Title:
Infertility and Women’s Age
by
Zohreh Nazemian
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science (IMS)
University of Toronto
© Copyright by Zohreh Nazemian (2011)
ii
Infertility and women's age
Zohreh Nazemian
Master of Science, IMS
University of Toronto
2011
Abstract:
In the first part of study, our objective was to determine the effect of CoQ10 supplementation of
culture media on preimplantation mouse and human embryo development. CoQ10
supplementation of culture media did not improve mouse or human embryo development in
vitro. Since the results appeared to be negative, we decided to move on to research the effect of
age on female infertility.
In the second part, we investigated the effect of female age and ovarian stimulation protocols on
IUI outcome in 411 infertile women. We found that the ongoing/live birth rate per cycle in
women ≤ 37 years was significantly higher than in older patients.
In the third section, we determined if very young age (≤25 yrs) has an impact on pregnancy
outcome in women undergoing IVF-ET. Our results demonstrating lower pregnancy rates in
very young patients and egg donors compared to the patients in their early thirties were
surprising.
iii
AKNOWLEDGEMENTS
There are so many people involved in making my graduate studies a rewarding experience. I
will take this opportunity to thank them all.
I would like to express sincere appreciation to my supervisor Dr. Robert Casper for allowing me
to do research in his clinic and for his continuous support and direction. I am also Thankful to
the members of my PAC committee, Dr. Julia Knight, Dr. Andrea Jurisicova, and Dr Ian Rogers
for their support and constructive suggestions.
I am obliged to the IVF and Andrology Laboratory staff of TCART who has contributed to this
project over the last few years. I would like to thank Murid Javed, PhD for his technical
assistance.
This project would not have been proceed without the support of Dr. Navid Esfandiari from IVF
laboratory, TCART for providing scientific and technical support that have made the studies
presented in this thesis possible.
Finally, I would like to thank my family, specifically my husband and my daughter for their
support and encouragement.
iv
Table of Contents
Abstract ii
Acknowledgements iv
Table of Contents iv
List of Abbreviations iv
List of Tables iv List of Figures iv
Chapter 1: Introduction and Literature Review 1
1.1 Cogenesis and Oocyte Biology 2
1.2 Oocyte Fertilization 3
1.3 Basics of Infertility 3
1.3.1 Male Infertility 4
1.3.2 Female Infertility 5
1.3.2.1 Age 5
1.3.2.2 Ovulation Disorder 7
1.3.2.3 Tubal Factor 8
1.3.2.4 Endometriosis 8
1.3.2.5 Unexplained Infertility 9
1.4 Infertility Treatment 9
1.4.1 Assisted Reproductive Technology 9
1.4.2 Intrauterine Insemination 10
1.4.3 In Vitro Fertilization 11
1.4.4 Intracytoplasmic Sperm Injection 14
1.4.4.1 Indication of ICSI 14
1.4.5 Human Embryo Culture and Development In Vitro 15
1.4.6 Embryo Cryopreservation 18
1.4.7 Children Born After IVF 19
1.5 Oocyte Maturation and Chromosomal Status 21
1.6 Mitochondria and Oocytes 22
v
1.7 Mitochondria and Reactive Oxygen Species 26
1.8 Mitochondrial Nutrients 26
1.8.1 Coenzyme Q10 27
Hypothesis 31
Objectives 31
Chapter 2: Effects of Mitochondrial Nutrients, CoQ10 On Human Early Embryo
Development In Vitro 33
2.1 Abstract 34
2.2 Introduction 35
2.3 Materials and Methods 37
2.3.1 Preparation of CoQ10 Solution 37
2.3.2 Preparation 0f Culture Dishes 37
2.3.3 Mouse Embryo Culture 37
2.3.4 Experimental Groups 38
2.3.5 Human Embryo Culture 39
2.3.6 Experimental Groups 41
2.3.7 Pronuclear Embryo Thawing 41
2.3.8 Blastocyst Culture and Scores 43
2.3.9 Different Alphanumeric Scores 45
2.3.10 Different Scores of Inner Cell Mass 46
2.3.11 Trophectoderm Grades 46
2.3.12 Blastocyst Vitrification 46
2.3.13 Blastocyst Thawing 47
2.4 Statistical Analysis 47
2.5 Results 49
2.5 1 Mouse Embryo Development Rate 49
2.5.2 Blastocyst Total Cell Number 49
2.5.3 Human Embryo Development Rates and Scores 49
2.5.4 Blastocyst Vitrification Rates 50
2.6 Discussion 51
2.7 Conclusion 56
2.8 Figures and Tables 57
vi
Chapter 3: Outcome of Intrauterine Insemination Cycles Using Various Ovarian
Stimulation Protocols 67
3.1 Abstract 68
3.2 Introduction 69
3.3 Materials and Methods 70
3.4 Statistical Analysis 73
3.5 Results 74
3.6 Discussion 77
3.7 Conclusion 81
3.8 Figures and Tables 82
Chapter 4: The Effect of Age on In Vitro Fertilization Outcome 91
4.1 Abstract 92
4.2 Introduction 93
4.3 Materials and Methods 94
4.3.1 Patients 94
4.3.2 Semen Analysis and Sperm Preparation 94
4.3.3 Ovarian Stimulation and Oocyte Retrieval 95
4.3.4 IVF and ICSI Procedures 95
4.4 Primary and Secondary Outcome Measures 96
4.5 Statistical Analysis 96
4.6 Results 98
4.7 Discussion 100
4.8 Figures and Tables 103
Chapter 5: Future Directions 108
Chapter 6: References 110
vii
LIST OF ABBREVIATIONS
AE Adverse event
ART Assisted reproductive technologies
CC Clomiphene citrate
COH Controlled ovarian hyperstimulation
CoQ10 Co-Enzyme Q10
E2 17--estradiol
FSH Follicle stimulating hormone
hCG Human chorionic gonadotropin
HTF Human tubal fluid
IVF in vitro fertilization
ICSI intracytoplasmic sperm injection
IU International units
IUI Intrauterine insemination
LH Luteinizing hormone
OHSS Ovarian hyperstimulation syndrome
P Progesterone
SOF Synthetic oviduct fluid
SSS Serum supplement substitute
TVS Transvaginal sonography
US Ultrasound/ultrasonography
viii
List of Tables
Table I.a: Mouse embryo development after 48 and 72 hours
Table II.a: Human embryo development on Day-2 and Day-3
Table I.b: Outcome of IUI treatment with reference to age
Table II.b. Pregnancy rate per cycle with reference to infertility diagnosis
Table III. Pregnancy rate per cycle with reference to ovarian stimulation protocol
Table III.b: Pregnancy rate per cycle with reference to ovarian stimulation protocol
Table IV.b: Semen characteristics in IUI patients
Table V.b: Pregnancy rate as reference to total motile sperm
Table VI.b: Pregnancy rate as reference to number of follicles bigger than 14 mm
Table VII.b: Pregnancy rate as reference to E2 level at the day of hCG
Table VIII.b: Pregnancy rate as reference to basal FSH level
Table I.c: Patient demographics of study groups
Table II.c: Semen characteristics in all groups
Table III.c: Cycle characteristics of study groups
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List of Figures
Figure 1a: Structure of Coenzyme Q 10
Figure 1b: intracytoiplasmic sperm injection
Figure 2: Mouse embryos at morula stage
Figure 3: Mouse embryos at early, expanding, expanded, and hatching blastocyst stage
Figure 4: Flourescence image of mouse blastocyst
Figure 5: Human embryo development in A) control, B) 10µL alcohol, C) 5µM CoQ10, D) 10
µM CoQ10 group.
Figure 6. Mouse embryo development in vitro after 24 hours
Figure 7. Mouse embryo development in vitro after 48 hours
Figure 8. Mouse embryo development in vitro after 72 hours
Figure 9. Human embryo development in culture media supplemented with CoQ10
Figure 10. Retrieved oocytes A) from a young infertility patient B) from a young egg donor,
and C) embryo quality on day-2 post retrieval in a young patient
1
Chapter 1: Introduction and Literature Review
Fertility is the capacity to produce offspring, and a couple is considered to be infertile if they
cannot conceive after 12 months of unprotected intercourse. A more Strict definition of
infertility is failure to achieve a pregnancy in a 12 month period for patients under 35 years of
age and failure to conceive in a 6 month period for the over 35 years (1). The prevalence of
infertility worldwide is estimated to be one in seven to one in five (14-20%) couples in their
reproductive age. The causes of infertility can be male, female or a combination of both and
include ovulatory disorders, tubal disease, endometriosis, chromosomal abnormalities, sperm
factors and unexplained infertility. The majority of infertility cases, both male and female
factors, are overcome through surgical and medical infertility treatment. Medical treatment
options include assisted reproductive techniques (ART) such as in vitro fertilization (IVF),
intracytoplasmic sperm injection (ICSI), and intrauterine insemination (IUI). Infertility
treatment has been dramatically advanced in recent years; however, age related infertility
remains as one of the most difficult challenges. Age must be taken into account when couples
are considering assisted reproductive technology. It is well known for years that the pregnancy
rate is inversely related to the age of the female (2 Some explanations for this include
diminished ovarian reserve, increased rate of aneuploidy, decreased frequency of sexual
intercourse, diminished desire for childbearing and increased rate of spontaneous abortion.
Among infertile women undergoing infertility treatment, advanced maternal age is also
associated with lower fertilization rates and higher risk of chromosomal abnormalities (3). In
humans, the age of the oocyte, not the age of the uterus, is the main cause of reproductive
failure in IVF and embryo transfer techniques.
2
1.1: Oogenesis and Oocyte Biology:
Oogenesis starts early in fetal life when primordial germ cells (oogonia) migrate to ovarian
cortex. Oogonia undergo many cycles of mitotic divisions and their number increase to several
million in the fetal ovary by 20 weeks of pregnancy. However, the majority of these oogonia are
degenerated in the process of atresia while some others enlarge and undergo nuclear changes
and called primary oocytes. Primary oocytes undergo the first meiotic division and arrest at
prophase of meiosis-I. During meiosis in addition to reduction in number of chromosomes from
diploid to haploid which only happens in germ cells, chromatin crossing over results in genetic
diversity of each gamete. By the 28th
week of pregnancy, the primary oocytes are become
encapsulated by follicular cells and form primordial follicles. Primordial follicles are the source
of gametes from which all mature oocytes will develop. It is at this point that meiosis arrests in
primary oocytes and will not resume until after sexual puberty (4).
At puberty, extensive growth in oocyte and surrounding follicular cells leads to completion of
first meiotic division forming a large secondary oocyte and a small polar body containing a
redundant set of chromosomes/chromatids plus cytoplasmic organelles. The polar body is a
result of asymmetric cell division as the majority of cytoplasm remains in secondary oocyte.
This asymmetry is very important as it keeps almost all the cytoplasm, organelles, and nutrients
that are critical for oocyte maturation, fertilization, and embryo development for the secondary
oocyte (5). At this stage, the meiosis arrest again and is not resumed until the oocyte is
penetrated by the sperm. Sperm penetration into the oocyte vestments activates the oocyte and
induces resumption of meiosis-II. The sperm haploid set of chromosomes combines with
haploid chromosomes of the oocytes and results in diploid zygote. The oocyte is now called a
fertilized egg.
3
1.2: Oocyte Fertilization:
Fertilization is the union of the male and female gametes in female oviducts that result in
formation of a zygote. In infertility patients where the gametes cannot meet in the female
oviduct, their union is made possible through in vitro fertilization (IVF) or intracytoplasmic
sperm injection (ICSI). If fertilization does not occur, the gametes lose their fertilization ability
and degenerate in the female reproductive tract.
Fertilization is a complex process in which sperm interacts with the homologous oocyte, to
create a new individual. It contains several steps which should occur successively (6). These
steps include 1) ovulation and oocyte transport into the oviduct, 2) Deposited spermatozoa are
actively transported from the vagina via the cervical canal and the uterine cavity to the oviducts,
where fertilization occurs, 3) Sperm capacitation and acrosome reaction, 4) adhesion of sperm
to the oocyte and penetration of the cumulus oophorus, 5) sperm penetration of zona pellucida
and fusion with oocyte membrane, 6) oocyte activation, cortical reaction, and block to
polyspermy, and 7) male pronucleus formation and genomic union. If any of the above steps is
compromised, the fertilization will fail. Recent advances in assisted reproductive technology
including IVF have provided new avenues to the understanding of normal fertilization and are
being used to overcome fertilization failure and treatment of infertility.
1.3: Basics of Infertility:
The incidence of infertility varies between populations mainly due their dietary and life-style,
environmental and occupational factors, and infectious diseases. However, infertility involves
both male and female partners and diagnosis requires a long list of diagnostic tests. The first
4
step in overcoming infertility is to perform on infertility evaluation. Evaluation of infertility
includes medical and sexual history of both partners, a physical examination including pelvic
ultrasound of the female and a semen analysis.
1.3.1: Male Infertility:
Infertility due to male factor accounts for at least 40% of infertility cases either as main or the
contributing factor (7). The first and most common test used to evaluate male infertility is
semen analysis. Semen analysis includes a set of descriptive measurements of sperm parameters
such as concentration, motility and morphology and some seminal plasma characteristics. The
predictive value of semen analysis is not ideal, however a significant amount of clinically useful
information can be obtained if the semen analysis is performed correctly (8). For instance a low
volume of ejaculate may indicate retrograde ejaculation and the absence of sperm may indicate
an obstruction in the male reproductive tract. Semen analysis is a very simple test to perform
and consists of macroscopic and microscopic evaluations. Macroscopic evaluation mainly
determines the seminal plasma characteristics such as liquefaction, viscosity, volume, color, and
pH. However, the microscopic evaluation of semen specimen provides information on sperm
characteristics including sperm count, motility and morphology.
A normal morphology of sperm consists of an oval-shaped head with a well defined acrosome
residing on the anterior head and covers up to 70% of the head area. The post-acrosomal region
is round and contains the nucleus. The tail is approximately 45 µm in length and is straight. The
neck and mid-piece are between the head and the tail and should contain no cytoplasmic
remnant. Sperm abnormalities can be seen in the head, mid-piece and/or tail. The world health
organization (WHO) provides the reference ranges for human sperm and semen, which are the
5
basis for diagnosis of the sub-fertile men (8). The most severe cases of male infertility are those
with no sperm in their ejaculate (azoospermia).
In addition to semen analysis, physical exam also provides valuable information about the
infertile man. For example varicocele which is a relatively common dilatation of the
pampiniform plexus of the spermatic vein affecting testicular function and sperm parameters.
1.3.2: Female infertility:
Similar to the male infertility, evaluation of an infertile woman consists of a detailed medical,
sexual and reproductive history, and a comprehensive physical examination.
1.3.2.1: Age: According to the statistics on female age and declining fertility, there is a slow
decline in pregnancy rates in the early 30's, while it becomes more substantial in the late 30's
and early 40's. In addition to decline in pregnancy, miscarriage rates also increase as the mother
ages and very few women over 44 are still fertile. The same trend is seen in pregnancy rate
following infertility treatment as in vitro fertilization success rates start dropping in the early
30's and fall faster starting at about age 38 (9). The following graph from US Government's
centers for disease control" (CDC), Society for Assisted Reproductive Technology" (SART)
2008 Report shows the impact of advancing female age on IVF success.
6
It can be seen from the chart above that pregnancy success rates declines with increasing age
starting at about age 30 and in about the age 38 the pregnancy rate drops faster. In addition, the
chance of having a successful pregnancy at age 44 and over by IVF using patient’s own eggs is
less than 5%. In contrast, the female age has no effect on pregnancy rates when the eggs from a
young and fertile donor are used. Thus, infertility in older women is directly associated with
poor developmental potential of aged oocytes (10).
Treating infertility due to late maternal age is difficult and debate is ongoing as to whether
transferring a high number of embryos in older patients is beneficial since it icreases the
possibility of having at least one chromosomally normal embryo transferred. Pre-implantation
genetic diagnosis (PGD) is a procedure which helps select chromosomally normal embryos for
transfer hence increasing the implantation rate and avoiding high rates of miscarriage in older
women. PGD can be done on polar body (PB), blastomeres from cleavage stage embryos, and
trophectoderm (TE) cells from blastocysts (11). During PGD using PB biopsy, the first PB is
removed from unfertilized oocytes and the second PB from the zygote shortly after fertilization.
Blastomere biopsy is generally performed by removing 1 or 2 cells from cellular stage embryo
that reach 8-cell stage (day-3 after fertilization). Blastocyst biopsy provides larger number of
7
cells and DNA material for testing and alleviates the difficulties related to the single cell
technique.
Although PGD has been used extensively in IVF centers all over the world, it comes with
disadvantages including additional cost to the IVF treatment, loss of some otherwise healthy
embryos after the biopsy, genetic misdiagnosis, and diminished embryo implantation potential
(12). The reliability of results obtained from blastomere biopsy is questioned due to high levels
of chromosomal mosaicism at cleavage stage. This may result in the tested cell not being
representative of the embryo. Moreover, taking 2 cells out of a cellular stage embryo
significantly compromises its implantation potential.
For women over 40, IVF success rates are extremely low due to reduced ability to obtain
enough oocytes following ovarian stimulation. The low quality of the retrieved oocytes also
results in poor fertilization and low quality embryos available for transfer.
1.3.2.2: Ovulation Disorders:The ovulatory factor refers to the ability of a woman to normally
undergo the process of ovulation. Ovulatory disorders represent a major cause of infertility.
Polycystic ovary syndrome (PCOS) is the most common cause of oligo-ovulation and
anovulation (13). Infertile patients with ovulatory dysfunction present more frequently with
primary infertility. They usually have a higher BMI. Oligo-menorrhoea, amenorrhoea,
hirsutism, and hormonal abnormalities are more frequent in overweight than infertile patients
with ovulatory dysfunction having a normal BMI (14). IVF is a successful treatment in patients
with PCO and although the response to follicular stimulation in PCO women is better than that
for women with normal ovaries, the outcome of pregnancy in vitro fertilization is similar (15).
8
1.3.2.3: Tubal Factor:
The fallopian tubes are two hollow structures connected to each side of the uterus and provide
the path for the sperm on its voyage to the site of fertilization of the oocyte. Tubal disease is a
disorder in which the fallopian tubes are blocked or damaged and is responsible for 25–35% of
female infertility (16). The primary cause of tubal factor infertility is pelvic inflammatory
disease which is generally caused by endometriosis, gonorrhea, or Chlamydia infection.
Evaluating tubal patency is an important part of infertility workup. Diagnosis can be achieved
by laparoscopy, hysterosalpingography, sonohysterosalpingography, salpingoscopy, and
chlamydial serology. In young patients when the damage to the tubes is minimal, tubal
reconstruction can be considered; however for patients having their tubes removed, IVF is the
only remaining option. IVF completely bypasses the tubal blockage as fertilization of oocytes
by sperm takes place in a culture dish in vitro. Recurrent ectopic pregnancy and hydrosalpinx
are situations that jeopardize the pregnancy outcome of IVF in patients with tubal factor.
1.3.2.4: Endometriosis:
Endometriosis is a chronic disease manifested by pelvic pain and infertility. It is characterized
by the presence of endometrial glandular epithelial and stromal cells on the pelvic peritoneum
and in other extra-uterine sites. In general population, endometriosis is thought to occur in 7-
10% of women, but random laparoscopic biopsies at the time of tubal ligation revealed evidence
of endometriosis in approximately 25% of women (17). The presence of this pathologic
condition in infertile women has been reported to range between 20 and 50% with a mean
prevalence of 38% (18). Infertility can be one of the consequences of Endometriosis. Where
endometriosis is minimal without tubal damage, intrauterine insemination with COH may be a
http://www.fertilityfactor.com/infertility_types.htmlhttp://www.fertilityfactor.com/infertility_types.html
9
reasonable option. For severe disease, the most cost effective management is in vitro
fertilization and the success rates are equivalent to those of other diagnostic groups. Fertilization
and implantation rates have been studied in women with minor endometriosis and compared
with controls. The results suggest reduced fertilization and cleavage rates in women with
endometriosis compared with controls (19).
1.3.2.5: Unexplained Infertility: Investigation of infertility is initiated after 1 year of attempts
to achieve spontaneous pregnancy. When the results of a thorough infertility evaluation are
normal and no clear cause of infertility found, the diagnosis of unexplained infertility is
assigned. The failure in finding the cause of infertility in part is due to insufficient testing or
investigation. Usually, IUI is tried as first line of treatment for couples with unexplained
infertility. Thus, insemination during three to four cycles can be offered while the couples are
on the waiting list for IVF. IVF is usually offered as a second treatment option, but the most
effective way of obtaining pregnancy for couples with unexplained infertility has not yet been
clearly demonstrated (20). The knowledge that fertilization has occurred after IVF is important
in that fertilization failure is a known cause of unexplained infertility.
1.4: Infertility Treatment
1.4.1: Assisted Reproductive Technology (ART):Assisted reproductive technology includes
all infertility treatment in which the male and female gametes are handled in vitro. The goal of
ART techniques along with ovarian stimulation is to increase the number of oocytes available
for fertilization or to process a sperm sample to inseminate oocytes. The main methods of ART
include IVF, ICSI, and intrauterine insemination (IUI). However there is no agreement on
whether IUI, which includes only sperm handling should be considered an ART technique. The
10
variety of ART treatments as modified versions of IVF have been used extensively in the past
however they are now obsolete mainly due to their need for invasive surgical procedures. These
include gamete intra-fallopian transfer (GIFT), zygote intra-fallopian transfer (ZIFT), and direct
intra-peritoneal insemination (DIPI). A prospective study was undertaken to evaluate the
relative efficacy of three in vivo methods of GIFT, combined intrauterine and direct
intraperitoneal insemination (IUI+DIPI), and controlled hyperstimulation (COH) alone in
infertile women with patent fallopian tubes. The clinical pregnancy rate was highest in the
IUI/DIPI (29.3%) and GIFT groups (28.6%), compared to COH (8.9%). The authors concluded
that controlled ovarian hyperstimulation combined with IUI and DIPI is a good alternative to
GIFT (21).
ART is indicated for those infertile patients that the diagnosis on the cause of infertility is made.
Cause of infertility and women’s age are major determinants of the ART procedure of choice. A
patient with blocked tubes and no male factor needs IVF while a severe male factor or men with
obstructive azoospermia need to be treated by ICSI. Men with sperm parameters slightly below
normal range can often achieve pregnancy with less aggressive techniques like IUI.
Choice of ART technique also largely depends on maternal age, where a young couple can be
given more time to establish pregnancy with other methods than ART such as timing of
intercourse or even IUI. The pregnancy rate following various methods of ART is different and
has been reported comprehensively in the literature. It is also published annually in Canada
(Canadian ART registry, CARTR) and the United States (Society for ART, SART) (22,9).
1.4.2: Intrauterine Insemination:
11
IUI is one of the most commonly performed treatments for infertile or sub-fertile patients with
male factor and consists of instrumental introduction of processed sperm into the uterus via the
cervix to deliver motile sperm to the fallopian tubes around ovulation time. In this procedure the
sperm sample is centrifuged, washed and kept in the incubator for capacitation prior to
insemination into the uterine cavity. This treatment is used for women with patent tubes and
considered the most cost-effective initial treatment for unexplained and moderate male factor
infertility (23). IUI can be the method of choice when normal sexual intercourse is not possible
in case of impotence, anejaculation, retrograde ejaculation, anatomic malformations, or
vaginismus. Although unexplained infertility is considered the major indication of IUI, patients
with cervical factor of infertility and minimal endometriosis can also benefit from this
procedure.IUI in combination with controlled ovarian hyperstimulation (COH) results in
acceptable pregnancy rates per cycle however the overall reported success rates of IUI are
variable and ranges from 5-70% (24). Female age is the most important predictor of IUI success
since age has negative impact on ovarian reserve and oocyte quality (25). Achieving maximum
pregnancy rate following IUI-COH requires a balance between an optimal number of follicles,
risk of ovarian hyper-stimulation, and multiple pregnancies. In patients with a large number of
follicles growing, conversion from IUI to IVF, avoids cancellation of the cycle and lowers the
risk of high order multiple pregnancy (26).
1.4.3: In Vitro Fertilization:
IVF is one of the most widely used treatments for infertility and is a process by which oocytes
are fertilized by sperm in vitro. The fertilized oocyte then kept in the culture medium for few
more days before being transferred into the patient’s uterus. The first successful pregnancy
following IVF was reported by Drs Robert Edwards and Patrick Steptoe in 1978. Few months
12
ago Dr Edwards was awarded the 2010 Nobel Prize in Medicine for his achievements in
infertility treatment.
There are a variety of indications for IVF including male and female factors. In its early days
IVF was considered primarily for patients with tubal factor infertility as it was providing the
chance of sperm and oocytes to stay in close vicinity and fertilize outside the human body.
However, IVF was quickly emerged as a treatment option for patients with severe male factor
infertility, ovulatory dysfunction, endometriosis, polycystic ovary disease, and unexplained
infertility. Since the first IVF baby was born in 1978, more than 3.5 million babies have been
conceived as a result of in vitro fertilization (27). During each menstrual cycle, the human
ovaries are designed to mature and ovulate a single oocyte. However, during infertility
treatment using ART, multiple follicles are usually recruited and multiple oocytes are formed.
The IVF process involves controlled ovarian stimulation, oocyte retrieval from the woman's
ovaries and inseminating them with partner sperm in culture medium. The inseminated oocytes
are checked and normally fertilized oocytes are kept in culture for two to five more days before
transferring to the patient's uterus in order to establish a successful pregnancy. The variability in
quality and maturation rate of human oocytes and developing embryos probably forms part of
the process of natural selection in that many oocytes are formed but only a few can reach the
stage of producing a viable foetus. However, understanding factors determining oocyte quality
is very important as it helps select the most viable embryos with high implantation potential in
order to achieve a singleton pregnancy and avoid high order multiple pregnancies. Where there
is a defect in sperm parameters that prevents sperm from penetrating and fertilizing the oocyte,
ICSI procedure is used. For ICSI, a sperm cell is injected directly into the cytoplasm of the
oocyte.
http://en.wikipedia.org/wiki/Ovaryhttp://en.wikipedia.org/wiki/Uterushttp://en.wikipedia.org/wiki/Sperm_quality
13
After the process of fertilization, the development of a human embryo to a blastocyst with a
high implantation potential depends on several factors. Normal development includes proper
cell division, absence or fragmentation of blastomeres and development to the morula and
blastocyst on day4 and day 5 or 6 respectively.
While produced in the same menstrual cycle of the same woman, it is very possible that the
produced oocytes vary significantly in their fertilization and developmental potential. This can
be in part the reason of failure to fertilize all mature oocytes in an ART cycle. The average
fertilization rate following insemination with sperm or ICSI is about 60-70% of mature oocyte
at metaphase-II (28). The developmental potential of fertilized oocytes is also not identical and
can vary drastically. If cultured to blastocyst stage, on average about half of the embryos arrest
or exhibit delayed development and fail to reach the blastocyst stage. Normal morphology
blastocysts are also different in their implantation potential as almost half of the transferred
blastocysts implant, and only a fraction of the implanted ones eventually go on to form a viable
foetus.
Although IVF has resulted in numerous pregnancies and healthy deliveries, total failure of
fertilization after IVF occurs in 10-25% of cycles even in cases where sperm and oocytes seem
to be normal (29). Sperm defects, oocyte abnormalities, and disturbances in sperm-oocyte
interactions have been proposed as possible causes. Several remedies have been attempted over
the years to salvage these cycles including re-insemination of unfertilized eggs and rescue ICSI
with discouraging results. Different techniques were developed in the past to assist the sperm to
penetrate oocyte vestments. These techniques mainly worked by thinning the zona pellucida
through exposure to enzymes, creating an opening in the zona using chemical digestions, or
14
mechanical breech. Although these modalities helped rescuing the cycle from total failure of
fertilization, they were accompanied by low fertilization and high incidence of polyspermy,
which was not compatible with normal development. The micromanipulation method of choice
that is currently used to achieve fertilization is ICSI.
1.4.4: Intracytoplasmic Sperm Injection (ICSI)
The ICSI procedure involves selecting a single spermatozoon, picking it up with a specialized
micro-needle, puncturing the zona and oolema, and direct deposition of spermatozoon into the
oocyte cytoplasm. The ability of ICSI to achieve fertilization regardless of sperm quality has
made this technique an ideal procedure to treat severe male factor infertility. In severe cases of
male infertility such as obstructive azoospermia or congenital absence of the vas deferens, ICSI
is the ideal treatment. With the use of ICSI, the fertilization and pregnancy outcome is
comparable when using ejaculated, epididymal or testicular sperm (28) (Figure 1c)
Figure 1b: intracytoiplasmic sperm injection (TCART archive)
.
1.4.4.1: Indications of ICSI
15
For men who required epididymal or testicular sperm extraction, conventional IVF results in
very poor fertilization and ICSI is method of choice. In addition, ICSI is performed for couples
with repeated IVF failures. In many infertility centers, ICSI is the method of choice regardless
of the quality and source of sperm to avoid any failed fertilization. However many other
infertility specialists strictly using ICSI for the most severe cases of male infertility. The
Practice Committee of American Society for Reproductive Medicine (ASRM) has published
guidelines on ICSI, as a component of in vitro fertilization (IVF), and regarded ICSI as an
standard clinical technique that being used for many years, and no longer should be considered
experimental. ICSI has dramatically increased the ability of males previously considered
infertile to father their own children (30). Since the first reports of successful pregnancies in
humans after treatment with intracytoplasmic sperm injection (ICSI), technical development of
ICSI has been significant (31).
1.4.5: Human Embryo Culture and Development in VitroEmbryo culture media are
designed to meet the metabolic needs of preimplantation embryos in vitro. The potential
requirements of human embryos include salts, sugar, amino acids, lipids, protein, vitamins,
hormones, and growth factors. Pyruvate has been shown to be the preferred energy substrate for
human embryos (32). Glucose however, is not necessary during the early stages of human
embryo development (33) although a surge in glucose uptake from 8-cell stage to blastocyst has
been shown in human embryos (32). In human embryos the uptake of pyruvate stays high
throughout the preimplantation development possibly due to higher demand for ATP for
compaction and blastocyst formation. Inclusion of amino acids in human embryo culture media
enhances the development to blastocysts. Within the embryo, amino acids are utilized for
http://www.ncbi.nlm.nih.gov/pubmed?term=%2522Practice%20Committee%20of%20American%20Society%20for%20Reproductive%20Medicine%2522%255BCorporate%20Author%255D
16
nucleic acid and protein production. They also act as chelators, pH buffers and antioxidants.
(34).
For IVF, retrieval of oocytes from mature follicles (18mm and bigger) is performed by
transvaginal ultrasound-guided aspiration 36 hours after hCG administration. Follicular fluid
along with the cumulus-oocyte-complex (COC) is aspirated to sterile tubes containing culture
media supplemented with heparin. Heparin avoids clot formation in follicular fluid which
contains red blood cells. Follicular aspirates are checked under the dissecting microscope and
COCs are identified. The COCs are washed in overnight equilibrated culture medium and kept
in a CO2 incubator at 37ºC for further insemination or ICSI.
Oocyte maturity is an important prerequisite for fertilization and is comprised of both nuclear
and cytoplasmic maturity. Nuclear maturity is checked by observing the first extruded polar
body (resumption of meiosis). The cytoplasmic maturity, however, is not easy to evaluate as it’s
related to the presence of cytoplasmic components that promote processes leading to
fertilization and embryo development. At present, cytoplasmic maturity can be assumed to some
degree by observing nuclear maturity. Oocyte quality can also be qualitatively described for the
cytoplasm (ie dark, heterogeneous, granular, inclusions, etc), appearance of zona pellucida
(thick, irregular, oval, dark, etc), and previtelline space (35).
For conventional insemination of oocytes in vitro, COCs are placed into cell culture dishes
containing culture medium and serum supplement with or without an oil overlay. The COCs are
usually inseminated by processed sperm in groups of 3-5 and kept in the incubator overnight.
For the ICSI procedurethe cumulus and corona cells are enuded 2-4 hrs following retrieval
17
using enzyme digestion, and oocytes are then washed in three consecutive washes culture
medium containing serum. ICSI is performed as above. Approximately 18 hrs after
conventional insemination or ICSI, oocytes are checked for signs of fertilization (two distinct
pronuclei and two polar bodies). Any oocytes with one pronucleus, or 3 and more pronuclei are
considered as abnormally fertilized and are segregated and not being used for transfer to the
uterus. Partial or total fertilization failures can occur following conventional insemination or
ICSI (28). Following ICSI, most cases of fertilization failure occur due to low number of mature
oocytes, failure of oocyte activation or non-availability of appropriate spermatozoa for injection.
After checking for fertilization, normally fertilized oocytes are moved to pre-equilibrated
culture medium and kept in the incubator. The embryo transfer is performed based on the clinic
policy and can be vary from Day-2, -3, or blastocyst stage. The first cell cycle lasts for about
30 hours. Two- to 4-cell stage embryos can usually be observed on day 2 after insemination,
whereas 6- to 8-cell stage embryos appear on day 3 after insemination. Compaction begins
during the fourth cell division (approximately 68 h after insemination) while expanded
blastocysts can be observed day-5 to day-7. The number of embryos transferred is determined
mainly by patient’s age, assessed embryo quality and developmental stage of embryos, and
reproductive history of the patient. With the patient in in the lithotomy position, the physician
inserts a speculum into the vagina to expose the cervix, and calls the lab to load the embryo(s).
Selected embryos are loaded into a catheter and the physician guides the catheter through the
cervix into the uterine cavity. When the catheter is in place the embryos are unloaded into the
uterus. The catheter is then returned back to the IVF laboratory to be checked under the
microscope to ensure that all the embryos are successfully transferred. The supernumerary
embryos with acceptable quality are cryopreserved for future use.
18
1.4.6: Embryo Cryopreservation
In theory, human embryos can be kept frozen in liquid nitrogen (-196ºC) at least for a thousand
years, and recently a successful pregnancy using embryos kept frozen for almost 20 years was
reported (36). The principal of cryopreservation is to dehydrate the embryo to avoid the
formation of intracellular ice crystals which can mechanically damage embryo mostly through
rupturing the cytoplasmic membrane. Cryoprotectants are compounds that protect mammalian
cells against freezing damage. They are all low molecular weight compounds (
19
water. In practice, the easiest and safest method is to store cryopreserved embryos in liquid
nitrogen at -196°C.
An alternative to the slow-freezing method is vitrification, which avoids the formation of ice
crystals in the intra- and extra-cellular space. Vitrification is achieved by a combination of a
high concentration of cryoprotectant (30 – 50% (v/v)) and an extremely high cooling rate that
result in an increased viscosity. Therefore, during the vitrification process, the molecules do not
have sufficient time to rearrange themselves into a crystal structure. (37). In slow- freezing the
procedure is based on a progressive dehydration of the embryo during the cooling process. In
contrast, in vitrification the cells are dehydrated rapidly before the cooling process starts, by
exposure to the high concentrations of cryoprotectants necessary to obtain a vitrified intra- and
extra-cellular state.
Cryopreservation of embryos, regardless of the method used, has several advantages besides
saving them for future use. Possibly the most important is to reduce the chance of high order
multiple pregnancy during IVF. Availability of embryo freezing has resulted in transferring less
number of embryos to the uterus in each ART cycle which in turn resulted in more embryos
being available for freezing. Since the first birth following transfer of frozen thawed embryo in
1984 (36) it is estimated that between 25-40% of the children born after ART are now born after
transferring frozen thawed embryos (27).
1.4.7: Children Born after IVF One of the major concerns of infertility experts as well as
infertile patients has been the safety of ART in terms of the health and reproductive life of the
babies born after IVF or ICSI. Although many consider the IVF and ICSI to be safe procedures,
20
the increased risks of prematurity and birth defects have been reported. A recent British study
shows that children conceived by IVF have increased health problems and spend almost double
the time in hospital than naturally conceived children (39). Another review article raised the
concern that children born after ICSI have an increased risk of major congenital deformity as
compared with children born naturally (40). Both paternal and maternal risk factors seem to
pose an increased risk of congenital malformations in the offspring born after ICSI although
ICSI technique per se is not an independent risk factor.
Preterm birth also increases following ART treatment. Preterm birth occurs when a woman
gives birth before 37 full weeks of pregnancy. Infants born preterm are at greater risk for death
in the first few days of life, as well as other adverse health outcomes including visual and
hearing impairments, intellectual and learning disabilities, and behavioral and emotional
problems throughout life. The bar graph below represents the percentages of preterm births
from ART cycles using fresh non-donor eggs or embryos, by number of infants born (9).
21
As mentioned for IVF and ICSI, the health of children born from frozen thawed embryos also
has been of concern, especially with the increasing use of the vitrification technique. This is
mainly because in vitrification, concentrations of potentially toxic cryoprotectants are used as
compared with the widely used slow-freezing technique. A recent systematic review of outcome
data has shown that for early cleavage embryos, there is better, or at least as good, obstetrical
outcomes for children born after cryopreservation as compared to children born after transfer of
fresh embryos. However they were unable to find long-term follow up data for children born
following slow-freezing of blastocysts or vitrifiction of oocytes, cleavage stage embryos, or
blastocysts (27).
1.5: Oocyte Maturation and Chromosomal Status
Immature oocytes in the ovarian cortex have 46 chromosomes arrested in prophase of the first
meiotic division. After follicle growth and maturation, the onset of the LH surge, or the hCG
trigger during infertility treatment, leads to resumption of meiosis in the oocyte and 23
chromosomes are extruded to form the first polar body. This involves alignment and separation
of the chromosomes by the nuclear spindle so that mature oocytes contain 23 chromosomes and
23 chromosomes are isolated outside the oolema in the first polar body (5). When penetrated by
a normal sperm, the oocyte extrudes 23 sister chromatids to form the second polar body and the
fertilized zygote has a normal diploid complement of 46 chromosomes once more (6).
The process of separating and pulling 23 chromosomes outside the egg into the first and second
polar bodies requires a significant amount of energy which is provided by ATP (adenosine
triphosphate). In the oocyte ATP is produced through anaerobic and aerobic pathways. In the
anaerobic pathway, pyruvate, a glucose metabolite, is converted to lactic acid. During anaerobic
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22
respiration, ATP is produced in the absence of oxygen. In this pathway glucose is not
metabolized completely, therefore ATP is produced in small amounts. The lactic acid must be
eliminated from the cell as to maintain the cytoplasmic pH. Second pathway of ATP production
is aerobic respiration and requires oxygen. Upon formation in cytoplasm, pyruvate enters the in
inner membrane of the mitochondria and completely metabolizes into water and carbon dioxide.
As glucose completely breaks down during the aerobic pathway, the amount of ATP produced
is significantly higher that anaerobic respiration. Therefore, the energy provided by ATP results
from oxidative phosphorylation in the mitochondria (41). Mitochondria are membrane-enclosed
organelles found in most eukaryotic cells. They are sometimes described as "cellular power
plants" because they generate energy through formation of ATP.
1.6: Mitochondria and the oocytes
The mitochondria is a membrane bound organelle in animal cells. It is enclosed within two
membranes, the outer membrane and the inner membrane. Mitochondrial functions in the cells
include energy production, regulation of membrane potential, signaling, cellular differentiation,
cell death, cell cycle control and cell growth (42). Mitochondria are the only providers of
energy for the metabolic requirements of the oocyte. The oocyte has the largest number of
mitochondria and mtDNA copies of any cell, (between 20,000 and 800,000 copies) (43), which
is even more than muscle cells and neurons that have high energy requirements. The ATP
produced by mitochondria is the energy source used for a variety of metabolic reactions. ATP is
produced by oxidative phosphorylation (OXPHOS), a process that uses energy derived from the
oxidation of nutrients to phosphorylated ADP (44). OXPHOS involves the action of the
mitochondrial respiratory chain consisting of four complexes located on the inner mitochondrial
membrane. Complex I and II oxidizes products of the tricarboxylic acid cycle (TCA) or Krebs
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23
cycle, nicotinamide dinucleotide (NADH) and flavine adenine dinucleotide (FADH2), and then
transfers the electrons to ubiquinone, also known as Coenzyme Q10 (CoQ10). CoQ10 transfers
the electrons to complex III. Complex III reduces cytochrome c and transfers the electrons to
complex IV with reduction of O2 to produce H2O. The energy produced by the transfer of
electrons along the respiratory chain is used to eject protons to the space between the inner and
outer mitochondrial membranes. The process of proton transfer to intermembrane space is the
result of the action of complexes I, III, IV and CoQ10 (45). The accumulation of protons creates
a gradient composed both of a charge and pH imbalance. Complex V, ATP synthase, provides a
channel for the entrance of protons back into the mitochondrial matrix and this influx of protons
supplies the energy needed for the phosphorylation of ADP to ATP. In addition to catabolism
of nutrients and production of energy, mitochondria play an important role in cell survival and
steroid biosynthesis. Although mitochondria produce ROS, they also participate in ROS
detoxification through a specific ROS-detoxifying enzymatic pathway (43).
Aging and age related pathologies are frequently associated with loss of mitochondrial function
mainly due to the accumulation of mtDNA mutations and deletions. This process of cumulative
DNA damage is attributed to the continuous exposure to ROS generated through normal
metabolism by the mitochondria themselves (46). The mammalian eggs and early embryos are
dependent on mitochondrial OXPHOS for their supply of energy since the alternative energetic
process of glycolysis is blocked due to suppression of the regulatory glycolytic enzyme,
phosphofructokinase (47). There is an increased rate of consumption of ATP in the mature
oocyte that is essential for its ability to undergo normal fertilization. The processes that follow
the fertilization of the egg, cortical granule exocytosis, and chromosome dysjunction for second
polar body extrusion all increase the energy demand even more. Therefore, mtDNA mutations
that diminish ATP production may result in polyspermy, chromosomal imbalance and arrest of
24
embryo development. The mitochondrial dysfunction of oocyte has been proposed as one of the
causes of high levels of developmental retardation and arrest that occur in preimplantation
embryos generated using Assisted Reproductive Technology (48). Failures in mitochondrial
replication during oogenesis may be responsible for the failure of mature oocytes to fertilize. In
fact, unfertilized oocytes were found to contain significantly fewer copies of mtDNA than
the
range described above, suggesting that mtDNA copy number, and by supposition, number of
mitochondria, is indicative of fertilization potential (49).
Maternally provided mitochondria act as the founding population of all daughter-cell
mitochondria of the developing embryo. It is shown that oocyte mitochondria make a necessary
physiological contribution to the cytoplasmic regulation of preimplantation embryos. Therefore
mitochondrial dysfunction is directly responsible for the early arrest of preimplantation embryos
in vitro (50), mostly due to the embryonic cell requirements for cytoplasmic energy production.
A functional link between age-related reduction in human oocyte mitochondrial membrane
potential (51) and an age-related decline in human preimplantation embryo developmental
potential (51,52) has been reported. The higher average age of infertility patients compared to
fertile couples is problematic since increased maternal age is accompanied not only by
mitochondrial DNA mutations (53) but also by reduced mitochondrial function (54) and
metabolic activity (55). Mitochondrial decay is a significant factor in aging, caused, in part, by
the release of reactive oxygen species (ROS) as by-products of mitochondrial electron transport.
One type of mitochondrial decay is oxidative modification of key mitochondrial enzymes.
Mitochondria are targets of their own oxidant by-products. Although oxygen is consumed
during preimplantation embryo development, higher oxygen tension during in vitro culture has
been suggested to cause developmental arrest as a result of the generation of reactive oxygen
25
species in cells. Thus the mitochondria of these early embryos might be damaged by in vitro
culture under high oxygen tension. This in turn results in a loss of ATP-generating capacity,
especially in times of greater energy demand.
Mitochondrial damage is a major contributor to aging and its associated degenerative diseases,
including cancer and neural decay. In a study, mitochondria from old rats compared with those
from young rats generated increased amounts of oxidant by-products (54) and have decreased
membrane potential, respiratory control ratio, cellular oxygen consumption, and cardiolipin (a
key lipid found only in mitochondria). Therefore, it is possible as women and oocytes age and
the mitochondrial energy production decreases, that many of the processes of oocyte
maturation, especially nuclear spindle activity and chromosomal segregation, may be impaired
(10,57,58). The result is the increased rate of aneuploidy, predominantly trisomies, that is
observed in older women. One of the main reasons for the poor performance of embryos from
older patients is an increased rate of chromosomal aberrations (43).
Thus, preservation of mitochondrial function is important for maintaining overall cell function
during aging. Inadequate dietary intakes of vitamins and minerals are widespread, most likely
due to excessive consumption of energy-rich, micronutrient-poor, refined food. Inadequate
intakes may result in chronic metabolic disruption, including mitochondrial decay. It is shown
that caloric restriction maintains mitochondrial function and lowers oxidant production (46). A
diet of restricted calorie might not be appropriate, and researchers are searching for other
alternative regimens to improve or maintain normal mitochondrial activities. Several dietary
supplements, including the mitochondrial cofactor and antioxidant lipoic acid (LA), increase
endogenous antioxidants or mitochondrial bioenergetics (54).
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1.7: Mitochondria and Reactive Oxygen Species
Within most mammalian cells, the mitochondrial electron-transport chain is the main source of
reactive oxygen species (ROS) (58). Reactive oxygen species generated from mitochondria have
been implicated in various forms of cell signaling in the vasculature and stimulation of cell
growth and migration through modulation of intracellular calcium, and activation
of
transcription factors such as NF- B (59).The rate of ROS production from mitochondria is
increased in a variety of pathologic conditions including hypoxia, ischemia, reperfusion, and
aging (60). Once mitochondrial enzymatic and nonenzymatic antioxidant systems are
overwhelmed by these ROS, oxidative damage and cell death can occur. Therefore possible
protective strategies would be to enrich tissue mitochondria with antioxidants thereby limiting
mitochondrial oxidative damage and cellular injury. The mitochondrial function can be
analyzed for membrane potential (MMP) (JC1 dye), ROS (Mitosox), ATP content
(luminescence assay), lisosomal activity (Lysotracker), metabolic mitochondrial measurements
(Mitotracker/NAD(P)H and FAD++.
1.8: Mitochondrial Nutrients
A group of endogenous substances that can directly or indirectly protect mitochondria from
oxidative damage and can increase mitochondrial function, including energy production, have
been termed “mitochondrial nutrients” (61). The direct protection mainly includes both
preventing from generation or scavenging free radicals, and elevating cofactors of defective
mitochondrial enzymes, and also protecting enzymes from further oxidation. The indirect
protection includes repairing oxidative damage by enhancing antioxidant defense systems(61).
27
These nutrients include the naturally occurring vitamins alpha-lipoic acid, coenzyme Q10, the
phytoalexin resveratrol, and L- or N-acetyl carnitine. The best studied of these nutrients to date
is alpha-lipoic acid which has been shown to have numerous benefits in neurodegenerative
diseases, cognition, ischemia/reperfusion injury, endothelial damage, and heart muscle function
(61), especially when paired with coenzyme Q10 (62,63).
1.8.1: Co-enzyme Q10
Co-enzymeQ10 (CoQ-10, Ubiquinone 50, Ubiquinone-10) is a lipid-soluble component of
virtually all cell membranes. All animals, including humans, can synthesize ubiquinones, hence,
coenzyme Q10 cannot be considered a vitamin (64.). The name ubiquinone refers to the
ubiquitous presence of these compounds in living organisms and their chemical structure. It
contains a functional group known as a benzoquinone. CoQ10, is a 1,4-benzoquinone, where Q
refers to the quinone chemical group, and 10 refers to the number of isoprenyl chemical
subunits in its tail. The CoQ10 Empirical formula is C59H90O4 with a molecular weight of
863.34. It appears in yellow to dark orange powder and should be kept in -20ºC (Sigma-Aldrich,
C9538). The most critical feature in biochemical function of CoQ10, is the ability of the
benzoquinone to accept and donate electrons. Coenzyme Q10 can exist in three oxidation states
(Figure 1a)
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28
Figure 1a: Structure of Coenzyme Q 10
Coenzyme Q can exist in three oxidation states: the fully reduced ubiquinol form (CoQH2), the
radical semiquinone intermediate (CoQH·), and the fully oxidized ubiquinone form (CoQ).
The biosynthesis of coenzyme Q10 involves three major steps: 1) synthesis of the
benzoquinone structure from either tyrosine or phenylalanine 2) synthesis of the isoprene side
chain from acetyl-coenzyme A (CoA) via the mevalonate pathway, and 3) the joining or
condensation of these two structures. The enzyme hydroxymethylglutaryl (HMG)-CoA
reductase plays a critical role in the regulation of coenzyme Q synthesis as well as the
regulation of cholesterol synthesis (65).It is shown that vitamin B6 is required in the first step of
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29
benzoquinone biosynthesis. Therefore it seems that adequate vitamin B6 nutrition is essential for
coenzyme Q10 biosynthesis (66).
The level of CoQ10 vary in different tissues and changes by age. In healthy young adult the
blood level ranges between 0.4 to 1.8 mg/mL (67). CoQ10 is measured using high performance
liquid chromatography (HPLC) (68). CoQ10 absorption is similar to other lipids and the uptake
mechanism appears to be similar to that of vitamin E. Coenzyme Q10 is primarily found in fish
and meat and levels over 50 mg/kg can be found in beef, pork and chicken heart, and liver.
Dairy products are much poorer sources of CoQ10 compared to animal tissues. Vegetable oils
are also quite rich in CoQ10. Within vegetables, parsley, and perilla are the richest CoQ10
sources. Broccoli, grape, and cauliflower are modest sources of CoQ10. Most fruit and berries
represent a poor to very poor source of CoQ10, with the exception of avocado, with a relatively
high CoQ10 content (69). Data on the metabolism of CoQ10 is scarce, but it seems that it is
metabolized in all tissues and mainly in liver (70) In regards to the safety, CoQ10 has very low
toxicity and no significant adverse side effects have been observed at doses as high as 1200
mg/d for up to 16 months (71) and 600 mg/d for up to 30 months ). Evidence from
pharmacokinetic studies suggest that exogenous CoQ10 does not influence the biosynthesis of
endogenous CoQ9/CoQ10 nor does it accumulate into plasma or tissues after cessation of
supplementation. Therefore it is highly safe to use COQ10 as dietary supplement (73). Primary
CoQ10 deficiency is an autosomal recessive condition with a clinical spectrum that
encompasses at least five major phenotypes: (1) encephalomyopathy characterized by the triad
of recurrent myoglobinuria, brain involvement and ragged red fibers; (2) severe infantile
multisystemic disease; (3) cerebellar ataxia; (4) Leigh syndrome with growth retardation, ataxia
and deafness; and (5) isolated myopathy (74).
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CoQ10 transports electrons from complex I and II to complex III. CoQ10 is essential for the
stability of complex III (75). It is also an antioxidant (76) and is involved in multiple aspects of
cellular metabolism (77). Deficiency states are characterized by a clinically heterogeneous
presentation that involves high energy consuming tissues such as the nervous system, skeletal
muscles and endocrine glands (78). CoQ10 is generally administered in dosages of 100 -300 mg
/ day. With both short and long term treatment with CoQ10 there was a significant increase in
its concentration in the plasma, muscle and sperm (79,80). Efficacy of coenzyme Q10 on semen
parameters, sperm function and reproductive hormones in infertile men has also been shown in
recent studies (81-84).
Several studies examined the efficacy of CoQ10 administration to healthy people and to patients
suffering from a primary or secondary deficiency of CoQ10 (78-80, 85, 86). In those studies
CoQ10 administration in the therapeutic dose was associated with a significant enhancement of
mitochondrial dependent functions. A meta analysis that reviewed RCTs involving CoQ10
administration noted that no side effects or adverse events were noted in any of the studies (86).
Zhipeng et al, had administered CoQ10 to rats in doses which are 1000 times higher than the
clinical therapeutic dose for 90 days, with generally good tolerability for male and female rats
(87). There are no reports in the literature of an association of CoQ10 during pregnancy and
birth defects. However, because of its intrinsic functions in cell growth and energy metabolism
(ATP synthesis), and its protective effects against oxidative stress, CoQ10 is a good candidate
for supporting growth of embryos in culture.
31
Hypothesis
Diminished oocyte quality is the cause of reproductive aging and decreased pregnancy and live
birth rates. Preliminary studies by Dr Casper’s team has shown that oocytes from older women
contain a higher percentage of dysfunctional mitochondria compared to the oocytes of young
women. We hypothesize that aging-associated oocyte mitochondrial abnormalities associated
with diminished energy production result in poor fertilization, poor embryo development, and
lower pregnancy outcome. We propose that dietary supplementation with coenzyme Q10 in
older women (in vivo) and supplementation of embryo culture media (in vitro) will improve
mitochondrial function in oocytes and embryos resulting in improved reproductive outcome.
Objectives
The overall goal of our research is to study the association between maternal age and oocyte and
embryo quality.
The objective in the first part of our study was to examine, both in mouse and human fertilized
eggs, whether CoQ10, as a mitochondrial nutrient, can improve mitochondrial function resulting
in higher embryo quality and blastocyst development rate.
Female age is a significant factor with intrauterine insemination (IUI) and in second part of our
study, we aim to investigate the effect of age and ovarian stimulation protocols on intrauterine
insemination outcome.
Despite our knowledge of the impact of advanced maternal age on fertility, the quality of
oocytes produced by very young patients or by young donors following IVF cycles has not been
carefully investigated. It is generally assumed that young women are fundamentally the best
32
category of patients for infertility treatment or for oocyte donation and should produce normal
healthy eggs with low rates of chromosomal abnormalities. The aim of our third section of the
study was to determine whether young age (25 years and younger) was associated with
improved oocyte and embryo quality and pregnancy outcome.
33
Chapter 2
Effects of mitochondrial nutrient, CoQ10, on human early embryo development in vitro
34
2.1: Abstract
Fertility decreases with age and maternal ageing is associated with diminished mitochondrial
activity in oocytes. The objective of this study was to determine the effect of mitochondrial
nutrient (CoQ10) supplementation of culture media on preimplantation mouse and human
embryo development. Ninety mouse embryos and 91 frozen thawed human zygotes were
cultured in different concentrations of CoQ10 or the solvent (alcohol). Blastocyst development
rates were calculated and the total cell number in mouse blastocysts was determined by staining
with Hoechst 33258 and survival rates after vitrification were assessed for human blastocysts.
Blastocyst development and total cell number of mouse embryos were similar between all
groups. The blastocyst hatching rate was slightly but not significantly higher in 5µM CoQ10
group than other groups. Human embryos cultured in presence of CoQ10 had similar
development rate and thaw survival rate after vitrification with control group. In conclusion,
CoQ10 supplementation of culture media does not improve embryo development in vitro.
35
2.2: Introduction
Assisted reproductive technologies have revolutionized the treatment of infertility. Because
assisted reproduction are costly and not universally successful, mainly in older women, attempts
have been made to determine the factors which predict a successful pregnancy outcome in a
given couple. Improvement of ART success rate is dependent on the oocyte quality and
quantity. Post-fertilization culture environment has a huge impact on embryo development that
is manifested at the level of morphology, metabolism, and gene expression (88,89). One of the
major differences between in vivo and in vitro environment for the embryo is oxygen tension.
The relatively high oxygen concentrations in the microenvironment surrounding
preimplantation embryos in vitro may disturb the balance between the formation of reactive
oxygen species and antioxidants, leading to oxidative stress. Oxidative stress has been
implicated in many different types of injuries, including membrane lipids peroxidation,
oxidation of amino acids and nucleic acids, and apoptosis. Although apoptosis takes place as a
normal part of embryonic development in vivo, it is more likely to occur during in vitro embryo
culture due to suboptimal conditions (88).
Coenzyme Q10 is an essential component of the plasma membrane ion transporter system and
of the electron transport chain in the inner mitochondrial membrane (90). Because of its intrinsic
functions in cell growth and energy metabolism (ATP synthesis), and its protective effects
against oxidative stress, CoQ10 is a good candidate for supporting growth of embryos in
culture. The hypothesis of our proposed study was that supplementation of embryo culture
systems with coenzyme Q10 would improve development to the blastocyst stage and post
vitrification survival of human embryos by improving mitochondrial energy production. To set
36
up the study and due to limitation of donated human embryos, mouse embryo culture system
was used and the development to blastocyst and total cell number in mouse blastocysts were
determined.
37
2.3: Materials and Methods
2.3.1: Preparation of CoQ10 Solution
Co enzyme CoQ10 was purchased from Sigma Chemical Co (Cat # C9538). It was dissolved in
100% Ethanol and used at 3 concentrations; 5 µM, 10µM, and 20µM. CoQ10 is lipophilic and
practically insoluble in water, therefore it needs to be dissolved in alcohol before being added to
the culture media. A working solution of CoQ10 was prepared after dissolving 0.001726 g in 2
mL ethanol to obtain 1 mM solution. From this working solution, 5, 10, and 20µL was added to
995, 990, and 980 µL culture medium to obtain a concentration of 5, 10, and 20 µM
respectively.
2.3.2: Preparation of culture dishes
Central well dishes (Falcon # 3037) were prepared and equilibrated over night at 37 °C in an
environment of 5.5% CO2 in air before placing embryos. The central well consisted of 1 mL
solution of each experimental group and the outer well contained 4 mL culture medium. The
dishes were not covered with oil as lipid soluble CoQ10 could be absorbed to the oil.
2.3.3: Mouse Embryo Culture
A two-cell mouse embryo culture system was used to evaluate the effects of CoQ10
supplementation in culture medium on pre-implantation mouse embryo development. Two-cell
mouse embryos were obtained from B6C3F1 mouse crossed with B6D2F1 mouse (Embryotech
Laboratories, Inc., Wilmington, MA, USA). Ninety cryopreserved 2-cell mouse embryos were
used. The straws were thawed by resting the straw horizontally on the bench in the room
temperature. After two minutes, the moisture on the straw is gently wiped and in order to mix
the content of the straw, the straw was shaken vigorously 3-4 times. The straw then transferred
38
into a 37°C water bath and then wiped out. The straw was cut at the end and the content
expelled onto the culture dish lid. The embryos recovered from the droplet and transferred into
an overnight equilibrated culture media. The embryos with intact blastomeres were used for the
study.
2.3.4: Experimental Groups
This mouse embryo study had following 6 experimental groups: human tubal fluid (HTF)+5%
synthetic serum substitute (SSS, Irvine Scientific, USA, Cat # 99193) (n = 14) as control, HTF-
SSS plus 10μL (1% v/v) solvent (alcohol) (n = 16) and 20 μL (2% v/v) solvent (alcohol) (n =
15), HTF- SSS supplemented with 5μM CoQ10 (n = 15), 10μM CoQ10 (n = 15), and 20μM
CoQ10 (n = 15). Before starting the culture, each cell-culture dish containing 1 mL of culture
medium was incubated overnight for equilibration at 37°C in a 5.5% CO2. Thawed embryos
were transferred into overnight equilibrated dishes of experimental treatments.
HTF Medium is intended for use in assisted reproductive procedures which include gamete and
embryo manipulation. HTF Medium is a synthetic, defined solution for use as a culture media
through Day 3 of human embryo development as well as the processing of gametes. HTF is
bicarbonate-based and is designed for use in a CO2 incubator and requires protein
supplementation. The components include;
- Sodium Chloride
- Potassium Chloride
- Magnesium Sulfate, Anhydrous
- Potassium Phosphate, Monobasic
- Calcium Chloride, Anhydrous
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- Sodium Bicarbonate
- Glucose
- Sodium Pyruvate
- Sodium Lactate
- Gentamicin
- Phenol Red
Embryos in all groups were monitored daily at the same time in the morning using bright field
inverted optics. The number of embryos cleaving to 4- to 8-cell, morula, blastocysts (early,
expanding, expanded), and hatching as endpoint was recorded (Figure 2, 3). To determine the
blastocyst quality, blastocysts were photographed and the total blastomere count per embryo
was determined by staining the embryos with bisbenzimide (Hoechst dye 33258, Sigma
Chemical Co., St. Louis, MO) (Figure 4). The embryos were washed extensively and mounted
with slight cover slip compression in Vectashield anti-bleaching solution (Vector Labs,
Burlingame, CA). The slides were sealed with clear nail polish and stored at 4°C in the dark
until immunoflorescence analysis.
2.3.5: Human Embryo Culture
To determine the effects of different concentrations of CoQ10 on cleavage, embryo
morphology, blastocyst formation, and hatching rate, donated Day-1 frozen embryos were
thawed and cultured in P1 medium (Irvine Scientific, USA, Cat # 90125) with 10% serum
substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA, USA) supplemented with
CoQ10 or with the corresponding concentrations of CoQ10 solvent at 37°C in a humidified
atmosphere of 5.5% CO2.
40
P1 culture medium is intended for use in culturing human gametes and embryos during
fertilization and growth through Day 3 of development. This medium is made based upon the
original human tubal fluid (HTF) formula but modified to provide a glucose-free and
phosphate-free environment. This formulation also contains taurine, an amino acid shown to
have protective and embryogenic functions. P-1 Medium is bicarbonate-based and is designed
for procedures utilizing a CO2 incubator. The components include:
Sodium Chloride
Potassium Chloride
Magnesium Sulfate, Anhydrous
Calcium Chloride, Anhydrous
Sodium Bicarbonate
Sodium Pyruvate
Sodium Lactate
Taurine
Sodium Citrate
Phenol Red
Gentamicin
Embryos in all groups were monitored daily at the same time in the morning using bright field
inverted optics. The number of embryos cleaving, developing to 4- to 8-cell, morula,
blastocysts (early, expanding, expanded), and hatching as endpoint were recorded. At cellular
stage, embryos were scored based on number and regularity of blastomeres, degree of
fragmentation, and blastomere multinucleation. In our grading system I represent the best and V
41
represents the lowest quality (highest degree of fragmentation). The resulting blastocysts were
vitrified and warmed and the survival rate was determined for each study group.
2.3.6: Experimental Groups
A total of 100 donated embryos at pronuclear stage (zygotes) were thawed and the 91 surviving
embryos were scored for intactness and morphology. Donated pronuclear stage embryos were
originated from women younger than 35 years. To minimize the effect of embryo origin on
blastocyst outcome, embryos originating from one patient were equally distributed for all 4
groups. For inclusion criteria, each embryo must meet the following criteria: 1) signed consent
form for donating excess embryos to research
, 2) Frozen at 2PN stage (day-1). Exclusion Criteria consisted of abnormal morphology of
thawed Day-1 embryos.
This study had 4 experimental groups. 1) Control (n=28), 2) 10 µL Ethanol (1% v/v) (n = 21),
3) 5 µM CoQ10 (n = 21), and 4) 10 µM CoQ10 (n = 21). Since we did not observe a positive
effect of 20 µM of CoQ10 in mouse embryo study and due to a limited number of donated
human embryos, we decided to culture human embryos in 5 and 10 µM of CoQ10. For the
control group, embryos were cultured in 1 mL P1 supplemented with 10% SSS. For 5 µM
CoQ10, 5 µL of the working solution was added to 995 µL of P1 + 10% SSS (culture medium)
and for 10 µM, 10 µL of the working solution of CoQ10 was added to 990 µL of the culture
medium. For the fourth group 10 µL of solvent (ethanol) was added to 990 µL of the culture
medium. After 8-cell stage the embryos were moved from P1 to blastocyst culture medium.
2.3.7: Pronuclear Embryo Thawing
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Pronuclear embryos were thawed using embryo thawing solutions from Vitrolife (THAW-KIT
1™ ,Vitrolife, Sweden, Ref # 10067). THAW-KIT 1™ consists of four physiological salt
buffers containing human serum albumin and pharmaceutical grade Penicillin G as a
preservative. THAW-KIT 1™ is supplemented with 1,2-Propanediol and sucrose as following
concentrations;
- ETS 1 contains 1.0 M 1,2-Propanediol and 0.2 M Sucrose
- ETS 2 contains 0.5 M 1,2-Propanediol and 0.2 M Sucrose
- ETS 3 contains 0.2 M Sucrose
-
Before thawing, the identity and location of straws containing donated embryos were
determined. The straws were kept under liquid nitrogen (LN2) in a small Dewar vessel until
actual thawing. 0.5-1mL volumes of Cryo-PBS, ETS 1, ETS 2 and ETS 3 were pipetted into
respective labeled sterile multi-well dishes and the dishes were pre-equilibrated 20 ± 5 °C.
The straws were thawed one at a time. The straw was removed from LN2 and kept in the air for
30 seconds. The condensation on the straw was gently wiped using a soft tissue and the straw
was checked for any dama
