Development of the Embryo

The union of individual male and female gametes represents an orderly and highly regulated sequence of events collectively termed fertilization.¹⁴ Fertilization involves maturation of spermatozoa^15,16,17 by capacitation, and their movement through the cumulus of the ovum,¹⁸ although human sperm need not undergo capacitation to traverse the cumulus.¹⁶ Sperm binding to zona pellucida proteins (ZP1-4), primarily ZP3,^19,20 precedes the acrosome reaction²¹ that is essential for sperm fusion with the oocyte membrane.²² After fusion of the egg and sperm membranes, release of cortical granules in the oocyte results in conformational changes in ZP2 and ZP3 that render the ovum impenetrable to additional sperm.¹⁶

At the 8-cell stage, blastomeres begin to form gap and tight junctions,^23,24 a process that initiates segregation of inner cells from outer cells and marks the onset of embryo differentiation.²⁵ After additional cell divisions, the embryo (now termed a morula) enters the uterus, approximately 4 days after ovulation in both humans and nonhuman primates.²⁶ After formation of the blastocoele, cells of the blastocyst differentiate into an inner cell mass destined to form the fetus and an outer mass of cells destined to become the placenta (Fig. 1).²⁷ This process appears to be modulated by cell adhesion molecules, since E-cadherin-null embryos fail to form a trophectoderm.²⁸ The developing blastocyst continues to grow and essentially floats in the uterine cavity for an additional 2 to 3 days during which time the blastocyst (embryo) hatches from the surrounding zona pellucida.^29,30,31 Hatching appears to be essential for embryo-uterine contact and for subsequent implantation that occurs between 7 and 9 days after ovulation in humans and most nonhuman primates. Enders^32,33 has concluded that prior to attachment to the uterine epithelium, mononucleated cytotrophoblast cells of the trophectoderm fuse to form a syncytiotrophoblast layer. The syncytiotrophoblast appears to initially interact with and adhere to the endometrium. Only after the embryo is totally embedded in the endometrium do cytotrophoblast cells begin to move from the trophoblastic shell to invade the uterus and the uterine vasculature.^34,35,36

Preparation of the Uterus for Implantation

As the embryo (blastocyst) is developing and moving through the fallopian tubes into the uterus, the endometrium undergoes extensive differentiation ultimately to permit embryo attachment (i.e., implantation). In virtually all mammals, preparation of the uterus for implantation is regulated by the coordinated actions of estrogen and progesterone³⁷ produced and secreted by the ovary-corpus luteum and perhaps by the developing embryo itself.^38,39 Estrogen and progesterone act on the endometrium and myometrium directly through estrogen-progesterone receptor-mediated events, and indirectly by stimulation of various growth factors (e.g., EGF, TGF-B, insulin-like growth factor I (IGF-I) and IGF-II), proteins (e.g., placental protein 14, 24 kd protein, CA-125), and cytokines (e.g., IL-1, CSF-1) (Fig. 2).⁴⁰ Uterine receptivity to a putative implantation signal is limited to a discrete period of time during the luteal phase of the menstrual cycle consistent with the concept of a window of implantation that has been advanced in several animal models and in the human.^41,42,43 However, despite intense investigation, the molecular basis for the transient nature of uterine receptivity, estimated to span days 19/20 to 24 of the menstrual cycle,^35,44,45 remains unknown. During this interval, and presumably under the influence of estrogen and progesterone, the uterine endometrium is thickened and highly secretory in nature and becomes rich in glycogen and lipids.³⁷ Morphologic studies have shown that uterine receptivity is heralded by the formation of pinopods on the apical surface of endometrial epithelial cells, a process that appears to be regulated by progesterone.⁴⁶ Pinopods may absorb fluid from the uterine cavity, thereby making the endometrium more accessible to the blastocyst. Changes in the composition of the uterine glycocalyx have also been observed during the peri-implantation period. For example, the levels of two transmembrane glycoproteins, mucin (MUC-1) and keratan sulfate, increase on the endometrial glandular surface during the early luteal phase,^9,47,48 then decrease as the window for implantation opens.^9,48 Because MUC-1 is a relatively large cell surface molecule, its down-regulation may unmask smaller molecules on the uterine surface, such as the cadherins and integrins, thereby mediating specific adhesion of the trophectoderm to the endometrium. Epithelial integrins have been proposed as markers for the window of implantation in the human.^49,50 Endometrial integrins are expressed in an “on/off” pattern,⁵¹ and both they and their ligands are expressed in trophectoderm.⁵² Taken together, these observations support the hypothesis that integrins may be involved in early implantation events. The β 3 integrin subunit is expressed on the endometrial surface after day 19 of the human menstrual cycle, just when the endometrial window of implantation opens.⁵³ The fact that this integrin is almost never expressed by epithelial cells, but is expressed by human trophectoderm,⁵⁴ supports a role for these molecules in implantation. A similar distribution of endometrial integrins and extracellular matrix proteins has been described across the menstrual cycle and early pregnancy in the baboon.⁵⁵ Human secretory phase endometrium also produces the glycoprotein leukemia-inhibiting factor(LIF).^56,57 LIF has the capacity to inhibit embryonic stem cell differentiation in vitro,⁵⁸ and LIF receptors are detected on human blastocysts.^56,57 Moreover, Stewart and colleagues⁵⁹ have demonstrated that LIF (production of which is regulated by estrogen and progesterone) is essential for implantation in the mouse since implantation of blastocysts did not occur in mutated mice lacking a functional LIF gene. A similar role for LIF in humans and nonhuman primates has not been confirmed.

In addition to expression of integrins, the human blastocyst/embryo secretes a number of factors at various times during its development that may be essential for pregnancy recognition or its normal progression. Almost immediately after fertilization, the embryo secretes platelet-activating factor (PAF), interleukins-1 and -6, and early pregnancy factor. Although inhibition of PAF activity in vivo prevented implantation in rodents,⁶⁰ a similar role in primate pregnancy has not been demonstrated. By the 8-cell stage, the blastocyst apparently secretes a number of cytokines and growth factors including chorionic gonadotropin(hCG), long recognized as one embryonic factor that is essential for early pregnancy in primate species.^{31,61,62,63,64}

Rescue of the Corpus Luteum by Human Chorionic Gonadotropin

hCG is a glycosylated protein heterodimer composed of noncovalent bound α and β subunits.⁶⁵ β-hCG is structurally similar to the β subunit of pituitary luteinizing hormone (LH), differing only in the terminal 28 amino acids. Whereas α subunit genes are expressed in the pituitary and placenta,⁶⁶ the 6 β subunit genes located on chromosome 19q13.3 are expressed only in the placenta.⁶⁷ The three-dimensional structure of hCG has also been determined⁶⁸; both the highly glycosylated α and β subunits⁶⁹ contain several disulfide linkages that form cysteine knot motifs.^68,70 Deglycosylation studies indicate that the carbohydrate moieties of the α, but not the β, subunit are critical for activation of the hCG receptor and its associated G-protein coupled signaling systems.⁷⁰ In human pregnancy, hCG levels in maternal blood are detectable approximately 3 days after implantation⁷¹ and rapidly rise thereafter with peak levels being achieved between weeks 8 and 12 of gestation (Fig. 3).⁷² Although in vitro studies suggest that hCG may be produced by hatched embryos prior to attachment, expression is generally assumed to occur after attachment.^26,73 In the absence of hCG, the newly formed corpus luteum will invariably regress; production of estrogen and progesterone declines resulting in endometrial shedding (i.e., menses). The hCG produced by the syncytiotrophoblast binds to the LH/CG receptor in the corpus luteum to stimulate progesterone and estradiol synthesis, presumably by increasing low-density lipoprotein receptor expression and thus facilitating uptake of cholesterol substrate for steroidogenesis (Fig. 4).³¹ Immunohistochemical studies also suggest that hCG may act to increase the expression of rate limiting steroidogenic enzymes (e.g., 3β-hydroxysteroid [-HSD] and aromatase; Fig. 5).⁷⁴ In some species, but perhaps not the human, rescue of the corpus luteum by hCG may also involve local inhibition of factors (e.g., prostaglandin) that promote luteal demise (e.g., luteolysis). hCG is also thought to stimulate corpus luteum relaxin production (see Fig. 4) and, as discussed later, appears to enhance testosterone production by the fetal testes that is required for differentiation of the internal duct system and external genitalia in males. The high levels of hCG secreted into the maternal circulation during early gestation apparently are sufficient to bind to the thyroid-stimulating hormone (TSH) receptor and thereby to increase maternal thyroid hormone production.⁷⁵ Although our current understanding of the regulation of hCG production remains incomplete, it has been proposed that gonadotropin-releasing hormone (GnRH) produced by the placental cytotrophoblast may modulate hCG production by binding to GnRH receptors in the syncytiotrophoblast.⁵ Interestingly, human placental trophoblast produces a variety of other hypothalamic-/pituitary-like neuropeptides,⁵ including corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), neuropeptide Y, oxytocin, somatostatin, and thyrotropin-releasing hormone (TRH). Although the physiologic role of most of these factors remains to be defined, CRH may play a role in parturition,^76,77 modulation of uterine blood flow,⁷⁸ and regulation of maternal pituitary ACTH production.^79,80 Finally, the trophoblast also produces several protein hormones (chorionic somatomammotropin [hCS] and growth hormone variant [GH-V]) and growth factors (IGF-I and IGF-II) that are considered essential to the regulation of maternal, and perhaps fetal, intermediary metabolism as well as growth and maturation of the placenta.⁸¹

Placental Development, Vascularization, and Intrauterine Endovascular Invasion

DECIDUALIZATION

Upon attachment of the embryo to the uterus, the uterine endometrial glycogen-containing stromal cells are rapidly transformed into large decidual cells that increase in size throughout the course of gestation. The human decidua that represents the maternal component of the placenta is composed primarily of the decidua basalis, underlying the site of implantation, the decidua capsularis that initially overlies the gestational sac but gradually disappears with advancing gestation, and the decidua vera that lines the remainder of the uterine cavity. In humans, as well as the baboon,⁸² the decidua produces and secretes a variety of factors that include the hormones relaxin and prolactin, the IGF-binding protein (IGF-BP-1), and a variety of other proteins (e.g., placental protein 14, also known as progesterone-associated endometrial protein [PEP], or glycodelin).

In the human, IGF-BP-1 is secreted by the stromal cells that surround the spiral arteries during the late luteal phase, whereas in the baboon, IGF-BP-1 is secreted by the endometrial glands in response to progesterone.^83,84 IGF-BP-1 is one of several proteins that bind IGF-I and IGF-II and thereby regulate the ability of these growth factors to interact with their receptors. Ritvos and associates⁸⁵ have suggested that decidual IGF-BP1 may act to control invasion and/or proliferation of trophoblast cells during implantation/placentation by sequestering the IGFs. IGF-BP-1 levels increase rapidly during early gestation in parallel with decidualization, then transiently decline before increasing again in late pregnancy.^86,87 Pregnancy termination with the progesterone receptor antagonist RU-486 is associated with a marked decline in IGF-BP-1 levels, an observation suggesting that decidual IGF-BP-1 production is progesterone dependent.⁵³ It has also been hypothesized that restructuring of the decidual cell cytoskeleton is essential for IGF-B1 gene expression.⁸⁸

Glycodelin, synthesized by the uterine glandular epithelium, shares homology with the B-lactoglobulins and retinol-binding proteins⁸⁹ and has been implicated as an immunosuppressive agent.^90,91 Close temporal relationships in serum profiles suggest that glycodelin production is regulated by progesterone^92,93 and/or relaxin.⁹⁴ Serum glycodelin levels are elevated in the luteal phase of the menstrual cycle and markedly increase during the first trimester of pregnancy. In the baboon, although the pattern of uterine glycodelin mRNA and protein expression mimics that in the human, glycodelin production is regulated by hCG,⁹⁵ perhaps by direct action on the endometrium.

Decidual prolactin production begins on day 22 of the idealized human menstrual cycle (approximately 8 days after ovulation)⁹⁶ and prolactin mRNA and protein expression is observed in the epithelial cells of the deep basal glands of the baboon uterus during the late luteal phase. Decidual prolactin expression increases markedly with advancing gestation and is stimulated by progesterone in both the human^97,98 and baboon.⁹⁹ Decidual prolactin is apparently secreted into the amniotic fluid in humans^100,101 and baboons,¹⁰² and levels increase with advancing gestation. Although the precise role(s) of decidual prolactin remains unclear, it has been proposed that the hormone may enhance uterine contractility, an action also apparently antagonized by decidual relaxin.^103,104

PLACENTAL TROPHOBLAST DEVELOPMENT AND VASCULARIZATION

During human and nonhuman primate pregnancy, the placenta simultaneously accesses the maternal blood and develops a vascular network for the transport of nutrients to and waste products from the fetus across the syncytiotrophoblast to ensure fetal growth and development. Both processes depend on the ability of the primordial stem-cell cytotrophoblasts to take either the villous pathway where they remain in the fetal compartment and differentiate morphologically into the syncytiotrophoblast or the extravillous pathway where they proliferate, aggregate into cell columns of the anchoring villi, and invade the endometrial stroma (Fig. 6).¹⁰⁵ The syncytiotrophoblast covers the floating chorionic villi that become highly vascularized, whereas the extravillous cytotrophoblasts infiltrate the walls of the spiral arterioles to facilitate the process of placentation.

The vascular network within the placental villous core develops by in situ differentiation of fetal mesenchymal cells into vessels (vasculogenesis) and proliferation of existing vessels (angiogenesis) resulting in secondary and tertiary villi equipped with a functional arterio-capillary-venous system.³⁴ Development of the uteroplacental circulation begins early in pregnancy with neovascularization of both fetal and maternal tissues.^106,107,108 Neovascularization results in increased blood flow and increases in the effective exchange area crucial to support fetal development. In humans, placental vasculogenesis begins at approximately 21 days of gestation¹⁰⁹ and continues through at least the 26th week of pregnancy.³⁴ Angiogenesis accompanies vasculogenesis and is critical not only early in gestation but also important for early widespread extension of the fetal capillary system and later growth of the fetal vascular compartment during the final third of pregnancy.^109,110 Several factors expressed by the placenta, including basic fibroblast growth factor,^111,112,113 platelet-derived growth factor,¹¹⁴ placental growth factor,¹¹⁵,¹¹⁶ and vascular endothelial growth/permeability factor (VEG/PF)^{115 ,117} have been proposed as regulators of placental angiogenesis. Among these, VEG/PF selectively stimulates endothelial cell proliferation and the formation of new blood vessels.^118,119 It also induces vascular permeability by actions on endothelial cells resulting in extravasation of plasma proteins that provide an extracellular fibrin matrix for angiogenesis.¹²⁰ VEG/PF is encoded from a single gene and expressed as 5 isoforms having 121, 145, 165, 189, or 206 amino acids,^121,122 the 121 and 165 isoforms exhibiting the greatest angiogenic activity.^123,124,125 VEG/PF mRNA and protein are expressed by cytotrophoblasts, syncytiotrophoblast, and Hofbauer macrophage cells within the villous human placenta.^{114,117,124,126} Inactivation of the VEG/PF gene in transgenic mice results in significant defects in the vasculature of embryonic tissues and organs that are lethal.¹²⁷

VEG/PF binds to two structurally related transmembrane tyrosine kinase receptors, VEG/PF flt-1 and KDR/flk-1,^128,129 which are expressed on placental villous vascular endothelial cells^126,129,130 and in villous and extravillous trophoblasts.^117,126,131 Both receptors are essential for vascular development. Homozygous KDR/flk-1 defective mice die in utero as a result of an early defect in hematopoietic and endothelial cell development,¹³² and mouse embryos homozygous for the flt-1 mutation die because of failure to organize normal vascular channels.¹³³

Two other closely related proteins, angiopoietin-1 and -2, work in concert with VEG/PF in signaling vascular morphogenesis by binding to the endothelial cell-specific transmembrane tyrosine kinase receptor Tie-2. In Tie-2 null mice, endothelial cells develop and assemble into tubes, but vessels are immature, lacking branching networks, encapsulation by periendothelial support cells, and proper organization into small and large vessels.^134,135 These associations indicate that the Tie-2 receptor may also mediate the capacity of endothelial cells to recruit stromal cells that encase endothelial tubes for vessel stabilization. Transgenic/gene knock-out studies further indicate that angiopoietin-1 signals Tie-2 to recruit vascular support cells and that angiopoietin-2 inhibits this action^136,137,138 by competitively inhibiting angiopoietin-1-induced kinase activation of the Tie-2 receptor. During early human pregnancy, angiopoietin-1 is localized to the cytotrophoblast and syncytiotrophoblast, angiopoietin-2 to the cytotrophoblast, and Tie-2 receptor to the endothelium, ^139,140 observations that are consistent with the proposed role of the angiopoietin-Tie-2 system in development of the placental circulation.

Despite the importance of angiogenesis to neovascularization of the developing placenta, very little is known about the regulation of the process and the expression of the VEG/PF-angiopoitin-1/-2 system during human pregnancy. Although hypoxia is a potent stimulus of VEG/PF expression,¹⁴¹ estrogen has also been shown to regulate VEG/PF expression in the rat uterus^142,143,144 and in human endometrial cells.¹⁴⁵ Moreover, chronic estrogen treatment induced uterine angiogenesis in normal but not estrogen receptor-null transgenic mice.¹⁴⁶ In the baboon, cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta increase with advancing gestation in parallel with increasing placental estrogen production.¹⁴⁷ Thus, the well-established role for estrogen in enhancing uteroplacental blood flow^{148, 149} may not only reflect changes in vascular reactivity but also enhanced angiogenesis.

At the same time the vascular system is developing within the chorionic villi, a select population of extravillous cytotrophoblasts migrate and invade the spiral arteries of the uterine endometrium at the placental-decidual junction (see Fig. 6). Histologic studies performed during the first half of human, baboon, and macaque pregnancy^{32, 150,151,152,153,154,155} demonstrate that cytotrophoblasts migrate to and colonize spiral arterioles/arteries by displacing endothelial cells from their basal lamina and partially or completely replacing the smooth muscle component within the tunica media. Consequently, the structure of spiral arteries and the dynamics of blood flow within them are modified by cytotrophoblast invasion, presumably to facilitate implantation and placentation.

As cytotrophoblasts differentiate into cells capable of invading the uterine stroma and blood vessels, their expression of adhesion molecules changes in the human,^{8,105,156,157,158,159} baboon,¹⁶⁰ and rhesus monkey.^154,161 As extravillous cytotrophoblasts migrate, expression of the integrin complex α5β1 and α6β4 is lost and that of the α1β1 laminin/collagen receptor is induced.^158,162,163 Moreover, interaction of the α1β1 receptor with collagen type IV promotes, whereas interaction of the α5β1 receptor with fibronectin inhibits, invasion of human cytotrophoblasts in vitro.^164,165 Zhou and associates¹⁰⁵ have suggested that cytotrophoblasts balance invasion-retraining and invasion-promoting adhesion mechanisms as they differentiate. Migratory cytotrophoblast cells also express specific adhesion molecules, specifically vascular cell adhesion molecule (VCAM) and cadherins (e.g., VE-cadherin), that appear to secure cytotrophoblasts to each other and to the endothelium, thereby facilitating their migration against arterial blood flow.^161,166,167 Interstitial and spiral arteriole invasion is also associated with the expression of matrix metalloproteinases (MMPs; e.g., MMP-9 collagenase), by intraluminal, extravasating, and intramural cytotrophoblasts,^168,169 apparently to disrupt the extracellular matrices in the tunica media to allow cytotrophoblasts to modify the vessel wall. MMP-9 promotes cytotrophoblast invasion; the capacity of human cytotrophoblast to invade is completely inhibited by MMP-9 antibody in vitro.¹⁷⁰ Clearly, the process of endovascular spiral artery invasion involves an intricately and temporally ordered expression of integrins, adhesion molecules, and proteinases by the extravillous cytotrophoblasts. This area of research is of intense interest and clinically relevant. Abnormal expression of several of these components has been observed in cytotrophoblasts of women who develop preeclampsia and in whom endovascular invasion is superficial.¹⁷¹ Unfortunately, our current understanding of the factors acting/interacting to regulate the timely expression of ECM and cell adhesion molecules by invading trophoblasts remains incomplete.

Estrogen Production

CONCENTRATIONS IN THE CIRCULATION

The placenta becomes the primary source of estrogens (Fig. 7) after approximately week 9 of human pregnancy (23% of gestational length¹⁷²). As a result of extensive 16-hydroxylation of c19 steroids within the fetus,¹⁷³ large quantities of estriol are produced by the placenta during human pregnancy. A fourth estrogen, estetrol, is also produced uniquely but in relatively low levels during human pregnancy.¹⁷⁴

Plasma concentrations of estrone, estradiol, and estriol increase as human pregnancy progresses (Fig. 8)¹⁷⁴ with daily excretion rates at term approximating 2, 1, and 40 mg, respectively.^172,174 In humans, plasma concentrations of estradiol near term range from 6 to 30 ng/mL.^175,176 In women with threatened first-trimester abortion, abnormal estradiol concentrations are highly associated with a subsequent pregnancy loss.¹⁷⁷ Moreover, a 50% spontaneous abortion rate has been observed among women having a mutation in the amino terminal region of the estrogen receptor involved in transcription.¹⁷⁸ In the baboon, reduction of maternal estrogen levels to less than 0.1 ng/mL by daily administration of an inhibitor of placental estrogen synthesis resulted in a 66% incidence of abortion during early gestation that was prevented by treatment with exogenous estradiol.¹⁷⁹ Low serum estradiol concentrations during the third trimester are also associated with poor obstetric outcome.^180,181 Taken together, these observations suggest that estrogen plays a critically important role in the maintenance of primate pregnancy, but others have refuted this notion¹⁸² because pregnancy is maintained in most women having low estrogen levels resulting from deficiencies in various placental enzymes. ^183,184 Interestingly, in those cases, although maternal estradiol levels are markedly reduced, concentrations approximate 0.45 ng/mL, or 10^-9 mol/L, a concentration that approximates the dissociation constant for estradiol binding to its receptor.¹⁸⁵ Differences in the outcome of pregnancy in various women with estrogen deficiency suggest that the important biologic effects of estradiol can be achieved with available receptor and concentrations of estrogen sufficient to interact with it. It would appear that in both human and nonhuman primate pregnancy, estrogen is produced in considerable excess.

FETAL-PLACENTAL UNIT

The primate placenta does not express the P450 17α-hydroxylase/17–20 lyase enzyme (P450c17) and thus cannot convert de novo those c21 progestogens (pregnenolone and progesterone) to the c19 androgens (dehydroepiandrosterone [DHA] and androstenedione).¹⁸⁶ As shown by Ryan¹⁸⁷ and Siiteri and MacDonald,¹⁸⁸ the placenta can convert c19 androgens into estrogen. Because pregnancy with an anencephalic fetus is associated with very low levels of urinary estrogen and fetal adrenal hypoplasia, Frandsen and Stakeman¹⁸⁹ suggested that the fetal adrenal may provide the requisite androgen precursors for placental estrogen production. Siiteri and MacDonald^188,190 demonstrated that DHAS of maternal and fetal origin contributed about equally to estrone and estradiol synthesis, whereas more than 90% of estriol was synthesized from 16-hydroxy DHAS of fetal origin (Fig. 9).¹⁹¹ They subsequently¹⁸⁸ proposed the existence of a fetal-placental unit for estrogen biosynthesis, a concept that was later confirmed by Diczfalusy and colleagues.^192,193 A similar fetal-placental unit for the formation of estrogen also exists in several nonhuman primates including the rhesus monkey and the baboon.¹⁹¹

ENZYMES FOR ESTROGEN BIOSYNTHESIS

Placental synthesis of estrogens by conversion of c19-steroid precursors requires the enzymes sulfatase, Δ⁵-3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase (3β-HSD), P450 aromatase(P450arom), and 17β-hydroxysteroid oxidoreductase (17β-HSD) (see Fig. 9). Once secreted by the fetal adrenal gland, DHA is rapidly sulfated in the fetal liver to form DHAS and subsequently hydroxylated at carbon 16 to form 16-hydroxy DHAS, the primary precursor for estriol formation. On arrival in the placenta, sulfurylated c19 steroids precursors are desulfurylated by the enzyme sulfatase to yield unconjugated DHA and 16-hydroxy DHA. Although the regulation of sulfatase is unknown, prolactin and oxytocin stimulate enzyme activity in decidual cells isolated before the onset of human labor.¹⁹⁴ Mitchell and colleagues¹⁹⁵ have also observed a significant increase in hydrolysis of estrone sulfate by fetal membranes at term that could increase free estrogen concentrations locally within tissues with no associated change in peripheral plasma estrogen concentrations.

Unconjugated Δ⁵ — c19 steroid precursors in the placenta are subsequently converted to androstenedione/testosterone or 16-hydroxyandrostenedione by the enzyme 3β-HSD. A family of closely related genes encode for 3β-HSD¹⁹⁶ and various 3β-HSD isoforms are expressed in a tissue-specific manner. The human type I 3β-HSD is expressed at high levels in placenta, whereas the type II isoform is almost exclusively expressed in the adrenal cortex and gonads. In the placenta, the multinucleated syncytiotrophoblast is the principal site of 3β-HSD expression.

The subsequent conversions of androstenedione and testosterone to estrone and estradiol and 16-hydroxyandrostenedione to estriol (see Fig. 9) are catalyzed by P450arom, an enzyme consisting of an aromatase cytochrome P450 and the flavoprotein, NADPH-cytochrome P450 reductase.^197,198P450arom is a member of the cytochrome P450 superfamily of enzymes that includes approximately 220 members.¹⁹⁹ The enzyme binds c19-steroid substrates and catalyzes a series of reactions resulting in the formation of the phenolic A ring that is characteristic of estrogens (see Fig. 7). The entire three-step process is catalyzed at one active site by a single cytochrome P450 species.²⁰⁰

Recent studies have shown that the CYP19 gene encoding human cytochrome P450arom is located on chromosome 15 and consists of 9 exons and two polyadenylation sites in the last coding exon downstream from the terminating stop codon that gives rise to the 3.4 and 2.9 kb transcripts that encode human P450arom. Although the entire intron sequences remain to be mapped, the CYP19 gene is at least 70 kb long and is the largest cytochrome P450 gene characterized to date (reviewed elsewhere¹⁹⁹).

The interconversions of androstenedione and testosterone and of estrone and estradiol are catalyzed by 17β-HSD. There are at least four isoforms of 17β-HSD, with the placenta expressing 17β-HSD-1 and -2.^201,202,203 Placental 17β-HSD-1 has been localized to chromosome 17 and is a cytosolic enzyme that preferentially catalyzes the reduction of estrone to estradiol but does not utilize androgens as substrates.²⁰¹ In contrast, placental 17β-HSD-2 has been localized to chromosome 16 and catalyzes the oxidation of estradiol to estrone^201,204,205 and of testosterone to androstenedione.^{204,205,206,207} Estrone and estradiol are extensively interconverted within the placenta, but these two estrogens are not secreted in equal amounts into the maternal and fetal compartments in the human,^208,209 rhesus monkey,²¹⁰ or baboon.²¹¹ Biologically active estradiol is secreted primarily into the maternal circulation, whereas the weaker estrogen, estrone, is preferentially released into the fetal circulation, perhaps to limit exposure of the developing fetus to estrogen. Although a carrier system specific for estradiol has been suggested to explain this selective secretion,²¹⁰ differential localization of the 17β-HSD-1 and -2 enzymes within the syncytiotrophoblast may be a more likely explanation, but remains to be confirmed.

REGULATION

The 3β-HSD, sulfatase, and aromatase enzymes are abundantly expressed in the primate placenta,¹⁹¹ and the production of estradiol and estrone per milligram placental protein remains relatively constant throughout pregnancy in women.²¹² Although cAMP and certain growth factors can modulate estrogen production by transformed trophoblast cell lines in vitro,^213,214 placental estrogen production appears most dependent on the amount of substrate provided by the fetal adrenal gland, uteroplacental blood flow, and placental trophoblast mass.

Role of Uteroplacental Blood Flow

The fraction of circulating maternal DHAS converted to estradiol increases significantly from 5% in the first trimester to more than 35% late in gestation,^190,215 reflecting increases in both placental mass³⁴ and uteroplacental blood flow. ²¹⁶ The metabolic clearance rate (MCR) of DHAS increases from approximately 7L/day in the nonpregnant state to more than 60L/day in the third trimester (Fig. 10).²¹⁵ The influence of uteroplacental blood flow on placental estrogen production has been confirmed by direct experiment. Novy and colleagues^217,218 demonstrated that reductions in uteroplacental perfusion caused by graded reductions in maternal distal aortic blood flow in the pregnant baboon resulted in corresponding decreases in the placental clearance of DHA through estradiol formation. Consistent with these observations in the baboon, the MCR of DHAS is 50% lower in women with pregnancy-induced hypertension, presumably reflecting the marked decrease in intervillous perfusion that characterizes this clinical syndrome.^219,220,221

Fetal Adrenal c19 Steroids

The fetal adrenal is a major source of the c19 steroids required for estrogen production; growth and function of the gland therefore greatly influence the production of estrogen. In fetal baboons, acute stress induced by hypoxemia results in a marked increase in fetal adrenal c19-steroid secretion and results in a corresponding rise in placental estrogen production.²²² As pregnancy advances, the primate fetal adrenal exhibits a marked increase in growth, accompanied by a corresponding increase in umbilical serum levels of DHAS (Fig. 11).^{223,224,225,226} Throughout most of gestation, 75% or more of the human fetal adrenal cortex⁶ consists of the fetal zone, which expresses the P450c17 and P450scc mRNAs and proteins essential for c19 steroid DHA and DHAS production (see Fig. 5). In contrast, the “transitional zone,” which expresses both the 3β-HSD and P450c17 enzymes for cortisol production, and the “definitive zone,” which expresses 3β-HSD but not P450c17 and makes aldosterone,^6,227 do not develop until very late in gestation. A number of studies have endeavored to define the mechanisms controlling growth and differential steroidogenesis in the three zones of the fetal adrenal gland (reviews available elsewhere^1,2,6). Suppression of fetal pituitary ACTH secretion by administration of synthetic glucocorticosteroids (e.g., betamethasone) during mid- to late gestation results in fetal adrenal atrophy in humans,²²⁸ baboons,^229,230 and rhesus monkeys.²³¹ These observations indicate that fetal pituitary ACTH has a critical role in the growth and maintenance of the fetal adrenal in primates. Indeed, fetal anencephaly, accompanied by absence of pituitary ACTH, is associated with fetal adrenal atrophy and a striking decline in mid- to late gestation estrogen production in humans²³² and rhesus monkeys.²³³ Clearly, by stimulating fetal adrenal growth and the overall capacity for c19-steroid formation, fetal ACTH has a major role indirectly in regulating placental estrogen biosynthesis. This tropic action of ACTH appears to be mediated in part by peptide growth factors (e.g., IGF-II, bFGF) in fetal adrenocortical cells.^{234,235,236,237}

ACTH also has an important role in regulating elements of the steroid biosynthetic pathway in the primate fetal adrenal gland. Receptor-mediated uptake of low-density lipoprotein (LDL) cholesterol produced in the fetal liver is a major source of cholesterol substrate for steroidogenesis within the human fetal adrenal.²³⁸ Addition of LDL to cultures of human fetal adrenal cells results in increased production of DHAS and cortisol.^239,240 ACTH has an important role in stimulating the receptor-mediated uptake and degradation of LDL within the human fetal adrenal to provide cholesterol substrate for steroidogenesis.^241,242 In addition, the de novo pathway for the formation of cholesterol may account for up to 30% of the daily secretion of DHAS and cortisol by the human fetal adrenal gland in culture.^243,244 Under these conditions, ACTH stimulates HMG CoA reductase, the rate-limiting enzyme for de novo teroidogenesis.²⁴⁴ The ACTH-stimulated LDL pathway appears to be the preferred mechanism for fueling steroidogenesis in the primate fetal adrenal gland, however, because the HMG CoA reductase enzyme is not stimulated in the presence of ACTH and LDL.

In addition to supplying cholesterol substrate to the fetal adrenal cell, ACTH regulates specific enzymes involved in c19-steroid formation. For example, the mRNAs for and activities of the P450 cholesterol side-chain (P450scc) and P450c17 enzymes, and the hydroxysteroid-sulfotransferase (HST) enzyme that catalyzes the sulfurylation of DHA, are stimulated by ACTH in cultures of human fetal adrenal cells. ^235,245,246 Moreover, betamethasone suppresses and ACTH restores expression of the mRNAs for the ACTH receptor, P450scc and P450c17, and serum estrogen levels in baboons.²²⁹ Thus, by stimulating fetal adrenal c19-steroid formation, fetal ACTH has a pivotal role in regulating placental estrogen biosynthesis during primate pregnancy.

Because estrogen production is only partially reduced after suppression of the fetal hypothalamic pituitary adrenal axis with synthetic cortico-steroids,^{172,247,248,249} it appears that factors other than ACTH may also be important to the regulation of fetal adrenal hormonogenesis. hCG,^250,251 prolactin, growth hormone, and several other peptides, including ACTH, CLIP, and MSH²⁵² stimulate DHAS production by incubates of human fetal adrenal slices. Similar observations have been made in the baboon fetal adrenal both in vitro²⁵³ and in vivo.²⁵⁴ Recently, it has also been shown that placental CRH stimulates DHAS production and P450c17 expression by human fetal adrenal cells through activation of a phospholipase C-inositol second messenger system.²⁵⁵ Placental CRH may also regulate the maternal pituitary-adrenocortical axis. Thus, the progressive increase in maternal plasma CRH levels in human pregnancy (Fig. 12)²⁵⁶ is accompanied by a corresponding rise in the concentrations of maternal ACTH and cortisol (Fig. 13)²⁵⁷ as well as levels of DHAS.²⁵⁸ Interestingly, in cultured human trophoblasts, glucocorticoids increase the expression of CRH,^80,259 and it has been proposed that a positive feed-forward loop involving the maternal pituitary-adrenocortical axis and placental CRH may operate during primate pregnancy (Fig. 14).²⁶⁰ Whether a comparable feed-forward axis operates in the primate fetus, as originally suggested by Robinson and associates,⁸⁰ remains to be determined.

Studies also suggest that estrogen itself modulates fetal and perhaps maternal adrenal steroidogenesis. The primate fetal adrenal gland expresses estrogen receptors α and β at mid- and late gestation thereby providing a mechanism for mediating the action of estrogen.²⁶¹ Estrogen stimulates ACTH-induced DHAS production and inhibits ACTH-induced cortisol synthesis in human fetal adrenal cells in culture.^{6,262,263,264} It has been proposed,⁶ therefore, that the availability of DHAS for placental estrogen production is controlled by a positive feedback loop in which estrogen alone and/or in conjunction with other factors enhances production of precursor DHAS by the fetal zone cells. In contrast to the stimulating effect of estrogen in long-term cultures of human fetal adrenal cells, estrogen inhibits ACTH-induced stimulation of DHA both in vivo²⁶⁵ and in short-term incubates of fetal baboon adrenal cells.²⁶⁶ Moreover, in the absence of any changes in MCR, maternal serum levels and production rates of DHA and DHAS are markedly reduced by estrogen treatment of intact or fetectomized baboons.²⁶⁷ Based on these observations, it has been proposed that placental estrogen feeds back to down-regulate the biosynthesis and secretion of DHA by the maternal and fetal adrenal glands to maintain a physiologically normal balance of estrogen production during primate pregnancy.^2,3

Progesterone Production

CONCENTRATIONS IN THE CIRCULATION

The placenta becomes a significant source of progesterone by approximately weeks 6 to 8 (15% to 20% of gestational length) of human pregnancy.²⁶⁸ In women, serum progesterone concentrations rise in a linear fashion with advancing gestation, ultimately attaining values of 150 to 175 ng/mL at term (Fig. 15).^176,269 Fetal progesterone levels are more than twofold greater than in the mother during human,²⁷⁰ baboon,²⁷¹ and rhesus monkey²⁷² pregnancy, in largest part a reflection of the lower MCR of progesterone in the fetus than in the mother.²⁷² In humans, the placental production rate of progesterone during the third trimester approximates 210 mg/day,²⁷³ a value more than 10-fold greater than in the luteal phase of the menstrual cycle.²⁷⁴ Progesterone levels in women with first-trimester threatened abortion are predictive of pregnancy outcome.¹⁷⁷ Abortion ultimately results in more than 80% of women with progesterone levels below 10 ng/mL; no viable pregnancy is associated with concentrations below 5 ng/mL.^177,275,276 Progesterone concentrations are also typically low in women with ectopic pregnancies.²⁷⁶ In contrast, progesterone levels are elevated in women with hydatidiform mole, particularly between the 10th and 20th weeks of gestation.²⁷⁷ In pregnancies complicated by Rh isoimmunization, progesterone levels are more than twofold higher than normal, presumably reflecting the marked increase in placental mass associated with erythroblastosis.¹⁷⁶ Finally, although maternal progesterone concentrations fall dramatically and levels rise in late gestation before the onset of parturition in rats,²⁷⁸ sheep,²⁷⁹ and other laboratory species,¹⁹¹ no such changes occur in primates including humans,¹⁷⁶ baboons,^{280,281,282,283} and rhesus monkeys.²⁸⁴

BIOSYNTHESIS OF PROGESTERONE—LDL PATHWAY

The primate placenta possesses abundant quantities of the P450scc and 3β-HSD enzymes required to convert substrate cholesterol into progesterone. Unlike estrogen formation, progesterone production does not require direct participation of the fetus; however, in contrast to other steroid-secreting organs, the human and nonhuman primate placenta exhibits a very limited capacity for de novo cholesterol and progesterone synthesis from acetate.^285,286 The elegant work of Simpson and co-workers^285,287 conclusively demonstrated that progesterone biosynthesis in human placental trophoblast cells follows the classical LDL pathway first described by Goldstein and Brown²⁸⁸ in fibroblasts. Earlier studies conducted in pregnant women^289,290 and with perfusion of the human fetoplacental unit in vitro^291,292 showed that cholesterol in the maternal circulation is taken up and used for progesterone formation by the placenta. Moreover, Winkel and associates^287,293 demonstrated that LDL uptake by human trophoblast cells in culture is mediated by high-affinity binding, is saturable and that LDL degradation is a consequence of cellular uptake and internalization. A receptor-mediated pathway, involving coated pits and vesicles, endosomes, and lysosome-like bodies has been identified in human placental trophoblasts for the uptake of colloidal-gold conjugated LDL.²⁹⁴ Henson and colleagues²⁹⁵ have also demonstrated the existence of high-affinity receptor-mediated LDL uptake (i.e., binding and internalization), as well as LDL degradation by trophoblasts isolated from the baboon placenta. High-density lipoprotein also increases progesterone secretion by human trophoblast cultures,^287,296 but to a much lesser extent than LDL.

REGULATION

Role of Estrogen

During rat and rabbit pregnancy, estrogen is the major luteotropic stimulus that maintains the corpus luteum and progesterone production.^297,298,299 Estrogen stimulates the uptake of high-density lipoprotein cholesterol substrate³⁰⁰ and P450scc expression in the rat³⁰¹ and rabbit³⁰² corpus luteum, thereby promoting progesterone production. During mid- to late primate pregnancy, when the placenta is the principal source of progesterone, estrogen has a similar regulatory role within trophoblasts. ^2,3,191 Placental progesterone formation and serum progesterone concentrations are decreased by administration of the estrogen receptor antagonist ethamoxytriphetol^283,303,304 in baboons, an effect that can be reversed by diethylstilbestrol.³⁰⁵ Moreover, placental progesterone production by human trophoblasts in culture is inhibited by treatment with an aromatase inhibitor and restored by estradiol.³⁰⁶ The increase in receptor-mediated LDL uptake³⁰⁷ and LDL receptor³⁰⁸ and P450scc³⁰⁹ mRNA expression in placental trophoblasts observed during the second half of baboon pregnancy when estrogen levels rise, can be suppressed by blocking the action or formation of estrogen.^{304,310,311,312} In contrast, placental 3β-HSD and adrenodoxin mRNA expression and 3β-HSD activity are not developmentally regulated or altered by antiestrogen treatment in baboons.^281,309 Therefore, inhibiting the action or levels of estrogen specifically blocks the developmental increase in placental LDL cholesterol uptake and expression of the P450scc enzyme essential for the metabolism of cholesterol to pregnenolone in baboons.

Effects of cAMP

In cultures of human trophoblast cells, cAMP increases the secretion of progesterone and hCG^313,314 and expression of the mRNAs for P450scc.^234,315 The observation that cAMP stimulates progesterone formation in cytotrophoblasts that are prevented from transforming into syncytiotrophoblasts suggests that the process is not dependent on syncytia formation. Strauss and co-workers have postulated that cAMP activates a protein kinase that phosphorylates a protein(s) integral to gene transcription of specific components of the progesterone pathway.^316,317

Other Factors

Translocation of cholesterol from the outer to the inner mitochondrial membrane accounts for the rapid increase in steroidogenesis within the adrenals and gonads in response to tropic stimulation and is mediated in large part by the steroidogenic acute regulatory protein (StAR; review available elsewhere³¹⁸). However, because human trophoblast cells do not express StAR,³¹⁹ other unique mechanisms may be involved in the intracellular trafficking of cholesterol within the placenta. Recent studies³¹⁷ indicate that the mitochondria of human syncytiotrophoblasts exhibit a morphology that is quite different from cytotrophoblast, adrenal, or gonadal cells. Their unique structure may in some way facilitate cholesterol entry into syncytiotrophoblast mitochondria without the mediation of factors such as StAR.

Various peptide growth factors (e.g., IGF-I and IGF-II) are expressed^320,321 and have stimulatory effects on P450scc activity³²² in human trophoblasts. EGF also has been observed to stimulate progesterone formation in JEG-3 choriocarcinoma cells in culture.^85,323 The relative physiologic significance of peptide growth factors in primate placental progesterone production and their potential interaction with the other factors that have been discussed above remains to be determined.

Estrogen Regulates the Developmental Pattern of Cortisol-Cortisone Metabolism by the Primate Placenta

Although steroid hormones and other lipophilic substances of maternal origin rapidly traverse the human and nonhuman primate placenta,² in most instances these compounds are catabolized to biologically inactive metabolites before entry into the fetal circulation.^{324,325,326,327} With regard to cortisol, it has generally been considered³²⁴ that the major role of the primate placenta was to catabolize biologically active cortisol (i.e., binds to the glucocorticoid receptor) to its inactive (does not bind to receptor) metabolite cortisone. However, in vivo isotope dilution studies in the baboon have shown that in contrast to the relatively constant pattern of cortisol-cortisone metabolism by maternal tissues during both baboon³²⁸ and human pregnancy,³²⁹ the pattern of placental cortisol-cortisone metabolism changes from preferential reduction(formation of cortisol from cortisone) early in pregnancy to oxidation(conversion of cortisol to cortisone) near term.³³⁰ The same may also occur in the human; indirect estimates of placental cortisol metabolism in vivo early in gestation indicate an extensive conversion of cortisone to cortisol across the placenta.³³¹ Moreover, the change in transplacental corticosteroid metabolism is prevented by inhibiting estrogen action in pregnant baboons,³³² and placental oxidation of cortisol to cortisone can be induced prematurely at midgestation by estradiol.³³³ It has therefore been proposed (Fig. 16)³³⁴ that estrogen regulates the ontogenetic change in placental glucocorticoid metabolism.²

Estrogen stimulation of placental oxidation of cortisol also appears to occur in fetal tissues. Conversion of cortisol to cortisone (29%) by the baboon fetus at midgestation³³⁵ increases twofold (60%) by the time of delivery,³³⁶ increases at midgestation (50%) in response to maternal estrogen administration,³³⁷ and decreases at term (21%)in neonates delivered to mothers treated with antiestrogen.³³⁸ Although the transfer constant measured in peripheral blood represents metabolic contributions of several individual organ systems, one of the sites of estrogen action appears to be the kidney.³³⁹

The interconversion of cortisol and cortisone is catalyzed by two 11β-hydroxysteroid dehydrogenase (11β-HSD-1 and -2) enzymes that are the product of two different genes.^340,341 In the human and baboon placenta, the mRNA and protein for both 11β-HSD-1 and -2 are primarily expressed in the syncytiotrophoblast.^{334,342,343,344} Recently, it was demonstrated that the mRNA and protein levels of 11β-HSD-1 and -2,³³⁴ and the activity of 11β-HSD oxidase,³⁴⁵ in syncytiotrophoblast increases with advancing gestation and that upregulation may involve a direct estrogen receptor-dependent action of estrogen on the promoters of these two genes.³⁴⁶ Estrogen has also recently been shown to regulate the levels of 11β-HSD-1 and -2 protein in rat uterine endometrium.³⁴⁷ It has been proposed, therefore, that estradiol acts through its receptor to regulate the functional maturation of baboon syncytiotrophoblast that is further manifested by an increase in the genomic expression of the 11β-HSD enzymes controlling cortisol-cortisone interconversion (see Fig. 16).

Although the estrogen-dependent up-regulation of 11β-HSD-2 is consistent with increased transplacental oxidation of cortisol to cortisone at term,³³⁰ the concomitant up-regulation of 11β-HSD-1 is surprising since the reduction of cortisone to cortisol across the placenta declined with advancing gestation; however, because the syncytiotrophoblast is a polarized cell, it is possible that once the levels of 11β-HSD-1 and -2 are increased by estrogen, the syncytiotrophoblast may undergo further development resulting in a spatial compartmentalization and functional separation of the two 11β-HSD enzymes.^334,348 For example, Burton and associates ³⁴⁷ have shown that 11β-HSD-1 and -2 exhibit marked differences in their expression between basal and labyrinth zones of the rat placenta.

Regulation of the Fetal Pituitary Adrenocortical Axis by Estrogen-Induced Changes in Placental Cortisol-Cortisone Metabolism

Because the transition zone of the primate fetal adrenal develops relatively late in gestation (see^1,6 for review), the fetal adrenal gland in human^349,350 and nonhuman primates ^351,352,353 does not synthesize cortisol de novo for most of intrauterine development. In contrast, it appears that the baboon hypothalamus^354,355 and pituitary gland³⁵⁶ develop relatively early in gestation, as previously shown in the human. ^357,358 Thus, development of the fetal adrenal appears to be “out-of-phase” with that of the hypothalamus and pituitary, and yet by term, the pituitary adrenocortical axis has become functionally integrated. Studies in the baboon, however, have demonstrated that at midgestation essentially all of the cortisol measured in the fetus is of maternal origin, whereas at term less than 50% derives from the mother.³³⁰ Estrogen-dependent regulation of placental metabolism of maternal cortisol and cortisone is an integral step in the sequence of events that control development of the fetal pituitary adrenocortical axis(see Fig. 16). Because placental glucocorticoid metabolism during early to midgestation favors reduction of cortisone to cortisol, the primary maternal corticosteroid reaching the fetal circulation appears to be biologically active cortisol that would inhibit fetal ACTH production and thus limit growth and maturation of the fetal adrenal transitional zone.^1,2 With advancing gestation, the estrogen-induced increase in placental 11β-HSD- catalyzed oxidation of cortisol to cortisone would decrease the concentration of maternal cortisol in the fetus causing activation of fetal pituitary ACTH production culminating in maturation of the fetal adrenal gland. Moreover, the concomitant increase in oxidation of cortisol to cortisone within the fetus could act, in concert with increased placental oxidation of maternal stores, to ensure continued synthesis and release of ACTH by the fetal pituitary gland.^{1,337,338,339} In support of this hypothesis, fetal pituitary ACTH expression,³⁵⁹ the specific activities of rate limiting enzymes,³⁶⁰ and the ontogenesis of de novo fetal baboon adrenal cortisol production³⁶¹ increase with advancing gestation and can be induced prematurely at midgestation by estrogen induction of placental NAD-dependent 11β-HSD oxidase activity.³⁴⁵

In humans, bipotential gonads develop as stratifications of the coelomic epithelium at approximately week 4 of gestation. Although most cell types comprising the gonads are derived from mesoderm of the urogenital ridges,¹⁰ primordial germ cells originate from ectoderm of the inner cell mass³⁶² and migrate to the coelomic epithelium of the gonadal ridges.^363,364,365 By week 5 of gestation, the bipotential gonad consists of germ cells, supporting cells of the coelomic epithelium that give rise to testicular Sertoli cells or ovarian granulosa, and stromal/interstitial cells of mesenchymal origin.¹⁰ It appears that two XX chromosomes are required for differentiation of the bipotential gonad into an ovary, whereas a single Y chromosome (e.g., 46XY) encoding the Sry gene and Sry-related transcription factors (e.g., SOX9) are necessary for testicular development.^366,367,368 Subsequent development of the gonads continues throughout gestation with histologic differentiation of the fetal testes preceding that of the ovary.^369,370

In the male, Leydig cells rapidly proliferate between weeks 12 and 18 of gestation,^369,371 accompanied by increasing fetal testicular production and fetal serum testosterone levels (Fig. 17).³⁷² At the same time, Sertoli cells produce androgen-binding protein (ABP)³⁷³ and the protein hormone müllerian-inhibiting hormone (MIH) in the developing seminiferous tubules.^374,375 Jost demonstrated that castration of rabbit embryos of either sex resulted in the development of the female phenotype,^376,377 and thereby established that the female urogenital tract develops spontaneously, whereas secretions of the fetal testes were necessary for male development. Presumably, testosterone binds to ABP to create a locally high concentration of androgen that induces differentiation of the Wolffian duct, whereas MIH causes the demise of the múllerian primordia. Differentiation of the external genitalia/prostate into the male phenotype is induced by testosterone secreted into the fetal circulation that is subsequently converted to 5α-dihydrotestosterone(DHT) by the enzyme 5α-reductase type I³⁷⁸ in the primordia of the external genitalia and by 5α-reductase type II in the prostate.^{379,380,381,382} Androgen treatment of female embryos induces male development of the internal and external genitalia,³⁸³ whereas administration of antiandrogenic agents during embryogenesis impairs male development.^384,385 The female fetus is usually protected from testosterone and androstenedione of maternal origin,^386,387 presumably because these androgens are either catabolized by the placenta and/or fetal liver,³⁸⁸ and/or are converted to estrogens by placental aromatase.^188,389

Testosterone synthesis in the fetal testes appears to be regulated in large part by increasing levels of placental hCG³⁸² and perhaps by fetal pituitary FSH/LH, levels of which peak at approximately weeks 12 to 20 of human pregnancy (see Fig. 17). Observations that testosterone synthesis begins before any significant increase in fetal gonadotropin levels³⁷² and that LH (i.e., hCG) receptors are detected in fetal testicular tissues suggests an important role for hCG.^390,391 The observation that male pseudohermaphroditism can result from a defect in testicular gonadotropin receptors provides further evidence.³⁹² LDL cholesterol provides the substrate for fetal testicular steroidogenesis,³⁹³ and LDL receptor levels decline after week 20 of gestation in association with the decrease in fetal serum levels of testosterone. Finally, the fetal testes expresses the mRNAs for P450scc, P450c17, and 3β-HSD, all required for formation of testosterone.^11,394

Histologic development of the fetal ovary is strikingly similar in humans and other nonhuman primates, such as rhesus monkeys.^369,395,396 In both species, mitotic activity in oogonia is maximal at approximately 45%of gestation, with the number of germ cells falling substantially thereafter. Meiosis is initiated in the largest oogonia (now called oocytes)by the end of the first third of gestation with more germ cells entering meiosis and proceeding through leptotene, zygotene, and pachytene stages as gestation advances (Fig. 18).³⁹⁷ As early as week 10 of gestation, human oocytes become surrounded by a single layer of presumptive granulosa cells, the unit now termed a primordial follicle. Ohno and Smith³⁹⁸ demonstrated that many oocytes regress during the early stages of meiosis unless enveloped by granulosa cells once they have entered the diplotene stage. At approximately 40% of gestation, the process of follicular development intensifies with maturation and subsequent development of primary follicles (i.e., flattened granulosa cells become cuboidal and begin to divide) occurring principally in the medullary (inner)component of the ovary. Preantral follicles containing an enlarged oocyte with a zona pellucida, multiple layers of granulosa cells, and a theca cell layer that originates from the stroma occasionally develop at approximately the 6th to 8th month of gestation in humans^11,399 and near term in nonhuman primates.^399,400 Finally, it has been shown that the number of degenerating oocytes increases rapidly during folliculogenesis, primarily at the expense of diplotene oocytes. By term, the human and nonhuman primate ovary is thus composed of a relatively wide cortex filled with oocytes and several primordial and growing primary follicles, including preantral follicles primarily located in the inner medullary region. Although the histologic features of fetal ovary development are well defined, our current understanding of the factors that regulate this process remains incomplete.

Throughout most of gestation, the uterus remains relatively quiescent. Although uterine contractions occur, they are of low amplitude, not synchronized, and very little uterine pressure is developed. This pattern of uterine activity generally classified as Braxton Hicks contractions in humans, and contractures in baboons, rhesus monkeys, and sheep^401,402 has been termed phase 0 (Fig. 19)⁴⁰³ of the parturition process.^12,404,405 At term, the uterus demonstrates well-coordinated contractions. The myometrium becomes highly excitable and generates contractions that increase in both frequency and amplitude. Transition from a quiescent to an active myometrium is termed activation or phase 1. During phase 2, an activated myometrium becomes increasingly responsive to various stimulatory factors.^405,406,407 In this sequence, the initiation of parturition corresponds to the transition from phase 0 to phase 1 or from uterine quiescence to uterine activation (see Fig. 19).

Progesterone is generally considered to be a gene suppressor and indeed does down-regulate a number of genes that are considered essential for parturition, including the gap junction protein connexin 43.^408,409 Moreover, progesterone alone and/or in conjunction with estradiol has been shown to restrain uterine contractility during pregnancy by (1) controlling calcium uptake/availability from external stores through both calcium channels and fast sodium channels; (2) mobilization of calcium from internal stores by regulation of α-1-adrenergic function; (3) removal of calcium from the uterine cytosol; and (4) coupling of β2-receptor to adenylate cyclase and the activation of protein kinase A to reduce calcium-calmodulin binding to myosin-light chain kinase (MLCK) and thus phosphorylation and activity of MLCK.^2,12 In species in which progesterone withdrawal normally precedes labor (e.g., rats, rabbits, sheep), administration of progesterone prevents delivery,⁴¹⁰ whereas treatment with antiprogestins late in gestation results in premature parturition similar to spontaneous labor induction. In contrast, in species in which progesterone production does not decline prior to labor (e.g., nonhuman primates and humans), exogenous progesterone does not prevent parturition at term⁴¹¹; however, treatment with the progesterone receptor antagonist RU-486 does lead to increased uterine activity and induction of premature labor.⁴¹² Moreover, suppression of progesterone production in late rhesus monkey gestation causes premature vaginal delivery that can be prevented by treatment with progesterone.⁴¹¹ Treatment with antiprogestins also increases myometrial responsiveness to contractile agents but does not necessarily result in preterm birth.⁴¹³ For example, in monkeys, although antiprogestin treatment alone during late gestation does not induce labor, concomitant administration of oxytocin facilitates parturition.^414,415 As discussed earlier, estrogen is also essential for maintenance of pregnancy¹⁷⁹ and presumably, therefore, phase 0 of uterine function. In baboons, suppression of placental estradiol production by treatment with an aromatase inhibitor between early and midgestation was associated with a high rate of miscarriage, effects prevented by concomitant treatment with estradiol.

Nitric oxide (NO) is a potent endogenous muscle relaxant that elicits its actions through cyclic guanosine monophosphate and calcium channels.⁴⁰⁹ The mRNA levels of inducible nitric oxide synthase(iNOS) are highest in myometrium of preterm human patients not in labor;mRNA and protein levels decrease in term myometrium.⁴¹⁶ Moreover, iNOS activity in myometrium of a variety of species prior to parturition appears to decrease progressively.⁴¹⁶ Therefore, several investigators have proposed that NO acts in a paracrine manner alone and/or in conjunction with progesterone to maintain uterine quiescence during pregnancy.⁴⁰⁹

Relaxin may have a dual role in pregnancy by inhibiting myometrial contractility⁴¹⁷ and regulating changes in cervical connective tissue.⁴¹⁸ Relaxin can suppress spontaneous uterine contractility in the rat,⁴¹⁷ and expression of relaxin is up-regulated in patients with premature rupture of membranes.⁴¹⁹ Relaxin receptors are localized to decidua and chorionic trophoblast cells, and relaxin acts through these receptors to up-regulate various metalloproteinases.⁴¹⁹ In the cervix, relaxin also enhances expression of metalloproteinses-1 and -3 and inhibits the activity of tissue inhibitor of metalloproteinases.⁴¹⁸

As discussed by Challis,^12,403 activation of the myometrium in phase 1 is associated with up-regulation of several contraction-associated proteins (CAPs) including connexin-43, oxytocin receptor, prostaglandin F receptor, and various ion channel proteins. It appears that uterine stretch as modified by the hormonal milieu is critical. Connexin-43 and oxytocin receptor mRNA expression are increased in rat myometrium after initiation of a quick stretch response.^407,420 Significantly, this response to stretch is diminished in rats treated with progesterone and in unilateral pregnant rats in which serum progesterone levels are still relatively high. Although these observations implicate the increase in fetal growth to induction of CAPs and may explain the gradual increase in connexin-43 mRNA levels in the human uterus with advancing gestation,¹² they further highlight the important role of progesterone in maintaining uterine quiescence. Recent observations indicate that the biochemical changes occurring in the uterus during phase 1 are associated with and are perhaps regulated by increased activity of the fetal pituitary-adrenocortical axis (i.e., increased production of cortisol and DHA/DHAS by the developing fetal adrenal gland).^2,12 Increased production of estrogen^421,422 and withdrawal of the production and/or action of progesterone appear to be important mediators of phase 1.^12,403

In phase 2, increased production of the uterotonins oxytocin and prostaglandins stimulates the uterus that was activated in phase 1. In humans, the likely source of prostaglandins that stimulate myometrial contractility is the decidua and/or the myometrium because the level of the prostaglandin-catabolizing enzyme prostaglandin dehydrogenase (PGDH) in trophoblast chorion is sufficiently high to prevent movement of bioactive prostaglandins produced in the amnion to the myometrium.⁴⁰³

In animals such as the sheep and goat, parturition is initiated by activation of the fetal hypothalamic-pituitary-adrenocortical axis⁴²³ that results in increased production of cortisol. Thus, lesions in the paraventricular nucleus, hypophysectomy, or adrenalectomy of the sheep fetus prolong gestation, whereas infusion of ACTH or glucocorticoid into the fetus induces premature parturition. Fetal-derived cortisol apparently acts on the sheep placenta to induce P450c17 expression that results in decreased output of progesterone, a rise in estrogen, and subsequently, a marked increase in prostaglandin production by intrauterine tissues.^424,425

The role of the human fetus in initiating parturition is not as well defined. In anencephalics and infants with other abnormalities that prevent cortisol synthesis, parturition is not significantly delayed⁴²⁶ ;however, in the rhesus monkey fetus, there is an increase in the distribution of both premature and postmature births after adrenalectomy⁴²⁷ or experimental anencephaly.²³³ Treatment of the monkey fetus with dexamethasone does not induce premature labor²³¹ ; however, chronic infusion of androstenedione to pregnant rhesus monkeys increases estrogen production and results in premature birth,⁴²² effects that are blocked by co-administration of an aromatase inhibitor.⁴²⁸ Interference with the conversion of androgen to estrogen locally within the placenta also blocks the patterns of myometrial contractility induced by administration of precursor androstenedione alone.⁴²⁸ In primates, androgens produced by the fetal adrenal as a source of substrate for placental estrogen synthesis may represent a link between the fetus and mother in the initiation of parturition.¹² The role of cortisol appears more tenuous. In the human, glucocorticoids have the capacity to increase PGDH activity in the chorion⁴²⁹ and thereby to increase prostaglandin production. As discussed earlier, it has been suggested that placental CRH, production of which is upregulated by cortisol,⁴³⁰ may play a role in the onset of labor in humans.⁴⁰³ Indeed, maternal CRH levels are increased in women with preterm labor,^431,432 and at 26 to 28 weeks of gestation, elevated maternal CRH levels may discriminate patients with apparent premature labor who go on to deliver within 24 to 48 hours from those who do not.⁴³³ CRH receptors are present in the myometrium and fetal membranes.⁴³⁴ CRH also stimulates the release of prostaglandins from human decidua and amnion in vitro⁴³⁵ and can potentiate the action of oxytocin and prostaglandins both in vitro^436,437 and in vivo.⁴³⁸ Thus, by regulating production of placental CRH and trophoblast chorion PGDH activity, fetal-derived cortisol may play a significant role in phase 1 and/or 2 of uterine contractility integral to the onset and progression of labor.

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