P A R T II FETAL DEVELOPMENT C H A P T E R Immunologic Basis of Placental Function 5 and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord Satyan Kalkunte, James F. Padbury, and Surendra SharmaComplex yet intricate interactions between maternal and invading trophoblast cells, and arterial endothelial cells.fetal systems promote fetal growth and normal pregnancy Pregnancy is considered an immunologic paradox, inoutcomes. Throughout embryonic development, organo- which paternal antigen-expressing placental cells interactgenesis and functional maturation are taking place. This directly with and coexist with the maternal immune systemperiod of development coincides with a high rate of cel- (Medawar, 1953). This anatomic distinction of the immu-lular proliferation and organ development, which creates nologic interface that arises from hemochorial placentationcritical periods of vulnerability. Adverse factors, disruption, that occurs in humans and rodents is distinct from epithe-or impairment during these critical periods of fetal devel- liochorial placentation as seen in marsupials, horses, andopment can alter developmental programming, which can swine or the endotheliochorial placentation seen in dogslead to permanent metabolic or structural changes (Baker, and cats. Understanding the anatomic and physiologic1998). For example, triggers such as undernutrition can events that occur during placentation is the key to appre-elicit placental and fetal adaptive responses that can lead ciate the uniqueness of human placentation in the phylo-to local ischemia and metabolic, hormonal, and immune genetic evolution. Typically, in hemochorial placentation,reprogramming, resulting in small for gestational age (SGA) maternal uterine blood vessels and decidualized endome-fetuses. Maternal health, dietary status, and exposure to trium are colonized by trophoblast cells, derived fromenvironmental factors, uteroplacental blood ﬂow, placental trophectoderm of the implanting blastocyst. These cellstransfer, and fetal genetic and epigenetic responses likely come in direct contact with maternal blood and uterineall contribute to in utero fetal programming (Figure 5-1). tissue. A similar phenomenon is evident in murine preg-Adult diseases such as coronary heart disorders, hyperten- nancy, except the trophoblast invasion is deeper in humanssion, atherosclerosis, type 2 diabetes, insulin resistance, (Moffett and Loke, 2006). In epitheliochorial placentation,respiratory distress, altered cell-mediated immunity, can- trophoblast cells of the placenta are in direct contact withcer, and psychiatric disorders are now thought to be a con- the surface epithelial cells of the uterus, but there is nosequence of in utero life (Sallour and Walker, 2003). It is a trophoblast-cell invasion beyond this layer. In endothelio-matter of considerable interest that, in addition to maternal chorial placentation, the trophoblast cells breach the uter-predisposing factors, cytokines, hormones, growth factors, ine epithelium and are in direct contact with endothelialand the intrauterine immune milieu also contribute to in cells of maternal uterine blood vessels.utero programming. Adaptations of the maternal immunesystem exist to modulate detrimental effects on the fetusand additional mechanisms and factors actively cross the EMBRYOLOGIC DEVELOPMENTplacenta and induce regulatory T cells in the fetus to sup- OF THE PLACENTApress fetal antimaternal immunity. These effects persist at Shortly after fertilization takes place in the ampullary por-least through adolescence (Burlingham, 2009; Mold et al, tion of the fallopian tube, the fertilized ovum or zygote2008). Excessive restraint of maternal immune responses begins dividing into a ball of cells called a morula. As thecould lead to a lethal infection in the newborn. On the other morula enters the uterus (by the fourth day after fertiliza-hand, too little modulation of maternal immune response to tion), it forms a central cystic area and is called a blastocystthe fetal allograft could lead to autoimmune-mediated fetal- (Figure 5-2). The blastocyst implants within the endome-placental rejection. Moreover, placental growth resembles trium by day seven (Moore, 1988).that of a tumor, evading immune surveillance and initiat- The blastocyst has two components: an inner cell mass,ing its own angiogenesis. Therefore a healthy mother with which becomes the developing embryo, and the outer cellhealthy placentation is critical to healthy fetal outcomes. layer, which becomes the placenta and fetal membranes. The cells of the developing blastocyst, which eventually become the placenta, are differentiated early in gestationMAMMALIAN PLACENTATION (within 7 days after fertilization). The outer cell layer, theThe immune tolerance of the semiallograft fetus and de trophoblast, invades the endometrium to the level of thenovo vascularization are two highly intriguing processes decidua basalis. Maternal blood vessels are also invaded.that involve direct interaction of maternal immune cells, Once entered and controlled by the trophoblast, these 37
38 PART II Fetal Development Maternal Placenta Fetus Outer cell layer Uteroplacental Metabolic Health status perfusion adaptation Inner cell mass Diet and Genetics and exposure status Ischemia epigenetics Hormonal Immune Immune status regulation regulation Placental-fetal A response Cytotrophoblast Syncytiotrophoblast Fetal pole Fetal ProgrammingFIGURE 5-1 Fetal programming. Maternal health and the placentainﬂuence fetal adaptations. Dietary status, exposure to environmentalfactors, uteroplacental blood ﬂow, placental transfer, and genetic andepigenetic changes contribute to the in utero fetal programming.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 isthought to represent the more immature form of tropho-blast. The syncytiotrophoblast, which is essentially acel- Blular, is the site of most placental hormone and metabolic FIGURE 5-2 A, The human blastocyst contains two portions: anactivity. Once the trophoblast has invaded the endome- inner cell mass, which develops into the embryo, and an outer celltrium, it begins to form outpouchings called villi, which layer, which develops into the placenta and membranes. B, The outerextend into the blood-ﬁlled maternal lacunae or further acellular layer is the syncytiotrophoblast, and the inner cellular layer isinvade the endometrium to attach more solidly with the the cytotrophoblast. (From Moore TR, Reiter RC, Rebar RW, et al, editors:decidua, forming anchoring villi. Gynecology and obstetrics: a longitudinal approach, New York, 1993, Churchill Livingstone.)PLACENTAL ANATOMY AND CIRCULATIONAt term, the normal placenta covers approximately onethird of the interior portion of the uterus and weighs EXAMINATION OF THE PLACENTAapproximately 500 g. The appearance is of a ﬂat circular A renaissance in placental pathology has led to a new rel-disc approximately 2 to 3 cm thick and 15 to 20 cm across evance of the placenta to neonatology and early infant(Benirschke and Kaufmann, 2000). Placental and fetal life, including issues of preterm birth, growth restriction,weights throughout gestation are presented in Table 5-1. and cerebral, renal, and myocardial diseases. The placentaDuring the ﬁrst trimester and into the second, the placenta can give some clues to the timing and extent of impor-weighs more than the fetus; after that period, the fetus out- tant adverse prenatal or neonatal events as well as to theweighs the placenta. With the formation of the tertiary villi relative effects of sepsis and asphyxia on the causation of(19 days after fertilization), a direct vascular connection neonatal diseases. Placental disorders can be noted imme-is made between the developing embryo and the placenta diately in the delivery room, and others can be diagnosed(Moore, 1988). Umbilical circulation between the placenta through detailed gross and microscopic examinations overand the embryo is evident by 51⁄2 weeks’ gestation. Figure the ensuing 48 hours. Every placenta should be examined5-4 demonstrates aspects of the maternal and fetal circula- at the time of birth regardless of whether the newborntion in the mature placenta. The umbilical arteries from the has any immediate problems. Most placentas invert withfetus reach the placenta and then divide repetitively to cover traction at the time of delivery, and the fetal membranesthe fetal surface of the placenta. Terminal arteries then pen- cover the maternal surface. It is important to reinvert theetrate the individual cotyledons, forming capillary beds for membranes and examine all surfaces of the placenta andsubstrate exchange within the tertiary villi. These capillaries membranes, looking for abnormalities. Table 5-2 liststhen reform into tributaries of the umbilical venous system, pregnancy complications or conditions that are diagnos-which carries oxygenated blood back to the fetus. able at birth through examination of the placenta.
CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord 39 Maternal lacunae TABLE 5-1 Fetal and Placental Weight Throughout Gestation Syncytiotrophoblast Gestational Placental Fetal Age (wk) Weight (mg) Weight (g) Cytotrophoblast 14 45 — 16 65 59 18 90 155 20 115 250 22 150 405 24 185 560 26 217 780 28 250 1000 Endometrium 30 282 1270 A 32 315 1550 Mesenchymal cells 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. 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 B stained, the passage of meconium by the fetus may have Fetal blood vessel 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 efﬁcient organ of gas and nutrient exchange and as a robust endocrine and metabolic organ. C Besides mediating the transplacental exchange of gasesFIGURE 5-3 A, The cytotrophoblast indents the syncytiotrophoblast and nutrients, the placenta also synthesizes glycogen withto form primary villi. B, Mesenchymal cells invade the cytotrophoblast a signiﬁcant turnover of lactate. Hormones secreted by2 days after formation of the primary villi to form secondary villi.C, Blood vessels arise de novo and eventually connect with blood the placenta have an important role for the fetus and thevessels from the embryo, forming tertiary villi. (From Moore TR, Reiter mother. Placental trophoblasts are a rich source of cho-RC, Rebar RW, et al, editors: Gynecology and obstetrics: a longitudinal lesterol and peptide hormones, including human chori-approach, New York, 1993, Churchill Livingstone.) 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 The initial placental examination should include check- as early as day 8 after conception, is secreted by syncytio-ing the edges for completeness. The membranes and trophoblasts into the maternal circulation, reaches maximalfetal surface should be shiny and translucent. An odor levels by week 8 of pregnancy and diminishes later duringmay suggest infection, and cultures of the placenta may gestation. HCG is essential to promote estrogen and pro-be beneﬁcial (Benirschke and Kaufmann, 2000). Green- gesterone synthesis during different stages of pregnancy.ish discoloration may represent meconium staining or old Human placental lactogen mobilizes the breakdown ofblood; placentas with such discoloration should be sent to maternal fatty acid stores and ensures an increased supplythe pathologist for complete histologic examination. The of glucose to the fetus. VEGF and PlGF are secreted byﬁnding of deep meconium staining of the membranes and trophoblasts and specialized natural killer (NK) cells in theumbilical cord suggests that the meconium was passed at decidua, and they promote angiogenesis and vascular activ-least 2 hours before delivery; this fact may be helpful in ity, particularly during early stages of pregnancy when spiral
40 PART II Fetal Development Fetal Umbilical vein circulation Umbilical arteries Decidua perietalis Amniochorionic membrane Smooth chorion Chorionic plate Intervillous space Amnion Mainstem Stump of villus mainstem villus Placental septum Anchoring villus Decidua Cytotrophoblastic shell basalis Myometrium Endometrial Endometrial veins arteries Maternal circulationFIGURE 5-4 Schematic drawing of a section of a mature placenta showing the relation of the villous chorion (fetal part of the placenta) to thedecidua basalis (maternal part of the placenta), the fetal placental circulation, and the maternal placental circulation. Maternal blood ﬂows into theintervillous spaces in funnel-shaped spurts, and exchanges occur with the fetal blood as the maternal blood ﬂows 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 cotyledonsare separated from each other by decidual septa of the maternal portion of the placenta. Each cotyledon consists of two or more mainstem villi andtheir 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 inﬂammatory response syndrome, cerebral palsy Chorangioma Hydrops, cardiac failure, consumptive coagulopathy Abnormal cord coiling IUGR, fetal intolerance of labor Maternal ﬂoor 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 and maturation. Placental transport is another importantaddition, the placenta is a rich source of estrogen, proges- function, efﬁciently transferring nutrients and solutes thatterone, and glucocorticoids. Whereas progesterone main- are essential for normal fetal growth. The syncytiotropho-tains a quiescent, noncontractile uterus, it also has a role in blast covering the maternal villous surface is a specializedprotecting the conceptus from immunologic rejection by epithelium that participates in the transport of gases, nutri-the mother. Glucocorticoids promote organ development ents, and waste products and the synthesis of hormones
CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord 41 Anchoring Villi ST Placenta CT IT Decidua ET ST: Syncytiotrophoblasts M NK CT: Columnar trophoblasts DC ET: Endovascular trophoblasts T IT: Interstitial trophoblasts Treg Treg Spiral GC: Giant cells Myometrium GC arteries NK: Natural killer cells T: T lymphocytes Treg: Regulatory T cells M: Macrophages DC: Dendritic cells Normal Preeclampsia/IUGRFIGURE 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, andsome of them fuse to form the multinucleated gaint cells. Endovascular transformation ensues as endovascular trophoblasts migrate into and colonizethe 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 intrauterinegrowth restriction.that regulate placental, fetal, and maternal systems. The TROPHOBLAST DIFFERENTIATIONsyncytiotrophoblast layer of the placenta is an importantsite of exchange between the maternal blood stream and AND REMODELING OF SPIRALthe fetus. In addition to simple diffusion, syncytiotropho- ARTERIESblasts facilitate exchange by transcellular trafﬁcking that The placental-decidual interaction through invading tro-utilizes transport proteins such as the water channels (aqua- phoblasts determines whether an optimal transformationporins). Facilitated diffusion for molecules such as glucose of the uterine spiral arteries is achieved. Trophoblast-and amino acids are performed by glucose transporters orchestrated artery remodeling is an essential feature of(GLUT) and amino acid transporters. In addition, adenos- normal human pregnancy. As shown in Figure 5-5, pro-ine triphosphate (ATP)-mediated active transport, such as genitor trophoblast cells from villi differentiate along twothe Na+, K+-ATPase or the Ca2+-ATPase, besides endocy- pathways: terminally differentiated syncytiotrophoblaststosis and exocytosis, participates in transplacental exchange and the extravillous cytotrophoblasts (EVTs) that migrate(Hahn et al, 2001; Malassiné and Cronier, 2002; Randhawa out in columns and anchor the placenta to the decidua.and Cohen, 2005; Siiteri, 2005). From these anchoring layers of EVTs further differ- In healthy women who are not pregnant, uterine blood entiation takes place into invasive or proliferative EVTs.vessels receive approximately 1% of the cardiac output to The invasive EVTs invade the decidua and shallow partsmaintain the uterus. During pregnancy, these same ves- of the myometrium and are known as interstitial EVTs.sels must support the rapidly growing and demanding pla- Thereafter endovascular transformation ensues as invasivecenta and fetus. This evolutionary challenge is addressed EVTs migrate into and colonize the spiral arteries, almostby remodeling of the spiral arteries, converting them into reaching the myometrium. These trophoblasts are knownlarge, thin-walled, dilated vessels with reduced vascular as endovascular EVTs. Insufﬁcient uteroplacental interac-resistance. tion characterized by shallow trophoblast invasion and
42 PART II Fetal Developmentincomplete transformation of spiral arteries is a common maternal and fetal cell interactive model under the preg-feature of preeclampsia and intrauterine growth restriction nancy milieu offers a potential approach to study cell-cell(IUGR) (Brosens et al, 1977; Meekins et al, 1994). The interactions and to decipher inﬂammatory componentsprecise period when trophoblast invasion of decidua and in the serum samples from adverse pregnancy outcomesspiral arteries ceases is not clear. Nevertheless it is widely (Kalkunte et al, 2010). One of the inimitable contributorsbelieved to be completed late in the second trimester. to trophoblast cell invasion is the specialized NK cell of Although our understanding of the molecular events the pregnant uterus.underlying spiral artery remodeling in pregnancy remainspoor, efﬁcient trophoblast invasion is an essential feature. IMMUNE PROFILE ANDThere are two waves of trophoblast invasion that followimplantation. The ﬁrst wave is during the ﬁrst trimester, IMMUNO VASCULAR BALANCEwhen the invasion is limited to the decidual part of the DURING PLACENTATIONspiral artery. The second wave is during the late second During pregnancy, trophoblast cells directly encountertrimester involving deeper trophoblast invasion, reaching maternal immune cells at least at two sites. One site is thethe inner third of myometrial segment. The initial inva- syncytiotrophoblasts covering the placental villi that aresion of EVTs into the endometrium initiates the decidual- bathed in maternal blood, and the other is by the invad-ization process, which is characterized by replacement of ing trophoblasts in the decidua. Although the syncytio-extracellular matrix, loss of normal musculoelastic struc- trophoblasts do not express MHC antigens, the invadingture, and deposition of ﬁbrinoid material. Displacement trophoblasts express nonclassic HLA-G and HLA-Cof the endothelial lining of spiral arteries by the invading and would elicit immune responses in the decidua. Thetrophoblasts further results in uncoiling and widening decidua is replete with innate immune cells includingof the spiral artery, ensuring the free ﬂow of blood and T cells, regulatory T cells, macrophages, dendritic cellsnutrients to meet the escalating demands of the growing and NK cells (Table 5-3). Interestingly, NK cells peak andfetus (Kham et al, 1999; Pijnenborg et al, 1983). A lack of constitute the largest leukocyte population in the earlyspiral artery remodeling with shallow trophoblast invasion pregnant uterus, accounting for 60% to 70% of total lym-has been associated with preeclampsia. During the pro- phocytes. These cells diminish in proportion as pregnancycess of invasion in a normal pregnancy, cytotrophoblasts proceeds.undergo phenotypic switching, with a loss of E-cadherinexpression, and they acquire vascular endothelial-cadherin,platelet-endothelial adhesion molecule-1, vascular endo- PHENOTYPIC AND FUNCTIONAL FEATURESthelial adhesion molecule-1, and α4 and αvβ3 integrins OF UTERINE NATURAL KILLER CELLS(Bulla et al, 2005; Zhau et al, 1997). Along with a repertoire Peripheral blood NK (pNK) cells constitute 8% to 10%of facilitators for invasion, trophoblasts express a nonclas- of the CD45+ population in circulation. All NK cells aresic major histocompatibility complex (MHC) human leu- characterized by a lack of CD3 and expression of CD56kocyte antigen (HLA) G, which has gained widespread antigen. Based on the intensity of CD56 antigen, NK cellsinterest because of providing noncytotoxic signals to uter- are further divided into CD56bright and CD56dim popula-ine NK cells. It still needs to be evaluated whether intrinsic tions. The presence or absence of FcγRIII or CD16 fur-HLA-G inactivation by polymorphic changes inﬂuences ther differentiates subpopulations of uterine NK (uNK)the dysregulated trophoblast invasion seen in preeclampsia cells. Thus the majority of peripheral NK cells are of the(Hiby et al, 1991; Le Bouteiller et al, 2007). CD56dimCD16+ phenotype (approximately 90%), and Although the exact gestational age at which tropho- the remaining cells are CD56brightCD16– (approximatelyblast invasion ceases is not known, recent studies have 10%). The majority of uterine NK cells (approximatelyshown that late pregnancy trophoblasts loose the ability 90%) are CD56brightCD16–. In the uterine decidua, uNKto transform the uterine arteries. Using a novel dual-cell cell numbers cyclically increase and decrease in tandemin vitro culture system that mimics the vascular remodel- with the menstrual cycle—low in the proliferative phaseing events triggered by normal pregnancy serum, we have (10% to 15%), which ampliﬁes during the early, middleshown that ﬁrst- and third-trimester trophoblasts respond and late secretory phases (25% to 30%)—falling to adifferentially to interactive signals from endothelial cells basal level with menstruation (Figure 5-7) (Kalkunte et al,when cultured on the extracellular matrix, matrigel. Term 2008a; Kitaya et al, 2007).trophoblasts not only fail to respond to signals from endo- With successful implantation, the uNK cell popula-thelial cells, but they inhibit endothelial cell neovascular tion further increases in the decidualized endometrium,formation. In contrast, trophoblast cells representing ﬁrst- reaches a peak in ﬁrst-trimester pregnancy, and dwindlestrimester trophoblasts with invasive properties undergo thereafter by the end of the second trimester. The originspontaneous migration and promote endothelial cells to of uNK cells that peak during the secretory phase of theform a capillary network (Figure 5-6). menstrual cycle and early pregnancy is currently not well This disparity in behavior was conﬁrmed in vivo using established, and the evidence indicates multiple differenta matrigel plug assay. Poor expression of VEGF-C and possibilities. These possibilities include recruitment ofVEGF receptors coupled with high E-cadherin expres- CD56brightCD16– pNK cells, recruitment and tissue spe-sion by term trophoblasts contributed to their restricted ciﬁc terminal differentiation of CD56dimCD16+ pNK cells,migratory and interactive properties. Furthermore, these development of NK cells from Lin–CD34+CD45+ pro-studies showed that the kinase activity of VEGF receptor 2 genitor cells, or proliferation of resident CD56brightCD16–is essential for proactive crosstalk by invading ﬁrst-trimes- NK cells. Comparative surface expression of antigens,ter trophoblast cells (Kalkunte et al, 2008b). This unique natural cytotoxicity receptors, inhibitory receptors, and
CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord 43 A B C D E F G H I J K LFIGURE 5-6 (Supplemental color version of this ﬁgure is available online at www.expertconsult.com.) Differential endovascular activity of ﬁrst- and third-trimester trophoblasts in response to normal pregnancy serum. A representative micrograph of trophoblasts-endothelial cell interactions on matrigelis 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 ﬁrst-trimester trophoblast HTR8 cells (C), third trimester trophoblast TCL1 cells (D), and primary term trophoblasts (E).However, in cocultures, HTR8 cells ﬁngerprint the HUtECs (F) and HUVECs (G), while TCL1 cells (H) and primary term trophoblasts (I) inhibit thetube formation by endothelial cells (magniﬁcation ×4). Panels J to L show the cocultures of HTR8 with HUVECs ( J), HUtECs ( K), and term trophoblastswith HUVECs (L) at higher magniﬁcation (×10). (Reproduced with permission from Kalkunte S, Lai Z, Tewari N, et al: In vitro and in vivo evidence for lack ofendovascular remodeling by third trimester trophoblasts, Placenta 29:871-878, 2008.) being replete with cytotoxic accessories of perforin, gran- TABLE 5-3 Comparison of Peripheral Blood and Decidual zymes A and B and the natural cytotoxicity receptors Immune Cell Proﬁles NKp30, NKp44, NKp46, NKG2D, and 2B4 as well as Peripheral LFA-1, uNK cells are tolerant cytokine-producing cells at Immune Cells blood (%) Decidua (%) the maternal-fetal interface (Kalkunte et al, 2008a). The T cells 65-70 9-12 temporal occurrence around the spiral arteries and timed ampliﬁcation of these specialized uNK cells observed dur- γδT cells 2-5 7-10 ing the ﬁrst trimester implicate its role in spiral artery Macrophages 7-10 15-20 remodeling. B cells 7-10 ND NK cell–deﬁcient mice display abnormalities in decid- NKT cells 2-5 0.5-1.0 ual artery remodeling and trophoblast invasion, possibly Tregs 2-4 6-10 because of a lack of uNK cell–derived interferon γ (Ashkar NK cells 7-12 65-70 (CD56brightCD16-) et al, 2000). Other studies have shown that unlike pNK cells, uNK cells are a major source of VEGF-C, Angio- ND, Not detected. poietins 1 and 2 and transforming grwoth factor (TGF- β1) within the placental bed that decrease with gestationalchemokines and cytokines on human pNK and uNK cells age (Lash et al, 2006). These observations implicate uNKare provided in Table 5-4. Furthermore, CD56bright uNK cells in promoting angiogenesis. Studies have providedcells are different from the CD56bright minor population further evidence that uNK cells, but not pNK cells, regu-of pNK cells because of the expression of CD9, CD103, late trophoblast invasion both in vitro and in vivo throughand killer immunoglobulin-like receptors (KIRs). Despite the production of interleukin-8 and interferon-inducible
44 PART II Fetal Development LH P4 E2 NK cell population NKG2D KIR NKp44 NK NKp46 NKp30 ~30% ~50–60% Inactive Proliferative Secretory First Second Term phase phase phase trimester trimester Menstrual cycle PregnancyFIGURE 5-7 Biologic pattern of natural killer (NK) cells in the human endometrium and the decidua. The uterine NK cell populationcharacterized 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. E2, Estradiol; LH, luteinizing hormone;P4, progesterone.protein-10, in addition to other angiogenic factors (Hanna The antiinﬂammatory cytokine interleukin (IL)-10et al, 2006). Recent studies suggest that VEGF-C, a pro- plays a critical role in pregnancy because of its regulatoryangiogenic factor produced by uNK cells, is responsible relationship with other intrauterine modulators and itsfor the noncytotoxic activity (Kalkunte et al, 2009). As wide range of immunosuppressive activities (Moore et al,noted previously, VEGF-C–producing uNK cells support 2001). IL-10 expression by the human placenta dependsendovascular activity in a coculture model of capillary tube on gestational age, with signiﬁcant expression through theformation on matrigel (Figure 5-8). Peripheral blood NK second trimester followed by attenuation at term (Hannacells fail to produce VEGF-C and remain cytotoxic. This et al, 2000). IL-10 expression is also found to be poor infunction can be reversed by recombinant human VEGF- decidual and placental tissues from unexplained spontane-C. Cytoprotection by VEGF-C is related to induction of ous abortion cases (Plevyak et al, 2002) and from deliveriesthe transporter associated with antigen processing 1 and associated with preterm labor (Hanna et al, 2006) and pre-MHC assembly in target cells. Overall, these ﬁndings sug- eclampsia (S. Kalkunte et al, unpublished observations).gest that expression of angiogenic factors by uNK cells However, the precise mechanisms by which IL-10 protectskeeps these cells noncytotoxic, which is critical to their the fetus remains poorly understood. IL-10–/– mice sufferpregnancy compatible immunovascular role during pla- no pregnancy defects when mated under pathogen-freecentation and fetal development (Kalkunte et al, 2009). conditions (White et al, 2004), but they exhibit exquisite Although uNK cells seem to play a role that is compat- susceptibility to infection or inﬂammatory stimuli com-ible with pregnancy, retention of their cytolytic abilities pared with wild type animals. It is then plausible that insuggests their role as sentinels at the maternal-fetal inter- addition to IL-10 deﬁciency, a “second hit” such as anface in situations that threaten fetal persistence. This facet inﬂammatory insult resulting from genital tract infec-of uNK cell function was elegantly demonstrated in animal tions, environmental factors, or hormonal dysregulationmodels when pregnant mice were challenged with toll- during gestation can lead to adverse pregnancy out-like receptor (TLR) ligands that mimic bacterial and viral comes (Tewari et al, 2009; Thaxton et al, 2009). Ourinfections. These observations raise an important question recent studies provide direct evidence that uNK cells canwhether uNK cells can harm the fetal placental unit and, if become adversely activated and mediate fetal demise andso, under what conditions? preterm birth in response to low doses of TLR ligands
CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord 45 to predominate over Th1 cellular immune response under TABLE 5-4 Phenotypic Characteristics of Surface Markers normal pregnancy. Recently the role of specialized T lym- and Receptors on Natural Killer Cells phocytes, termed regulatory T cells (Tregs), in tolerogenic Antigen Peripheral blood Decidua mechanisms has emerged. Tregs are potent suppressors CD56 Dim (>90%) Bright of T cell–mediated inﬂammatory immune responses and prevent autoimmunity and allograft rejection. Tregs act CD16 + – by controlling the autoreactive T cells that have escaped CD45 + + negative selection from the thymus, and they restrain the CD7 + + intensity of responses by T cells reactive with alloantigens CD69 – + and other exogenous antigens. This unique functional capa- L-Selectin – –/+ bility to suppress responses to tissue-speciﬁc self-antigens NK Receptors that escape recognition by T cells during maturation is due to tissue speciﬁc expression and alloantigens, particularly in KIR + + the epithelial surfaces where tolerance to nondangerous for- NKp30 + + eign antigen is essential to normal function. This capability NKp44 –* + enables Tregs to play a unique role at the maternal-fetal NKp46 + + interface. Tregs are typically characterized by a CD4+CD25+ NKG2D + + surface phenotype and expression of the hallmark suppres- CD94/NKG2A –/+ + sive transcription factor Foxhead Box P3 (Foxp3+). Their cell numbers increase in blood, decidual tissue, and lymph Chemokine Receptors nodes draining the uterus during pregnancy. These cells are CXCR1 + + implicated in successful immune tolerance of the conceptus, CXCR2 + – mainly by producing IL-10 and TGF-β. Recent evidence CXCR3 – + suggests that fetal Tregs also play a vital role in suppress- CXCR4 + + ing fetal antimaternal immunity against maternal cells that CX3CR1 + – cross the placenta (Mold et al, 2008). CCR7 – + In the absence of Tregs the allogeneic fetus is rejected, suggesting their critical role in normal pregnancy. Unex- Data from Kalkunte S, Chichester CO, Sentman CL, et al: Evolution of non-cytotoxic plained infertility, spontaneous abortion, and preeclampsia uterine natural killer cells, Am J Reprod Immunol 59:425-432, 2008. +, Present; –, absent; –/+, variable expression; KIR, killer immunoglobulin receptor; are associated with proportional deﬁcience, functional Treg CXCR, CX-chemokine receptor; CX3CR1, CX3-C–chemokine receptor 1; CCR7, CC- deﬁciency, or both. In the context of pregnancy, the local chemokine receptor 7. *Expression seen on activation with interleukin 2. milieu, particularly during the ﬁrst 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+ Tregsresulting in placental pathology (Murphy et al, 2005; with Foxp3 expression with immunosuppressive features.Murphy et al, 2009). Moreover, spontaneous abortion is This induction is thought to occur through the immatureassociated with an increase in CD56dimCD16+ cells and a dendritic cells. In addition to immune suppressive and anti-decrease in CD56brightCD16– NK cells in the preimplan- inﬂammatory properties, TGF-β is recognized as inducingtation endometrium during the luteal phase (Michimata differentiation of naïve CD4 T cells into suppressor T-cellet al, 2002; Quenby et al, 1999). Therefore a ﬁne bal- phenotype, expressing Foxp3, and promoting the prolifera-ance between maternal activating and inhibitory KIRs tion of mature Tregs. In addition to the suppressive effects ofand their ligand HLA-C on fetal cells seems to be main- cytokines produced by these cells, contact-mediated immunetained in normal pregnancy. Insufﬁcient inhibition of suppression by Tregs results from ligation of inhibitoryuNK cells can activate the cytolytic machinery, resulting cytotoxic T-lymphocyte antigen (CTLA-4) and its ability toin spontaneous abortion, intrauterine growth restriction, induce tolerogenic dendritic cells and inﬂuence T-cell pro-or preterm labor, depending on the timing of the insult duction of IL-10 (Aluvihare et al, 2004; Schumacher et al,(Varla-Leftherioti et al, 2003). In the setting of IVF, the 2009; Shevach et al, 2009). Therefore the pregnant uterusimplantation failure has been associated with high uNK may be a natural depot for Tregs.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 EPIGENETIC REGULATIONcells retain the ability to become foes to pregnancy under IN THE PLACENTAthe axis of genetic stress and inﬂammatory trigger. The regulation of gene expression is a crucial process that deﬁnes phenotypic diversity. Switching off or turning on genes as well as tissue-speciﬁc variation in gene expressionREGULATORY T CELLS AND PREGNANCY contributes to this diversity. Besides the genetic make-up (i.e,The existence of regulatory mechanisms that suppress the sequence) of the individual, the regulation of gene expressionmaternal immune system was proposed in the early 1950 is also inﬂuenced by epigenetic factors. Epigenetic changes(Medawar, 1953). For several years, maternal tolerance include the noncoding changes in DNA and chromatin,toward fetal alloantigens was explored in the context of or both, that mediate the interactions between genes andTh1/Th2 balance, with Th2 cells and cytokines proposed their environment. Epigenetic regulation generates a wider
46 PART II Fetal Development DECIDUA Non cytotoxic NK cells Cytotoxic NK cells CD16 CD56 CD56brightCD16Ϫ CD56dimCD16ϩրϪ Intrauterine infections, VEGF C Inflammation, TLR activation, Loss of IL-10 Promotes endovascular activity Poor endovascular activity Upregulates TAP-1 and MHC Trophoblast lysis molecules on trophoblats Poor trophoblast invasion Efficient trophoblast invasion Normal pregnancy Adverse pregnancyFIGURE 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 endovascularactivity in a coculture of endothelial cells and ﬁrst-trimester trophoblast HTR8 cells on matrigel. By contrast, cytotoxic uterine NK cell clones similarto peripheral blood NK cells disrupted the endovascular activity because of endothelial and trophoblast cell lysis. This distinct functional featuredetermines whether optimal trophoblast invasion takes place and can result in normal or adverse pregnancy outcomes.diversity of cell types during mammalian development and with tumorigenesis, including rapid mitotic cell division,sustains the stability and integrity of the expression proﬁles migration, angiogenesis and invasion, and escape fromof different cell types and tissues. This regulation is choreo- immune surveillance. Indeed, there are striking similaritiesgraphed by changes in cytosine-phosphate-guanine (CpG) in the DNA methylation pattern of tumor-associated genesislands of the DNA promoter region by methylation, his- between invasive trophoblast cell lines and ﬁrst-trimestertone modiﬁcation, genomic imprinting, and expression of placenta and tumors (Christensen et al, 2009). This ﬁndingnoncoding RNAs such as micro RNA (miRNA). suggests that a distinct pattern of tumor-associated meth- Gene-environment interactions resulting in epigenetic ylation can result in a series of epigenetic silencing eventschanges in the placenta during the critical window of devel- necessary for normal human placental invasion and func-opment can inﬂuence fetal programming in utero, with tion (Novakovic et al, 2008). Other studies using the pla-predisposing health consequences later in life. Using a micro- centa as a source suggest that the speciﬁc loss of imprintingarray-based approach to compare chorionic villous samples because of altered methylation and subsequent gene expres-from the ﬁrst trimester of pregnancy with gestational age– sion can result in small for gestational age (SGA) newborns.matched maternal blood cell samples, recent studies show Moreover, unbalanced expression of imprinted genes intissue-speciﬁc differential CpG methylation patterns that IUGR placenta when compared with non-IUGR placentaidentify numerous potential biomarkers for the diagnosis was observed suggesting a differential expression pattern ofof fetal aneuploidy on chromosomes 13, 18 and 21 (Chu imprinted genes as a possible biomarker for IUGR (Guoet al, 2009). Human placentation displays many similarities et al, 2008; McMinn et al, 2006).
CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord 47 The unique cytokine and hormonal milieu in utero may gestation. Proteinuria is deﬁned by excretion of 300 mginﬂuence the trophoblast function and differentiation as or more of protein every 24 hours or 300 mg/L or morewell as immune cell regulation through histone posttrans- in two random urine samples taken at least 4 to 6 hourslational modiﬁcation. In this regard, interferon γ produced apart (ACOG Committee on Practice Bulletins, 2002).by uNK cells and essential for spiral artery remodeling The fetal problems most commonly associated with pre-fails to induce MHC class II expression in trophoblast eclampsia include fetal growth restriction, reduced amni-cells because of hypermethylation of regulatory class II otic ﬂuid, and abnormal oxygenation (Sibai et al, 2005).MHC transactivator (CIITA) regions (Morris et al, 2002). However, the onset of clinical signs and symptoms canThis inability to upregulate classical MHC class II mol- result in either near-term preeclampsia without affect-ecules by trophoblasts is essential for maintaining immune ing the fetus or its severe manifestation that is associatedtolerance at the maternal-fetal interface. Moreover, the with low birthweight and preterm delivery (Vatten andtranscription factor regulating trophoblastic fusion pro- Skjaerven, 2004). The heterogeneous manifestation oftein syncytin, which is essential for the syncytialization of this disease is further confounded by preexisting maternaltrophoblasts, is regulated by histone acetyl transferase and vascular disease, multifetal gestation, metabolic syndrome,histone deacetylase activity (Chuang et al, 2006). miRNAs obesity, or previous incidence of the disease. In addition,are small regulatory RNA molecules that can alter gene the pathophysiology of the disorder could differ from theand protein expression without altering the underlying onset before 24 weeks’ gestation and its diagnosis at latergenetic code. Expression of miRNA is tissue speciﬁc, and stages of pregnancy:several are expressed in the placenta. In placental pathol- Abnormal remodeling of spiral arteries and shallow tro-ogy associated with preeclampsia, there is differential phoblast invasion are two hallmark features of preeclamp-expression of miRNA (such as miR-210 and miR-182) sia. Preeclampsia is considered a two-stage disease wherecompared with normal pregnancy placenta. This ﬁnding a poorly perfused placenta (stage I) causes the release ofsuggests that signature differences in placental miRNA factors leading to maternal symptoms (stage II). However,and their detection in maternal serum may potentially be it is also now being recognized that the maternal factorsused as a biomarker for preeclampsia. Because implanta- may contribute to programming of stage I of preeclamp-tion and early placentation is under the regulation of low sia, suggesting that the intrinsic maternal factors stem-oxygen tension, it is possible that miRNA are differentially ming from genetic, behavioral, and physiologic conditionsexpressed under different oxygen levels, as suggested by may contribute to placental pathology. Stage I initiatedrecent observations (Maccani and Marsit, 2009; Pineles pathology may be particularly apparent in the oxidativeet al, 2007). stress-induced release of causative factors from the poorly perfused placenta and their effects on the maternal syn- drome (Roberts and Hubel, 1999).PLACENTAL DISEASES Despite a poor mechanistic understanding of placentalThe placenta provides a wealth of retrospective informa- pathology leading to preeclampsia, several critical featurestion about the fetus and prospective information regarding are common to this disease. Multiple studies have shownthe infant. Healthy development of the placenta requires that reduced vascular activity could be a major factor con-efﬁcient metabolic, immune, hormonal, and vascular tributing to preeclampsia. In normal pregnancy, the circu-adaptation by the maternal system as well as the fetus. lating PlGF levels steadily increase in the ﬁrst and secondAbnormal placentation and placental infections can lead trimesters, peak at 29 to 32 weeks, and decline thereafter.to maternal or fetal anomalies as seen in preeclampsia, However, free VEGF remains low and unchanged dur-preterm birth, and SGA, which can have lifelong bearing ing this window. Reduced placental expression of VEGFon the development and health of infants. Maternal fac- and PlGF is consistently observed in preeclampsia. Fur-tors such as ascending infections, obesity, hypertension, thermore, preeclampsia is frequently accompanied bygenetic predisposition such as gene polymorphism of the enhanced circulation and placental expression of the anti-pregnancy-compatible cytokine milieu, and environmental angiogenic soluble VEGF receptor 1 (sFlt-1), which is aexposure could also contribute to the placental pathology. decoy receptor titrating out VEGFs and PlGF (LevineThe following sections contain an abbreviated discussion et al, 2004; Romero et al, 2008; Thadhani et al, 2004).of the pathogenesis of some of these placenta-associated A lack of available VEGF and increased sFlt-1 expres-disorders. 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-PREECLAMPSIA genic effects of sFlt-1. Soluble endoglin has been foundHypertensive disorders of pregnancy are enigmatic. They to be elevated in the serum of preeclamptic women and ispose a major public health problem and affect 5% to 10% accompanied by an increased ratio of sFlt-1:PlGF and cor-of human pregnancies. Preeclampsia is clinically associ- relates with the severity of the disease. Soluble endoglin isated with maternal symptoms of hypertension, protein- thought to inhibit TGF-β1 signaling in endothelial cellsuria, and glomeruloendotheliosis. This disorder is strictly and blocks activation of endothelial nitric oxide synthasea placental condition because of its clearance after deliv- and vasodilatation (Venkatesha et al, 2006). Several recentery. It causes morbidity and mortality in the mother, fetus, studies have suggested an increase in apoptosis within vil-and newborn. Pregnancy-associated hypertension is deﬁned lous trophoblast from preeclampsia and IUGR deliveriesas blood pressure greater than 140/90 mm Hg on at least (Allaire et al, 2000; Heazell and Crocker, 2008; Levy et al,two occasions and at 4 to 6 weeks apart after 20 weeks’ 2002). Unlike normal pregnancy, villous placental explants
48 PART II Fetal Developmentfrom preeclamptic placenta have an increased sensitiv- substance abuse, maternal age (<18 or >40 years), obesity,ity and susceptibility to apoptosis on exposure to proin- diabetes, thrombophilia, ethnicity, anemia, and fetal fac-ﬂammatory cytokines, suggesting altered programming tors such as congenital anomalies and growth restriction.of apoptotic cascade pathway (Crocker et al, 2004; Levy Activation of the hypothalamic-pituitary-adrenal (HPA)et al, 2002). It is possible that incomplete spiral artery as a result of major maternal physical or psychologicaltransformation resulting in reduced placental perfusion stress is thought to increase the release of corticotrophin-(stage I) in preeclampsia leads to focal regions of hypoxia releasing hormone. In addition to the hypothalamus as awith increase in apoptosis, oxidative stress, shedding of source of corticotrophin-releasing hormone, placentalvillous microparticles, and release of antiangiogenic fac- trophoblasts, amnion, and decidual cells also express thistors such as sFlt-1.64 (Hung et al, 2002; Nevo et al, 2006; hormone during pregnancy. Corticotrophin hormoneRedman and Sargent, 2000). regulates the release of adrenocorticotropic hormone Another pathway that may contribute to the etiology of from pituitary and cortisol from adrenal glands, and itpreeclampsia is unscheduled and excessive activation of the can also inﬂuence the activity of matrix metalloprotein-complement cascade; this is highly likely as a result of the ases (MMPs). Premature activation of the HPA axis canmaternal immune system responding to paternal antigens eventually stimulate the prostaglandins, ultimately result-and inﬂammation. However, in normal pregnancy the ing in parturition via activation of proteases. In addition,placenta expresses complement regulatory proteins such activation of the HPA axis promotes the release of estrone,as DAF, CD55, and CD59 and may control activation of estradiol, and estriol that can activate the myometrium bycomplement factors (Tedesco et al, 1993). Despite the increasing oxytocin receptors, prostaglandin activity, andpositioning of complement inhibitory proteins for protec- enzymes such as myosin light chain kinase and calmodu-tive roles, increasing evidence supports the involvement of lin, which are responsible for muscle contraction. Con-complement activation in the pathogenesis of preeclamp- comitantly, progesterone withdrawal is expected with thesia (Lynch et al, 2008). Interestingly, recent in vitro studies raising concentration of myometrial estrogen receptors,suggest that hypoxia enhances placental deposition of the further enhancing estrogen-induced myometrial activa-membrane attack complex and apoptosis in cultured tro- tion and preterm birth (Dole et al, 2003; Grammatopoulosphoblasts (Rampersad et al, 2008). The upstream factors and Hillhouse, 1999; McLean et al, 1995).that trigger complement activation are not yet known. There is increasing evidence that approximately 50% of Recent studies also suggest increased serum levels preterm births are associated with infection of the decidua,of agonistic autoantibodies against angiotensin type 1 amnion, or chorion and amniotic ﬂuid caused by eitherreceptor (AT-1-AA) in preeclampsia as compared with systemic or ascending genital tract infection. Both clinicalhealthy women (Zhou et al, 2008). Importantly, studies and subclinical chorioamnionitis are implicated in pretermfrom our laboratory have shown that the full spectrum of birth. Maternal or fetal inﬂammatory responses to cho-preeclampsia-like symptoms can be reproduced in mice rioamniotic infection can trigger preterm birth. Activatedby injecting human preeclampsia serum containing sub- neutrophils and macrophages and the release of cytokinesthreshold levels of AT-1-AA immunoglobulin G, sug- IL-1β, IL-6, IL-8, tumor necrosis factor alpha (TNF-α)gesting that pregnancy serum contains some unknown and granulocyte colony-stimulating factor can lead to ancausative factors. Therefore serum can be used as a blue- enhanced cascade of signaling activity, causing release ofprint to identify functional biomarkers for preeclampsia prostaglandins and expression of various MMPs of fetal(Kalkunte et al, 2009). membranes and the cervix. Furthermore, elevated levels of TNF-α and apoptosis are associated with PPROM. Non–infection-related inﬂammation caused by placentalPRETERM BIRTH insufﬁciency and apoptosis can also cause preterm birth.Preterm birth is the leading cause of infant morbidity In addition to augmented inﬂammatory responses toand mortality in the world. Babies born before 37 weeks’ infections, pathogenic microbes (e.g. Staphylococcus, Strep-gestation are considered premature. In the United States, tococcus, Bacteroides, and Pseudomonas spp.) are thought toapproximately 12.8% of births are preterm, and the rate directly degrade fetal membranes by releasing proteases,of premature birth has increased by 36% since early 1980s collagenases, and elastases, produce phospholipase A2,(Martin et al, 2009). Babies from preterm birth face an and release endotoxin that stimulate uterine contractionsincreased risk of lasting disabilities such as mental retarda- and cause preterm birth (Goldenberg et al, 2000, 2008;tion, learning and behavioral problems, autism, cerebral Romero et al, 2006; Slattery and Morrison, 2002).palsy, bronchopulmonary dysplasia, vision and hearing The innate immune system and trophoblasts duringloss, and risk for diabetes, hypertension, and heart disease pregnancy recognize bacterial and viral infections usingin adulthood. The majority of preterm deliveries are due TLRs. Placental transcripts for TLRs 1 to 10 have beento preterm labor. Other factors leading to premature birth detected in human placental tissue, and placental chorio-are preterm premature rupture of membranes (PPROM), carcinoma cell lines reportedly express TLR-2, TLR-4,intervention for maternal or fetal problems, preeclampsia, and TLR-9 (Abrahams and Mor, 2005). Studies have dem-fetal growth restriction, cervical incompetence, and ante- onstrated functionality for TLR-2, TLR-3, and TLR-4partum bleeding. Additional risk factors for preterm birth in ﬁrst- and third-trimester placental tissue (Patni et al,include stress, occupational fatigue, uterine distention by 2007). Decidual expression in humans has demonstratedpolyhydramnios or multifetal gestation, systemic infec- functional receptors in term decidua of TLR-1, TLR-2,tion such as periodontal disease, intrauterine placental TLR-4, and TLR-6 (Canavan and Simhan, 2007). Ourpathology such as abruption, vaginal bleeding, smoking, recent studies using mice have shown that extremely small
CHAPTER 5 Immunologic Basis of Placental Function and Diseases: the Placenta, Fetal Membranes, and Umbilical Cord 49doses of the TLR-4 ligand lipopolysaccharide can cause toxins such as warfarin, anticonvulsants, folic acid antag-preterm birth or fetal demise in pregnant IL-10–deﬁcient onists, and caffeine; and pregnancies conceived throughmice by activating and promoting inﬁltration of uterine assisted reproductive techniques have a higher prevalenceNK cells into the placenta and inducing apoptosis by of IUGR. IUGR results in the birth of an infant who issecretion of TNF-α (Murphy et al, 2005, 2009). Similarly, SGA. Mortality and morbidity are increased in SGAactivation of TLR-3 or TLR-9 has been shown to induce infants compared with those who are appropriate for ges-spontaneous abortion or preterm birth in IL-10–deﬁcient tational age. SGA infants at birth have many clinical prob-pregnant mice that is attributed to immune inﬁltration and lems that include impaired thermoregulation; difﬁculty inproinﬂammatory cascade in the placenta (Thaxton et al, cardiopulmonary transition with perinatal asphyxia, pul-2009). monary hypertension, hypoglycemia, polycythemia and Decidual hemorrhage leading to vaginal bleeding hyperviscosity; impaired cellular immune function; andincreases the risk for preterm birth and PPROM. Increased increased risk for perinatal mortality. SGA infants in theiroccult decidual hemorrhage, hemosiderin deposition, and childhood and adolescence are at higher risk for impairedretrochorionic hematoma formation is seen between 22 physical growth and neurodevelopment. Adolescentsand 32 weeks’ gestation as a result of PPROM and preterm born SGA at term were reported to have learning difﬁ-birth after preterm labor. The development of PPROM in culties with attention deﬁcits. Cognitive performance isthe setting of abruption could be caused by high decidual generally lower in SGA infants at the ages of 1 to 6 yearsconcentration of tissue factors, which eventually gener- compared with those whoe are appropriate for gestationalate thrombin. Thrombin activation as measured by serum age. Adults who were SGA infants could be at higher riskthrombin-antithrombin III complex levels are elevated for ischemic heart diseases and essential hypertensionon preterm birth. Thrombin binds to decidual protease- (Figueras et al, 2007; Kaijser et al, 2008; Lapillonne et al,activated receptors (PAR1 and PAR2), induces the produc- 1997; Norman and Bonamy, 2005; O’Keefe et al, 2003;tion of IL-8 in decidua, attracts neutrophils, and promotes Spence et al, 2007).degradation of the fetal membrane MMPs that can resultin PPROM (Lockwood et al, 2005; Salaﬁa et al, 1995). Polyhydramnios is also a high risk factor for preterm FETAL MEMBRANES AND THEIRbirth. It was shown recently that exposure of IL-10– PATHOLOGYdeﬁcient pregnant mice to polychlorinated biphenyls, an The fetal tissue–derived membrane structure surroundsenvironmental toxicant, can lead to preterm birth with the fetus and forms the amniotic cavity. This membrane,IUGR. The IUGR was due to increased amniotic ﬂuid which lacks both vascular and nerve cells, is composed ofvolume (polyhydramnios) and placental insufﬁciency an inner layer adjacent to the amniotic ﬂuid and is calledcaused by poor spiral artery remodeling associated with the amnion. The outer layer that is attached to the deciduareduced expression of water channel aquaporin-1 in the is called the chorion. Amnion is composed of inner epithe-placenta (Tewari et al, 2009). Increasing evidence also sug- lial cells, and the mesenchymal cell layer is composed ofgests impaired vascular activity because of an increase in ﬁbroblast and an outer spongy layer. Intact, healthy fetalantiangiogenic factors such as sFlt-1 and decreased VEGF membranes are required for normal pregnancy outcome.in PPROM and preterm birth (Kim et al, 2003). Chorion is composed of an outer reticular cell layer com- posed of ﬁbroblasts and macrophages and an inner cyto- trophoblast layer. The elasticity and strength of theseINTRAUTERINE GROWTH RESTRICTION membranes are maintained by extracellular matrix proteinsIUGR is used to designate a fetus that has not reached such as collagens, ﬁbronectin, laminins, and the activity ofits growth potential; it can be caused by fetal, placental, MMP-2 and MMP-9 and their inhibitors until the initia-or maternal factors. Disparities between fetal nutritional tion of parturition when the membranes are susceptibleor respiratory demands and placental supply can result to rupture. During parturition, when contractions beginin impaired fetal growth. Chromosomal abnormalities or membranes rupture, MMP activity in the amnion and(aneuploidy, partial deletions, gene mutation particu- chorion increases with a concurrent fall in tissue inhibitorslarly on the gene for insulin-like growth factors), con- of metalloproteinases. This change is followed by apopto-genital abnormalities, multiple gestation, and infections sis in the amnion epithelial and chorion trophoblast layerscan also result in IUGR. Preterm birth, preeclampsia, of fetal membrane. Interestingly, some evidence suggestsand abruption because of placental ischemia can result in that fetal membranes have antimicrobial activity and areIUGR. Reduced placental weight with identiﬁable pla- known to express TLR-2 and TLR-4, which are patterncental histologic abnormalities (e.g, impaired develop- recognition receptors and help in initiating a protectivement or obstruction in uteroplacental vasculature, chronic host response to infection.abruption, chronic infections, maternal ﬂoor infarction, The histopathology of amnion and chorion includesthrombosis in uteroplacental vasculature or fetoplacen- infections, amniotic ﬂuid contaminants, and fetal diseases.tal vasculature) are common ﬁndings in IUGR. In addi- In addition to the membranes, whose infection can lead totion, a single umbilical artery, velamentous umbilical cord chorioamnionitis, another vulnerable portal for infectioninsertion, bilobate placenta, circumvallate placenta, and to occur is the placental intervillous space and fetal villiplacental hemangioma are some of the other structural that provide hematogenous access. Hematogenous sourcesanomalies seen in the placenta. Maternal factors such as of infection are typically associated with inﬂammation ofnutritional deﬁciency; severe anemia; pulmonary disease villi (villitis) and intervillous space (intervillositis). Viralleading to maternal hypoxemia; smoking; exposure to pathogens (cytomegalovirus, HIV, herpes simplex virus)
50 PART II Fetal Developmentcommonly produce hematogenous infection of the pla- oligohydramnios, low Apgar scores, and signiﬁcant neuro-centa in addition to bacteria, spirochetes, fungi, and pro- developmental delay. Interruption of normal blood ﬂow intozoa (Gersell, 1993; Goldenberg et al, 2000; Lahra and the cord can cause prolonged hypoxia in utero. ClampingJeffery, 2004). 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-UMBILICAL CORD pitals showed that infants whose cord clamping occurredThe connecting cord from the developing embryo or fetus later than 60 seconds after birth had a signiﬁcantly higherto the placenta is the umbilical cord, or funiculus umbili- risk of neonatal jaundice requiring phototherapy. How-calis. During prenatal development in humans, the nor- ever, randomized, controlled studies have shown thatmal umbilical cord contains two umbilical arteries and delayed cord clamping in preterm infants reduces theone umbilical vein buried within Wharton’s jelly. The incidence of intraventricular hemorrhage and late-onsetumbilical vein supplies the fetus with oxygenated blood sepsis. Furthermore, premature clamping can increase thefrom the placenta while the arteries return the deoxygen- risk of ischemia and hypovolemic shock, which can leadated, nutrient-depleted blood to the placenta. In the fetus, to fetal complications (McDonald and Middletone, 2008;the umbilical vein branches into the ductus venosus and Mercer et al, 2006).another branch that joins the hepatic portal vein. Shortlyafter parturition, physiologic processes cause the Whar-ton’s jelly to swell with the collapse of blood vessels, result- SUGGESTED READINGSing in a natural halting of the ﬂow of blood. Within the Aluvihare VR, Kallikourdis M, Betz AG: Regulatory T cells mediate maternal tolerance to the fetus, Nat Immunol 5:266-271, 2004.infant, the umbilical vein and ductus venosus close and Ashkar AA, Di Santo JP, Croy BA: Interferon γ contributes to initiation of uterinedegenerate into remnants known as the round ligament of vascular modification, decidual integrity, and uterine natural killer cell matura- tion during normal murine pregnancy, J Exp Med 192:259-269, 2000.the liver and the ligamentum venosum, while the umbilical Baker DJP: In utero programming of chronic disease, Clin Sci 95:115-128, 1998.arteries degenerate into what is known as medial umbilical Christensen BC, Houseman EA, Marsit CJ, et al: Aging and environmentalligaments. exposures alter tissue-specific DNA methylation dependent upon CpG island context, PLoS Genet 5:e1000602, 2009. Abnormalities associated with the umbilical cord can Goldenberg RL, Culhane JF, Iams JD, et al: Epidemiology and causes of pretermaffect both the mother and the child. Pathology of umbili- birth, Lancet 371:75-84, 2008.cal cord is generally grouped as congenital remnants, Hanna J, Goldman-Wohl D, Hamani Y, et al: Decidual NK cells regulate key developmental processes at the human fetal-maternal interface, Nat Medinfections, meconium, and masses. Abnormalities that 12:1065-1074, 2006.have clinical signiﬁcance are nuchal cord, single umbili- Kalkunte S, Mselle TF, Norris WE, et al: VEGF C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal-fetalcal artery, umbilical cord prolapse, umbilical cord knot, interface, J Immunol 182:4085-4092, 2009.umbilical cord entanglement, vasa previa, and velamen- Moffett A, Loke C: Immunology of placentation in eutherian mammals, Nat Revtous cord insertion. Common intrauterine infections can Immunol 6:584-594, 2006. Murphy SP, Hanna NN, Fast LD, et al: Evidence for participation of uterineresult in the umbilical cord being invaded by fetal cells natural killer cells in the mechanisms responsible for spontaneous pretermand bacteria inﬁltrated from the decidua to amniotic ﬂuid, labor and delivery, Am J Obstet Gynecol 200:308, 2009.or they can elicit fetal inﬂammatory response. Umbilical Paria BC, Reese J, Das SK, et al: Deciphering the cross-talk of implantation: advances and challenges, Science 296:2185-2188, 2002.cord inﬂammation, known as funisitis or vasculitis, poses a Roberts JM, Hubel CA: Is oxidative stress the link in the two–stage model of preec-higher risk for development of neurologic compromise in lampsia? Lancet 354:788-789, 1999. Slattery MM, Morrison JJ: Preterm delivery, Lancet 360:1489-1497, 2002.the fetus. Funisitis is predictive of a lower median Bayley Thadhani R, Sachs BP, Epstein FH, et al: Circulating angiogenic factors and thepsychomotor developmental index in infants. Meconium risk of preeclampsia, N Engl J Med 350:672-683, 2004.pigment at high concentrations can damage the umbili- Complete references and supplemental color images used in this text can be found online atcal cord by triggering apoptosis of smooth muscle cells. www.expertconsult.comVascular necrosis caused by meconium is associated with