Williams Obstetrics, 24th Edition

CHAPTER 7. Embryogenesis and Fetal Morphological Development

GESTATIONAL AGE VARIOUSLY DEFINED

EMBRYO-FETAL GROWTH AND DEVELOPMENT

PLACENTAL PHYSIOLOGY AND FETAL GROWTH

FETAL NUTRITION

FETAL ORGAN SYSTEM DEVELOPMENT

DEVELOPMENT OF GENITALIA

Contemporary obstetrics includes physiology and pathophysiology of the fetus, its development, and its environment. An important result is that fetal status has been elevated to that of a patient who, in large measure, can be given the same meticulous care that obstetricians provide for pregnant women. Normal fetal development is considered in this chapter. Anomalies, injuries, and diseases that affect the fetus and newborn are addressed in Chapter 33 and others.

GESTATIONAL AGE VARIOUSLY DEFINED

Several terms are used to define pregnancy duration, and thus fetal age (Fig. 7-1). Gestational age or menstrual age is the time elapsed since the first day of the last menstrual period, a time that actually precedes conception. This starting time, which is usually about 2 weeks before ovulation and fertilization and nearly 3 weeks before blastocyst implantation, has traditionally been used because most women know their last period. Embryologists describe embryo-fetal development in ovulation age, or the time in days or weeks from ovulation. Another term is postconceptional age, nearly identical to ovulation age.

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Figure 7-1 Terminology used to describe the pregnancy duration.

Clinicians customarily calculate gestational age as menstrual age. Approximately 280 days, or 40 weeks, elapse on average between the first day of the last menstrual period and the birth. This corresponds to 9 and 1/3 calendar months. A quick estimate of a pregnancy due date based on menstrual data can be made as follows: add 7 days to the first day of the last period and subtract 3 months. For example, if the first day of the last menses was July 5, the due date is 07–05 minus 3 (months) plus 7 (days) = 04–12, or April 12 of the following year. This calculation has been termed Naegele rule. Many women undergo first- or early second-trimester sonographic examination to confirm gestational age. In these cases, the sonographic estimate is usually a few days later than that determined by the last period. To rectify this inconsistency—and to reduce the number of pregnancies diagnosed as postterm—some have suggested assuming that the average pregnancy is actually 283 days long and that 10 days be added to the last menses instead of 7 (Olsen, 1998). The period of gestation can also be divided into three units, each 13 to 14 weeks long. These three trimesters are important obstetrical milestones.

EMBRYO-FETAL GROWTH AND DEVELOPMENT

The complexity of embryo-fetal development is almost beyond comprehension. Shown in Figure 7-2 is a schematic sequence of various organ systems as they develop. New information regarding organ development continues to accrue using modern technologies. For example, imaging techniques help evaluate the role of gene regulation and tissue interaction on eventual 3-dimensional organ morphology (Mohun, 2011). And, Williams and colleagues (2009) described the sequence of gene activation that underlies cardiac development.

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Figure 7-2 Embryo-fetal development according to gestational age determined by the first day of the last menses. Times are approximate.

Image Zygote and Blastocyst Development

During the first 2 weeks after ovulation and then fertilization, the zygote develops to the blastocyst stage, which undergoes implantation 6 or 7 days following fertilization. The 58-cell blastocyst differentiates into five embryo-producing cells—the inner cell mass—and the remaining 53 cells form the placental trophoblast. Details of implantation and early development of the blastocyst and placenta are described in Chapter 5 (p. 88).

image Embryonic Period

The conceptus is termed an embryo at the beginning of the third week after ovulation and fertilization. Primitive chorionic villi form, and this coincides with the expected day of menses. The embryonic period lasts 8 weeks, during which organogenesis takes place. The embryonic disc is well defined, and most pregnancy tests that measure human chorionic gonadotropin (hCG) become positive by this time. As shown in Figures 7-3 and 7-4, the body stalk is now differentiated, and the chorionic sac is approximately 1 cm in diameter. There are villous cores in which angioblastic chorionic mesoderm can be distinguished and a true intervillous space that contains maternal blood. During the third week, fetal blood vessels in the chorionic villi appear. In the fourth week, a cardiovascular system has formed, and thereby, a true circulation is established both within the embryo and between the embryo and the chorionic villi. By the end of the fourth week, the chorionic sac is 2 to 3 cm in diameter, and the embryo is 4 to 5 mm in length (Figs. 7-5 and 7-6). Partitioning of the primitive heart begins in the middle of the fourth week. Arm and leg buds are present, and the amnion is beginning to unsheathe the body stalk, which thereafter becomes the umbilical cord.

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FIGURE 7-3 Early human embryos. Ovulation ages: A. 19 days (presomite). B. 21 days (7 somites). C. 22 days (17 somites). (After drawings and models in the Carnegie Institute.)

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FIGURE 7-4 Drawing of an 18-day Mateer-Streeter embryo shows the amnionic cavity and its relations to chorion and yolk sac. (Redrawn from Streeter, 1920.)

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FIGURE 7-5 Three- to four-week-old embryos. A, B. Dorsal views of embryos during 22 to 23 days of development showing 8 and 12 somites, respectively. C–E. Lateral views of embryos during 24 to 28 days, showing 16, 27, and 33 somites, respectively. (Redrawn from Moore, 1988.)

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Figure 7-6 Embryo photographs. A. Dorsal view of an embryo at 24 to 26 days and corresponding to Figure 7-5C. B. Lateral view of an embryo at 28 days and corresponding to Figure 7-5E. C. Lateral view of embryo-fetus at 56 days, which marks the end of the embryonic period and the beginning of the fetal period. The liver is within the fine, white circle. (From Werth, 2002, with permission.)

At the end of the sixth week, the embryo is 22 to 24 mm long, and the head is large compared with the trunk. The earliest synapses in the spinal cord develop at 6 to 7 weeks (Kadic, 2012). The heart is completely formed. Fingers and toes are present, and the arms bend at the elbows. The upper lip is complete, and the external ears form definitive elevations on either side of the head. Three-dimensional images and videos of human embryos from the MultiDimensional Human Embryo project are found at: http://embryo.soad.umich.edu/.

image Fetal Period

Transition from the embryonic period to the fetal period is arbitrarily designated by most embryologists to begin 8 weeks after fertilization—or 10 weeks after onset of last menses. At this time, the embryo-fetus is nearly 4 cm long (see Fig. 7-6C).

Development during the fetal period consists of growth and maturation of structures that were formed during the embryonic period. Crown-to-rump measurements, which correspond to the sitting height, are most accurate for dating (Table 7-1).

TABLE 7-1. Criteria for Estimating Age During the Fetal Period

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12 Gestational Weeks

The uterus usually is just palpable above the symphysis pubis, and the fetal crown-rump length is 6 to 7 cm. Centers of ossification have appeared in most fetal bones, and the fingers and toes have become differentiated. Skin and nails have developed, and scattered rudiments of hair appear. The external genitalia are beginning to show definitive signs of male or female gender. The fetus begins to make spontaneous movements.

16 Gestational Weeks

The fetal crown-rump length is 12 cm, and the weight is 110 g. By 14 weeks, gender can be determined by experienced observers by inspection of the external genitalia. Eye movements begin at 16 to 18 weeks, coinciding with midbrain maturation.

20 Gestational Weeks

This is the midpoint of pregnancy as estimated from the beginning of the last menses. The fetus now weighs somewhat more than 300 g, and weight increases in a linear manner. From this point onward, the fetus moves about every minute and is active 10 to 30 percent of the time (DiPietro, 2005). The fetal skin has become less transparent, a downy lanugo covers its entire body, and some scalp hair has developed. Cochlear function develops between 22 and 25 weeks, and its maturation continues for six months after delivery.

24 Gestational Weeks

The fetus now weighs approximately 630 g. The skin is characteristically wrinkled, and fat deposition begins. The head is still comparatively large, and eyebrows and eyelashes are usually recognizable. The canalicular period of lung development, during which the bronchi and bronchioles enlarge and alveolar ducts develop, is nearly completed. A fetus born at this time will attempt to breathe, but many will die because the terminal sacs, required for gas exchange, have not yet formed. By 26 weeks, nociceptors are present over all the body, and the neural pain system is developed (Kadic, 2012).

28 Gestational Weeks

The crown-rump length is approximately 25 cm, and the fetus weighs about 1100 g. The thin skin is red and covered with vernix caseosa. The pupillary membrane has just disappeared from the eyes. Isolated eye blinking peaks at 28 weeks. The otherwise normal neonate born at this age has a 90-percent chance of survival without physical or neurological impairment.

32 and 36 Gestational Weeks

At 32 weeks, the fetus has attained a crown-rump length of about 28 cm and a weight of approximately 1800 g. The skin surface is still red and wrinkled. In contrast, by 36 weeks, the fetal crown-rump length averages about 32 cm, and the weight is approximately 2500 g. Because of subcutaneous fat deposition, the body has become more rotund, and the previous wrinkled facial appearance has been lost.

40 Gestational Weeks

This is considered term from the onset of the last menstrual period. The fetus is now fully developed. The average crown-rump length is about 36 cm, and the weight is approximately 3400 g.

PLACENTAL PHYSIOLOGY AND FETAL GROWTH

The placenta is the organ of transfer between mother and fetus. At this maternal-fetal interface, there is transfer of oxygen and nutrients from the mother to the fetus and carbon dioxide and metabolic wastes from fetus to mother. There are no direct communications between fetal blood, which is contained in the fetal capillaries of the chorionic villi, and maternal blood, which remains in the intervillous space. Instead, bidirectional transfer depends on the processes that permit or aid the transport through the syncytiotrophoblast that line chorionic villi.

That said, there are occasional breaks in the chorionic villi, which permit escape of fetal cells into the maternal circulation. This leakage is the mechanism by which some D-negative women become sensitized by the erythrocytes of their D-positive fetus (Chap. 15p. 306). It can also lead to chimerism from entrance of allogeneic fetal cells, including trophoblast, into maternal blood (Sunami, 2010). These are estimated to range from 1 to 6 cells/mL around midpregnancy, and some are “immortal” (Lissauer, 2007). A clinical corollary is that some maternal autoimmune diseases may be provoked by such chimerism (Bloch, 2011; Boyon, 2011). This is also discussed in Chapter 59 (p. 1168).

image The Intervillous Space

Maternal blood within the intervillous space is the primary unit of maternal–fetal transfer. Blood from the maternal spiral arteries directly bathes the trophoblasts. Substances transferred from mother to fetus first enter the intervillous space and are then transported to the syncytiotrophoblast. Thus, the chorionic villi and intervillous space function together as the fetal lung, gastrointestinal tract, and kidney.

Circulation within the intervillous space is described in Chapter 5 (p. 95). Intervillous and uteroplacental blood flow increases throughout the first trimester of normal pregnancies (Mercé, 2009). At term, the residual volume of the intervillous space measures about 140 mL. Before delivery, however, the volume of this space may be twice this value (Aherne, 1966). Uteroplacental blood flow near term has been estimated to be 700 to 900 mL/min, with most of the blood apparently going to the intervillous space.

Active labor contractions reduce blood flow into the intervillous space. The degree of reduction depends on the contraction intensity. Blood pressure within the intervillous space is significantly less than uterine arterial pressure, but somewhat greater than venous pressure. The latter, in turn, varies depending on several factors, including maternal position. When supine, for example, pressure in the lower part of the inferior vena cava is elevated, and consequently, pressure in the uterine and ovarian veins, and in turn in the intervillous space, is increased.

image Placental Transfer

Substances that pass from maternal to fetal blood must first traverse the syncytiotrophoblast, then villous stroma, and finally, the fetal capillary wall. Although this histological barrier separates maternal and fetal circulations, it is not a simple physical barrier. First, throughout pregnancy, syncytiotrophoblast actively or passively permits, facilitates, and adjusts the amount and rate of substance transfer to the fetus. The maternal-facing syncytiotrophoblast surface is characterized by a complex microvillous structure. The fetal-facing basal cell membrane is the site of transfer to the intravillous space. Finally, the villous capillaries are an additional site for transport from the intravillous space into fetal blood, or vice versa. In determining the effectiveness of the human placenta as an organ of transfer, at least 10 variables are important, as shown in Table 7-2 and described next.

TABLE 7-2. Variables of Maternal-Fetal Substance Transfer


Maternal plasma concentration and carrier-protein binding of the substance

Maternal blood flow rate through the intervillous space

Trophoblast surface area size available for exchange

Physical trophoblast properties to permit simple diffusion

Trophoblast biochemical machinery for active transport

Substance metabolism by the placenta during transfer

Fetal intervillous capillary surface area size for exchange

Fetal blood concentration of the substance

Specific binding or carrier proteins in the fetal or maternal circulation

Villous capillary blood flow rate

Mechanisms of Transfer

Most substances with a molecular mass less than 500 Da pass readily through placental tissue by simple diffusion. These include oxygen, carbon dioxide, water, most electrolytes, and anesthetic gases (Carter, 2009). Some low-molecular-weight compounds undergo transfer facilitated by syncytiotrophoblast. These are usually those that have low concentrations in maternal plasma but are essential for normal fetal development.

Insulin, steroid hormones, and thyroid hormones cross the placenta, but very slowly. The hormones synthesized in situ in the trophoblasts enter both the maternal and fetal circulations, but not equally (Chap. 5p. 101). Examples are concentrations of chorionic gonadotropin and placental lactogen, which are much lower in fetal plasma than in maternal plasma. Substances of high molecular weight usually do not traverse the placenta, but there are important exceptions. One is immunoglobulin G—molecular weight 160,000 Da—which is transferred by way of a specific trophoblast receptor-mediated mechanism.

Transfer of Oxygen and Carbon Dioxide

Placental oxygen transfer is blood-flow limited. Using estimated uteroplacental blood flow, Longo (1991) calculated oxygen delivery to be approximately 8 mL O2/min/kg of fetal weight. Normal values for oxygen, carbon dioxide, and pH in fetal blood are presented in Figure 7-7. Because of the continuous passage of oxygen from maternal blood in the intervillous space to the fetus, its oxygen saturation resembles that in maternal capillaries. The average oxygen saturation of intervillous blood is estimated to be 65 to 75 percent, with a partial pressure (Po2) of 30 to 35 mm Hg. The oxygen saturation of umbilical vein blood is similar, but with a somewhat lower oxygen partial pressure.

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FIGURE 7-7 Umbilical venous oxygen pressure (Po2(A); carbon dioxide pressure (Pco2(B); and pH (C) from cordocentesis performed in fetuses being evaluated for possible intrauterine infections or hemolysis, but who were found to be healthy at birth and appropriately grown. (From Ramsay, 1996, with permission.)

The placenta is highly permeable to carbon dioxide, which traverses the chorionic villus by diffusion more rapidly than oxygen. Near term, the partial pressure of carbon dioxide (Pco2) in the umbilical arteries averages about 50 mm Hg, or approximately 5 mm Hg more than in the maternal intervillous blood. Fetal blood has less affinity for carbon dioxide than does maternal blood, thereby favoring carbon dioxide transfer from fetus to mother. Also, mild maternal hyperventilation results in a fall in Pco2 levels, favoring a transfer of carbon dioxide from the fetal compartment to maternal blood (Chap. 4p. 63).

Selective Transfer and Facilitated Diffusion

Although simple diffusion is an important method of placental transfer, the trophoblast and chorionic villus unit demonstrate enormous selectivity in transfer. This results in different metabolite concentrations on the two sides of the villus. Importantly, the levels of many substances that are not synthesized by the fetus are several times higher in fetal than in maternal blood. Ascorbic acid is one example. This relatively low-molecular-weight substance might be expected to traverse the placenta by simple diffusion. The concentration of ascorbic acid, however, is two to four times higher in fetal plasma than in maternal plasma (Morriss, 1994). Another example is the unidirectional transfer of iron. Typically, the mother’s plasma iron concentration is much lower than that of her fetus. Even with severe maternal iron deficiency anemia, the fetal hemoglobin mass is normal.

FETAL NUTRITION

Because of the small amount of yolk in the human ovum, growth of the embryo-fetus is dependent on maternal nutrients during the first 2 months. During the first few days after implantation, blastocyst nutrition comes from the interstitial fluid of the endometrium and the surrounding maternal tissue.

Maternal adaptations to store and transfer nutrients to the fetus are discussed in Chapter 4 and summarized here. Three major maternal storage depots—the liver, muscle, and adipose tissue—and the storage-hormone insulin are intimately involved in the metabolism of the nutrients absorbed from the maternal gut.

Insulin secretion is sustained by increased serum levels of glucose and amino acids. The net effect is storage of glucose as glycogen primarily in liver and muscle, retention of some amino acids as protein, and storage of the excess as fat. Storage of maternal fat peaks in the second trimester and then declines as fetal energy demands increase in the third trimester (Pipe, 1979). Interestingly, the placenta appears to act as a nutrient sensor, altering transport based on the maternal supply and environmental stimuli (Fowden, 2006; Jansson, 2006b).

During times of fasting, glucose is released from glycogen, but maternal glycogen stores cannot provide an adequate amount of glucose to meet requirements for maternal energy and fetal growth. Augmentation is provided by cleavage of triacylglycerols, stored in adipose tissue, which result in free fatty acids and activation of lipolysis.

image Glucose and Fetal Growth

Although dependent on the mother for nutrition, the fetus also actively participates in providing for its own nutrition. At midpregnancy, fetal glucose concentration is independent of and may exceed maternal levels (Bozzetti, 1988). Glucose is the major nutrient for fetal growth and energy. Logically, mechanisms exist during pregnancy to minimize maternal glucose use so that the limited maternal supply is available to the fetus. Human placental lactogen (hPL), a hormone normally abundant in the mother but not the fetus, is believed to block peripheral uptake and use of glucose, while promoting mobilization and use of free fatty acids by maternal tissues (Chap. 5p. 104).

Glucose Transport

The transfer of d-glucose across cell membranes is accomplished by a carrier-mediated, stereospecific, nonconcentrating process of facilitated diffusion. There are 14 glucose transport proteins (GLUTs) encoded by the SLC2A gene family and characterized by tissue-specific distribution (Leonce, 2006). GLUT-1 and GLUT-3 primarily facilitate glucose uptake by the placenta and are located in the plasma membrane of the syncytiotrophoblast microvilli (Korgun, 2005). DNA methylation regulates expression of placental GLUT genes, with epigenetic modification across gestation (Novakovic, 2013). GLUT-1 expression is essential for decidualization. It increases as pregnancy advances and is induced by almost all growth factors (Frolova, 2011; Sakata, 1995).

Lactate is a product of glucose metabolism and is also transported across the placenta by facilitated diffusion. By way of cotransport with hydrogen ions, lactate is probably transported as lactic acid.

Glucose and Insulin Role in Fetal Macrosomia

The precise biomolecular events in the pathophysiology of fetal macrosomia are not defined. Nonetheless, it seems clear that fetal hyperinsulinemia is one driving force (Schwartz, 1994). As discussed in Chapter 44 (p. 872), insulin-like growth factor and fibroblast growth factor are important regulators of placental development and function (Forbes, 2010; Giudice, 1995). In addition, recent work has shown that placental corticotropin-releasing hormone (CRH) stimulates trophoblastic production of GLUT-1 and inhibits expression of GLUT-3 through interaction on the CRH-R1 receptor. This suggests a role for CRH in the nutritional regulation of fetal growth and development (Gao, 2012).

image Leptin

This polypeptide hormone was originally identified as a product of adipocytes and a regulator of energy homeostasis. It also contributes to angiogenesis, hemopoiesis, osteogenesis, pulmonary maturation, and neuroendocrine, immune, and reproductive functions (Henson, 2006; Maymó, 2009). Leptin is produced by the mother, fetus, and placenta. It is expressed in syncytiotrophoblast and fetal vascular endothelial cells. Of placental production, 5 percent enters fetal circulation, whereas 95 percent is transferred to the mother (Hauguel-de Mouzon, 2006). As a result, the placenta greatly contributes to maternal leptin levels.

Fetal leptin levels begin rising at approximately 34 weeks and are correlated with fetal weight. Abnormal levels have been associated with growth disorders and preeclampsia. Postpartum, leptin levels decline in both the newborn and mother (Grisaru-Granovsky, 2008). Perinatal leptin is associated with the development of metabolic syndromes later in life (Granado, 2012).

image Free Fatty Acids and Triglycerides

The newborn has a large proportion of fat, which averages 15 percent of body weight (Kimura, 1991). Thus, late in pregnancy, a substantial part of the substrate transferred to the human fetus is stored as fat. Although maternal obesity affects placental fatty acid uptake, it appears to have no effect on fetal growth (Dube, 2012). Neutral fat in the form of triacylglycerols does not cross the placenta, but glycerol does. There is preferential placental-fetal transfer of long-chain polyunsaturated fatty acids (Gil-Sanchez, 2012). Lipoprotein lipase is present on the maternal but not on the fetal side of the placenta. This arrangement favors hydrolysis of triacylglycerols in the maternal intervillous space while preserving these neutral lipids in fetal blood. Fatty acids transferred to the fetus can be converted to triacylglycerols in the fetal liver.

The placental uptake and use of low-density lipoprotein (LDL) is an alternative mechanism for fetal assimilation of essential fatty acids and amino acids (Chap. 5p. 109). The LDL binds to specific receptors in the coated-pit regions of the syncytiotrophoblast microvilli. The large—about 250,000 Da—LDL particle is taken up by a process of receptor-mediated endocytosis. The apoprotein and cholesterol esters of LDL are hydrolyzed by lysosomal enzymes in the syncytium to give: (1) cholesterol for progesterone synthesis; (2) free amino acids, including essential amino acids; and (3) essential fatty acids, primarily linoleic acid. Indeed, the concentration of arachidonic acid, which is synthesized from linoleic acid in fetal plasma, is greater than that in maternal plasma. Linoleic acid or arachidonic acid or both must be assimilated from maternal dietary intake.

image Amino Acids

The placenta concentrates many amino acids in the syncytiotrophoblast, which are then transferred to the fetal side by diffusion (Lemons, 1979). Based on data from cordocentesis blood samples, the amino acid concentration in umbilical cord plasma is greater than in maternal venous or arterial plasma (Morriss, 1994). Transport system activity is influenced by gestational age and environmental factors. These include heat stress, hypoxia, and under- and overnutrition, as well as hormones such as glucocorticoids, growth hormone, and leptin (Fowden, 2006). Trophoblastic mammalian target of rapamycin complex 1 (mTORC1) regulates placental amino acid transporters and modulates transfer across the placenta (Jansson, 2012). In vivo studies suggest an upregulation of transport for certain amino acids and an increased fetal delivery in women with gestational diabetes associated with fetal overgrowth (Jansson, 2006a).

image Proteins

Placental transfer of larger proteins is limited, but there are exceptions. Immunoglobulin G (IgG) crosses the placenta in large amounts via endocytosis and trophoblast Fc receptors. IgG transfer depends on maternal levels of total IgG, gestational age, placental integrity, IgG subclass, and antigen nature (Palmeira, 2012). IgA and IgM of maternal origin are effectively excluded from the fetus (Gitlin, 1972).

image Ions and Trace Metals

Iodide transport is clearly attributable to a carrier-mediated, energy-requiring active process. And indeed, the placenta concentrates iodide. The concentrations of zinc in the fetal plasma also are greater than those in maternal plasma. Conversely, copper levels in fetal plasma are less than those in maternal plasma. This fact is of particular interest because important copper-requiring enzymes are necessary for fetal development.

Placental Sequestration of Heavy Metals

The heavy-metal–binding protein metallothionein-1 is expressed in human syncytiotrophoblast. This protein binds and sequesters a host of heavy metals, including zinc, copper, lead, and cadmium. Lead enters the fetal environment at a level 90 percent of maternal concentrations, but placental transfer of cadmium is limited (Kopp, 2012). The most common source of environmental cadmium is cigarette smoke. Cadmium levels in maternal blood and placenta are increased with maternal smoking, but there is no increase in cadmium transfer into the fetus. In the rat, data suggest that cadmium reduces the number of trophoblasts, leading to poor placental growth (Lee, 2009).

Metallothionein also binds and sequesters copper (Cu2+) in placental tissue. This accounts for the low levels of Cu2+ in cord blood (Iyengar, 2001). Several enzymes require Cu2+, and its deficiency results in inadequate collagen cross-linking and, in turn, diminished tensile strength of tissues. This may be important because the concentration of cadmium in amnionic fluid is similar to that in maternal blood. The incidence of preterm membrane rupture is increased in women who smoke. It is possible that cadmium provokes metallothionein synthesis in the amnion. This may cause sequestration of Cu2+, a pseudocopper deficiency, and in turn, a weakened amnion.

Calcium and Phosphorus

These minerals also are actively transported from mother to fetus. Calcium is transferred for fetal skeletal mineralization (Olausson, 2012). A calcium-binding protein is produced in placenta. Parathyroid hormone-related protein (PTH-rP), as the name implies, acts as a surrogate PTH in many systems (Chap. 5p. 105). PTH is not demonstrable in fetal plasma. However, PTH-rP is present, suggesting that PTH-rP is the fetal parathormone. The expression of PTH-rP in cytotrophoblasts is modulated by the extracellular concentration of Ca2+ (Hellman, 1992). It seems possible, therefore, that PTH-rP synthesized in decidua, placenta, and other fetal tissues is important in fetal Ca2+ transfer and homeostasis.

image Vitamins

The concentration of vitamin A (retinol) is greater in fetal than in maternal plasma and is bound to retinol-binding protein and to prealbumin. Retinol-binding protein is transferred from the maternal compartment across the syncytiotrophoblast. The transport of vitamin C—ascorbic acid—from mother to fetus is accomplished by an energy-dependent, carrier-mediated process. The levels of the principal vitamin D—cholecalciferol–metabolites, including 1,25-dihydroxycholecalciferol, are greater in maternal plasma than are those in fetal plasma. The 1β-hydroxylation of 25-hydroxyvitamin D3 is known to take place in placenta and in decidua.

FETAL ORGAN SYSTEM DEVELOPMENT

image Amnionic Fluid Formation

In early pregnancy, amnionic fluid is an ultrafiltrate of maternal plasma. By the second trimester, it consists largely of extracellular fluid that diffuses through the fetal skin and thus reflects the composition of fetal plasma (Gilbert, 1993). After 20 weeks, the cornification of fetal skin prevents this diffusion, and amnionic fluid is composed largely of fetal urine. Fetal kidneys start producing urine at 12 weeks, and by 18 weeks, they are producing 7 to 14 mL per day. Fetal urine contains more urea, creatinine, and uric acid than fetal plasma. Amnionic fluid also contains desquamated fetal cells, vernix, lanugo, and various secretions. Because these are hypotonic, the net effect is that amnionic fluid osmolality decreases with advancing gestation. Pulmonary fluid contributes a small proportion of the amnionic volume, and fluid filtering through the placenta accounts for the rest. Aquaporins 8 and 9 play a role in water flow via intramembranous absorption and placental water transfer (Jiang, 2012)

The volume of amnionic fluid at each week is variable (Chap. 11p. 231). In general, the volume increases by 10 mL per week at 8 weeks and increases to 60 mL per week at 21 weeks, then peaks at 34 weeks (Brace, 1989).

Amnionic fluid serves to cushion the fetus, allowing musculoskeletal development and protecting it from trauma. It also maintains temperature and has a minimal nutritive function. Epidermal growth factor (EGF) and EGF-like growth factors, such as transforming growth factor-β, are present in amnionic fluid. Ingestion of fluid into the gastrointestinal tract and inhalation into the lung may promote growth and differentiation of these tissues. Animal studies have shown that pulmonary hypoplasia can be produced by draining amnionic fluid, by chronically draining pulmonary fluid through the trachea, or by physically preventing the prenatal chest excursions that mimic breathing (Adzick, 1984; Alcorn, 1977). Thus, intrapulmonary fluid formation and, at least as important, the alternating egress and retention of fluid in the lungs by breathing movements are essential to normal pulmonary development.

image Cardiovascular System

The fetal circulation is substantially different from that of the adult and functions until birth, when it is required to change dramatically. For example, because fetal blood does not need to enter the pulmonary vasculature to be oxygenated, most of the right ventricular output bypasses the lungs. In addition, the fetal heart chambers work in parallel, not in series, which effectively supplies the brain and heart with more highly oxygenated blood than the rest of the body.

Oxygen and nutrient materials required for fetal growth and maturation are delivered from the placenta by the single umbilical vein (Fig. 7-8). The vein then divides into the ductus venosus and the portal sinus. The ductus venosus is the major branch of the umbilical vein and traverses the liver to enter the inferior vena cava directly. Because it does not supply oxygen to the intervening tissues, it carries well-oxygenated blood directly to the heart. In contrast, the portal sinus carries blood to the hepatic veins primarily on the left side of the liver, and oxygen is extracted. The relatively deoxygenated blood from the liver then flows back into the inferior vena cava, which also receives less oxygenated blood returning from the lower body. Blood flowing to the fetal heart from the inferior vena cava, therefore, consists of an admixture of arterial-like blood that passes directly through the ductus venosus and less well-oxygenated blood that returns from most of the veins below the level of the diaphragm. The oxygen content of blood delivered to the heart from the inferior vena cava is thus lower than that leaving the placenta.

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FIGURE 7-8 The intricate nature of the fetal circulation is evident. The degree of blood oxygenation in various vessels differs appreciably from that in the postnatal state. aa = arteries; LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle; v = vein.

In contrast to postnatal life, the ventricles of the fetal heart work in parallel, not in series. Well-oxygenated blood enters the left ventricle, which supplies the heart and brain, and less oxygenated blood enters the right ventricle, which supplies the rest of the body. These two separate circulations are maintained by the right atrium structure, which effectively directs entering blood to either the left atrium or the right ventricle, depending on its oxygen content. This separation of blood according to its oxygen content is aided by the pattern of blood flow in the inferior vena cava. The well-oxygenated blood tends to course along the medial aspect of the inferior vena cava and the less oxygenated blood flows along the lateral vessel wall. This aids their shunting into opposite sides of the heart. Once this blood enters the right atrium, the configuration of the upper interatrial septum—the crista dividens—preferentially shunts the well-oxygenated blood from the medial side of the inferior vena cava through the foramen ovale into the left heart and then to the heart and brain (Dawes, 1962). After these tissues have extracted needed oxygen, the resulting less oxygenated blood returns to the right atrium through the superior vena cava.

The less oxygenated blood coursing along the lateral wall of the inferior vena cava enters the right atrium and is deflected through the tricuspid valve to the right ventricle. The superior vena cava courses inferiorly and anteriorly as it enters the right atrium, ensuring that less well-oxygenated blood returning from the brain and upper body also will be shunted directly to the right ventricle. Similarly, the ostium of the coronary sinus lies just superior to the tricuspid valve so that less oxygenated blood from the heart also returns to the right ventricle. As a result of this blood flow pattern, blood in the right ventricle is 15 to 20 percent less saturated than blood in the left ventricle.

Almost 90 percent of blood exiting the right ventricle is shunted through the ductus arteriosus to the descending aorta. High pulmonary vascular resistance and comparatively lower resistance in the ductus arteriosus and the umbilical–placental vasculature ensure that only about 15 percent of right ventricular output—8 percent of the combined ventricular output—goes to the lungs (Teitel, 1992). Thus, one third of the blood passing through the ductus arteriosus is delivered to the body. The remaining right ventricular output returns to the placenta through the two hypogastric arteries, which distally become the umbilical arteries. In the placenta, this blood picks up oxygen and other nutrients and is recirculated through the umbilical vein.

Circulatory Changes at Birth

After birth, the umbilical vessels, ductus arteriosus, foramen ovale, and ductus venosus normally constrict or collapse. With the functional closure of the ductus arteriosus and the expansion of the lungs, blood leaving the right ventricle preferentially enters the pulmonary vasculature to become oxygenated before it returns to the left heart. Virtually instantaneously, the ventricles, which had worked in parallel in fetal life, now effectively work in series. The more distal portions of the hypogastric arteries, which course from the level of the bladder along the abdominal wall to the umbilical ring and into the cord as the umbilical arteries, undergo atrophy and obliteration within 3 to 4 days after birth. These become the umbilical ligaments, whereas the intraabdominal remnants of the umbilical vein form the ligamentum teres. The ductus venosus constricts by 10 to 96 hours after birth and is anatomically closed by 2 to 3 weeks, resulting in formation of the ligamentum venosum (Clymann, 1981).

image Hematological Development

Hemopoiesis

In the early embryo, hemopoiesis is demonstrable first in the yolk sac, followed by the liver and finally bone marrow. The first erythrocytes released into the fetal circulation are nucleated and macrocytic. Mean cell volumes are expressed in femtoliters (fL), and one femtoliter equals one cubic micrometer. The mean cell volume is at least 180 fL in the embryo and decreases to 105 to 115 fL at term. The erythrocytes of aneuploid fetuses generally do not undergo this maturation and maintain high mean cell volumes—130 fL on average (Sipes, 1991). As fetal development progresses, more and more of the circulating erythrocytes are smaller and nonnucleated. With fetal growth, both the blood volume in the common fetoplacental circulation and hemoglobin concentration increase. Hemoglobin content of fetal blood rises to approximately 12 g/dL at midpregnancy and to 18 g/dL at term (Walker, 1953). Because of their large size, fetal erythrocytes have a short life span, which progressively lengthens to approximately 90 days at term (Pearson, 1966). As a consequence, red blood cell production is increased. Reticulocytes are initially present at high levels, but decrease to 4 to 5 percent of the total at term. Fetal erythrocytes differ structurally and metabolically from those of the adult. They are more deformable, which serves to offset their higher viscosity. They also contain several enzymes with appreciably different activities (Smith, 1981).

Erythropoiesis is controlled primarily by fetal erythropoietin because maternal erythropoietin does not cross the placenta. Fetal hormone production is influenced by testosterone, estrogen, prostaglandins, thyroid hormone, and lipoproteins (Stockman, 1992). Serum erythropoietin levels increase with fetal maturity. Although the exact production site is disputed, the fetal liver appears to be an important source until renal production begins. There is a close correlation between the erythropoietin concentration in amnionic fluid and that in umbilical venous blood obtained by cordocentesis. After birth, erythropoietin normally may not be detectable for up to 3 months.

In contrast, platelet production reaches stable levels by mid-pregnancy, although there is some variation across gestation (Fig. 7-9). The fetal and neonatal platelet count is subject to various agents as discussed in Chapter 15 (p. 313).

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Figure 7-9 Platelet counts by gestational age obtained the first day of life. Mean values and 5th and 95th percentiles are shown. (Data from Christensen, 2012.)

Fetoplacental Blood Volume

Although precise measurements of human fetoplacental blood volume are lacking, Usher and associates (1963) reported values in term normal newborns to average 78 mL/kg when immediate cord clamping was conducted. Gruenwald (1967) found the fetal blood volume contained in the placenta after prompt cord clamping to average 45 mL/kg of fetal weight. Thus, fetoplacental blood volume at term is approximately 125 mL/kg of fetal weight.

Fetal Hemoglobin

This tetrameric protein is composed of two copies of two different peptide chains, which determine the type of hemoglobin produced. Normal adult hemoglobin A is made of α and β chains. During embryonic and fetal life, various α and β chain precursors are produced. This results in the serial production of several different embryonic hemoglobins. Genes for β-type chains are on chromosome 11 and for α-type chains on chromosome 16. This sequence is shown in Figure 7-10. Each of these genes is turned on and then off during fetal life, until the α and β genes, which direct the production of hemoglobin A, are permanently activated.

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FIGURE 7-10 Schematic drawing of the arrangement of the α and β gene precursors on chromosomes 11 and 16 and the hemoglobin types made from them. (Modified after Thompson, 1991.)

The timing of the production of each of these early hemoglobins corresponds to the site of hemoglobin production. Fetal blood is first produced in the yolk sac, where hemoglobins Gower 1, Gower 2, and Portland are made. Erythropoiesis then moves to the liver, where fetal hemoglobin F is produced. When hemopoiesis finally moves to the bone marrow, adult-type hemoglobin A appears in fetal red blood cells and is present in progressively greater amounts as the fetus matures (Pataryas, 1972).

The final adult version of the α chain is produced exclusively by 6 weeks. After this, there are no functional alternative versions. If an α-gene mutation or deletion occurs, there is no alternate α-type chain that could substitute to form functional hemoglobin. In contrast, at least two versions of the β chain—δ and γ—remain in production throughout fetal life and beyond. In the case of a β-gene mutation or deletion, these two other versions of the β chain often continue to be produced, resulting in hemoglobin A2 or hemoglobin F, which substitute for the abnormal or missing hemoglobin.

Genes are turned off by methylation of their control region, which is discussed in Chapter 13 (p. 272). In some situations, methylation does not occur. For example, in newborns of diabetic women, hemoglobin F may persist due to hypomethylation of the γ gene (Perrine, 1988). With sickle-cell anemia, the γ gene remains unmethylated, and large quantities of fetal hemoglobin continue to be produced (Chap. 56p. 1108). Increased hemoglobin F levels are associated with fewer sickle-cell disease symptoms, and pharmacological modification of these levels by hemoglobin F-inducing drugs is one approach to disease treatment (Trompeter, 2009).

There is a functional difference between hemoglobins A and F. At any given oxygen tension and at identical pH, fetal erythrocytes that contain mostly hemoglobin F bind more oxygen than do those that contain nearly all hemoglobin A (Fig. 47-2p. 945). This is because hemoglobin A binds 2,3-diphosphoglycerate (2,3-DPG) more avidly than does hemoglobin F, thus lowering the affinity of hemoglobin A for oxygen (De Verdier, 1969). During pregnancy, maternal 2,3-DPG levels are increased, and because fetal erythrocytes have lower concentrations of 2,3-DPG, the latter has increased oxygen affinity.

The amount of hemoglobin F in fetal erythrocytes begins to decrease in the last weeks of pregnancy. At term, approximately three fourths of total hemoglobin is hemoglobin F. During the first 6 to 12 months of life, the hemoglobin F proportion continues to decrease and eventually reaches the low levels found in adult erythrocytes. Glucocorticosteroids mediate the switch from fetal to adult hemoglobin, and the effect is irreversible (Zitnik, 1995).

Coagulation Factors

There are no embryonic forms of the various hemostatic proteins. With the exception of fibrinogen, the fetus starts producing normal, adult-type procoagulant, fibrinolytic, and anticoagulant proteins by 12 weeks. Because they do not cross the placenta, their concentrations at birth are markedly below the levels that develop within a few weeks of life (Corrigan, 1992). In normal neonates, the levels of factors II, VII, IX, X, and XI, as well as those of prekallikrein, protein S, protein C, antithrombin, and plasminogen, are all approximately 50 percent of adult levels. In contrast, levels of factors V, VIII, XIII, and fibrinogen are closer to adult values (Saracco, 2009). Without prophylactic treatment, the vitamin K-dependent coagulation factors usually decrease even further during the first few days after birth. This decline is amplified in breast-fed infants and may lead to newborn hemorrhage (Chap. 33p. 644).

Fetal fibrinogen, which appears as early as 5 weeks, has the same amino acid composition as adult fibrinogen but has different properties (Klagsbrun, 1988). It forms a less compressible clot, and the fibrin monomer has a lower degree of aggregation (Heimark, 1988). Plasma fibrinogen levels at birth are less than those in nonpregnant adults, however, the protein is functionally more active than adult fibrinogen (Ignjatovic, 2011).

Levels of functional fetal factor XIII—fibrin-stabilizing factor—are significantly reduced compared with those in adults (Henriksson, 1974). Nielsen (1969) described low levels of plasminogen and increased fibrinolytic activity in cord plasma compared with that of maternal plasma. Platelet counts in cord blood are in the normal range for nonpregnant adults (see Fig. 7-9).

Despite this relative reduction in procoagulants, the fetus appears to be protected from hemorrhage because fetal bleeding is rare. Excessive bleeding does not usually occur even after invasive fetal procedures such as cordocentesis. Ney and coworkers (1989) have shown that amnionic fluid thromboplastins and a factor(s) in Wharton jelly combine to aid coagulation at the umbilical cord puncture site.

Various thrombophilias may cause thromboses and pregnancy complications in adults (Chap. 52p. 1029). If the fetus inherits one of these mutations, thrombosis and infarction can develop in the placenta or in fetal organs. This is usually seen with homozygous inheritance.

Plasma Proteins

Liver enzymes and other plasma proteins are produced by the fetus, and these levels do not correlate with maternal levels (Weiner, 1992). Concentrations of plasma proteins, albumin, lactic dehydrogenase, aspartate aminotransferase, γ-glutamyl transpeptidase, and alanine transferase all increase, whereas prealbumin levels decrease with gestational age (Fryer, 1993). At birth, mean total plasma protein and albumin concentrations in fetal blood are similar to maternal levels (Foley, 1978).

image Immunology

Infections in utero have provided an opportunity to examine mechanisms of the fetal immune response. Evidence of immunological competence has been reported as early as 13 weeks (Kohler, 1973; Stabile, 1988). In cord blood at or near term, the average level for most components is approximately half that of the adult values (Adinolfi, 1977).

In the absence of a direct antigenic stimulus such as infection, fetal plasma immunoglobulins consist almost totally of transferred maternal immunoglobulin G (IgG). Thus, antibodies in the newborn are most often reflective of maternal immunological experiences.

Immunoglobulin G

Maternal IgG transport to the fetus begins at approximately 16 weeks and increases thereafter. The bulk of IgG is acquired during the last 4 weeks of pregnancy (Gitlin, 1971). Accordingly, preterm neonates are endowed relatively poorly with protective maternal antibodies. Newborns begin to slowly produce IgG, and adult values are not attained until 3 years of age. In certain situations, the transfer of IgG antibodies from mother to fetus can be harmful rather than protective to the fetus. The classic example is hemolytic disease of the fetus and newborn resulting from D-antigen alloimmunization (Chap. 15p. 306).

Immunoglobulin M and A

In the adult, production of immunoglobulin M (IgM) in response to an antigenic stimulus is superseded in a week or so predominantly by IgG production. In contrast, very little IgM is produced by normal fetuses, and that produced may include antibody to maternal T lymphocytes (Hayward, 1983). With infection, the IgM response is dominant in the fetus and remains so for weeks to months in the newborn. And because IgM is not transported from the mother, any IgM in the fetus or newborn is that which it produced. Increased levels of IgM are found in newborns with congenital infection such as rubella, cytomegalovirus infection, or toxoplasmosis. Serum IgM levels in umbilical cord blood and identification of specific antibodies may be useful in intrauterine infection diagnosis. In infants, adult levels of IgM are normally attained by age 9 months.

Immunoglobulin A (IgA) ingested in colostrum provides mucosal protection against enteric infections. This role as an immune barrier against infection may explain the small amount of fetal secretory IgA found in amnionic fluid (Quan, 1999).

Lymphocytes and Monocytes

The immune system develops early. B lymphocytes appear in fetal liver by 9 weeks and in blood and spleen by 12 weeks. T lymphocytes begin to leave the thymus at approximately 14 weeks. Despite this, the newborn responds poorly to immunization, and especially poorly to bacterial capsular polysaccharides. This immature response may be due to either deficient response of newborn B cells to polyclonal activators, or lack of T cells that proliferate in response to specific stimuli (Hayward, 1983). In the newborn, monocytes are able to process and present antigen when tested with maternal antigen-specific T cells. DNA methylation patterns are developmentally regulated during monocyte-macrophage differentiation and contribute to the antiinflammatory phenotype in macrophages (Kim, 2012).

image Skull

Fetal head size is important because an essential feature of labor is the adaptation between the head and the maternal bony pelvis. The firm skull is composed of two frontal, two parietal, and two temporal bones, along with the upper portion of the occipital bone and the wings of the sphenoid. These bones are separated by membranous spaces termed sutures (Fig. 7-11).

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FIGURE 7-11 Fetal head at term showing fontanels and sutures.

The most important sutures are the frontal, between the two frontal bones; the sagittal, between the two parietal bones; the two coronal, between the frontal and parietal bones; and the two lambdoid, between the posterior margins of the parietal bones and upper margin of the occipital bone. The term fontanel describes the irregular space enclosed by a membrane at the junction of several sutures (see Fig. 7-11). The greater, or anterior, fontanel is a lozenge-shaped space situated at the junction of the sagittal and the coronal sutures. The lesser, or posterior, fontanel is a small triangular area at the intersection of the sagittal and lambdoid sutures. The localization of these fontanels gives important information concerning the presentation and position of the fetus during labor.

It is customary to measure certain critical diameters and circumferences of the newborn head (Fig. 7-12). These include:

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FIGURE 7-12

1. The occipitofrontal (11.5 cm), which follows a line extending from a point just above the root of the nose to the most prominent portion of the occipital bone.

2. The biparietal (9.5 cm), the greatest transverse diameter of the head, which extends from one parietal boss to the other.

3. The bitemporal (8.0 cm), which is the greatest distance between the two temporal sutures.

4. The occipitomental (12.5 cm), which extends from the chin to the most prominent portion of the occiput.

5. The suboccipitobregmatic (9.5 cm), which follows a line drawn from the middle of the large fontanel to the undersurface of the occipital bone where it joins the neck.

The greatest circumference of the head, which corresponds to the plane of the occipitofrontal diameter, averages 34.5 cm, a size too large to fit through the pelvis. The smallest circumference, corresponding to the plane of the suboccipitobregmatic diameter, is 32 cm. The cranial bones are normally connected only by a thin fibrous tissue layer. This allows considerable shifting or sliding of each bone to accommodate the size and shape of the maternal pelvis. This process is termed molding. The head position and degree of skull ossification result in a spectrum of cranial plasticity. In some cases, this contributes to fetopelvic disproportion, a leading indication for cesarean delivery (Chap. 23p. 463).

image Central Nervous System and Spinal Cord

Brain Development

There is a steady gestational-age-related change in the fetal brain appearance so that it is possible to identify fetal age from its external appearance (Dolman, 1977). Neuronal proliferation and migration proceed along with gyral growth and maturation (Fig. 7-13). Sequential maturation studies have characterized the developing fetal brain imaged using magnetic resonance (MR) imaging (Fig. 10-41p. 224). (Manganaro, 2007). Recent studies also using MR imaging have quantified development of subcortical brain structures from 12 to 22 weeks (Meng, 2012). Myelination of the ventral roots of the cerebrospinal nerves and brainstem begins at approximately 6 months, but most myelination occurs after birth. This lack of myelin and incomplete skull ossification permit fetal brain structure to be seen with sonography throughout gestation.

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FIGURE 7-13 Neuronal proliferation and migration are complete at 20 to 24 weeks. During the second half of gestation, organizational events proceed with gyral formation and proliferation, differentiation, and migration of cellular elements. Approximate gestational ages are listed. A. 20 weeks. B. 35 weeks. C. 40 weeks.

Spinal Cord and Sensory Organs

The spinal cord extends along the entire length of the vertebral column in the embryo, but after that it grows more slowly. By 24 weeks, the spinal cord extends to S1, at birth to L3, and in the adult to L1. Spinal cord myelination begins at midgestation and continues through the first year of life. Synaptic function is sufficiently developed by the eighth week to demonstrate flexion of the neck and trunk (Temiras, 1968). During the third trimester, integration of nervous and muscular function proceeds rapidly.

The internal, middle, and external components of the ear are well developed by midpregnancy (see Fig. 7-2).

image Gastrointestinal System

Swallowing begins at 10 to 12 weeks, coincident with the ability of the small intestine to undergo peristalsis and transport glucose actively (Koldovsky, 1965; Miller, 1982). Much of the water in swallowed fluid is absorbed, and unabsorbed matter is propelled to the lower colon (Fig. 7-14). It is not clear what stimulates swallowing, but the fetal neural analogue of thirst, gastric emptying, and change in the amnionic fluid composition are potential factors (Boyle, 1992). The fetal taste buds may play a role because saccharin injected into amnionic fluid increases swallowing, whereas injection of a noxious chemical inhibits it (Liley, 1972).

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Figure 7-14 Radiograph of a 115-g fetus at about 16 weeks’ gestational age showing radiopaque dye in the lungs, esophagus, stomach, and entire intestinal tract after injection into the amnionic cavity 26 hours before delivery. These reflect inhalation as well as active swallowing of amnionic fluid. (From Davis, 1946, with permission.)

Fetal swallowing appears to have little effect on amnionic fluid volume early in pregnancy because the volume swallowed is small compared with the total. Late in pregnancy, however, amnionic fluid regulation is substantially affected by fetal swallowing. For example, if swallowing is inhibited, hydramnios is common (Chap. 11p. 233). Term fetuses swallow between 200 and 760 mL per day—an amount comparable to that of the neonate (Pritchard, 1966).

Hydrochloric acid and some digestive enzymes are present in the stomach and small intestine in very small amounts in the early fetus. Intrinsic factor is detectable by 11 weeks, and pepsinogen by 16 weeks. The preterm neonate, depending on the gestational age when born, may have transient deficiencies of these enzymes (Lebenthal, 1983).

Stomach emptying appears to be stimulated primarily by volume. Movement of amnionic fluid through the gastrointestinal system may enhance growth and development of the alimentary canal. That said, other regulatory factors likely are involved because anencephalic fetuses, in whom swallowing is limited, often have normal amnionic fluid volumes and normal-appearing gastrointestinal tracts. Gitlin (1974) demonstrated that late in pregnancy, approximately 800 mg of soluble protein is ingested daily by the fetus.

Several anomalies can affect normal fetal gastrointestinal function. Hirschsprung disease—known also as congenital aganglionic megacolon, prevents the bowel from undergoing parasympathetic-mediated relaxation and thus from emptying normally (Watkins, 1992). It may be recognized prenatally by grossly enlarged bowel during sonography. Obstructions such as duodenal atresia, megacystis-microcolon syndrome, or imperforate anus can also prevent normal bowel emptying. Meconium ileus, commonly found with fetal cystic fibrosis, is bowel obstruction caused by thick, viscid meconium that blocks the distal ileum.

Meconium

Fetal bowel contents consist of various products of secretion, such as glycerophospholipids from the lung, desquamated fetal cells, lanugo, scalp hair, and vernix. It also contains undigested debris from swallowed amnionic fluid. The dark greenish-black is caused by pigments, especially biliverdin. Meconium can pass from normal bowel peristalsis in the mature fetus or from vagal stimulation. It can also pass when hypoxia stimulates arginine vasopressin (AVP) release from the fetal pituitary gland. AVP stimulates colonic smooth muscle to contract, resulting in intraamnionic defecation (DeVane, 1982; Rosenfeld, 1985).

Liver

Serum liver enzyme levels increase with gestational age. However, the fetal liver has a gestational-age-related diminished capacity for converting free unconjugated bilirubin to conjugated bilirubin (Chap. 33p. 644). Because the life span of normal fetal macrocytic erythrocytes is shorter than that of adult erythrocytes, relatively more unconjugated bilirubin is produced. As noted, the fetal liver conjugates only a small fraction, and this is excreted into the intestine and ultimately oxidized to biliverdin. Most of the unconjugated bilirubin is excreted into the amnionic fluid after 12 weeks and transferred across the placenta (Bashore, 1969).

Importantly, placental transfer is bidirectional. Thus, a pregnant woman with severe hemolysis from any cause has excess unconjugated bilirubin that readily passes to the fetus and then into the amnionic fluid. Conversely, conjugated bilirubin is not exchanged to any significant degree between mother and fetus.

Most fetal cholesterol is from hepatic synthesis, which satisfies the large demand for LDL cholesterol by the fetal adrenal glands. Hepatic glycogen is present in low concentration during the second trimester, but near term there is a rapid and marked increase to levels two to three times those in the adult liver. After birth, glycogen content falls precipitously.

Pancreas

Insulin-containing granules can be identified by 9 to 10 weeks, and insulin is detectable in fetal plasma at 12 weeks (Adam, 1969). The pancreas responds to hyperglycemia by secreting insulin (Obenshain, 1970). Glucagon has been identified in the fetal pancreas at 8 weeks. In the adult rhesus monkey, hypoglycemia and infused alanine cause an increase in maternal glucagon levels. Although, similar stimuli do not evoke a fetal response in the human, by 12 hours after birth, the newborn is capable of responding (Chez, 1975). At the same time, however, fetal pancreatic α cells do respond to L-dopa infusions (Epstein, 1977). Therefore, nonresponsiveness to hypoglycemia is likely the consequence of failure of glucagon release rather than inadequate production. This is consistent with developmental expression of pancreatic genes in the fetus (Mally, 1994).

Most pancreatic enzymes are present by 16 weeks. Trypsin, chymotrypsin, phospholipase A, and lipase are found in the 14-week fetus, and their concentrations increase with gestation (Werlin, 1992). Amylase has been identified in amnionic fluid at 14 weeks (Davis, 1986). The exocrine function of the fetal pancreas is limited. Physiologically important secretion occurs only after stimulation by a secretogogue such as acetylcholine, which is released locally after vagal stimulation (Werlin, 1992). Cholecystokinin normally is released only after protein ingestion and thus ordinarily would not be found in the fetus.

image Urinary System

Two primitive urinary systems—the pronephros and the mesonephros—precede the development of the metanephros, which forms the final kidney (Chap. 3p. 36). The pronephros has involuted by 2 weeks, and the mesonephros is producing urine at 5 weeks and degenerates by 11 to 12 weeks. Failure of these two structures either to form or to regress may result in anomalous urinary system development. Between 9 and 12 weeks, the ureteric bud and the nephrogenic blastema interact to produce the metanephros. The kidney and ureter develop from intermediate mesoderm. The bladder and urethra develop from the urogenital sinus. The bladder also develops in part from the allantois.

By week 14, the loop of Henle is functional and reabsorption occurs (Smith, 1992). New nephrons continue to be formed until 36 weeks. In preterm neonates, their formation continues after birth. Although the fetal kidneys produce urine, their ability to concentrate and modify the pH is limited even in the mature fetus. Fetal urine is hypotonic with respect to fetal plasma and has low electrolyte concentrations.

Renal vascular resistance is high, and the filtration fraction is low compared with values in later life (Smith, 1992). Fetal renal blood flow and thus urine production are controlled or influenced by the renin-angiotensin system, the sympathetic nervous system, prostaglandins, kallikrein, and atrial natriuretic peptide. The glomerular filtration rate increases with gestational age from less than 0.1 mL/min at 12 weeks to 0.3 mL/min at 20 weeks. In later gestation, the rate remains constant when corrected for fetal weight (Smith, 1992). Hemorrhage or hypoxia generally results in a decrease in renal blood flow, glomerular filtration rate, and urine output.

Urine usually is found in the bladder even in small fetuses. The fetal kidneys start producing urine at 12 weeks. By 18 weeks, they are producing 7 to 14 mL/day, and at term, this increases to 27 mL/hr or 650 mL/day (Wladimiroff, 1974). Maternally administered furosemide increases fetal urine formation, whereas uteroplacental insufficiency, fetal-growth restriction, and other types of fetal disorders decrease it. Obstruction of the urethra, bladder, ureters, or renal pelves can damage renal parenchyma and distort fetal anatomy. With urethral obstruction, the bladder may become sufficiently distended that it ruptures or dystocia results. Kidneys are not essential for survival in utero, but are important in the control of amnionic fluid composition and volume. Thus, abnormalities that cause chronic anuria are usually accompanied by oligohydramnios and pulmonary hypoplasia. Pathological correlates and prenatal therapy of urinary tract obstruction are discussed in Chapter 16 (p. 330).

image Lung Development

Lung maturation and the biochemical indices of functional fetal lung maturity are of considerable interest to the obstetrician. Morphological or functional immaturity at birth leads to the development of the respiratory distress syndrome (Chap. 34p. 653). A sufficient amount of surface-active materials—collectively referred to as surfactant—in the amnionic fluid is evidence of fetal lung maturity. As Liggins (1994) emphasized, however, the structural and morphological maturation of fetal lung also is extraordinarily important to proper lung function.

Anatomical Maturation

Like the branching of a tree, lung development proceeds along an established timetable that apparently cannot be hastened by antenatal or neonatal therapy. The limits of viability appear to be determined by the usual process of pulmonary growth. There are four essential lung development stages as described by Moore (2013). First, the pseudoglandular stage entails growth of the intrasegmental bronchial tree between 6 and 16 weeks. During this period, the lung looks microscopically like a gland. Second, during the canalicular stage, from 16 to 26 weeks, the bronchial cartilage plates extend peripherally. Each terminal bronchiole gives rise to several respiratory bronchioles, and each of these in turn divides into multiple saccular ducts. Next, the terminal sac stage begins at 26 weeks. During this stage, respiratory bronchioles give rise to primitive pulmonary alveoli—the terminal sacs. Last, the alveolar stage begins at 32 weeks, although the exact transition from the terminal sac stage is unclear. During the alveolar stage, the alveolar epithelial lining thins to improve gas exchange. Simultaneously, an extracellular matrix develops from proximal to distal lung segments until term. An extensive capillary network is built, the lymph system forms, and type II pneumonocytes begin to produce surfactant. At birth, only approximately 15 percent of the adult number of alveoli is present. Thus, the lung continues to grow, adding more alveoli for up to 8 years.

Various insults can upset this process, and their timing determines the sequelae. With fetal renal agenesis, for example, there is no amnionic fluid at the beginning of lung growth, and major defects occur in all three stages. A fetus with membrane rupture before 20 weeks and subsequent oligohydramnios usually exhibits nearly normal bronchial branching and cartilage development but has immature alveoli. Membrane rupture after 24 weeks may have little long-term effect on pulmonary structure.

Pulmonary Surfactant

After the first breath, the terminal sacs must remain expanded despite the pressure imparted by the tissue-to-air interface, and surfactant keeps them from collapsing. Surfactant is formed in type II pneumonocytes that line the alveoli. These cells are characterized by multivesicular bodies that produce the lamellar bodies in which surfactant is assembled. During late fetal life, at a time when the alveolus is characterized by a water-to-tissue interface, the intact lamellar bodies are secreted from the lung and swept into the amnionic fluid during respiration-like movements that are termed fetal breathing. At birth, with the first breath, an air-to-tissue interface is produced in the lung alveolus. Surfactant uncoils from the lamellar bodies, and it then spreads to line the alveolus to prevent alveolar collapse during expiration. Thus, it is the capacity for fetal lungs to produce surfactant, and not the actual laying down of this material in the lungs in utero, that establishes lung maturity.

Surfactant Composition. Gluck and associates (1967, 1970, 1972) and Hallman and coworkers (1976) found that approximately 90 percent of surfactant dry weight is lipid. Proteins account for the other 10 percent. Approximately 80 percent of the glycerophospholipids are phosphatidylcholines (lecithins). The principal active component of surfactant is a specific lecithin—dipalmitoylphosphatidylcholine (DPPC or PC)—which accounts for nearly 50 percent. Phosphatidylglycerol (PG) accounts for another 8 to 15 percent. Its precise role is unclear because newborns without phosphatidylglycerol usually do well. The other major constituent is phosphatidylinositol (PI). The relative contributions of each component are shown in Figure 7-15.

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Figure 7-15 Relation between the levels of lecithin, or dipalmitoylphosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylglycerol (PG) in amnionic fluid as a function of gestational age.

Surfactant Synthesis. Biosynthesis takes place in the type II pneumocytes. The apoproteins are produced in the endoplasmic reticulum, and the glycerophospholipids are synthesized by cooperative interactions of several cellular organelles. Phospholipid is the primary surface tension-lowering component of surfactant, whereas the apoproteins aid the forming and reforming of a surface film. The surface properties of the surfactant phospholipids are determined principally by the composition and degree of saturation of their long-chain fatty acids.

The major apoprotein is surfactant A (SP-A), which is a glycoprotein with a molecular weight of 28,000 to 35,000 Da (Whitsett, 1992). It is synthesized in the type II cells, and its content in amnionic fluid increases with gestational age and fetal lung maturity. SP-A may also play a role in the onset of parturition (Mendelson, 2005). Synthesis of SP-A is increased by treatment of fetal lung tissue with cyclic adenosine monophosphate (AMP) analogues, epidermal growth factors, and triiodothyronine. Increased apoprotein synthesis precedes surfactant glycerophospholipid synthesis.

SP-A gene expression is demonstrable by 29 weeks (Snyder, 1988). There are two separate genes on chromosome 10. These are SP-A1 and SP-A2, and their regulation is distinctive and different (McCormick, 1994). Specifically, cyclic AMP is more important in SP-A2 expression, whereas dexamethasone decreases SP-A2 expression.

Several smaller apoproteins such as SP-B and SP-C are likely important in optimizing the action of surfactant. For example, deletions in SP-B gene are incompatible with survival despite production of large amounts of surfactant.

Corticosteroids and Fetal Lung Maturation. Since Liggins (1969) first observed lung maturation in lamb fetuses given glucocorticosteroids before preterm delivery, many suggested that fetal cortisol stimulates lung maturation and surfactant synthesis. It is unlikely that corticosteroids are the only stimulus for augmented surfactant formation. There is evidence, however, that glucocorticosteroids administered at certain critical times during gestation improve fetal lung maturation. The use of betamethasone and dexamethasone to accelerate fetal lung maturity, as well as neonatal replacement surfactant therapy, is discussed in Chapter 34 (p. 653).

Breathing

Within a few minutes after birth, the respiratory system must provide oxygen as well as eliminate carbon dioxide. Respiratory muscles develop early, and fetal chest wall movements are detected by sonography as early as 11 weeks (Boddy, 1975). From the beginning of the fourth month, the fetus is capable of respiratory movement sufficiently intense to move amnionic fluid in and out of the respiratory tract.

image Endocrine Gland Development

Pituitary Gland

The fetal endocrine system is functional for some time before the central nervous system reaches maturity (Mulchahey, 1987). The anterior pituitary gland develops from oral ectoderm—Rathke pouch, whereas the posterior pituitary gland derives from neuroectoderm.

Anterior and Intermediate Lobes. The adenohypophysis, or anterior pituitary, differentiates into five cell types that secrete six protein hormones: (1) lactotropes produce prolactin—PRL; (2) somatotropes produce growth hormone—GH; (3) corticotropes produce corticotropin—ACTH; (4) thyrotropes produce thyrotropin or thyroid-stimulating hormone—TSH; and (5) gonadotropes produce luteinizing hormone—LH and follicle-stimulating hormone—FSH.

ACTH is first detected in the fetal pituitary gland at 7 weeks, and GH and LH have been identified by 13 weeks. By the end of the 17th week, the fetal pituitary gland synthesizes and stores all pituitary hormones. Moreover, the fetal pituitary is responsive to hormones and is capable of secreting these early in gestation (Grumbach, 1974). The fetal pituitary secretes β-endorphin, and cord blood levels of β-endorphin and β-lipotropin increase with fetal Pco2(Browning, 1983).

There is a well-developed intermediate lobe in the fetal pituitary gland. The cells of this structure begin to disappear before term and are absent from the adult pituitary. The principal secretory products of the intermediate lobe cells are α-melanocyte–stimulating hormone (α-MSH) and β-endorphin.

Neurohypophysis. The posterior pituitary gland or neurohypophysis is well developed by 10 to 12 weeks, and oxytocin and arginine vasopressin (AVP) are demonstrable. Both hormones probably function in the fetus to conserve water by actions largely at the lung and placenta rather than kidney. AVP levels in umbilical cord plasma are strikingly higher than maternal levels (Chard, 1971; Polin, 1977). Elevated fetal blood AVP appears to be associated with fetal stress (DeVane, 1982).

Thyroid Gland

The pituitary–thyroid system is functional by the end of the first trimester. The thyroid gland is able to synthesize hormones by 10 to 12 weeks, and thyrotropin, thyroxine, and thyroxine-binding globulin (TBG) have been detected in fetal serum as early as 11 weeks (Ballabio, 1989). The placenta actively concentrates iodide on the fetal side, and by 12 weeks and throughout pregnancy, the fetal thyroid concentrates iodide more avidly than does the maternal thyroid. Thus, maternal administration of either radioiodide or appreciable amounts of ordinary iodide is hazardous after this time. Normal fetal levels of free thyroxine (T4), free triiodothyronine (T3), and thyroxine-binding globulin increase steadily throughout gestation (Ballabio, 1989). Compared with adult levels, by 36 weeks, fetal serum concentrations of TSH are higher, total and free T3 concentrations are lower, and T4 is similar. This suggests that the fetal pituitary may not become sensitive to feedback until late in pregnancy (Thorpe-Beeston, 1991; Wenstrom, 1990).

Fetal thyroid hormone plays a role in the normal development of virtually all fetal tissues, especially the brain. Its influence is illustrated by congenital hyperthyroidism, which occurs when maternal thyroid-stimulating antibody crosses the placenta to stimulate the fetal thyroid. These fetuses develop tachycardia, hepatosplenomegaly, hematological abnormalities, craniosynostosis, and growth restriction. As children, they have perceptual motor difficulties, hyperactivity, and reduced growth (Wenstrom, 1990). Neonatal effects of fetal thyroid deficiency are discussed in Chapter 58 (p. 1147).

The placenta prevents substantial passage of maternal thyroid hormones to the fetus by rapidly deiodinating maternal T4 and T3 to form reverse T3, a relatively inactive thyroid hormone (Vulsma, 1989). Several antithyroid antibodies cross the placenta when present in high concentrations. Those include the long-acting thyroid stimulators (LATS), LATS-protector (LATS-P), and thyroid-stimulating immunoglobulin (TSI). It was previously believed that normal fetal growth and development, which occurred despite fetal hypothyroidism, provided evidence that T4 was not essential for fetal growth. It is now known, however, that growth proceeds normally because small quantities of maternal T4 prevent antenatal cretinism in fetuses with thyroid agenesis (Vulsma, 1989). The fetus with congenital hypothyroidism typically does not develop stigmata of cretinism until after birth. Because administration of thyroid hormone will prevent this, all newborns are tested for high serum levels of TSH (Chap. 32p. 631).

Immediately after birth, there are major changes in thyroid function and metabolism. Cooling to room temperature evokes sudden and marked increase in TSH secretion. This in turn causes a progressive increase in serum T4levels that are maximal 24 to 36 hours after birth. There are nearly simultaneous elevations of serum T3 levels.

Adrenal Glands

The fetal adrenal glands are much larger in relation to total body size than in adults. The bulk is made up of the inner or fetal zone of the adrenal cortex and involutes rapidly after birth. This zone is scant to absent in rare instances in which the fetal pituitary gland is congenitally absent. The function of the fetal adrenal glands is discussed in detail in Chapter 5 (p. 108).

DEVELOPMENT OF GENITALIA

image Embryology of Uterus and Oviducts

The uterus and tubes arise from the müllerian ducts, which first appear near the upper pole of the urogenital ridge in the fifth week of embryonic development (Fig. 7-16). This ridge is composed of the mesonephros, gonad, and associated ducts. The first indication of müllerian duct development is a thickening of the coelomic epithelium at approximately the level of the fourth thoracic segment. This becomes the fimbriated extremity of the fallopian tube, which invaginates and grows caudally to form a slender tube at the lateral edge of the urogenital ridge. In the sixth week, the growing tips of the two müllerian ducts approach each other in the midline. One week later, they reach the urogenital sinus. At that time, the two müllerian ducts fuse to form a single canal at the level of the inguinal crest. This crest gives rise to the gubernaculum, which is the primordium of the round ligament.

image

Figure 7-16 A. Cross section of an embryo at 4 to 6 weeks. B. Large ameboid primordial germ cells migrate (arrows) from the yolk sac to the area of germinal epithelium, within the genital ridge. C.Migration of sympathetic cells from the spinal ganglia to a region above the developing kidney. (Redrawn from Moore, 1988.)

Thus, the upper ends of the müllerian ducts produce the fallopian tubes, and the fused parts give rise to the uterus. The vaginal canal is not patent throughout its entire length until the sixth month (Koff, 1933). Because of the clinical importance of anomalies that arise from abnormal fusion and dysgenesis of these structures, their embryogenesis is discussed in detail in Chapter 3 (p. 37).

image Embryology of the Ovaries

At approximately 4 weeks, gonads form on the ventral surface of the embryonic kidney at a site between the eighth thoracic and fourth lumbar segments. The coelomic epithelium thickens, and clumps of cells bud off into the underlying mesenchyme. This circumscribed area is called the germinal epithelium. By the fourth to sixth week, however, there are many large ameboid cells in this region that have migrated into the embryo body from the yolk sac (see Fig. 7-16). These primordial germ cells are distinguishable by their large size and certain morphological and cytochemical features.

When the primordial germ cells reach the genital area, some enter the germinal epithelium and others mingle with groups of cells that proliferate from it or lie in the mesenchyme. By the end of the fifth week, rapid division of all these cell types results in development of a prominent genital ridge. The ridge projects into the body cavity medially to a fold in which there are the mesonephric (wolffian) and paramesonephric (müllerian) ducts (Fig. 7-17). By the seventh week, this ridge is separated from the mesonephros except at the narrow central zone, the future hilum, where blood vessels enter. At this time, the sexes can be distinguished, because the testes can be recognized by well-defined radiating strands of cells termed sex cords. These cords are separated from the germinal epithelium by mesenchyme that is to become the tunica albuginea. The sex cords, which consist of large germ cells and smaller epithelioid cells derived from the germinal epithelium, develop into the seminiferous tubules and tubuli rete. The latter establishes connection with the mesonephric tubules that develop into the epididymis. The mesonephric ducts become the vas deferens.

image

image

FIGURE 7-17 Continuation of embryonic sexual differentiation. TDF = testis-determining factor. (Redrawn from Moore, 1988.)

In the female embryo, the germinal epithelium proliferates for a longer time. The groups of cells thus formed lie at first in the region of the hilum. As connective tissue develops between them, these appear as sex cords. These cords give rise to the medulla cords and persist for variable times (Forbes, 1942). By the third month, medulla and cortex are defined (see Fig. 7-17). The bulk of the ovary is made up of cortex, a mass of crowded germ and epithelioid cells that show some signs of grouping, but there are no distinct cords as in the testis. Strands of cells extend from the germinal epithelium into the cortical mass, and mitoses are numerous. The rapid succession of mitoses soon reduces germ-cell size to the extent that these no longer are differentiated clearly from the neighboring cells. These germ cells are now called oogonia.

By the fourth month, some germ cells in the medullary region begin to enlarge. These are called primary oocytes at the beginning of the growth phase that continues until maturity. During this cell growth period, many oocytes undergo degeneration, both before and after birth. A single layer of flattened follicular cells that were derived originally from the germinal epithelium soon surrounds the primary oocytes. These structures are now called primordial follicles and are seen first in the medulla and later in the cortex. Some follicles begin to grow even before birth, and some are believed to persist in the cortex almost unchanged until menopause.

By 8 months, the ovary has become a long, narrow, lobulated structure that is attached to the body wall along the line of the hilum by the mesovarium. The germinal epithelium has been separated for the most part from the cortex by a band of connective tissue—tunica albuginea. This band is absent in many small areas where strands of cells, usually referred to as cords of Pflüger, are in contact with the germinal epithelium. Among these cords are cells believed by many to be oogonia that resemble the other epithelial cells as a result of repeated mitosis. In the underlying cortex, there are two distinct zones. Superficially, there are nests of germ cells in meiotic synapsis, interspersed with Pflüger cords and strands of connective tissue. In the deeper zone, there are many groups of germ cells in synapsis, as well as primary oocytes, prospective follicular cells, and a few primordial follicles.

image Fetal Gender

Theoretically, there should be a primary gender ratio of 1:1 at the time of fertilization because there are equal numbers of X-and Y-bearing spermatozoa. This is not the case, however, and many factors have been shown to contribute to gender ratios at conception. These include differential susceptibility to environmental exposures as well as medical disorders. Also, couples with a large age discrepancy are more likely to have a male offspring (Manning, 1997). Whatever the cause, it is impossible to determine the primary gender ratio because this would require gender assignment to zygotes that fail to cleave, blastocysts that fail to implant, and other early pregnancy losses.

The secondary gender ratio pertains to fetuses that reach viability and is usually held to be approximately 106 males to 100 females. The unbalanced secondary gender ratio is explicable by the loss of more female than male embryo-fetuses during early pregnancy. That said, Davis and colleagues (1998) report a significant decline in male births since 1950 in Denmark, Sweden, The Netherlands, the United States, Germany, Norway, and Finland. Similarly, Allan and associates (1997) reported that male births in Canada since 1970 dropped by 2.2 per 1000 live births.

image Gender Assignment at Birth

In the delivery room, parents first want to know the gender of their newborn. If the external genitalia of the newborn are ambiguous, the obstetrician faces a profound dilemma. In these cases, assignment requires knowledge of the karyotypic sex, gonadal sex, hormonal milieu to which the fetus was exposed, exact anatomy, and all possibilities for surgical correction. In the past, newborns with a small or likely insufficient phallus were often assigned to the female gender. Based on what is now known of the role of fetal exposure to hormones in establishing gender preference and behavior, it can be seen why such a policy may have caused gender identity disorder (Slijper, 1998). Thus, it seems best to inform the parents that although their newborn appears healthy, the gender will need to be determined by a series of tests. To develop a plan that can assist in determining the cause of ambiguous genitalia, the mechanisms of normal and abnormal sexual differentiation must be considered.

image Sexual Differentiation

Phenotypic gender differentiation is determined by the chromosomal complement acting in conjunction with gonadal development.

Chromosomal Gender

Genetic gender—XX or XY—is established at fertilization, but for the first 6 weeks, development of male and female embryos is morphologically indistinguishable. The differentiation of the primordial gonad into testis or ovary heralds the establishment of gonadal sex.

Gonadal Gender

As described earlier and shown in Figure 7-16, primordial germ cells that originate in the yolk sac endoderm migrate to the genital ridge to form the indifferent gonad. If a Y chromosome is present, at about 6 weeks after conception, the gonad begins developing into a testis (Simpson, 1997). Testis development is directed by a gene located on the short arm of Y—testis-determining factor (TDF), also called sex-determining region (SRY). This gene encodes a transcription factor that acts to modulate the transcription rate of a number of genes involved in gonadal differentiation. The SRY gene is specific to the Y chromosome and is expressed in the human single-cell zygote upon ovum fertilization. It is not expressed in spermatozoa (Fiddler, 1995; Gustafson, 1994). In addition, testis development requires a dose-dependent sex reversal (DDS) region on the X chromosome, as well as other autosomal genes (Brown, 2005).

The contribution of chromosomal gender to gonadal gender is illustrated by several paradoxical conditions. The incidence of 46,XX phenotypic human males is estimated to be approximately 1 in 20,000 male births (Page, 1985). These infants apparently result from translocation of the Y chromosome fragment containing TDF to the X chromosome during meiosis of male germ cells (George, 1988). Similarly, individuals with XY chromosomes can appear phenotypically female if they carry a mutation in the TDF/SRY gene. There is evidence that genes on the short arm of the X chromosome are capable of suppressing testicular development, despite the presence of the SRY gene. Indeed, this accounts for a form of X-linked recessive gonadal dysgenesis.

The existence of autosomal sex-determining genes is supported by several genetic syndromes in which disruption of an autosomal gene causes, among other things, gonadal dysgenesis. For example, camptomelic dysplasia, localized to chromosome 17, is associated with XY phenotypic sex reversal. Similarly, male pseudohermaphroditism has been associated with a mutation in the Wilms tumor suppressor gene on chromosome 11.

Phenotypic Gender

Urogenital tract development in both sexes is indistinguishable before 8 weeks. Thereafter, development and differentiation of the internal and external genitalia to the male phenotype is dependent on testicular function. In the absence of a testis, female differentiation ensues irrespective of genetic gender. The fundamental experiments to determine the testis role in male sexual differentiation were conducted by the French anatomist Alfred Jost. Ultimately, he established that the induced phenotype is male and that secretions from the gonads are not necessary for female differentiation. Specifically, the fetal ovary is not required for female sexual differentiation. Jost and associates (1973) found that if castration of rabbit fetuses was conducted before differentiation of the genital anlagen, all newborns were phenotypic females with female external and internal genitalia. Thus, the müllerian ducts developed into uterus, fallopian tubes, and upper vagina.

If fetal castration was conducted before differentiation of the genital anlagen, and thereafter a testis was implanted on one side in place of the removed gonad, the phenotype of all fetuses was male. Thus, the external genitalia of such fetuses were masculinized. On the side of the testicular implant, the wolffian duct developed into the epididymis, vas deferens, and seminal vesicle. With castration, on the side without the implant, the müllerian duct developed but the wolffian duct did not.

Wilson and Gloyna (1970) and Wilson and Lasnitzki (1971) demonstrated that testosterone action was amplified by conversion to 5α-dihydrotestosterone (5α-DHT). In most androgen-responsive tissues, testosterone is converted by 5α-reductase to 5α-DHT. This hormone acts primarily and almost exclusively in the genital tubercle and labioscrotal folds.

Mechanisms of Gender Differentiation

Based on these observations, the physiological basis of gender differentiation can be summarized. Genetic gender is established at fertilization. Gonadal gender is determined primarily by factors encoded by genes on the Y chromosome, such as the SRY gene. In a manner not yet understood, differentiation of the primitive gonad into a testis is accomplished.

image Fetal Testicles and Male Sexual Differentiation

The fetal testis secretes a proteinaceous substance called müllerian-inhibiting substance, a dimeric glycoprotein with a molecular weight of approximately 140,000 Da. It acts locally as a paracrine factor to cause müllerian duct regression. Thus, it prevents the development of uterus, fallopian tube, and upper vagina. Müllerian-inhibiting substance is produced by the Sertoli cells of the seminiferous tubules. Importantly, these tubules appear in fetal gonads before differentiation of Leydig cells, which are the cellular site of testosterone synthesis. Thus, müllerian-inhibiting substance is produced by Sertoli cells even before differentiation of the seminiferous tubules and is secreted as early as 7 weeks. Müllerian duct regression is completed by 9 to 10 weeks, which is before testosterone secretion has commenced. Because it acts locally near its site of formation, if a testis were absent on one side, the müllerian duct on that side would persist, and the uterus and fallopian tube would develop on that side. Female external genital differentiation is complete by 11 weeks, whereas male external genital differentiation is complete by 14 weeks (Sobel, 2004).

Fetal Testosterone Secretion

Apparently through stimulation initially by human chorionic gonadotropin (hCG), and later by fetal pituitary LH, the fetal testes secrete testosterone. This hormone acts directly on the wolffian duct to effect the development of the vas deferens, epididymis, and seminal vesicles. Testosterone also enters fetal blood and acts on the external genitalia anlagen. In these tissues, however, testosterone is converted to 5α-DHT to cause virilization of the external genitalia.

image Genital Ambiguity of the Newborn

Ambiguity of the neonatal genitalia results from excessive androgen action in a fetus that was destined to be female or from inadequate androgen representation for one destined to be male. Rarely, genital ambiguity indicates true hermaphroditism. Several transcription factors—SOX9, SF1, and WT1, and disruptions of signaling molecules—hedgehog, WNT, cyclin-dependent kinase, and Ras/MAP kinase—cause disorders of sexual development. Examples are congenital adrenal hyperplasia and androgen insensitivity syndrome (Larson, 2012). Abnormalities of gender differentiation causing genital ambiguity can be assigned to one of four clinically defined categories: (1) female pseudohermaphroditism; (2) male pseudohermaphroditism; (3) dysgenetic gonads, including true hermaphroditism; and rarely (4) true hermaphroditism (Low, 2003).

Category 1: Female Pseudohermaphroditism

In this condition, müllerian-inhibiting substance is not produced. Androgen exposure is excessive, but variable, for a fetus genetically predestined to be female. The karyotype is 46,XX and ovaries are present. Therefore, by genetic and gonadal gender, all are predestined to be female, and the basic abnormality is androgen excess. Because müllerian-inhibiting substance is not produced, the uterus, fallopian tubes, and upper vagina develop.

If affected fetuses were exposed to a small amount of excess androgen reasonably late in fetal development, the only genital abnormality will be slight to modest clitoral hypertrophy, with an otherwise normal female phenotype.

With somewhat greater androgen exposure, clitoral hypertrophy will be more pronounced, and the posterior labia will fuse. If androgen levels increase earlier in embryonic development, then more severe virilization can be seen. This includes labioscrotal fold formation; development of a urogenital sinus, in which the vagina empties into the posterior urethra; and development of a penile urethra with scrotal formation—the empty scrotum syndrome (Fig. 7-18A).

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FIGURE 7-18 Ambiguous genitalia. A. Female pseudohermaphroditism caused by congenital adrenal hyperplasia. Infant was 46,XX and has severe virilization with scrotal formation without a testis and has a penile urethra. B.Male pseudohermaphroditism caused by incomplete androgen insensitivity syndrome. Infant is 46,XY with external genitalia demonstrating clitoral hypertrophy. C. True hermaphroditism in an infant with 46,XX/46,XY. A hemiscrotum is seen and there are different skin tones on each side. (Photographs contributed by Dr. Lisa Halvorson.)

Congenital Adrenal Hyperplasia. This is the most common cause of androgenic excess in fetuses with female pseudohermaphroditism. The hyperplastic glands synthesize defective enzymes that cause impaired cortisol synthesis. This leads to excessive pituitary ACTH stimulation of the fetal adrenal glands with secretion of large amounts of cortisol precursors, including androgenic prehormones. These prehormones, for example, androstenedione, are converted to testosterone in fetal extraadrenal tissues.

Mutations may involve any of several enzymes, but the most common are steroid 21-hydroxylase, 11β-hydroxylase, and 3β-hydroxysteroid dehydrogenase. Deficiency of the last prevents synthesis of virtually all steroid hormones. Deficiency of either 17β- or 11β-hydroxylase results in increased deoxycorticosterone production to cause hypertension and hypokalemic acidosis. These forms of congenital adrenal hyperplasia thus constitute medical emergencies in the newborn (Fritz, 2011). Currently, 21-hydroxylase deficiency can be diagnosed in utero through molecular fetal DNA analysis. Prenatal maternal dexamethasone administration has been used to reduce fetal genital virilization, however, this is controversial (Miller, 2013; New, 2012) This fetal therapy is discussed in Chapter 16 (p. 323).

Excessive Androgen from Maternal Sources. Transfer of androgen from the maternal compartment may arise from the ovaries with hyperreactio luteinalis or theca-lutein cysts or from Leydig cell and Sertoli-Leydig cell ovarian tumors (Chap. 63p. 1228). In most of these conditions, the female fetus does not become virilized. This is because during most of pregnancy, the fetus is protected from excess maternal androgen by the extraordinary capacity of the syncytiotrophoblast to convert most C19-steroids, including testosterone, to estradiol-17β. The only exception to this generalization is fetal aromatase deficiency, which produces both maternal and fetal virilization (Chap. 5p. 110). Some drugs also can cause female fetal androgen excess. Most commonly, the drugs implicated are synthetic progestins or anabolic steroids (Chap. 12p. 249). Importantly, except those with aromatase deficiency, all with female pseudohermaphroditism can be normal, fertile women if the proper diagnosis is made and appropriate and timely treatment initiated.

Category 2: Male Pseudohermaphroditism

This is characterized by incomplete and variable androgenic exposure of a fetus predestined to be male. The karyotype is 46,XY, and there are either testes or no gonads. In some cases, incomplete masculinization follows inadequate production of testosterone by the fetal testis. It also may arise from diminished responsiveness of the genital anlage to normal quantities of androgen—including failure of the in situ formation of 5α-DHT in androgen-responsive tissue. Because testes were present for at least some time in embryonic life, müllerian-inhibiting substance is produced. Thus, the uterus, fallopian tubes, and upper vagina do not develop.

Fetal testicular testosterone production may fail if there is an enzymatic defect of steroidogenesis that involves any one of four enzymes in the biosynthetic pathway for testosterone synthesis. Impaired fetal testicular steroidogenesis can also be caused by an abnormality in the LH-hCG receptor and by Leydig cell hypoplasia.

With embryonic testicular regression, the testes regress during embryonic or fetal life, and there is no testosterone production thereafter (Edman, 1977). This results in a phenotypic spectrum that varies from a normal female with absent uterus, fallopian tubes, and upper vagina, to a normal male phenotype with anorchia.

Androgen resistance or deficiencies in androgen responsiveness are caused by an abnormal or absent androgen receptor protein or by enzymatic failure of conversion of testosterone to 5α-DHT in appropriate tissues (Wilson, 1978).

Androgen Insensitivity Syndrome. Formerly called testicular feminization, this is the most extreme form of the androgen resistance syndrome, and there is no tissue responsiveness to androgen. More than 800 mutations that cause this syndrome have been reported in the androgen receptor, which is an eight-exon gene on the long arm of the X chromosome (Hughes, 2012; The Lady Davis Institute for Medical Research, 2013).

These result in a female phenotype with a short, blind-ending vagina, no uterus or fallopian tubes, and no wolffian duct structures. At the expected time of puberty, testosterone levels in affected individuals increase to values for normal men. Nonetheless, virilization does not occur, and even pubic and axillary hair does not develop because of end-organ resistance. Presumably, because of androgen resistance at the level of the brain and pituitary, LH levels also are elevated. In response to high concentrations of LH, there is increased testicular secretion of estradiol-17β compared with that in normal men (MacDonald, 1979). Increased estrogen secretion and absence of androgen responsiveness act in concert to cause feminization in the form of breast development.

Individuals with incomplete androgen insensitivity are slightly responsive to androgen. They usually have modest clitoral hypertrophy at birth (see Fig. 7-18B). And at the expected time of puberty, pubic and axillary hair develops but virilization does not occur. These patients also develop feminine breasts, presumably through the same endocrine mechanisms as in those with the complete form of the disorder (Madden, 1975). Insensitivity may also be due to germline missense mutations in the X-linked androgen-receptor gene in the highly structured DNA and ligand binding domains (Lagarde, 2012).

Another group has been referred to as familial male pseudohermaphroditism, type I (Walsh, 1974). Also commonly referred to as Reifenstein syndrome, it constitutes a spectrum of incomplete genital virilization. Phenotypes can vary from a phenotype similar to that of individuals with incomplete androgen insensitivity to that of a male phenotype with only a bifid scrotum, infertility, and gynecomastia.

The gene encoding the androgen-receptor protein is located on the X chromosome. More than 100 different mutations have been demonstrated. This accounts for the wide variability in androgen responsiveness among persons in whom the androgen-receptor protein is absent or abnormal and for the many different mutations associated with one disorder (McPhaul, 1991; Patterson, 1994).

An alternate form of androgen resistance is caused by 5α-reductase deficiency in androgen-responsive tissues. Because androgen action in the external genitalia anlagen is mediated by 5α-DHT, persons with this enzyme deficiency have external genitalia that are female but with modest clitoral hypertrophy. But because androgen action in the wolffian duct is mediated directly by testosterone, there are well-developed epididymides, seminal vesicles, and vas deferens, and the male ejaculatory ducts empty into the vagina (Walsh, 1974).

Category 3: Dysgenetic Gonads

In affected individuals, karyotype varies and is commonly abnormal. As the name describes, most have abnormally developed gonads, and streak gonads are typically found. As a result, müllerian-inhibiting substance is not produced and fetal androgen exposure is variable. The uterus, fallopian tubes, and upper vagina are present.

The most common form of gonadal dysgenesis is Turner syndrome (46,X). The phenotype is female, but secondary gender characteristics do not develop at the time of expected puberty, and genital infantilism persists. In some persons with dysgenetic gonads, the genitalia are ambiguous, a finding indicating that an abnormal gonad produced androgen, albeit in small amounts, during embryonic and fetal life. Generally, there is mixed gonadal dysgenesis—one example is a dysgenetic gonad on one side and an abnormal testis or dysontogenetic tumor on the other. Recombinant human growth hormone (hGH) is approved by the Food and Drug Administration for treatment of short stature in Turner syndrome (Chacko, 2012).

Category 4: True Hermaphroditism

In most cases, the guidelines for category 3 are met. External genitalia of such a case are shown in Figure 7-18C. In addition, true hermaphrodites have both ovarian and testicular tissues with germ cells for both ova and sperm in the abnormal gonads.

Preliminary Diagnosis of the Cause of Genital Ambiguity

A preliminary diagnosis of genital ambiguity can be made at the birth of an affected child. By history, during physical and sonographic examination of the newborn, an experienced examiner can ascertain a number of important findings. These include gonad location, phallus length and diameter, urethral meatus position, degree of labioscrotal fold fusion, and identification of a vagina, vaginal pouch, or urogenital sinus (Fritz, 2011). If the uterus is present, the diagnosis must be female pseudohermaphroditism, testicular or gonadal dysgenesis, or true hermaphroditism. A family history of congenital adrenal hyperplasia is helpful. If the uterus is not present, the diagnosis is male pseudohermaphroditism. Androgen resistance and enzymatic defects in testicular testosterone biosynthesis are often familial.

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