refraction caused by the curved lateral walls of the cyst. Excessive
attenuation of the beam by a highly reflecting or absorbing structure such as bone will
result in decreased amplitude of the received echoes behind the structure. As a result a
dark shadow is displayed posterior to the structure. Enhancement is the opposite effect to
shadowing and occurs with a structure that only attenuates the beam to a small degree
resulting in the echoes behind the structure being displayed with excessively high
amplitudes. Enhancement artifact is also used excessively to identify cystic structures in
the breast as the fluid within the cyst presents minimum attenuation of the ultrasound
beam compared to surrounding structures in the breast. The ultrasound beam has a finite
thickness perpendicular to the scan plane and this results in a slice or section thickness
artifact. Echoes that are received from within the beam width are displayed as if they
originate from the centre of the ultrasound beam. For example echoes maybe displayed
within a small cyst that have arisen from surrounding tissue.
2.1.2.3 Basic principles of breast ultrasound
The breast is a very superficial structure therefore a high frequency transducer in
the 7.5-12 MHz range is used. These frequencies provide good image resolution and
adequate penetration of the breast tissues. Linear array transducers are used to give good
66
contact with the breast and a wider field of view as compared to a phased array. The
woman is place in the oblique position turned away from the side to be examined and
supported by a foam pad and her arm is raised above the head. This position reduces the
breast thickness allowing better visualization of the deeper tissues. Often multiple foci
are used depending on the depth of the breast tissue. Compression of the tissue is useful
to reduce refraction and scattering from normal structures such as the Cooper’s ligaments
however too much compression may displace lesions or deform structures thereby
hindering image interpretation.
2.1.3 Ultrasound equipment
The mothers were scanned at the Breast Feeding Centre of Western Australia
using a dedicated ultrasound machine (Acuson XP10, Acuson Corp, USA) with a linear
array transducer (10-5 MHz). Although a dedicated stand-off pad was not available for
this ultrasound machine attempts were made to use a specialized mobile stand-off pad
however due the position of the mother (upright) and the size of the pad problems
associated with movement and compression of the milk duct impeded imaging. The
length of the transducer face is 40mm. The superficial organ preset was utilized and
adjustments were made to the gain, dynamic range and time gain compensation to
optimise the image. Average values for the ultrasound setting are given in Table 2.3. All
ultrasound scans were videotaped for later analysis and play back facilities on the
ultrasound machine were used to analyze ultrasound images.
67
Table 2.3 B-mode settings for the Acuson XP10 Ultrasound Machine
Setting Value Focus Single for milk ducts
3 for anatomical scans Gain 15 db Dynamic Range 57 db Persistence 3 Power 9 db Time Gain Compensation Curve Gentle slope
2.1.4 Validation of ultrasound equipment
The performance of the ultrasound system with the linear array transducer (10-5
MHz) was verified using a multi purpose phantom (model 539, 1992, ATS Laboratories,
Inc Connecticut, USA).
The dead zone refers to the distance from the transducer at which the first echo
within the phantom is imaged. In practice this relates to the first structure that can be
resolved under the transducer. The dead zone measured 2 mm (limit 10 mm).
The ability of the ultrasound system to resolve two closely spaced reflectors is
termed the resolution. Axial resolution refers to resolution along the axis of the sound
beam (depth resolution) whereas lateral resolution is the resolution perpendicular to the
axis of the sound beam (beam width). The system was capable of imaging the most
closely spaced reflectors (1.0 mm apart) within the phantom in both the axial and lateral
planes.
The functional resolution is the ability of the system to display the depth of an
anechoic (absence of echoes) structure within the test phantom. This transducer and
ultrasound system was able to resolve all of the anechoic structures (2, 3, 4, 6 and 8 mm
in diameter) in the phantom at a depth of 20 mm. Therefore the axial, lateral and
68
functional resolution of this transducer was at least as sensitive as the lowest sensitivity
calibrations in the ultrasound phantom.
The focal zone is the portion of the sound beam at which the lateral resolution is
the greatest. The focal zone width was 1.6 mm at a depth of 10 mm and 1.8 mm at a
depth of 20 mm.
2.1.5 Ultrasound gel
Parker Ultrasonic Gel (Fairfield, USA) was applied to the breast for all ultrasound
scans to enhance transmission of the ultrasound beam through the skin.
2.1.6 Condoms
In order to determine the echogenicity of breast milk Ansell non-lubricated
condoms (Ansell Medical Ltd., Richmond, New South Wales, Australia) were used to
hold samples of breast milk and a mixture of olive oil and water for a comparison of the
echogenicity of milk fat and olive oil emulsions
.
2.1.7 Electric Breast Pump
For studies investigating the milk ejection response to breast pump an
experimental computer driven electric breast pump (B2000, Medela AG, Baar,
Switzerland) equipped with standard breast shield and bottle was used. This pump was
able to provide an adjustable peak vacuum of 0 to –280 mm Hg with cycling times of
20–120 cycles per minute.
69
For the study of the response of the breast to seven different stimulation patterns
(Chapter 5) the characteristics of the patterns are given in Table 2.3.
Table 2.4 Characteristics of the stimulation patterns* for Chapter 5 A B C D E F G Frequency (cycles/min) 45 76 105 110 125 125 105 Vacuum setting (mm Hg) 0% -136 -87 -85 -57 -45 -80 -85 24% -174 -128 -112 -84 -67 -104 -112 50% -204 -152 -132 -99 -80 -120 -132 100% -273 -239 -188 -166 -138 -166 -188
*The vacuums were recorded at the nadir of each cycle using a closed system
.
For the study investigating the use of ultrasound to characterise milk ejection in women
using an electric breast pump (Chapter 6) the pump was programmed to provide either
the commercially available Medela Classic pumping pattern (47 cycles/minute; Figure
2.1) or an experimental 3-phase pumping pattern consisting of a stimulation phase (pre-
milk ejection, 120 cycles/minute) and expression phases A (0-2 minutes post-milk
ejection, 20 cycles/minute) and B (2-10 minutes post-milk ejection, range 55-78
cycles/minute; Figure 2.2).
70
-140
-120
-100
-80
-60
-40
-20
0
0 1 2 3 4 5 6 7 8 9 10Time (s)
Vacu
um
(m
m H
g)
Time (s) 0 1 2 3 4 5 6 7 8 9 10
0
20
40
60
80
100
120
140
Vacuum (mm Hg)
Figure 2.1 The vacuum profile of the Classic pumping pattern (47 cycles/minute) for Chapter 6
-250
-200
-150
-100
-50
0
0 2 4 6 8 10 12 14 16 18Time (s)
122 124 126 128
)m
m H
gcu
um
(V
a
A B C
Vacuum (mmHg)
C B
250
200 A
150
100
0
50
Time (s) 0 2 4 6 8 10 122 124 126 128
Figure 2.2 The vacuum profile of the 3-Phase pumping pattern consisting of A: stimulation phase
(pre-milk ejection, 120 cycles/minute) and B: expression phase A (0-2 minutes post-milk ejection, 20 cycles/minute) and C: expression phase B (2-10 minutes post-milk ejection, range 55-78 cycles/minute). This pattern was tested in Chapter 6
71
2.1 Methods
2.2.1 Ultrasound imaging of breast milk
The echogenicity of breast milk was determined by placing three samples of
breast milk in Ansell non-lubricated condoms (Ansell Medical Ltd., United Kingdom)
and scanning before and after the milk fat (cream) had separated under the influence of
gravity. After separation, the cream layer was measured in the condom using both
vernier and ultrasound calipers. In addition, three condoms containing olive oil
emulsions and water were scanned as described above.
2.2.2 Ultrasound imaging of the breast tissues
On the ultrasound image the skin (SK) appears as an echogenic (bright echoes, white)
line at the top of the image where as the adipose tissue appears as hypoechoic (less
echoes, dark grey) tissue. The adipose tissue is located directly under the skin
(subcutaneous fat; S), within the glandular tissue (intraglandular fat; I) and behind the
glandular tissue in front of the pectoral muscles (retromammary fat; R). The glandular
tissue (G) appears as echogenic tissue (bright echoes) within the breast (Figure 2.3)
and its intensity decreases as the breast is drained of milk presumably due to changes
in the overall content of milk fat and the rearrangement of the structures of the
secretory tissue in the drained breast.
72
* *N SFSK
10 mm
▲
S
G
G
R
SK
10 mm
G
R
SKS
G
I ID D
Figure 2.3 Ultrasound image of tissues of the lactating breast. The skin (SK) is shown as an
echogenic (bright) line at the top of the image. The subcutaneous fat (S) is less echogenic and situated below the skin. The intraglandular fat (I) is of similar echogenicity to the subcutaneous fat. The glandular tissue is echogenic (G) while the milk duct (←D) appears as a hypeoechoic (low echo) tubular structure. The retromammary fat (R) is a thin hypoechoic band along the chest wall.
The mothers were seated comfortably for all ultrasound scans and their arm was
placed on the top of their head to enable scanning of the axillary region and inferior
portion of the breast. Initially a real time scan was performed of the whole breast to
subjectively estimate the distribution of glandular and adipose tissue and ensure there
were no obvious abnormalities. Using the clock face method, which divides the breast
into sectors according to the face of the clock as the observer is looking at the breast,
(Mendelson, 1998; Figure 2.4a) images of the breast tissue were documented along 8
radii (12.00, 1.30, 3.00, 4.30, 6.00, 7.30, 9.00 and 10.30 o’clock) of the breast. The
images were taken sequentially along the particular axis from the base of the nipple to
73
the outer portion of the breast until the glandular tissue was no longer visualised
(Figure 2.4b). Three to four images were documented along the radius, which
corresponds with scans measuring up to 120 to 150 mm from the base of the nipple
depending on the extent of the extension of the glandular tissue. Each image included
all of the breast tissue from the skin to the chest wall (pectoral muscles overlying the
ribs) and the full extent of glandular tissue from the nipple to the periphery of the
breast.
1.30
4.30 7.30
10.30
12
9
6
3
(a) (b)
Figure 2.4 (a) The right breast divided into sectors according to the face of the clock. (b) The arrows show the four images documented along the 3 o’clock axis. The number of images was dependent on the size of the breast, the axis measured and the extent of the glandular tissue.
The ultrasound images were recorded on videotape for later analysis and
annotated using the clock face method. Measurements were made of the depth of
glandular tissue (G), subcutaneous (S), intraglandular (I) and retromammary fat (R) at 30
mm intervals along the 8 radii of the breast from the base of the nipple 0 mm (Figure
2.5).
74
120 mm
90 mm
60 mm
30 mm
0 mm
Figure 2.5 A sagittal section of the breast showing the intervals at which measurements were made of the depths of breast tissues along the 12 o’clock axis.
(a)
Glandular tissue (G)
Nipple
Skin (SK)
Intraglandular fat (I) Subcutaneous fat (S)
Retromammary fat (R)
Pectoral muscle
75
(b)
R
(c)
S
I1
I2
G1
G2
G3
76
XX
(d)
Figure 2.6 (a) Sagittal section of the breast demonstrating the breast structures (b) Diagram demonstrating the measurements of depths of the subcutaneous fat (S) and retromammary fat (R) at a 30 mm radius from the base of the nipple (c) Diagram demonstrating multiple measurements of glandular tissue at a 30 mm radius from the base of the nipple (d) diagram showing the measurement (X) which represents the total of the glandular tissue and intraglandular fat measured at a 30 mm radius from the base of the nipple.
The subcutaneous fat was measured as shown in Figure 2.6b and the total depth of the
subcutaneous fat (above the glandular tissue) at 30 mm intervals along each radius
was calculated as follows (example 12 o’clock with glandular tissue extending out to
150 mm):
77
(1) S12o’c = S0 + S30 + S60 + S90 + S120 + S150
The cumulative total of subcutaneous fat in the entire breast was therefore the sum of
the total subcutaneous fat of each axis as follows:
(2) STOT = S12o’c + S1.30o’c + S3o’c + S4.30 o’c + S6o’c + S7.30o’c + S9o’c + S10.30o’c
The retromammary fat was measured as shown in Figure 2.6b and the total depth of
the retromammary fat at 30 mm intervals along each axis was calculated by
substituting S with R in equation (1). Similarly, the cumulative total of retromammary
fat in the entire breast was calculated by substituting S with R in equation (2).
The glandular tissue and intraglandular fat was measured as shown in Figure 2.6c,
however, an additional measurement X as shown in Figure 2.6d was made to check
for operator error introduced by the placement of measurement calipers on the
ultrasound image. The measurement “X” was made from the posterior border of the
subcutaneous fat to the anterior border of the retromammary fat (Figure 2.6b) and
compared to the sum of the glandular and intraglandular tissue measured separately
(equation 3). If there was a discrepancy of more than 5 mm the measurements were
repeated.
(3) X – [(G1 + G2 ... Gn) + (I1 + I2 ... In))] ≤ 5 mm
78
Where for example G1 + G2 ... Gn represents the depth of the glandular
tissue at the particular 30 mm intersection measured and I1 + I2 ... In
represents the depth of the intraglandular fat at the same 30 mm
intersection measured (Figure 2.6c).
The total depth of the glandular tissue at 30 mm intervals along each axis was
then calculated by substituting S with G in equation (1) and the cumulative total of
glandular tissue in the entire breast was calculated by substituting S with G in equation
(2). The total depth of the intraglandular fat at 30 mm intervals along each axis and the
cumulative total of intraglandular tissue in the entire breast was calculated using
equations (1) and (2) and substituting S with I.
To describe the distribution of tissues within the breast, cumulative totals of all
tissues were calculated (T) by summing all of the measurements made in the breast at 0,
30, 60 and at 90,120 and 150 mm distances from the base of the nipple depending on the
size of the breast, the axis measured and the extent of the glandular tissue.
(4) T = GTOT + STOT + ITOT + RTOT
Where GTOT, STOT, ITOT and RTOT represent the sum of all depth
measurements for all of the breast tissues made at 30 mm intervals for all
8 radii
To obtain an estimate of the tissues of the breast (GTOT, STOT, ITOT and RTOT ) the
results were expressed as totals of the tissue in mm and as a percentage of the total tissue
of the breast.
79
2.2.1 Ultrasound imaging of the milk ducts
Milk ducts were identified as tubular structures with echogenic walls and hypoechoic
(low level echoes) lumen (Figure 2.7).
N
↑ →
5mm
N
↑
5mm
→
(a) (b)
Figure 2.7 (a) Ultrasound image of milk duct in the lactating breast. The duct is traced from deep in
the breast to the nipple (N). The walls are echogenic (↑) and the lumen hypoechoic (→). Note the echogenic flecks within the duct that are consistent with the appearance of fat globules in the breast milk. A small branch is noted ( ). (b) The milk duct is outlined.
80
The main milk ducts of the breast were identified at the base of the nipple and
then traced back into the parenchyma of breast to the limits of detection of the ultrasound
equipment. Milk ducts above 0.5mm in diameter were readily imaged. When scanning
ductal systems care was taken to apply minimum pressure with the transducer to avoid
either compression or complete obliteration of the milk ducts and when necessary colour
Doppler flow imaging was used to discriminate between milk ducts and blood vessels
(Chersevani, 1995). The number of main ducts in each breast was counted and duct
diameters measured at the base of the nipple. In addition 1-3 ducts in each breast were
traced into the periphery of the breast. Measurements were made of the depth of the main
duct and the distance of the first branch from the nipple. The diameter and depth from the
skin of the branches of the ducts were also measured. The nipple diameter and areola
radius was measured for each breast.
2.2.4 Ultrasound imaging of resting milk ducts
Resting milk ducts were defined as ducts monitored in the absence of factors that
may have induced the milk ejection reflex. Milk ducts were identified for repeated
measurements by their position in the breast and branching patterns. When necessary
colour Doppler flow imaging (Chersevani et al., 1995) was used to discriminate between
milk ducts and blood vessels. Details of the duct scanned were recorded
diagrammatically in a work book and prior to the second scan of the duct the videotape of
the first scan was reassessed to ensure that the same milk duct was monitored. The
ultrasound transducer was initially positioned on the areola next to the nipple on the
lateral portion of the right breast and orientated to display a duct in sagittal section. This
duct was scanned continuously for a 5-minute period on two separate days, at least 7 days
81
apart. Measurements of duct diameter were made repeatedly in a portion of the duct that
was perpendicular to the ultrasound beam at 3 to 20 second intervals, that is, at times
when the breast had stabilized from movement of either the mother or the positioning of
the transducer. The measurement calipers were placed from the middle of the anterior
duct wall to the middle of the posterior duct wall. In connection with this the zoom
feature of the ultrasound machine was not used and fundamental frequencies were
applied. The resting mean, standard deviation and coefficients of variation of the duct
diameter were calculated for both monitoring periods for each mother.
2.2.5 Ultrasound imaging of milk ducts during breastfeeding
To detect changes in the diameter of the milk duct during a breastfeed a duct in
the unsuckled breast was visualized as previously described (See section 2.2.4). The scan
commenced when the baby attached to the mothers breast and ended when the baby
finished feeding. Mothers were asked to indicate either when they sensed milk ejection
or, if they did not sense milk ejection, when the baby changed its sucking pattern. Each
scan was videotaped and after each breastfeeding session, the tape was analyzed by
measuring the changes in duct diameter every 3 to 20 seconds, that is, at times when the
breast had stabilized from movement of either the mother, baby or the positioning of the
transducer.
82
2.2.6 Ultrasound imaging of milk ducts during either stimulation or expression
of the breast using an electric breast pump
The stimulation and expression patterns applied to the breast by the electric breast
pump are described in section 2.1.6. To detect changes in the diameter of the milk duct
during either stimulation of milk ejection or expression of milk with an electric breast
pump, the breast not being pumped was visualized as previously described (See section
2.2.4). During stimulation of the breast the duct was scanned for 60 seconds after duct
dilation was observed. If duct dilation did not occur the duct was scanned for a maximum
of 240 seconds. During expression of milk using an electric breast pump a duct was
monitored in the breast not being pumped for 10 minutes after the initial duct dilation
was observed. Each scan was videotaped and after each session, the tape was analyzed by
measuring the changes in duct diameter every 8 to 12 seconds, that is, at times when the
breast had stabilized from either movement of the mother or the positioning of the
transducer.
2.2.7 Measurement of 24-hour milk production
Milk yield was determined for each breast by the test weighing of the infant
(Arthur et al., 1987). Test weighing was carried out over a 24-28 hour period, that is one
breastfeed longer than the 24-hour period, using an electronic balance (Medela
BabyWeighScales, Medela AG, Baar Switzerland). The milk productions were
normalized to 24 hours as described by Arthur et al. (1987). All 24-hour milk productions
were carried out in the mother’s home approximately one week prior to undertaking the
study. Mothers were instructed on the use of the balance, provided with written
83
instructions and were able to demonstrate how to use the balance to the visiting
researcher. No correction for infant insensible water loss was made, therefore milk
production may be underestimated by 10 ± 12 % (mean ± SD)(Arthur et al., 1987). In
addition, milk samples (< 1ml) were hand expressed from each breast into 5 ml
polypropylene plastic vials (Disposable Products, Adelaide, Australia) before and after
each breastfeed. The samples were frozen immediately and kept at –15ºC for analysis.
2.2.8 Calculation of degree of fullness and storage capacity
The degree of fullness of the breast (Cox et al., 1996) is a measure of how much
milk is stored in the breast at any point in time and was determined using the relationship
between the fat content of the milk and the degree of emptying of the breast (Daly et al.,
1993, Mitoulas et al., 2002). The milk samples collected from each breast before and
after each breast feed over a 24-28 hour sampling period were warmed to 37ºC and
gently mixed. Subsamples were taken up into hematocrit tubes (Chase Scientific Glass,
Inc, Rockwood, Tenn, USA) and centrifuged at 15,700 g for 6 minutes (Mikro 12-24,
Hettich centrifuge, HD Scientific, Blacktown, Australia). The fat content of each sample
was determined using the creamatocrit technique (Lucas et al., 1978; Mitoulas et al.
2002). Samples containing the maximum and minimum content of fat were designated as
having degrees of emptying of one and zero, respectively. The remaining samples
collected during the 24-28 hour sampling period were then used to determine the
relationship between fat content and degree of emptying for that mother by fitting the
curve described by Daly and coworkers (Daly et al., 1993). The fat content of any
subsequent milk sample was then used to determine the degree of emptying of the breast
84
at the time the sample was collected for that mother. Degree of fullness was calculated as
1-degree of emptying (Cox et al., 1996).
The storage capacity of the breast is the amount of milk available to the infant
when the breast is at its maximum volume during the 24-hour period. It was determined
using a regression line relating change in degree of fullness at each feed to the amount of
milk removed from the breast at that feed. A change in degree of fullness of zero is taken
to represent an infant milk intake of zero from the breast therefore the regression line was
forced to pass through the origin. Storage capacity was then calculated as the amount of
milk corresponding to a change in degree of fullness of 1.
2.2.9 Measurement of milk intake during at a breastfeed
For the purposes of these studies a breastfeed was defined as an uninterrupted
sucking session at one breast. The amount of milk consumed by the baby during a
breastfeed was determined by test weighing the baby (Arthur et al., 1987). This involved
weighing the infants using an electronic balance (Medela Electronic Baby-Weigh Scales,
Medela AG, Switzerland; accuracy 1 g) prior to and after breastfeeding. Milk intake was
calculated by subtracting the initial weight of the baby from the final weight of the baby.
2.2.10 Statistical Analysis
During a breastfeed milk ejection was determined to have occurred when the duct
diameter increased by more than 3 standard deviations (determined for each mother by
the 5 minute monitoring of a resting milk duct) over the initial duct diameter calculated
as a progressive mean. The duration of increased duct diameter was the time during
85
which the duct diameter was greater than 3 standard deviations from the mean initial duct
diameter. In a proportion of scans (58%) duct diameter did not return to the initial value.
On these occasions a mean from the lower duct diameters was calculated to provide a
new initial duct diameter.
All Students paired t-tests, correlations, partial correlations and ANOVA analyses
were performed using SPSS for Windows Standard Version, Release 10.0.1, 27 October
1999 (SPSS Inc., 1989-1999, Chicago, Illinois, USA). Regression relationships were
calculated using Microsoft Excel 1997 (Microsoft Corporation, 1985-1996). For trends
over time, SuperANOVA (Abacus Concepts Inc., USA, 1987) was used for repeated
measures analysis. All values are reported as mean ± SD unless otherwise stated.
86
Chapter 3
Anatomy of the Lactating Human Breast Redefined with Ultrasound
Imaging
3.1 Introduction Anatomical diagrams and descriptions of the gross anatomy of the lactating
human breast have changed little over the last 160 years and are based on the meticulous
dissections of the breasts of lactating cadavers (Figure 3.1) by Sir Astley Cooper (1840).
Furthermore, many of the current descriptions and illustrations do not correspond with
Cooper’s original work. For example, the glandular tissue in Gray’s Anatomy is depicted
as 15-20 lobes radiating out from the nipple (Bannister, 1995) whereas Cooper stated that
he observed up to 22 ducts leading to the nipple in some women but considered that
many of these ducts were not functional and that there were normally less than 12 patent
ducts opening at the nipple.
The lactating breast is described in Grays Anatomy (Bannister et al., (1995) as
being composed of glandular and adipose tissue held together by a loose framework of
fibres called suspensory ligaments of Cooper. There is a wide variation in the distribution
of breast tissues between women (Chersevani, 1995; Sohn, Blohomer and Hamper, 1999)
but not between breasts within women (Bomalaski et al., 2001). Histological studies
show that the lobes are composed of lobules, which consist of clusters of alveoli
containing lactocytes that synthesise breastmilk (Tobon and Salazar, 1975; Fawcett,
87
1986). The alveoli are connected to very small ducts that join to form larger ducts
draining the lobules. These larger ducts finally merge into one milk duct for each lobe.
Then under the areola this single milk duct is depicted to widen into a lactiferous sinus
(Vorherr, 1974; Bannister, 1995) before narrowing at the base of the nipple and
terminating at its orifice on the surface of the nipple. The adipose tissue of the breast is
typically situated between lobes rather than within lobules.
In developed countries successful positioning and attachment of the baby to the
breast is considered a major factor for the establishment and success of lactation (Righard
and Alade, 1992). The rationale for correct attachment of the infant to the breast is based
on the assumption that it is important that the infant should accept a significant
proportion of the areola so that milk can be stripped from the lactiferous sinuses (Righard
and Alade, 1992; Jensen, Wallace and Kelsay, 1994).
Apart from dissections performed by Cooper (1840) there has been little
investigation into the distribution of ductal structures and duct diameter in the lactating
breast since diagnostic methods such as mammogaphy, galactography (the injection of
radio-opaque contrast media and subsequent radiography), ductoscopy (insertion of a
small telescopic camera through a nipple orifice) are normally contraindicated in the
healthy lactating woman.
Ultrasound is used extensively to investigate the non-lactating breast to distinguish
between abnormalities (e.g. cysts, fibroadenomas and malignant changes) and normal
structures (e.g. milk ducts, Cooper’s ligaments, lymph nodes, glandular and adipose
tissue) (Stavros, 1995; Sohn, Blohmer and Hamper, 1999). In addition, recent
technological advances have increased the ability of ultrasound to resolve very fine
88
structures such as ducts as small as 0.5 mm (Chung and Chun, 1994), therefore, I have
used ultrasound as a noninvasive approach to re-examining the gross anatomy of the
lactating breast.
Figure 3.1 Artists impression of the lobes of the breast. The ducts were injected with coloured wax prior to dissection (from Cooper, 1840)
89
3.1 Materials
3.1.1 Participants
Twenty-two Caucasian mothers of healthy term infants were as described in
Chapter 2 section 2.1.1. These mothers were also participants in a study of the
stimulation of milk ejection using an electric breast pump (Kent et al., 2003).
3.2.2 Ultrasound equipment
3.2.2uipment
The ultrasound machine and appropriate settings used to scan the breast structures
are detailed in Chapter 2 section 2.1.2.
3.3 Methods
3.3.1 Measurement of 24-hour milk production
The 24-hour production for each mother was determined by the method described in
Chapter 2 section 2.2.7 within a week of commencing the study.
3.3.2 Calculation of storage capacity of the breast
Calculation of the storage capacity of both breasts for each mother was calculated
using the methods described in Chapter 2 section 2.2.8.
90
3.3.3 Ultrasound imaging of the anatomy of the breast 3.3.3 Ultrasound imaging of the anatomy of the breast
The ultrasound technique used to image the anatomy of the breast was performed
and documented according to methods described in Chapter 2 sections 2.2.2 and 2.2.3.
3.3.4 Statistical analysis
Student paired t-tests were performed to determine differences between breasts.
Correlation coefficients were used to determine relationships between the breast tissues,
external anatomy, milk production and storage capacity and were calculated using the
statistical software package SPSS for Windows Standard Version, Release 10.0.1, 27
October 1999 (SPSS Inc., Chicago, Illinois, USA, 1989-1999). P values of less than 0.05
were taken to be significant. Unless otherwise stated the results are presented as mean
and standard deviation (mean ± SD).
3.4 Results
3.4.1 Participant Characteristics
The 24-hour milk production was 387 ± 101 g for the left breast and 407 ± 121 g
for the right breast and the storage capacity of the left breast was 169 ± 51 mL and 175 ±
54 mL for the right breast. The mean areola radius was 27.8 ± 5.5 mm and 25.6 ± 5.5 mm
for the left and right breasts (n=14) respectively. Nipple diameter for the left breast was
15.7 ± 1.8 mm and 15.8 ± 2.4 mm for the right breast (n=14). There were no significant
differences between left and right breasts for any of the above measurements. Further
details for the participants are recorded in Chapter 2 Table 2.1 (mothers 1-22).
91
3.4.2 Milk duct system
The mean number of main ducts (<0.5 mm diameter) at the base of the nipple was
9.6 ± 2.9 (range 6-18) and 9.2 ± 2.9 (range 4-14) for the left breast and right breast,
respectively, and was not significantly different. The mean diameter of the main ducts at
the base of the nipple was 1.9 ± 0.6 mm (range 1.0 - 4.4 mm) and 2.1 ± 0.7 mm (range
1.0 – 4.0 mm) for the left and right breast, respectively, and was not significantly
different. Although there was usually an increase in duct diameter at multiple branch
points, the ‘typical’ sac like appearance of lactiferous sinuses under the areola was not
observed during scanning. Both the mean number of ducts and the diameter of the main
ducts was not related to either nipple diameter, areola radius or milk production for
individual breasts. Furthermore, there was no relationship between the number and
diameter of the milk ducts in each breast.
The depth of the first collecting branch from the base of the nipple for the left and
right breasts was 4.50 ± 1.98 mm and 4.74 ± 1.59 mm, respectively. The distance of the
first branch of the main duct from the nipple base was 8.20 ± 6.27 mm in the left breast
and 7.00 ± 3.98 mm in the right breast and the mean diameter of first milk duct branch
was 1.35 ± 0.58 mm and 1.25 ± 0.68 mm, respectively. The depth of the first branch was
8.21 ± 3.96 mm in the left breast and 7.20 ± 2.43 mm in the right breast. Of the mothers
with areola radius measurements (n = 14) the first branch of all of the ducts imaged (n =
33) occurred beneath the areola (Figure 3.2). There was no significant difference between
breasts for any of these measurements.
92
N N
*
10 mm 10 mm
* *
Figure 3.2 Ultrasound image of milk duct in the lactating breast. The duct is traced from the nipple (N) to the periphery of the breast. The walls are echogenic (↑) and the lumen hypoechoic (*). The first branch of this duct (→) is imaged almost directly under the nipple.
3.4.3 Breast tissues N N
3.43.4.33333Breast tissues
The distribution of tissues within the breast is shown in Table 3.1. There were no
significant differences between either, the amount of glandular tissue, subcutaneous,
intraglandular, retromammary, total fatty tissue or total tissue in the left and right breast
for each mother. When the glandular and fatty tissues were expressed as percentages of
the total amount of tissue measured, the glandular tissue represented 63 ± 9% (range 46-
83%) of the breast tissue and the fatty tissue 37 ± 9% (range 16-51%) for the left breast.
93
94
For the right breast the glandular tissue represented 65 ± 11% (range 45-83%) of the
breast tissue and the fat 35 ± 12% (range 9-54%). The subcutaneous fat represented 24 ±
7 %, the intraglandular fat 7 ± 5% and the retromammary fat 7 ± 3% of the breast tissue
in the left breast. The subcutaneous fat represented 22 ± 7%, the intraglandular fat 6 ± 4
% and the retromammary fat 9 ± 3 % of the breast tissue in the right breast. In addition
in 10 women glandular tissue was identified extending more than 120 mm from the
nipple to the axilla. Of these women, this was observed to be bilateral in six women and
unilateral in the remaining four women.
The subcutaneous fat was minimal at the base of the nipple (left: 5.5 ± 3.4%;
right: 6.3 ± 4.2% of the total depth of tissue) and increased gradually to a 30 mm radius
of the base of the nipple (left: 17.0 ± 5.3%; right: 18.0 ± 7.6% of the total depth of
tissue), whereas the retromammary fat changed little and was displayed as a relatively
uniform fat pad (left: 59.7 ± 14.7%; right: 55.7 ± 11.0% of the total depth of tissue) under
the glandular tissue and was indistinguishable from the subcutaneous fat peripherally. A
large proportion of the glandular tissue was located within a 30 mm radius of the base of
the nipple (left: 72 ± 9%; right: 70 ± 8% of the total depth of tissue), whereas
approximately half the intraglandular fat was situated amongst the glandular tissue within
a 30 mm radius of the base of the nipple (left: 60 ± 17%; right: 53 ± 24% of the total
tissue; Figure 3.3). These estimates were consistent with subjective observations of the
proportional distribution of glandular and fatty tissue during real time scanning. *
■ ■ 95
0
100
200
300
400
0 2 4 6 8 10 1
Distance from nipple (cm)
Cum
lativ
e de
pth
of ti
ssue
(mm
)
2
(a)
0
100
200
300
400
0 2 4 6 8 10
Distance from nipple (cm)
Cum
lativ
e de
pth
of ti
ssue
(mm
)
X – Subcutaneous fat ▲- Intraglandular fat
- Glandular tissue - Retromammary fat
(b)
Figure 3.3 The distribution of different tissues in the right breast of 2 different lactating women. Cumulative totals of each tissue were calculated by summing all of the measurements made in the breast at 0, 3 ,6 ,9 and 12 cm distances from the base of the nipple. (a) mother 1 had a total tissue sum of 1465 mm and a 24-hour milk production of 299 g . (b) mother 9 had a total tissue sum of 790 mm and a 24-hour milk production of 176 g.
96
The amount of glandular tissue was not related to the amount of subcutaneous,
intraglandular, retromammary fat or total fat content in either breast. However, the
amount of glandular tissue was related to the total amount of tissue in both breasts (left: r
=0.69, P=0.001; right: r = 0.68, P=0.001;) as was the subcutaneous fat (left: r = 0.60,
P<0.01; right: r =0.75, P=0.001), intraglandular fat (left: r =0.48, P<0.05; right: r =0.519,
P=0.016) and total fatty tissue (left: r =0.53, P<0.05; right: r = 0.73, P<0.01). The
retromammary fat in both breasts was not related to the total tissue in each breast.
There was no correlation between milk production and the amount of glandular
tissue, number of ducts or mean diameter of the milk ducts. Nor was there a correlation
between the amount of glandular tissue and the storage capacity of the breast.
3.5 Discussion
Based on the ultrasound findings of this study, we have created an artist’s
impression of the gross anatomy of the lactating breast (Figure 3.4).
Ultrasound imaging identified approximately nine milk ducts (range 4-18) in each
lactating breast whereas Gray’s Anatomy (1995) and many other texts describe 15-20
lobes and milk ducts in the female breast. However, there are some recent studies that are
in agreement with our results. Love (2003) has found an observed (method not stated) of
an average of 5 ducts conveying milk (range 1-12) in lactating women and Going and
Moffatt (2004) identified up to 27 ducts in the mastectomy nipple of a non-lactating
breast with only 7 having a patent lumen. In the one lactating nipple that Going and
Moffatt (2004) investigated they found only 4 patent ducts. Interestingly Cooper (1840)
97
Figure 3.4 Gross anatomy of the lactating breast based on ultrasound observations made of the milk duct system and distribution of different tissues within the breast.
98
injected wax into a maximum of 12 ducts in a lactating cadaver and more often 7-10
ducts. On one occasion he observed up to 22 openings on the nipple but not all opened
into milk ducts. Our observation of an average of nine milk ducts corresponds with two
descriptions in the literature that appear to be based on observation (Kopans, 1989; Love,
1990). In addition we found no difference in both the number of milk ducts and milk duct
diameter between the right and left breast within individual mothers suggesting the ductal
anatomy is similar in both breasts. However, there was a wide range of mean diameters
of milk ducts between mothers (1 - 4.4 mm). Furthermore, as the number and mean
diameter of milk ducts were not related to either nipple or areola radius, it was evident
that the breast morphology was not predictive of the internal anatomy of the breast.
The milk ducts measured by ultrasound for both breasts had a mean diameter of
2.0 mm ± 0.8 mm (range 1.0-4.4 mm) and is similar to that observed for of non-lactating
women (Chersevani et al., 1995; Mendelson, 1998). However, the ducts I observed were
in a resting state and are capable of increasing in diameter temporarily to accommodate
the increase in milk volume at milk ejection (Chapter 4). It is of note that I have
occasionally observed milk ducts as large as 10mm in diameter in mothers not involved
in this study. Numerous reports state that the milk ducts increase in size during lactation.
(Cooper, 1840; Chersvani et al., 1995; Sohn et al., 1999). In contrast, our results imply
that during lactation the milk ducts are not enlarged compared to the non-lactating state.
In this connection, it should be noted that Cooper injected coloured wax to visualize the
ductal system and thus was observing forcibly expanded milk ducts.
The milk ducts at the base of the nipple were superficial (Figure 3.2; mean depth
of 4.6 ±1.8 mm; range 0.7-7.9 mm), small (2.0 mm ± 0.8 mm) and easily compressed
99
(Chapter 4). These features make them easy to occlude and difficult to palpate. Since the
milk ducts were easily occluded by gentle pressure anecdotal evidence suggesting that
prolonged compression of the milk ducts may cause milk stasis leading to blocked ducts
is plausible. In addition, some mothers have milk ducts that are immediately below areola
and, at milk ejection, duct dilation is visible (in the unsuckled breast) for a period of 2 to
3 minutes, that is, until milk flow subsides and the dilation reverses (Chapter 4).
All of the ducts imaged on ultrasound branched under the areola, very close to the
nipple and did not display the typical sac like appearance commonly referred to as the
‘lactiferous sinus’ (Figure 3.2). The branches appeared to emanate from the glandular
tissue located directly beneath the nipple. Enlargement of the ducts also occurred at
points where multiple branches merged and at these points ducts in the periphery of the
breast were sometimes the same size as those near the nipple. Currently it is assumed that
the milk ducts near the nipple act as reservoirs for milk and this milk is available to the
infant before and during suckling (Woolridge, 1986; Riordan, 1993). However, recent
studies have shown that only small amounts of milk (1-10ml) can be expressed prior to
milk ejection when using an electric breast pump (Mitoulas et al., 2002; Kent et al., 2003)
and that the breastfeeding infant consumes little milk prior to milk ejection (Chapter 4).
After milk is ejected into the main milk ducts, duct dilation occurs. When milk is not
removed from the breast the diameter decreases back to a resting level within
approximately 2 minutes due to the backward flow of milk (Chapter 4). The smaller
number, size and shape of the ducts observed in my study suggests that the main function
of the ducts is the transport rather than storage of milk.
100
The course of the ducts imaged by ultrasound from the nipple into the breast was
diverse and complicated. The ducts were not always arranged systematically in a radial
pattern and often the main ducts lay under one another. Cooper (1840) had already noted
the erratic course of the ducts in his dissections and likened the milk ducts to the
intertwined roots of a tree. In order to illustrate the ducts, however, he separated them and
laid them out in an ordered, radial manner for the artist to draw and it is this image of the
ducts that is presented in current illustrations. My observations are in accordance with
Bannister (1995) who describes the lobes to be merged and difficult to separate surgically
and this is reflected in Figure 3.4.
I am unaware of any attempts to quantify the amount of glandular and adipose
tissue in the lactating breast. Ultrasound is a non-invasive and relatively inexpensive
method of assessing the breast. Clinically only subjective estimates are made of the
proportion of glandular and adipose tissue in the scanned breast. Quantitative
measurements are not provided because two dimensional real time ultrasound imaging
producing images that are essentially very thin ‘slices’ of the breast. These physical
properties of ultrasound make it to determine whether all of the tissue has been scanned
or if some areas have been scanned more than once. Therefore, in my attempt to quantify
and describe the distribution of tissues I chose consistently reproducible planes to
perform measurements (Chapter 2, section 2.2.2).
I found no difference in the amounts of glandular and adipose tissue in either
breast for each woman indicating relative symmetry of the breasts. Many texts state that
the lactating breast is composed predominately of glandular tissue (Vorherr 1974; Cowie,
Forsyth and Hart, 1983; Lawrence, 1999). In addition, histological studies show that
101
during lactation the proportion of glandular tissue increases relative to the adipose tissue
(Tobon and Salazar, 1975). The calculated ratio of glandular tissue to adipose tissue is
almost 2:1 for the lactating women in this study compared to 1:1 for non-lactating women
(as determined by mammography) (Heggie, 1996; Klein et al., 2002; Jamal et al., 2004)
supporting a two-fold increase in glandular tissue during lactation. However, my results
suggest that in some women there is an abundance of adipose tissue and it may constitute
up to half of the breast tissue. This is in contrast to other species such as the cow and goat
where the adipose tissue of the mammary gland is situated predominately in the
subcutaneous and abdominal regions with very little interspersed between the alveoli
(Patton and Jensen, 1976). Also, I have observed that the fat located within the glandular
tissue shows the greatest variation between women (CV 94% and 72% for the left and
right breast, respectively). It is of interest that within a 30 mm radius of the base of the
nipple there is a large amount of glandular tissue relative to adipose tissue (approximately
2.5 times as much). The predominance of glandular tissue, the minimal subcutaneous fat
and absence of lactiferous sinuses in this area obviously does not impede the infant’s
ability to remove milk successfully from the breast. In fact a shallow latch may well
occlude the milk ducts close to the nipple impeding milk flow. Furthermore, the absence
of a relationship between milk production and storage capacity with the amount of
glandular tissue, number of milk ducts and size of the milk ducts is consistent with milk
production being controlled by the infant’s appetite (Daly et al., 1992).
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Chapter 4
Ultrasound Imaging of Milk Ejection
in the Breast of
Lactating Women
4.1 Introduction
Suckling stimulates the neurohypopysis to release oxytocin, which causes the
contraction of the myoepithelial cells surrounding the alveoli forcing milk from the
alveoli into the milk ducts. This is termed the milk ejection reflex. In species whose
mammary glands contain milk cisterns (e.g. cows, ewes and goats), milk can usually be
removed prior to milk ejection, whereas those that do not have milk cisterns (e.g. humans
and sows) milk removal is almost totally dependent upon the effective stimulation of the
milk ejection reflex (Mepham, 1983).
Women may sense milk ejection by a range of sensations in the breast such as
pins and needles, pressure, pain as well as milk leakage, and thirst (Renfrew, Fisher and
Arms, 1990). Also, in the immediate period after birth uterine contractions may be felt as
a consequence of milk ejection reflex. However, some women never sense milk ejection
(Renfrew, Fisher and Arms, 1990) while in others the sensation either weakens or is lost
as lactation advances. Milk ejection in women has been objectively assessed by either
measuring oxytocin in serial samples of blood (McNeilly et al., 1983; Leake et al., 1983;
103
Yokoyama et al., 1994) or by monitoring changes in intra-ductal pressure by cannulating
a milk duct through its nipple pore (Cobo et al., 1967; Sala et al., 1974; Luther et al.,
1974). These studies showed a pulsatile response in both plasma oxytocin and intra-
ductal pressure during both breastfeeds and expression of milk with a breast pump.
However, both serial sampling of blood and measurement of intra-ductal pressure are
invasive procedures and the associated stress may inhibit the milk ejection reflex
(Newton and Newton, 1948; Udea et al., 1994).
Diagnostic ultrasound has been used extensively for the detection of breast
abnormalities in non-lactating women (Chung and Chun, 1994). Recent advances have
enabled this non-invasive technology to image and measure small structures (1-2mm in
size) such as milk ducts in the breasts of non-lactating and lactating women (Chersevani
et al., 1995; Mendelson, 1998). In lactating women, both increased pressure in the milk
ducts due to the expulsion of milk from the alveoli and the widening and shortening of
the ducts due to the action of oxytocin at milk ejection (Linzell, 1955) are likely to cause
duct dilation. We hypothesize that milk ejection will result in changes in duct
morphology that can be viewed by ultrasound imaging and that these changes can be used
to investigate the physiology of milk ejection in women.
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4.2 Materials
4.2.1 Participants
Twenty-four mothers (Table 4.1) of healthy term infants were recruited as
described in Chapter 2 section 2.1.1. These mothers were also participants in a study on
the stimulation of milk ejection using an electric breast pump (Chapter 5).
4.2.2 Ultrasound equipment
The ultrasound machine and appropriate settings used to scan the breast structures
are detailed in Chapter 2 section 2.1.2.
4.2 Methods
4.3.1 Measurement of 24-hour milk production
The 24-hour milk production for each mother was determined by the method
described in Chapter 2 section 2.2.7 within a week of commencing the study.
4.3.2 Measurement of milk intake
For the purposes of this study a breastfeed was defined as an uninterrupted
sucking session at one breast. The amount of milk consumed by the infant during a
breastfeed was determined by test weighing, previously described in Chapter 2 section
2.2.9.
105
4.3.3 Ultrasound imaging of breastmilk
The echogenicity of breast milk was determined by scanning three samples of
breast milk and three samples of olive oil and water before and after separation as
described in Chapter 2 section 2.2.1.
4.3.4 Ultrasound monitoring of resting milk ducts
Resting milk ducts were defined as ducts monitored in the absence of factors that
may have induced the milk ejection reflex. Twenty-four ducts were scanned as described
in Chapter 2 section 2.2.4. On 7 occasions, due to either the mother’s commitments or an
unsettled baby it was not possible to obtain a second 5-minute observation period.
4.3.5 Ultrasound imaging during the entire breastfeed
The mothers were scanned in the same manner as described in Chapter 2 section
2.2.5 for the duration of a breastfeed (1 mother was monitored for 6 breastfeeds, 4
mothers for 4 breastfeeds, 5 mothers for 3 breastfeeds, 11 mothers for 2 breastfeeds and 3
mothers for 1 breastfeed).
4.2.6 Statistical analysis and determination of changes in milk duct diameter
The variation of repeated measurements is expressed as the coefficient of
variation (CV). T=0 was taken as the time when the baby first attached to the breast. A
significant increase in duct diameter was determined to have occurred according to the
criteria in Chapter 2 section 2.2.10.
106
The mean amount of milk removed per milk ejection was calculated by dividing
the amount of milk consumed by the baby by the number of milk ejections observed at
that breastfeed.
Student’s paired t-tests were used to determine differences between breastfeeds.
Correlation and partial correlation coefficients were calculated using the statistical
software package SPSS for Windows Student Version, Release 6.1.3, 29 January 1996
(SPSS Inc., 1989-1995) and SPSS for Windows Standard Version, Release 10.0.1, 27
October 1999 (SPSS Inc., 1989-1999). Regression relationships were calculated using
Microsoft Excel 1997 (Microsoft Corporation, 1985-1996). For trends over time,
SuperANOVA (Abacus Concepts Inc., USA, 1987) was used for repeated measures
analysis. P values of less than 0.05 were taken to be significant. Results are presented as
mean and standard deviation (mean ± SD) unless otherwise stated.
4.3 Results
4.3.1 Ultrasound echogenicity of breastmilk
When mixed breast milk was scanned, evenly dispersed echoes (bright flecks)
were observed throughout the milk. After the milk had separated, the cream layer
appeared as a hyperechoic (highly reflected sound waves giving a bright image) band.
The skim milk appeared hypoehoic (darker image). The thickness of the top hyperechoic
layer of three milk samples measured by ultrasound was 1.70, 3.00 and 1.00 mm. These
values were in close agreement with the cream layer measured using vernier calipers,
1.75, 2.73 and 1.03 mm, respectively. Scans were also performed on a mixture of olive
107
oil and water. After separation of the oil layer, similar ultrasound appearances to
breastmilk were observed. Measurements of the thickness of the oil layer with ultrasound
were 6.00, 4.30 and 13.40 mm and were in close agreement with the measurements made
using vernier calipers, 6.04, 4.09 and 12.80 mm, respectively.
4.3.2 Ultrasound monitoring of resting milk ducts
Milk ducts were identified as tubular structures with hyperechoic walls and a
hypoechoic lumen (Figure 4.1) similar to that of the non-lactating breast (Chersevani et
al., 1995; Chung and Chun, 1994; Mendelson, 1998). However, echoes were observed
within the ducts, similar to the echoes observed in mixed milk and the cream layer of
separated breast milk, and were attributed to the milk fat globules. The ducts readily
collapsed under slight pressure similar to that required to compress a superficial vein on
the back of the hand and therefore accurate images of the ducts could only be obtained
when minimal pressure was applied to the ultrasound transducer. Ducts with diameters
greater than 0.5mm were readily identified.
108
N
B G
Figure 4.1 An ultrasound image of two main milk ducts in the lactating breast. The ducts in the
nipple (N) are narrow. The more superficial duct (→) displays a constant diameter for approximately 20 mm from the nipple. The deeper duct branches almost immediately before entering the nipple. Note the glandular tissue (G, bright echoes) directly below the nipple.
The diameter of resting milk ducts was 2.82 ± 0.09 mm (range 1.09 mm - 5.85
mm, n =51). The variation in the duct diameter during the 5-minute observation period
was small with coefficients of variation for individual mothers ranging from 1.4% to
8.3%. No significant difference was found between the mean duct diameter on the first
and second days. The duct diameters for the two days were in close agreement regardless
of either the duct size or the time since the last breastfeed (Figure 4.2).
109
Figure 4.2 Monitoring of milk ducts for 5 minute. A relatively large (4.2mm) duct ( ! and ∀) and
small (1.9mm) duct (% and 3) monitored for 5 minutes at least 7 days apart are shown. 4.3.4 Ultrasound imaging of the entire breastfeed
The mean length of a breastfeed was 6 minutes 40 seconds ± 3 minutes 29
seconds and the amount of milk consumed by the baby was 75 ± 40 g. The amount of
milk consumed at the monitored breastfeed was not significantly different from the mean
amount of milk consumed at each breastfeed during a 24-hour period (left breast: 73 ± 26
g; right breast: 76 ± 29 g). The total 24-hour milk productions for the mothers was 779 ±
181 g. Further details of the mothers breastfeeding characteristics are given in Chapter 2,
Table 2.1 (mothers 1-17 and 22-28).
5
4
Duct diameter
(mm) 3
2
1
N
1 3 4 5 6 2
Time (min)
110
Initial milk duct diameter in the unsuckled breast was 2.49 ± 0.84 mm and an
increase in duct diameter of 58 ± 41% was detected within 56 ± 30 seconds of the start of
the breastfeed on the other breast. This increase in duct diameter coincided with the
sensation of milk ejection and a change in the sucking pattern of the infant. In those
mothers who could not sense milk ejection the increase in duct diameter occurred when
the sucking pattern of the infant changed. The increase in duct diameter was usually
associated with a flow of small hyperechoic flecks towards the nipple. One mother did
not show a significant increase in duct diameter during one breastfeed lasting 3 minutes
and 37 seconds and the baby received 5 g of milk. For all breastfeeds that showed an
initial increase in duct diameter a significant decrease in duct diameter followed.
Multiple increases in milk duct diameter (duct dilations, Figure 4.3) were detected
in 74% of breastfeeds, whereas only one duct dilation was observed in 26% of
breastfeeds. There was an average of 2.5 ± 1.5 duct dilations per breastfeed (range=0-9, n
= 62). Twenty-one of the 24 mothers reported sensing milk ejection at the time of the
initial increase in duct diameter, however, none of the mothers reported sensing milk
ejection for the remainder of the breastfeed. The maximum diameter of the duct for each
significant increase during a breastfeed was consistent (mean CV = 4.5%). The mean
increase in duct diameter was 1.15 ± 0.22 mm, in cross sectional area of the duct was
4.66 ± 3.69 mm2 and percentage increase of the duct diameter was 49 ± 12% (n = 61).
The initial increase in duct diameter to a maximum occurred in 34 ± 22 seconds (range 4-
88 seconds) and then returned to either the initial diameter or greater in all but one
breastfeed. Of the mothers who had multiple duct dilations, 39% of babies finished
feeding at the time of an increase in duct diameter (Figure 4.4).
111
Time (min)
0 2 4 6 8 10 12
Duct diameter (mm)
1
2
3
4
5
Figure 4.3 Multiple milk ejections. 4 milk ejections are detected in this mother during an 11.5 min breastfeed. The maximum diameter for each milk ejection was consistent. The duration of each duct dilation was between 90 and 120 seconds. The infant finished feeding as the duct diameter reached a maximum (↓).
Partial correlation analysis demonstrated a significant correlation between the
number milk duct dilations that occurred during a breastfeed and the amount of milk the
baby consumed when controlling for the length of the breastfeed (r2 = 0.365, P < 0.01, n
= 57, Figure 4.4). Importantly there was no relationship between milk intake and length
of the breastfeed when controlling for the number duct dilations. However, there was a
positive relationship between the number of duct dilations and the length of the
breastfeed when controlling for the effect of milk intake (r2 = 0.353, P < 0.01, n = 52).
112
Number of Milk Ejections
Milk Intake
(g)
0 1 2 3 4 5 6 7 8 9 10
20
40
60
80
100
120
140
160
180
Figure 4.4 Milk intake of the infant in relation to the number of milk ejections detected during a breastfeed.
The duration of the initial milk duct dilation was not significantly different to
subsequent duct dilations (Figure 4.3) and the duration of duct dilations were not
significantly different either between breastfeeds or between mothers. The mean time the
duct was dilated was 86 ± 51 seconds (n = 146) and the time between the beginning of
each milk duct dilation was 122 ± 56 seconds (range 45 to 356 seconds). Some women
displayed extreme responses during the breastfeed, for example, one mother had one duct
dilation that lasted 231 seconds and the baby consumed 140g of milk (Figure 4.5)
whereas another mother had 3 duct dilations in 270 seconds and the baby consumed 100g
113
of milk (Figure 4.5). The total milk consumed divided by the number of duct dilations
during a breastfeed gave a mean milk intake per duct dilation of 35 ± 22g. Milk intake
was not related to either the degree of duct dilation or the maximum duct diameter.
5
Figure 4.5 Variation of the duration of milk duct dilation. Single milk ejection (∀). One duct dilation lasted 231s during this mothers’ breastfeed and her infant consumed 140g of milk. Multiple milk ejections (%). Three duct dilations of 45, 64 and 89 seconds were observed during this mothers’ breastfeed of 4 minutes and 49 seconds and her infant consumed 100g of milk.
Time (min)
1 2 3 4 5 6 7
2
3
4
Duct diameter (mm)
1
0
114
4.4 Discussion
In 62 successful breastfeeds changes detected by ultrasound (increased duct
diameter (Figure 4.3) and movement of fat globules coincided with the mother sensing
milk ejection and/or a change in sucking pattern of the baby. Furthermore, the time to
milk ejection (56 ± 30 seconds) was similar to previous clinical observations (Gunther,
1973; Renfrew, Fisher and Arms, 1990), the increase in the concentration of oxytocin in
the blood (McNeilly et al., 1993; Leake et al., 1983), and intra-ductal pressure in the
unsuckled breast (Cobo et al., 1967; Sala et al., 1974; Luther et al., 1974). Therefore, it
was concluded the changes in duct morphology associated with breastfeeding were a
consequence of milk ejection. This ultrasound technique is therefore a valuable non-
invasive method of objectively assessing milk ejection in women who are either
breastfeeding or expressing breastmilk.
During this study one mother showed no duct changes during a breastfeed (3.5
minutes) and the infant consumed only 5g of milk. This mother had a normal 24-hour
milk production (674g) and we had previously monitored 3 breastfeeds in which the
infant consumed 60, 65 and 115g of milk and increases in milk duct diameter were
detected. Thus ultrasound suggests the absence of milk ejection as an explanation for the
low milk intake by the infant. Ultrasound monitoring of multiple breastfeeds could,
therefore, be used to assess milk ejection in infants that appear to be attaching well to the
breast but consume little milk.
Although spontaneous milk ejections have been reported between breastfeeds
(Cobo, 1993) we did not observe these during monitoring periods (Figure 4.2). If a
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spontaneous milk ejection had occurred it appeared to have no persistent effect on resting
duct diameter. The very small variations in duct diameter (SD = 0.09 mm) were attributed
to either movement of the mother, or ultrasound transducer and measurement error.
Accurate monitoring of a milk duct with ultrasound was ensured by applying only
enough pressure to make contact with the breast and produce an ultrasound image. This
was assisted by the application of extra ultrasound gel.
The increase in milk duct diameter at milk ejection in the unsuckled breast is
likely to be caused by a combination of shortening and widening of the duct (Linzell,
1955) and increased pressure within the duct due to the expulsion of milk from the
alveoli. A recent study of lactating human breast (Kimura et al., 1998) suggests oxytocin
receptors are more prevalent in the ductal and glandular epithelium than the
myoepithelial cells. However, Ramsay (2004) showed that the increase in duct diameter
was less when the second breast was suckled compared to first. This suggests that the
response of the ducts may be affected by the degree of fullness of the monitored breast.
Contraction of the myoepithelial cells surrounding alveoli that have been partially
emptied may result in lower intraductal pressure and subsequently cause smaller duct
dilation.
The increase in milk duct diameter during a breastfeed was not related to either
milk intake or time to milk ejection and is in agreement with Ramsay (2004) who also
showed no affect of either the time since last breastfeed or stage of lactation. The increase
in duct diameter and time to milk ejection were not related to either 24-hour milk
production or milk intake, suggesting that the milk ejection reflex operates independently
of the synthetic activity of the breast and/or the appetite of the infant (Daly et al., 1992).
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The subsequent decrease in duct diameter after milk ejection (Figure 4.3) suggests
milk is not stored in the larger ducts close to the nipple but flows back into the smaller
collecting ducts and ductules, a phenomenon we have observed as a reversal in flow of
the echogenic fat globules within the duct. Reversal of milk flow has previously been
demonstrated in vitro in the rat, mouse, guinea pig and rabbit (Linzell, 1955) and is likely
to occur in the sow (Kent, Kennaugh and Hartmann, 2003). Also, both women and the
above-mentioned species do not have milk cisterns (Cooper, 1840), therefore, little milk
would be expected to be available prior to milk ejection. This is consistent with the
observation of the mother who did not display a milk ejection and yielded only 5g of
breast milk.
In this study 88% of the mothers were able to sense the initial milk ejection
however, none sensed subsequent milk ejections this may be due to decreased sensitivity
after the first release of oxytocin as oxytocin has been shown to low the pain threshold.
During 74% of breastfeeds multiple milk ejections were demonstrated by further peaks in
duct diameter as the feed progressed (Figure 4.3). This is comparable to the pulsatile
release of oxytocin (McNeilly et al., 1983; Leake et al., 1983; Yokoyama et al., 1994) as
well as increases in intraductal pressure during breastfeeding (Cobo et al., 1967; Sala et
al., 1974; Luther et al., 1974) and breast expression (Alekseev, Omel’yanyuk and
Talalaeva, 2000). The number of milk ejections detected during a breastfeed was the only
factor related to the amount of milk the baby consumed for that breastfeed regardless of
the length of the breastfeed (Figure 4.4). However, in 39% of the mothers that had
multiple milk ejections (Figure 4.3), the baby terminated the breastfeed during a milk
ejection, consistent with the baby’s appetite regulating milk intake (Daly et al, 1992).
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Maximum milk duct diameters were similar during a breastfeed (Figure 4.3)
inferring consistent ductal responses to multiple releases of oxytocin. Peak concentrations
of oxytocin in the blood are inconsistent during breastfeeding (Drewett, Bowen-Jones and
Dogterom, 1982) and the first increase in intraductal pressure during a breastfeed and
breast expression usually displays a higher maximum than subsequent increases (Luther
et al, 1974; Cobo, 1993; Alekseev, Omel’yanyuk and Talalaeva, 2000). The increased
pressure associated with the first milk ejection may be due to the availability of a greater
number of oxytocin receptors at the beginning of the suckling compared to the rest of the
breastfeed or it is possible that the first release of oxytocin may be the highest. Our
observations suggest there is a maximum duct expansion within a breastfeed regardless of
pressure or oxytocin release.
Duct dilation at milk ejection lasted approximately one and a half minutes as
measured by ultrasound (Figure 4.3), and is similar to the duration of oxytocin pulses in
the blood (Lucas, Drewett and Mitchell, 1980; McNeilly et al., 1983) and increases in
intraductal pressure (Cobo, 1993; Alekseev, Omel’yanyuk and Talalaeva, 2000).
Duration of duct dilation was similar for all milk ejections, breastfeeds and mothers.
However, extreme responses did occur, for example in one mother a single duct dilation
lasted 231 seconds (Figure 4.4) suggesting a sustained response to oxytocin. Sustained
increases of oxytocin in the blood have been shown during a breastfeed (McNeilly et al.,
1983), and hand breast expression (Yokoyama et al., 1994). Also, we found a minimum
latency period of 45 seconds between milk ejections, which is similar to the time between
pulses of oxytocin during a breastfeed (<1 minute) (Drewett, Bowen-Jones and
Dogterom, 1982). Thus it is apparent that a large number of milk ejections can occur in a
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short period of time. Indeed we have observed 3 duct dilations in 3 minutes (Figure 4.4)
corresponding with similar reports of oxytocin release (Lucas, Drewett and Mitchell,
1980). It is therefore conceivable that babies who consume a large amount of milk in a
short time are stimulating frequent milk ejections.
The results of this study support the use of ultrasound imaging as a non-invasive,
familiar, non-threatening procedure for assessing milk ejection as the sensation of milk
ejection is not a reliable indicator of the physiological response of the breast to oxytocin
during a breastfeed.
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Chapter 5
Response of the Breast to Different Stimulation Patterns of an
Electric Breast Pump
5.1 Introduction
For mothers with premature infants and those returning to work outside of the
home, breastfeeding is not always either possible or practical. It is important that the
expression of milk from the breast be as efficient and comfortable as possible so that
these mothers can provide expressed breast milk for their infants.
Whether breast milk is removed by a breastfed infant, removed manually,
removed by a mechanical breast pump or an electric breast pump, little milk can be
withdrawn from the human breast unless a milk ejection has occurred (Hartmann, 1991).
During breastfeeding, milk ejection is triggered by neural impulses from infant sucking
stimulating the release of oxytocin from the neurohypopysis. Oxytocin causes the
contraction of the myoepithelial cells that surround the alveoli in the breasts and results in
the expulsion of milk from the alveoli and an increase in intra-ductal pressure, facilitating
the withdrawal of milk from the breast by the infant. Milk ejection also can occur without
the physical stimuli of the infant sucking the breast (Cobo et al., 1967; McNeilly and
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McNeilly, 1978) and can be inhibited by stress (Newton and Newton, 1948; Udea et al.,
1994).
An infant stimulates the milk ejection reflex at the beginning of a feed by sucking
rapidly, between 72 and 120 sucks/minute, before slowing to 60 sucks/minute once milk
starts to flow (Drewett and Woolridge, 1979; Woolridge, 1986; Luther et al., 1974).
However, currently available electric breast pumps make no specific provision for the
stimulation of the milk ejection reflex. Although the ISIS mechanical breast pump
(Avent, Glemsford, United Kingdom) is designed to provide pulsatile pressure around the
areola to simulate an infant’s compressive action on the areola during breastfeeding, the
effect this has on milk ejection has not been quantitatively assessed (Fewtrell, 2001). I
wished to assess the effectiveness of different stimulation patterns provided by an electric
breast pump. Because milk ejection occurs simultaneously in both breasts and can be
visualized using ultrasound (Chapter 4), I used this technique to measure the time taken
for the patterns to elicit milk ejection. In addition, I compared the patterns with respect to
the volume of milk removed in the short term, the changes in milk ducts of the breasts,
and the mother’ perceptions of the patterns.
5.2 Materials
5.2.1 Participants
Twenty-eight mothers of healthy term infants who were exclusively breastfeeding
were recruited using the method described in Chapter 2 section 2.1.1.
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5.2.2 Ultrasound equipment
5.2.2 Ultrasound equipment
The ultrasound machine and appropriate settings used to scan the breast structures
are detailed in Chapter 2 section 2.1.2.
5.2.3 Electric breast pump
The breast pump used for this study is described in Chapter 2 section 2.1.6. The
pump was programmed to produce seven stimulation patterns (A-G) with frequencies of
45 to 125 cycles/min and adjustable vacuums (0-100%) ranging from –45 to –273 mm
Hg of which characteristics are detailed in Chapter 2 Table 2.1.
5.3 Methods
5.3.1 Measurement of 24-hour milk production
The 24-hour milk production of each mother was determined by the method described
in Chapter 2 section 2.2.7 within a week of commencing the study.
5.3.2 Calculation of degree of fullness and storage capacity of the breast
Calculation of the degree of fullness and storage capacity of both breasts for each
mother was calculated using the methods described in Chapter 2 section 2.2.8.
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5.3.3 Protocol
5.3.3.1 Prestudy protocol
The mothers attended the Breast Feeding Centre at King Edward Memorial
Hospital for Women on 3 occasions commencing between 9 AM and 11 AM. During the
first visit, the milk ducts of the right breast were located using ultrasound according to the
method described in Chapter 2 section 2.2.4 and one duct was chosen to be monitored
during all subsequent sessions. At the beginning of each session, the transducer was
positioned similarly on the right breast and the duct relocated by its unique topography.
Measurements were made perpendicular to the long axis the duct. This duct was
monitored (1) during a 5-minute period to establish variations in duct diameter while the
breasts were not being stimulated (Chapter 4) as described in Chapter 2 section 2.2.4, (2)
while the infant was breastfeeding from the left breast, and (3) while the left breast was
being pumped using pattern A. In addition, if the infant was fed from the right breast, a
duct on the left breast was monitored. This visit also allowed the mothers to become
familiar with the investigators, the experimental room, and the research pump.
5.3.3.2 Study Protocol
During the second and third visits to the Breast Feeding Centre, the 7 different
stimulation patterns of the breast pump were tested in a predetermined random order such
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that each pattern was tested in each position an equal number of times. During each
session, there were no other psychological stimuli for milk ejection (e.g. baby pictures) in
the room, the mothers’ infants were cared for in another room, the sound of the
ultrasound machine provided white noise, the mothers were instructed to concentrate on
the sensations within their breasts during pumping, and apart from adjusting the vacuum
to the comfort of the mother, the investigators remained silent.
The degree of fullness of both breasts was calculated prior to stimulation. The
first stimulation commenced when both breasts had a degree of fullness of at least 0.3.
The mother applied the breast shield to the left breast, the ultrasound transducer was
positioned over the milk duct previously selected in the right breast and pumping
commenced. The vacuum was initially set to 24% of maximum and adjusted as soon as
possible to the mother’s comfort. Pumping continued until 60 seconds after milk ejection
was visually detected by ultrasound, or for 240 seconds if milk ejection did not occur.
The amount of milk removed was measured by reweighing the tared collection bottle and
a subsample collected for the measurement of creamatocrit and the calculation of the
average degree of fullness of the left breast during that expression. The time of milk
ejection was defined as the time at which the duct dilated beyond three standard
deviations (determined from the 5-minute period without stimulation) of the mean
baseline diameter. Because the quantification of the duct diameter was done
retrospectively from videotape recordings, there was occasionally a discrepancy between
the visual and quantitative detection of the time of milk ejection. Quantitative data were
used for all analyses. If milk ejection did not occur, a time of 240 seconds was recorded.
The data for the amount of milk removed during pumping were used only when the
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actual collection period was between 50 and 70 seconds after milk ejection (determined
quantitatively). The increase in cross-sectional area of a duct was calculated, assuming
the cross-section of the duct to be circular.
The mother gave a qualitative evaluation of each pattern by rating the frequency
and strength on a scale of 1 (slow, soft) to 10 (fast, strong) and rating on a scale of 1
(dislike) to 5 (like) her approval of the frequency and strength of each pattern. She was
also asked to comment on how the pattern compared to her infant when it first attached to
the breast and on the milk ejection (if any) that she experienced. The mother was then
allowed to rest for 20 minutes before the testing of the next stimulation pattern. We
aimed to test 4 patterns on the first day and 3 on the second. The selected duct was
monitored for the 5-minute period between 2 stimulations on each day and during a
breastfeed after the last stimulation if possible. A small milk sample (< 1 mL) was
collected by manual expression into a 5-mL polypropylene tube from the both breasts
after each test, the creamatocrit was measured, and the degree of fullness of the breast
was calculated.
5.3.4 Statistical analysis
The primary goal of the analysis was to examine the effects of different breast
pump stimulation patterns on the milk ejection reflex. Several performance measures
were considered, with the primary performance indicator measured being the time to milk
ejection. Other evaluations of stimulation patterns included (1) analysis of the absence or
presence of milk ejection, (2) the total amount of milk removed over 50 to 70 seconds
after milk ejection, (3) the mother’s perception of the strengths and speeds, and (4)
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comparisons of strength and speed of the infant’s sucking. Supplementary analyses were
also performed to assess factors influencing increases in the duct’s cross-sectional area
during the milk ejection. A number of potential covariates were considered in all
analyses, including estimated breast storage capacity, degree of fullness of the breast,
feeding an infant immediately before the test, time since the last stimulation, and mean
vacuum chosen by the mother for each stimulation pattern.
Repeated-measures analyses of the time for milk ejection and the amount of milk
removed for the stimulation patterns and modeling of the duct diameter were
implemented using PROC MIXED. Repeated-measures analysis of variance (ANOVA)
of vacuum setting and mean vacuum was carried out using SuperANOVA. Evaluations of
mothers’ ratings and perceptions of the stimulation patterns as well as the comparisons of
the stimulation patterns with the infants’ sucking action were conducted using
Friedman’s ANOVA.
Two-side P values are quoted and a P value < 0.05 is regarded a statistically
significant. Multiple contrasts in the repeated-measures analyses were performed at the
overall significant level of 0.05. Results are presented as mean and standard deviation
(mean ± SD) unless otherwise stated.
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5.3 Results
5.3.1 Participant Characteristics
Twenty-eight mothers (22 to 38 years old, parity 1 to 4) participated. Three of the
mothers had no previous experience of breast pumps, whereas some expressed regularly.
The pumps used by the mothers before the study were Ameda Egnell Purely Yours,
Avent ISIS, Boots, Cannon Babysafe, Kaneson, Medela (electric, minielectric, or
manual), Pigeon, and Tommee Tippee. The total 24-hour milk production of the mothers
ranged from 372 to 1101g, and the 24-hour milk production of the left breast ranged from
196 to 566 g. The storage capacities of the left and right breasts were 169 ± 50 mL and
173 ± 54 mL, respectively. Further details of maternal and infant characteristics are given
in Chapter 2 section 2.1.1 Table 2.1 (mothers 1-28). Tests were commenced between 16
and 225 minutes after the last stimulation of either breast by breast pump or breastfeed.
The mean degree of fullness before each test for the left breast was 0.51 ± 0.22 (range
0.04-1.00) and for the right breast 0.46 ± 0.23 (range 0.01-0.98).
5.3.2 Milk ejection
When the infants were put to the breast, milk ejection occurred with the exception
of 1 occasion. During pumping, milk ejection occurred in response to all patterns for 14
mothers. For the remaining mothers, milk ejection occurred in response to between 1 and
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6 patterns (Table 5.1). Neither parity nor previous experience with breast pumps affected
the occurrence or timing milk ejection. Three mothers never sensed milk ejection, and
overall, 21% of milk ejections detected by ultrasound were not sensed by the mothers.
When mothers did sense milk ejection, the sensation coincided with the dilation of the
duct monitored by ultrasound.
The time taken for each pattern to elicit milk ejection as detected by ultrasound,
the vacuum settings chosen, and the resulting vacuums are presented in Table 5.1. The
time for milk ejection to occur in response to breastfeeding was faster than for all
stimulation patterns of the pump (all P < 0.001). Further comparisons were made only
between different stimulations patterns. The time for milk ejection was affected by the
time since last stimulation of either (P < 0.001), with a longer time since last stimulation
being associated with a shorter time for milk ejection. The degree of fullness of the left
breast also had a significant effect (P = 0.002), whereas the degree of fullness of the right
breast had only a marginal effect (P = 0.059). After adjusting for the time since last
stimulation and degree of fullness of the left breast, the time for milk ejection was not
related to the stimulation pattern used (P = 0.630) or the applied vacuum (P = 0.795).
Although statistical significance was not reached, after adjusting for the time since the
last stimulation and degree of fullness, patterns C, G, and D had the shortest times for
milk ejection, with estimated means of 120, 121 and 123 seconds, respectively. Similar
results were obtained considering only the data when milk ejection occurred within 240
seconds.
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Table 5.1 Time for milk ejection, vacuums chosen and amount of milk removed for each stimulation pattern*
A B C D E F G BF** Time for milk ejection† (s)
149±12a 129±12a 120±13a 123±9a 125±10a 129±9a 121±11a 56±4b
Number of mothers‡ 21 22 23 25 25 23 23 58 Vacuum setting (%) 25.8±4.6a 35.3±3.8b 42.6±4.6b,d 56.1±4.0c 56.8±4.2c 54.4±4.4c 46.9±3.5c,d Vacuum (mm Hg) Initial -148 -96 -83 -56 -41 -76 -83 Mean -139±8a -113±6b -99±6c -87±4d -70±6c -99±4c -105±4b,c Milk removed (g) 24.0±4.7a 15.3±2.3a 14.6±3.0a 13.8±2.8a 9.9±1.8a 15.2±2.2a 8.7±1.4a *Values are presented as x ± SEM. Values in the same row with the same superscripts are not significantly different (univariate analysis). **BF = breastfeed †Adjusted for the time since last stimulation and degree of fullness ‡Number of mothers who experience milk ejection within 240 seconds for the stimulation patterns or number of breastfeeds.
5.3.3 Milk Removal
Because milk ejection was one of the responses on which this study was focused,
we did not want to disturb the mothers by changing collection bottles during the
application of the stimulation patterns. Therefore, we only have estimates of the amount
of milk removed before milk ejection occurred. This ranged from 0 to 37.5 g, with a
mean of 2.7 g, and is comparable to the quantitative data of Mitoulas et al., (2002) of
6.5g, who used a stimulation pattern similar to pattern C.
The amount of milk that was removed from the breast by the pump up to 50 to 70
seconds after milk ejection (detected by ultrasound) ranged from 0.1 to 69.5 g and is
shown in Table 5.2. The total time of milk collection ranged from 96 to 249 seconds and
was not significantly different between patterns. Univariate analysis indicated that there
was no significant difference between the patterns in the amount of milk removed (P =
0.118). However, the mean vacuum chosen by the mother significantly influenced the
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amount of milk removed over 50 to 70 seconds (P < 0.001), with a stronger vacuum
chosen associated with a higher volume of milk removed. After adjustment for the mean
vacuum chosen, there was a significant association between stimulation patterns and the
amount of milk removed over 50 to 70 seconds (P = 0.016), with significant differences
between patterns E and G (P = 0.004) and D and G (P = 0.009). The estimated mean
differences were 8.35 mL (95% confidence interval (CI), 4.13-12.57) and 7.12 mL (95%
CI, 1.95-12.30) for differences between patterns D and G and between patterns E and G,
respectively. There was no relationship between either the initial duct diameter or the
increase in cross-sectional area of the duct measured and the amount of milk removed.
5.3.4 Changes in milk ducts
The mean diameter of the milk ducts before each stimulation test was 2.83 ± 0.99
mm (range, 1.1-5.9 mm). During the 5-minute periods without stimulation, there was
little variation in duct diameter (mean ± SD of coefficient of variation was 3.74 ± 1.73%).
In response to stimulation by the pump, the increase in cross-sectional area of the duct
when milk ejection occurred was 6.15 ± 0.52 mm2 (mean ± SEM; n = 163; range, 0.9-
50.6 mm2). There was no difference between the 7 different patterns (P = 0.158). The
increase in cross-sectional area of the duct in response to breastfeeding was 6.45 ± 0.98
mm2 (mean ± SEM) for 58 breastfeeds, which was not different from the response to
stimulation by the pump (P = 0.945). No significant predictors of change in cross-
sectional area were found.
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5.3.5 Mothers’ perceptions
The mothers’ perceptions of the frequencies and strengths of the patterns are
presented in Figure 5.1. There were highly significant differences between the
stimulation patterns (P < 0.001) in the mothers’ ratings of the frequencies and strengths
of the patterns. Pairwise comparisons of frequency ratings indicated that pattern A was
judged to be different from all other patterns (all P < 0.001), pattern E was judged to be
faster than patterns B (P < 0.001) and G (P < 0.001), and pattern F was different from
pattern G (P < 0.001). Pairwise comparisons of strength ratings indicated that pattern A
was judged to be stronger than pattern E (P < 0.001), and there was a significant
difference between patterns A and G (P < 0.003; Figure 5.1).
There were differences between the perceptions of the frequencies and strengths
of stimulation patterns compared to the mothers’ own infants (Table 5.2) (Friedman’s
ANOVA, P < 0.001, P < 0.001, respectively). Pattern C was most often judged to be
similar in frequency and strength to an infant, whereas pattern A was the most often
judged to be slower and stronger.
There was no significant difference in the approval ratings of the stimulation
patterns according to their frequencies (P = 0.544) or strengths (P = 0.289), with all
patterns having a median approval rating of 4 out of 5 for both frequency and strength.
The mothers’ descriptions of the strength of the sensation of milk ejection
compared to that elicited by breastfeeding ranged from weak (1) to very strong (5).
However, there was no relationship between the perceived strength of milk ejection and
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the stimulation pattern or the vacuum used. Nor was there any relationship between the
perceived strength of milk ejection and the degree of fullness of either breast, the dilation
of the milk ducts, or the amount of milk removed.
Table 5.2 Comparison of perceived frequency and strength of each pattern with the infant’s frequency and strength (% of responses)
Frequency Strength
Pattern Slower Similar Faster Softer Similar Stronger A 92 8 0 15 40 45 B 43 48 9 17 57 26 C 24 56 20 32 64 4 D 8 46 46 39 52 9 E 7 37 56 50 41 9 F 12 50 38 38 50 12 G 52 43 4 29 59 12
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A
B
A B C D E F G0
2
4
6
8
10
A B C D E F G0
2
4
6
8
10
St imulat ion Pat t er n
Rat
ing
Rat
ing
Figure 5.1 Mothers’ ratings of the frequencies (A) and strengths (B) of the stimulation patterns on a scale of 1 (slow, soft) to 10 (fast, strong). Bars indicated the range of responses, boxes indicate the first and third quartiles, and the dashed line indicates the median
133
5.4 Discussion
All the mothers were fully breastfeeding their infants when they commenced their
participation in the study, although some mothers had experienced difficulties at the start
of their lactation. The mother who had the lowest milk production (372 g/d) was taking
full-strength oral contraceptive pill, which has been associated with reduced milk
production (Tankeyoon et al., 1984). The infant of the mother with the next lowest milk
production (398 g/d) was under the care of a pediatrician for low weight gain. The milk
production of the remaining mothers ranged from 535 to 1101 g/d, consistent with normal
milk production, as discussed by Kent et al. (1999).
We have shown that the stimulation patterns applied by the electric breast pump
in this study elicited an increase in the cross-sectional area of the milk ducts that was
similar to the response during breastfeeding (Chapter 4). Other workers have also found
that the response of the breast is independent of the mechanism of stimulation. Zinaman
et al. (1992) demonstrated a physiologic response in oxytocin concentration to
stimulation by an electric, battery-operated, or manually operated breast pump.
Moreover, Sandholm (1967) observed an increase in intra-ductal pressure both in
response to artificial stimulation of the nipple and after intranasal administration of
oxytocin.
Although some of the stimulation patterns mimicked the physical characteristics
(frequency and strength of sucking) of an infant when it first latches on to the breast, the
faster time taken for milk ejection to occur when breastfeeding compared to pumping is
consistent with the fact that milk ejection is, at least in part, a conditioned response
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(Hartmann, 1991). This conditioning could also reduce the time taken for milk ejection to
occur in response to a breast pump when the mother expresses her breasts routinely. The
only stimulation pattern to which the mothers would have had had the opportunity to
become conditioned to was A, because those mothers who had previously used electric
pumps would have experienced patterns similar to A, and all mothers experienced
pumping with A on their first visit to the Breast Feeding Centre. Therefore, the testing of
the other stimulation patterns is rigorous because there was no opportunity for any of the
mothers to become accustomed to these patterns. Furthermore, the sound and the
appearance of the B2000 experimental breast pump was different to available commercial
breast pumps.
The frequency of pattern A was least similar to that of nonnutritive sucking at the
beginning of a breastfeed (Woolridge, 1986) and was perceived as such by the mothers.
Although all mothers had experienced pattern A at least once before, 7 of the mothers had
no milk ejection after pumping with A, and the time for the milk ejection reflex in
response to A was the longest. The frequencies of patterns C, D, E, and F were within the
range reported for nonnutritive sucking at the beginning of a breastfeed (Woolridge,
1986). Mothers were more sensitive to the differences in frequency of the patterns than
the significant differences in strength of vacuum (Figure 5.1). The higher frequency,
lower strength patterns were as good as, if not better than, pattern A in facilitating milk
ejection. Although some infants pause at variable intervals in their stimulation of the
breast, the response to pattern G indicates that a regular pause in the pump pattern gave
no added advantage. Moreover, the perception of some mothers that an increase in the
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applied vacuum would “get the milk ejection reflex going” was not supported by the data
(Table 5.1).
Because mothers were able to have the vacuum adjusted to their comfort, it is
interesting that there were significant differences between the patterns both in the vacuum
chosen (Table 5.2) and in the perception of the strength of the applied vacuum. The lower
initial vacuum of the pattern, the higher the final setting (percentage of maximum)
chosen, but the same vacuum was not achieved. The perception of the strength of vacuum
may be related to the frequency of the pattern because the stronger vacuums were used
with the slower patterns A and B.
Because the milk ducts remain dilated for 86 ± 51 seconds after the
commencement of duct dilation during milk ejections that occur during breastfeeding
(Chapter 4), the breast pump should be able to remove milk from the breast without
restriction for up to at least 70 seconds. Pumping with pattern A resulted in the highest
amount of milk (24.0 g ± 17.5) removed from the breast up to 50 to 70 seconds after milk
ejection. This is comparable with the breastfeeding data (Chapter 4) in that the average
milk yield for each milk ejection was 35 g, resulting at a calculated flow rate of 24.4
g/minute.
The amount of milk removed from the breast could depend on either the degree of
dilation of the ducts and/or the vacuum applied to the breast. However, there was no
relationship between the initial duct diameter or the degree of dilation of the ducts
measured and the amount of milk removed. This is similar to the findings in Chapter 4 in
that during breastfeeding there was no relationship between the increase in cross-
sectional area of the ducts and the amount of milk taken by the infant from the other
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breast. Assuming that the duct that was monitored is representative of the ducts in the
breast being milked, this suggests that dilation of the ducts plays a permissive rather than
controlling role in the rate of milk transfer.
The current findings of a relationship between the applied vacuum and the milk
yield are in contrast with those of Mitoulas et al. (2002) who found no relationship
between applied vacuum and total milk yield up to 5 minutes after milk ejection in a
study on different expression patterns of a Medela electric breast pump. In addition,
Prieto et al., (1996) measured peak intra-oral pressures in different infants of –60 to –170
mm Hg, but there was no obvious relationship between the degree of suction applied by
the infant and the total amount of milk the infant received during the breastfeed.
However, further analysis of the data of Mitoulas et al., (2002) reveals a relationship
between the applied vacuum and the amount of milk removed by a pattern similar to C
before milk ejection (r2 = 0.137, P = 0.0001, n =174). These data suggest that in the short
term, strength of suction is a more important variable than magnitude of duct dilation in
the determination of milk flow. In addition, the lower milk yield of pattern G
demonstrates that the pause at zero vacuum results in milk not being removed while it is
available. In Chapter 4 it was found that milk intake was related to the number of milk
ejections experienced by the mother rather than the time spent at the breast. Combined
with the current findings, we suggest that each milk ejection makes a certain amount of
milk available, and the applied vacuum affects how fast this amount is removed.
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Chapter 6
The Use of Ultrasound to Characterise Milk Ejection in
Women using an Electric Breast Pump
6.1 Introduction
Stimulation of the milk ejection reflex is essential for the adequate removal of
milk from the mammary gland particularly in species that do not possess cisterns
(Cowie et al, 1980; Mepham, 1983; Scott Young et al., 1996). Poor removal of milk
from the gland eventually leads to reduced milk synthesis (Wilde and Knight, 1990)
as a result of local inhibition. The decrease in milk production is likely to be due to
the accumulation of Feedback Inhibitior Protein (Wilde et al., 1995), which
eventually leads to the involution of secretory tissue (Wilde, Calvert and Peaker,
1988; Li et al., 1999).
In breastfeeding women suckling provides the main stimulus for the milk
ejection reflex causing the release of oxytocin from the neurohypophysis into the
bloodstream. The stimulation of a breast pump can efficiently induce a milk ejection
in expressing mothers, however, the time from the initiation of pumping to milk
ejection was slightly longer than that from the beginning of a breastfeed to milk
ejection (Chapter 5). Ultrasound imaging demonstrated that milk ejection could be
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detected during breastfeeding and that breastfeeding mothers had multiple milk
ejections during a breastfeed (Chapter 4). Furthermore, the volume of milk removed
during a breastfeed was related to the number of milk ejections (mean = 2.5)
stimulated and not the duration of the feed. It was not possible to calculate the change
in the rate of milk transfer associated with a milk ejection during a breastfeed without
interrupting the breastfeed. However, the pattern of milk ejections during a pumping
session in relation to milk removal has not yet been investigated. Therefore, the aims
of this study were to: (a) use ultrasound imaging to investigate the milk ejection
responses of the milk ducts to two markedly different vacuum patterns of an electric
breast pump, and (b) determine if there is a relationship between changes in duct
diameter and milk flow.
6.2 Materials
6.2.1 Participants
Participants for this study (n = 11) were recruited as described in Chapter 2
section 2.1.1. Mothers were asked to refrain from breastfeeding from the selected breast
for at least 2 hours before reporting for the study.
6.2.2 Ultrasound equipment
The ultrasound machine and appropriate settings used to scan the non-expressed
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breast are detailed in Chapter 2 section 2.1.2.
6.2.3 Electric breast pump and vacuum patterns
An electric breast pump as described in Chapter 2 section 2.1.6 was used for milk
expression in this study. Parameters of the two different vacuum patterns applied to the
breast are detailed in Chapter 2 section 2.1.6.
6.3 Methods
6.3.1 Measurement of 24-hour milk production
The 24-hour production for each mother was determined by the method described
in Chapter 2 section 2.2.7 within a week of commencing the study.
6.3.2 Degree of fullness and the storage capacity of the breast
Calculations of the degree of fullness and storage capacity of the each beast for
each mother were calculated using the methods described in Chapter 2 section 2.2.8.
6.3.3 Ultrasound imaging of milk ducts in the non-expressed breast
The technique used to scan the non-expressed breast during pumping is described
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in Chapter 2 section 2.2.6.
6.3.4 Expression parameters
The mothers expressed milk for a 10-minute period after milk ejection (increase
in milk duct diameter determined by ultrasound) from either the left or right breast on
two separate occasions, using the two different (Classic and 3-Phase), breast pump
vacuum patterns. The order of application of the two patterns was randomized. The
same breast was expressed on both occasions. Mothers were able to adjust the vacuum to
their own comfort throughout the pumping session.
6.3.5 Milk sampling during the expression period
Expressed milk was collected into a pre-weighed empty bottle that was replaced
every 30 seconds during the 10 minute expression period. A sub-sample (≤ 2 mL) of
milk from each 30-second period was collected and analysed immediately for fat content
by creamatocrit.
The total volume of milk expressed was the sum of the volumes of milk expressed
in each 30-second period. The volume of milk expressed whilst a duct in the contra-
lateral breast was dilated was the sum of the volumes of milk expressed for each 30-
second period that the duct was dilated even though the duct may not have been dilated
for the entire 30-second period.
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6.3.6 Statistical analysis
The time to the first milk ejection was taken as the time from when the pump was
first activated to the last point before duct diameter increased significantly. The duct
diameter was determined to have increased significantly by the methods described in
Chapter 2 section 2.2.10.
Comparisons between expression patterns were made using Students’ paired t-
tests. Characteristics of milk ejections were made using repeated measures ANOVA
using SPSS for Windows Standard Version, Release 10.0.1, 27 October 1999 (SPSS Inc.,
1989-1999 Chicago, Illinois, USA). P values of less than 0.05 were taken to be
significant. Values are reported as mean ± SD unless otherwise stated.
6.3 Results
6.3.1 Participant characteristics
The mean age of all mothers (n = 11) was 31.9 ± 2.5 years (range 28 – 35) with a
mean parity of 1.8 ± 0.6 children. Ten of the mothers stated that they had sensed milk
ejection during the current lactation however, only four stated that they had sensed
multiple milk ejections during a breastfeed. Ten of the mothers had used a breast pump in
either this or a previous lactation. Mean 24-hour milk production from left and right
breasts was 332.3 ± 101.9 g and 452.6 ± 124.3 g, respectively, and differed significantly
between breasts (P = 0.012). All infants were exclusively breastfed and had a mean age
of 16.54 ± 6.56 weeks (range 7 – 24). Further details of the participant’s characteristics
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are given in Chapter 2 section 2.1.1 Table 2.2.
For this study 6 mothers expressed from the left breast and 5 from the right breast.
The mean intital degree of fullness of the expressed breast was 0.75 ± 0.15 and 0.72 ±
0.12 for the Classic and 3-Phase pattern, respectively, whereas the mean degree of
fullness of the scanned breast was 0.28 ± .06 and 0.26 ± 0.07 % for the Classic and 3-
Phase pattern respectively.
6.3.2 Expression characteristics
The calculated amount of available milk in the expressed breast was 191.5 ± 96.8
g and 191.5 ± 96.6 g for the Classic and 3-Phase pattern, respectively. The mean volume
of milk expressed was 89.5 ± 32.1 g with the Classic pattern and 95.1 ± 45.5 g with the 3-
Phase pattern. These volumes represent 49.1 ± 19.4% and 52.7 ± 20.7% of the available
milk for the Classic and 3-Phase pattern, respectively. Mean milk flow rates for the full
expression period were 4.56 ± 4.52 g/30s (range 1.40-18.2) for the Classic pattern and
4.75 ± 3.18 g/30s (range 1.48-11.5) for the 3-Phase pattern. Mean milk flow rates during
the first milk ejection were 6.77 ± 3.65 g/30s (range 0.28-3.30) for the Classic pattern and
6.44 ± 3.09 g/30s (range 1.28-11.5 g/30s) for the 3-Phase pattern. For the period of
expression after the first milk ejection mean milk flow rates were 4.29 ± .50 g/30s (range:
1.74-5.99 g/30s) and 3.87 ± 2.68 g/30s (range 0.37-8.99 g/30s) for the Classic and 3-
Phase patterns, respectively. There was no difference in mean flow rates both during the
first milk ejection and for the period after the first milk ejection or between the Classic
and 3-Phase patterns. However, there was a significant decrease in the milk flow rate
after the first milk ejection for both the Classic (P=0.025) and the 3-Phase pattern
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(P=0.037).
6.3.3 Vacuum characteristics
The mean vacuum chosen by the mothers prior to milk ejection was significantly
softer for the 3-Phase pattern (P=0.003; –64 ± 33 mmHg) compared to the Classic pattern
(-117 ± 9 mmHg). There was no significant difference in the mean vacuum chosen for the
expression phase (after milk ejection) for the Classic pattern (–128 ± 32 mmHg) and 3-
Phase pattern (-148 ± 36 mmHg).
6.3.4 Ultrasound monitoring of milk duct diameter for the initial milk ejection
Initial milk duct diameter in the non-expressed breast was 2.24 ± 0.27 mm and
2.33 ± 0.23 mm for the Classic and 3-Phase patterns, respectively, and was not related to
degree of fullness of the breast.
For both patterns acute increases in duct diameter, consistent with milk ejection
(Chapter 4) occurred during the expression period and were not related to the degree of
fullness of the non-expressed breast. The initial increase in duct diameter was not
different for both patterns (Classic: 33.8 ± 5.67 % and 3-Phase: 36.6 ± 6.0 %) and
coincided with the sensation of milk ejection. For the 3-Phase pattern the increase in duct
diameter coincided with an increase in milk flow within 30 seconds in ten of the mothers
whereas for one mother flow increased within 60 seconds of milk ejection. For the
Classic pattern 5 mothers showed an increase in milk flow up to 60 seconds prior to milk
ejection, 5 mothers showed increased flow within 30 seconds post milk ejection and one
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mother showed increased flow within 60 seconds post milk ejection.
The time to the initial increase in duct diameter (time to milk ejection) was 124 ±
68.5 seconds and 84.7 ± 54.5 seconds for the classic and the 3-Phase pattern,
respectively. Time to milk ejection was not related to the degree of fullness of either the
non-expressed or expressed breast for either the Classic or 3-Phase pattern. Prior to milk
ejection the Classic pattern removed significantly more milk (P = 0.009; 9.53 ± 8.78 g;
range 0-29.6g) than the 3-Phase pattern (1.10 ± 1.97 g; range 0-6.3 g)
The time taken for the duct to reach its maximum diameter was 35.8 ± 25.4
seconds and 26.5 ± 11.2 seconds for the Classic and the 3-Phase patterns, respectively,
and was not related to the degree of fullness of either the non-expressed or expressed
breast. The time that the duct was dilated for the initial milk ejection was 150 ± 72
seconds for the Classic pattern and 167 ± 50 seconds for the 3-Phase pattern and was not
related to the degree of fullness of the breast. The volume of milk removed from the
pumped breast during this time was 38.4 ± 20.4 mL and 42.1 ± 25.3 mL for the Classic
and 3-Phase pattern, respectively, and was not significantly different. These volumes
represent 41.4 ± 21.5 % and 46.7 ± 26.6 % of the total volume of milk expressed for the
Classic and 3-Phase patterns, respectively, and equates to 21.9 ± 4.0% and 23.9 ± 4.4%
of the available milk in the breast for the Classic and 3-Phase patterns. Neither the
percentage of milk removed nor the rate at which the milk was removed for the first milk
ejection was related to the degree of fullness of the expressed breast.
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6.3.5 Ultrasound monitoring of milk duct diameter for the entire expression
period
Multiple acute increases in duct diameter (milk ejections) were detected in 91 %
of the expression periods for the Classic pattern and 100 % of the expression periods for
the 3-Phase pattern and were often associated with increases in milk flow (Figure 6.1).
The mean number of milk ejections detected during the expression period was 3.27 ±
2.05 (range 1-9) for the Classic pattern and 3.72 ± 1.19 (range 2-6) for the 3-Phase
pattern. The number of milk ejections during the expression period did not correlate with
the total volume of milk removed for either pattern (Classic: 89.5 ± 9.3 mL, 3-Phase:
95.2 ± 13.2 mL) or the percentage of available milk removed (Classic: 49.1 ± 5.6%, 3-
Phase: 52.7 ± 6.0%). With the 3-Phase pattern milk ejection coincided with the pump
switching from expression phase A to expression phase B in only two mothers.
In 15 expression periods there was a significant correlation between the mean
duct diameter and volume of milk measured for each 30-second period. On 5 occasions
significance was not achieved as milk was removed between milk ejections during 3
expression periods and only small volumes of milk were removed on 2 occasions (0.5-2.8
g and 1.8-5.7 g).
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60
0
10
20
30
40
50
Duct diameter
(mm)
60
50
40
30
20
10 Volume
(ml)
Milk ejection Milk ejection Milk ejection
5 6 7 8 9 10 11 0 1 2 3 4
Time (min)
Figure 6.1 Changes in milk duct diameter (times 10) in 10 second periods (x) and changes in the volume of expressed milk in 30 second periods (+) over a full 10 minute expression period using the 3-Phase pattern.
6.4 Discussion
Acute changes in milk duct diameter, consistent with milk ejection (Chapter 4),
were observed by ultrasound in the non-expressed breast of women using two different
vacuum patterns of an electric breast pump (Classic and 3-Phase). These acute increases
in milk duct diameter were associated with increased milk flow from the expressed breast
(Figure 6.1). However, I observed a time lag of up to 60 seconds between the detection of
milk ejection and the measurement of an increased milk flow on two occasions and this
was attributed to the low rates of milk flow (range 0.5-3.9 g/30s) exhibited during the
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entire expression period. This phenomenon may also be affected by the amount
intraductal pressure generated at milk ejection.
The time taken for milk ejection to occur during expression tended to be shorter
for the 3-Phase pattern (84.7 ± 16.4 seconds) compared to the Classic pattern (124 ± 20.6
seconds) although statistical significance was not reached (P=0.069). During
breastfeeding the infant sucks rapidly (72-120 sucks/minute) (Drewett & Woolridge,
1979; Woolridge, 1986) and elicits a milk ejection in approximately 60 seconds (Cobo et
al., 1967, Luther et al., 1974; Sala et al., 1974a; Sala et al., 1974b; Chapter 4). The
stimulation phase of the 3-Phase pattern more closely resembles this rapid sucking action
and therefore the shorter time taken by this pattern to stimulate milk ejection is consistent
with the pattern more closely resembling the infants’ sucking action. In addition, we
found that the time taken for milk ejection to occur was not related to the degree of
fullness of either breast. Therefore, to increase the efficiency of the time taken for milk
expression, the stimulation and conditioning of the milk ejection reflex is likely to play a
greater role than the amount of milk in the breast.
During expression, the duct diameter in the non-expressed breast took between 10
and 82 seconds to reach its maximum value and this is in agreement with values obtained
during breastfeeding (Chapter 4). It is interesting that the degree of fullness of the non-
expressed breast did not affect either the initial duct diameter or the time that the duct
took to reach its peak. Previously it has been assumed that increases in intraductal
pressure are positively related to the amount of milk in the breast (Sala et al., 1974a;
Sandholm, 1968), however, the rate at which the duct increased had not been quantified.
Our data suggests that the ducts reach a threshold diameter at milk ejection relatively
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quickly regardless of the degree of fullness of the breast and pressure of milk flow.
Multiple milk ejections were detected by ultrasound in all mothers during
expression with the 3-Phase pattern and 10 out of the 11 mothers during expression with
the Classic pattern. The increase in duct diameter was often associated with an increase in
milk flow (Figure 6.1) similar to that observed with the initial milk ejection. However,
accurate measurements of milk flow during these times was difficult due to increases in
duct diameter often not coinciding with the beginning of a milk sampling period and
because some women expressed very small amounts of milk. Indeed, a more frequent
measurement of milk flow would enable a more accurate association between changes in
duct diameter and milk flow, particularly in women where very small volumes of milk
are removed. In addition, milk ejection was observed near the end of the expression
period with no subsequent increases in milk flow in 3 women who had low degrees of
fullness (38, 17 and 5%) at this point of the expression period. This suggests that the milk
ejection reflex still occurs as a result of stimulation even when the breast is almost
drained of milk.
The number of milk ejections detected during the expression period was not
affected by the expression pattern (Classic: 3.27 ± 2.05; 3-Phase: 3.72 ± 1.19) nor did the
change from expression phase A to B in the 3-Phase pattern consistently stimulate a milk
ejection. Furthermore, the number of milk ejections observed during a breastfeed was 2.5
(Chapter 4), therefore, the breast pump appeared to be as efficient as the breastfeeding
infant in stimulation of multiple milk ejections. These data suggest that it is not necessary
to change the vacuum pattern of the electric breast pump during a 10-minute expression
period in order to stimulate multiple milk ejections and increased milk output.
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The Classic pattern removed significantly more milk (9.53 ± 8.78 g) prior to milk
ejection compared to the 3-Phase pattern (1.1 ± 1.97 g). These volumes are comparable
to volumes recorded in Chapter 5 (mean 2.7 g). Also Alekseev et al., (2000) was able to
express milk prior to milk ejection using an experimental pump that applied a vacuum at
a frequency of 60 cycles/minute. In Chapter 5 it was shown that a higher frequency
vacuum of 120 cycles/min removed less milk than a lower frequency vacuum pattern of
47 cycles/min. Furthermore, the lower frequency pattern more closely resembles that of
the breastfeeding infant after milk ejection (60 sucks/minute) (Drewett & Woolridge,
1979; Woolridge, 1986). Therefore, the ability of the Classic pattern to remove more milk
prior to milk ejection than the 3-Phase pattern may be due to the cycle time and/or the
stronger vacuum used with this pattern. However, the volume expressed before milk
ejection was significantly less than that obtained after milk ejection and most likely
represent only the milk contained within the main milk ducts.
The duration of the initial milk ejection during this study was not different for
either pattern nor was it related to the degree of fullness of the breast. This suggests that
once stimulated, the milk ejection response is similar regardless of the either the vacuum
pattern or strength of vacuum applied. In addition the volume of milk removed during the
initial milk ejection was similar for both expression patterns and represented almost half
the volume expressed during the pumping period (Classic: 41.4 ± 6.47 %; 3-Phase: 46.7
± 8.02 %). Interestingly the degree of fullness of the expressed breast did not affect either
the volume or the rate at which milk was removed for the initial milk ejection. However,
a significant decline in the rate at which milk was removed after the initial milk ejection
was observed for both patterns suggesting that the rate of milk flow decreased as milk
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was removed from the breast i.e. as less milk is available. These results suggest that the
flow rate is not limited at least by the amount of milk remaining in the breast and that in
order to increase flow rate during expression and hence efficacy of pumping other factors
such as the method of milk removal and breast/nipple anatomy should be considered. For
example, Gunther (1974) was able to measure 70g milk dripping from the unsuckled
breast for one woman by warming the nipple during a 10 minute breastfeed. In addition,
5 increases in flow rate were observed during this period. Further study is necessary to
determine if the application of heat to the nipple would enhance milk flow.
The number of milk ejections detected during the expression period was not
related to either the total volume or the percentage of available milk removed for either
pattern. This is in contrast to observations of milk ejection made during breastfeeding
where it was found that the number of milk ejections was related to the volume of milk
removed by the infant when analysis controlled for the length of the breastfeed (Chapter
4). These differences may be due to the mechanism of milk removal by the pump
compared to the infant and that the pumping time was restricted to 10 minutes in this
study. Although alterations of the expression pattern did not produce a change in the milk
ejection profiles of the women in this study this maybe due to limited numbers of women
recruited for the study. Therefore, further investigation of these and different expression
patterns along with extended expression periods would be recommended to determine if
milk removal could be further optimised during milk ejection.
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Chapter 7
Clinical Implications of Ultrasound Imaging of the Anatomy of the
Lactating Human Breast and Milk Ejection
The ability to successfully lactate depends on both the breast to completely
develop in order to produce milk and the occurrence of milk ejection. In the 164 years
since Sir Astley Coopers (1840) work, there had been few attempts to investigate the
anatomy of the human breast in its fully developed state (lactation). Nevertheless, the
rationalization of current experienced based clinical procedures used to facilitate the
positioning and attachment of the baby for breastfeeding are based on concepts of the
anatomy of the lactating breast derived from Cooper’s original dissections. My study
(Chapter 3) has shown that the ductal anatomy in the region of the areola and nipple is
different to that depicted in standard anatomical textbooks. The early branching of the
milk ducts and absence of the lactiferous sinuses suggests the current explanation for the
importance of the positioning and attachment of the baby to the breast requires revision.
It is believed that for a ‘good latch’ (attachment to the breast) the infant needs to take
both the nipple and a substantial proportion of the areola into to its mouth to allow the
peristaltic motion of the tongue to strip the milk out of the lactiferous sinuses during
suckling (Woolrdidge, 1986). Although I have found no evidence of lactiferous sinuses,
clinical experience supports the importance of a ‘good latch’ for effective and
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comfortable breastfeeding. Therefore, further investigations are required to determine the
importance of the positioning of the nipple and areola in the infant’s mouth. It may be
that a good ‘latch’ is required so that the infant the nipple is appropriately positioned to
allow the infant to co-ordinate the complex suck /breathe /swallow reflex
The milk ducts are readily occluded by light pressure (Chapter 4) therefore
variation in the compressibility along the length of a duct should permit assessment of
both the extent and location of the blockage in women diagnosed with blocked ducts. In
addition ultrasound monitoring of the blocked ducts during a breastfeed would determine
if the ducts responded normally to oxytocin. Lack of expansion of the duct would
indicate that there is a reduced response to oxytocin resulting in ineffective milk ejection
and subsequently poor milk removal from this portion of the breast. Furthermore,
localisation of the blockage would enable treatment (for example with therapeutic
ultrasound) to be effectively focused on the site of the blockage. Blocked ducts have been
shown to be a major contributor to mastitis (Fetherston, 1998) which may lead to
premature weaning of the infant. Hence early identification and treatment may not only
dislodge the blockage but also prevent the development of mastitis.
The prevalence of mastitis in Western societies is higher (81% of women in the
first 6 months of lactation) (Fetherston, 1997) than that of traditional societies (2.8%)
(Prentice et al, 1985). It has been suggested that the integrity of the nipple pore is integral
in the resistance of bacterial invasion of the breast that is responsible for infective
mastitis. During a breastfeed I have observed retrograde milk flow in the milk ducts after
peak duct dilation at milk ejection in the unsuckled breast. It is therefore conceivable that
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this backward flow of milk may assist in the spread of bacteria through the breast tissue.
In traditional societies infants are breastfed frequently therefore engorged breasts are
uncommon resulting in perhaps fewer or no spontaneous milk ejections. This in
combination with unrestricted clothing such as bras may contribute to the substantially
lower incidence of mastitis.
Although the assessment of the amount of glandular tissue in the breast with
ultrasound is generally qualitative rather than quantitative, a more systematic approach,
as I have taken in my study (Chapter 3), may be useful in assessing the amount of
glandular tissue in the breasts of mothers with either very low milk productions or those
demonstrating asymmetry of the breasts associated with unilateral lactation failure. The
latter condition is assumed to be due to a lack of glandular tissue (Neifert, Seacat and
Jobe, 1985). Therefore the comparison of the percentage of glandular tissue to overall
tissue may provide information for women with either, low or no milk production.
Furthermore, the standard treatment for women with low milk supply is to encourage
them to stimulate their breasts with more frequent breastfeeds and pumping the breast
after breastfeeds. However, not all women respond to this treatment. Therefore,
assessment of the proportion of glandular tissue in the breast may be predictive of the
effectiveness of such recommendations.
My results (Chapter 3) are also important in relation to breast surgery. The co-
distribution of glandular and fatty tissue within the breast suggests that it would be
difficult to preferentially remove fatty tissue. Furthermore, the retention of tissue within
the first 30mm of the nipple would be predicted to conserve approximately 50% of the
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potential lactating (glandular) tissue in women undergoing reduction mammoplasty.
Almost all women had less than the assumed 15-20 ducts and one woman only had 4
milk ducts leading to the nipple. Therefore, ablation of as few as 4 ducts could
completely impair the subsequent lactation potential of a breast. In those women who
have had breast surgery prior to lactation and are experiencing difficulties initiating
lactation, the use of ultrasound to tracing of the main ducts to the nipple could determine
patency. Since not all women sense milk ejection, ultrasound confirmation of milk
ejection (Chapter 4) could be used to show that either the nerve supply to the nipple was
intact or that milk ejection in the mother had become conditioned to her infant’s
breastfeeding behaviour.
There was a large variation in the amount of adipose tissue in the breast with
some women having almost half the breast composed of adipose tissue (Chapter 3). There
is evidence suggesting that obese women have difficulty in both initiating lactation
(Hilson, Rasmussen and Kjolhede, 1997; Rasmussen, Hilson and Kjolhede, 2001) and
attaching the infant successfully to the breast. Similarly obesity in animals has been
found to be detrimental to lactation (Vernon and Flint, 1984; Rasmussen, Hilson and
Kjolhede, 2002) and a recent study has shown that a low fat diet fed to obese lactating
rats improved lactation performance (Rasmussen, Wallace and Gournis, 2001). It would
be of interest to examine the distribution of the adipose tissue in the breasts of obese
women to determine if there is an abundance of both the subcutaneous and intraglandular
fat particularly within a 30 mm radius of the nipple. An increase in the amount of fat in
this region may make it difficult for the infant to successfully attach to the breast and
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remove milk. Poor milk removal may lead to decreased milk synthesis and either a delay
or inhibition of the initiation of lactation in these women.
The ultrasound imaging of the milk ejection has enabled the definition of the milk
ejection reflex in women (Hensyl, 1990) to be more precisely defined and to differentiate
between the milk ejection reflex and process of milk ejection (Chapters 4, 5 and 6). The
“Milk Ejection Reflex” in women is a neurohormonal reflex resulting from the tactile
stimulation of the nipple sending neural impulses through the spinothalamic tract causing
the neurohypophysial release of oxytocin into the blood stream. Oxytocin causes
contraction of the myoepithelial cells surrounding the alveoli in the breast, moving milk
into the larger milk ducts and expanding these ducts as the milk flows toward the nipple.
Whereas “Milk Ejection” in women may occur from one to eight times during a
breastfeed and is the period of time that there is an increased availability of milk from the
nipple as a result of the stimulation of the milk ejection reflex. Prior to milk ejection little
milk is available and the average duration of a milk ejection during a breastfeed in
women is approximately 1.5 minutes. The duration of milk ejection is defined by a
period of expansion of the milk ducts and increased intra-ductal pressure in the unsuckled
breast and by a transitory increase in the rate of milk flow during breast expression.
Ultrasound monitoring of milk ejection (Chapters 4, 5 and 6) in the clinical
setting has many applications. The number of milk ejections during a breastfeed was
related to the amount of milk consumed by the infants, therefore, ultrasound monitoring
of the number of milk ejections could be used to assess whether or not milk ejection is
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occurring in mothers whose infants have consistently low milk intakes and slow growth
rates. Similarly ultrasound confirmation of milk ejection during expression for mothers of
premature infants may exclude inadequate oxytocin release as a cause of low milk
production at a time of considerable stress. My studies have provided a non-invasive
method to measure successful milk ejection in women and therefore will provide an
opportunity for future investigations of the factors that may inhibition milk ejection and,
indirectly milk synthesis, in women.
I have found that only a small amount of milk was available prior to milk ejection
in most women (Chapters 4, 5 and 6) and differences in these volumes appeared to
depend upon the size and number of ducts in the breast. Further ultrasound assessment of
ductal anatomy of the lactating breast is required to may confirm these preliminary
observations. In addition I have shown that different expression patterns result in similar
milk ejection profiles within women (Chapter 6) so future research aimed at improving
breast pumps may need to focus on determining factors that limit milk flow during
periods of milk ejection. Women with similar milk ejections profiles often express
different proportions of their available milk at a breast expression session. Since the milk
ejection reflex per sae does not appear to explain these differences, ultrasound
investigations are required to determine whether women that have large increases in duct
diameter are able to release more milk at milk ejection than those that have small
increases. Furthermore, assessment of the anatomy of the breast in relation to the number
of ducts and their anatomical architecture within the areola and nipple area may provide
information to explain individual differences in the efficiency of breast expression
between exclusively breastfeeding mothers.
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Suckling, mechanical stimulation and the application of negative pressure (Cobo
et al, 1967; Luther et al, 1974; Sala et al, 1974) has been shown to be effective in
stimulating the initial milk ejection. However, little is known about the stimulus of
multiple milk ejections. My data suggests (Chapter 6) that continual stimulation of the
breast produces multiple milk ejections. Therefore, all infants are likely to induce and
benefit from the initial milk ejection. However, weaker infants may not provide the
continuous stimulation required to invoke multiple milk ejections resulting in less milk
intake which will lead to both poor growth and a reduction in milk production. Thus very
frequent breastfeeding (e.g every hour) may be necessary until the infant grows stronger
and is able to stimulate multiple milk ejections to remove more milk.
In conclusion, my studies using ultrasound imaging have extended Cooper’s
(1840) descriptions of the gross anatomy of the lactating breast defining the morphology
of the main milk ducts and the distribution of glandular and adipose tissues within the
lactating breast. These findings bring into question the existence of lactiferous sinuses
and emphasize the importance of glandular tissue within a 30mm radius of the nipple.
The morphology of the milk ducts in the areola area is not consistent with the current
explanation of the mechanism of milk removal by the breastfeeding infant, suggesting
that further investigation of the infant’s sucking mechanism is required. Furthermore,
ultrasound imaging of the milk ducts was able to detect increases in duct diameter that
were associated with milk ejection. This non-invasive technique was then used to study
the physiology of milk ejection in women during both breastfeeds and breast expression.
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The presence of multiple milk ejections during both breastfeeds and breast expression
was observed and the physiological importance of multiple milk ejections, the number
and diameter of the milk ducts as well as the distribution of adipose tissue in relation to
milk intake of the breastfed baby requires further investigation. The ultrasound
techniques developed in my research provide a new and non-invasive methods for further
the investigation of the physiology of lactating breast and the clinical assessment of
impaired lactation in women.
.
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References
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