Multiple Gestation: The Biology of Twinning

The incidence of multiple births has increased dramatically over the past 5 decades. In the United States, twin birth rates increased almost twofold between 1971 and 2014. This sharp increase in twin births has been linked to two related factors: older maternal age distribution and expanded use of fertility-enhancing therapies (assisted reproductive technologies [ART] and non-ART treatments such as ovulation stimulation). Improved reproductive technology has led to a steady 1% decline in twin birth rates since 2014 and twins currently account for about 3% of all births. The perinatal mortality and morbidity rates of twins are three to seven times higher than those of singletons, mainly as a result of higher prematurity rates. The risk is highest for monochorionic twins, which constitute approximately 20% to 25% of all twin pregnancies. Monochorionic twin pregnancies are predisposed to a specific set of complications, including twin-to-twin transfusion syndrome (TTTS), twin anemia-polycythemia sequence (TAPS), twin reversed arterial perfusion (TRAP) sequence, malformations, and intertwin growth discordance. This chapter reviews the basic mechanisms underlying the twinning process, the relationship between zygosity and chorionicity, and the various types of twinning. The major complications of monochorionic and dichorionic twinning in association with their reported placental characteristics are also described.

Zygosity and Chorionicity

Twins and twin pregnancies can be categorized according to zygosity or chorionicity. Zygosity refers to the type of conception. Dizygotic (nonidentical, fraternal) twins (approximately 70% of all twins) result from multiple ovulations with (near-)synchronous fertilization of two separate ova by two separate sperm cells. Dizygotic twins thus share the same genetic relationship as nontwin siblings and share approximately 50% of genes. Monozygotic twins (so-called identical twins; approximately 30% of all twins) are generated by division of a zygote that originated from the fertilization of one single ovum by one single sperm cell. Dizygotic twinning is more common than monozygotic twinning and shows large regional differences, ranging from 6 per 1000 births in Asia to 40 per 1000 births in sub-Saharan Africa. In contrast, monozygotic twinning occurs at a constant rate of approximately 3 to 4 per 1000 births worldwide.

Chorionicity refers to the type of placentation and is closely related to zygosity. In dizygotic pregnancies, each zygote develops its own amnion, chorion, and placental circulations. Therefore dizygotic twins almost always have a dichorionic placenta. The appearance of the dichorionic placenta depends on the sites of blastocyst implantation. Distant implantation sites are more likely to result in separate placentas; when the blastocysts implant close to each other, the placentas may be fused as a single placental mass (with separate fetoplacental circulations).

According to the widely accepted—but mechanistically unproven—fission or splitting model of monozygotic twinning, the type of placentation is believed to depend primarily on the timing of division of the zygote. Early division, within the first 3 days after fertilization (70% of monozygotic twins), usually results in dichorionic placentation. Division in the blastocyst stage, after formation of the chorion but before formation of the amnion (3 to 9 days after fertilization; 25%) results in diamniotic-monochorionic placentation. Late division (8 to 12 days after fertilization; 2%) leads to monoamniotic-monochorionic placentation, whereas even later zygotic splitting (13 to 16 days after fertilization; 1:100,000) results in conjoined monoamniotic-monochorionic twinning. Mirror-image twinning, defined as the presence of asymmetric phenotypic features, may occur in 25% of monozygotic twins. Such mirror asymmetries include direction of hair whorl, unilateral eye and ear defects, and bony and central nervous system abnormalities. According to the traditional fission model of monozygotic twinning, mirror-image twinning is the result of late zygotic splitting at days 9 to 12 after fertilization, immediately before formation of conjoined twins.

Cases of atypical twinning such as chimeric twins, phenotypically discordant monozygotic twins, mirror-image twins, and polar body twins have prompted critical reexamination of the traditional postzygotic fission models of monozygotic twinning. Alternative theories such as a fusion model have been proposed. According to the fusion model, monozygotic twinning occurs at the first zygotic cleavage division, and chorionicity and amnionicity are determined by the degree of subsequent fusion of embryonic membranes within the zona pellucida. Currently, both the fission and the fusion models remain controversial and unsubstantiated.

Knowledge of chorionicity is important for optimal prenatal management of twin pregnancies, as monochorionic twins are notoriously at risk for specific complications such as TTTS, TAPS, fetal growth discordance, and malformations. Chorionicity is usually assessed early in pregnancy by ultrasound examination between 10 and 14 weeks’ gestation. Ultrasound clues to chorionicity include sex (fetal sex discordance indicates dichorionicity), placental site (two clearly distinct placental sites indicate dichorionicity), thickness and layering of the dividing membrane (thick four-layered dividing membrane indicates dichorionicity), and shape of the junction between dividing membrane and placenta. The “lambda” or “twin peak” sign, a triangular projection of echodense chorionic villi and trophoblast extending up to the base of the dividing membrane, reflects dichorionicity. The “T” sign, created by the dividing membrane approximating the placenta at a 90-degree angle, suggests monochorionicity. Postnatally, chorionicity is assessed or confirmed by pathologic examination of the placenta, as described later.

Although knowledge of chorionicity is important for the course and management of the pregnancy, accurate zygosity diagnosis is important for postnatal and lifelong health care of twins with respect to medical issues such as organ transplantation and inheritance of specific genetic diseases. It has traditionally been asserted that monochorionicity implies monozygosity and that dizygotic twinning results in dichorionic placentation. However, the unequivocal existence of sporadic dizygotic monochorionic (diamniotic) twins is well documented, usually, but not always, in pregnancies achieved by ART. ^, More than 20 cases of dizygotic monochorionic twins have been reported to date, usually associated with confined tissue or blood chimerism or both. Because assessments of zygosity based on antenatal ultrasound findings or postnatal placental examination may be unreliable, definitive diagnosis depends on genetic markers such as blood group testing (a difference in blood groups is proof of dizygosity) or, preferably, DNA studies using skin biopsy, umbilical cord tissue, or a buccal smear.

Mechanisms Underlying Dizygotic and Monozygotic Twinning

Dizygotic Twinning

Dizygotic twinning is defined as the concomitant conception and development of two independent zygotes during one pregnancy. The basic mechanism underlying spontaneous dizygotic twinning is the concurrent release of two or more oocytes during the same menstrual cycle (polyovulation), followed by fertilization by two separate sperm. Major factors influencing spontaneous dizygotic twinning are maternal age, parity, and genetic inheritance. Compared with other women, mothers of dizygotic twins are taller, have a higher body mass index, and more often smoke before the pregnancy. There are marked geographic differences in dizygotic twinning rates, likely reflecting populations with a predisposition to high follicle-stimulating hormone (FSH) levels and polyovulation.

Mothers of spontaneously conceived dizygotic twins (without ART) have a predisposition to multiple ovulation events resulting from interference with single dominant follicle selection. Factors controlling ovarian folliculogenesis and ovulation include circulating FSH and intraovarian growth factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 and their receptors. Genetic mapping studies in humans and other species are beginning to unravel the genes and pathways contributing to dizygotic twinning. Associations between dizygotic twinning and variants in factors controlling ovarian folliculogenesis and ovulation have been reported, such as GDF9, ^, FSH beta subunit (FSHB), and SMAD3, the product of which plays a major role in gonadal responsiveness to FSH. However, these variants are infrequent and therefore account for only a small portion of the genetic contribution to twinning.

Monozygotic Twinning

Monozygotic (so-called identical) twins (approximately 30% of twins) are the product of division of a zygote created by fertilization of one ovum by one sperm cell. The stimuli for monozygotic twinning are incompletely understood. The incidence of monozygotic twins is constant worldwide and seems independent of environmental factors. However, evidence suggests at least a twofold to fivefold increase in the incidence of monozygotic (and, even more, dizygotic) twinning in pregnancies achieved by ART compared with spontaneous conception. ^, This supports a role for environmental influences, possibly related to micromanipulation techniques, ^, length of cultivation, ^, ^, or glucose or glutathione concentration in cultivation media, in combination with maternal age and intrinsic anomalies associated with infertility. ^, ^, In addition, genetic factors are implicated, suggested by higher prevalence of females in many monozygotic twinning disorders, the abnormally high monozygotic twinning rates in some genetic syndromes such as Beckwith-Wiedemann and Opitz G/BBB syndromes, and the occurrence of familial monozygotic twinning.

Monozygotic twins are generally assumed to be genetically identical. However, it is now well established that phenotypic and genotypic differences between monozygotic twins are common. ^, The original zygotic genome may be modified via a number of mechanisms, including unequal blastomere allocation and postzygotic genetic, epigenetic, or environmental events. ^, ^, ^, Missegregation of genetic material in the course of the monozygotic twinning process may result in two distinct cell populations secondary to discordant postzygotic nondisjunction or crossing over, imprinting differences, activation or expression of selected genes, X-inactivation, and differences in telomere size. ^, ^, Depending on the timing of the genetic event relative to zygotic cleavage, the genetic difference may be seen in multiple somatic tissues or may be mosaic.

Postzygotic nondisjunction in one twin (confined twin mosaicism) can result in heterokaryotypia for autosomal trisomies and for the gonosomes (chromosomes involved in sex determination). The most common, although still very rare, form of genetic discordance in heterokaryotic monozygotic twins involves the sex chromosomes. This condition is most evident in male twins where one becomes monosomy X (Turner syndrome), presumably resulting from loss of the Y chromosome by nondisjunction early in development. ^, This mechanism has also been implicated in the extremely rare occurrence of sex-discordant monozygotic twins, caused by generation of monozygotic 46,XY and 46,XX twins with varying degrees of mosaicism in solid tissues or blood from a 47,XXY zygote. ^, Monozygotic twins discordant for (postzygotic) single-gene point mutations have also been reported. The occurrence and importance of heteroplasmy in mitochondrial DNA remain to be determined. ^,

Epigenetic mechanisms may mediate the effects of twin-specific environmental factors ^, and can be discordant in monozygotic twins. A large survey identified age-dependent epigenetic differences such as discordant X-inactivation, DNA methylation, and histone acetylation in about one-third of monozygotic twin pairs. ^, An example of twin discordance by epigenetic mechanisms is the relatively high frequency of discordant Beckwith-Wiedemann syndrome in monozygotic twins, usually, but not always, seen in female twin pairs.

Twin Placentation Types

The twin placentation type is determined based on the presence and number of amnions and chorions. Twin placentas may be diamniotic-dichorionic, diamniotic-monochorionic, or monoamniotic-monochorionic ( Fig. 5.1 ).

Dichorionic Placentation

The diamniotic-dichorionic twin placenta is characterized by the presence of a chorion (and amnion) for each twin. Dichorionic twin placentas may be separate or fused, in roughly equal proportions, depending on the implantation sites of the blastocysts. Close implantation of the blastocysts results in fusion of the placentas and formation of an apparently single disk with dividing septum and separate fetoplacental circulations ( Fig. 5.2 ). The intertwin membrane of fused dichorionic placentas is relatively thick and opaque owing to the presence of chorion between the two amniotic layers (see Fig. 5.2 ). The chorionic tissue is continuous with the chorion of the underlying placenta and forms a ridge along the base of the intertwin membrane attachment, corresponding to the sonographic twin peak or lambda sign. Chorionicity is confirmed by microscopic examination of the layers of the intertwin membrane in sections of the rolled septal membrane. The intertwin membrane of the diamniotic-dichorionic twin placenta is composed of two amnion layers separated by a fused layer of two chorions (see Fig. 5.2 inset).

Figure 5.2, Diamniotic-dichorionic twin placenta.

In dichorionic placentas, the location of the septum corresponds to the fused borders of the twin placentas and thus defines the vascular equator of the two placentas. In virtually all cases, fused diamniotic-dichorionic twin placentas have separate chorionic vascular beds. Exceedingly rare exceptions have been described where fetal chorionic vessels cross the area of fusion ; the existence of such small anastomoses may explain the rare finding of blood group chimerism in dizygotic twins of opposite sex.

Twins with dichorionic placentas, whether separate or fused, may be dizygotic or monozygotic. Sex discordance virtually always corresponds to dizygosity, although monozygotic twins of different sex have been described. ^, In same-sex dichorionic twins, further investigations are required to determine zygosity.

Monochorionic Placentation

The monochorionic twin placenta is a single-disk placenta characterized by the presence of a single chorion. Monochorionic twins may have separate amnions (diamniotic-monochorionic placentation) or a common single amniotic cavity (monoamniotic-monochorionic placentation). In the presence of an intertwin membrane, a diamniotic-monochorionic twin placenta is distinguished from a fused diamniotic-dichorionic twin placenta by examination of the intertwin membrane and chorionic plate vasculature. A thin, semitranslucent, two-layered intertwin membrane, loosely attached to the chorionic plate, is diagnostic of diamniotic-monochorionic placentation ( Fig. 5.3 ). Microscopic examination of the intertwin membrane allows confirmation of monochorionicity. The dividing membrane of diamniotic-monochorionic placentas consists of two amnion layers, without interposed chorion (see Fig. 5.3B inset).

Figure 5.3, Diamniotic-monochorionic twin placenta.

In contrast to fused dichorionic placentas, almost all monochorionic placentas (>95%) exhibit intertwin vascular anastomoses crossing the intertwin membrane ( Fig. 5.4 ). Vascular communications between monochorionic twins can be artery-to-artery (AA), vein-to-vein (VV), or artery-to-vein (AV). These intertwin anastomoses can be categorized based on the near-constant anatomic relationships between the different vessel types: chorionic arteries virtually always course superficial to their accompanying veins (see Fig. 5.4 ). AA and VV anastomoses are superficial; they form a direct communication between homonymous vessels from each twin without penetrating the chorionic plate (see Fig. 5.4A ). In contrast to these superficial anastomoses, AV anastomoses occur deep within the parenchyma at the villous capillary level and are recognized by the chorionic penetration of an unpaired artery of one twin in close proximity to an unpaired vein of the opposite twin (see Fig. 5.4B ). AV anastomoses are obligatorily unidirectional. AA and VV anastomoses are bidirectional and allow flow in either direction, depending on pressure gradients between twins. Superficial AA and VV anastomoses are thus believed to be able to compensate for flow imbalances generated by nonequilibrated AV anastomoses.

Figure 5.4, Diamniotic-monochorionic twin placenta following vascular dye injection.

Although monochorionicity remains an excellent proof of monozygosity, rare exceptions have been described involving dizygotic monochorionic twinning. ^, Therefore determination of monochorionic placentation status should be regarded as a screening tool, rather than unequivocal evidence of monozygosity. Further genotyping is especially recommended when monochorionic twins have a dissimilar phenotype and following artificial reproduction. Definitive zygosity determination relies on genetic markers such as blood group testing or, preferably, polymerase chain reaction analysis of variable microsatellite markers using DNA extracted from a skin biopsy specimen, umbilical cord tissue, or buccal smear. Possible pitfalls in interpretation must be taken into account, such as those created by postzygotic mutations and blood mosaicism.

Complications of Monochorionic Twinning and Their Associated Placental Characteristics

Monochorionicity is associated with a higher perinatal mortality and with a higher incidence of preterm birth, low birth weight, and prolonged stay in the neonatal intensive care unit compared with dichorionic twin pregnancies. The overall perinatal mortality is approximately 12% in monochorionic twins compared with 2% to 5% in dichorionic twins, and mortality is even higher in monoamniotic twins. ^, In addition, monochorionic twin pregnancies are susceptible to a specific set of complications, including TTTS, TAPS, TRAP sequence, discordant growth restriction, and malformations.

Because nearly all monochorionic pregnancies have connections between the two choriovascular beds, death of one twin affects the outcome of the surviving co-twin. These vascular disruptions are usually seen following the death of one co-twin but may occur in monochorionic twins with two surviving infants. Consequences for the surviving co-twin include survival with cerebral impairment, preterm delivery with its sequelae, or intrauterine death. Many organ systems may be affected including brain (hypoxic-ischemic encephalopathic brain disruptions with microcephaly, hydrocephalus, or porencephaly/hydranencephaly), gastrointestinal system (intestinal atresia), and skin (aplasia cutis). Proposed mechanisms to explain injury to the co-twin following twin fetal demise include the embolic theory, in which thromboplastin-like material is transferred through open placental vascular anastomoses to the survivor, and the hypovolemic shock-ischemic theory, in which blood is shunted into the low-resistance circulation of the dead or dying fetus.

In addition to the structural or growth anomalies associated with specific complications of monochorionic twinning such as TTTS, TAPS, TRAP sequence, and conjoined twinning, twin pregnancies are susceptible to other malformations, deformations, or disruptions that may or may not be related to their twin status. Primary structural malformations, chromosomal effects, and genetic syndromes seen in singletons may also occur in twins. The overall odds ratio for congenital anomalies in twins compared with singletons is estimated at 1.3, ³⁸ with a significantly higher frequency in monozygotic twins compared with dizygotic twins. ^, The placental findings in most congenital anomalies are either nonspecific or similar to those seen in singleton pregnancies.

Twin-to-Twin Transfusion Syndrome

Definition

TTTS is a complication of monochorionic twinning, characterized by chronic fetofetal blood transfusion from one twin (donor) to the other (recipient) through placental vascular communications. This unbalanced shift of blood volume results in hemodynamic imbalance and oligohydramnios in the donor and polyhydramnios in the recipient (so-called twin oligohydramnios-polyhydramnios sequence).

TTTS traditionally refers to an often severe, chronic condition and needs to be distinguished from several acute forms of intertwin transfusion. Acute perimortem TTTS occurs following intrauterine death of one monochorionic twin and is caused by exsanguination from the surviving twin into the low-pressure circulation of the dead or dying co-twin. This form of perimortem acute twin-to-twin transfusion is mediated mainly through large-sized AA or VV anastomoses. Acute peripartum (or perinatal) TTTS may occur during birth and is caused by acute shifts of blood volume between twins resulting from blood pressure differences associated with uterine contractions, delayed cord clamping, or changes in fetal position around the time of delivery. The clinical presentation of acute peripartum TTTS may range from subtle intertwin differences in hemoglobin levels without obvious effects to frank hypovolemic shock in the donor twin and polycythemia in the recipient. Similar to other forms of acute TTTS, acute peripartum TTTS is believed to be facilitated by large superficial AA and VV anastomoses. Acute peripartum TTTS is distinct from the more common postpartum placentofetal (as opposed to twin-to-twin, or fetofetal) transfusion that occurs when cord clamping of one twin directs blood from the entire placenta to the remaining twin through vascular anastomoses.

Pathogenesis

The pathogenesis of TTTS is incompletely understood. TTTS is a complex and multifactorial condition with both placental and fetal contributions. An intuitive, albeit simplistic, model of TTTS proposes that the primary event is flow imbalance from donor to recipient across unbalanced unidirectional AV anastomoses. If this flow imbalance is significant and not fully compensated by bidirectional AA anastomoses, the donor becomes hypovolemic and anemic, whereas the recipient develops polycythemia and hypervolemia. These volume changes are believed to induce modulation of a variety of hormonal and biochemical regulators in both twins. The renin-angiotensin system is upregulated in the donor twin and downregulated in the recipient twin. Both twins are likely exposed to equally high renin levels through their shared circulation, which may contribute to cardiovascular anomalies in some recipient twins. ^, In addition, concentrations of atrial natriuretic peptide, brain natriuretic peptide, and endothelin-1 are higher in the amniotic fluid of recipient twins compared with donor twins. Although their exact mechanisms of action remain incompletely understood, dysregulation of these and other unidentified biochemical and related mediators likely plays a role in a proposed exaggerated hemodynamic response to hypervolemia and hypovolemia in TTTS.

Placental Findings

TTTS has no pathognomonic placental or choriovascular signature. Nevertheless, several anatomic placental features have been linked to an increased risk for development of TTTS in diamniotic-monochorionic twin gestations. ^, ^, Pregnancies complicated by TTTS have a lower frequency of intertwin AA anastomoses than uncomplicated, non-TTTS monochorionic pregnancies (25% to 57% in TTTS versus >85% in non-TTTS placentas) ( Fig. 5.5 ). ^, ^, The relative paucity of AA anastomoses in TTTS placentas has contributed to the notion that these potentially bidirectional anastomoses have a protective role against the development of TTTS in monochorionic twin gestations by compensating for hemodynamic imbalances created by uneven AV anastomoses. Mathematical computer models of TTTS support the protective role of AA anastomoses. However, this theory does not account for the presence of AA anastomoses, often large, in a substantial portion of cases with TTTS.

Figure 5.5, Placenta of twin pregnancy complicated by twin-to-twin transfusion syndrome.

In contrast to AA anastomoses, the frequency of VV anastomoses is higher in TTTS pregnancies than in non-TTTS pregnancies (38% versus 15% to 25%). ^, ^, ^, ^, In the subgroup of placentas without AA anastomoses, this difference is even more striking. In the absence of AA anastomoses, the prevalence of VV anastomoses is 32% in TTTS placentas versus 8% in non-TTTS placentas. This suggests that VV anastomoses may play an adverse role in TTTS, especially in the absence of AA anastomoses, perhaps by acting as low-resistance functional AV anastomoses.

The contribution of AV anastomoses to the onset or continuation of TTTS is less clear. In contrast to AA and VV anastomoses, AV anastomoses are deep and obligatorily unidirectional. In the absence of compensating AA anastomoses, AV imbalance directed from donor to recipient strongly correlates with the development of TTTS. ^, This specific combination of absent AA anastomoses and severe AV imbalance is virtually diagnostic of TTTS but is seen in only a small minority of TTTS placentas (14%). The role of AV anastomoses in the vast remainder of monochorionic gestations is incompletely understood. TTTS has been described even in the absence of identifiable AV anastomoses. ^, ^, It has been speculated that in such cases an AA anastomosis may have been converted into a functional AV anastomosis; for instance, by arterial stenosis.

In addition to choriovascular features, both peripheral cord insertion and uneven placental sharing have been linked to an increased risk for TTTS development in monochorionic gestations. The reported frequency of peripheral (marginal or velamentous) cord insertion of at least one twin is significantly higher in TTTS gestations than in non-TTTS gestations (approximately 50% versus approximately 30%). When cord insertion is discordant, it is virtually always the donor twin that has a peripheral cord insertion. ^, ^, Markedly uneven placental sharing, traditionally defined as greater than 25% intertwin difference in distribution of placental choriovascular territory, is seen in approximately 50% of TTTS gestations versus 25% of non-TTTS gestations ; the donor twin almost always has the smaller share.

Diagnosis, clinical characteristics, management, and outcome are described in Chapter 37 .

Twin Anemia-Polycythemia Sequence

Definition

TAPS is a recently described form of chronic twin-to-twin transfusion in monochorionic pregnancies, characterized by the presence of a large intertwin difference in hemoglobin and reticulocyte levels in the absence of oligohydramnios and polyhydramnios. ^, TAPS may occur spontaneously (spontaneous TAPS; estimated incidence 3% to 6% of monochorionic twin pregnancies) or iatrogenically following laser treatment for TTTS (postlaser TAPS). In contrast to TTTS, which involves a severe discordance in amniotic fluid, TAPS is characterized by a severe discordance in hemoglobin levels.

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