Presented by John DeSesso and Joseph F. Holson in Symposium I ("A Detective Story: Is the Prenatal Toxicity of a Therapeutic in Rats Relevant to Human Risk?", J.F. Holson and L. B. Pearce, co-chairpersons) at the Forty-Third Annual Meeting of the Teratology Society, Philadelphia, PA, June 26, 2003.

1. 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 ATS
Mitretek Systems
Joseph F. Holson, PhD, DABFE
WIL Research Laboratories

2. Examples of Uterine Structure
Ramsey, 1982

3. Gross Anatomy of the Human Female
Reproductive Tract
Netter, 1998

4. Events in the Female Reproductive
Tract from Fertilization to Implantation
Moore, Persaud & Siota, 1997

5. Changes in Uterine Wall During
Menstrual Cycle

6. 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

7. Placenta
Any apposition of embryonal to parental
tissues for the purpose of physiological
exchange

8. Implantation of Human Embryo
Carlson, 1999

9. Establishment of the
Uteroplacental Circulation
Carlson, 1999

10. 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

11. Classifications of Placentae
• Gross shape
• Mode of implantation
• Fetal membranes
• Extent of invasiveness

12. Types of Placentae: Shape
Diffuse
(Placenta
Diffusa)
Multiplex
(Placenta
Cotyledonaria)
Banded
(Placenta
Zonaria)
Discoid
(Placenta
Discoidalis)
Pig
Horse
Rhinoceros
Ruminants
e.g. Cow
Sheep
Goat
Deer
Carnivores
e.g. Dog
Cat
Seal
Insectivores
Rodents
Higher Primates

13. Types of Placentae: Mode of Implantation
Central
(Superficial)
Eccentric
Interstitial
Lumen of Uterus
Chorion
Lumen of Uterus
Chorion
DeSesso, 1997

14. Placentae Are Formed from Different
Fetal Membranes
TRUE CHORIONIC CHORIOVITELLINE
CHORIOALLANTOIC CHORIOVITELLINE/
CHORIOALLANTOIC
DEVELOPING COMPLETE
NON-
VASCULAR
VASCULAR
Ramsey, 1982

15. Placentae Differ With Respect to
Invasiveness

16. Classification of Placentae

17. 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

18. 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?

19. Development of Human Embryo is Rapid
3 weeks
5 weeks
6.5 weeks
11 weeks

20. Developmentally Susceptible Periods
Differentiation
Organogenesis
Tissue Development
Functional Maturation
Implantation
Fertilization
Parturition
Time in Gestation
Embryonic Period Fetal Period Post Natal
Period
Relative
Susceptibility
0 CA D EB
DeSesso, 1997

21. Gestational Milestones for Mammals
Primitive Early Organogenesis Usual
Species 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
1
In gestational days; day of confirmed mating = gestational day 0
2
Letters refer to positions on Conceptual Roadmap of Embryonic Development
B C D E
Gestational Milestone1
DeSesso, 1997

22. Rodent Inverted Yolk Sac Placenta
Ramsey, 1982

23. Placentation in Rats:
Development of the Inverted
Yolk Sac Placenta
Gestational Day 7 Gestational Day 8 Gestational Day 10
Jollie, 1990

24. Placentation in Rats:
Establishment of the
Chorioallantoic Placenta
Gestational Day 11.5
Modified from Jollie, 1990

25. Visceral Yolk Sac and Early
Chorioallantoic Placenta
Ida Smoak, UNC

26. Gestational Day 10 Rat Conceptus

27. Gestational Day 12 Rat Conceptus

28. Oviparous
Yolk Sac
Viviparous
Allantois Chorion
Maternal Uterine Tissue
Cytotrophoblast
Syncytiotrophblast
Chorioallantoic
Yolk Sac
Chorion
Cytotrophoblast
Syncytiotrophblast
Maternal Uterine Tissue
Choriovitelline
Routes of Embryonic Nutrient Uptake

29. Inverted Yolk Sac
Maternal Uterine Tissue
Uterine Milk
Parietal YS
and Reichert’s
Membrane

30. 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

31. Countercurrent Blood Flow

32. 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

33. Tissue Development
Functional Maturation
Fertilization
Parturition
Time in Gestation
Relative
Susceptibility
22
Organogenesis
Developmentally Susceptible Periods:
Rat
Differentiation
Implantation
Embryonic Period Fetal Period Post Natal
Period
0 105 158.5

34. Developmentally Susceptible Periods:
Rat
Tissue Development
Functional Maturation
Fertilization
Parturition
Time in Gestation
Fetal Period Post Natal
Period
Relative
Susceptibility
15 22
Implantation
0 105 8.5
CAPYSP
Organogenesis
Differentiation

35. Types of Placentae Found in Animals
Used in Research
Primate Rodent
Dog Sheep Ramsey, 1987

36. Colorado State Website
Term Canine Conceptus Dissected

37. 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

38. 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

39. 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

40. Points to Remember for Modeling
Purposes
The size of extraembryonic fluid
compartments is large compared to the size
of the embryo during organogenesis

41. 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

42. Exocoelomic Sampling Technique
Jauniaux, et al., 1993

43. Vascularized Yolk Sac and
Chorioallantoic Placenta of Human
Mark Hill, UNSW

44. 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

45. 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

46. 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

48. END
• The following are extra slides

49. 1. amnion
2. chorion laeve
3. chorionic villi
4. embryonic
surface
5. umbilical
vessels
Gestational Day 26
England, 1996

50. Gestational Day 12 Rat Conceptus

51. Term Canine Zonary Placenta
Rob Foster 2002

52. Diagram of Integrin
Intra- and Extracellular Relationships
Gilbert, 1997

53. 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

54. Rat Implantation Chamber
A. Blastocyst
a. Embryoblast
b. Trophoblast
B. Epithelial
depression
C. Subepithelial
fibroblasts
showing decidual
reaction
Hebel and Stromberg, 1986

55. Species Differences in Developmental
Toxicity Studies
• Plasma protein binding
• Metabolic and biotransformational
capabilities
• Genotypic susceptibility
• Developmental schedules

56. 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 Yolk
Sac
Placenta
Chorioallantoic
Placenta
(20 Somites)
Days of Gestation

57. Vascular Flow in Mammals

58. Vascular Flow in Pregnant Mammals

59. Conceptual Roadmap of
Embryonic Development
Embryonic
Cellular Potency
Cellular
Differentiation

60. Interspecies Differences among Embryos
Increase with Age
Gilbert, 1997

61. Comparative Definitive Placentation
Amniotic Cavity
Extra-Embryonic
Coelom
Decidua
Yolk Sac
Uterine Artery
Decidua
Re-Established
Uterine Lumen
Amniotic Cavity
Visceral Yolk Sac
Vascular
Lacuna
Human Conceptus at the Time of
Chorioallantoic Placental Establishment Day 12 Rat Conceptus
Chorioallantoic
Placenta
Chorioallantoic
Placenta
Modified from Holson, 1973

62. 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

63. 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

64. 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

65. 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

66. 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
Millimeters
O’Rahilly and Muller, 1987

67. Conceptual Roadmap of
Embryonic Development
Embryonic
Cellular Potency
Cellular
Differentiation
DeSesso, 1997

68. Conceptual Roadmap of
Embryonic Development
Embryonic
Cellular Potency
Cellular
Differentiation

69. Gestational Stage and
Developmental Susceptibility
Usually Not
Affected
Highly
Susceptible:
Malformations
Readily Induced
Increasingly
Resistant;
Functional
Deficits Possible
DeSesso, 1997 after Wilson

70. Does the Embryo Occupy a Privileged
Site in an Impregnable Uterus?
After Wilson

71. 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

72. Considerations about the Placental
Interface and Toxicity
• Regardless of anatomical differences, all
placentae serve to transport nutrients,
metabolites, and gases between parent and
offspring

73. 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

75. 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.