Before the development of ultrasound, twins were often diagnosed at birth, after the delivery of one neonate. In fact, a multiple gestation was clinically suspected in only 25–50 %. In the famous Routine Antenatal Diagnostic Imaging with Ultrasound Study (RADIUS), 38 % of twins were recognized after 26 weeks and 13 % were not diagnosed until delivery [40]. In the Helsinki Ultrasound Trial, 25 % twins were not recognized until 21 weeks [41]. These two studies, however, were not really about first trimester ultrasound but rather about scanning at mid-trimester (16–24 weeks). The diagnosis should be obtainable, with ultrasound, from very early in gestation. When ultrasound is performed for an indication (e.g., the uterus is larger than expected), the accuracy is about 75 %. When ultrasound is performed routinely, this climbs to 90 % [42], with better outcomes in women known to carry multiple gestations [43]. The first ultrasound indication of a multiple gestation may be the presence of multiple corpora lutei (see Fig. 14.1). While routine ultrasound is still not the official rule, as recommended in low risk pregnancies by the American College of Obstetricians and Gynecologists (ACOG), the American College of Radiology (ACR) or the American Institute of Ultrasound in Medicine (AIUM), the advantages of a policy of routine scanning in the first trimester include, among others, the early detection of multiple gestations, allowing for early determination of chorionicity and amnionicity [44, 45]. Another clear advantage is accurate assessment of gestational age (GA). When ultrasound is ordered “to date” the pregnancy, in cases of unknown or unclear last menstrual period, fetal biometry is used to determine GA. In twins, however, there may be growth discordancy, for instance with one twin measuring 1 week more than the other. The published literature does not provide evidence-based data on whether dating should be based on the smaller twin, the larger or an average. It is important, however, to avoid missing early growth restriction in one twin, thus, the majority will date the pregnancy based on biometry of the larger twin [46]. An important consideration is whether growth nomograms for singleton gestations can be used for twins or higher order gestations [47]. It appears that during the first trimester, there are no major differences in fetal biometry between singleton and multiple pregnancies [46]. Hence, crown-rump-length (CRL) curves published for singletons may be used in the assessment of twins and triplets [48, 49]. Furthermore, there is no difference in placental mass between singletons, monochorionic (MC) and dichorionic (DC) twins and trichorionic triplets between 11 and 13 6/7 weeks [50]. Growth curves for singletons may be used in the assessment of biometry in twins until approximately 34 weeks GA [51]. For triplets, the upper limit may be lower, e.g., 25 weeks [52].

Determining the number of chorionic sacs is important because prognosis is much better in DC than MC twin pregnancies [53]. Mortality (stillbirth, perinatal, and neonatal death) is 3–4 times higher in MC twins [53–58]. The major reason is the presence of vascular anastomoses between the two placental circulations [59–61]. They are at risk of twin-to-twin transfusion syndrome or TTTS [62–66], twin anemia–polycythemia syndrome or TAPS [67–69], twin reversed arterial perfusion or TRAP syndrome [70, 71], unequal placental sharing with discordant twin growth or selective intrauterine fetal growth restriction [72], and, if also monoamniotic (MA), cord entanglement with the added risk of demise of one twin and embolization of thromboplastin from the demised fetus to the healthy twin [73–75]. Additionally, there is the risk of conjoining, an event occurring in 1/50,000 births [76]. Mortality is 8–10 % in DCDA, 25 % in MCDA, 50–60 % in MCMA, and perhaps 90 % in conjoined twins [77–79] with fetal loss under 24 weeks, 1.8 % in DC twins, and 12 % in MC twins [53].

As described above, approximately 70 % of twins delivered and conceived naturally, result from the fertilization of two independent oocytes, i.e., dizygotic (DZ) twins; the remaining 30 % are the result of the division of a single zygote, i.e., monozygotic (MZ) twins. Interestingly, the rate of MZ twins is three times higher in pregnancies conceived with the help of ART, compared to spontaneous conceptions [80, 81]. If the division of the zygote occurs at the two-cells stage (0–4 days), before the morula stage, this results in two morulas, two blastocysts, two chorions, and two amnions (dichorionic–diamniotic or DCDA placentation), about one-third of monozygotic twins. In about two-thirds of monozygotic twins, the split occurs after the morula stage (4–7 days) and the single morula split will result in MCDA placentation. If it occurs at 7–14 days, the embryonic disc was already formed and the result will be two embryos in the same sac (MCMA). If after day 13–14, conjoined twins will result. A combination of both may also exist, when one of two dizygotic twins splits, in a monozygotic fashion, resulting in various combinations of chorionicity and amnionicity.

Ultrasound plays an important, if not the major, role in the determination of chorionicity and amnionicity early in pregnancy [44, 72, 82–94]. Various algorithms themes can be used, based on what is the known (or assumed) gestational age [95–97]. With appropriate training, reproducibility of the results has been shown to be excellent [98].

From 4 to 6 weeks, the number of sacs determine the chorionicity: two sacs means twins are DC (Fig. 14.2).

From 6 to 8 weeks, if the number of sacs is the same as the number of yolk sacs and the number of fetuses, this is a DCDA pregnancy. If the pregnancy is MC, two fetuses will be visualized within the sac and the number of yolk sacs will help distinguish between DA and MA placentation. Observing two yolk sacs or two clear amniotic cavities (Fig. 14.3) allows one to make the diagnosis of diamniotic twins [99]. Visualization of two fetal poles with a single yolk sac is diagnostic of monoamnionicity (Fig. 14.4). Once the membranes can be visualized, ultrasound imaging can distinguish between MC and DC twin pregnancies with more than 90 % accuracy [93]. The “twin peak,” also called lambda sign, at the level of the attachment of the chorionic membranes to the placenta is formed by projection of the trophoblast from a fused dichorionic placenta between the layers of the membranes and indicates a DC twin pregnancy (Fig. 14.5), with 100 % accuracy, while the “T sign” at the site where the thin inter-twin membrane composed of two amnions with no chorions leaves the placenta at a 90° angle (Fig. 14.6) indicates a monochorionic–diamniotic (MCDA) twin pregnancy [100]. In a study of 55 cases, sensitivity of the twin-peak sign for dichorionicity was 94 %, specificity 88 %, positive predictive value 97 %, and negative predictive value 78 % [101]. In another study of 506 DC and 154 MC twin pregnancies, between 11 and 14 weeks of gestation, use of the twin-peak and T signs and the number of placentas had sensitivity of 100 % specificity of 99.8 % for monochorionicity, with only one DC pregnancy incorrectly assigned as MC [102].

From 8 to 14 weeks, the number of placental masses and/or the lambda or T sign can be assessed, as above, but at that stage, membrane thickness can also be analyzed [88]. A dichorionic membrane is typically well defined and easy to visualize with ultrasound. It consists of four layers (i.e., two layers of both amnion and chorion) and its width will be greater than 2 mm (Fig. 14.7). The presence of a thick dividing membrane indicated a dichorionic–diamniotic gestation in 38 (90 %) of 42 cases in which it was identified [103]. The number of dividing membranes can also, occasionally, be counted: four means DCDA (Fig. 14.8), two means MCDA [95].

For women presenting after 14 weeks 0 days, all of the above features should be used and, in addition, evaluation of fetal gender, since discordancy would, obviously, signify dizygocity [104]. In the second and third trimesters, membrane thickness is much less useful [105].

If transabdominal views are poor because of elevated BMI or retroverted uterus, transvaginal ultrasound is recommended. In a study by Bora and colleagues [106], chorionicity and amnionicity were documented in 67 viable twin pregnancies at both 7–9 and 11–14 weeks’ gestation. There was agreement in the chorionicity and amnionicity reported at each of the two scans in 65 out of 67 (97 %) cases. Of the DCDA pregnancies reported at 7–9 weeks, 53 out of 54 (98 %) were confirmed at the 11- to 14-week scan and one (2 %) was found to be MCDA. At birth, however, these twins were of different sex, confirming DCDA twins as initially diagnosed at 7–9 weeks. Of the 12 pregnancies diagnosed as MCDA at 7–9 weeks, all were found to be MCDA at the 11- to 14-week scan. In the (rare) case when chorionicity cannot be established, management should be based on the assumption that the gestation is monochorionic, until proved otherwise. After ultrasound diagnosis and characterization of twins, the risk of spontaneous loss of both fetuses before 22 weeks of gestation is significantly higher in MC than in DC pregnancies, and is significantly higher in MCMA pregnancies than in MCDA pregnancies [107]. Hence, no ultrasound report on twins should be considered finalized without details of the type of placentation [108]. Another sign has been described in triplet pregnancy: the ipsilon zone, the junction of the three interfetal membranes with 100 % success in determining chorionicity in 19 sets of triplets [109].

Another important role for ultrasound in multiple gestation is observation of the umbilical cord insertions in the placenta. Abnormal cord insertions such as marginal and velamentous insertions are much more frequent in multiple gestation (Fig. 14.9). In addition, single umbilical artery is also much more frequent in twins [110, 111].

Several complications are unique to multiple gestations: vanishing twin, death of one fetus, discordant fetal growth, discordance for genetic/structural anomaly, and partial mole. Some will only be found in monochorionic gestations (TTTS, TAPS, TRAP) while conjoined twins and cord entanglement are specific for MCMA gestations, which have been called “the most precarious of twin pregnancies” [112].

There is, often, unequal sharing of the placenta, which may cause grave problems: discordant fetal growth with IUGR, metabolic compromise and death [156]. In addition, chronic unidirectional blood shunting through placental vascular anastomoses may occur and result in TTTS or twin reverse arterial perfusion [TRAP] and death). Furthermore, for MCMA twins, additional risks include conjoined twinning and cord entanglement. Risk of cerebral injury and subsequent cerebral palsy is seven times higher than in DC, most likely secondary to vascular anastomoses. If TTTS is present, this risk climbs to 21 % [56, 126]. After single intrauterine demise it is up to 18 % [157–159].

Twins are at increased risk for genetic anomalies, as clearly documented in a study of 5.4 million births, from 14 European countries, of which 3 % were multiple [200]. The risk of karyotypic anomalies is different between monozygotic and dizygotic twins. For MZ twins, the age-related risk to simultaneously be abnormal is the same as in a singleton gestation, although, from a maternal standpoint, the risk to the pregnancy to have one affected fetus is twice the risk of a singleton in cases of twins, three times in the case of triplets, etc. For dizygotic twins, however, the risk of one being affected is similar to a singleton but the risk of both being affected is much lower. In fact it is the square of the risk of a singleton: for instance if the age related risk of the mother is 1:250, the risk of both twins, if dizygotic, to be affected is 1:250 × 1:250 or 1:62,500 [201]. Screening for trisomy 21 in multiple gestations is complicated [202, 203]. Local prevalence of aneuploidy in twins needs to be taken into account for all calculations of risk [204]. Serum screening alone is of limited value because a high value may indicate elevated risk but with no determination of which or how many fetuses are affected, since it is possible that an unaffected co-twin may “mask” the abnormal serum results of an affected one and the fact that for DZ or MZ twins the interpretation may need to be different [205–207]. Specific references may need to be utilized [208]. An acceptable screening test for aneuploidy in the first trimester twin pregnancy includes fetal nuchal translucency, combined with maternal age. Structural (as opposed to maternal serum) first trimester markers (including NT, nasal bone, tricuspid valve flow, and ductus venosus waveform) may be helpful in risk assessment for aneuploidy as they are independent measurements for each fetus, regardless of chorionicity [209]. Nuchal translucency (NT) screening is effective and is an excellent modality (when cell free fetal DNA is not available, see below) for twin pregnancies. When screening is done by nuchal translucency and maternal age, a pregnancy-specific risk should be calculated in MC twins. In DC twins, a fetus-specific risk is calculated [203]. Among twins, NT alone has a 69 % trisomy 21 detection rate [210]. Screening with first trimester serum analytes, combined with nuchal translucency (also known as First Screen) may also be considered. It decreases the false-positive rate. In a 2014 systematic review of first trimester combined risk assessment (nuchal translucency and maternal serum analytes) in twin pregnancies, test sensitivity in DC and MC twins was 86 % and 87 %, respectively [211]. Integrated screening with First Screen and second trimester serum screening is an option. Naturally, in addition to trisomy 21, increased nuchal translucency is a marker for other aneuploidies, congenital malformations, and a sign of early development of TTTS [65, 161]. First-trimester combined NT and serum biochemistry has a 72 % DS detection rate, and an integrated screen will have an 80 % DS detection rate at a 5 % FPR [210]. The issue of “vanishing twin” (see below) is specifically problematic since early loss of one or more embryos of a multiple gestation may affect analyte levels [212]. In known cases, NT screening may be the preferred option. When screening is done by nuchal translucency and maternal age, a pregnancy-specific risk should be calculated in MC twins. In DC twins, a fetus-specific risk is calculated [203]. Some laboratories that perform noninvasive DNA screening (NIDS) with MPS methodology offer testing for twin gestations after it has been validated for twins. Testing for monozygotic twins is expected to perform similarly to a singleton gestation, although testing in dizygotic twin and higher-order multiple gestations is complicated by the fact that the per-fetus fetal fraction may be lower [213]. In fact, the non-reportable rate is higher (7.4 %) than that for singleton pregnancies (2 %). Additionally, if one fetus is euploid while the other is aneuploid, there is a dilution of the cell free fetal DNA from the aneuploidy fetus resulting in decreased detection rates compared to singleton gestations. Based solely on NIDS results, it is impossible to determine which twin is abnormal. Therefore, invasive testing (CVS or amniocentesis) is required to distinguish which twin is affected. Several false-positive results have been reported with biological basis, such as confined placental mosaicism (CPM), maternal chromosome abnormality and vanishing twin. Additional unexpected information such as undiagnosed molar pregnancy or vanishing twin may be detected by some NIDS methods [214].

The incidence of congenital anomalies is much higher in MZ twins, in fact three to five times higher than in DZ twins [215, 216]. Although this has been party correlated with assisted reproductive technologies [217], there seems to also be a direct relation with the twinning phenomenon itself, whether spontaneous or induced with a common etiology for both the MZ twinning and the early sequence of the malformation [218]. Most common structural anomalies in twins include anencephaly, facial clefts, holoprosencephaly, VATER association (vertebral defects, imperforate anus, esophageal fistula with tracheoesophageal fistula, radial and renal dysplasia), exstrophy of the cloaca malformation sequence, and sacrococcygeal teratoma, all of which should be recognized early by detailed ultrasound anatomy scan. In a large study by Glinianaia and colleagues 2329 twin pregnancies (4658 twins) and 147,655 singletons were compared. The rate of congenital anomalies in twins was 405.8 per 10,000 twins versus 238.2 per 10,000 singletons (rate ratios [RR] = 1.7, 95 % confidence interval [CI] 1.5-2.0]. In twins with known chorionicity (84.8 % of all twins), the prevalence of congenital anomalies in MC twins (633.6 per 10,000) was nearly twice that in DC (343.7 per 10,000; RR = 1.8, 95 % CI 1.3-2.5). There was an increased rate of congenital anomalies for all major types of anomalies in twin compared with singleton pregnancies, except chromosomal abnormalities [215]. Monozygocity specifically MCDA twinning, seems to be an independent factor for an increase in congenital heart disease (CHD) with a 9.18 relative risk increase in one report of 40 fetuses with CHDs among 830 fetuses from MCDA twin gestations [219]. Congenital heart disease, however, is also more common in DZ twins than in singleton [220]. Thus fetal echocardiography is, in fact, indicated in all wins. In a study of 844 pairs of twins, the prevalence of major congenital malformations was 2.7 % for MZ twins, 1.0 % for DZ twins, and 0.6 % for singletons. The concordance rate of major congenital malformations was 18 % for MZ twins, but no DZ pair was concordant for any major congenital malformation [221].

Are monozygotic twins “really” identical? They are very similar but genetically, most often, not “exactly” the same [222–224]. In fact, hundreds (360 by one estimate) of genetic differences may occur very early in fetal life. These may be due to post-fertilization events, such as chromosomal mosaicism, skewed X-inactivation, imprinting mechanisms, as well as DNA point mutations or copy errors, taking place early after blastocyst splitting [224]. There are also, genetic differences due to mutations which may occur later in life as well as epigenetic² modifications, due to environmental factors [225, 226]. Another phenomenon explaining a difference in the karyotype of two MCMA twins is heterokaryotypia: a discordance in karyotype due to either an early postzygotic chromosomal rescue in one fetus or a mitotic error that leads to one trisomic fetus with a normal co-twin [227]. Discordance for a congenital anomaly is extremely problematic, from a moral, ethic, religious, philosophical, and, often, medical standpoints [228, 229]. Until intrauterine therapy is effective and safe (it may already be for a very small number of anomalies), the options include expectant management [230], termination of the entire pregnancy or selective feticide [231–233]. Selective termination of an anomalous DC twin is relatively safe with intravascular injection of potassium chloride or digoxin, although there is some increased risk of miscarriage or preterm delivery [234, 235]. In monochorionic twins, selective feticide needs to result in complete separation of the circulations [236, 237] and is, thus, best accomplished by sealing one umbilical cord with ligation [238], bipolar coagulation [239, 240], radiofrequency [241], or laser ablation [236].

As described in the introduction, maternal morbidity (and mortality) is increased in multiple pregnancies. Multiple pregnancy is the most powerful predictive factor for adverse maternal, obstetrical, and perinatal outcomes [242]. Pregnancy induces physiological stress to the maternal body and multiple gestations provide even additional strain and nutritional demands [243]. Most of the complications do not become clinically apparent in the first trimester but later in pregnancy, such as preeclampsia and diabetes [244]. Among women with 684 twin and 2946 singleton gestations enrolled in multicenter trials, rates for both gestational hypertension and preeclampsia were significantly higher among women with twin gestations than among those with singleton gestations. Furthermore, adverse neonatal outcomes were more frequent in women with twin pregnancies and hypertensive complications [16]. In a study of over 23,000 women, 553 of whom had twins, after adjusting for age, race/ethnicity, body mass index, maximal systolic and diastolic blood pressure, smoking and parity, multiple regression analysis showed that twin pregnancy was associated with an approximately twofold increase in the risk for developing gestational diabetes. The risk was highest among African-American and young women [245]. Thromboembolic disorders are major causes of morbidity and mortality in the pregnant patient. Contributing factors are increased blood coagulability [246], elevated BMI, maternal age above 35 and, specifically, multiple gestation with an incidence rate of 6.3/10,000 year in singletons versus 18.2/10,000 year among women with multiple pregnancies [247]. Other complications more common in women carrying multiple gestations, most likely secondary to increased levels of various hormones, in particular βHCG, include hyperemesis gravidarum [248]—although this is not universally accepted as a more frequent complication in multiple pregnancies [249]—iron deficiency anemia [250], intrahepatic cholestasis of pregnancy [251] and pruritic urticarial papules and plaques of pregnancy or PUPPP. This is the most common specific dermatosis of pregnancy, with an incidence is 1/160–1/300 pregnancies [252]. The majority of patients are nulliparous and PUPPP is 8- to 12-fold more common in women with multiple gestations, possibly due to increased hormones levels, as stated above, or increased abdominal distension [253]. An additional complication is acute fatty liver. This is a rare condition, usually of the third trimester, complicating approximately 1 in 10,000 singleton gestations [254] but, of all the published cases, 14 % have been reported in twin gestations [255]. The rate seems to be 7 % in triplet pregnancies [256].

These pregnancies (triplets, quadruplets, etc.) are at extremely high risk of complications [257]. The classic teachings are that the prevalence for triplets is 1:90² and 1:90³ for quadruplets. Numbers have greatly changed with the introduction of ART [258–260]. Classification is based on chorionicity and amnionicity [261] (Figs. 14.18 and 14.19). In a study of 49 consecutive sets of triplets, including 18 sets of spontaneously conceived triplet pregnancies and 31 sets resulting from ART the rate of MZ twin pairs was 48 % among spontaneously conceived triplet pregnancies; 30 % of DC triplet pregnancies were MZ and 70 % DZ; 20 % of trichorionic (TC) triplet pregnancies were DZ and 80 % trizygotic (TZ). For triplet pregnancies conceived using ART, the rate of MZ twin pairs was 6.5 %; 100 % of DC triplet pregnancies were DZ; 4 % of TC triplet pregnancies were DZ and 96 % TZ [261]. Early complications, such as genetic anomalies, growth discordancy, TTTS are similar to twin pregnancies, depending on placentation, although, naturally, much more challenging from a management standpoint [258, 262, 263]. Incidence of congenital anomalies is not increased, compared to twins [259]. The complications are mostly later in pregnancy. In a study of 316,696 twin, 12,193 triplet, and 778 quadruplet pregnancies, compared with mothers of twins, mothers of triplets and quadruplets were more likely to be diagnosed with preterm premature rupture of membranes (AORs, 1.53, 1.74, respectively), pregnancy-associated hypertension (AORs, 1.22, 1.27), and excessive bleeding (AORs, 1.50, 2.22), to be delivered by cesarean section (AORs, 6.55, 7.38) at <29 weeks of gestation (AORs, 3.76, 7.96), and to have one or more infants die (AORs, 3.02, 4.07). The rate of maternal complications is also increased compared to twin pregnancies where it is already increased compared to singletons. In a retrospective study of 57 triplet gestations, preterm labor occurred in 86.0 %, anemia in 58.1 %, preeclampsia in 33.3 %, preterm premature rupture of the membranes in 17.5 %, postpartum hemorrhage in 12.3 %, and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome in 10.5 % [264].