Sample chapter title - avery’s diseases of the newborn, 9e with expert consult print by gleason - elsevier

37

Complex yet intricate interactions between maternal and fetal systems promote fetal growth and normal pregnancy outcomes. Throughout embryonic development, organo-genesis and functional maturation are taking place. This period of development coincides with a high rate of cel-lular proliferation and organ development, which creates critical periods of vulnerability. Adverse factors, disruption, or impairment during these critical periods of fetal devel-opment can alter developmental programming, which can lead to permanent metabolic or structural changes ( Baker, 1998 ). For example, triggers such as undernutrition can elicit placental and fetal adaptive responses that can lead to local ischemia and metabolic, hormonal, and immune reprogramming, resulting in small for gestational age (SGA) fetuses. Maternal health, dietary status, and exposure to environmental factors, uteroplacental blood fl ow, placental transfer, and fetal genetic and epigenetic responses likely all contribute to in utero fetal programming ( Figure 5-1 ). Adult diseases such as coronary heart disorders, hyperten-sion, atherosclerosis, type 2 diabetes, insulin resistance, respiratory distress, altered cell-mediated immunity, can-cer, and psychiatric disorders are now thought to be a con-sequence of in utero life (Sallour and Walker, 2003). It is a matter of considerable interest that, in addition to maternal predisposing factors, cytokines, hormones, growth factors, and the intrauterine immune milieu also contribute to in utero programming. Adaptations of the maternal immune system exist to modulate detrimental effects on the fetus and additional mechanisms and factors actively cross the placenta and induce regulatory T cells in the fetus to sup-press fetal antimaternal immunity. These effects persist at least through adolescence (Burlingham, 2009; Mold et al, 2008). Excessive restraint of maternal immune responses could lead to a lethal infection in the newborn. On the other hand, too little modulation of maternal immune response to the fetal allograft could lead to autoimmune-mediated fetal-placental rejection. Moreover, placental growth resembles that of a tumor, evading immune surveillance and initiat-ing its own angiogenesis. Therefore a healthy mother with healthy placentation is critical to healthy fetal outcomes.

MAMMALIAN PLACENTATION The immune tolerance of the semiallograft fetus and de novo vascularization are two highly intriguing processes that involve direct interaction of maternal immune cells,

invading trophoblast cells, and arterial endothelial cells. Pregnancy is considered an immunologic paradox, in which paternal antigen-expressing placental cells interact directly with and coexist with the maternal immune system (Medawar, 1953). This anatomic distinction of the immu-nologic interface that arises from hemochorial placentation that occurs in humans and rodents is distinct from epithe-liochorial placentation as seen in marsupials, horses, and swine or the endotheliochorial placentation seen in dogs and cats. Understanding the anatomic and physiologic events that occur during placentation is the key to appre-ciate the uniqueness of human placentation in the phylo-genetic evolution. Typically, in hemochorial placentation, maternal uterine blood vessels and decidualized endome-trium are colonized by trophoblast cells, derived from trophectoderm of the implanting blastocyst. These cells come in direct contact with maternal blood and uterine tissue. A similar phenomenon is evident in murine preg-nancy, except the trophoblast invasion is deeper in humans ( Moffett and Loke, 2006 ). In epitheliochorial placentation, trophoblast cells of the placenta are in direct contact with the surface epithelial cells of the uterus, but there is no trophoblast-cell invasion beyond this layer. In endothelio-chorial placentation, the trophoblast cells breach the uter-ine epithelium and are in direct contact with endothelial cells of maternal uterine blood vessels.

EMBRYOLOGIC DEVELOPMENT OF THE PLACENTA Shortly after fertilization takes place in the ampullary por-tion of the fallopian tube, the fertilized ovum or zygote begins dividing into a ball of cells called a morula . As the morula enters the uterus (by the fourth day after fertiliza-tion), it forms a central cystic area and is called a blastocyst ( Figure 5-2 ). The blastocyst implants within the endome-trium by day seven (Moore, 1988).

The blastocyst has two components: an inner cell mass, which becomes the developing embryo, and the outer cell layer, which becomes the placenta and fetal membranes. The cells of the developing blastocyst, which eventually become the placenta, are differentiated early in gestation (within 7 days after fertilization). The outer cell layer, the trophoblast, invades the endometrium to the level of the decidua basalis. Maternal blood vessels are also invaded. Once entered and controlled by the trophoblast, these

Immunologic Basis of Placental Function

and Diseases: the Placenta, Fetal

Membranes, and Umbilical Cord

Satyan Kalkunte , James F. Padbury, and Surendra Sharma

C H A P T E R

5

P A R T II

FETAL DEVELOPMENT

38 PART II Fetal Development

maternal blood vessels form lacunae, which provide nutri-tion and substrates for the developing products of concep-tion. The trophoblast differentiates into two cell types, the inner cytotrophoblast and the outer syncytiotropho-blast ( Figure 5-3 ); the former has distinct cell walls and is thought to represent the more immature form of tropho-blast. The syncytiotrophoblast, which is essentially acel-lular, is the site of most placental hormone and metabolic activity. Once the trophoblast has invaded the endome-trium, it begins to form outpouchings called villi, which extend into the blood-fi lled maternal lacunae or further invade the endometrium to attach more solidly with the decidua, forming anchoring villi.

PLACENTAL ANATOMY AND CIRCULATION At term, the normal placenta covers approximately one third of the interior portion of the uterus and weighs approximately 500 g. The appearance is of a fl at circular disc approximately 2 to 3 cm thick and 15 to 20 cm across (Benirschke and Kaufmann, 2000). Placental and fetal weights throughout gestation are presented in Table 5-1 . During the fi rst trimester and into the second, the placenta weighs more than the fetus; after that period, the fetus out-weighs the placenta. With the formation of the tertiary villi (19 days after fertilization), a direct vascular connection is made between the developing embryo and the placenta (Moore, 1988). Umbilical circulation between the placenta and the embryo is evident by 51⁄2 weeks’ gestation. Figure 5-4 demonstrates aspects of the maternal and fetal circula-tion in the mature placenta. The umbilical arteries from the fetus reach the placenta and then divide repetitively to cover the fetal surface of the placenta. Terminal arteries then pen-etrate the individual cotyledons, forming capillary beds for substrate exchange within the tertiary villi. These capillaries then reform into tributaries of the umbilical venous system, which carries oxygenated blood back to the fetus.

EXAMINATION OF THE PLACENTA A renaissance in placental pathology has led to a new rel-evance of the placenta to neonatology and early infant life, including issues of preterm birth, growth restriction, and cerebral, renal, and myocardial diseases. The placenta can give some clues to the timing and extent of impor-tant adverse prenatal or neonatal events as well as to the relative effects of sepsis and asphyxia on the causation of neonatal diseases. Placental disorders can be noted imme-diately in the delivery room, and others can be diagnosed through detailed gross and microscopic examinations over the ensuing 48 hours. Every placenta should be examined at the time of birth regardless of whether the newborn has any immediate problems. Most placentas invert with traction at the time of delivery, and the fetal membranes cover the maternal surface. It is important to reinvert the membranes and examine all surfaces of the placenta and membranes, looking for abnormalities. Table 5-2 lists pregnancy complications or conditions that are diagnos-able at birth through examination of the placenta.

Outer cell layer

Inner cell mass

A

Syncytiotrophoblast

Cytotrophoblast

Fetal pole

B

FIGURE 5-2 A, The human blastocyst contains two portions: an inner cell mass, which develops into the embryo, and an outer cell layer, which develops into the placenta and membranes. B, The outer acellular layer is the syncytiotrophoblast, and the inner cellular layer is the cytotrophoblast. ( From Moore TR, Reiter RC, Rebar RW, et al, editors: Gynecology and obstetrics: a longitudinal approach, New York, 1993, Churchill Livingstone. )

Maternal

Health status

Diet andexposure status

Immune status

Placenta

Uteroplacentalperfusion

Ischemia

Hormonalregulation

Placental-fetalresponse

Fetal Programming

Fetus

Metabolicadaptation

Genetics andepigenetics

Immuneregulation

FIGURE 5-1 Fetal programming. Maternal health and the placenta infl uence fetal adaptations. Dietary status, exposure to environmental factors, uteroplacental blood fl ow, placental transfer, and genetic and epigenetic changes contribute to the in utero fetal programming.

39CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord

The initial placental examination should include check-ing the edges for completeness. The membranes and fetal surface should be shiny and translucent. An odor may suggest infection, and cultures of the placenta may be benefi cial (Benirschke and Kaufmann, 2000). Green-ish discoloration may represent meconium staining or old blood; placentas with such discoloration should be sent to the pathologist for complete histologic examination. The fi nding of deep meconium staining of the membranes and umbilical cord suggests that the meconium was passed at least 2 hours before delivery; this fact may be helpful in

cases of meconium aspiration syndrome, for which legal questions may arise as to whether the aspiration occurred before or during labor. If the membranes are deeply stained, the passage of meconium by the fetus may have predated onset of labor; therefore aspiration could have occurred before labor. The umbilical cord should also be examined for the number of vessels and their inser-tion into the placenta. Vessels on the fetal surface of the placenta should be examined for evidence of clotting or thrombosis.

FUNCTIONS OF THE PLACENTA To ensure normal fetal growth and development, the placenta behaves as an effi cient organ of gas and nutrient exchange and as a robust endocrine and metabolic organ. Besides mediating the transplacental exchange of gases and nutrients, the placenta also synthesizes glycogen with a signifi cant turnover of lactate. Hormones secreted by the placenta have an important role for the fetus and the mother. Placental trophoblasts are a rich source of cho-lesterol and peptide hormones, including human chori-onic gonadotrophin (HCG), human placental lactogen, cytokines, growth hormones, insulin-like growth factors, corticotrophin-releasing hormones, and angiogenic fac-tors such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). HCG, which is detected as early as day 8 after conception, is secreted by syncytio-trophoblasts into the maternal circulation, reaches maximal levels by week 8 of pregnancy and diminishes later during gestation. HCG is essential to promote estrogen and pro-gesterone synthesis during different stages of pregnancy. Human placental lactogen mobilizes the breakdown of maternal fatty acid stores and ensures an increased supply of glucose to the fetus. VEGF and PlGF are secreted by trophoblasts and specialized natural killer ( NK) cells in the decidua, and they promote angiogenesis and vascular activ-ity, particularly during early stages of pregnancy when spiral

Maternal lacunae

Syncytiotrophoblast

Cytotrophoblast

Endometrium

Mesenchymal cells

A

BFetal blood vessel

C FIGURE 5-3 A, The cytotrophoblast indents the syncytiotrophoblast to form primary villi. B, Mesenchymal cells invade the cytotrophoblast 2 days after formation of the primary villi to form secondary villi. C, Blood vessels arise de novo and eventually connect with blood vessels from the embryo, forming tertiary villi. (From Moore TR, Reiter RC, Rebar RW, et al, editors: Gynecology and obstetrics: a longitudinal approach, New York, 1993, Churchill Livingstone.)

TABLE 5-1 Fetal and Placental Weight Throughout Gestation

Gestational Age (wk)

Placental Weight (mg)

Fetal Weight (g)

14 45 — 16 65 59 18 90 155 20 115 250 22 150 405 24 185 560 26 217 780 28 250 1000 30 282 1270 32 315 1550 34 352 1925 36 390 2300 38 430 2850 40 470 3400

Adapted from Benirschke K, Kaufmann P: Pathology of the human placenta, ed 4, New York, 2000, Springer-Verlag.

40 PART II Fetal Development

Umbilical arteries

Cytotrophoblastic shell

Anchoring villus

Fetalcirculation

Endometrialveins

Endometrialarteries

Maternal circulation

Chorionic plate

Umbilical vein

Placentalseptum

Deciduabasalis Myometrium

Amniochorionic membrane

Intervillous space

Mainstemvillus

Stump ofmainstem villus

Amnion

Smooth chorion

Decidua perietalis

FIGURE 5-4 Schematic drawing of a section of a mature placenta showing the relation of the villous chorion (fetal part of the placenta) to the decidua basalis (maternal part of the placenta), the fetal placental circulation, and the maternal placental circulation. Maternal blood fl ows into the intervillous spaces in funnel-shaped spurts, and exchanges occur with the fetal blood as the maternal blood fl ows around the villi. Note that the umbil-ical arteries carry deoxygenated fetal blood to the placenta, and the umbilical vein carries oxygenated blood to the fetus. In addition, the cotyledons are separated from each other by decidual septa of the maternal portion of the placenta. Each cotyledon consists of two or more mainstem villi and their main branches. In this drawing, only one mainstem villus is shown in each cotyledon, but the stumps of those that have been removed are shown. ( From Moore KL: The developing human: clinically oriented embryology, ed 5, Philadelphia, 1993, WB Saunders.)

TABLE 5-2 Pregnancy Conditions Diagnosable at Birth by Gross Placental Examination and Associated Neonatal Outcomes

Pregnancy Conditions Fetal/ Neonatal Outcomes Monochorionic twinning TTT syndrome donor/recipient status, pump twin in TRAP, survivor status after fetal demise,

selective termination, severe growth discordance without TTT Dichorionic twinning Less likelihood of survivor brain disease in the event of demise of one fetus Purulent acute chorioamnionitis Risk of fetal sepsis, fetal infl ammatory response syndrome, cerebral palsy Chorangioma Hydrops, cardiac failure, consumptive coagulopathy Abnormal cord coiling IUGR, fetal intolerance of labor Maternal fl oor infarction IUGR, cerebral disease Abruption Asphyxial brain disease Velamentous cord IUGR, vasa previa Cord knot Asphyxia Chronic abruption oligohydramnios syndrome IUGR Single umbilical artery Malformation, IUGR Umbilical vein thrombosis Asphyxia Amnion nodosum Severe oligohydramnios leading to pulmonary hypoplasia Meconium staining Possible asphyxia, aspiration lung disease Amniotic bands Fetal limb reduction abnormalities Chorionic plate vascular thrombosis Asphyxia, possible thrombophilia Breus mole Asphyxia, IUGR

IUGR, Intrauterine growth retardation; TRAP, twin-reversed arterial perfusion; TTT, twin-to-twin transfusion.

artery transformation and trophoblast invasion occurs. In addition, the placenta is a rich source of estrogen, proges-terone, and glucocorticoids. Whereas progesterone main-tains a quiescent, noncontractile uterus, it also has a role in protecting the conceptus from immunologic rejection by the mother. Glucocorticoids promote organ development

and maturation. Placental transport is another important function, effi ciently transferring nutrients and solutes that are essential for normal fetal growth. The syncytiotropho-blast covering the maternal villous surface is a specialized epithelium that participates in the transport of gases, nutri-ents, and waste products and the synthesis of hormones

41CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord

Placenta

Decidua

Myometrium SpiralSpiralarteriesarteries

STST

CTCT

NKNK

ETET

ITIT

M

DCDC

T

GCGC

Tregreg

NormalNormal Preeclampsia/IUGRPreeclampsia/IUGR

Spiralarteries

ST

CT

NK

ET

IT

M

DC

T

GC

Treg

Normal Preeclampsia/IUGR

ST: Syncytiotrophoblasts

CT: Columnar trophoblasts

ET: Endovascular trophoblasts

IT: Interstitial trophoblasts

GC: Giant cells

NK: Natural killer cells

T: T lymphocytes

Treg: Regulatory T cells

M: Macrophages

DC: Dendritic cells

AnchoringAnchoringVilliVilli

AnchoringVilli

FIGURE 5-5 Trophoblast differentiation and spiral artery remodeling. Progenitor trophoblast cells from villi differentiate into syncytiotropho-blasts and the extravillous cytotrophoblasts (EVTs). EVTs migrate out in columns as columnar trophoblasts and anchor the placenta to the decidua. Further differentiation takes place into invasive or proliferative EVTs. The invasive EVTs invade the decidua known as interstitial trophoblasts, and some of them fuse to form the multinucleated gaint cells. Endovascular transformation ensues as endovascular trophoblasts migrate into and colonize the spiral arteries, almost reaching the myometrium. This results in wide-bore, low-resistant capacitance blood vessels as observed in normal preg-nancy. In contrast, shallow trophoblast invasion and incomplete transformation of spiral arteries is a common feature of preeclampsia and intrauterine growth restriction.

that regulate placental, fetal, and maternal systems. The syncytiotrophoblast layer of the placenta is an important site of exchange between the maternal blood stream and the fetus. In addition to simple diffusion, syncytiotropho-blasts facilitate exchange by transcellular traffi cking that utilizes transport proteins such as the water channels (aqua-porins). Facilitated diffusion for molecules such as glucose and amino acids are performed by glucose transporters (GLUT) and amino acid transporters. In addition, adenos-ine triphosphate (ATP)-mediated active transport, such as the Na + , K + -ATPase or the Ca 2+ -ATPase, besides endocy-tosis and exocytosis, participates in transplacental exchange (Hahn et al, 2001; Malassiné and Cronier, 2002; Randhawa and Cohen, 2005; Siiteri, 2005).

In healthy women who are not pregnant, uterine blood vessels receive approximately 1% of the cardiac output to maintain the uterus. During pregnancy, these same ves-sels must support the rapidly growing and demanding pla-centa and fetus. This evolutionary challenge is addressed by remodeling of the spiral arteries, converting them into large, thin-walled, dilated vessels with reduced vascular resistance.

TROPHOBLAST DIFFERENTIATION AND REMODELING OF SPIRAL ARTERIES The placental-decidual interaction through invading tro-phoblasts determines whether an optimal transformation of the uterine spiral arteries is achieved. Trophoblast-orchestrated artery remodeling is an essential feature of normal human pregnancy. As shown in Figure 5-5 , pro-genitor trophoblast cells from villi differentiate along two pathways: terminally differentiated syncytiotrophoblasts and the extravillous cytotrophoblasts (EVTs) that migrate out in columns and anchor the placenta to the decidua.

From these anchoring layers of EVTs further differ-entiation takes place into invasive or proliferative EVTs. The invasive EVTs invade the decidua and shallow parts of the myometrium and are known as interstitial EVTs. Thereafter endovascular transformation ensues as invasive EVTs migrate into and colonize the spiral arteries, almost reaching the myometrium. These trophoblasts are known as endovascular EVTs . Insuffi cient uteroplacental interac-tion characterized by shallow trophoblast invasion and

42 PART II Fetal Development

incomplete transformation of spiral arteries is a common feature of preeclampsia and intrauterine growth restriction (IUGR) (Brosens et al, 1977; Meekins et al, 1994). The precise period when trophoblast invasion of decidua and spiral arteries ceases is not clear. Nevertheless it is widely believed to be completed late in the second trimester.

Although our understanding of the molecular events underlying spiral artery remodeling in pregnancy remains poor, effi cient trophoblast invasion is an essential feature. There are two waves of trophoblast invasion that follow implantation. The fi rst wave is during the fi rst trimester, when the invasion is limited to the decidual part of the spiral artery. The second wave is during the late second trimester involving deeper trophoblast invasion, reaching the inner third of myometrial segment. The initial inva-sion of EVTs into the endometrium initiates the decidual-ization process, which is characterized by replacement of extracellular matrix, loss of normal musculoelastic struc-ture, and deposition of fi brinoid material. Displacement of the endothelial lining of spiral arteries by the invading trophoblasts further results in uncoiling and widening of the spiral artery, ensuring the free fl ow of blood and nutrients to meet the escalating demands of the growing fetus (Kham et al, 1999; Pijnenborg et al, 1983). A lack of spiral artery remodeling with shallow trophoblast invasion has been associated with preeclampsia. During the pro-cess of invasion in a normal pregnancy, cytotrophoblasts undergo phenotypic switching, with a loss of E-cadherin expression, and they acquire vascular endothelial-cadherin, platelet-endothelial adhesion molecule-1, vascular endo-thelial adhesion molecule-1, and α 4 and α v β 3 integrins (Bulla et al, 2005; Zhau et al, 1997). Along with a repertoire of facilitators for invasion, trophoblasts express a nonclas-sic major histocompatibility complex (MHC) human leu-kocyte antigen (HLA) G, which has gained widespread interest because of providing noncytotoxic signals to uter-ine NK cells. It still needs to be evaluated whether intrinsic HLA-G inactivation by polymorphic changes infl uences the dysregulated trophoblast invasion seen in preeclampsia (Hiby et al, 1991; Le Bouteiller et al, 2007).

Although the exact gestational age at which tropho-blast invasion ceases is not known, recent studies have shown that late pregnancy trophoblasts loose the ability to transform the uterine arteries. Using a novel dual-cell in vitro culture system that mimics the vascular remodel-ing events triggered by normal pregnancy serum, we have shown that fi rst- and third-trimester trophoblasts respond differentially to interactive signals from endothelial cells when cultured on the extracellular matrix, matrigel. Term trophoblasts not only fail to respond to signals from endo-thelial cells, but they inhibit endothelial cell neovascular formation. In contrast, trophoblast cells representing fi rst-trimester trophoblasts with invasive properties undergo spontaneous migration and promote endothelial cells to form a capillary network ( Figure 5-6 ).

This disparity in behavior was confi rmed in vivo using a matrigel plug assay. Poor expression of VEGF-C and VEGF receptors coupled with high E-cadherin expres-sion by term trophoblasts contributed to their restricted migratory and interactive properties. Furthermore, these studies showed that the kinase activity of VEGF receptor 2 is essential for proactive crosstalk by invading fi rst-trimes-ter trophoblast cells (Kalkunte et al, 2008b). This unique

maternal and fetal cell interactive model under the preg-nancy milieu offers a potential approach to study cell-cell interactions and to decipher infl ammatory components in the serum samples from adverse pregnancy outcomes (Kalkunte et al, 2010). One of the inimitable contributors to trophoblast cell invasion is the specialized NK cell of the pregnant uterus.

IMMUNE PROFILE AND IMMUNO VASCULAR BALANCE DURING PLACENTATION During pregnancy, trophoblast cells directly encounter maternal immune cells at least at two sites. One site is the syncytiotrophoblasts covering the placental villi that are bathed in maternal blood, and the other is by the invad-ing trophoblasts in the decidua. Although the syncytio-trophoblasts do not express MHC antigens, the invading trophoblasts express nonclassic HLA-G and HLA-C and would elicit immune responses in the decidua. The decidua is replete with innate immune cells including T cells, regulatory T cells, macrophages, dendritic cells and NK cells ( Table 5-3 ). Interestingly, NK cells peak and constitute the largest leukocyte population in the early pregnant uterus, accounting for 60% to 70% of total lym-phocytes. These cells diminish in proportion as pregnancy proceeds.

PHENOTYPIC AND FUNCTIONAL FEATURES OF UTERINE NATURAL KILLER CELLS Peripheral blood NK (pNK) cells constitute 8% to 10% of the CD45 + population in circulation. All NK cells are characterized by a lack of CD3 and expression of CD56 antigen. Based on the intensity of CD56 antigen, NK cells are further divided into CD56 bright and CD56 dim popula-tions. The presence or absence of Fc γ RIII or CD16 fur-ther differentiates subpopulations of uterine NK (uNK) cells. Thus the majority of peripheral NK cells are of the CD56 dim CD16 + phenotype (approximately 90%), and the remaining cells are CD56 bright CD16 – (approximately 10%). The majority of uterine NK cells (approximately 90%) are CD56 bright CD16 – . In the uterine decidua, uNK cell numbers cyclically increase and decrease in tandem with the menstrual cycle—low in the proliferative phase (10% to 15%), which amplifi es during the early, middle and late secretory phases (25% to 30%)—falling to a basal level with menstruation ( Figure 5-7 ) (Kalkunte et al, 2008a; Kitaya et al, 2007).

With successful implantation, the uNK cell popula-tion further increases in the decidualized endometrium, reaches a peak in fi rst-trimester pregnancy, and dwindles thereafter by the end of the second trimester. The origin of uNK cells that peak during the secretory phase of the menstrual cycle and early pregnancy is currently not well established, and the evidence indicates multiple different possibilities. These possibilities include recruitment of CD56 bright CD16 – pNK cells, recruitment and tissue spe-cifi c terminal differentiation of CD56 dim CD16 + pNK cells, development of NK cells from Lin – CD34 + CD45 + pro-genitor cells, or proliferation of resident CD56 bright CD16 – NK cells. Comparative surface expression of antigens, natural cytotoxicity receptors, inhibitory receptors, and

43CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord

chemokines and cytokines on human pNK and uNK cells are provided in Table 5-4 . Furthermore, CD56 bright uNK cells are different from the CD56 bright minor population of pNK cells because of the expression of CD9, CD103, and killer immunoglobulin-like receptors (KIRs). Despite

being replete with cytotoxic accessories of perforin, gran-zymes A and B and the natural cytotoxicity receptors NKp30, NKp44, NKp46, NKG2D, and 2B4 as well as LFA-1, uNK cells are tolerant cytokine-producing cells at the maternal-fetal interface (Kalkunte et al, 2008a). The temporal occurrence around the spiral arteries and timed amplifi cation of these specialized uNK cells observed dur-ing the fi rst trimester implicate its role in spiral artery remodeling.

NK cell – defi cient mice display abnormalities in decid-ual artery remodeling and trophoblast invasion, possibly because of a lack of uNK cell – derived interferon γ ( Ashkar et al, 2000 ). Other studies have shown that unlike pNK cells, uNK cells are a major source of VEGF-C, Angio-poietins 1 and 2 and transforming grwoth factor (TGF- β 1) within the placental bed that decrease with gestational age (Lash et al, 2006). These observations implicate uNK cells in promoting angiogenesis. Studies have provided further evidence that uNK cells, but not pNK cells, regu-late trophoblast invasion both in vitro and in vivo through the production of interleukin-8 and interferon-inducible

AA B C

D E F

G H I

J K L

A B C

D E F

G H I

J K L FIGURE 5-6 (Supplemental color version of this fi gure is available online at www.expertconsult.com.) Differential endovascular activity of fi rst- and third-trimester trophoblasts in response to normal pregnancy serum. A representative micrograph of trophoblasts-endothelial cell interactions on matrigel is shown. Endothelial cells and trophoblasts are labeled with red and green cell tracker respectively, were independently cultured ( A to E ) or cocultured ( F to I ) on matrigel. Capillary-like tube structures were observed with human uterine endothelial cells (HUtECs) ( A ) and umbilical vein endothelial cells (HUVECs) ( B ) , but not with fi rst-trimester trophoblast HTR8 cells ( C ) , third trimester trophoblast TCL1 cells ( D ) , and primary term trophoblasts ( E ). However, in cocultures, HTR8 cells fi ngerprint the HUtECs ( F ) and HUVECs ( G ) , while TCL1 cells ( H ) and primary term trophoblasts ( I ) inhibit the tube formation by endothelial cells (magnifi cation ×4). Panels J to L show the cocultures of HTR8 with HUVECs ( J ) , HUtECs ( K ) , and term trophoblasts with HUVECs ( L ) at higher magnifi cation (×10). (Reproduced with permission from Kalkunte S, Lai Z, Tewari N, et al: In vitro and in vivo evidence for lack of endovascular remodeling by third trimester trophoblasts, Placenta 29:871-878, 2008.)

TABLE 5-3 Comparison of Peripheral Blood and Decidual Immune Cell Profi les

Immune CellsPeripheral blood (%) Decidua (%)

T cells 65-70 9-12 γ δ T cells 2-5 7-10

Macrophages 7-10 15-20 B cells 7-10 ND NKT cells 2-5 0.5-1.0 Tregs 2-4 6-10 NK cells 7-12 65-70 (CD56 bright CD16 - )

ND, Not detected.

44 PART II Fetal Development

protein-10, in addition to other angiogenic factors ( Hanna et al, 2006 ). Recent studies suggest that VEGF-C, a pro-angiogenic factor produced by uNK cells, is responsible for the noncytotoxic activity (Kalkunte et al, 2009). As noted previously, VEGF-C – producing uNK cells support endovascular activity in a coculture model of capillary tube formation on matrigel ( Figure 5-8 ). Peripheral blood NK cells fail to produce VEGF-C and remain cytotoxic. This function can be reversed by recombinant human VEGF-C. Cytoprotection by VEGF-C is related to induction of the transporter associated with antigen processing 1 and MHC assembly in target cells. Overall, these fi ndings sug-gest that expression of angiogenic factors by uNK cells keeps these cells noncytotoxic, which is critical to their pregnancy compatible immunovascular role during pla-centation and fetal development ( Kalkunte et al, 2009 ).

Although uNK cells seem to play a role that is compat-ible with pregnancy, retention of their cytolytic abilities suggests their role as sentinels at the maternal-fetal inter-face in situations that threaten fetal persistence. This facet of uNK cell function was elegantly demonstrated in animal models when pregnant mice were challenged with toll-like receptor (TLR) ligands that mimic bacterial and viral infections. These observations raise an important question whether uNK cells can harm the fetal placental unit and, if so, under what conditions?

The antiinfl ammatory cytokine interleukin (IL)-10 plays a critical role in pregnancy because of its regulatory relationship with other intrauterine modulators and its wide range of immunosuppressive activities (Moore et al, 2001). IL-10 expression by the human placenta depends on gestational age, with signifi cant expression through the second trimester followed by attenuation at term (Hanna et al, 2000). IL-10 expression is also found to be poor in decidual and placental tissues from unexplained spontane-ous abortion cases (Plevyak et al, 2002) and from deliveries associated with preterm labor ( Hanna et al, 2006 ) and pre-eclampsia (S. Kalkunte et al, unpublished observations). However, the precise mechanisms by which IL-10 protects the fetus remains poorly understood. IL-10 – / – mice suffer no pregnancy defects when mated under pathogen-free conditions (White et al, 2004), but they exhibit exquisite susceptibility to infection or infl ammatory stimuli com-pared with wild type animals. It is then plausible that in addition to IL-10 defi ciency, a “second hit” such as an infl ammatory insult resulting from genital tract infec-tions, environmental factors, or hormonal dysregulation during gestation can lead to adverse pregnancy out-comes (Tewari et al, 2009; Thaxton et al, 2009). Our recent studies provide direct evidence that uNK cells can become adversely activated and mediate fetal demise and preterm birth in response to low doses of TLR ligands

KIRNKG2D

NKp44

NKNKp46

NKp30

NK cellpopulation

LHP4

E2

~30% ~50–60%

Pregnancy

Inactivephase

Proliferativephase

Secretoryphase

Firsttrimester

Secondtrimester

Term

Menstrual cycle FIGURE 5-7 Biologic pattern of natural killer (NK) cells in the human endometrium and the decidua. The uterine NK cell population characterized by natural cytotoxicity receptors (Nkp30, NKp44, NKp46, NKG2D), killer immunoglobulin-like receptors, and cytolytic machinery (perforin and granzyme) cyclically increases and decreases in tandem with the hormonal changes during menstrual cycle. With successful implanta-tion, uterine NK cells further increase in the decidua and dwindle thereafter by the end of second trimester. E 2 , Estradiol; LH, luteinizing hormone; P4, progesterone.

45CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord

resulting in placental pathology ( Murphy et al, 2005; Murphy et al, 2009 ). Moreover, spontaneous abortion is associated with an increase in CD56 dim CD16 + cells and a decrease in CD56 bright CD16 – NK cells in the preimplan-tation endometrium during the luteal phase ( Michimata et al, 2002; Quenby et al, 1999). Therefore a fi ne bal-ance between maternal activating and inhibitory KIRs and their ligand HLA-C on fetal cells seems to be main-tained in normal pregnancy. Insuffi cient inhibition of uNK cells can activate the cytolytic machinery, resulting in spontaneous abortion, intrauterine growth restriction, or preterm labor, depending on the timing of the insult (Varla- Leftherioti et al, 2003). In the setting of IVF, the implantation failure has been associated with high uNK cell numbers, but direct evidence for their role in abnor-mal implantation is not clear (Quenby et al, 1999). Never-theless current understanding strongly implies that uNK cells retain the ability to become foes to pregnancy under the axis of genetic stress and infl ammatory trigger.

REGULATORY T CELLS AND PREGNANCY The existence of regulatory mechanisms that suppress the maternal immune system was proposed in the early 1950 (Medawar, 1953). For several years, maternal tolerance toward fetal alloantigens was explored in the context of Th1/Th2 balance, with Th2 cells and cytokines proposed

to predominate over Th1 cellular immune response under normal pregnancy. Recently the role of specialized T lym-phocytes, termed regulatory T cells (Tregs), in tolerogenic mechanisms has emerged. Tregs are potent suppressors of T cell – mediated infl ammatory immune responses and prevent autoimmunity and allograft rejection. Tregs act by controlling the autoreactive T cells that have escaped negative selection from the thymus, and they restrain the intensity of responses by T cells reactive with alloantigens and other exogenous antigens. This unique functional capa-bility to suppress responses to tissue-specifi c self-antigens that escape recognition by T cells during maturation is due to tissue specifi c expression and alloantigens, particularly in the epithelial surfaces where tolerance to nondangerous for-eign antigen is essential to normal function. This capability enables Tregs to play a unique role at the maternal-fetal interface. Tregs are typically characterized by a CD4 + CD25 + surface phenotype and expression of the hallmark suppres-sive transcription factor Foxhead Box P3 (Foxp3 + ). Their cell numbers increase in blood, decidual tissue, and lymph nodes draining the uterus during pregnancy. These cells are implicated in successful immune tolerance of the conceptus, mainly by producing IL-10 and TGF- β . Recent evidence suggests that fetal Tregs also play a vital role in suppress-ing fetal antimaternal immunity against maternal cells that cross the placenta (Mold et al, 2008).

In the absence of Tregs the allogeneic fetus is rejected, suggesting their critical role in normal pregnancy. Unex-plained infertility, spontaneous abortion, and preeclampsia are associated with proportional defi cience, functional Treg defi ciency, or both. In the context of pregnancy, the local milieu, particularly during the fi rst trimester, that includes hCG, TGF- β , IL-10, granulocyte-macrophage colony-stimulating factor, and indoleamine 2,3-dioxygenase expres-sion has now been shown to induce CD4 + CD25 + Tregs with Foxp3 expression with immunosuppressive features. This induction is thought to occur through the immature dendritic cells. In addition to immune suppressive and anti-infl ammatory properties, TGF- β is recognized as inducing differentiation of naïve CD4 T cells into suppressor T-cell phenotype, expressing Foxp3, and promoting the prolifera-tion of mature Tregs. In addition to the suppressive effects of cytokines produced by these cells, contact-mediated immune suppression by Tregs results from ligation of inhibitory cytotoxic T-lymphocyte antigen (CTLA-4) and its ability to induce tolerogenic dendritic cells and infl uence T-cell pro-duction of IL-10 ( Aluvihare et al, 2004; Schumacher et al, 2009 ; Shevach et al, 2009). Therefore the pregnant uterus may be a natural depot for Tregs.

EPIGENETIC REGULATION IN THE PLACENTA The regulation of gene expression is a crucial process that defi nes phenotypic diversity. Switching off or turning on genes as well as tissue-specifi c variation in gene expression contributes to this diversity. Besides the genetic make-up (i.e, sequence) of the individual, the regulation of gene expression is also infl uenced by epigenetic factors. Epigenetic changes include the noncoding changes in DNA and chromatin, or both, that mediate the interactions between genes and their environment. Epigenetic regulation generates a wider

TABLE 5-4 Phenotypic Characteristics of Surface Markers and Receptors on Natural Killer Cells

Antigen Peripheral blood Decidua

CD56 Dim (>90%) Bright CD16 + – CD45 + + CD7 + + CD69 – + L-Selectin – – /+

NK Receptors

KIR + + NKp30 + + NKp44 – * + NKp46 + + NKG2D + + CD94/NKG2A – /+ +

Chemokine Receptors

CXCR1 + + CXCR2 + – CXCR3 – + CXCR4 + + CX3CR1 + – CCR7 – +

Data from Kalkunte S, Chichester CO, Sentman CL, et al: Evolution of non-cytotoxic uterine natural killer cells, Am J Reprod Immunol 59:425-432, 2008. +, Present; – , absent; – /+, variable expression; KIR, killer immunoglobulin receptor; CXCR, CX-chemokine receptor; CX3CR1, CX3-C – chemokine receptor 1; CCR7, CC-chemokine receptor 7. * Expression seen on activation with interleukin 2.

46 PART II Fetal Development

diversity of cell types during mammalian development and sustains the stability and integrity of the expression profi les of different cell types and tissues. This regulation is choreo-graphed by changes in cytosine-phosphate-guanine (CpG) islands of the DNA promoter region by methylation, his-tone modifi cation, genomic imprinting, and expression of noncoding RNAs such as micro RNA (miRNA).

Gene-environment interactions resulting in epigenetic changes in the placenta during the critical window of devel-opment can infl uence fetal programming in utero, with predisposing health consequences later in life. Using a micro-array-based approach to compare chorionic villous samples from the fi rst trimester of pregnancy with gestational age – matched maternal blood cell samples, recent studies show tissue-specifi c differential CpG methylation patterns that identify numerous potential biomarkers for the diagnosis of fetal aneuploidy on chromosomes 13, 18 and 21 (Chu et al, 2009). Human placentation displays many similarities

with tumorigenesis, including rapid mitotic cell division, migration, angiogenesis and invasion, and escape from immune surveillance. Indeed, there are striking similarities in the DNA methylation pattern of tumor-associated genes between invasive trophoblast cell lines and fi rst-trimester placenta and tumors ( Christensen et al, 2009 ). This fi nding suggests that a distinct pattern of tumor-associated meth-ylation can result in a series of epigenetic silencing events necessary for normal human placental invasion and func-tion (Novakovic et al, 2008). Other studies using the pla-centa as a source suggest that the specifi c loss of imprinting because of altered methylation and subsequent gene expres-sion can result in small for gestational age (SGA) newborns. Moreover, unbalanced expression of imprinted genes in IUGR placenta when compared with non-IUGR placenta was observed suggesting a differential expression pattern of imprinted genes as a possible biomarker for IUGR (Guo et al, 2008; McMinn et al, 2006).

Non cytotoxic NK cells

VEGF C

CD56brightCD16�

Promotes endovascular activityUpregulates TAP-1 and MHC

molecules on trophoblatsEfficient trophoblast invasion

Intrauterine infections,Inflammation,TLR activation,Loss of IL-10

Normal pregnancy

Cytotoxic NK cells

CD56dimCD16���

CD56CD16

Poor endovascular activityTrophoblast lysis

Poor trophoblast invasion

Adverse pregnancy

DECIDUA

FIGURE 5-8 (See also Color Plate 1.) Angiogenic features of natural killer (NK) cells render them immune tolerant at the maternal-fetal interface. Vascular endothelial growth factor (VEGF) C – producing noncytotoxic uterine NK cell clones similar to decidual NK cells support endovascular activity in a coculture of endothelial cells and fi rst-trimester trophoblast HTR8 cells on matrigel. By contrast, cytotoxic uterine NK cell clones similar to peripheral blood NK cells disrupted the endovascular activity because of endothelial and trophoblast cell lysis. This distinct functional feature determines whether optimal trophoblast invasion takes place and can result in normal or adverse pregnancy outcomes.

47CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord

The unique cytokine and hormonal milieu in utero may infl uence the trophoblast function and differentiation as well as immune cell regulation through histone posttrans-lational modifi cation. In this regard, interferon γ produced by uNK cells and essential for spiral artery remodeling fails to induce MHC class II expression in trophoblast cells because of hypermethylation of regulatory class II MHC transactivator (CIITA) regions (Morris et al, 2002). This inability to upregulate classical MHC class II mol-ecules by trophoblasts is essential for maintaining immune tolerance at the maternal-fetal interface. Moreover, the transcription factor regulating trophoblastic fusion pro-tein syncytin, which is essential for the syncytialization of trophoblasts, is regulated by histone acetyl transferase and histone deacetylase activity (Chuang et al, 2006). miRNAs are small regulatory RNA molecules that can alter gene and protein expression without altering the underlying genetic code. Expression of miRNA is tissue specifi c, and several are expressed in the placenta. In placental pathol-ogy associated with preeclampsia, there is differential expression of miRNA (such as miR-210 and miR-182) compared with normal pregnancy placenta. This fi nding suggests that signature differences in placental miRNA and their detection in maternal serum may potentially be used as a biomarker for preeclampsia. Because implanta-tion and early placentation is under the regulation of low oxygen tension, it is possible that miRNA are differentially expressed under different oxygen levels, as suggested by recent observations (Maccani and Marsit, 2009; Pineles et al, 2007).

PLACENTAL DISEASES The placenta provides a wealth of retrospective informa-tion about the fetus and prospective information regarding the infant. Healthy development of the placenta requires effi cient metabolic, immune, hormonal, and vascular adaptation by the maternal system as well as the fetus. Abnormal placentation and placental infections can lead to maternal or fetal anomalies as seen in preeclampsia, preterm birth, and SGA, which can have lifelong bearing on the development and health of infants. Maternal fac-tors such as ascending infections, obesity, hypertension, genetic predisposition such as gene polymorphism of the pregnancy-compatible cytokine milieu, and environmental exposure could also contribute to the placental pathology. The following sections contain an abbreviated discussion of the pathogenesis of some of these placenta-associated disorders.

PREECLAMPSIA Hypertensive disorders of pregnancy are enigmatic. They pose a major public health problem and affect 5% to 10% of human pregnancies. Preeclampsia is clinically associ-ated with maternal symptoms of hypertension, protein-uria, and glomeruloendotheliosis. This disorder is strictly a placental condition because of its clearance after deliv-ery. It causes morbidity and mortality in the mother, fetus, and newborn. Pregnancy-associated hypertension is defi ned as blood pressure greater than 140/90 mm Hg on at least two occasions and at 4 to 6 weeks apart after 20 weeks’

gestation. Proteinuria is defi ned by excretion of 300 mg or more of protein every 24 hours or 300 mg/L or more in two random urine samples taken at least 4 to 6 hours apart (ACOG Committee on Practice Bulletins, 2002). The fetal problems most commonly associated with pre-eclampsia include fetal growth restriction, reduced amni-otic fl uid, and abnormal oxygenation (Sibai et al, 2005). However, the onset of clinical signs and symptoms can result in either near-term preeclampsia without affect-ing the fetus or its severe manifestation that is associated with low birthweight and preterm delivery (Vatten and Skjaerven, 2004). The heterogeneous manifestation of this disease is further confounded by preexisting maternal vascular disease, multifetal gestation, metabolic syndrome, obesity, or previous incidence of the disease. In addition, the pathophysiology of the disorder could differ from the onset before 24 weeks’ gestation and its diagnosis at later stages of pregnancy:

Abnormal remodeling of spiral arteries and shallow tro-phoblast invasion are two hallmark features of preeclamp-sia. Preeclampsia is considered a two-stage disease where a poorly perfused placenta (stage I) causes the release of factors leading to maternal symptoms (stage II). However, it is also now being recognized that the maternal factors may contribute to programming of stage I of preeclamp-sia, suggesting that the intrinsic maternal factors stem-ming from genetic, behavioral, and physiologic conditions may contribute to placental pathology. Stage I initiated pathology may be particularly apparent in the oxidative stress-induced release of causative factors from the poorly perfused placenta and their effects on the maternal syn-drome ( Roberts and Hubel, 1999 ).

Despite a poor mechanistic understanding of placental pathology leading to preeclampsia, several critical features are common to this disease. Multiple studies have shown that reduced vascular activity could be a major factor con-tributing to preeclampsia. In normal pregnancy, the circu-lating PlGF levels steadily increase in the fi rst and second trimesters, peak at 29 to 32 weeks, and decline thereafter. However, free VEGF remains low and unchanged dur-ing this window. Reduced placental expression of VEGF and PlGF is consistently observed in preeclampsia. Fur-thermore, preeclampsia is frequently accompanied by enhanced circulation and placental expression of the anti-angiogenic soluble VEGF receptor 1 (sFlt-1), which is a decoy receptor titrating out VEGFs and PlGF ( Levine et al, 2004; Romero et al, 2008; Thadhani et al, 2004 ). A lack of available VEGF and increased sFlt-1 expres-sion has been associated with trophoblast injury. The soluble form of endoglin (CD105), a coreceptor involved in TGF- β signaling is reported to enhance the antiangio-genic effects of sFlt-1. Soluble endoglin has been found to be elevated in the serum of preeclamptic women and is accompanied by an increased ratio of sFlt-1:PlGF and cor-relates with the severity of the disease. Soluble endoglin is thought to inhibit TGF-β1 signaling in endothelial cells and blocks activation of endothelial nitric oxide synthase and vasodilatation (Venkatesha et al, 2006). Several recent studies have suggested an increase in apoptosis within vil-lous trophoblast from preeclampsia and IUGR deliveries (Allaire et al, 2000; Heazell and Crocker, 2008; Levy et al, 2002). Unlike normal pregnancy, villous placental explants

48 PART II Fetal Development

from preeclamptic placenta have an increased sensitiv-ity and susceptibility to apoptosis on exposure to proin-fl ammatory cytokines, suggesting altered programming of apoptotic cascade pathway (Crocker et al, 2004; Levy et al, 2002). It is possible that incomplete spiral artery transformation resulting in reduced placental perfusion (stage I) in preeclampsia leads to focal regions of hypoxia with increase in apoptosis, oxidative stress, shedding of villous microparticles, and release of antiangiogenic fac-tors such as sFlt-1.64 (Hung et al, 2002; Nevo et al, 2006; Redman and Sargent, 2000).

Another pathway that may contribute to the etiology of preeclampsia is unscheduled and excessive activation of the complement cascade; this is highly likely as a result of the maternal immune system responding to paternal antigens and infl ammation. However, in normal pregnancy the placenta expresses complement regulatory proteins such as DAF, CD55, and CD59 and may control activation of complement factors (Tedesco et al, 1993). Despite the positioning of complement inhibitory proteins for protec-tive roles, increasing evidence supports the involvement of complement activation in the pathogenesis of preeclamp-sia (Lynch et al, 2008). Interestingly, recent in vitro studies suggest that hypoxia enhances placental deposition of the membrane attack complex and apoptosis in cultured tro-phoblasts (Rampersad et al, 2008). The upstream factors that trigger complement activation are not yet known.

Recent studies also suggest increased serum levels of agonistic autoantibodies against angiotensin type 1 receptor (AT-1-AA) in preeclampsia as compared with healthy women (Zhou et al, 2008). Importantly, studies from our laboratory have shown that the full spectrum of preeclampsia-like symptoms can be reproduced in mice by injecting human preeclampsia serum containing sub-threshold levels of AT-1-AA immunoglobulin G, sug-gesting that pregnancy serum contains some unknown causative factors. Therefore serum can be used as a blue-print to identify functional biomarkers for preeclampsia ( Kalkunte et al, 2009 ).

PRETERM BIRTH Preterm birth is the leading cause of infant morbidity and mortality in the world. Babies born before 37 weeks’ gestation are considered premature. In the United States, approximately 12.8% of births are preterm, and the rate of premature birth has increased by 36% since early 1980s (Martin et al, 2009). Babies from preterm birth face an increased risk of lasting disabilities such as mental retarda-tion, learning and behavioral problems, autism, cerebral palsy, bronchopulmonary dysplasia, vision and hearing loss, and risk for diabetes, hypertension, and heart disease in adulthood. The majority of preterm deliveries are due to preterm labor. Other factors leading to premature birth are preterm premature rupture of membranes (PPROM), intervention for maternal or fetal problems, preeclampsia, fetal growth restriction, cervical incompetence, and ante-partum bleeding. Additional risk factors for preterm birth include stress, occupational fatigue, uterine distention by polyhydramnios or multifetal gestation, systemic infec-tion such as periodontal disease, intrauterine placental pathology such as abruption, vaginal bleeding, smoking,

substance abuse, maternal age (<18 or >40 years), obesity, diabetes, thrombophilia, ethnicity, anemia, and fetal fac-tors such as congenital anomalies and growth restriction.

Activation of the hypothalamic-pituitary-adrenal (HPA) as a result of major maternal physical or psychological stress is thought to increase the release of corticotrophin-releasing hormone. In addition to the hypothalamus as a source of corticotrophin-releasing hormone, placental trophoblasts, amnion, and decidual cells also express this hormone during pregnancy. Corticotrophin hormone regulates the release of adrenocorticotropic hormone from pituitary and cortisol from adrenal glands, and it can also infl uence the activity of matrix metalloprotein-ases (MMPs). Premature activation of the HPA axis can eventually stimulate the prostaglandins, ultimately result-ing in parturition via activation of proteases. In addition, activation of the HPA axis promotes the release of estrone, estradiol, and estriol that can activate the myometrium by increasing oxytocin receptors, prostaglandin activity, and enzymes such as myosin light chain kinase and calmodu-lin, which are responsible for muscle contraction. Con-comitantly, progesterone withdrawal is expected with the raising concentration of myometrial estrogen receptors, further enhancing estrogen-induced myometrial activa-tion and preterm birth (Dole et al, 2003; Grammatopoulos and Hillhouse, 1999; McLean et al, 1995).

There is increasing evidence that approximately 50% of preterm births are associated with infection of the decidua, amnion, or chorion and amniotic fl uid caused by either systemic or ascending genital tract infection. Both clinical and subclinical chorioamnionitis are implicated in preterm birth. Maternal or fetal infl ammatory responses to cho-rioamniotic infection can trigger preterm birth. Activated neutrophils and macrophages and the release of cytokines IL-1 β , IL-6, IL-8, tumor necrosis factor alpha (TNF- α ) and granulocyte colony-stimulating factor can lead to an enhanced cascade of signaling activity, causing release of prostaglandins and expression of various MMPs of fetal membranes and the cervix. Furthermore, elevated levels of TNF- α and apoptosis are associated with PPROM. Non – infection-related infl ammation caused by placental insuffi ciency and apoptosis can also cause preterm birth. In addition to augmented infl ammatory responses to infections, pathogenic microbes (e.g. Staphylococcus, Strep-tococcus, Bacteroides, and Pseudomonas spp.) are thought to directly degrade fetal membranes by releasing proteases, collagenases, and elastases, produce phospholipase A2, and release endotoxin that stimulate uterine contractions and cause preterm birth ( Goldenberg et al, 2000, 2008; Romero et al, 2006; Slattery and Morrison, 2002 ).

The innate immune system and trophoblasts during pregnancy recognize bacterial and viral infections using TLRs. Placental transcripts for TLRs 1 to 10 have been detected in human placental tissue, and placental chorio-carcinoma cell lines reportedly express TLR-2, TLR-4, and TLR-9 (Abrahams and Mor, 2005). Studies have dem-onstrated functionality for TLR-2, TLR-3, and TLR-4 in fi rst- and third-trimester placental tissue (Patni et al, 2007). Decidual expression in humans has demonstrated functional receptors in term decidua of TLR-1, TLR-2, TLR-4, and TLR-6 (Canavan and Simhan, 2007). Our recent studies using mice have shown that extremely small

49CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord

doses of the TLR-4 ligand lipopolysaccharide can cause preterm birth or fetal demise in pregnant IL-10 – defi cient mice by activating and promoting infi ltration of uterine NK cells into the placenta and inducing apoptosis by secretion of TNF- α ( Murphy et al, 2005, 2009 ). Similarly, activation of TLR-3 or TLR-9 has been shown to induce spontaneous abortion or preterm birth in IL-10 – defi cient pregnant mice that is attributed to immune infi ltration and proinfl ammatory cascade in the placenta (Thaxton et al, 2009).

Decidual hemorrhage leading to vaginal bleeding increases the risk for preterm birth and PPROM. Increased occult decidual hemorrhage, hemosiderin deposition, and retrochorionic hematoma formation is seen between 22 and 32 weeks’ gestation as a result of PPROM and preterm birth after preterm labor. The development of PPROM in the setting of abruption could be caused by high decidual concentration of tissue factors, which eventually gener-ate thrombin. Thrombin activation as measured by serum thrombin-antithrombin III complex levels are elevated on preterm birth. Thrombin binds to decidual protease- activated receptors (PAR1 and PAR2), induces the produc-tion of IL-8 in decidua, attracts neutrophils, and promotes degradation of the fetal membrane MMPs that can result in PPROM (Lockwood et al, 2005; Salafi a et al, 1995).

Polyhydramnios is also a high risk factor for preterm birth. It was shown recently that exposure of IL-10 – defi cient pregnant mice to polychlorinated biphenyls, an environmental toxicant, can lead to preterm birth with IUGR. The IUGR was due to increased amniotic fl uid volume (polyhydramnios) and placental insuffi ciency caused by poor spiral artery remodeling associated with reduced expression of water channel aquaporin-1 in the placenta (Tewari et al, 2009). Increasing evidence also sug-gests impaired vascular activity because of an increase in antiangiogenic factors such as sFlt-1 and decreased VEGF in PPROM and preterm birth (Kim et al, 2003).

INTRAUTERINE GROWTH RESTRICTION IUGR is used to designate a fetus that has not reached its growth potential; it can be caused by fetal, placental, or maternal factors. Disparities between fetal nutritional or respiratory demands and placental supply can result in impaired fetal growth. Chromosomal abnormalities (aneuploidy, partial deletions, gene mutation particu-larly on the gene for insulin-like growth factors), con-genital abnormalities, multiple gestation, and infections can also result in IUGR. Preterm birth, preeclampsia, and abruption because of placental ischemia can result in IUGR. Reduced placental weight with identifi able pla-cental histologic abnormalities (e.g, impaired develop-ment or obstruction in uteroplacental vasculature, chronic abruption, chronic infections, maternal fl oor infarction, thrombosis in uteroplacental vasculature or fetoplacen-tal vasculature) are common fi ndings in IUGR. In addi-tion, a single umbilical artery, velamentous umbilical cord insertion, bilobate placenta, circumvallate placenta, and placental hemangioma are some of the other structural anomalies seen in the placenta. Maternal factors such as nutritional defi ciency; severe anemia; pulmonary disease leading to maternal hypoxemia; smoking; exposure to

toxins such as warfarin, anticonvulsants, folic acid antag-onists, and caffeine; and pregnancies conceived through assisted reproductive techniques have a higher prevalence of IUGR. IUGR results in the birth of an infant who is SGA. Mortality and morbidity are increased in SGA infants compared with those who are appropriate for ges-tational age. SGA infants at birth have many clinical prob-lems that include impaired thermoregulation; diffi culty in cardiopulmonary transition with perinatal asphyxia, pul-monary hypertension, hypoglycemia, polycythemia and hyperviscosity; impaired cellular immune function; and increased risk for perinatal mortality. SGA infants in their childhood and adolescence are at higher risk for impaired physical growth and neurodevelopment. Adolescents born SGA at term were reported to have learning diffi -culties with attention defi cits. Cognitive performance is generally lower in SGA infants at the ages of 1 to 6 years compared with those whoe are appropriate for gestational age. Adults who were SGA infants could be at higher risk for ischemic heart diseases and essential hypertension (Figueras et al, 2007; Kaijser et al, 2008; Lapillonne et al, 1997; Norman and Bonamy, 2005; O’Keefe et al, 2003; Spence et al, 2007).

FETAL MEMBRANES AND THEIR PATHOLOGY The fetal tissue – derived membrane structure surrounds the fetus and forms the amniotic cavity. This membrane, which lacks both vascular and nerve cells, is composed of an inner layer adjacent to the amniotic fl uid and is called the amnion . The outer layer that is attached to the decidua is called the chorion . Amnion is composed of inner epithe-lial cells, and the mesenchymal cell layer is composed of fi broblast and an outer spongy layer. Intact, healthy fetal membranes are required for normal pregnancy outcome. Chorion is composed of an outer reticular cell layer com-posed of fi broblasts and macrophages and an inner cyto-trophoblast layer. The elasticity and strength of these membranes are maintained by extracellular matrix proteins such as collagens, fi bronectin, laminins, and the activity of MMP-2 and MMP-9 and their inhibitors until the initia-tion of parturition when the membranes are susceptible to rupture. During parturition, when contractions begin or membranes rupture, MMP activity in the amnion and chorion increases with a concurrent fall in tissue inhibitors of metalloproteinases. This change is followed by apopto-sis in the amnion epithelial and chorion trophoblast layers of fetal membrane. Interestingly, some evidence suggests that fetal membranes have antimicrobial activity and are known to express TLR-2 and TLR-4, which are pattern recognition receptors and help in initiating a protective host response to infection.

The histopathology of amnion and chorion includes infections, amniotic fl uid contaminants, and fetal diseases. In addition to the membranes, whose infection can lead to chorioamnionitis, another vulnerable portal for infection to occur is the placental intervillous space and fetal villi that provide hematogenous access. Hematogenous sources of infection are typically associated with infl ammation of villi (villitis) and intervillous space (intervillositis). Viral pathogens (cytomegalovirus, HIV, herpes simplex virus)

50 PART II Fetal Development

commonly produce hematogenous infection of the pla-centa in addition to bacteria, spirochetes, fungi, and pro-tozoa (Gersell, 1993; Goldenberg et al, 2000; Lahra and Jeffery, 2004).

UMBILICAL CORD The connecting cord from the developing embryo or fetus to the placenta is the umbilical cord, or funiculus umbili-calis. During prenatal development in humans, the nor-mal umbilical cord contains two umbilical arteries and one umbilical vein buried within Wharton’s jelly. The umbilical vein supplies the fetus with oxygenated blood from the placenta while the arteries return the deoxygen-ated, nutrient-depleted blood to the placenta. In the fetus, the umbilical vein branches into the ductus venosus and another branch that joins the hepatic portal vein. Shortly after parturition, physiologic processes cause the Whar-ton’s jelly to swell with the collapse of blood vessels, result-ing in a natural halting of the fl ow of blood. Within the infant, the umbilical vein and ductus venosus close and degenerate into remnants known as the round ligament of the liver and the ligamentum venosum, while the umbilical arteries degenerate into what is known as medial umbilical ligaments .

Abnormalities associated with the umbilical cord can affect both the mother and the child. Pathology of umbili-cal cord is generally grouped as congenital remnants, infections, meconium, and masses. Abnormalities that have clinical signifi cance are nuchal cord, single umbili-cal artery, umbilical cord prolapse, umbilical cord knot, umbilical cord entanglement, vasa previa, and velamen-tous cord insertion. Common intrauterine infections can result in the umbilical cord being invaded by fetal cells and bacteria infi ltrated from the decidua to amniotic fl uid, or they can elicit fetal infl ammatory response. Umbilical cord infl ammation, known as funisitis or vasculitis, poses a higher risk for development of neurologic compromise in the fetus. Funisitis is predictive of a lower median Bayley psychomotor developmental index in infants. Meconium pigment at high concentrations can damage the umbili-cal cord by triggering apoptosis of smooth muscle cells. Vascular necrosis caused by meconium is associated with

oligohydramnios, low Apgar scores, and signifi cant neuro-developmental delay. Interruption of normal blood fl ow in the cord can cause prolonged hypoxia in utero. Clamping of the umbilical cord within minutes of birth is hospital-based obstetric practice. A Cochrane review studying the effects of the timing of umbilical cord clamping in hos-pitals showed that infants whose cord clamping occurred later than 60 seconds after birth had a signifi cantly higher risk of neonatal jaundice requiring phototherapy. How-ever, randomized, controlled studies have shown that delayed cord clamping in preterm infants reduces the incidence of intraventricular hemorrhage and late-onset sepsis. Furthermore, premature clamping can increase the risk of ischemia and hypovolemic shock, which can lead to fetal complications (McDonald and Middletone, 2008; Mercer et al, 2006).

SUGGESTED READINGS Aluvihare V R , Kallikourdis M , Betz A G : Regulatory T cells mediate maternal

tolerance to the fetus , Nat Immunol 5 : 266 - 271 , 2004 . Ashkar A A , Di Santo J P , Croy B A : Interferon γ contributes to initiation of uterine

vascular modification, decidual integrity, and uterine natural killer cell matura-tion during normal murine pregnancy , J Exp Med 192 : 259 - 269 , 2000 .

Baker D J P : In utero programming of chronic disease , Clin Sci 95 : 115 - 128 , 1998 . Christensen B C , Houseman E A , Marsit C J , et al: Aging and environmental

exposures alter tissue-specific DNA methylation dependent upon CpG island context , PLoS Genet 5 : e1000602 , 2009 .

Goldenberg R L , Culhane J F , Iams J D , et al: Epidemiology and causes of preterm birth , Lancet 371 : 75 - 84 , 2008 .

Hanna J , Goldman-Wohl D , Hamani Y , et al: Decidual NK cells regulate key developmental processes at the human fetal-maternal interface , Nat Med 12 : 1065 - 1074 , 2006 .

Kalkunte S , Mselle T F , Norris W E , et al: VEGF C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal-fetal interface , J Immunol 182 : 4085 - 4092 , 2009 .

Moffett A , Loke C : Immunology of placentation in eutherian mammals , Nat Rev Immunol 6 : 584 - 594 , 2006 .

Murphy S P , Hanna N N , Fast L D , et al: Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery , Am J Obstet Gynecol 200 : 308 , 2009 .

Paria B C , Reese J , Das S K , et al: Deciphering the cross-talk of implantation: advances and challenges , Science 296 : 2185 - 2188 , 2002 .

Roberts J M , Hubel C A : Is oxidative stress the link in the two – stage model of preec-lampsia? Lancet 354 : 788 - 789 , 1999 .

Slattery M M , Morrison J J : Preterm delivery , Lancet 360 : 1489 - 1497 , 2002 . Thadhani R , Sachs B P , Epstein F H , et al: Circulating angiogenic factors and the

risk of preeclampsia , N Engl J Med 350 : 672 - 683 , 2004 .

Complete references and supplemental color images used in this text can be found online at www . expertconsult . com