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

357, no. 1–2 (2012): 108–18

2 Caroline E. Gargett, Hong P. T. Nguyen, and Louie Ye, “Endometrial Regeneration

and Endometrial Stem/Progenitor Cells,” Reviews in Endocrine & Metabolic

Disorders 13, no. 4 (2012): 235–51

3 Birgit Gellersen, Ivo A. Brosens, and Jan J. Brosens, “Decidualization of the Human

Endometrium: Mechanisms, Functions, and Clinical Perspectives,” Seminars in

Reproductive Medicine 25, no. 6 (2007): 445–53

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 -