34 - Kidney

Editors: Mills, Stacey E.

Title: Histology for Pathologists, 3rd Edition

Copyright 2007 Lippincott Williams & Wilkins

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43

Placenta

Steven H. Lewis

Kurt Benirschke

Introduction

The placenta is problematic for the pathologist. Many normal histologic variations may be mistaken for pathology and conversely, important pathologic alterations may be difficult to discern. Unique in pathology, the placenta, as a specimen, provides data about two patients and has three different anatomic sources. It is fetal, yet mostly extraembryonic in its differentiation and has maternal attachments. The placenta may be pivotal in adjudicating the etiology of bad babies in litigation (1,2) but more importantly, an objective, thorough, well-documented analysis can provide data important for both maternal and neonatal care. Acknowledging the placenta's many histologic and pathologic varients, its complicated derivation and its role in legal cases, the pathologist should become accustomed to obtaining information from both pediatric and obstetrical providers. The relevance of varients and alterations viewed by the pathologist can thereby be better understood.

These complex considerations cannot be addressed without a thorough understanding of the placenta's normal structure, and it is to this end that this chapter is devoted. Pathologic entities are discussed to better demonstrate normal anatomy and histology. For more encyclopedic and complete discussions regarding placental pathology, the reader is directed elsewhere (1,2). It is easiest and most appropriate to describe (and for that matter, report ) the principal structural components of the placenta in a compartmentalized manner. These consist of the umbilical cord, the membranes (amnion and chorion), the villous parenchyma, and the maternal decidual tissue.

Routine Storage, Examination, and Processing

After obstetric delivery, placentas may be stored at 4 C in a refrigerator before examination. The period of time for this storage generally should not exceed 1 week. Placentas should not be frozen before evaluation as freezing renders the gross examination difficult and histologic features are obscured. For refrigeration, suitable containers include

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cardboard buckets or styrofoam storage cups. It has been advocated by some to immediately fix the placenta in 10% buffered formalin for later examination (3). It should be noted that when this method of processing is used, placental weights increase by a factor of approximately 10% (4).

It should be underscored that although assessment of the placenta is considered in the pathologist's domain, it is the obstetrical provider who first visualizes the specimen. An educated clinician may aid the pathologist by submitting it. Otherwise it becomes discarded. Most institutions do not perform histologic evaluation of all placentas from all deliveries and a discarded placenta can be of no use when problems are associated with pregnancy, labor and delivery, and the neonatal period. Labor and delivery suites should have a list of appropriate clinical and pathologic entities that require pathologic examination of the placenta (5). Because of the frequent turmoil in labor and delivery areas and because problems may often arise in neonates within the first few days of life, the week of storage of all placentas is highly important. It is common for academic institutions to perform histologic examination on approximately 10% to 20% of all delivered placentas.

The gross morphologic assessment of the placenta should be approached in a thorough, routine fashion. Our procedure for the gross assessment and sectioning of placentas is illustrated in Table 43.1.

The placenta is removed from its container and its shape is described. It is usually discoid, but additional lobes may be present. Next, it is convenient to note the location of insertion of the umbilical cord, describe its length and diameter, note irregularities in its contour and texture, describe its color, and note the number of vessels it contains. The cord is then amputated at its base, and representative sections are immersed in fixative.

Attention is next directed to the membranes (amnion and chorion), which are inspected for completeness. Usually, the placenta is delivered vaginally with the membranes surrounding the placental maternal surface ( Schultze ). The membranes are then manually reflected to their normal anatomic position and the smallest distance from the point of rupture to the placental disk (the narrowest width of membranes) is measured. When this measurement equals zero (after vaginal delivery), a low-lying or marginal placenta previa is implicated. The membranes are then assessed for their color, transparency, sheen, and surface irregularities, as well as for the presence of membranous vessels or accessory lobes. The membranes are then removed from the placental disk margin, keeping track of the point of rupture. This point is grasped with a toothed forceps and rolled in a concentric fashion to produce a membrane roll. With the membranes rolled in such a fashion, the point of rupture can be identified histologically. The presence of inflammatory cells confined to this region suggests early mild chorioamnionitis. Representative sections are immersed in fixative.

The fetal surface of the placenta is next examined. Chorionic vascular thrombi, if present, and nodules or irregularities of the amnion are noted. The maternal surface of the placenta is inspected, and any blood clot that has settled in the storage container with the dependent portions of the organ is removed. Areas of blood clot that are adherent or discolored brown (indicating chronicity) and that are depressing the maternal surface are considered indicative of retroplacental hemorrhage (clinically designated as abruptio). Should this be noted, the dimensions or percentage of the maternal surface involvement are recorded. The organ is next weighed free of its cord and associated membranes. The average weight of the term placenta is approximately 400 to 600 g. Placental weight varies with neonatal weight and normal weights have been reviewed for all gestational ages (1). The average dimensions of the term placenta are approximately 18 16 2.3 cm.

The villous parenchyma is then inspected by sectioning the placenta at 1- to 2-cm intervals looking for irregularities in the parenchyma that indicate infarction, thrombi, or other pathologic entities. There are normally 16 to 20 cotyledonous units that do not have distinct functional correlates. An absent cotyledon may indicate a portion of retained placenta in utero. Representative sections of abnormal areas are blocked out, and areas of normal-appearing parenchyma (usually three) are placed into fixative along with the already sectioned membrane roll and umbilical cord.

The fixation of the materials for study is routine. We prefer to fix tissues in Bouin's solution for a period of 24 hours before trimming and submission for final processing. This process of fixation allows for excellent tissue penetration and ease of sectioning. In addition, it provides superior cytologic detail. The drawbacks to the Bouin's fixation are twofold. One is that if certain special studies are of interest (i.e., immunohistochemistry and in situ hybridization), Bouin's fixation may interfere with antigen antibody reactions or nucleic acid hybridization. This problem may be eliminated by fixing tissues desired for such studies in Bouin's solution for only a limited period of time. For small uterine cervix biopsies, it has been shown that Bouin's fixation for less than 8 hours produces good results with in situ hybridization (6). The second problem with Bouin's fixation is that if in the final preparation lithium carbonate is omitted, increased extraneous pigment formation occurs.

Pathologists are accustomed to using 10% buffered formalin solution for the processing of most tissues, and this is not contraindicated in the processing of placentas. We find that when using formalin, the period of time required to create sufficient tissue hardness for proper sectioning delays processing and histologic resolution is somewhat inferior to that obtained with Bouin's fixation. It is our

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procedure to stain tissue sections with hematoxylin and eosin (H&E) or hematoxylin-phloxine and saffron (HPS). Other standard special stains may be used for the detection of specific infectious agents, secretory activity, or structural composition (silver stains, periodic acid-Schiff (PAS), Masson's trichrome, etc.). Furthermore, a host of immunohistochemical stains have been used to elucidate functions of specific placental cell types.

Table 43.1 Recording Format^a

		Unit No:
		Name:
		Date of Birth:	Sex:
		Location:
		Path. No:
		Date of Delivery:
		Date Received:
		Physician:
		Baby's Unit No:
Previous Specimens:
SPECIMEN: Placenta
CLINICAL INFORMATION: (Circle and fill in pertinent information)
NSVD C-section	GA: ________ wk	DM class _______
Chorioamnionitis	Preeclampsia	Fetal distress
Newborn wt. _______ grams	5 min Apgar <7	Other: _________
GROSS DESCRIPTION:
Cord:	____ ____ cm	Insertion:
	# pieces: ______	Vessel #: ______
Membranes:	complete/incomplete	Narrowest width: ______ cm
	clear/opaque	Meconium: old/recent/none
	vascular thrombi: present/none	Calcification: present/none
Parenchyma:	red/pale/friable	Abruptio: __________ %
	Infarct: ______ %	Weight: __________ grams
Dimensions:	____ ____ ____ cm
Other:
MICROSCOPIC DESCRIPTION:(___) slides evaluated.
DIAGNOSIS(ES):
Umbilical cord:
Membranes:
Villi:
Decidua:
		Reviewed by:
Date Dictated:
Date Typed:
Print Date:
a This format is easily converted to a computerized final report that includes final microscopic diagnoses.

Umbilical Cord

Embryology

Specific embryologic considerations are germane to the understanding of the normal umbilical cord structure, including its frequent possession of embryologic remnants. The open region on the ventral surface of the developing

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embryo diminishes in size and then forms the early umbilicus. Through this structure extend both the yolk stalk and the body stalk, as well as the allantois. This cylindrical structure elongates, and its surface becomes covered by the expanding amnion. This is a single-layered epithelium on a layer of connective tissue. Therefore, the developing umbilical cord contains the yolk stalk, a pair of vitelline blood vessels, the allantois, and the allantoic blood vessels (two arteries and one vein) and is covered by amnionic epithelium. These anatomic relationships explain the presence of the omphalomesenteric duct (the connection between developing endoderm and the yolk sac) and the allantoic duct (which has its communication in early gestation with the urachus) within sections of proximal (fetal) umbilical cord.

Gross Morphology

The gross anatomic features of the umbilical cord that are of importance are the location of its insertion in the placental disk, its length, and the number of vessels. The presence of true knots (Figure 43.1) may be considered normal when there is no adverse outcome, yet this occurrence may lead to fetal demise when the knot is tight. The presence of vascular tortuosities (false knots) is common and rarely of clinical significance (Figure 43.2). The finding of meconium staining and the presence of surface plaques are definitely abnormal and are described below.

The normal umbilical cord is pearly white and somewhat translucent. The length of the umbilical cord has great significance, principally when it is excessively long or excessively short. Cord length has been shown to vary with gestational age, and measurements indicate that the cord elongates as gestation proceeds. At approximately 20 weeks' gestational age, the average cord length is 32 cm (7). The normal length of the umbilical cord at term has been determined to be, on average, between 55 and 65 cm (1,7,8,9,10) (Figure 43.3). The literature contains many articles that relate the significance of abnormal cord lengths with both in utero fetal activity and neonatal outcome. The reader is referred to an extensive review of the subject (1).

Figure 43.1 True knot, in this case, resulted in intrauterine fetal death. Reprinted with permission from:

Benirschke K, Kaufmann P. Pathology of the Human Placenta. 3rd ed. New York: Springer-Verlag; 1995.

Figure 43.2 False knot. Note the unrelated abnormal membranous vessels connecting placental lobes.

Histology

Histologic examination of the umbilical cord shows several distinct layers. On the surface is a well-defined single layer of amnionic epithelium. The epithelium is squamoid and, in the region of fetal cord insertion, often becomes multilayered and closely resembles its epidermal contiguity. Electron microscopy studies performed on cord amnionic cells

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have suggested that the epithelium is responsible for fluid equilibrium activities (11). True squamous metaplasia of the umbilical cord is considered a normal variant, and ultrastructural studies of this epithelium have shown morphologic similarities between this epithelium and the fetal epidermis (12).

Figure 43.3 Normal cord length dimensions associated with changes in gestational age. Reprinted with permission from:

Benirschke K, Kaufmann P. Pathology of the Human Placenta. 3rd ed. New York: Springer-Verlag; 1995.

Deep to the amnionic epithelium that comprises the surface of the cord is the substance of Wharton's jelly. This material largely is composed of mucopolysaccharides (hyaluronic acid and chondroitin sulfate). Ultrastructural examination of this material shows the presence of delicate interlacing microfibrils and sparse collagen. Mast cells are prominent. Their frequency is increased in the near periphery of the cord vasculature (13). In this same region, and in the cord in general, macrophages are rarely identified.

Embedded within the substance of Wharton's jelly are the umbilical vessels. There have been considerable interest and discussion focused on the identification of vasa vasorum and vascular neuronal innervation. Although the vasculature of the umbilical cord is of a considerable caliber, there are no vasa vasorum or lymphatic channels present in this structure. Studies investigating vascular innervation have concluded that no nerves are present within the umbilical cord. This has been borne out by electron microscopic studies (14). Occasionally, however, autonomic nerves are identified using acetylcholinesterase thiocholine techniques in the proximal (fetal) end of the cord (15). Such findings are compatible with the persistence of peripheral vagal neuronal elements associated with the ductus venosus, which are entrapped in the proximal portion of the umbilical cord. Certainly any neuronal vestiges found within the umbilical cord are best considered as remnants, and to date there has been no demonstration of their functional significance (16).

Since the yolk sac connects to the primitive midgut through the body stalk in early development, vestiges of this epithelium-lined duct are common in the umbilical cord. The persistence of the omphalomesenteric duct is characterized by a tubular structure present within Wharton's jelly and lined by a single layer of low cuboidal to columnar, mucin-secreting epithelium (Figure 43.4A D). Remnants of the duct may form cystic structures that contain a variety of endodermally-derived epithelia, including pancreatic, intestinal (small and large), and gastric components. Such findings are rarely of any clinical significance, although secretory products of gastric origin resulted in umbilical vascular ulceration, hemorrhage, and fetal death in a case report described by Blanc and Allan (17).

The allantois differentiates as a protuberance from the yolk sac into the body stalk and is essential for the development of the umbilical vessels. This structure is incorporated into the anterior aspect of the hindgut, where it communicates with the urachus. Remnants of the allantoic duct are often found in sections of proximal umbilical cords. Its intimate relationship with the formation of umbilical vessels explains its presence between the two umbilical arteries, when it is identified. These remnants rarely have clinical significance. The lining of this tract is often devoid of a lumen and consists of aggregates of epithelial cells with a variety of epithelia represented (transitional, bladder, and yolk sac derived endodermally classified cells) (Figure 43.5).

The vasculature of the umbilical cord is composed of two arteries and a single vein. The arteries possess no internal elastic lamina and have a double-layered muscular wall. Each of these muscular layers is composed of a network of interlacing smooth muscle bundles. The vein does have an inner elastic lamina. As noted, no vasa vasorum are present. Remnants of the vitelline vasculature in the proximal portion of the cord sometimes may be observed in sections taken from this region. The umbilical vein, which generally has a larger diameter, possesses a thinner muscular coat consisting of a single layer of circular smooth muscle (Figure 43.6A, B).

Of further interest, distinguishing umbilical vasculature from other systemic vessels, is that no true vascular adventitia is found. Near the placental insertion, it is common to identify anastomotic channels between the two umbilical arteries (18,19) (Figure 43.7).

Transverse serial sections confirm that two umbilical arteries spiral in parallel around the umbilical vein. Often, multiple twists in the cord occur. The proposed origin of this spiraling has been extensively discussed; however, its true functional significance and origin remain to be definitively elucidated (1).

Pathologic Alterations

Distinguishing normal anatomy from pathologic entities is the essence of proper understanding of the normal anatomy and histology of the umbilical cord. Most pathology of the cord may be seen in the gross sense. Histology is confirmatory. A tight knot with notching indicating stricture associated with proximal vascular dilatation may result in fetal death. Interestingly, although true knots occur frequently and are associated with long cords, adverse outcomes are rare events; therefore, in most instances a true knot can be considered a normal variant. The absence of an umbilical artery is a well-established observation and easy to identify grossly or in histologic sections (Figure 43.8). This phenomenon has been found in approximately 1% of neonates. The association of this finding with congenital anomalies is well known, and these malformations often take the form of urinary tract malformations.

Persistence of a second (right) umbilical vein is an unusual phenomenon. The pathologist is cautioned in this regard. It is not unusual to find histologic sections identifying more than three vessels in an umbilical cord. This finding is related to commonly identified tortuosities. These tortuosities have been termed false knots (Figure 43.2) and have

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little clinical significance. An exception is that these vessels rarely may be prone to thrombosis.

Figure 43.4 Omphalomesenteric duct remnant (A) with enteric epithelia (B). Omphalomesenteric duct adjacent to umbilical vein (C) with unusual finding of hepatic tissue (D).

Figure 43.5 Allantoic duct remnant hematoxylin and eosin (H&E stain).

The presence of thrombotic material in the vasculature of the umbilical cord is truly pathologic. The process may be related to the genesis of a single umbilical artery when it occurs in early gestation. Abnormal umbilical insertions may cause thrombosis.

Velamentous cord insertions are abnormal and are characterized by the presence of the umbilical vasculature implanting in the placental membranes as opposed to the usual implantation over the placental disk (Figure 43.9). These vessels course independently within the chorion and are unguarded by the protective substance of the umbilical cord (Wharton's jelly). Thrombosis thus results from pressure on those vessels by fetal parts, and these vessels are subject to injury at the time of spontaneous or, more commonly, artificial membrane rupture.

Figure 43.6 Umbilical vein (A) and artery (B) [hematoxylin-phloxine and saffron (HPS) stain].

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Other abnormalities and pathologic findings of the umbilical cord (certainly to be distinguished from normal morphology and histology) that are of clinical importance are umbilical cord vascular rupture, complete absence of Wharton's jelly (Figure 43.10), and neoplasms of the umbilical cord [hemangioma (Figure 43.11) and teratoma, both unusual findings].

Most significant when considering normal histologic changes in the umbilical cord is the presence of hemorrhagic material in the perivascular region, which would suggest umbilical cord vascular rupture. Although true cord hematomas do occur on occasion (Figure 43.12), the presence of hemorrhage in this region is common and generally attributed to the mode of delivery of the placenta, with traction or clamping of the umbilical cord producing this artifactual finding (Figure 43.13).

Umbilical torsion and stricture are associated with excessive fetal movement and focal absence of Wharton jelly,

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respectively. Both are associated with adverse outcomes (1). In the former, the normal twist or coiling of the cord becomes excessive. There is an association with long cords. The latter is less well understood but may at times be a function of torsion (Figure 43.14). Additionally, excessive coiling has been associated with increased fetal activity, cocaine use, abnormal fetal heart rate tracings and preterm deliveries (20). Nascent dimensions of cord width are therefore germane. There is little literature that actually defines the dimensions of normal cords, although published data correlate abnormalities associated with fat and thin cords (7,8) (Figure 43.15).

Figure 43.7 Normal proximal anastomosis of umbilical arteries rendering the appearance of a single umbilical artery.

Figure 43.8 Single umbilical artery (HSP stain).

Figure 43.9 Velamentous insertion of umbilical cord. Umbilical vessels insert in membranes adjacent to the chorionic plate. In this case, the fetus exsanguinated after amniotomy and rupture of membranous vessels.

Figure 43.10 Furcate insertion of umbilical cord. Umbilical cord vessels insert into placental substance individually (UA, left; UA and UV, right) not surrounded by Wharton's jelly.

Another definitively pathologic entity that must be distinguished from normal histology is the presence of leukocytes within the cord substance. Such findings are indicative of funisitis and are the result of inflammatory response to infectious antigens and recruitment through inflammatory pathways (Figure 43.16). When the process is prominent (severe) and with calcifications, the term necrotizing funisitis is applicable. Such severe pathology is indicative of chronic inflammation and may be seen in syphilis as well as other infections (1) (Figure 43.17). The identification of fungal elements about the umbilical cord are often difficult to discern from an overgrowth storage phenomenon. In this regard, the difficulty lies in the usual absence of associated inflammatory infiltrate. The cord, when involved, has white surface plaques. Fungal elements (i.e., Candida albicans) may be identified merely with hematoxylin and eosin stains, although special stains for fungi can be helpful when such pathology is suspect (1).

Figure 43.11 Dilated vascular lumina of umbilical cord hemangioma (HPS stain).

Figure 43.12 Hematoma of umbilical cord. This placenta was delivered by cesarean section and there was no traction or clamping of this segment of cord.

Figure 43.13 Perivascular hemorrhage located adjacent to umbilical artery found in the region of a cord clamp (HPS stain).

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Figure 43.14 Excessive spiraling (A) leading to torsion (B) and stricture resulting in fetal death.

Figure 43.15 Mean cord width (as determined by ultrasound 2 cm from umbilical insertion) versus advancing gestational age (n = 100, p < 0.05). Reprinted with permission from: Lewis SH, Starr C. Cord width. In Utero (Unpublished).

Figure 43.16 Acute funisitis. Polymorphonuclear leukocytes are present within the umbilical vein muscularis and adjacent Wharton's jelly.

Last, a finding in the cord that is notably pathologic is meconium-induced medial destruction (Figure 43.18A, B), which results from direct meconium toxicity and necrobiosis of vascular media (21). Associated vascular spasm and medial degeneration may adversely affect hemodynamics in the cord and chorionic vasculature.

Ramification of Chorionic Vasculature

At this point it is convenient to discuss the ramification of the umbilical vessels in the chorionic plate. The umbilical cord inserts in a central or eccentric fashion. Although abnormal insertion at the margin (Battledore) and in the membranes (velamentous) comprises a small portion of cord insertions, both should be considered pathologic

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and not normal variants insofar as they have been attributed to adverse outcomes when extensively analyzed (22).

Figure 43.17 Necrotizing funisitis with intense perivascular malformations associated with calcification (peripheral white circular bands) may be seen in the gross.

Figure 43.18 Globular degenerated necrobiotic medial cells so effected by meconium. The process is focal and contrasts with adjacent normal myocytes. Luna Ishak stain (A). Intensified magnification for delineation (B). Reprinted with permission from:

Rana J, Ebert GA, Kappy KA. Adverse perinatal outcome with an abnormal umbilical coiling index. Obstet Gynecol 1995;85:573 578.

The pattern of vascular ramification within the chorion is described as either magistral (characterized by large-diameter vessels, radially diminishing in caliber to the periphery of the placenta) or disperse (characterized by multiple small vessels emanating directly from the cord insertion site). It is of interest that in the chorionic vasculature, no distinction can be made between branches of the umbilical vein and umbilical arteries using histologic criteria (in counterdistinction to the aforementioned description of differentiation between vein and artery in the umbilical cord). The only means of identifying which vessels are branches of arteries and which are veins is by noting their gross anatomic distribution. Arteries always cross over veins when observed from the fetal surface (Figure 43.19). The notation of such vascular relationships is of extreme significance when considering vascular anastomoses, as may be seen in some twin pregnancies (23).

Figure 43.19 As early as the 1600s, Nicholas Hoboken recognized that chorionic arteries (H) overlie veins, and this was beautifully depicted in his painstaking drawings. These are the earliest accurate drawings of the human placenta known to exist.

The primary branches of the umbilical vasculature that course through the chorionic plate periodically dive beneath this stratum to establish the circulation of primary vascular ramifications ending in the terminal villi.

Pathologic Alterations of the Chorionic Vasculature

Abnormalities in the chorionic vasculature are similar to those found in the umbilical cord, the most significant being thrombosis of a chorionic vessel. During the gross examination of placentas so affected, the presence of thrombotic material may readily be identified by noting dilated vessels containing firm thrombotic substance. On the other hand, vascular thrombi may be more subtle and their appearance characterized only by the presence of faintly highlighted linear white streaks that parallel the peripheral

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margin of vessels involved (Figure 43.20). These findings can be confirmed histologically.

Figure 43.20 Chorionic vascular thrombosis characterized by a linear streak paralleling the vascular course (in this case a chorionic vein). Reprinted with permission from:

Benirschke K, Kaufmann P. Pathology of the Human Placenta. 3rd ed. New York: Springer-Verlag; 1995.

The presence of polymorphonuclear leukocytes migrating from the chorionic vasculature and from the umbilical vasculature is pathognomonic of chorionitis and umbilical cord funisitis, respectively. Findings of chorionitis are histologically similar to those aforementioned in acute funisitis (Figure 43.16).

Membranes

Embryology

The placental membranes consist of the amnion and chorion. The amnion, which constitutes the innermost aspect of the embryonic cavity, develops from the margin of the embryonic disk. As the embryonic disk begins to take the form of a tube, the amnionic periphery also folds inward and its attachment to the ventral body is defined. The amnionic cavity subsequently develops by the process of cavitation. Elongation of the body stalk coincides with embryonic prolapse into the amnionic compartment. As development proceeds, the resultant cavity expands, and by 12 weeks from the last menstrual period the amniotic cavity completely occupies the chorionic sac. At this point, fusion occurs with the chorionic wall. This event is commonly identified by clinicians via routine ultrasonographic analysis of advancing gestations. At this gestational age, the potential space between the chorion and amnion is visibly obliterated. The amnionic cavity remains filled with amnionic fluid, which by the end of gestation amounts to approximately 1 L.

The chorion forms the base for the peripherally radiating villi and serves to encapsulate the embryo and developing amnion. As the early implantation embryo develops, the embryonic tissues (the trophoblast and its mesodermal investments) continue to expand in a spherical fashion. The inner aspect of the condensation of mesoderm, which forms the inner capsular structure deep to the peripheral trophoblast, is also termed the chorion. In the region that becomes the placental disk proper, chorionic villi continue to develop beneath these structures, and the placenta proper or the chorion frondosum is defined. The region of the chorion that covers the expanded amnionic cavity forms what has been termed the chorionic laeve. This constitutes the reflected membranes and is discerned from the membranous covering of the chorionic plate. Chorionic villi in the region of the laeve (which delimits the sac containing amnionic fluid) atrophy by pressure, although remnants of villous tissue may be found in association with this structure. In the region of the chorion frondosum, the fetal blood vessels invest the chorionic plate. Such vessels only occur in the chorion; the amnion is an avascular structure.

Amnion and Chorion

Gross Morphology

The fetal membranes have a particular and characteristic appearance in normal deliveries. The sac, when viewed from the fetal surface, is clear and often has a bluish hue, and the amnion is devoid of vasculature. Remnants of atrophied vasculature may be seen in the overlying chorion and appear as filamentous streaks. The chorionic plate also has a characteristic blue sheen and, as described previously, the distribution of chorionic vessels has a characteristic appearance. The membranes of the chorionic plate are distinguished from the laeve as described above. It is not infrequent to find a peripheral nodule on the surface of the disk membranes. This normal nodule is the remnant of the fetal yolk sac (Figure 43.21).

Figure 43.21 White nodule is the residua of the fetal yolk sac.

Figure 43.22 A. Circumvallate placenta. B. Note loose association of amnion peripheral to marginal subamnionic fibrin deposition. Reprinted with permission from:

Benirschke K, Kaufmann P. Pathology of the Human Placenta. 3rd ed. New York: Springer-Verlag; 1995.

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Gross Morphologic Alterations

Although chorionic vessels are normal in the chorion of the chorionic plate overlying the disk, the persistence of functional vasculature in the chorion laeve is aberrant and equates to membranous vessels. These vessels may connect lobes of placenta or relate to the membranous insertion of the umbilical cord (velamentous insertion as described above).

On occasion, the chorionic plate may possess a ring of fibrin that forms a concentric ridge between the insertion of the cord and the margin of the placental disk. This fibrin ring, which lies deep to the amnion, is indicative of an extrachorial placentation. Such a placentation is characterized by two forms: the circumvallate placentation and the circumarginate placentation. In the former, the membranes are reflected upon themselves at the ridge of the fibrin deposition. They then cover the remaining margin of the placental disk in a loose fashion (Figure 43.22A, B). In the circumarginate placenta, the ring of fibrin is present over the chorion, and the overlying amnion is not reflected upon itself at this fibrinous ring (Figure 43.23). The amnion thus extends to the margin of the placental disk, and its departure to form the amnionic sac occurs at this margin. It is currently felt that this fibrinous ring represents placental migration in conjunction with an enlarging uterus during the second trimester (so-called trophotropism) (1).

Figure 43.23 Circumarginate placenta. Note the close association of amnion to the disk peripheral to the fibrin ring.

The common occurrence of squamous metaplasia on the amnionic surface can be identified grossly by its characteristic appearance. It is a normal finding unless its presence is extensive. Immersion of the placental membranes in water generally defines this area by its failure to become moist as opposed to the normal surrounding amnion. Thus, these areas are more clearly defined. In pathologic conditions, metaplasia

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in these regions may be pronounced, and large plaques and nodules may form (Figure 43.24). These nodules are distinguished from the truly pathologic condition of amnion nodosum by their failure to be easily denuded from the surface of the amnion by slight mechanical pressure.

Figure 43.24 Extensive squamous metaplasia on the amnionic surface from a fetus with an encephalocele. It is believed that irritation from the encephalocele in this region produced the extensive metaplasia.

The presence of amnion nodosum is characterized in the gross sense by the presence of multiple small papules on the amnionic surface (Figure 43.25). The clinical history is suggestive, and oligohydramnios characterizes these gestations. The small papules are easily removed from the amnionic surface by excoriation, and their substances are confirmed histologically by the presence of debris and degenerated squames. The origin of these cells is fetal epidermal, and their presence on the amnionic surface is related to apposition of this membrane and fetal skin in conditions where there is diminished amnionic fluid.

Amnionic bands are rare. The condition is responsible for in utero fetal part amputation and trauma and is a phenomenon that occurs in approximately one in 10,000 births. The occurrence is important because it demonstrates potential difficulties from abnormal amnionic membrane development. The precise mechanism is not known in most cases, but rupture of the amnion (most probably in the first trimester) allows the fetus to enter the chorionic sac. The remnants of amnion form the substance of the resulting amnionic bands. At term these placentas have highly opaque chorionic surfaces that reflect hyperplasia of this uncovered layer. The remaining amnionic epithelium is densely adherent to the umbilical cord from which it cannot be stripped. Only small amounts of amnion are present, which distinguishes this condition from artifactual disruption of the amnion from the chorionic plate during the delivery process (1,24,25). Occasionally, an abnormal web will be present at the base of the cord insertion, and this may limit normal cord movement (5) (Figure 43.26).

Figure 43.25 Multiple papules of amnion nodosum stipple the amnionic surface of this placenta from a gestation characterized by oligohydramnios.

Figure 43.26 Amniotic web partially immobilizes the cord by limiting its movement at the cord base.

Amnion Histology

The amnion, the innermost layer of the amnionic cavity, is lined by a single layer of epithelial cells that resides on a basement membrane. The basement membrane is attached to an underlying thin layer of connective tissue (26) (Figure 43.27). The amnion, although adjacent to the chorion, is not truly fused to it and may be separated with minimum effort. This juxtaposition of the two membranous layers occurs at 12 weeks' gestational age (27) (Figure 43.28). Before this time, as the amnion develops, it is separated from the chorion by the so-called magma reticulare, which is a viscous and thixotropic gelatinous fluid. Stellate mesenchymal cells may be found within this subtance. These cells also have epithelial characteristics and have been stained immunohistochemically and found to be cytokeratin and vimentin positive (28).

The epithelial cell layer of the amnion is composed of one distinct cytologic type (29). The epithelium is a single layer, squamoid to cuboidal, and devoid of secretory activity. Ultrastructural studies show extensive microvillous projections (30). Multiple vesicular structures have been identified at the base of these epithelia. It has been postulated that these vesicles represent pinocytotic activity (11). This observation is important because, as stated earlier, the amnion possesses no vasculature. This also pertains to its mesenchymal component. Therefore, the cytologic components of this layer gain their nutrition from adjacent amnionic fluid, which in turn is rich in nutrients from transudation (from fetal vasculature) and fetal excretory products. In early gestation, this nutrition is derived from the magma reticulare. Channels that also have been considered responsible for fluid transmission (31) are felt to be the residua of epithelial cell loss. A postulate relating to cell loss may invoke a newly revitalized theoretical discussion (initiated by Virchow) that describes apoptosis as a form of programmed cell death. Such epithelial loss or cell death by suicide also has been noted in placental

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components, including the wall of the yolk sac and in the endothelium of umbilical veins. Further investigation in these areas may enhance understanding of developmental biology and with refinement may be used for addressing future methodologies for the treatment of neoplasia (32,33).

Figure 43.27 Flattened to cuboidal amnionic epithelial cells (E) adhere to their basement membrane (B). Beneath this is the compact layer of the amnion (C), which is acellular and may form a barrier to PMNs. The compact layer is rarely affected by edema and is probably the strongest amnionic layer. A fibroblastic layer (F) lies beneath the compact layer, and macrophages may be found. A spongy layer (S), relatively devoid of fibroblasts, separates the amnion from the chorion, although the two may merge imperceptibly. Often, an artifactual separation may be present near the plane of true fusion. The amnion usually measures from 0.2 to 0.5 mm in thickness (1). The most superficial layer of chorion is usually an incomplete cellular zone (l) that overlies a thick reticular layer (R). This layer is composed of fibroblasts and macrophages. Beneath the reticular layer is a pseudo basement membrane (PB) overlying trophoblastic X cells (X) and then maternal decidua (D) (HPS stain).

Figure 43.28 The separation between amnion and chorion is more apparent in early gestations as seen in this section from the chorionic plate of a 10- to 12-week placenta. The amnion is readily distinguished from the underlying chorion, which contains the easily identifiable chorionic vessels. Mesynchymal components are prominent (H&E stain).

Amnionic epithelial cells are attached to one another by desmosomes in freeze-fracture experiments (34). Furthermore, the amnionic epithelium attaches to the underlying basement membrane by hemidesmosomes (35).

Amnionic epithelial cells divide by mitosis (36). On occasion, multinucleated cells are identified. Morphometry studies have demonstrated that polyploid cells exist in this layer (37). Other karyotypic anomalies occur, and amniocentesis for chromosomal defects may yield false-positive results when these amnionic cells contaminate preparations (38).

Although the epithelium of the amnion does not actively secrete, lipid droplets have been noted within these cells, an observation that correlates with increasing gestational age (36,39,40). Glycogen also has been found within amnionic cells.

Squamous metaplasia is a common occurrence in the amnionic epithelium, especially near the insertion of the umbilical cord (Figure 43.29). This epithelium may become keratinized, and keratohyalin granules can be identified. Although this appears to result from irritation of the amnionic epithelial surface, these changes can be found in more than half of all term placentas (1).

Beneath the basement membrane of the amnionic epithelium, an additional component of the amnion is identified. This layer principally is divided into a compact and a fibroblastic region. The connective tissue within this region may harbor macrophages, which have been identified within the first trimester of pregnancy (41).

Figure 43.29 Squamous metaplasia of the amnion with hyperkeratosis (HPS stain).

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Amnion Histopathology

Histologic abnormalities of the amnion are heralded by an abnormal gross appearance. For example, membranes that are stained green may reflect deposition of meconium. Amnionic membranes that are white may be indicative of polymorphonuclear leukocyte infiltration and acute chorioamnionitis (26) (Figure 43.30A, B).

Abnormalities of amnionic epithelial cells, although suggested by gross examination, can be confirmed by histologic assessment. Abnormalities that reflect degenerative changes are characterized by the presence of vacuolated cytoplasm and elongation to columnar forms. A rather rare and unusual finding associated with gastroschisis carries a pathognomonic histologic aberrancy of the amnion whereby amnionic epithelial cells contain innumerable vacuoles (1) (Figure 43.31).

Amnionic epithelial degeneration (Figure 43.32) is characteristic when meconium is present. Such findings may be confirmed when macrophages, present within the amnionic layer, contain meconium (a coarse brown pigment), which does not stain for iron (Figure 43.33). On the other hand, hemosiderin deposition may be found within the amnionic layer in macrophages, and this can be confirmed by the use of iron stains (i.e., Prussian blue).

Although there are no true tumors of the amnion, occasional cysts representing edema may be identified. Although they may be striking in their gross appearance, it must be recognized that no clinical significance can be identified. Occasional cysts of ectodermal and mesodermal tissue have been identified deep to the amnionic layer, but such findings are not considered true neoplasms. Teratomas have been described (42). These lesions probably represent degenerative acardiac twins (1), but the lack of directed differentiation from pluripotential stem cells could account for the former.

The papules of amnion nodosum are clearly pathologic and reflect, in most cases, decreased amnionic fluid. In such

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instances, vernix (desquamated epithelia from the fetus) forms minute nodules on the amnionic surface in this characteristic fashion (Figure 43.34). The lesions are composed of acellular debris and remnants of cells (43). The material within these papules is PAS and alcian blue positive.

Figure 43.30 Acute chorioamnionitis in membrane roll (A) (HPS stain) and in chorionic plate (B) (H&E stain).

Figure 43.31 Unusual vacuolated elongated amnionic epithelial cells pathognomonic of gastroschisis. The pathophysiology is uncertain (H&E).

Figure 43.32 A twisted membrane roll with amnionic epithelial degeneration (above) and normal amnionic epithelium (below) (HPS stain).

Figure 43.33 Amnionic epithelial degeneration overlies pigment-laden macrophages containing meconium (HPS stain).

Chorion Histology

The chorion is composed of a connective tissue membrane that carries the fetal vasculature. Its inner aspect is bounded by the outer layer of the amnion, and the outer aspect is directly associated with the trophoblastic villi that sprout from its surface. There are two distinct aspects of the chorion: the chorion frondosum and the chorion laeve. The chorion of the reflected membranes (chorion laeve) is composed of an inner cellular layer, a reticular layer, a pseudo-basement membrane, and an outer trophoblastic layer (44) (Figure 43.27). The precise origin of the mesenchymal component of the chorion is not clear, but it is believed that this connective tissue is derived from the primitive streak and not the trophoblast (45,46) (Figure 43.28). Electron microscopy studies have shown the connective tissue cells adjacent to the amnion to be rich in endoplasmic reticulum (47,48). Macrophages and degenerative endothelial cells also have been described as part of the cytologic makeup of this layer (48). Acid mucopolysaccharides are prominent within the connective tissue matrix of the chorion (49). Although present in other regions of the placenta, type VI collagen is a prominent constituent of the chorionic layer (50). The chorion frondosum is similarly constituted but contains functional chorionic vessels and is bordered deeply by functional villi.

Figure 43.34 Amnion nodosum (H&E stain).

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Chorion Histopathology

Pathology of the chorionic vasculature (described above in the context of its umbilical cord continuity) includes chorionitis and thromboses. The fetal chorionic vessels allow permeation of polymorphonuclear cells (PMNs) in response to intraamnionic bacterial antigens. Maternal PMNs also may be seen in the chorion laeve. In both cases, the amnion is later affected (Figure 43.30).

Chorionic cysts, which do not truly arise from the chorion proper, are derived from trophoblastic cytologic components (X cells). Multiple cysts may be present, bulging the fetal surface of the disk. These may appear terribly abnormal but in fact carry no significance in the form of true pathology (Figure 43.35). These cysts, which are commonly found in the chorionic plate and within placental septa, are discussed further below.

Multiple Gestation

The normal relationship of the placental membranes are germane to the understanding of twin or multiple-gestation placentations. These relationships are described briefly, but for a more complete discussion of twinning and associated pathological conditions, readers are referred to an extensive and detailed review of the topic (1).

The majority of twin placentas (incidences show a dependence on geographic location and ethnic background) are dizygotic. The dizygotic twin placenta has a variety of presentations: separate placentas or fused placentas. In the latter, the intervening membrane should be studied to distinguish dichorionic from monochorionic twin placentas. The intervening membrane of about 70% of monozygotic twin placentas is devoid of a chorion, and the term diamnionic monochorionic (DiMo) is applicable (Figure 43.36). All DiMo placentas are monozygotic. In these gestations (DiMo), the shared chorion invests only the chorionic plate and is not present in the intervening membrane. In DiMo placentas, shared vascular districts between placentas are possible, and vein-to-vein and artery-to-artery anastomoses are the most common. Artery-to-vein anastomoses are relatively infrequent and are the etiology of the twin twin transfusion syndrome (Figure 43.37A D).

Figure 43.35 Multiple normal chorionic cysts.

Figure 43.36 Intervening membrane from diamnionic monochorionic twin placenta. Note the absence of an intervening chorionic layer (HPS stain).

The diamnionic dichorionic (DiDi) placenta is distinguished morphologically by examining the intervening membrane and noting the presence of two fused chorions beneath the two amnionic layers (Figure 43.38A, B). Most of these placentas are dizygotic; however, approximately 30% of DiDi twin placentas result from monozygotic twin implantations and are the result of splitting within 3 days of fertilization. Vascular anastomoses are reportable.

The complete absence of an intervening membrane (monoamnionic monochorionic) in a twin gestation is also diagnostic of monozygotic twins. There, splitting of the embryo occurs later in gestation (at approximately 7 days of age), and although twin twin transfusions can occur, these are less common than in DiMo placentations. The significant pathologic problems from these placentations result from cord entanglements, and fetal death is common. Even later separations result in fused twin fetuses (Siamese twins).

Villi

Embryology

After formation of the blastocyst, the trophectoderm gives rise to extraembryonic trophoblastic villi. The organization of the inner cell mass gives rise to the embryo proper. The trophoblastic derivatives of the early implantation embryo

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are best characterized by discussing their structures and cell types. The trophoblastic villus forms the functional unit of the placenta. In the first trimester, trophoblastic villi are composed of an outer syncytiotrophoblastic layer and an inner cytotrophoblastic layer encompassing villous mesenchyme in which the fetal vasculature differentiates. Although the majority of the villus is surrounded by the characteristic two-cell layers, a polarity to the villi can be identified and their basal implantation regions are composed of additional trophoblastic constituents, which make up the trophoblastic cell columns (Figure 43.39). Cytotrophoblast gives rise to syncytiotrophoblast. The origin of X cells (which contribute to the trophoblastic cell columns and percolate into the maternal decidua along with syncytiotrophoblast) is less clearly understood.

Figure 43.37 A. Twin twin anastomosis characterized by artery-to-vein transfusion. B. Note pale anemic and edematous parenchyma of donor (left) and dark congested parenchyma of recipient (right). The arrowheads mark the vascular equator along the maternal surface. C. Villi from the anemic twin are edematous with abundant macrophages, and vascular spaces contain nucleated hematologic precursors denoting high-output failure and increased red cell production, respectively. D. Villi from plethoric twin are markedly congested (HPS stain). Characteristic classic twin twin transfusion outcomes may not always occur. Although one twin may be smaller, hemoglobin may be increased in a paradoxical fashion suggesting shifts in flow before analysis. Reprinted with permission from:

Benirschke K, Kaufmann P. Pathology of the Human Placenta. 3rd ed. New York: Springer-Verlag; 1995.

Figure 43.38 A. Diamnionic dichorionic intervening membrane, site of fusion at chorionic plate ( T zone ) (H&E stain). B. Note the more cellular intervening chorion separating the two layers of amnion (A) (HPS stain).

Figure 43.39 Polar trophoblastic proliferation comprising trophoblastic cell column. Many vacuolated X cells are identified (H&E stain).

As gestation progresses, the characteristic elements of the trophoblastic villus, as described from the early implantation, differentiate and develop to form a more functionally efficient unit. This occurs with gradual diminution in the size of peripheral branching villi. The tertiary villi, which stem from secondary villi, which in turn are derived from major stem villi, have a characteristic appearance (Figure 43.40A, B). The previously noted two-cell layer is less apparent, and the cytotrophoblast becomes much more difficult to identify. The overlying syncytiotrophoblast of the villus thins so that a syncytiovascular membrane forms the villous interface with the maternal intervillous blood. Deep to the trophoblast lies a basement membrane (also present in earlier villi), which in turn surrounds the villous mesenchyme. Deep to the villous mesenchyme, fetal blood courses within capillaries lined by endothelial cells supported on a basement membrane. These are the constituents of the syncytial vascular membrane separating fetal from maternal blood across which transport of essential nutrients and waste products must occur.

The course of development also has an impact on the mesenchyme. Early in development, when villi have a pronounced double trophoblastic cell layer, the mesenchyme is prominent. The cytologic constituents of this region alter with development. Before 6 weeks from the last menstrual period, capillary lumina are not readily identified (Figure 43.41). After this point, the development of vascularization within villous tissue becomes more pronounced. The vasculature is derived from branches of the stem vessels that connect with the vasculature of the chorionic plate. By about 8 weeks' gestational age (from the last menstrual period), only nucleated hematologic precursors are evident

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within these villous capillary spaces (Figure 43.42). Many of these primitive blood cell precursors have their origin within the yolk sac. As gestational age progresses, there is a decrease in the number of nucleated hematologic precursors, such that between 10 and 12 weeks' gestational age (from the last menstrual period) only approximately 10% of these blood cells are nucleated. The near absence of nucleated hematologic precursors after 12 weeks is readily apparent in histologic sections (Figure 43.43). Surrounding the early villous vasculature, the villous mesenchyme is also prominent early in gestation. The mesenchymal structural units (primitive fibroblastic cells) form about 50% of the cells in this region. The remainder of cells are composed of members of the macrophage family. These cells bear antigens that characterize them as such (CD4, LeuM3, and a variety of other macrophage markers can be identified) (51).

Figure 43.40 Third trimester villi. Secondary villus (A) and tertiary villus (B). Note that capillary lumina are more peripherally located in the tertiary villus. Furthermore, there is less prominent villous mesenchymal substance (HPS stain).

Figure 43.41 Villus from gestation of less than 6 weeks. No capillary lumina are present and no embryonic erythropoiesis is identified (H&E stain).

The villous macrophages or so-called Hofbauer cells lose their prominence as gestation proceeds. The maturing tertiary villus, although it possesses this cell type, has fewer of them at term. The function of Hofbauer cells, although not completely understood, is important in water regulation activities, the transport of various nutrients and waste, and villous homeostasis. This cell type also may be important to immune regulatory functions as an intermediate in the processing of infectious agents that are blood borne.

Chorionic villi may be useful in prenatal diagnosis. Karyotype analysis may be assessed at 10 to 12 weeks' gestational age through chorionic villus sampling (CVS). It should be noted that although early diagnosis is often preferable (to later amniocentesis), several concerns exist. There have been controversies regarding limb reduction abnormalities, but the vast majority of evidence does not confirm this adverse event, especially in experienced hands. Of genuine concern, CVS is not useful in the diagnosis of neural tube defects (amniotic fluid for -fetoprotein determination is required) and fragile X syndrome, which also requires amniotic fluid due to alterations in methylation patterns found in trophoblast as compared with fetal squames (52).

Figure 43.42 Trophoblastic villus at 8 weeks' gestation. Note only nucleated hematologic precursors present within villous capillary spaces (H&E stain).

Gross Morphology

The villous parenchyma is discoid and occupies the space beneath the chorionic plate. The substance of the parenchyma is red and beefy. On sectioning, it appears homogeneous in contour and texture. Irregularities within the substance denote abnormalities within the villous parenchyma. Many abnormalities so identified are common, and in most instances, due to their frequent occurrence, they should be considered as normal varients (unless they are unduly prominent). Such entities include infarcts and perivillous fibrin deposition.

Figure 43.43 Near absence of nucleated hematologic precursors within villous capillaries after 12 weeks' gestation (H&E stain).

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In addition to the perivillous fibrin deposition, other fibrinous depositions are common and are generally considered normal within the placenta. The so-called Langhans' stria, located below the chorionic plate, is probably related to alterations of materal intervillous blood flow. By virtue of its distance from the decidual vasculature, it tends to be more static.

Nitabuch's fibrin is present between the floor of the placenta and the maternal decidua. This layer was once believed to prevent allograft rejection, but now the precise nature and functional significance of this fibrin deposition are not clear.

On occasion, the placental parenchyma have spherical defects (usually 1 to 2 cm in greatest dimension), which represent so-called jet lesions. These cleared areas within the villous parenchyma represent pressure heads from maternal decidual vascular flow. It is not uncommon to histologically identify a small zone of acute infarction peripheral to these lesions.

Calcification is a common phenomenon in the mature placenta. Calcification has been used to diagnose placental maturity by ultrasonographic evaluation during pregnancy. Third-trimester gestations have an increase in the amount of calcium present in the placenta, and when calcifications are prominent, placentas are considered grade 3. A mature placenta detected by ultrasound does not necessarily denote fetal maturity. The appearance of calcium deposition in the gross sense is that of fine, pinhead-sized deposits of yellow white, gritty material. Calcification of the placenta is a normal physiologic response to development and aging (53,54).

Gross Morphologic Alterations

Many placentas normally have some degree of infarction. When infarction roughly exceeds 10 to 15% of the placental surface, or when it is more central than peripheral, this should be considered pathologic. Infarcts are characterized grossly as either acute or old. Acute infarcts are pale, poorly demarcated regions that are slightly granular on palpation. Old infarcts are white, often triangular, and they too are granular (Figure 43.44).

Infarcts evaluated grossly are distinguished from perivillous fibrin deposition. Upon palpation, infarcts are granular and firm. Perivillous fibrin, on the other hand, tends to be nodular and smooth. Further distinctions are made histologically, and these are described in detail below.

Intervillous thrombi also may be identified during the gross examination of the placenta. These triangular or diamond-shaped lesions within the placenta may consist of soft gelatinous red to white (depending on age of lesion) aggregates of collected blood. The lamellations of fibrin within this thrombotic material may be observed grossly. It is this aspect, as well as the gelatinous soft makeup of this material, that distinguishes these lesions from infarcts and perivillous fibrin deposition upon gross examination.

Figure 43.44 Multiple old infarcts from a hypertensive pregnancy.

Histology

The histologic variations in placental architecture are largely dependent on the developmental state at which observations are made. The histology of the individual cell types involved directly in villous implantation is now described.

X cells, also termed intermediate trophoblasts, are major constituents of the cell columns that form the deepest structural components of the implantation site. These cytologic components of trophoblastic origin are unique with respect to other trophoblastic derivatives (syncytiotrophoblasts and cytotrophoblasts). X cells are secretorily distinct and produce human placental lactogen and major basic protein, and are electron microscopically distinct insofar as they contain large numbers of mitochondria with tubular cristae (55,56,57). Similar cell types are identified along with the chorion laeve (Figure 43.45A, B), and these cells have been characterized histologically as being eosinophilic or vacuolated; it was suggested that they represent two distinct subpopulations in this region (58). In the implantation site, these cells are morphologically distinguished from decidual elements in that their cytoplasm is generally darker and they

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are occasionally multinucleated. Furthermore, these cells tend to be more vacuolated then their neighboring decidual cells. Difficulty in distinguishing these cell types has resulted in some problems with the diagnosis of intrauterine pregnancy when these are the only trophoblastic cells present in currettage specimens. In the absence of villi, most pathologists confirm their diagnosis of intrauterine pregnancy (and hence generally exclude the possibility of a tubal pregnancy) based on the presence of multinucleated trophoblastic derivatives, which are more readily distinguished from the surrounding decidua. Should difficulty arise with morphologic assessment of the true nature of the uninuclear cells in question, immunohistochemical localization of human placental lactogen within X cells is helpful (59). This cell type is of important immunologic interest because it may be responsible for the synthesis of fibronectin (1). An excess of immature intermediate trophoblasts has been associated with preeclampsia and eclampsia (60).

Figure 43.45 Prominent nonvacuolated X cells within the decidua of the chorionic laeve. Note their darker cytoplasm and occasional multinucleation (A) and vacuolated X cells deep in the chorion (B) (HPS stain).

An interesting normal finding within the placenta is the so-called chorionic cyst. These cysts occur in placental septae and are composed largely of decidua and X cells. The cysts are entirely lined by X cells. As noted above, such cysts also may be seen in the chorionic plate (Figure 43.35). On sectioning, gelatinous fluid may be present, and the major constituent of this fluid is one of the substances produced by X cells: major basic protein. The function of these cysts is not known. They should not be considered pathologic when present (1).

The syncytiotrophoblast, the outer cell layer, possesses a brush border. Microvilli that constitute this border are felt to be involved in pinocytotic activity. Vacuoles within the cytoplasm of these cells are indicative of the absorptive and secretory activities of the syncytiotrophoblast. Syncytiotrophoblasts are composed of pyknotic (often multiple) nuclei that are hyperchromatic. In addition to the multiple vesicles present within this cell type, ultrastructural examinations show a cytoplasm that is rich in endoplasmic reticulum, mitochondria, lipid droplets, and Golgi bodies (61).

The cytotrophoblastic nuclei are more round and open. Tritiated thymidine incorporation experiments have shown that uptake and incorporation are confined to the cytotrophoblastic layer and not the syncytiotrophoblastic layer (62). On occasion, cytotrophoblasts may possess mitotic figures (Figure 43.46). Ultrastructurally, the cytotrophoblast has fewer organelles than does the syncytiotrophoblast. Most prominent are large mitochondria, which may be numerous.

Human placental lactogen may be localized within X cells and syncytiotrophoblasts. Human chorionic gonadotropin can be identified within syncytiotrophoblast cells, but not within cytotrophoblast cells (55,63).

Figure 43.46 Sparse cytotrophoblast with rare mitotic figure in terminal villus from a third trimester placenta (HPS stain).

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Histopathology

Various pathologic entities, which may be identified within the villous parenchyma at the histologic level, serve to better illustrate the normal histology of the placenta.

Infarcts, which are common in the placental substance, have a characteristic gross appearance. Acute infarcts are characterized histologically by the presence of faint staining villi, which are aggregated, compressed, or agglutinated to one another and have interspersed polymorphonuclear leukocytes within the intervillous space (Figure 43.47). (As described below, it is crucial to distinguish this inflammation from that which occurs in an intravellous fashion). Earlier forms of infarction may be characterized by the presence of villous agglutination and congestion, with lysis of intervillous maternal blood (Figure 43.48). In very advanced ( old ) infarcts, complete absence of villous architecture is noted and a fuzzy outline of remaining villous constituents can be identified (Figure 43.49). No viable staining cells are identified, and an acute inflammatory infiltrate may persist. The placenta rarely undergoes organization or fibrosis. When acute infarcts resolve and

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become old, fibroblasts are generally lacking. It is for this reason that organization is not a term applied to the placenta. Classic organization as might apply to other organ systems undergoing ischemic change does not occur. The variance probably relates to the two distinct vascular supplies (maternal and fetal). Disturbance in maternal flow results in placental infarction.

Figure 43.47 Acute infarct (HPS stain).

Figure 43.48 Villous vascular congestion may indicate early infarction or need to be more closely examined to exclude chorangiosis (HPS stain).

Figure 43.49 Old infarct (HPS stain).

Figure 43.50 Increased perivillous fibrin. Note trophoblastic nuclear remnants encased in fibrin (HPS stain).

Infarcts are distinguished from perivillous fibrin deposition histologically in that regions of the latter contain cytotrophoblastic nuclear remnants that are often prominent (Figure 43.50). Intervillous thrombi are identified histologically by the presence of lamellated thrombotic material displacing neighboring villi (Figure 43.51).

Figure 43.51 Intervillous thrombus. Note lamellated lines of Zahn (HPS stain).

Figure 43.52 Focal edema (H&E stain).

Other abnormalities of the villous parenchyma include villous edema, where edema fluid displaces intravillous cytologic architecture; it is considered pathologic, especially in premature gestations (Figure 43.52) and has been reported as a cause of fetal ischemia. Its etiology is not clear (22). Tenney-Parker change is characteristic of placentas from preeclamptic gestations. This change is characterized by increased syncytial knotting on villi (Figure 43.53). These syncytial knots are best considered failed adaptive responses to low oxygen tension within the intervillous space. As noted above, increased intermediate trophoblasts also may be seen (60). Infarcts are not uncommon (1), and in preeclamptic/eclamptic gestations, decidual vasculopathy is also encountered and is discussed below.

Unusual appearances of villous vasculature take three unrelated forms. First, villous vasculature may be congested (Figure 43.48), and this may have no pathologic significance or may be indicative of early infarction. Second, increased numbers of villous capillaries, so-called chorangiosis, has been considered to be indicative of chronic hypoxic changes. The definition is precise, and in histologic terms three criteria

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(10 vessels/10 villi/10 fields at 10 ) need to be met (64). Third, abnormal proliferations of villous vessels that form histologically hemangiomatous nodules, are termed chorioangiomas when present in the placenta (Figure 43.54A, B). These lesions have pathologic significance when prominent and may result in high-output cardiac failure in the fetus or microangiopathic hemolytic phenomena.

Figure 43.53 Increased syncytial knots in a hypertensive pregnancy (HPS stain).

Dysmaturity of villi is also considered pathologic. Villi that show the characteristic two-cell layer of the first-trimester implantation, when present in third-trimester placentas, are indicative of abnormal developmental events. This change has been noted to occur in mothers who are diabetic.

Nucleated red blood cells are common in very early gestations, but as gestation proceeds, and especially in the third trimester, nucleated red blood cells should not be present. When present, fetal anemia with increased erythroid production should be suspected. Additionally, recent investigations have addressed the presence of nucleated red blood cells (in advanced-gestation placentas), correlating such findings with erythropoietin secretion by the fetus and concomitant fetal hypoxia (1) (Figure 43.55).

Figure 43.54 Chorioangioma (A) characterized by hemangiomatous proliferation (B) (HPS stain).

Figure 43.55 Fetal nucleated red blood cells are abnormal at term. There is associated focal villous edema (H&E stain).

In patients with sickle cell disease, it is not unusual to find sickling of maternal erythrocytes within the intervillous spaces. This is promoted by diminished oxygen tension in the setting of abnormal maternal hemoglobin S. It is wise to correlate histopathologic findings with clinical status in that it has been reported that hypoxia during and after placental separation causes sickling (65) (Figure 43.56).

Inflammatory changes within villi are probably one of the most interesting aspects of placental pathology. These inflammatory changes herald the presence of infectious disease agents in many cases. The presence of syphilis and cytomegalovirus (CMV) should be considered when

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infiltrates of lymphocytes and plasma cells are found within trophoblastic villi. Special stains may be confirmatory. It is important to distinguish this chronic inflammation from that of the acute inflammatory infiltrate (surrounding villi) associated with acute infarction. An acute inflammatory infiltrate within villi may suggest Listeria infection with extensive microabscess formation throughout the placenta. This often can be seen in the gross as innumerable white dots. The odor has been characterized as sweet (1). In many instances, inflammatory changes within the villi are limited to an increase in the villous cellularity (increased Hofbauer cells), and no specific infectious disease agent can be identified. Increased numbers of inflammatory cells (without plasma cells) also signify chronic villitis; however, in most instances when this occurs, no infectious disease organism can be identified. These inflammatory changes within the placenta have been termed villitis of unknown etiology (VUE) (64,66) (Figure 43.57A, B). The infiltrate has been identified as maternal in origin (67).

Figure 43.56 Intervillous maternal red cell sickling in patient with sickle cell disease (H&E stain).

Figure 43.57 Villitis of unknown etiology (VUE) in which a chronic inflammatory infiltrate devoid of plasma cells is present (A) and chronic villitis due to known syphilis infection characterized by the presence of a mononuclear infiltrate containing plasma cells (B) (HPS stain).

Figure 43.58 Parvovirus B19 infection associated with villous edema and nucleated fetal red cells with characteristic inclusions (H&E).

The absence of inflammation does not always coincide with the absence of infection. For example, in parvovirus B19 infection, hydropic villi and the presence of villous vascular nucleated red blood cells (occasionally with smudged amphophilic intranuclear inclusion bodies) may herald the presence of severe congenital infection even in the setting of absence of maternal symptoms (1) (Figure 43.58).

Confounding the topic of placental and neonatal infection is the entirely normal placenta, by routine examination. Only special molecular studies will detect antigens and nucleic acids indicating the presence of infectious materials (68) (Figure 43.59A, B).

Figure 43.59 In situ hybridization with S³⁵ probe detects HIV-1 nucleic acids in Hofbauer (H) cells (A) and trophoblast (C, T) of immature villi (Cv) (B). Reprinted with permission from:

Redline RW, Patterson P. Villitis of unknown etiology is associated with major infiltration of fetal tissue by maternal inflammatory cells. Am J Pathol 1993;143:473 479.

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Decidua

Histology

The hypersecretory glandular epithelium of the endometrium, which is progestationally induced, affords the proper environment for implantation. At times, these hypersecretory glands may exhibit the Arias-Stella reaction in which cytologic atypia is noted (Figure 43.60). In this condition, nuclei are often polyploid. However, nuclear cytoplasmic ratios remain low, distinguishing this normal finding from neoplasia. In this continued progestational influence, endometrial glands become secretorily exhausted. The endometrial stroma has undergone its characteristic decidualization, and decidual cells of the endometrium are characterized as epithelioid and polygonal. Their small rounded nuclei are generally situated centrally in abundant pale eosinophilic, often vacuolated, cytoplasm (Figure 43.61). The cytoplasm is rich in glycogen and glycoproteins. In regions of decidual tissue, where trophoblastic derivatives are not present, nuclear content is diploid (69). Ultrastructural examination of the decidua shows that tight junctions separate these cells (70).

Figure 43.60 Arias-Stella reaction (H&E stain).

In addition to the decidual cells, an admixture of fibroblasts and lymphocytes is also identified. An additional cell type, the granular cell, has been shown to produce relaxin (71).

The intercellular matrix contains abundant type IV collagen and laminin. Fibronectin and heparin sulfate proteins also have been identified (72). Other collagens are also present throughout the decidual matrix, and these include types I, III, and V.

Figure 43.61 Decidua with centrally placed open nuclei and abundant pale cytoplasm with prominent cell borders. Note sparse normal lymphocytic infiltrate and occasional X cell with cytoplasm darker than decidual cytoplasm (HPS stain).

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Although prior reports suggested that the secretory activity of the decidua included the production of prolactin and human placental lactogen, it is known that this hormone is produced not by decidual cells but by invading trophoblastic derivatives (principally intermediate trophoblast or X cells) (55). Much of the difficulty in studying decidual tissues and their hormonal production has resulted from the inability of many investigators to distinguish decidual components from invading trophoblastic contaminants (1).

The vascularization of the decidual component of the implantation site is critical to the developing gestation. The major branches of the uterine arteries extend deeply into the myometrial substance, resulting in arcuate arteries that then branch to form radial arteries. These become the spiral arterioles, the terminal components of the endometrial vasculature. These spiral vessels have been shown by injection studies to be responsible for the intervillous blood flow within cotyledonary units (73). The precise number of spiral arterioles that serve to perfuse the placenta is a debated topic. Estimates range from 25 to 300 vascular openings (74,75).

Figure 43.62 Decidual vascular atherosis (A) and concentric medial hypertrophy (B) from a hypertensive gestation (HPS stain).

That trophoblastic cells invade the underlying decidual vasculate is a well-known phenomenon. This finding has been documented within the decidual bed, as well as in systemic maternal vascular compartments, most notably the lung (76). It is important to distinguish these decidual vascular changes from those abnormalities of the decidual vasculature that indicate pathologic conditions.

Gross inspection of the decidua is generally unrevealing, and important attributes and diagnoses are identified histologically. In through-and-through sections of the placenta disk, which includes the chorionic plate and the villous parenchyma, the underlying decidua is often denuded from the placental implantation site during the delivery process. Therefore, it is not uncommon to find sections of parenchyma devoid of decidual tissue. One region in which decidua is often prominent is on the chorion laeve. Sections of membrane rolls, which include amnion and chorion, often include sections of adherent decidua, which represent fusion of the decidua capsularis and the decidua vera during development. In some patients in whom decidua abnormalities are suspect, decidual bed biopsies have been performed at the time of delivery. Such biopsies often reveal aberrant vascular relationships of the more proximal vascular tree (radial and basal arteries). The more proximal vessels are characterized by thicker vascular walls, and their arteries often have internal elastic laminae as evidenced by silver stains. The more distal arteries (the spiral arterioles) are thin walled and do not possess any internal elastic lamina.

Histopathology

Principal among decidual pathology is the so-called atherosis of the decidual vascular bed, which is known to accompany preeclampsia and hypertensive conditions of gestation (77). These lesions are characterized by fibrinoid necrosis and hyalinization of the vascular wall along with the deposition of foamy macrophages and should be readily distinguishable from normal trophoblastic vascular invasion. Another abnormality that may characterize hypertensive pregnancies is concentric arteriolar mural hypertrophy (Figure 43.62A, B).

Bleeding (retroplacental) is responsible for premature placental separation or abruptio placenta. This has been related to two maternal conditions: increased maternal blood pressure and decidual vascular necrosis (due to vasculopathies or inflammatory-bacterial decidual infections) (Figure 43.63).

Figure 43.63 Decidual vascular necrosis, in this case due to a severe acute deciduitis. Note vascular wall hyalinization and necrosis, which resulted in adjacent abruptio (H&E stain).

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When trophoblastic villi implant directly on myometrial tissue and intervening decidua is absent, the pathologic condition of placenta accreta is present (Figure 43.64). This finding is more common in low implantations of the placenta, especially when prior cesarean sections have been performed. Variations in which trophoblastic villi implanted within the myometrium (placenta increta) and when implantation results in the presence of villous tissue protruding through the uterine serosal surface (placenta percreta) are more severe forms of this condition. Generally, all placental tissue cannot be removed from the uterus at delivery and severe hemorrhage ensues. Heroic measures only rarely save the uterus and may increase morbidity due to persistant hemorrhage (and in latter stages bacterial infection) by delay of definitive therapy, which is hysterectomy. Occasionally, accreta are confirmed by studying the decidua of the chorion laeve. Failure of resolution of villi during capsular expansion may result in myometrial implantation without intervening decidua. Also occasionally, myometrium adherent to frondosum is found without intervening decidua. In both cases the diagnosis of accreta can be made by the pathologist in the absence of the hysterectomy specimen.

Within the decidua attached to the delivered placenta rare leukocytes may be seen. If these cytological constituents are plasma cells, syphilis or CMV infection should be considered.

In the absence of pathologic alterations (placental, maternal or neonatal) these rare leukocytes within the decidua are likely physiologic or nonspecific.

More destinctive inflammatory processes involving the decidua are deciduitis and can be associated with inflammation elsewhere. Acute chorioamnionitis and acute or chronic villitis should be sought in the further examination of the placenta.

Gestational Trophoblastic Disease

Although the discussion of neoplastic trophoblastic disease is beyond the scope of this chapter, several normal or exaggerated findings of the implantation site or the placenta proper are discussed to distinguish them from more significant pathologic entities.

Degenerative changes of early trophoblastic villi are not uncommon, especially in the presence of incomplete abortus material. Trophoblastic villi from such gestations are often seen in histologic sections to be swollen (hydropic). These findings are distinguished from the gestational trophoblastic neoplasm (complete hydatidiform mole) in several aspects. Principal among these distinguishing characteristics is that no trophoblastic atypia or proliferation is present along the surface of the villi (Figure 43.65A, B). When degenerative changes have taken place and fetal components are blighted, the chorionic vasculature may be absent within the villi. However, if remnants of these vessels persist, the presence of complete hydatidiform mole is essentially not possible. The ease of identifying nucleated hematologic precursors facilitates this observation. Furthermore, should any fetal parts be identified, this too excludes the presence of complete hydatidiform mole. On the other hand, hydropic villi in the presence of fetal parts may herald genotypically abnormal gestations. This form of

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incomplete mole, which is rarely neoplastic, is characterized by scalloped trophoblastic borders and occasional trophoblastic island inclusions (representing tangential cuts of scalloped borders within the villous stroma) (Figure 43.66A, B).

Figure 43.64 Placenta accreta. No decidua is present between the implanting trophoblastic villi and the uterine myometrium. The presence of small amounts of fibrin does not alter the diagnosis.

Figure 43.65 Degenerative (nonneoplastic) focal hydropic degeneration (A) and (neoplastic) complete mole (B) with trophoblastic hyperplasia and atypia (C) (H&E stain).

Figure 43.66 Triploid incomplete mole with scalloped trophoblastic borders (A) and trophoblastic inclusions (B), which are actually tangential cuts of scalloped borders (H&E stain).

Figure 43.67 Exaggerated implantation site (A) and invading choriocarcinoma (B) (H&E stain).

Abundant trophoblastic derivates, prominent within the nidus of the implantation site, may from time to time need to be distinguished from choriocarcinoma. The so-called syncytial endometritis a poor term because it is not an inflammatory or infectious condition refers to an exaggerated implantation site where syncytiotrophoblastic and other trophoblastic derivative counterparts are prominant (Figure 43.67A, B). In the absence of included trophoblastic villi during examination, the histologic appearance is similar to that of invading choriocarcinoma. The chief distinction is the lack of pronounced cytologic and nuclear atypia that characterizes choriocarcinoma.

Last, an unusual lesion that is neoplastic is the placental site trophoblastic tumor. This neplasm is composed solely of X cells (or, as described by Kurman et al. (55), intermediate trophoblasts) (Figure 43.68).

Figure 43.68 Placental site trophoblastic tumor (H&E stain).

References

1. Benirschke K, Kaufmann P. Pathology of the Human Placenta. 3rd ed. New York: Springer-Verlag; 1995.

2. Lewis SH, ed. Pathology of the Placenta. New York: Churchill Livingstone; Elsivier; 1999.

3. Bartholomew RA, Colvin ED, Grimes WH Jr, Fish JS, Lester WM, Galloway WH. Criteria by which toxemia of pregnancy may be diagnosed from unlabeled formalin-fixed placentas. Am J Obstet Gynecol 1961;82:277 290.

4. Schremmer BN. Gewichtsver nderungen verschiedener Gewebe nach Formalinfixierung. Frank Z Pathol 1967;77:299 305.

5. College of American Pathologists Conference XIX. The examination of the placenta: patient care and risk management. Arch Pathol Lab Med 1991;115:660 721.

6. Nuovo G, Richart RM. Buffered formalin is the superior fixative for the detection of HPV DNA by in situ hybridization analysis. Am J Pathol 1989;34:837 842.

7. Naeye RL. Umbilical cord length: clinical significance. J Pediatr 1985;107:278 281.

8. Lewis SH, Starr C. Cord width. In Utero (Unpublished).

9. Grosser O. Fr hentwicklung, Eihautbildung und Placentation des Menschen und der S ugetiere. Munich: JF Bergman; 1927.

10. Gardiner JP. The umbilical cord: normal length; length in cord complications; etiology and frequency of coiling. Surg Gynecol Obstet 1922;34:252 256.

11. Wynn RM, French GL. Comparative ultrastructure of the mammalian amnion. Obstet Gynecol 1968;31:759 774.

12. Hoyes AD. Ultrastructure of the epithelium of the human umbilical cord. J Anat 1969;105(pt 1):145 162.

13. Moore RD. Mast cells of the human umbilical cord. Am J Pathol 1956;32:1179 1183.

14. Nadkarni BB. Innervation of the human umbilical artery: an electron microscope study. Am J Obstet Gynecol 1970;107:303 312.

15. Ellison JP. The nerves of the umbilical cord in man and the rat. Am J Anat 1971;132:53 60.

16. Lauweryns JM, De Bruyn M, Peuskens J, Bourgeois N. Absence of intrinsic innervation of the human placenta. Experientia 1969;25:432.

17. Blanc WA, Allan GW. Intrafunicular ulceration of the persistent omphalomesenteric duct with intra-amniotic hemorrhage and fetal death. Am J Obstet Gynecol 1961;82:1392 1396.

18. Priman J. A note on the anastomosis of the umbilical arteries. Anat Rec 1959;134:1 5.

19. Arts NF. Investigations on the vascular system of the placenta. I. General introduction and the fetal vascular system. Am J Obstet Gynecol 1961;82:147 158.

20. Rana J, Ebert GA, Kappy KA. Adverse perinatal outcome with an abnormal umbilical coiling index. Obstet Gynecol 1995;85:573 578.

21. Altshuler G, Hyde S. Meconium-induced vasocontraction: a potential cause of cerebral and other hypoprofusion and of poor pregnancy outcome. J Child Neurol 1989;4:137 142.

P.1126

22. Naeye RL. Disorders of the Placenta, Fetus and Neonate: Diagnosis and Clinical Significance. St. Louis: Mosby Year Book; 1992.

23. Vonthobitien N. Anatomia Suc Undina Human. 1668.

24. Garza A, Cordero JF, Mulinare J. Epidemiology of the early amnion rupture spectrum of defects. Am J Dis Child 1988;142:541 544.

25. Torpin R. Fetal Malformations Caused by Amnion Rupture During Gestation. Springfield, IL: Charles C Thomas; 1968.

26. Danforth D, Hull RW. The microscopic anatomy of the fetal membranes with particular reference to the detailed structure of amnion. Am J Obstet Gynecol 1958;75:536 550.

27. Boyd JD, Hamilton WJ. The Human Placenta. Cambridge, MA: Heffer & Sons; 1970.

28. Michael H, Ulbright TM, Brodhecker C. Magma reticulare-like differentiation in yolk sac tumor and its pluripotential nature [Abstract]. Mod Pathol 1988;1:63.

29. King BF. Developmental changes in the fine structure of rhesus monkey amnion. Am J Anat 1980;157:285 307.

30. Mukaida T, Yoshida K, Kikyokawa T, Soma H. Surface structure of the placental membranes. J Clin Electron Microsc 1977;10:447 448.

31. Bourne GL. The microscopic anatomy of the human amnion and chorion. Am J Obstet Gynecol 1960;79:1070 1073.

32. Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995;146:3 15.

33. Tsukada T, Eguchi K, Migita K, et al. Transforming growth factor beta 1 induces apoptotic cell death in cultured human umbilical vein endothelial cells with down-regulated expression of bcl-2. Biochem Biophys Res Commun 1995:210:1076 1082.

34. Bartels H, Wang T. Intercellular junctions in the human fetal membranes. A freeze-fracture study. Anat Embryol (Berl) 1983; 166:103 120.

35. Robinson HN, Anhalt GJ, Patel HP, Takahashi Y, Labib RS, Diaz LA. Pemphigus and pemphigoid antigens are expressed in human amnion epithelium. J Invest Dermatol 1984;83:234 237.

36. Schwarzacher HG, Klinger HP. Die Entstehung mehrkerniger Zellen durch Amitose im Amnionepithel des Menschen und die Aufteilung des chromosomalen Materials auf deren einzelne Zellkerne. Z Zellforsch 1963;60:741 754.

37. Schindler PD. Nuclear deoxyribonucleic acid (DNA) content, nuclear size and cell size in the human amnion epithelium. Acta Anat (Basel) 1961;44:273 285.

38. Kalousek DK, Fill FJ. Chromosomal mosaicism confined to the placenta in human conceptions. Science 1983;221:665 667.

39. Bautzmann H, Hertenstein C. Zur Histogenese und Histologie des menschlichen fetalen und Neugeborenen-Amnions. Z Zellforsch 1957;45:589 611.

40. Schmidt W. Struktur und Funktion des Amnionepithels von Mensch und Huhn. Z Zellforsch 1963;61:642 660.

41. Schwarzacher HG. Beitrag zur histogenese des menschilichen amnion. Acta Anat 1960;43:303 311.

42. Nickell KA, Stocker JT. Placental teratoma: a case report. Pediatr Pathol 1987;7:645 650.

43. Salazar H, Kanbour AI. Amnion nodosum: ultrastructure and histopathogenesis. Arch Pathol 1974;98:39 46.

44. Bourne GL. The Human Amnion and Chorion. London: Lloyd-Luke; 1962.

45. Luckett WP. The origin of extraembryonic mesoderm in the early human and rhesus monkey embryos [abstract]. Anat Rec 1971; 169:369 370.

46. Rossant J, Croy BA. Genetic identification of tissue of origin of cellular populations within the mouse placenta. J Embryol Exp Morphol 1985;86:177 189.

47. Hoyes AD. Ultrastructure of the mesenchymal layers of the human amnion in early pregnancy. Am J Obstet Gynecol 1970;106:557 566.

48. Hoyes AD. Ultrastructure of the mesenchymal layers of the human chorion laeve. J Anat 1971;109(pt 1):17 30.

49. Sala MA, Matheus M. Histochemical study of the fetal membranes in the human term pregnancy. Gegenbaurs Morphol Jahrb 1984; 130:699 705.

50. Hessle H, Engvall E. Type VI collagen. Studies on its localization, structure, and biosynthetic form with monoclonal antibodies. J Biol Chem 1984;259:3955 3961.

51. Goldstein J, Braverman M, Salafia C, Buckley P. The phenotype of human placental macrophages and its variation with gestational age. Am J Pathol 1988;133:648 659.

52. American College of Obstetricians and Gynecologists. ACOG Committee Opinion (Committee on Genetics) no. 160: Chorionic villus sampling; 1995.

53. Pitkin RM, Reynolds WA, Williams GA, Hargis GK. Calcium metabolism in normal pregnancy: a longitudinal study. Am J Obstet Gynecol 1979;133:781 790.

54. Tsang RC, Donovan EF, Steichen JJ. Calcium physiology and pathology in the neonate. Pediatr Clin North Am 1976;23:611 626.

55. Kurman RJ, Main CS, Chen HC. Intermediate trophoblast: a distinctive form of trophoblast with specific morphological, biochemical and functional features. Placenta 1984;5:349 370.

56. Wasmoen TL, Benirschke K, Gleich GJ. Demonstration of immunoreactive eosinophil granule major basic protein in the plasma and placentae of non-human primates. Placenta 1987;8:283 292.

57. Wynn RM. Cytotrophoblastic specializations: an ultrastructural study of the human placenta. Am J Obstet Gynecol 1972;114:339 355.

58. Yeh IT, O'Connor DM, Kurman RJ. Vacuolated cytotrophoblast: a subpopulation of trophoblast in the chorion laeve. Placenta 1989;10:429 438.

59. O'Connor DM, Kurman RJ. Utilization of intermediate trophoblast in the diagnosis of an in utero gestation in endometrial curettings without chorionic villi [Abstract]. Mod Pathol 1988;1:68.

60. Redline RW, Patterson P. Pre-eclampsia is associated with an excess of proliferative immature intermediate trophoblast. Hum Pathol 1995;26:594 600.

61. Wislocki GB, Dempsey EW. Electron microscopy of the human placenta. Anat Rec 1955;123:133 167.

62. Richart R. Studies of placental morphogenesis. I. Radioautographic studies of human placenta utilizing tritiated thymidine. Proc Soc Exp Biol Med 1961;106:829 831.

63. Pierce GB Jr, Midgley AR Jr. The origin and function of human syncytiotrophoblastic giant cells. Am J Pathol 1963;43:153 173.

64. Altshuler G. Placental infection and inflammation. In: Perrin EVDK, ed. Pathology of the Placenta. New York: Churchill Livingstone; 1984.

65. Fujikura T, Froehlich L. Diagnosis of sickling by placental examination. Geographic differences in incidence. Am J Obstet Gynecol 1968;100:1122 1124.

66. Knox WF, Fox H. Villitis of unknown aetiology: its incidence and significance in placentae from a British population. Placenta 1984;5:395 402.

67. Redline RW, Patterson P. Villitis of unknown etiology is associated with major infiltration of fetal tissue by maternal inflammatory cells. Am J Pathol 1993;143:473 479.

68. Lewis SH, Reynolds-Kohler C, Fox HE, Nelson JA. HIV-1 in trophoblastic and villous Hofbauer cells, and hematologic precursors in eight-week fetuses. Lancet 1990;355:565 568.

69. Sachs H. Quantitativ histochemische Untersuchung des Endometrium in der Schwangerschaft und der Placenta (Cytophotometrische Messungen). Arch Gynecol Obstet 1968; 205:93 104.

70. Lawn AM, Wilson EW, Finn CA. The ultrastructure of human decidual and predecidual cells. J Reprod Fertil 1971;26:85 90.

71. Dallenbach FD, Dallenbach-Hellweg G. Immunohistologische Untersuchungen zur Lokalisation des Relaxins in menschlicher Placenta und Decidua. Virchows Arch A 1964;337:301 316.

72. Wewer UM, Faber M, Liotta LA, Albrechtsen R. Immunochemical and ultrastructural assessment of the nature of pericellular basement membrane of human decidual cells. Lab Invest 1985; 53:624 633.

73. Freese UE. The uteroplacental vascular relationship in the human. Am J Obstet Gynecol 1968;101:8 16.

74. Borell U, Fernstrom I, Ohlson L, Wiqvist N. The influence of uterine contractions on the uteroplacental blood flow at term. Am J Obstet Gynecol 1965;93:44 57.

75. Haller U. Beitrag zur Morphologie der Utero-placentargefaesse. Arch Gynaekol 1968;205:185 202.

76. Attwood HD, Park WW. Embolism to the lungs by trophoblast. J Obstet Gynaecol Br Commonw 1961;68:611 617.

77. Hertig AT. Vascular pathology in hypertensive albuminuric toxemias of pregnancy. Clinics 1945;4:602 614.