The Placenta of the RatComparative morphology of the placenta 29.08.2014 Cutting Edge Pathology 2014 2 Modified from: Chavatte-Palmer & Guillomot (2007) Comparative Implantation and

The Placenta of the Rat Histology and Pathology

Cutting Edge Pathology 2014

Second Joint European Congress of the ESTP, ESVP and ECVP

Dr. Maria Cecilia Rey Moreno

Comparative morphology of the placenta

2 Cutting Edge Pathology 2014 29.08.2014

Modified from: Chavatte-Palmer & Guillomot (2007)

Comparative Implantation and Placentation;

Bernirschke, Kaufmann & Baergen (2006)

Pathology of the human placenta

Diffuse

Horse, pig, whale

Zonary Cotyledonary Discoidal

Ruminants Carnivores

Human

Monkeys (bidiscoidal)

Rodents

General characteristics shared by all species

3 29.08.2014

Transient organ: supplies embyonal/fetal nutrition, growth and protection

Vascular organ: close proximity of maternal and fetal vascular systems with large transfer

contact surface between each other

Interhemal membrane (placental barrier): separates maternal and fetal blood separated

in their own compartments; site of transfer

General functions: O2/CO2 transfer, water balance, synthesis and secretion of steroid

hormones, paracrine and growth factors, metabolic and immunologic functions (immune

tolerance)

Origin: trophectoderm of the blastocyst

Trophoblast cell: parenchymal cells of the placenta; different subtypes with different

locations and functions


Gestational period and life-span of the rat

placenta

4 29.08.2014 Cutting Edge Pathology 2014

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 / 22

Fertilization Implantation Birth

days

Placenta

GD21/22

Yolk sac

Decidua

Placenta

Placentation

Blastocyst

Gestational

day (GD) 5/6

Trophectoderm→

Histology of the placental development

in the rat


GD8 GD10 GD12 Non-pregnant uterus

GD14 GD16 GD18 GD21


GD8/9: Decidualization and anastomosis

of maternal vasculature

Mesometrial side

Mesometrial

triangle

Uterine

lumen

Antimesometrial side

Decidua

Implantation

*

*

Decidualization (*): proliferation and

differentiation of stromal cells

Site of developing placenta:

ectoplacental cone (EPC)

Embryo

Allantois

Giant

trophoblast

cell

Yolk sac

Anastomosis of maternal vessels

around the embryo

GD10: Yolk sac and placental

cone differentiation


Ectoplacental

Cone

Yolk sac

membrane

Decidua

(D)

Chorion

Uterus

lumen

2

3

D

D Mesometrial

triangle

D

GD12: Chorio-allantoic placentation:

interhemal membrane formation


Actin smooth muscle IHC:

fetal blood vessel walls

Morphogenesis of

fetal vessels

and maternal sinuses

A

C

A: Allantois

C: Chorion


First half of gestation:

Uterine natural killer cells (uNK)

Mesometrial triangle:

sheaths of uNK (or granulated mesometrial

cells) around spiral arteries

GD10/12: Remodeling of spiral arteries

GD10

Second half of gestation:

trophoblast cell invasion

Decidua:

Intravascular and interstitial trophoblast cell

invasion; apoptosis of uNK

GD12

GD10/18: Remodeling of spiral arteries


β-Actin:

smooth muscle

Cytokeratin

Pan-26:

trophoblast cell

GD18 GD10

GD18 GD10


Vimentin: fetal vessels

GD14/16: Maturation of materno-fetal

interhemal membrane

HE

Alignment of

fetal vessels

GD14

GD14

GD16

GD16


GD18: Mature and fully functional:

differentiation completed

Fetal demands

Efficiency

Placental size unchanged

Branching + dilation of fetal capillaries

Thickness of interhemal membrane

Remodeling:

Rat placenta at GD 21

General characteristics

13 29.08.2014

Shape

Interhemal membrane

Fetal blood

(capillary)

Maternal blood

(sinus)

I

II

III

I: Cytotrophoblast cells

II + III: Syncytiotrophoblast


Discoidal

Hemotrichorial

Interdigitation

Labyrinthine

Rat placenta at GD 21

General structure

14 29.08.2014

Kaufmann P., 2005

http.//placentation.u

csd.edu/rat.htm

L

T

D

UW

UC

UM

Fetal side

Maternal side

SP

MT

Labyrinth (L)

Trophospongium (T)

Decidua (D)

Uteromaternal channel (UM)

Mesometrial triangle (MT)

Spiral arteries (SP)

Uterine wall (UW)

Umbilical cord (UC)

Yolk sac (YS)

Amniotic membrane (AM)


YS

AM

Histology of the normal labyrinth at GD21

15 29.08.2014

Maternal sinus (M)

Fetal capillary (F)

Cytotrophoblast (C)

Syncytiotrophoblast (S)

M

F

S

C


Interhemal membrane

Histology of the normal trophospongium

at GD21

16 29.08.2014

(L)

Sp

Gly

(UM)


Spongiotrophoblast cells (Sp)

Glycogen trophoblast cells (Gly)

Giant trophoblast cells

Uteromaternal channel (UM), lined by parietal trophoblast cells (Pa)

Pa

Histology of the normal decidua at GD21

17 29.08.2014

Spiral arteries (S)

Glycogen trophoblast cells (Gly)

Decidual cells (D)

Uterine natural killer cells (uNK) (or granulated

mesometrial cells)

uNK

Gly

D


S

Research by BASF SE


Birth Defects Research Part B (2013)

98 3 208-221, 230-246, 247-259

Epoxiconazole (EPX)

19 29.08.2014

Inhibits CYP51 (lanosterol 14α-

demethylase), a key fungal enzyme

aromatase estradiol testosterone

CYP 19

CYP 51

ergosterol lanosterol

Epoxiconazole

Antifungal agrochemical: azole

class

Proven effectiveness against

fungi which resist other classes

Inhibits CYP19 (aromatase) in

mammals



Mechanistic studies with Epoxiconazole

in rats

Two Modified Prenatal Developmental Toxicity Studies (OECD 414) in rats

Hormone measurements

Hematology and clinical chemistry

Pathology (organ weights, histopathology)

Study 2 Study 1

Administration of Epoxiconazole (EPX)

Maternal adverse effects (↓ weight,

↓maternal serum estrogen, anemia, liver

parameters altered, etc.)

↑ late fetal resorptions

↑ placental size

Histopathological changes in placenta

Pathology:

Material + Methods


Uterus extracted in toto

Uterus opening at the antimesometrial

side

Placentas remained attached to the

uterus

Fixation: 10% neutral buffered formalin

(24 - 48 h); transferred to 70% ethanol

Trimming: transverse cut at the center

of the disc (mid-sagittal section)

Routine HE histotechnical processing

Mean/median grading of

degeneration/litter (statistical analysis)

Macroscopic findings

22 29.08.2014

Control

Treated (50 mg/kg bw/d

EPX)

Late resorption Placenta with

live fetus


EPX : Epoxiconazole

Histopathology of the labyrinth


Control, GD 21; HE, 2.5x

Normal architecture of the labyrinth

Treated, GD 21, HE, 2.5x

Degeneration, labyrinth, grade 4

Cystic dilation of vascular spaces filled with blood

and/or fibrin replace the normal structure of the

labyrinth (50 - 70%).

Rey Moreno MC et al. (2013)

Birth Def Res (Part B) 98:208-211

Histopathology of the labyrinth


Control, GD 21, HE, 20x

Normal architecture of the labyrinth

Treated, GD 21, HE, 20x

Disruption of interhemal membrane

Filiform and irregular remnants of interhemal

membranes; degeneration of trophoblast cells.

Rey Moreno MC et al. (2013). Birth Def Res (Part B) 98:208-211





Histopathology of the trophospongium


Control, GD 21, HE, 2.5x

Normal architecture of the

trophospongium

Treated, GD 21, HE, 2.5x

Degeneration, trophospongium, grade 2

Increased thickness of trophospongium (2-fold),

disorganized aspect, increased eosinophilia, dilation

of maternal venous spaces

L

T

T

L





26 29.08.2014

Degeneration in a placenta with a live

fetus

**

*


Labyrinth: Treated, GD21, HE, 4x

Degeneration, labyrinth, grade 2

Cystic dilations **

Mineralization of fetal vessels *

Trophospongium: Treated, GD21, HE, 4x

Degeneration, trophospongium, grade 3

Disorganization of architecture

Necrosis (↑)

Degeneration in a placenta with a late

resorption

27 29.08.2014

m

*

*

*


Labyrinth: Treated, GD21, HE, 4x

Degeneration, grade 5

Cystic dilations * (> 70%)

Mineralization of fetal vessels (↑)

Trophospongium: Treated, GD21, HE, 4x

Degeneration, grade 4

Extensive necrosis (*) and fibrinoid deposit

* *

*

UA

D

Severity of degeneration was dose-dependent

and associated with the fetal outcome


(Live Fetus)

(Late Resorption) (Late Resorption)

(Live Fetus) = control

= low dose EPX

(23 mg/kg bw/day)

= high dose EPX

(50 mg/kg bw/day)


Severity of degeneration was dose-dependent


(Live Fetus)


(Live Fetus) = control

= low dose EPX

(23 mg/kg bw/day)

= high dose EPX

(50 mg/kg bw/day)

Mechanistic studies with Epoxiconazole

in Rats


Two Modified Prenatal Developmental Toxicity Studies (OECD 414) in Rats

Hormone measurements

Hematology and clinical chemistry

Pathology (organ weights, histopathology)

Study 1

Administration of EPX

↓ rat maternal serum estrogen

↑ late fetal resorptions

↑ placental size

Histopathological changes in placenta

(labyrinth and trophospongium)

Study 2

Administration of EPX + estradiol

↓ rat maternal serum estrogen

↓ late fetal resorptions

↓ placental size

↓ severity of histopathological

changes in placenta (labyrinth and

trophospongium)


(Live Fetus) (Live Fetus)

Severity of degeneration decreased with

increasing estrogen supplementation

= control

= 50mg/kg bw/d EPX

= 50mg/kg bw/d EPX

+ 0.5 µg/d estrogen

= 50mg/kg bw/d EPX

+ 1.0 µg/d estrogen




(Live Fetus) (Live Fetus)


Severity of degeneration decreased with

increasing estrogen supplementation

= control

= high dose EPX

= high dose EPX +

0.5 µg/d estrogen

= high dose EPX +

1.0 µg/d estrogen


Conclusions

Maternal estrogen depletion caused by EPX aromatase inhibition was

associated with rat placental degeneration

Placental degeneration appeared to be critical for the viability of fetus

and was associated with late fetal resorptions

Supplementation of estrogen ameliorated placental degeneration

leading to a lower number of late resorptions


Guinea pig exposed to EPX (prenatal and pre-postnatal toxicity studies)

revealed: no histopathological findings in placenta and no late resorptions

Conclusions


Regulation of pregnancy Rat Guinea pig Human

Aromatase expression maternal ovaries placenta placenta

Source: Estrogen maternal ovaries placenta placenta

Source: Progesterone corpus luteum corpus luteum/placenta corpus luteum/placenta

Guinea pigs and humans have a similar placental hormonal synthesis

Rat Human Guinea Pig


Lab technicians and colleagues:

Pathology & Reprotox

Vinzenz Stahl

Thank you !

Documents

The Placenta of the RatComparative morphology of the placenta 29.08.2014 Cutting Edge Pathology 2014 2 Modified from: Chavatte-Palmer & Guillomot (2007) Comparative Implantation and