Master Thesis
Trophoblast-Invasion of uterine lymphatic
vessels in the early human placenta
submitted by
Michaela Lichtensteiner, BSc.
for the Academic Degree of
Master of Science
(MSc)
at the
Medical University of Graz
Institute for Cell Biology, Histology and Embryology
under the supervision of
Mag.a rer.nat. Gerit Moser, PhD
2017
II
ACKNOWLEDGEMENT
I would like to express my deepest gratitude to my supervisor Gerit for supporting
and encouraging me throughout the last year. Always teaching and offering me broad
knowledge so that you have increased my interests in science in a way that I could
not have for seen. You are a patient, cooperative and open-minded guide who has
given me the chance for my further personal development at the congress in
Eisenstadt. Thank you that I could be a part of the placenta-community for a short
time so that I hopefully could advance the research field a bit!
I want to thank Monika, Moni and Astrid for familiarizing me with lab skills and helping
me in all intents and purposes. Thanks Rudi and Daniel for solving technical
obstacles and a special thank to Daniel for supporting me in statistical assessment. I
want to thank Moni and Rudi again for giving first aid after my accident at lab. I want
to thank Fr. Scheiber and the whole secretary team for administrative stuff. I want to
thank all my colleagues and friends for our tight relationship that turned into
everlasting friendship. Especially I want to thank Michelle, my work-buddy, for always
keeping me smiling inside and outside of the lab and Helí, my #friendshipneverende,
for staying by my side through all ups and downs. I want to thank my family for being
there! No writing can express my deep feelings and love about you! I am just grateful
to have you in my life!
I want to thank Prof. Dohr for giving me the opportunity to carry out my master thesis
at the Institute of Cell Biology, Histology and Embryology! I want to thank all
members of the Histo-Team for the great and warm-hearted atmosphere. It was a
pleasure to be a part of your team.
Thank you!
III
ABSTRACT
Lymphatic vessels play a major role for maintenance of tissue balance by draining
and absorbing macromolecules, plasma or cells. In collaboration with secondary
lymphatic organs efficient immune-defense can be achieved. In early human
pregnancies fetal cells (Extravillous trophoblasts - EVTs) invade into decidual
endometrium to anchor the placenta to the uterus. Thereby EVTs pass spiral arteries,
veins as well glands and penetrate their endothelial and epithelial layer until reaching
the inner lumen. This master thesis aimed to investigate whether EVTs invade into
uterine lymphatic vessels. First trimester placentas (7-8 weeks of gestational age)
were obtained from elective terminations of pregnancies and were subsequently fixed
and embedded in paraffin. Serial sections were immunohistochemically double
stained with the antibodies D2-40 Podoplanin, Cytokeratin 7, HLA-G and VWF to
examine invaded and non-invaded decidual tissue by light microscopic techniques.
Trophoblast invasion into lymphatic vessels was semi-quantitatively determined with
the Vis Visiopharm Software. In early human placentas a lymphatic vasculature is
prominent and is invaded by EVTs. Until now spiral arteries, veins and uterine glands
are known to be invaded by endovascular and endoglandular trophoblasts. Results of
this master thesis show that EVTs migrate towards endothelial basement membrane
and are finally situated within the lumen of lymphatic vessels. Now it has to be
determined whether lymphatic vessels play a pivotal role in interstitial fluid
homeostasis of decidua or even support maternal immune-response in first trimester.
IV
ZUSAMMENFASSUNG
Lymphgefäße spielen eine wichtige Rolle bei der Erhaltung des Gewebeausgleichs
indem sie Makromoleküle, Plasma oder Zellen filtern und aufnehmen. Gemeinsam
mit den sekundären lymphatischen Organen kann eine geeignete Immunabwehr
erreicht werden. In der frühen humanen Schwangerschaft invadieren fetale Zellen
(Extravillöse Trophoblasten – EVTs) in deziduales Endometrium um die Plazenta im
Uterus zu verankern. Dabei passieren EVTs Spiralarterien, Venen sowie Drüsen und
penetrieren deren Endothel- und Epithelschicht bis sie in das innere Lumen
gelangen. Diese Masterarbeit zielt darauf ab die Invasion von EVTs in Lymphgefäße
zu untersuchen. Ersttrimester Plazenten (7. bis 8. Schwangerschaftsswoche) von
freiwilligen Schwangerschaftsabbrüchen wurden fixiert und in Paraffin eingebettet.
Davon angefertigte Serienschnitte wurden immunhistochemisch mit den Antikörpern
D2-40 Podoplanin, Cytokeratin 7, HLA-G und VWF doppelt gefärbt um invadiertes
und nicht invadiertes Deziduagewebe mit lichtmikroskopischen Methoden zu
untersuchen. Die Trophoblast Invasion in die Lymphgefäße wurde semi-quantitativ
mit der Vis Visiopharm Software bestimmt. In frühen humanen Plazenten ist ein
Lymphgefäßsystem vorhanden und dieses wird von EVTs invadiert. Bis jetzt sind
Spiralarterien, Venen und uterine Drüsen dafür bekannt von endovaskulären und
endoglandulären Trophoblasten invadiert zu werden. Die Ergebnisse zeigen, dass
die EVTs in Richtung endothelialer Basalmembran wandern und letztlich im Lumen
von Lymphgefäßen lokalisiert sind. Nun muss bestimmt werden, ob Lymphgefäße in
der Dezidua eine essentielle Rolle bei der interstitiellen Flüssigkeitshomöostase
spielen oder sogar die mütterliche Immunantwort im ersten Trimester unterstützen.
V
TABLE OF CONTENTS
ACKNOWLEDGEMENT ............................................................................................. II
ABSTRACT ............................................................................................................... III
ZUSAMMENFASSUNG ............................................................................................. IV
1 INTRODUCTION.................................................................................................. 7
1.1 Modification of Human Endometrium after Implantation ................................ 7
1.2 Development of Early Human Placenta ......................................................... 7
1.3 Subpopulation of EVTs in the First Trimester: Routes of Invasion ................. 9
1.4 Expression Patterns of EVTs, Uterine Glands and Vessels......................... 10
1.5 The Lymphatic System in the Human Body: Main Functions....................... 11
1.6 Composition and Structural Features of Lymphatics ................................... 12
1.7 Development of Lymphatic Vessel Network ................................................ 12
1.8 Lymphatic Vessels in the Human Endometrium:pregnant and non-pregnant
state ............................................................................................................. 13
2 AIM..................................................................................................................... 15
3 METHODS ......................................................................................................... 16
3.1 Tissue Collection ......................................................................................... 16
3.2 Preparation of Tissue .................................................................................. 16
3.3 Immunohistochemistry ................................................................................. 17
3.4 Testing of antibodies ................................................................................... 18
3.5 Optimiziation of GBI double staining Kits ..................................................... 19
3.6 Immunofluorescence ................................................................................... 19
3.7 Image Acquisition and Quantification .......................................................... 20
3.8 Statistical analysis ....................................................................................... 22
4 RESULTS .......................................................................................................... 23
4.1 Histological observations ............................................................................. 23
4.2 Trophoblast invasion into lymphatic vessels ................................................ 24
4.3 Antibodies for immunostaining of lymphatic vessels .................................... 26
4.4 Relative frequency of lymphatic vessels in first trimester placenta .............. 27
4.5 Frequency of vessels and glands in decidua basalis and decidua ..................
parietalis ...................................................................................................... 28
4.6 Invasion of EVTs into lymphatic vessels ...................................................... 29
4.7 The relation between glands and vessels within invaded decidua............... 30
VI
4.8 Immunofluorescent double staining ............................................................. 30
4.9 Optimization of GBI double staining Kits ..................................................... 31
4.10 Investigation of Spiral arteries .................................................................. 34
5 DISCUSSION ..................................................................................................... 36
6 CONCLUSIO ...................................................................................................... 40
7 ABBREVIATIONS .............................................................................................. 42
8 REFERENCES .................................................................................................. 43
9 PROTOCOLS .................................................................................................... 49
9.1 Embedding .................................................................................................. 49
9.2 Dissection .................................................................................................... 50
9.3 Deparaffination and Antigen Retrieval ......................................................... 51
9.4 Immunohistochemistry ................................................................................. 52
9.5 GBI-Double Staining Kits ............................................................................. 54
9.6 Immunofluorescense ................................................................................... 60
7
1 INTRODUCTION
1.1 Modification of Human Endometrium after Implantation
Development of conceptus takes place in mother’s reproductive organ: the uterus.
Pear-shaped human uterus is composed of two zones: the endometrium and the
myometrium.1 The endometrium is lined by the regenerating layer stratum
functionalis and the permanent maintaining stratum basalis.2 The lower basal layer
consists of dense stromal fibroblast cells that transform into specialized secretory
decidua cells when blastocyst implants within uterine wall.3 The onset of
decidualization is described by successive cell enlargement, rounding of the nucleus,
accumulation of glycogen and lipid droplets as well as oedematous stroma.3,4 As a
consequence, cells alter fundamentally gene expression patterns and affect therefore
immune cells, composition of extracellular matrix and angiogenesis. Decidualization
is an intricate hormone driven process so that its induction and enhancement is
highly regulated by progesterone, estrogen and cAMP signalling pathways.
Promoting proliferation and differentiation provide a nutritive microenvironment for
implanting embryo as well developing and sustaining of placenta in early
pregnancy.1,5
On day 6-7 post conception blastocyst attaches and implants into the uterine
epithelial mucosa of the endometrium.6,7 The blastocyst comprises of an inner cell
mass that establishes the growing embryo and a coating layer that differentiates to
primitive trophoblast cells. In later stages trophoblast cells start to develop two
different cell lineages: the cytotrophoblast and syncytiotrophoblast.4,6 The poly-
nucleated syncytiotrophoblast displays the only fetal tissue that connects intimately
maternal parts by deep invasion. The underneath lying cytotrophoblast remains
mononucleated but act as stem cells which proliferate and fuses continuously with
the syncytiotrophoblast.6,8
1.2 Development of Early Human Placenta
At implantation pole the trophoblastic wall is thicker compared to the antiimplantation
pole. The thicker part is then transformed to the placenta, whereas the thinner parts
establish smooth chorion and the membranes. After attachment, the blastocyst is
8
completely embedded by maternal decidua therefore three types of decidua can now
be differentiated. Decidua capsularis closes over the blastocyst and surrounds
developing fetus. Decidua basalis is the part of endometrium which generates the
maternal component in developing early placenta. Decidua parietalis is the residual
endometrial mucosa which plays no role regarding placenta attachment and is not in
contact with blastocyst 9 (Fig. 1).
The placenta is an extracorporeal organ that establishes the feto-maternal interface
and enables the exchange of gas, the selective transport of nutrients and waste
products as well as protecting the developing fetus.10
Figure 1: Pregnant human uterus 10 weeks after fertilization. On days 6-7 post conception blastocyst
implants into endometrial wall that undergoes decidual reaction. Two poles evolve, at the implantation site
develops early placenta consisting of fetal part (villous structures) and maternal part (decidua basalis). At
antiimplantation site establishes decidua capsularis that capsulate the blastocyst and then the developing fetus.
Remaining endometrial mucosa differentiates to decidua parietalis.11
(modified by Michaela Lichtensteiner)
The fetal side develops from the chorionic plate and is smoothly covered by the
amnion while the maternal part is built up by the basal plate also referred to decidua
basalis. At the chorionic plate the syncytiotrophoblast; followed by the
cytotrophoblastic cells, spreads and forms villous structures; now referred to villous
trophoblast. Based on these main placental villi several side branches are sprouting
to form villous trees protruding through the wide luminal intervillous space within the
placenta. Floating villi in advanced stages are able to attach to the basal plate as
anchoring villi that form finally cell columns. At the distal ends of columnar tips,
9
proliferative cytotrophoblasts accumulate and serve as source of the extravillous
trophoblasts (EVTs) (Fig. 2).6,10,12 EVTs follow different routes of invasion; the so
called interstitial trophoblasts are able to invade decidual interstitium and reach the
inner third of the myometrium but lose their proliferative properties.12,13 Aggressive
invasion, migration and proliferation of EVTs is a highly regulated process which is
triggered by metalloproteinases (MMPs) besides of prior successful decidualization.
Extracellular matrix (ECM) proteins of decidual stroma, such as collagen, laminin and
fibronectin, are proteolytically degraded by MMPs that are expressed on EVTs’
surface.3,14,15 EVT invasion is crucial to anchor the placenta to the uterus, but failures
of invasion cause placenta accreta, percreta or increta, which are characterized by
deep and non-stopping invasion until reaching myometrium or bladder.16
1.3 Subpopulation of EVTs in the First Trimester:
Routes of Invasion
The placenta supports a hemochorial fetal-maternal interface but until the end of the
first trimester early developmental stages operate under a low physiological oxygen
condition.10,12 In early pregnancy endometrial spiral arteries encounter remodeling
adaptations, from highly contractile smooth muscle walls into dilated tubes with wide
lumen.3,12,17 EVTs are main drivers in transforming vasculature to supply optimal
nourishment of fetus. A specialized phenotype - the endovascular trophoblast -
develops during the first trimester and is able to invade vascular endothelium of
blood vessels, such as spiral arteries, capillaries or veins. EVTs massively infiltrate
spiral arteries and reach the lumen to form endovascular plugs for blocking the
maternal blood flow towards the placenta.17 At the end of the first trimester
disintegration of plugs marks the onset of uteroplacental perfusion by filling the
intervillous space with maternal blood, prior to that only blood plasma is seeping
through occluding EVTs.17,18,19
Apart from interstitial and endovascular trophoblast another subpopulation can be
presented that is characterized by invasion into the endometrial glands – referred to
as endoglandular trophoblast. A few years ago Moser et al. could show that this
trophoblast type is responsible for establishing histiotrophic nutrition during early
pregnancy. In this case EVTs migrate to the uterine glands and replace their
10
epithelial layer so that the glandular lumen starts to open towards the intervillous
space to enable the release of secretion products.18 Glandular fluids are composed
of a mixture of carbohydrate and lipid rich substances such as glycogen, glycodelin
A, and lipid droplets.20,21 Recently Uteroferrin presence could be determined in
glandular epithelium and may contribute to histiotrophic nutrition in prior stages of
pregnancy.22
Figure 2 Schematic cross section of human early placenta (6-11 weeks). The placenta establishes a feto-
maternal interface. From the chorionic plate protruding anchoring villi attach to maternal decidua and develop cell
columns. There cytotrophoblasts accumulate, turn into extravillous trophoblasts (EVTs) and start to invade into
maternal parts: The interstitial trophoblast (1) is able to migrate into decidua basalis and reaches the inner third of
myometrium. Spiral arteries and uterine glands are invaded by endovascular trophoblast (2) and endoglandular
trophoblast (3).23 (modified by Michaela Lichtensteiner)
1.4 Expression Patterns of EVTs, Uterine Glands and Vessels
Determination of single extravillous trophoblasts within decidual stroma and
associated with endometrial luminal structures can be routinely assessed by classical
immunohistochemistry. Double immunohistochemistry is a possible method for
discrimination of decidua basalis (EVTs present) and decidua parietalis (EVTs
absent).24 The indirect immunohistochemical staining is characterized by labeling cell
specific epitopes that are recognized by primary mono/polyclonal antibodies. But only
a second antibody targeting the first antibody allows visualization within histological
sections.
11
HLA-G molecules belong to the MHC class type I and are only expressed in fetal
cells thus EVTs can be identified easily.25 HLA-G at the maternal-fetal interface could
be responsible for biochemical reprogramming of the local maternal immune
response and therefore seems to play an immunological role in tolerance of the fetal
semi-allograft to protect the fetus of external events.26,27,28
Cytokeratin 7 displays a marker for all types of trophoblasts (Syncytiotrophoblast,
Cytotrophoblast, EVTs) as well for glandular epithelial cells within the early
placenta.25
Von Willebrand Factor specifically marks vascular endothelial cells of different vessel
types including spiral arteries, capillaries, veins and lymphatic vessels. Double
immunostaining with HLA-G allows representative discrimination of invading vessels
and EVTs.29
1.5 The Lymphatic System in the Human Body: Main Functions
The human body consists of an intricate cardiovascular system but apart from that a
secondary vascular system is present as well.30 The lymphatic vasculature is
composed of a branched vessel network that has great impact on physiological
processes within the body to sustain healthy conditions.31 Lymphatic system has
diverse functions and several main roles: the maintaining of interstitial fluid
homeostasis, fluid and lipid transport and immune surveillance.19,32,33 Misdirected
regulation leads to deficiencies and their respective diseases, such as lymphedema,
lymphangitis or types of cancer.34 Lymphatic vessels can be found in almost all
vascularised tissues so that they keep on staying in tight contact with the blood
vessels to enable resorption and draining of substances.35 Lymphatic-capillary
connections enable distribution of oxygen, nutrients and hormones whereas
lymphatico-venous junctions collect carbon dioxide and eliminate waste. Interstitial
fluid and cells in tissues are filtered by lymphatics and return protein-rich extravasate
back to capillaries and venous system. The lymph that contains antigens, immune
cells and plasma components passes through the lymph nodes with sieving
functions. There foreign particles are recognized by antigen presenting cells to
induce efficient immune responses. Moreover lymphatic vessels are present in
mucous membranes therefore lymphatics within intestine absorb digested lipids and
transport packaged chylomicrons through the mesenteric lymphatic system.30,31,35
12
1.6 Composition and Structural Features of Lymphatics
The lymphatic vascular network is made up of different tube-like structures such as
lymphatic capillaries, precollecting and collecting vessels.
Blind ended lymphatic capillaries are thin-walled and consist of a single layer of
endothelial cells (LEC).30 Fine capillaries exhibit no or perforated basement
membrane and are not covered by pericytes or smooth muscle cells.31 Additionally
interendothelial gaps emerge due to discontinuous structure that contributes to high
permeability of lymph components. Because lymphatics are embedded within tissue
lymphatic capillaries are linked to ECM by anchoring filaments. Due to interaction of
collagen fibers microenviromental changes can be sensed and accordingly
responded to stimuli, such as tissue swelling and pressure ratio. Thus anchoring
filaments avoid vessel collapse when interstitial pressure is rising.30,31 Side branches
of capillaries are connected to precollecting vessels that fuse with large collecting
lymphatic vessels.30,35 Collectors show a continuous basement membrane and are
ensheathed by contractile smooth muscle wall that supports lymph propulsion. In
addition big collecting lymphatic vessels resemble small veins because they have
bileaflet valves to prevent lymph backflow.30,31 Lymph microcirculation is sustained by
many extrinsic and intrinsic factors such as arterial pulsation, lymph formation and
flow rate, nitric oxide, inflammatory mediators and a combination of oncotic and
hydrostatic pressure.35,36
1.7 Development of Lymphatic Vessel Network
Lymphangiogenesis - the process of developing lymphatic linage – starts with the
expression of prox1 (prospero related homeobox-1) upon endothelial cells that derive
from the cardinal vein.35 The transcription factor prox1 initiates the biochemical
remodeling of venous endothelial cells to lymphatic progenitors (LECs).32
Beside initial factor prox1 the second key player vascular endothelial growth factor
VEGF-C is involved and functions during adulthood as well. In addition to that
angiogenic molecules, like angiopoietin-2, are characterized by stimulating postnatal
vessel growth.32,35,37 Upon cell surface of LECs further different molecules are highly
expressed that functions as useful markers, such as LYVE1 or Podoplanin.39
13
Podoplanin is a 38-kD mucin-type transmembrane glycoprotein that was first found
on the surface of podocytes and controls platelet aggregation.19,38 One benefit of
podoplanin is that it is expressed in lymphatic capillaries as well in collecting
lymphatic vessels.32 Thus it serves well as marker for lymphatic vessels in
immunohistochemical stainings.
LYVE1 (Lymphatic vessel endothelial receptor-1) is as well a transmembrane
receptor and interacts with the glycosaminoglycan hyaluron.28,39 Hyaluron is a 105-
107 Da mucoploysaccharide that is ubiquitous present in extracellular matrix of
tissues.40
1.8 Lymphatic Vessels in the Human Endometrium:
pregnant and non-pregnant state
Donoghue et al. stated very well that “there are conflicting reports on the distribution
of endometrial lymphatics, with some studies reporting lymphatics in the functional
zone of human endometrium, others only in the endometrial basalis, and some
reporting none at all.”41
Immunohistochemical staining revealed that lymphatic vessels of non pregnant
endometrium are present in all layers at which the higher situated stratum
functionalis has a lower amount than the deeper situated stratum basalis.41 Red-
Horse et al. described that lymphatic vasculature is not a prominent feature within the
non-pregnant endometrium but is only initialized during decidualization. Lymphatic
vessels are ubiquitous in decidua. The group stated that lymphangiogenesis
establishes only in decidua basalis and in decidua parietalis at the feto-maternal
interface.28 Murine models showed that cytotrophoblasts stimulate
lymphangiogenesis within decidua because they express lymphangiogenic
molecules.28,33 However, Volchek et al. claimed that lymphatic vessels are regressed
during remodeling of uterine endometrium.39
Apart from challenging presence or absence of lymphatics during decidualization
both groups could not find any evidence for a physical interaction between EVTs and
lymphatic vessels. Red Horse et al. described that invasive trophoblasts are
consistently detected in decidua and stay in contact with lymphatics but do not reach
their lumen.33 Very recently published data of two other groups (Windsperger et al.
14
and He et al. 2017) confirmed that EVTs enter lymphatic vessels within decidua
basalis.42,43
15
2 AIM
Extravillous trophoblast (EVT) invasion during early human pregnancy into uterine
glands and blood vessels is well examined and secures supply with nutrients of the
developing embryo. Histiotrophic nutrition is enabled by EVT into uterine glands.
EVTs invade and replace the glandular epithelium and thereby open the uterine
glands towards the intervillous space. Until the end of first trimester spiral arteries are
blocked by endovascular trophoblasts plugs before the establishment of the feto-
maternal blood flow. This master thesis wants to extend the general concept of EVT
invasion. There is a significant knowledge gap on how lymphatic vasculature can be
affected by EVTs. Main focus of this thesis was to investigate EVT invasion into
lymphatic vessels.
Determination of hypothesis was addressed by using immunohistochemistry. Based
on bright field and fluorescence microscopy the quantification of lymphatic vessels
was assessed. Therefore commercially available double staining kits and specific
antibodies against lymphatics were tested and optimized with formalin fixed paraffin
embedded first trimester placenta.
16
3 METHODS
3.1 Tissue Collection
First trimester placentas were obtained from elective terminations of pregnancies and
fixed in 4% formalin with subsequent paraffin embedding (Tissue-Tek® VIP™;
Sakura, USA).
Additional archival tissue samples of the Institute Histology, Cell Biology and
Embryology, Medical University Graz, were provided for investigation. For analysis of
EVT invasion in lymphatic vessels 19 placentas were used. Especially decidua
basalis and decidua parietalis were examined. Placentas in all experiments had a
gestational age (GA) of 7 to 8 weeks.
3.2 Preparation of Tissue
Dissection
Tissue samples were sectioned in single and serial 5µm sections with the slide
microtome (HM 440E, MICROM, Zeiss, Germany) and rotational microtome (HM
355S, MICROM, Zeiss, Germany). Cut tissues were unfolded in a 37°C warm water
bath and then applied to Superfrost Plus glass slides (Menzel-Gläser, Thermo
Scientific, Germany). For adhesion of tissues the slides were incubated overnight at
52°C.
Deparaffination and Antigen Retrieval
Microscope slides were deparaffinized by a rehydrating graded alcohol serial session
beginning with the xylene subsitute Tissue Clear (Tissue-Tek, Sakura, USA).
Then sections were slewed in a 1:1 mixture of Tissue Clear and 100% ethanol
followed by a 70% and 50 % alcohol purification step and placed in distilled Aqua.
After removal of paraffin Heat-induced antigen retrieval was performed. Sections
were boiled with antigen retrieval solutions at pH 6, 9 (Leica, Germany) with a
pressure cooker for 7 min at 120°C (Decloaking chamber, BioCarta, Germany).
Slides were cooled down for 20 min and still remain in distilled boiled Aqua until
beginning of the immunohistochemistry.
17
3.3 Immunohistochemistry
Immunohistochemistry was carried out in a moist chamber. Different detection
systems were used and are listed in chapter 9.
Single Immunohistochemistry
Single stainings were performed with the Ultravision LP detection system (Thermo
Scientific, USA) containing UV Hydrogen Peroxidase Block and Ultra V Block.
Primary antibodies, described in Table 1, were diluted with antibody dilution solution
(Dako, USA). Slides were counterstained with Mayer’s hemalaun and mounted with
the aqueous mounting medium Aquatex (Merck, Germany).
Table 1: Overview of antibodies. Lists the characterisation of using antibodies and their working instructions.
Antibodies/
Clones
Host/
isotype
Company Target Stocks
mg/ml
pH value Working
Dilution
HLA-G
(4H84)
Anti-mouse
mc
Bioscience EVT 0,5 9 1:1000
VWF
(F3520)
Anti-rabbit
pc
Sigma
Aldrich
Vessels 7,1 9 1:3000
CK7
(APO6204PU-N)
Anti-rabbit
pc
Acris Glands
EVT
1 9 1:1000
Podoplanin
(D2-40)
Anti-mouse mc
Dako Lymphatic vessels
0,2 9 1:1000
LYVE1 Anti-mouse
mc
Angio Bio Lymphatic
vessels
0,5 9
6
1:500
1:1000
HLA-G, human leukocyte antigen G; VWF, von Willebrand Factor; CK7, Cytokeratin 7;
mc, monoclonal; pc, polyclonal; EVT, extravillous trophoblast
Double Immunohistochemistry
Double immunostaining was the main technique in this master thesis. Therefore the
GBI Labs double staining system (Golden Bridge International, USA) was used. Kits
instructions and handling details are described in chapter 9.
Per placenta three serial sections were double stained with various antibody cocktails
(Table 2).
18
Table 2: Double immunohistochemistry. Shows three different double staining procedures using during master
thesis.
Double IHC Anti-mouse (mc) Anti-rabbit (pc) Target
1 HLA-G VWF EVT, Vessels
2 Podoplanin Cytokeratin 7 Lymphatic vessels,
Glands, EVT
3 Cytokeratin7 HLA-G Glands, EVT
mc, monoclonal; pc, polyclonal
3.4 Testing of antibodies
Antibodies with different hosts were used for single and double
immunohistochemistry (Table 1). To determine the optimal working condition of the
antibodies two parameters were examined: Antigen Retrieval buffer with different pH
values (pH 6, pH 9, without treatment) and diverse antibody concentrations. For the
analysis of lymphatic vessels staining procedures were compared with two
antibodies: Podoplanin and LYVE1 (Table 3). In addition positive and negative IgG
controls were assessed within respective tissues, such as Colon, Appendix and
Lymphnode.
Table 3: Lymphatic vessel binding antibodies. Describes the improving working conditions of the antibodies
LYVE1 and Podoplanin.
Different dilution factors Different pH values
LYVE1 1:25
6 9 without
treatment
1:50
1:100
1:500
1:1000
Podoplanin 1:100
6 9 without
treatment
1:200
1:500
1:750
1:1000
Positive control: Colon, Appendix
Negative control: Lymphnode
19
Finally, Podoplanin antibody with dilution factor 1:1000 and the antigen retrieval
buffer at pH 9 was used for labeling lymphatic vessels throughout master thesis.
3.5 Optimization of GBI double staining Kits
Three GBI double staining kits Polink DS-MR-Hu A1, B1 and C1 were tested and
optimized. This system consists of an anti-Mouse HRP-Polymer and an anti-Rabbit
AP-Polymer with two alterable chromogens (Table 4).
For an optimal staining result, various conditions were tested to increase the
sensitivity, stability and reproducibility. Amongst others dilution factors, different pH
buffers, incubation steps, substrate enhancement through repeating steps, different
chromogens, counterstainings, various water soluble mounting media and the
presence of cover slips were tested. Based on these changeable features the best kit
was chosen to use it throughout the whole experimental double stainings. Finally, the
improved GBI Labs A1 staining kit (Golden Bridge International, USA) with Vector
Blue Substrate as chromogen was selected for the experiments.
Table 4: GBI double staining kits.
IHC mix Chromogen Staining appears
A1 Kit catalog no DS201A-6
HLA-G DAB brown (HRPP)
CK7 / VWF GBI-Permanent-Red
[Vector Blue]
red
[blue] (APP)
B1 Kit catalog no DS201B-6/(D63-6)
HLA-G AEC red (HRPP)
CK7 / VWF BCIP/NBT
[Vector Blue]
blue/purple
[blue] (APP)
C1 Kit catalog no DS201C-6
HLA-G Emerald green (HRPP)
CK7 / VWF GBI-Permanent-Red
[Vector Blue]
red
[blue] (APP)
HRPP, horseradish peroxidase-Polymer; APP, alkaline phophatase-Polymer;
[ ], improved protocol with Vector Blue substrate
3.6 Immunofluorescence
Additionally, an immuno fluorescence double staining was performed. Deparaffinized
slides at pH 9 were incubated with Ultra V Block (Thermo Scientific, Germany) before
20
primary antibodies were applied for 30 min at room temperature. After three washing
steps with PBS-T, the secondary antibodies were incubated for 30 min at room
temperature. Secondary antibodies Alexa Fluor 555 goat anti-mouse and Alexa Fluor
488 goat anti-rabbit (Invitrogen, Austria) were diluted in PBS and served as
fluorescent-labeled antibodies. Slides were counterstained with in PBS diluted Dapi
and terminally rinsed in PBS three times again. Air dried glass slides were sealed
with ProLong Gold Antifade reagent (Invitrogen, Austria). Table 5 shows using
antibodies for immunofluorescence.
Table 5: Immunofluorescence. Shows the using antibodies in double fluorescent staining and their working
conditions.
IgGs Host/Isotype Company Stock mg/ml
Dilution Target
Primary
antibodies
HLA-G Mouse IgG
(mc)
Bioscience 0,5 1:500 EVT
CK7 Rabbit IgG
(pc)
Acris 1 1:500 Glands
EVT
VWF Rabbit IgG
(pc)
Sigma
Aldrich
7,1 1:1500 Vessels
Podo Mouse IgG
(mc)
Dako 0,2 1:500 Lymphatic
vessels
Fluorescent
labeled
antibodies
Alexa
Fluor 488
(green)
Goat anti-
mouse (mc)
Invitrogen 2 1:200 HLA-G
Podoplanin
Alexa
Fluor 555
(red)
Goat anti-
rabbit (pc)
Invitrogen 2 1:200 Cytokeratin 7
VWF
Nuclear counterstaining with Dapi (diluted 1:2000)
3.7 Image Acquisition and Quantification
In an attempt to determine the importance of lymphatic vessels in early human
placentas images of immunostained histological sections at 10x and 20x
magnification were taken. Both bright-field and fluorescence microscopic analysis
was performed with the microscope Leica (model DM6000B) with integrated high
definition digital camera (model DP72; Olympus Austria GmbH, Vienna, Austria).
21
For evaluation a semi-quantitative analysis was accomplished that was divided in two
quantifying modes (VIS Visiopharm Software): For the first quantification five regions
of interest (ROI) within decidua basalis (invaded) were selected to analyze the
invasion into lymphatic vessels (n=5 images/invaded region). Therefore three serial
sections with different immunodouble staining of 19 placentas were investigated. For
the quantification of these images three groups (EVT-invaded, EVT-attached and
EVT-non invaded) were classified to count lymphatic vessels, blood vessels and
glands within the ROI. Totally 285 images were explored (n=3 slides).
For the second quantification five regions within decidua basalis (invaded) and
decidua parietalis (non-invaded) were randomly selected to quantify the relative
amount of lymphatic vessels compared to blood vessels and glands (n=5 images per
invaded and non-invaded region). Therefore two serial sections with different double
IHC of 15 placentas were analyzed. All visible lymphatic vessels, blood vessels and
glands within these regions were counted. In sum 300 images were evaluated (n=2
slides). Both quantifying modes are demonstrated in Fig. 3.
Figure 3: Image acquisition and quantification of assessed first trimester placentas. Chart represents two
modes for semi-quantitative evaluation.
22
3.8 Statistical analysis
Data was analyzed with Microsoft Excel and figures were created with GraphPad
Prism7.
23
4 RESULTS
4.1 Histological observations
Uterine glands, blood vessels and lymphatic vessels can be observed within decidua
of first trimester placentas. The epithelial layer of glands is composed of a single row
of mononuclear columnar cells. The vascular system consists of capillaries and
arteries that have a single layer of mononuclear flattened cells. Lymphatic vessels in
the assessed first trimester placentas showed the same characteristics like vessels
(veins). Lymphatic endothelia appeared often thin-walled and unstructured. Their
lumen profiles varied in architecture and size, depending on higher or deeper portion
of sections lymphatics had a wide or narrow lumen. Lymphatics appeared mostly with
a discrete lumen but occasionally collapsed lumina occurred too.
Decidual tissue can be divided in invaded (Decidua basalis) and non-invaded
(Decidua parietalis) tissue. The main discrimination is the presence or absence of
extravillous trophoblast cells (EVTs). However EVTs can be observed as enlarged
cells throughout the whole stroma of decidua basalis (invaded). EVTs are also found
associated to epithelia of glands and endothelia of vessels. They were able to
penetrate and replace their basal membrane. When EVTs attached to or invaded
lymphatic vasculature it was mostly destructed. Lumina appeared loose and
disrupted. Furthermore, EVTs invaded repeatedly vascular smooth muscle walls of
blood vessels, stayed within the lumen and formed endovascular plugs. Sometimes
spiral arteries showed some peculiarities within invaded tissue. In some sections
EVTs seem to accumulate around spiral arteries but did not invade smooth muscle
walls. However, in deeper proportions of the same vessel EVTs clearly invaded the
spiral artery.
24
4.2 Trophoblast invasion into lymphatic vessels
Beside blood vessels and uteroplacental glands, lymphatic vasculature was observed
in decidual tissue of early placentas. In some placentas lymphatic vessels appeared
more frequent than in others, although the placentas had the same gestational age.
Lymphatics occurred ubiquitous in decidua, but emerged conspicuously often in
spatial proximity of glands. Embedded glands were surrounded repeatedly by
weaving lymphatic vessels. Appearance of lymphatics differed between decidua
basalis and parietalis. Lymphatic vessels in non-invaded parts of decidua were
apparently more compact, whereas in invaded parts endothelial walls seemed often
dissolved (Fig. 4).
Figure 4: Lymphatic vessels within decidua. Sections of first trimester placenta (GA 7-8 weeks) were double
stained with Podoplanin (brown, marker for lymphatics) and CK7 (blue, marks here epithelium of glands and
EVT). (a) In non-invaded decidua lymphatic vessels (brown) were found frequently beside uterine glands. (b) In
invaded decidua EVTs (blue) were located in stroma and attached to lymphatic vessels (red circle). EVTs also
invade into lymphatic vessels (arrows). Abbrv: L, [lymphatic vessels]; G, [uterine glands]; V, [vessels]; arrows, [for
EVT]; O, [EVT attached]
EVTs were found repeatedly in close vicinity to the lymphatic vessels, attached to the
basal side of the lymphatic endothelium, replaced the endothelium and were situated
in the lumen of lymphatic vessels (Fig. 5a-f).
25
Figure 5: EVTs infiltration into lymphatic vessels within decidua basalis. Histological sections of first
trimester placenta (GA 7-8 weeks) (a-f) were immuno double stained with Podoplanin (specific marker against
lymphatics, brown) and CK7 (binds to EVTs & glands, blue). (a) EVTs penetrated lymphatic vessels and stayed
within the lumen. (b) EVTs started to degrade endothelium of lymphatic vessel. (c) Lymphatics with narrow
endothelial lumen is invaded by EVTs. (d) Lymphatic vessels can be identified precisely with Podoplanin-
Immunostaining and distinguished from blood vessels. (e,f) Lymphatic endothelial layers are disintegrated. EVTs
(arrows) are situated within the lumen of lymphatic vessels. Abbrv: V, [vessels]; L, [lymphatic vessels]; G, [uterine
glands]; arrows [for EVT]
26
4.3 Antibodies for immunostaining of lymphatic vessels
Two antibodies for immunostaining of lymphatic vessels were tested: Podoplanin and
LYVE1. The LYVE1 antibody reacted positive not only with lymphatic vessels within
placental tissue. Also the endothelium of spiral arteries was stained by LYVE1. This
non-specific interaction with lymphatic vessels was confirmed with colon tissue as
positive control. Intestine glands within colon tissue reacted unspecific positive with
LYVE1 antibody (Fig. 6a,b).
The Podoplanin antibody stained specifically lymphatic vessels within placental
decidua and colon (Fig. 6c,d). No unspecific staining was visible within spiral arteries
or crypts of colon.
Figure 6: Proof of antibodies’ specificity against lymphatic vessels. First trimester placental (a,c) and colon
tissue (b,d) were immuno stained with LYVE1 (a,b) and Podoplanin (c,d) antibody. Colon served as positive
control. LYVE1 binds non-specific to lymphatic vessels. Spiral arteries and crypts of colon are stained
unspecifically red (a,b).Podoplanin stains specifically lymphatic vessels (red) without co-staining of spiral arteries
(green rectangles) and crypts (c,d). Nuclei were counterstained with hematoxylin. Abbrv: L, [lymphatic vessels];
G, [glands]; D, [decidua]; C, [crypts]; □, [spiral arteries]; ∆, [artifact]
27
4.4 Relative frequency of lymphatic vessels in first trimester
placenta
Embedded in decidual stroma various common structures, like spiral arteries, veins,
glands and lymphatic vessels, were present in the first trimester placenta, but their
frequency differed (Fig. 7). Vessels dominated within placental bed during
microscopy assessment. Semi-quantitative analysis confirmed this and demonstrated
that vessels had the highest amount, especially blood vessels were most frequent.
Lymphatic vessels were commonly found in decidua, but not as often as blood
vessels. Apart from vessels uteroplacental glands were present and had the smallest
amount within decidual tissue. In addition analysis showed that the quantity of glands
and lymphatic vessels differed slightly.
Figure 7: Frequency of blood vessels, lymphatics and glands in early placenta. Decidua basalis (invaded)
and decidua parietalis (non-invaded) of 15 placentas (GA 7-8 weeks) were investigated and semi-quantitative
assessed. Analysis revealed that vessels were the highest fraction within decidual tissue: Arteries and veins (68,2
± 40,7%) and lymphatic vessels (18 ± 16,9%) were prominent in first trimester placentas. Uterine glands (13,7 ±
9,6%) appeared not as often. Data is shown as mean ± SEM [%].
28
4.5 Frequency of vessels and glands in decidua basalis and
decidua parietalis
Semi-quantitative evaluation revealed that the amount of blood vessels, lymphatic
vessels and glands showed differences in EVT-invaded (decidua basalis) and non-
invaded (decidua parietalis) areas. Uteroplacental structures within non-invaded
decidua exhibited constantly a higher quantity compared to structures in invaded
decidua. Depending on EVTs presence or absence the frequency of luminal
structures varied (Fig. 8): Arteries and veins showed in non-invaded regions the
highest amount (48 ± 19,9%) but in invaded regions their amount was smaller (20,4 ±
9,5%). Furthermore the quantity of lymphatic vessels differed in non-invaded (10,9 ±
9%) and invaded (7,1 ± 7,7%) areas. Beside vessels also the number of glands was
higher in non-invaded decidua (9,7 ± 4,8%) and fewer in invaded decidua (3,9 ±
2,8%).
Non-
invaded 48 ± 19,9% 10,9 ± 9% 9,7 ± 4,8%
Invaded 20,4 ± 9,5% 7,1 ± 7,7% 3,9 ± 2,8 %
Figure 8: Regression of luminal structures due to influence of EVTs. The amount of vessels and glands in
placental bed (GA 7-8 weeks) differed in decidua basalis and decidua parietalis. In non-invaded regions the
amount of blood vessels, glands and lymphatic vessels was higher than their quantity in EVT-invaded parts of
decidua. Therefore the number of luminal structures within decidua basalis (invaded) was constantly low. Data is
shown as mean ± SEM [%].
0
20
40
60
80
100
Am
ou
nt
of
str
uctu
res
wit
hin
fir
st
trim
este
r d
ecid
ua [
%]
Non-invaded region
Invaded region
Arteries &
Veins
Lymphatics Glands
29
4.6 Invasion of EVTs into lymphatic vessels
Quantification showed that EVTs were predominantly attached and in close vicinity to
blood vessels, lymphatic vessels and glands but deep invasion occurred as well.
Although the relative frequency of that invasion varied (Fig. 9): The invasion of EVTs
into lymphatic vessels (32,3 ± 25,8%) and glands (32,5 ± 39,2%) differed slightly but
compared to that, arteries and veins (22,9 ± 20,5%) were commonly penetrated less.
Beside invaded vessels and glands also EVT-unaffected structures appeared within
decidua basalis (invaded). Non-invaded blood vessels had the highest amount,
whereas unaffected lymphatic vessels and glands displayed similar quantities.
Arteries &
Veins
22,9 ± 20,5% 43,6 ± 46,4% 33,5 ± 38,3%
Lymphatic
Vessels 32,3 ± 25,8% 38 ± 40% 29,7 ± 35,6%
Glands 32,5 ± 39,2% 38,5 ± 48,6% 29 ± 47,7%
Figure 9: Infiltration capacity of extravillous trophoblasts in decidua basalis (GA 7-8 weeks). Different
groups (invaded-attached-non-invaded) were determined to analyze the interaction of EVTs with blood vessels,
lymphatic vessels and glands. The observation of decidua basalis (N=19) exposed dissimilarities between and
inside the different groups. Quantitative analysis revealed that the amount of EVT-attached structures had the
highest level (bars in the middle). EVT invasion into lymphatics and glands was widely balanced. Compared to
that, arteries and veins showed a lower rate of invasion (bars in the left). Not EVT-affected vessels and glands are
present within invaded areas of decidua too (bars in the right). Data is shown as mean ± SEM [%].
invaded attached non-invaded0
20
40
60
80
100
Ac
tio
n o
f E
VT
s [
%]
Arteries & Veins
Lymphatics
Glands
30
4.7 The relation between glands and vessels within invaded
decidua
Quantification of EVT invasion revealed that glands were invaded more often than
vessels, as shown in the right bars of Fig. 10. When comparing uterine glands with
blood vessels, evaluation demonstrated that arteries and veins had a smaller EVT
invasion than glands. However, rate of EVT invasion into lymphatic vessels was
nearly equal to the rate of invasion into uterine glands.
Figure 10: Comparison of glands and vessels in decidua basalis. The rate of EVT invasion into glands and
vessels within placental bed (GA 7-8 weeks) showed dissimilarities. Arteries and veins (22,9 ± 20,5%) were less
penetrated than uterine glands (32,5 ± 39,2%). EVT invasion into lymphatic endothelium (32,3 ± 25,8%) was
similar to the invasion into glandular epithelium (32,5 ± 39,2%). Comparing the whole vessel amount (26,2 ±
15,5%) with the quantity of glands (32,5 ± 39,2%) evaluation showed that the epithelial layer was more invaded
by EVTs. Data is shown as mean ± SEM [%].
4.8 Immunofluorescent double staining
Fluorescence imaging demonstrated trophoblast cells within decidual stroma and
associated with glands and vessels. Single EVTs appeared enlarged and infiltrated
the epithelium of glands and replaced it (Fig. 11a). The presence of lymphatic
vessels was confirmed with immunofluorescent double staining using Podoplanin and
VWF antibodies (Fig. 11b). Thereby, lymphatic vessels can be distinguished from
blood vessels. Lymphatic vessels displayed a dismantled and destructed
endothelium wall, as already seen in bright field microscopy. Counterstaining with
DAPI reflected all nuclei within decidual tissue.
0
20
40
60
80
100
Comparing Groups
Invasio
n o
f E
VT
s [
%]
Arteries & Veins
Glands
Lymphatics
Total Vessel
31
Figure 11: Fluorescence microscopy of early decidua. Histological sections of placentas (GA 7-8 weeks) were
double fluorescent stained. Image (a) was stained with HLA-G (marker for EVT) and CK7 (binds here to glands
and EVT). Glandular epithelium (red) can be clear identified and differentiated from invading EVTs (green). EVT
appears in a yellow merge as well. Image (b) shows the presence of disintegrated lymphatic vessels (green)
within decidua. VWF binding allows localizing the endothelium of vessels (red). Nuclear counterstain with DAPI
was done (blue). Abbrv: G, [glands]; L, [lymphatics]; V, [vessels]; circle, [EVTs]
4.9 Optimization of GBI double staining Kits
Clear identification of maternal and fetal components, such as vessels, glands and
fetal EVTs within decidua, was enabled with various antibody cocktails for double
immunohistochemistry: HLA-G and Cytokeratin 7 (CK7) allowed identification of
EVTs. CK7 visualized additionally the epithelial layer of glands. Vessels were
detected with endothelial cell marker Von Willebrand Factor (VWF).
GBI staining kits varied in performing double labeling, substrate conversion and color
mixes: Double immunohistochemistry with A1 kit represented a red and brown color
mix. The kit enabled an efficient differentiation between uterplacental glands, vessels
and penetrating EVTs (Fig. 12a,b).
A clear discrimination of EVTs and tubular lumina was achieved by using B1 kit.
Endothelia and epithelia were colored in purple. EVT cells were stained red or dark
red depending on the combination of antibodies (Fig. 12c,d).
C1 kit stained vessels pink and EVTs green by using the antibodies against HLA-G
and VWF. Epitope co-expression of EVTs caused by CK7 and HLA-G was
challenging (Fig. 12e,f). Differentiation was often not sufficient with original color
merges of GBI kits. A better distinction was obtained with improved A1 kit combined
with Vector Blue substrate. This staining procedure reflected high contrast of EVTs
and their proper invasion into vascular endothelium and glandular epithelium (Fig.
12g,h).
32
Figure 12: Testing of double staining kits within invaded decidua. First trimester placental tissues (GA 7-8
weeks) were used for double immunohistochemistry. Images from the left column showed staining with VWF
33
(against endothelial cells) and HLA-G (marker for EVT). Images in the right column showed double labeling with
CK7 (binds to epithelia of glands) and HLA-G (binds to EVT). (a,b) Sections in the upper row are immunodouble
stained with A1 Kit. The endothelium of vessels (red) and the epithelium of glands (red) can be distinguished by
EVTs (brown). (c,d) Sections are double stained with B1 kit. EVTs (dark red) invade vessels (purple) and glands
(purple). (e,f) The discrimination of EVTs, vessels and glands was more difficult when using C1 kit. In section (e)
EVTs (green) are observed within stroma and vessels (red). In section (f) EVTs (violet) invade epithelium of
glands (red). (g,h) Clear differentiation was enabled with improved A1 kit. Epithelial and endothelial cells appear
blue whereas EVTs are stained brown. Strong replacement of glandular epithelial cells is obvious (*). Nuclei were
counterstained in sections (a-d). Abbrv: G, [uterine glands]; V, [vessels]; arrows, [for EVT]
Optimized A1 kit was used for immunodouble staining of placental sections
throughout this master thesis (Fig. 13). Different antibody cocktails (Table 2) allowed
comparison of EVT invasion into lymphatic vessels, blood vessels and glands at
identical areas within serial sections.
Figure 13: Deep EVT invasion into lymphatic vessels within decidua basalis. Serial sections of placental
tissue (GA 7-8 weeks) were immunohistochemically double stained. Rows (a-c) and (d-f) are composed of serial
sections and allow direct comparison of invasion into vessels (a,d) (including arteries, veins and lymphatics),
lymphatics (b,e) and glands (c,f). (a,d) Sections show EVT (brown) invasion (arrows) into vessels (blue). VWF
served as marker for all endothelial cells. (b,e) Strong CK7 (blue, EVT & glands) and Podoplanin (brown,
lymphatics) staining confirms the presence of lymphatic vessels and reveals their infiltration. Invasion of glands
could not be shown with this staining procedure. (c,f) Only double staining with CK7 (marker for glands and EVT)
and HLA-G (serves as marker for EVT) enables visualization of EVT invasion (arrows) into glands. Single
trophoblast cells (brown) migrate into the layer of glandular epithelium. Abbrv: V, [vessels]; L, [lymphatic vessels];
G, [uterine glands]; arrows [for EVT]
34
4.10 Investigation of Spiral arteries
During microscopic analysis it was observed that oftentimes the muscle wall of spiral
arteries was not penetrated by EVTs although the arteries were surrounded by EVTs.
However, in deeper segments of the same artery trophoblast cells were situated
inside the arterial lumen. Tracking of spiral arteries within higher or deeper portions
revealed that the morphology changed progressively. Profile varied in size, shape
and appearance regarding to each section. In some portions the surrounding arterial
vascular smooth muscle wall was not affected by EVTs. In other portions EVTs are
located within the muscle wall and/or lumen of the artery (Fig. 14a-f).
35
Figure 14: Observation and tracking of spiral arteries in decidua basalis. Serial sections of first trimester
placenta (GA 7-8 weeks) are shown. Every tenth section was immunodouble stained with VWF (binds to vessels,
blue) and HLA-G (serves as marker for EVT, brown) to track the invasion of EVT into spiral arteries. (a-f). The
images (a) and (b) highlight the progressive trend of EVT migration and the alteration of spiral artery. After ten
cuts (b) EVTs penetrate into muscle layer (*) and some cut sides of the artery are already fused. In section (c)
and (d) the conformation of the artery changes so that new lumina of the same artery appear. In deeper portions
of slices the spiral artery cannot be tracked anymore (e,f). Abbrv: V, [vessels]; L, [lymphatic vessels]; G, [uterine
glands]; arrows [for EVT]; *, [unaffected vascular muscle wall]
36
5 DISCUSSION
In this master thesis it was determined whether or not lymphatic vessels were
present and invaded by extravillous trophoblasts (EVTs) within human placenta of
early pregnancy (gestational age 6-11 weeks). Trophoblast invasion is not a rare
event in first trimester of human pregnancy. Results of this master thesis reveal that
EVTs invade more structures in decidua than was known until recently. According to
Moser et al. uteroplacental glands are invaded and replaced by endoglandular
trophoblasts to provide histiotrophic nutrition.18 Images of double
immunohistochemistry (Fig. 12h) confirm that the epithelial layer of glands is
substituted by trophoblast cells. Apart from that, EVTs are the main driver in
remodeling of spiral arteries during early pregnancy.44 Recently, Weiss et al. verified
that endovascular trophoblasts accumulate and form plugs within the lumen until the
end of first trimester.45 Histological analysis confirms infiltration into blood vessels
(Fig. 12c,e,g). This master thesis demonstrates that lymphatic vessels are prominent
in decidua basalis and decidua parietalis of first trimester placentas. EVTs are
repeatedly situated within the endothelium and the lumen of lymphatic vessels. It is
likely that, EVTs migrate through the decidual interstitium, attach to the basal side of
endothelial wall of lymphatic vessels, penetrate the vascular basement membrane
and are finally inside the lumen of the lymphatic vessel (Fig. 5a-f). Consistent to our
findings very recently two other groups have also shown evidence of EVT invasion
into lymphatics.42,43
For studying trophoblast invasion in first trimester placental decidua single and
double immunohistochemistry is a useful method; as already used in current
papers.18,46,47 Double immuno staining enables co-localization of two different
morphological features within histological sections. Thereby interaction between both
cell types can be investigated. Analysis needs an appropriate staining kit protocol to
visualize cells of interest in proper manner. GBI Labs double staining kits have
already been used in current publications.48,49,50 Li et al. assessed pathological
relevant patterns of basal cell adenoma.48 Similar proteins of adrenal cortex
producing aldosterone or cortisol could be distinguished by Gomez-Sanchez et al.49
The presence of endothelial progenitor cells in vital myocardic tissue and their
correlation to cardiovascular diseases could be observed by Barsotti et al.50 Process
of trophoblast invasion into glands, blood vessels and lymphatic vessels within first
37
trimester placenta can be shown clearly when approaching A1 and B1 kit. C1 cannot
be recommended for optimal discrimination. However, results indicate that the best
possibility to represent placental bed is the optimized staining protocol of A1 Kit in
combination with Vector Blue substrate as described in the results 4.9 (Fig. 12g,h).
High reproducibility of data can be achieved.
Trophoblast invasion within double stained sections can be analyzed with bright field
microscopy. Immunofluorescent double staining is an alternative method to visualize
co-expressions of Human Leukocyte Antigen G (HLA-G) and Cytokeratin 7 (CK7)
epitopes on extravillous trophoblasts.23,42,43 If one cell expresses both antigens,
double staining shows a color merge in light and fluorescence imaging. The intensity
of color correlates with the expression profile of epitopes. Additionally fluorescent
staining enables clear distinction of both epitopes at EVTs (Fig. 11). Due to the better
screening of larger tissue areas, bright field microscopy was used for this master
thesis.
For screening of serial sections of placental tissues, cell specific antibodies are
required. HLA-G and CK7 are accepted antibodies to identify trophoblastic cells.25
However, CK7 also reacts with glandular epithelial cells. Only a double staining with
both antibodies allows a clear discrimination between invading EVT and glandular
epithelial cells. Regarding histology, blood and lymphatic vessels cannot be
discriminated easily when no erythrocytes are located within the lumen of blood
vessels. In particular veins and capillaries share same similarities like lymphatics.
Therefore all vessel types can be identified by the endothelial marker Von Willebrand
Factor (VWF) due to specific epitopes on endothelial surface. But single staining with
VWF is not sufficient to differentiate between blood and lymphatic vessels. Only
double immuno labeling with a specific lymphatic marker allows discrimination of
arteries and veins from lymphatic vessels. Lymphatic vessels express various
antigens on surface that can be exploited for simple identification. Antibodies like
lymphatic endothelial hyaluronan receptor-I (LYVE1) and Podoplanin recognize them
and react positive with the lymphatic endothelium.39 Referring to the performed
experiments, Podoplanin determines precisely lymphatic vessels within decidua of
first trimester. Double staining with Podoplanin and CK7 allows an elegant possibility
to discriminate clearly between lymphatic vessels, glands and EVTs. Based on this,
presence of lymphatic vasculature within decidua parietalis and decidua basalis was
38
confirmed. Lymphatic vessels are still present during dezidualization. Therefore
previous data of Volchek et al. cannot be confirmed.39 The experiments of this master
thesis demonstrate that lymphatic vessels did not completely disappear in first
trimester placenta. Our evaluation reveals that lymphatics are regressed in invaded
decidua compared to non-invaded decidua (Fig. 8). We suggest that this is due to
invasion of EVTs.
Volchek et al. claims that trophoblasts have no influence on the appearance of
lymphatic endothelium and do not interact with vessel.39 Referring to our data this
statement cannot be shared. Trophoblasts affect obviously lymphatic vessels and
disintegrate their endothelial layer (Fig. 5a-f). EVTs can be found associated to the
vascular wall and the structure of lumen appears dissolved. It is likely that due to
disruption of basal membrane EVTs can cross towards the lumen of the lymphatic
vessel. The picture of endovascular trophoblast invasion has been recently expanded
and stratified into endoarterial and endovenous trophoblast.51 Our results suggest a
further extension of this stratification: beside endoarterial and endovenous
trophoblast, the ‘endolymphatic trophoblast’ is present. All routes of trophoblast
invasion are demonstrated in Fig. 15.
39
Figure 15: Schematic overview of human first trimester placenta. This illustration shows possible EVT
pathways in invaded decidua. Extravillous trophoblasts origin from the distal end of anchoring villi and form cell
columns. EVTs invade the decidua basalis until they stop after the first third of myometrium (interstitial trophoblast
(1)). Beside that, EVTs invade the vascular endothelium of vessels (red) and are referred to endovascular
trophoblasts (2). Within the lumen of spiral arteries, they form endovascular plugs. Only blood plasma can seep
through these plugs. Uteroplacental glands (green) are invaded by the endoglandular trophoblasts (3). The EVTs
penetrate the epithelium of glands, replace it and open them towards the intervillous space. Thereby, histiotrophic
nutrition (green direct arrow) is enabled before establishment of the uteroplacental blood flow (hemotrophic
nutrition). At the end of the first trimester the endovascular plugs disintegrate and maternal blood can reach the
intervillous space for hemotrophic nutrition of the embryo. Finally, a novel route of EVTs can be determined.
Lymphatic vessels (yellow) are invaded by the endolymphatic trophoblast (4) during first trimester of pregnancy.
The endothelial layer of the invaded lymphatic vessels appears disintegrated. Apart from invaded decidua a non-
invaded decidua is present as well. In non-invaded decidua there are no fetal cells. All luminal structures (glands,
arteries, veins, lymphatics) appear compact.18
(modified by Michaela Lichtensteiner)
Quantitative measurement shows that vessels have the largest majority in first
trimester placenta (Fig. 7). Compared to vessels, uteroplacental glands are mostly
invaded by trophoblasts. This strengthens the prior assumption that histiotrophic
nutrition occurs until the establishing of feto-maternal blood flow.18 Due to the fact
that blood vessels have a tubular system more truncated sides appear within the
same section. Depending on tissue positioning during embedding process more or
less capillaries can be cut and thereby semi-quantitatively evaluated. This may be
the reason for the highest amount of blood vessels within decidua. In accordance to
statistical assessment lymphatic vessels and glands are penetrated by EVTs with
similar frequency. This could be due to the spatial proximity of glands and lymphatic
40
vessels that was often observed in decidua. This finding has already been described
by Volchek et al.39 Therefore EVTs can reach consistently both structures.
Before EVTs invade a structure, they attach to endothelium of vessels and epithelium
of glands from the basal side.52,53 Our results show that EVT-attachment to vessels
and glands is predominantly compared to EVT-invasion into them (Fig. 9). However,
vessels and glands that are EVT-attached in higher portions can be EVT-invaded in
deeper portions. This was shown on spiral arteries within placental bed (Fig. 14a-f).
Spiral arteries are modified by invading EVTs, so that their lumen widens for
hemotrophic nutrition from the beginning of second trimester.18,54,55 Observations
indicate that at some proportions of the arteries, EVTs accumulate around the spiral
arteries, but do not invade smooth muscle walls. However in higher or deeper
portions of the same vessel EVTs invade the spiral artery. Fig. 16 demonstrates a
schematic representation of a tracked spiral artery that changes conformation due to
EVTs influence.
Figure 16: Transformation of spiral arteries during early pregnancy. This represents a schematic cross
section of first trimester placental tissue. Figure illustrates a possible change of morphology of endothelial layer
and coating muscle wall of spiral arteries (red circles). Image (a) illustrates that the smooth muscle wall of spiral
arteries is not affected by EVTs in higher sections. Some sections later (b) EVTs accumulate in prior non-invaded
space around muscle wall. In deeper portion of sections (c) spiral artery undergoes morphological alteration.
EVTs penetrate smooth muscle wall and induce conformational changes of artery. In addition epithelial layer of
glands is replaced by EVTs. Abbrv: V, [vessels]; L, [lymphatic vessels]; G, [uterine glands]; D, [decidua]; arrows
[for EVT]
6 CONCLUSIO
Until recently three different routes of extravillous trophoblasts were described.
Based on cell columns of anchoring villi the first interstitial trophoblasts migrate
rapidly into basal part of decidua until they reach the first third of the myometrium.
Endoglandular trophoblasts penetrate epithelium of glands and ensure histiotrophic
41
nutrition. Endovascular trophoblasts invade through smooth muscle wall of blood
vessels, replace the vascular endothelium and are responsible for the formation of
endovascular plugs within spiral arteries. Now, the endovascular route of trophoblast
invasion can be further stratified, the endolymphatic trophoblast, beside endoarterial
and endovenous trophoblast. Possible heading directions can be determined with
double immunohistochemistry. Bright field as well fluorescence microscopy enable
clear discrimination of morphological features within decidua. For their identification
specific antibodies are required. Podoplanin or HLA-G; are valuable monoclonal
antibodies to mark specifically lymphatic vessels and extravillous trophoblasts.
Spiral arteries may show in higher portions no invasion by EVTs; however in deeper
portion of the same artery invasion has already occurred. Invasion of trophoblasts
into luminal structures has an effect on development of pregnancy. Well known,
failure of remodeling steps of spiral arteries leads to miscarriages or pregnancy
disorders such as preeclampsia or intrauterine growth restriction (IUGR).56,57
Results of master thesis raise the question of what physiological relevance has
lymphatic vasculature within early decidua (gestational age of 6 to 12 weeks).
According to function of lymphatics, trophoblast invasion into lymphatic vessels may
contribute to fulfill interstitial fluid balance within decidua. Another reason could be
that lymphatic vessels play a role in supporting histiotrophic nutrition. According to
Volchek et al.39 lymphatic vessels are not adjacent to blood vessels, especially spiral
arteries, therefore important functions of lymphatic vessels cannot be assured, such
as filtering waste products and debris of cells. Thereby it could be that, in early
stages of pregnancy, the connection between blood system and lymphatic systems is
yet not fully developed. Our data show that lymphatic vessels are present in close
vicinity of uterine glands, this has already been described by Volchek et al.39
Therefore an interaction of glands and lymphatic vessels may take control over this
absent connection and may support circulation of histiotrophic nutrition.
Extravillous trophoblast invasion into lymphatic vessels may have more impact on the
outcome of early pregnancy than assumed up to now. Whether or not lymphatic
vessels play a role in healthy pregnancies still remains to be determined. Actually,
this new route of trophoblast invasion opens an avenue for further exploration such
as identification of the components of lymphatic fluid and their function.
42
7 ABBREVIATIONS
AEC 3-amino-9-ethylcarbazole
AP-Polymer Alkaline Phophatase-Polymer
BCIP/NBT 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium
CK7 Cytokeratin 7
DAB 3,3’-diamminobenzidine
EVTs Extravillous Trophoblasts
ECM Extracellular Matrix
HLA-G Human leukocyte antigen G
G Glands
GA Gestational Age
HIER Heat induced antigen retrieval
HRP-Polymer Horseradish Peroxidase-Polymer
IgG Immunoglobulin G
IHC Immunohistochemistry
IVS Intervillous space
L Lymphatic vessel
LEC Lymphatic endothelial cell
LYVE1 Lymphatic vessel endothelial receptor-1
mc Monoclonal
pc Polyclonal
Podo Podoplanin
prox1 Prospero related homeobox-1
V Vessel
VEGF-C Vascular endothelial growth factor C
VWF Von Willebrand Factor
43
8 REFERENCES
1 Margeaux Wetendorf and Francesco J. DeMayo, “The Progesterone Receptor
Regulates Implantation, Decidualization, and Glandular Development via a
Complex Paracrine Signaling Network,” Molecular and Cellular Endocrinology
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2 Caroline E. Gargett, Hong P. T. Nguyen, and Louie Ye, “Endometrial Regeneration
and Endometrial Stem/Progenitor Cells,” Reviews in Endocrine & Metabolic
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3 Birgit Gellersen, Ivo A. Brosens, and Jan J. Brosens, “Decidualization of the Human
Endometrium: Mechanisms, Functions, and Clinical Perspectives,” Seminars in
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4 W. J. Hamilton and J. D. Boyd, “Development of the Human Placenta in the First
Three Months of Gestation,” Journal of Anatomy 94 (1960): 297–328
5 Kameliya Vinketova, Milena Mourdjeva, and Tsvetelina Oreshkova, “Human
Decidual Stromal Cells as a Component of the Implantation Niche and a
Modulator of Maternal Immunity,” Journal of Pregnancy (2016)
6 B. Huppertz, “The Anatomy of the Normal Placenta,” Journal of Clinical Pathology
61, no. 12 (2008): 1296–1302
7 J. D. Aplin, “The Cell Biological Basis of Human Implantation,” Bailliere’s Best
Practice & Research. Clinical Obstetrics & Gynaecology 14, no. 5 (2000): 757–64
8 Kurt Benirschke and Peter Kaufmann, “Pathology of the Human Placenta,” in
Chapter 5 S.44, 4th Edition (Springer Verlag, 2000)
9 Kurt Benirschke and Peter Kaufmann, “Pathology of the Human Placenta,” in
Chapter 5 S.44 & Chapter 11 S.282, 4th Edition (Springer Verlag, 2000)
10
Berthold Huppertz, Debabrata Ghosh, and Jayasree Sengupta, “An Integrative
View on the Physiology of Human Early Placental Villi,” Progress in Biophysics
and Molecular Biology 114, no. 1 (2014): 33–48
11
Kurt Benirschke and Peter Kaufmann, “Pathology of the Human Placenta,” in
Chapter 11 S.285, 4th. Edition (Springer Verlag, 2000)
44
12 Graham J. Burton and Eric Jauniaux, “What Is the Placenta?,” American Journal
of Obstetrics and Gynecology 213, no. 4 Suppl (2015): S6.e1, S6-8
13
Berthold Huppertz, Gregor Weiss, and Gerit Moser, “Trophoblast Invasion and
Oxygenation of the Placenta: Measurements versus Presumptions,” Journal of
Reproductive Immunology 101–102 (2014): 74–79
14
Ursula Hiden et al., “Membrane-Type Matrix Metalloproteinase 1 Regulates
Trophoblast Functions and Is Reduced in Fetal Growth Restriction,” The
American Journal of Pathology 182, no. 5 (2013): 1563–71
15
B. Gellersen et al., “Invasiveness of Human Endometrial Stromal Cells Is
Promoted by Decidualization and by Trophoblast-Derived Signals,” Human
Reproduction (Oxford, England) 25, no. 4 (2010): 862–73
16
Stewart F. Cramer and Debra S. Heller, “Placenta Accreta and Placenta Increta:
An Approach to Pathogenesis Based on the Trophoblastic Differentiation
Pathway,” Pediatric and Developmental Pathology: The Official Journal of the
Society for Pediatric Pathology and the Paediatric Pathology Society 19, no. 4
(2016): 320–33
17
Gerit Moser et al., “A Revised Picture of Extravillous Trophoblast Invasion,”
Journal of Reproductive Health and Medicine, Emerging Concepts on Female
Reproduction, 2, Supplement 2 (2016): S9–14
18
G. Moser et al., “Evidence from the Very Beginning: Endoglandular Trophoblasts
Penetrate and Replace Uterine Glands in Situ and in Vitro,” Human
Reproduction (Oxford, England) 30, no. 12 (2015): 2747–57
19
Y. Wang et al., “D2-40/Podoplanin Expression in the Human Placenta,” Placenta
32, no. 1 (2011): 27–32
20
Graham J. Burton et al., “Uterine Glands Provide Histiotrophic Nutrition for the
Human Fetus during the First Trimester of Pregnancy,” The Journal of Clinical
Endocrinology and Metabolism 87, no. 6 (2002): 2954–59
21
G.J. Burton, E. Jauniaux, and D.S. Charnock-Jones, “Human Early Placental
Development: Potential Roles of the Endometrial Glands,” Placenta 28 (2007):
S64–69
45
22 Michelle Neumann, “Uteroferrin in the Early Human Placenta” (Masterthesis, Karl
Franzens University/Medical University Graz, 2017).
23
G. Moser et al., “Endoglandular Trophoblast, an Alternative Route of Trophoblast
Invasion? Analysis with Novel Confrontation Co-Culture Models,” Human
Reproduction (Oxford, England) 25, no. 5 (2010): 1127–36
24
V. S. Talaulikar et al., “A Novel Hysteroscopic Technique for the Accurate Biopsy
of Decidua Parietalis and Basalis,” Placenta 33, no. 6 (2012): 473–79
25 G. Moser et al., “The Art of Identification of Extravillous Trophoblast,” Placenta 32,
no. 2 (2011): 197–99
26
S. Kovats et al., “A Class I Antigen, HLA-G, Expressed in Human Trophoblasts,”
Science (New York, N.Y.) 248, no. 4952 (1990): 220–23
27
M. T. McMaster et al., “Human Placental HLA-G Expression Is Restricted to
Differentiated Cytotrophoblasts,” Journal of Immunology (Baltimore, Md.: 1950)
154, no. 8 (1995): 3771–78
28
Kristy Red-Horse et al., “Cytotrophoblast Induction of Arterial Apoptosis and
Lymphangiogenesis in an in Vivo Model of Human Placentation,” The Journal of
Clinical Investigation 116, no. 10 (2006): 2643–52
29
J. J. Sixma and P. G. de Groot, “Von Willebrand Factor and the Blood Vessel
Wall,” Mayo Clinic Proceedings 66, no. 6 (1991): 628–33
30
Tuomas Tammela and Kari Alitalo, “Lymphangiogenesis: Molecular Mechanisms
and Future Promise,” Cell 140, no. 4 (2010): 460–76
31
Kari Alitalo, Tuomas Tammela, and Tatiana V. Petrova, “Lymphangiogenesis in
Development and Human Disease,” Nature 438, no. 7070 (2005): 946–53
32
Satoshi Hirakawa, Michael Detmar, and Sinem Karaman, “Lymphatics in
Nanophysiology,” Advanced Drug Delivery Reviews 74 (2014): 12–18
33
K. Red-Horse, “Lymphatic Vessel Dynamics in the Uterine Wall,” Placenta 29
(2008): S55-59
46
34 Inho Choi, Sunju Lee, and Young-Kwon Hong, “The New Era of the Lymphatic
System: No Longer Secondary to the Blood Vascular System,” Cold Spring
Harbor Perspectives in Medicine 2, no. 4 (2012)
35
Cristina T. Kesler et al., “Lymphatic Vessels in Health and Disease,” Wiley
Interdisciplinary Reviews. Systems Biology and Medicine 5, no. 1 (2013): 111–
24
36
Virginia H. Huxley and Joshua Scallan, “Lymphatic Fluid: Exchange Mechanisms
and Regulation,” The Journal of Physiology 589, no. Pt 12 (2011): 2935–43
37
Nicholas W. Gale et al., “Angiopoietin-2 Is Required for Postnatal Angiogenesis
and Lymphatic Patterning, and Only the Latter Role Is Rescued by
Angiopoietin-1,” Developmental Cell 3, no. 3 (2002): 411–23
38
S. Breiteneder-Geleff et al., “Angiosarcomas Express Mixed Endothelial
Phenotypes of Blood and Lymphatic Capillaries: Podoplanin as a Specific
Marker for Lymphatic Endothelium,” The American Journal of Pathology 154,
no. 2 (1999): 385–94
39 Mila Volchek et al., “Lymphatics in the Human Endometrium Disappear during
Decidualization,” Human Reproduction (Oxford, England) 25, no. 10 (2010):
2455–64
40
David G. Jackson, “The Lymphatics Revisited: New Perspectives from the
Hyaluronan Receptor LYVE-1,” Trends in Cardiovascular Medicine 13, no. 1
(2003): 1–7
41
P. a. W. Rogers, J. F. Donoghue, and J. E. Girling, “Endometrial
Lymphangiogensis,” Placenta 29 (2008): S48-54
42
Karin Windsperger et al., “Extravillous Trophoblast Invasion of Venous as Well as
Lymphatic Vessels Is Altered in Idiopathic, Recurrent, Spontaneous Abortions,”
Human Reproduction (Oxford, England), (2017): 1–10
43
Nannan He et al., “Human Extravillous Trophoblasts Penetrate Decidual Veins
and Lymphatics before Remodeling Spiral Arteries during Early Pregnancy,”
PloS One 12, no. 1 (2017)
47
44 I. Brosens, H. G. Dixon, and W. B. Robertson, “Fetal Growth Retardation and the
Arteries of the Placental Bed,” British Journal of Obstetrics and Gynaecology
84, no. 9 (1977): 656–63
45
Gregor Weiss et al., “The Trophoblast Plug during Early Pregnancy: A Deeper
Insight,” Histochemistry and Cell Biology 146, no. 6 (2016): 749–56
46
R. Demir et al., “Structural Differentiation of Human Uterine Luminal and Glandular
Epithelium during Early Pregnancy: An Ultrastructural and
Immunohistochemical Study,” Placenta 23, no. 8–9 (2002): 672–84
47
Joanne Hempstock et al., “Endometrial Glands as a Source of Nutrients, Growth
Factors and Cytokines during the First Trimester of Human Pregnancy: A
Morphological and Immunohistochemical Study,” Reproductive Biology and
Endocrinology: RB&E 2 (2004): 58 48
Bin-Bin Li, Chuan-Xiang Zhou, and Sheng-Nan Jia, “Basal Cell Adenoma of
Salivary Glands with a Focal Cribriform Pattern: Clinicopathologic and
Immunohistochemical Study of 19 Cases of a Potential Pitfall for Diagnosis,”
Annals of Diagnostic Pathology 18, no. 1 (2014): 5–9
49
Celso E. Gomez-Sanchez et al., “Development of Monoclonal Antibodies against
Human CYP11B1 and CYP11B2,” Molecular and Cellular Endocrinology 383,
no. 1–2 (2014): 111–17
50
M. C. Barsotti et al., “Endothelial Progenitor Cell Homing in Human Myocardium in
Patients with Coronary Artery Disease,” International Journal of Cardiology 172,
no. 2 (2014): 516–17
51 Gerit Moser and Berthold Huppertz, “Implantation and Extravillous Trophoblast
Invasion: From Rare Archival Specimens to Modern Biobanking,” Placenta,
2017
52
C. M. Craven, L. Zhao, and K. Ward, “Lateral Placental Growth Occurs by
Trophoblast Cell Invasion of Decidual Veins,” Placenta 21, no. 2–3 (2000): 160–
69
53 Gerit Moser et al., “Extravillous Trophoblasts Invade More than Uterine Arteries:
Evidence for the Invasion of Uterine Veins,” Histochemistry and Cell Biology
147, no. 3 (2017): 353–66
48
54 G.J. Burton et al., “Rheological and Physiological Consequences of Conversion of
the Maternal Spiral Arteries for Uteroplacental Blood Flow during Human
Pregnancy,” Placenta 30, no. 6 (2009): 473–82
55 F Beck, “Comparative Placental Morphology and Function.,” Environmental Health
Perspectives 18 (1976): 5–12
56
Judith E. Cartwright et al., “Remodelling at the Maternal–fetal Interface: Relevance
to Human Pregnancy Disorders,” Reproduction 140, no. 6 (2010): 803–13
57
Peter Kaufmann, Simon Black, and Berthold Huppertz, “Endovascular Trophoblast
Invasion: Implications for the Pathogenesis of Intrauterine Growth Retardation
and Preeclampsia,” Biology of Reproduction 69, no. 1 (2003): 1–7
49
9 PROTOCOLS
9.1 Embedding
Equipment Company/Catalogue no Intended Use
PBS - Tissue purification and
storage
Formalin (4%) - Fixation reagent
Paraffin -
Embedding
Embedding cassettes -
Embedding automate Tissue-Tek® VIP™,
Sakura, USA
Molds -
Cooling plate -
Steps Approach Time
Tissue Preparation Purify in PBS (1x) -
Cut into pieces -
Fixing with formalin (4%) at room
temperature
over night
Transfer in embedding cassettes and
put into the embedding automate
-
Embedding Program 60% alcohol 60 min
80 % alcohol 60 min
96 % alcohol 60 min
100 % alcohol 60 min (3x)
Tissue Clear 60 min (3x)
Paraffin 60 min (3x)
Paraffin Block Transferring and positioning in pre-
heated molds
-
Cover with molten paraffin (60°C) and
gently press tissue flat with the backing
of cassette
-
Place on cooling plate (-15°C) 30 min
When wax completely hardened block -
50
can be popped out easily and is ready to use
9.2 Dissection
Equipment Company/Catalogue no Intended Use
Sliding microtome HM 440E, MICROM,
Zeiss, Germany Sectioning of paraffin
block Rotational microtome HM 355S, MICROM,
Zeiss, Germany
Blades Feather/R35, Japan Trimming and cutting
Brushes - Catching of sections
Superfrost Plus glass slide Menzel-Gläser, Thermo
Scientific, Germany
Adhesion of section
Cooling trough TUC1 Tube Cooler,
Pathisto®, Germany
Cooling of paraffin block
Water bath Sanova, MEDAX, Austria Unfolding of sections
Filter paper - Eliminating of air bubbles
Heating plate TFP 40, MEDITE,
Germany
Drying of sections
Steps Approach Time
Dissection Precooling of paraffin block (-9°C)
when using sliding microtome
20 min
Insertion of paraffin block into the
microtome chuck and positioning
-
Cutting of serial sections in 5µm -
Unfold sections in water bath (38°C) 2 min
Float sections onto glass slides -
Drain slides and filter water 10 min
Dry slides on heating plate (52°C) over night
51
9.3 Deparaffination and Antigen Retrieval
Equipment Company/Catalogue no Intended Use
Tissue Clear-Xylene
Substitute
Tissue-Tek® VIP™, Sakura, USA, 1335002002
Deparaffination of sections Alcohol 100 %, 96 %,
70%, 50 %
-
Aqua distilled -
Epitope Retrieval Solution
pH 9 buffer (10x)
pH 6 citrate buffer
Leica, Novocastra™, Germany, RE7119
Heat-induced Epitope
Retrieval (HIER) Pressure cooker
Decloaking chamber, BioCarta, Germany
Steps Approach Time
Deparaffination and
Rehydration
Tissue Clear 4x5 min
1:1 mix tissue clear and 100 % alcohol
Slewing and
draining
several times
100 % alcohol
96 % alcohol
70 % alcohol
50 % alcohol
Aqua distilled (3x)
HIER Transfer into antigen retrieval buffer
(pH 9 or pH 6)
-
Cook in pressure cooker at 120°C 7 min
Cool down in buffer 20 min
Transfer into boiled distilled water 5 min
Ready for IHC/FIHC -
52
9.4 Immunohistochemistry
General Equipment for immunohistochemistry
Equipment Company/Catalogue no Intended Use
Moist chamber - Maintenance of humidity
Dako Pen Dako, Denmark, S2002 Tissue encircling
Antibody diluent Dako, Denmark, S3022 Dilutes antibodies
TBS-T (0,05%) [TBS (20x)
+Tween]
Merck, Germany,
9005-64-5 Washing buffer
Aqua distilled - Washing
Aquatex Merck, Germany Aqueous mounting agent
Cover slip Menzel-Gläser, Thermo
Scientific, Germany Protects tissue
Single Staining
Additional Equipment Company/Catalogue no Intended Use
Kit
(Lab Vision™
UltraVision™ LP Detection
System)
Thermo Scientific,
Germany, TL-125-HL
Single IHC
Ultra V Protein Block Thermo Scientific,
Germany, TA-125-PBQ
Reduces nonspecific
background staining
Primary Antibody
Enhancer
Thermo Scientific,
Germany, TL-125-PB
Enhances signal of
antibody
HRP Polymer Thermo Scientific,
Germany, TL-125-PH
Provides increased
sensitivity
Hydrogen Peroxide Block Thermo Scientific,
Germany, TA-125-HP
Quenches endogenous
peroxidase
AEC Substrate Thermo Scientific,
Germany, TA-125-SA Chromogen
Mayer’s Hemalaun - Nuclear counterstaining
Ammonium water -
53
Steps Approach Time
Preparation Redraw tissues with Dako Pen and
place in moist chamber
-
Use Peroxidase Block 10 min
Single Staining Wash with distilled water 3 times
Drip Ultra V Block on tissues 5 min
Tilt and incubate with primary antibody
at room temperature (RT)
45 min
Wash with TBS-T 3 times
Incubate with Enhancer (only when
using anti-mouse antibodies) at RT
10 min
Wash with TBS-T 3 times
Incubate with HRP-Polymer at RT 15 min
Wash with TBS-T 3 times
Add AEC Chromogen 10 min
Nuclear Counterstaining and Mounting
Place in acidic hemalaun 10 min
Rinse with distilled water 3 times
Slew in ammonium water until visible
blue staining
-
Rinse with distilled water again 3 times
Cover with Aquatex -
Double Staining
Additional Equipment Company/Catalogue no Intended Use
Dako Dual Endogenous Enzyme Block
Dako, Denmark, S2003 Suppresses endogenous
alkaline phosphatase and
peroxidase
Vector® Blue AP Substrate
Kit
Vector Laboratories,
Burlingame, USA,
SK-5300
Chromogen
GBI-Staining Kits Golden Bridge
International, USA
Double IHC
54
9.5 GBI-Double Staining Kits
Polink DS-MR-Hu
A1Kit
GBI Labs, USA
DS201A-6
Polink DS-MR-Hu
B1Kit
GBI Labs, USA
DS201B-6/(D63-6)
Polink DS-MR-Hu
C1Kit
GBI Labs, USA
DS201C-6
Rea
ge
nts
HRP-Polymer
anti-Mouse
HRP-Polymer
anti-Mouse
HRP-Polymer
anti-Mouse
AP-Polymer anti-Rabbit AP-Polymer anti-Rabbit AP-Polymer anti-Rabbit
DAB Substrate
DAB Chromogen
BCIP/NBT GBI-Permanent Red
Substrate
GBI-Permanent Red
Activator
GBI-Permanent Red
Chromogen
GBI-Permanent Red
Substrate
GBI-Permanent Red
Activator
GBI-Permanent Red
Chromogen
AEC Substrate
AEC Chromogen
Hydrogen Peroxide
Emerald Chromogen
Simpo Mount Simpo Mount U-Mount
A1 Kit
(DAB-HRP-anti-Mouse-brown | GBI-Permanent Red-AP-anti-Rabbit-red)
Steps Approach Time
Preparation Redraw tissues with Dako Pen and
place in moist chamber
-
Use blocking reagent and cover tissue
completely
10 min
Wash with TBS-T (1x) 3 times
Double Staining Incubate with primary antibody mix 30 min
Wash with TBS-T (1x) 3 times
Prepare secondary antibody 1:1 mix 30 min
55
per slide
(50µl Reagent 1+ 50µl Reagent 2)
Wash with TBS-T (1x) 3 times
Add 1 drop of Reagent 3B to 1ml of
Reagent 3A
Mix well and cover tissue
Attention
protect from light
use within 7h
DAB may be carcinogenic
5 min
Rinse with distilled water 1x
Wash with TBS-T (1x) 3 times
Prepare GBI-Permanent Red staining
Add 100 µl Reagent 4B to 500µl
Reagent 4A and mix well.
Supplement 5µl Reagent 4C, shake
well and apply
Attention
make fresh working solution and
use immediately
To increase AP signal make new
solution and incubate additional 10
min
10 min
Rinse with distilled water 1x
Counterstaining Counterstain with hematoxylin -
Rinse with tap water 2-3 min
Put slides in ammonium water until
blue coloring
-
Rinse thoroughly in distilled water 1x
Mounting Apply enough Simpo-Mount
(Reagent 5) as long as tissue is wet
-
Rotate slides until completely covering
Attention
No cover slip
-
Bake slides in oven (52°C) 30 min
56
A1 Kit Improved
(Vector Blue Substrate-HRP-anti-Mouse-brown | GBI-Permanent Red-AP-anti-
Rabbit-red)
Steps Approach Time
Preparation Redraw tissues with Dako Pen and
place in moist chamber
-
Use blocking reagent and cover tissue
completely
10 min
Wash with TBS-T (1x) 3 times
Double Staining Incubate with primary antibody mix 30 min
Wash with TBS-T (1x) 3 times
Prepare secondary antibody 1:1 mix
per slide
(50µl Reagent 1+ 50µl Reagent 2)
30 min
Wash with TBS-T (1x) 3 times
Add 1 drop of Reagent 3B to 1ml of
Reagent 3A
Mix well and cover tissue
Attention
protect from light
use within 7h
DAB may be carcinogenic
5 min
Rinse with distilled water 1x
Wash with TBS-T (1x) 3 times
Prepare 100 mM-200 mM Tris-HCl, pH
8,2-8,5 buffer
Add to 2,5 ml Tris-HCl
1 drop Reagent 1
1 drop Reagent 2
1 drop Reagent 3
mix well between each supplement
and apply
Attention
Protect from light
10 min
Wash with TBS-T (1x) 3 times
57
Rinse with distilled water 1x
Mounting Apply Aquatex and cover slip -
B1 Kit
(AEC-HRP-anti-Mouse-red | BCIP/NBT-AP-anti-Rabbit-purple)
Steps Approach Time
Preparation Redraw tissues with Dako Pen and
place in moist chamber
-
Use blocking reagent and cover tissue
completely
10 min
Wash with TBS-T (1x) 3 times
Double Staining Incubate with primary antibody mix 30 min
Wash with TBS-T (1x) 3 times
Prepare secondary antibody 1:1 mix
per slide
(50µl Reagent 1+ 50µl Reagent 2)
30 min
Wash with TBS-T (1x) 3 times
Apply enough volume (100µl/slide) of
BCIP/NBT (Reagent 3)
5-10 min
Rinse with distilled water 1x
Wash with TBS-T (1x) 3 times
Prepare AEC staining
Add 50µl of Reagent 4A to 1ml distilled
water.
Supplement 100µl of Reagent 4B and
50µl of Reagent C.
Shake well between steps and apply
Attention
Protect from light
Use within 1h
10 min
Rinse with distilled water
Attention
Do not dehydrate
1x
58
Counterstaining Counterstain with hematoxylin -
Rinse with tap water 2-3 min
Put slides in ammonium water until
blue coloring
-
Rinse thoroughly in distilled water 1x
Mounting Apply enough Simpo-Mount
(Reagent 5) as long as tissue is wet
-
Rotate slides until completely covering
Attention
No cover slip
-
Bake slides in oven (52°C) 30 min
C1 Kit
(Emerald-HRP-anti-Mouse-green | GBI-Permanent Red-AP-anti-Rabbit-red)
Steps Approach Time
Preparation Redraw tissues with Dako Pen and
place in moist chamber
-
Use blocking reagent and cover tissue
completely
10 min
Wash with TBS-T (1x) 3 times
Double Staining
Step 1
Incubate with primary antibody mix 30 min
Wash with TBS-T (1x) 3 times
Prepare secondary antibody 1:1 mix
per slide
(50µl Reagent 1+ 50µl Reagent 2)
30 min
Wash with TBS-T (1x) 3 times
Prepare GBI-Permanent Red staining
100 µl Reagent 3B into 500µl Reagent
3A per slide
Supplement 5µl Reagent 3C
Shake well between steps and apply
Attention
10 min
59
make fresh working solution and
use immediately
To increase AP signal make new
solution and incubate additional 10
min
Rinse with distilled water 1x
Wash with TBS-T (1x) 3 times
Rinse with distilled water 1x
Counterstaining (Optional)
Counterstain with hematoxylin -
Rinse with tap water 2-3 min
Put slides in ammonium water until
blue coloring
-
Rinse thoroughly in distilled water 1x
Double Staining
Step 2
Apply 50µl of Reagent 4 (Emerald) per
slide and cover tissue
Attention
Emerald is water soluble
Do counterstain first
Stain after GBI-Permanent Red
5 min
Rinse slides with tap water 1x
Rinse thoroughly with distilled water 1x
Dehydrate Section Wipe off extra water -
Dehydrate with 85 % alcohol 20 sec
Dehydrate with 95 % alcohol 20 sec
Dehydrate with 100 % alcohol 3x 20 sec
Dehydrate with xylene 20 sec
Mounting Apply enough U-Mount
(Reagent 5) as long as tissue is wet
-
Rotate slides until completely covering
Attention
cover slip
-
60
9.6 Immunofluorescense
Equipment Company/Catalogue no Intended Use
Dako Pen Dako, Denmark, S2002 Tissue encircling
Moist chamber - Maintenance of humidity
Ultra V Protein Block Thermo Scientific,
Germany, TA-125-UB
Reduces nonspecific background staining
Antibody diluent Dako, Denmark, S3022 Dilutes primary antibodies
PBS - Dilutes fluorescent
antibodies
PBS-T
(0,05% Tween)
Merck, Germany,
9005-64-5
Washing buffer
Goat anti-Mouse Alexa
Fluor 488
Invitrogen, Austria,
A 11001 Fluorescent secondary antibody Goat anti-Rabbit Alexa
Fluor 555
Invitrogen, Austria,
A 21428
DAPI - Nuclear counterstaining
ProLong Gold Antifade
reagent
Invitrogen, Austria, P
36930
Mounting medium
Covering slip Menzel-Gläser, Thermo
Scientific, Germany
Protects tissue
Steps Approach Time
Preparation Redraw tissues with Dako Pen and
place in moist chamber
-
Incubate with Ultra V Block 5 min
Fluorescent Staining Tilt and incubate with primary antibody
at room temperature
30 min
Wash with PBS-T 3 times
Incubate with second fluorescent
antibody at room temperature
30 min
Wash with PBS-T 3 times
Nuclear Staining and Mounting
Counterstain with Dapi at room
temperature
5 min
Wash with PBS-T 3 times
Let slides dry at room temperature -
61
Cover with ProLong Gold -