An Analysis and Review of the Relative Morphology of Extraembryonic

Membranes in Mammals: Their Roles in Histiotrophic Nutrition and Possible Sites of Developmental Insult

John M. DeSesso, PhD, Fellow ATSMitretek Systems

Joseph F. Holson, PhD, DABFEWIL Research Laboratories

Examples of Uterine Structure

Ramsey, 1982

Gross Anatomy of the Human Female Reproductive Tract

Netter, 1998

Events in the Female Reproductive Tract from Fertilization to Implantation

Moore, Persaud & Siota, 1997

Changes in Uterine Wall During Menstrual Cycle

Nutrition During Early Development

• Follows Ficke’s Law of Diffusion• Proportional to surface areas and/or

efficiency of exchange• Becomes increasingly inefficient when the

diameter of the conceptus exceeds 0.2 mm

Placenta

Any apposition of embryonal to parental tissues for the purpose of physiological

exchange

Implantation of Human Embryo

Carlson, 1999

Establishment of theUteroplacental Circulation

Carlson, 1999

Uteroplacental Vasculature Begins

• Early on gestational day 8 in the mouse• Late gestational day 8/early day 9 in the rat • Gestational day 13 in humans

Onset of Embryonic Heartbeat

• Gestational day 8½ - ¾ in the mouse• Gestational day 9½ in the rat • ~ Gestational day 23 in humans

Classifications of Placentae

• Gross shape• Mode of implantation• Fetal membranes• Extent of invasiveness

Types of Placentae: Shape

Diffuse(Placenta Diffusa)

Multiplex(Placenta

Cotyledonaria)

Banded(Placenta Zonaria)

Discoid(Placenta

Discoidalis)

PigHorseRhinoceros

Ruminants e.g. Cow

SheepGoatDeer

Carnivores e.g. Dog

CatSeal

InsectivoresRodentsHigher Primates

Types of Placentae: Mode of Implantation

Central(Superficial)

EccentricInterstitial

Lumen of Uterus

Chorion

Lumen of Uterus

Chorion

DeSesso, 1997

Placentae Are Formed from Different Fetal Membranes

TRUE CHORIONIC CHORIOVITELLINE

CHORIOALLANTOIC CHORIOVITELLINE/CHORIOALLANTOIC

DEVELOPING COMPLETENON-

VASCULAR VASCULAR

Ramsey, 1982

Placentae Differ With Respect to Invasiveness

Classification of Placentae

Placental Characteristics Affecting Transfer of Substances

• Placental morphology– Grosser classification– Number of layers between maternal

and embryonic circulation

• Placental metabolism• Placental age

– Thickness

– Surface area

Placental Transfer of Chemical Substances

Assume that EVERY chemical is transferred across the placenta

The ensuing questions are• HOW MUCH reaches the fetus?• HOW RAPIDLY does it cross to the fetal

circulation?• HOW LONG does it remain in the fetus?

Development of Human Embryo is Rapid

3 weeks

5 weeks

6.5 weeks

11 weeks

Developmentally Susceptible Periods

Differentiation

Organogenesis

Tissue Development

Functional Maturation

Implantation

Fertilization

Parturition

Time in Gestation

Embryonic Period Fetal Period Post NatalPeriod

RelativeSusceptibility

0 CA D EB

DeSesso, 1997

Gestational Milestones for Mammals

Primitive Early Organogenesis UsualSpecies Implantation Streak Differentiation Ends Parturition

Rat 5-6 8.5 10 15 21-22

Mouse 5 6.5 9 15 19-20

Rabbit 7.5 7.25 9 18 30-32

Hamster 4.5-5 7 8 13 16

Guinea Pig 6 12 14.5 ~29 67-68

Monkey 9 17 21 ~44-45 166

Human 6-7 13 21 ~50-56 266

A2

1In gestational days; day of confirmed mating = gestational day 02Letters refer to positions on Conceptual Roadmap of Embryonic Development

B C D E

Gestational Milestone1

DeSesso, 1997

Rodent Inverted Yolk Sac Placenta

Ramsey, 1982

Placentation in Rats: Development of the Inverted

Yolk Sac Placenta

Gestational Day 7 Gestational Day 8 Gestational Day 10

Jollie, 1990

Placentation in Rats: Establishment of the

Chorioallantoic Placenta

Gestational Day 11.5

Modified from Jollie, 1990

Visceral Yolk Sac and Early Chorioallantoic Placenta

Ida Smoak, UNC

Gestational Day 10 Rat Conceptus

Gestational Day 12 Rat Conceptus

Oviparous

Yolk Sac

Viviparous

Allantois Chorion

Maternal Uterine Tissue

Cytotrophoblast

Syncytiotrophblast

Chorioallantoic

Yolk SacChorion

Cytotrophoblast

Syncytiotrophblast

Maternal Uterine Tissue

Choriovitelline

Routes of Embryonic Nutrient Uptake

Inverted Yolk Sac

Maternal Uterine Tissue

Uterine Milk

Parietal YS and Reichert’s

Membrane

Definitions

• Histiotroph: Total nutrients supplied to the embryo in viviparous animals from sources other than the maternal blood

• Hemotroph: Total nutrients supplied to the embryo from the maternal blood

Countercurrent Blood Flow

CAP

CAP

Temporal Comparison of Early Development: Rat and Human

Rat

Human

Conception

Day 0

Day 0

5.5 - 7

6-13

Implantation

Primitive Streak

Appears

13.5

8.5 9

18

Neural Folds

To reach equivalent lengths – 3 mm – Human: 25 days vs. Rat: 9 days (From: O’Rahilly & Muller, 1987)

27

11.511

26

InvYSP InvYSP

First Somite Formed

First Heartbeat

9.5

19 23

Chorioallantoic Placenta

Circulation Begins

10

Forelimb Bud

Tissue Development

Functional Maturation

Fertilization

Parturition

Time in Gestation

RelativeSusceptibility

22

Organogenesis

Developmentally Susceptible Periods:Rat

Differentiation

Implantation

Embryonic Period Fetal Period Post NatalPeriod

0 105 158.5

Developmentally Susceptible Periods:Rat

Tissue Development

Functional Maturation

Fertilization

Parturition

Time in Gestation

Fetal Period Post NatalPeriod

RelativeSusceptibility

15 22

Implantation

0 105 8.5

CAPYSP

Organogenesis

Differentiation

Types of Placentae Found in Animals Used in Research

Primate Rodent

Dog Sheep Ramsey, 1987

Colorado State Website

Term Canine Conceptus Dissected

Extraembryonic Membranes and Placentation in the Dog

Modified from Noden and de Lahunta (1985)

Chorioallantoic Placenta

Choriovitelline Placenta

Amniotic Cavity

Allantoic Cavity

Allantois

Chorion

Yolk Sac

Chorionic, Amniotic, and Yolk Sac Cavities Develop Early

Drawings at the same scale of human embryos from stage 2 to stage 5c illustrating implantation. Asterisk, primary yolk sac cavity.

O’Rahilly and Muller, 1987

Chorionic Cavity Expands Rapidly During Early Gestation

The relative size of the embryo and the chorion at weekly intervals. The stages shown are 6, 10, 13, 16, 17, 20, and 23.

O’Rahilly and Muller, 1987

Points to Remember for Modeling Purposes

The size of extraembryonic fluid compartments is large compared to the size of the embryo during organogenesis

Points to Remember for Modeling Purposes

As gestation proceeds:– Surface area for exchange expands dramatically– Distance between maternal and offspring blood

decreases– Maternal plasma volume increases up to 50%– Maternal protein binding decreases

Exocoelomic Sampling Technique

Jauniaux, et al., 1993

Vascularized Yolk Sac and Chorioallantoic Placenta of Human

Mark Hill, UNSW

Recent Reports Regarding Human Uteroplacental Circulation (Jauniaux et al.)

• Based on in vivo Doppler ultrasound and dynamic oxygen tension measurements– Erosion of maternal capillaries (week 3) allows blood

into intervillous space (IVS), but sluggish movement

– No “connections” between spiral arteries and IVS until week 4 (presence of cytotrophoblast plugs)

– Minimal maternal blood flow through IVS until week 6

– Fully established uteroplacental circulation by week 10

Comparative Developmental Milestones

Species Fertilization Blastocyst Implantation Begins InvYSP CAP

Neural Tube

Closure

Mouse 0 3-6 5 7.2 9.1 9.1

Rat 0 3.5-5.5 5.5 9.5 11.5 10.75

Rabbit 0 3-6 7.5 9 10 9.75

Dog 0 12-16 16 ---* 22 21

Rhesus 0 5-6 9 --- ~28 31

Human 0 4-6.5 6.5 --- 27 27

DeSesso, 1997

* Yolk sac of the dog abuts chorion ~19.5 day of gestation

Hypothetical Impact of Two Concepts of Early Embryonal Nutrition on Interpretation

of Data for Potential Human Risk

• Classic Anatomical Model: – Uteroplacental circulation begins on gestational day 13– Hemotrophic nutrition begins– No impact on embryonic nutrition

• Recent Clinical Reports:– Spiral arteries are ‘plugged’ until 8th week, preventing

uteroplacental circulation– Product unlikely to reach trophoblast cells– No impact on embryonic nutrition

END

• The following are extra slides

1. amnion

2. chorion laeve

3. chorionic villi

4. embryonic surface

5. umbilical vessels

Gestational Day 26

England, 1996

Gestational Day 12 Rat Conceptus

Term Canine Zonary Placenta

Rob Foster 2002

Diagram of IntegrinIntra- and Extracellular Relationships

Gilbert, 1997

Possible Mechanism for Control of Adhesion

• Ovarian steroids (progesterone) elicit– Expression of β-integrins on surface of endometrial cells for

a window of time– Secretion of signal molecules, including the cytokine

leukemia inhibitory factor (LIF), into uterine lumen

• Blastocyst responds to LIF– Expresses the glycoprotein L-secretin on trophoblast cells

• Expression of both glycoproteins occurs in discrete areas

• Carbohydrate moieties of the glycoproteins interact

Rat Implantation Chamber

A. Blastocyst

a. Embryoblast

b. Trophoblast

B. Epithelial depression

C. Subepithelial fibroblasts showing decidual reaction

Hebel and Stromberg, 1986

Species Differences in Developmental Toxicity Studies

• Plasma protein binding• Metabolic and biotransformational

capabilities• Genotypic susceptibility• Developmental schedules

Chronology of Early Events During Gestation of Mouse Embryos

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Fertilization

Blastocyst

Implantation

Inv YolkSacPlacenta

ChorioallantoicPlacenta(20 Somites)

Days of Gestation

Vascular Flow in Mammals

Vascular Flow in Pregnant Mammals

Conceptual Roadmap ofEmbryonic Development

EmbryonicCellular Potency

CellularDifferentiation

Interspecies Differences among Embryos Increase with Age

Gilbert, 1997

Comparative Definitive Placentation

Amniotic Cavity

Extra-Embryonic Coelom

DeciduaYolk Sac

Uterine Artery

Decidua

Re-EstablishedUterine Lumen

Amniotic Cavity

Visceral Yolk Sac

VascularLacuna

Human Conceptus at the Time of Chorioallantoic Placental Establishment Day 12 Rat Conceptus

ChorioallantoicPlacenta

ChorioallantoicPlacenta

Modified from Holson, 1973

Comparative Early Placentation

Amniotic Cavity

Extra-Embryonic Coelom

Decidua

Yolk Sac

Uterine Lumen

Uterine Artery

Decidua

Ectoplacenta

Allantois

Visceral Yolk Sac

Vascular Lacuna

Human Conceptus (Pre-Chorioallantoic Placental Stage) Day 10 Rat Conceptus

Modified from Holson, 1973

Mechanisms of Placental Transfer

• Diffusion (e.g., nearly all drugs and foreign substances)– No metabolic energy– With concentration gradient– Affected by molecular size and charge

• Facilitated diffusion (e.g., glucose)– Involves carrier substance– Rate greater than that expected by diffusion– No metabolic energy– With concentration gradient

Mechanisms of Placental Transfer

• Active transport (e.g., essential amino acids, iron)– Against concentration gradient– Saturable

– Inhibited by metabolic poisons

– Competition exists

• Pinocytosis / receptor-mediated endocytosis (e.g., immunoproteins)– Vacuolizations

• Leakage (e.g., erythroblastosis fetalis)– Discontinuities

1. abdomen

2. amnion

3. amnion on umbilical cord

4. back

5. chorionic villi

6. embryo

7. fetus

8. head

9. leg

10. leg bud

11. umbilical cord

12. umbilical vessels

Week 8

England, 1996

Diameter of Chorion Greatly Exceeds Length of Embryo During First 8 Weeks

The length of the embryo from stage 8 to stage 23, approximately 2-1/2 to 8 postovulatory weeks, based on the measurements of more than 100 specimens that had been graded as excellent in quality. The maximum diameter of the shaded band includes approximately 80 percent of the specimens. At 4 weeks the embryo is about 5mm in length and the chorion about 25mm in diameter. At 8 weeks the embryo is about 30 mm in length and the chorion is about 65mm in diameter.

Weeks

Mill

imet

ers

O’Rahilly and Muller, 1987

Conceptual Roadmap ofEmbryonic Development

EmbryonicCellular Potency

CellularDifferentiation

DeSesso, 1997

Conceptual Roadmap ofEmbryonic Development

EmbryonicCellular Potency

CellularDifferentiation

Gestational Stage andDevelopmental Susceptibility

Usually Not Affected

Highly Susceptible: Malformations Readily Induced

Increasingly Resistant; Functional Deficits Possible

DeSesso, 1997 after Wilson

Does the Embryo Occupy a Privileged Site in an Impregnable Uterus?

After Wilson

Is There a “Placental Barrier”?

• Virtually all substances can and do cross the placenta

• Closest correlations to a “barrier”– Expression of the mdr gene in trophoblast cells– Presence of p-glycoprotein on placental trophoblast

Considerations about the Placental Interface and Toxicity

• Regardless of anatomical differences, all placentae serve to transport nutrients, metabolites, and gases between parent and offspring

Considerations about the Placental Interface and Toxicity

• Placentae are established early and continue to develop throughout gestation

• Placentae exhibit wide interspecies differences in morphology

• In contrast to humans, many experimental animals (e.g., rat, mouse, rabbit) possess an inverted visceral yolk sac placenta that is established earlier than the chorioallantoic placenta, transports materials by a different mechanism, and remains functional until (nearly?) term

Generalized Implications from our Studies and Analysis

There should be no doubt that the InvYSP can be a target for toxicity leading to serious developmental disruption. To the contrary, it has not been demonstrated that the noninverted yolk sac is a similar target.

Caution should be exercised In generalizing too broadly the findings of studies of this product, which by design, was given at high doses (mass) of hemoglobin protein, 6 g/kg.

Large and/or proteinaceous agents 1) with no pharmacologic action on the biochemical modalities of the InvYSP or 2) which do not contain a moiety with toxic properties would not be expected to exert similar effects.

The former types of agents would appear to represent a small number of the universe of xenobiotics and no broad sense lessens the value of current models.