Determining Chorionicity and Amnionicity in Multifetal Pregnancies

Multiple pregnancies compromise 2-3% of live births. They are characterized by the presence of more than one fetus in the uterus, posing unique challenges to maternal and fetal health. Chorionicity refers to the number of placentas, while amnionicity describes the number of amniotic sacs. Approximately 30% of twin pregnancies are monozygotic (MZ), arising from cleavage of a single embryo early in development, the remaining 70% are dizygotic (DZ), resulting from two embryos with fertilization of two ova by two sperms. Essentially all DZ twins are dichorionic diamniotic (DCDA), whereas MZ twins may be dichorionic diamniotic DCDA (25-30%), monochorionic diamniotic MCDA (70-75%) or monochorionic monoamniotic MCMA (1-2%) depending on the timing of cleavage (1). Determining the risk of twin pregnancy is primarily related to chorionicity and amnionicity rather than zygosity. However, detecting zygosity in twin pregnancy might be useful in some situations, for example when ultrasound assessment of chorionicity is unreliable or equivocal, especially when there are concerns whether both fetuses sharing variable expressed disease or not.  Detecting zygosity is not possible by ultrasound, however, it can be assessed using either invasive testing or non-invasive prenatal testing (NIPT) using cell free fetal DNA (cfDNA) (2). Interestingly, MC twins are not always monozygotic, Dizygotic MC twins were reported very rarely in literature, and this was explained by possible early fusion of trophoblast of the two zygotes, this is particularly more common in pregnancies conceived by in vitro fertilization due to proximity of blastocysts (3,4). NIPT using cfDNA has many applications in multiple pregnancy that will not be covered in this article. Since monochorionity and monoamnionicity are both associated with substantially increased risks for pregnancy complications and adverse outcomes, accurate determination of both chorionicity and amnionicity is essential for determining the risks, prenatal monitoring, management strategies, and outcomes in multiple pregnancies (5).

1. Chorionicity refers to number of placental masses

1.1 Monochorionic pregnancies (MC) account for approximately 20% of twin pregnancies, but their perinatal mortality rates are considerably higher than dichorionic (DC) pregnancies. Complications unique to monochorionic pregnancies are related to vascular anastomosis between both fetuses; these include twin-to-twin transfusion syndrome (TTTS), twin anemia-polycythemia sequence (TAPS), selective intrauterine growth restriction (sIUGR) and twin reversed arterial perfusion sequence (TRAP-S) (6,7).

1.2. Dichorionic pregnancies: Dichorionic pregnancies (DC) involve each fetus having its own separate placenta. Dichorionic pregnancies are generally associated with a lower risk of complications compared to monochorionic pregnancies (5).

2. Amnionicity refers to the number of amniotic sacs in multiple pregnancies and is categorized as follows

2.1. Monoamniotic pregnancies compromise approximately 1% of MC twins. The absence of a inter-twin membrane leads to cord entanglement in almost all cases of MCMA twins; this arrangement can be associated with complications such as cord accidents and sudden fetal demise (5).

2.2. Diamniotic pregnancies: Diamniotic pregnancies involve each fetus having its own separate amniotic sac. This is the most common type of amnionicity in multiple pregnancies (5).

Determining chorionicity and amnionicity can be reliably detected by ultrasound in the first trimester and ideally should be confirmed before 14 weeks of gestation. However, it is best done at 10-14 weeks’ gestation when detecting a twin or triplet pregnancy (8,9). This can be achieved by looking at:

• the number of placental masses
• the presence of amniotic membrane(s) and membrane thickness
• T sign and lambda (λ) sign (Figure 1-5)

Chorionicity determination at 10-14 weeks (8,9):

1. Separate placentas: indicates DC twins.
2. T sign and lambda (λ) sign at the inter-twin membrane-placental junction: MC twins will show T sign due to single placenta, whereas DC twins will show lambda (λ) sign instead.
3. Thickness and number of layers in the inter-twin membrane: a thin inter-twin membrane (two amnion layers) is suggestive of MC twins, whereas a thick and more echogenic membrane (multiple layers including chorion and amnion for both twins) indicates DC twins (Figure 6).

Other methods of detecting chorionicity (4):

Determining fetal sex: discordant fetal sex indicates dizygosity and therefore indicates DC twins. This is particularly helpful in late gestation.

Detecting chorionicity can be checked earlier than 10 weeks by number of gestational sacs: a single gestational sac containing two live fetuses suggests MC twins, whereas two distinct gestational sacs, with a fetal pole in each of them are likely DC twins (figures 7-9).

Amnionicity determination (10):

1. Visualization of an inter-twin membrane indicates diamniotic (DA) twins, whereas non-visualization suggests monoamniotic (MA) twins (Figure 10). However, even when the inter-twin membrane is not visualized, the most likely scenario is diamniotic twins since these are more common, and the dividing membrane may be difficult to visualize. Poor maternal imaging characteristics or polyhydramnios/oligohydramnios with a “stuck twin” may lead to mischaracterizing DA twins as MA.
2. The presence of umbilical cord entanglement can be confirmed using both colour and pulsed wave Doppler ultrasound; this generally indicates MA twins. Nevertheless, as detailed later in this article, cord entanglement might occur in diamniotic pregnancies in certain circumstances (Figure 11).
3. The number of yolk sacs detected earlier in the first trimester is not reliable in confirming amnionicity and the presence of one yolk sac does not necessarily mean the twin pregnancy is monoamniotic. Fenton et al found that two yolk sacs were present in 32% of monochorionic-monoamniotic (MCMA) twins in their study, this refutes that a single yolk sac is diagnostic for MCMA twin pregnancy (11).

Although the accuracy of detecting chorio-amnionicity at 10-14 weeks is as high as 95–100% using the above-described methods (4,12,13,14), challenges are not uncommon, especially at either early, late gestation or in twins with complications. Here we describe errors in classification of twins that might occur in each of the above situations and how to avoid them.

The presence of two placental masses does not always indicate DC twins. Two apparently separate placental masses may exist when the placenta is bilobed or bipartite. Moreover, 3% of MC twins pregnancies have two placental masses. The diagnosis of MC twins when placenta seems to have more than one mass can be supported by development of MC specific complications like twin-twin transfusion syndrome TTTS in a pregnancy diagnosed initially as DC (4).

The lambda (λ) sign or T signs may be misdiagnosed by unexperienced clinicians or during trans-abdominal scans. Using transvaginal scans might be helpful in detecting lambda (λ) sign or T signs, especially earlier than 10 weeks (4,15).

Occasionally, MC pregnancies may be misdiagnosed as being DC by a false lambda (λ) sign (Figure 12). Careful examination of the entire length of the placental inter-twin membrane junction and use of colour Doppler will avoid this misdiagnosis. Furthermore, the lambda (λ) sign gradually becomes less prominent or even disappears completely later in gestation. Sepulveda et al. suggested that approximately 7% of DC pregnancies lose the characteristic lambda (λ) sign by 20 weeks (16). In late gestation, when there are no available early pregnancy scan images to determine chorionicity, one should check other scan signs like discordant fetal sexes rather than T and lambda (λ) signs. If chorionicity is not possible to determine, and there is a same-sex twin pair, the pregnancy should be considered a MC pregnancy and managed as such.

Partial MC pregnancy is a very rare condition but has been reported in literature (17). This hybrid placentation is described in rare case reports with cases had both T and lambda signs evident in the first trimester, while other cases developed TTTS in a twin pregnancy previously identified as DC twins. Postnatal placental histopathology only can confirm these conditions with the inter-twin membrane clearly showing transitional zone between a MC placental part, showing vascular anastomosis, and a DC part placental part (4).

Non visualization of inter-twin membrane does not necessarily indicate MA pregnancy. False labelling of twins as MA is not uncommon, this can occur either at early gestation when the amniotic membrane is very thin, or when there are complications like TTTS where the membrane is tightly wrapped on the donor twin (10).

In early gestation, the following imaging tip can be adopted to improve visualization of the inter-twin membrane: Using a high-resolution transducer; staying perpendicular to the membrane; and increasing gain (4,13,15). If in doubt, it is always good practice to repeat the scan or seek a second opinion.

The inter-twin membrane might also rupture spontaneously or iatrogenically after surgical intrauterine interventions like fetoscopic laser treatment for TTTS. This is identified on scan by interruption of the continuity of the inter-twin membrane, accumulation of amniotic fluid on one sac, presence of both twins on one side of the membrane or cord entanglement. If inter-twin membrane rupture is confirmed, a diagnosis of pseudo-monoamniotic twins is made and the management of the pregnancy will change accordingly, hence the importance of meticulous examination during each scan (6,7,10).

The thickness of the inter-twin membrane is not a reliable indicator for detecting chorionicity, especially in late gestation (Figure 13). Few studies suggested use of cut offs of less than 2mm for membrane thickness to identify chorionicity. However, this showed considerably inferior accuracy than standard methods in earlier gestation due to multiple limiting factors.  

Occasionally, intrauterine amniotic bands (Figures 14 A and B), adhesions or synechia (Figures 15 A and B) can be misdiagnosed as inter-twin membrane. This can again be avoided by examining the entire inter-twin membrane placental junction for its entire length (4).

Finally, although discordant fetal sex usually suggests dichorionic twin pregnancy, there are situations where monochorionic pregnancies can have discordant fetal sex. Lu et al (4) summarized the possible causes as follows:

• Sex chromosome aberrations including X/XY and XX/XY mosaicism.
• Dizygotic MC twins as described above in this article.
• Malformations of genitalia of one twin which are more common in MC pregnancies, for example cloacal malformation or hypospadias. 

In multifetal gestation, detecting chorionicity and amnionicity can be achieved by ultrasound in the first trimester and ideally between 10-13+6 weeks of gestation.

If transabdominal ultrasound is inconclusive, high-resolution transducers including transvaginal ultrasound should be used. The ultrasound signs to look for include number of placental masses, T and lambda signs, thickness and number of layers of the inter-twin membrane and identifying fetal sex. Misdiagnosing chorio-amniocity can occur due to various factors and hence, careful assessment of the entire length of the placental inter-twin membrane junction and use of both colour and pulsed wave Doppler ultrasound is recommended. Also, combination of as many of the above ultrasound features as possible is better than using only one (18). If chorionicity is not possible to be confirmed, the pregnancy should be considered monochorionic and managed as such.

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