**3.2. 10th–14th week of gestation**

As the pregnancy continues, the ultrasound signs that help in the determination of chorionicity and amnionicity are changing: gestational sacs are now fused and the intertwin membrane is formed. As a result, four other ultrasound figures set the diagnosis of chorionicity and amnionicity. These are: (1) sex discordance, (2) distinct placentas number, (3) intertwin membrane characteristics and (4) chorionic peak sign—'λ' sign.

#### *3.2.1. Sex discordance*

in the identification of the membrane rupture are the location of the fetuses in the same side of the warped membrane, the equal quantity of amniotic fluid in both sides of the dividing membrane in a pregnancy, which was complicated with TTTS, and of course a previous diag-

**Picture 3.** (a) Dichorionic diamniotic pregnancy with one of the pregnancies having miscarried. The size of the empty sac has been measured. (b) 3D imaging of DCDA pregnancy in which one of the sacs appears "empty" due to miscarriage.

nosis of a monochorionic-diamniotic twin pregnancy [5].

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If a male and a female fetus are identified in the late first or early second trimester, a dichorionic twin pregnancy is the rule. However, gender discordance is the biggest pitfall for the diagnosis of chorionicity. Discordant fetal sex phenotype can be present in monochorionic twins, leading to a false diagnosis of dichorionic twins.

A false diagnosis of dichorionic twins might be the result of a postzygotic sex chromosome aneuploidy. For instance, there is a 46,XY zygote which splits, but a postzygotic anaphase lag can cause the loss of the Y chromosome in one of the twins. The karyotype of one of the fetuses will be 46,XY which corresponds to a normal male fetus, while the other karyotype will be 45,XO which is a female fetus with Turner syndrome (**Figure 1**)*.* If we want to take our example a step forward, postzygotic nondisjunction after the anaphase lag can lead to mosaicism in the monozygotic twins leading to two embryos with a variety of proportion of 45,XO and 46,XY cells. The phenotype of this individual will correspond to the amount of cells having the abnormal karyotype (**Figure 2**) [19, 20].

A sex discordance in monozygotic twins can also be caused by a trisomic 47,XXY zygote. A process known as trisomy rescue can lead to either the production of a normal 46,XY male fetus (loss of X chromosome) or a normal 46,XX female fetus (loss of Y chromosome) Hence, this mechanism causes the production of two euploids fetuses from a trisomic zygote (**Figure 3**) [21]. In addition, confusion might be caused if a 46,XY zygote splits with nondisjunction of the Y chromosome, producing a male fetus with a 47,XYY karyotype and a female fetus with a 45,XO karyotype, Turner syndrome, and female sex phenotype (**Figure 4**) [22].

Beyond sex chromosome abnormalities, sex discordance may be the result of epigenetic single gene defects in only one of the monoygotic twins, effecting testis-determining genes such as SOX9 which inhibits the expression of SRY gene [23, 24].

**Figure 1.** Postzygotic anaphase lag causing sex discordance due to loss of Y chromosome in one of the fetuses.

IUGR [26, 27]. As hypospadias might lead to female sex phenotype, confusion about chorionicity is expected, as the IUCR male fetus will present with female external genitalia, while the normally developing twin will be present as a normal male fetus. Cloacal malformation in one of the female fetuses (karyotype 46,XX) leads to phallus-like structure, causing phenotypically

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A very rare mechanism can cause the transverse situation: a dizygotic twin pregnancy is been diagnosed as monochorionic because of the fusion of the trophoblasts. Two distinct blastocysts produce two distinctive trophoblasts. If these trophoblasts fuse before the implantation, the result is the creation of a placental mass. The fused placenta will form vascular anastomoses, and the twins can exchange blood cells. As a result, blood chimerism of two populations of blood cells will be present in both fetuses [29, 30]. This mechanism is present more frequently in pregnancies carried out from ART because of the disruption of the zona pellucida and spatial proximity of multiple embryos [29, 31]. Dizygotic twins forming a monochorionic placenta have significant importance because these twins are genetically and phenotypically normal and they have to be distinguished from the pathological

male external genitalia. The outcome is again confusion of chorionicity [28].

sex discordance [5].

**Figure 3.** Trisomy rescue.

**Figure 2.** Postzygotic nondisjunction leading to both fetuses with gonadal mosaicism.

Nonetheless, sex discordance may be caused by malformed genitalia unrelated to chromosomal or genetic disorders. It is well established that a monochorionic twin pregnancy is complicated frequently with selective growth restriction [25], and hypospadias is a known complication of Early Pregnancy Ultrasound Assessment of Multiple Pregnancy http://dx.doi.org/10.5772/intechopen.81498 71

**Figure 3.** Trisomy rescue.

Nonetheless, sex discordance may be caused by malformed genitalia unrelated to chromosomal or genetic disorders. It is well established that a monochorionic twin pregnancy is complicated frequently with selective growth restriction [25], and hypospadias is a known complication of

**Figure 1.** Postzygotic anaphase lag causing sex discordance due to loss of Y chromosome in one of the fetuses.

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**Figure 2.** Postzygotic nondisjunction leading to both fetuses with gonadal mosaicism.

IUGR [26, 27]. As hypospadias might lead to female sex phenotype, confusion about chorionicity is expected, as the IUCR male fetus will present with female external genitalia, while the normally developing twin will be present as a normal male fetus. Cloacal malformation in one of the female fetuses (karyotype 46,XX) leads to phallus-like structure, causing phenotypically male external genitalia. The outcome is again confusion of chorionicity [28].

A very rare mechanism can cause the transverse situation: a dizygotic twin pregnancy is been diagnosed as monochorionic because of the fusion of the trophoblasts. Two distinct blastocysts produce two distinctive trophoblasts. If these trophoblasts fuse before the implantation, the result is the creation of a placental mass. The fused placenta will form vascular anastomoses, and the twins can exchange blood cells. As a result, blood chimerism of two populations of blood cells will be present in both fetuses [29, 30]. This mechanism is present more frequently in pregnancies carried out from ART because of the disruption of the zona pellucida and spatial proximity of multiple embryos [29, 31]. Dizygotic twins forming a monochorionic placenta have significant importance because these twins are genetically and phenotypically normal and they have to be distinguished from the pathological sex discordance [5].

*3.2.3. Intertwin membrane characteristics*

*3.2.4. The chorionic peak sign—the "λ" sign*

possibility of monochorionic pregnancy is about 90% [4].

two cord insertions in the placenta [4], and the short intercord distance [5].

The intertwin membrane of a dichorionic pregnancy comprises three layers of three membranes: amnion-chorion-amnion, as the monochorionic pregnancy consists only two layers of amnion. Therefore, intertwin membrane in a dichorionic pregnancy is thicker and more echogenic than the intertwin membrane in monochorionic pregnancies. Measuring the thickness of the membrane can help us define chorionicity: a membrane thicker than 2 mm indicates dichorionicity (positive predictive value: 95%), and if the membrane is thinner than 2 mm, the

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The intertwin membrane has to be carefully detected and if it cannot be visualized, a transvaginal ultrasound scan has to be performed, to set the definitive diagnosis of monoamniotic pregnancy [4]. When a single placental mass is visualized and chorionicity is identified as monochorionic, evaluation of the intertwin membrane characteristics is the key to determine amnionicity. The most significant sonographic figure that demonstrates monoamniocity is the demonstration of cord enlargement from the placental or umbilical origin and it is identified easier via color Doppler. Other important findings intimating monoamnionicity are the entanglement of limbs or observation of a limb circumscribing the other, the failure to find the membrane between the

However, intertwin membrane thickness difference between monochorionic and dichorionic pregnancy decreases during gestation [36]. In addition, the measurement of the thickness of the membrane is not widely accepted since this parameter can be affected by many factors such as the position and the quality of the probe, and as a result, it has poor reproducibility [37]. A rare but significant pitfall may lead to a wrong determination of a monochorionic pregnancy as dichorionic is the intrauterine synechiae in twin pregnancy with a fetus with anencephaly. Intrauterine synechiae can mimic the thick dichorionic membrane [38]. This septum is not the intertwin membrane and does not include the layer of chorion between the layers of amnion.

The chorionic peak sign or the "λ" sign supports strongly dichorionicity, with an accuracy of 99% [5]. It shows a projecting zone of tissue which is as echogenic as the placenta; it has a triangular shape in cross-section; and it is wider at the chorionic surface of the placenta, extending into, and tapering to a point within, the intertwin membrane [39, 40]. The absence of the "λ" sign or the presence of "T" sign indicates monochorionicity. The "T" sign represents

The chorionic peak sign is ideally evaluated during the late first trimester or the very early second trimester, as in second trimester, it is more difficult to be visualized and it might be disappeared at 16–20 weeks of gestation, leading to a false negative "λ" sign. As a result, the impossible depiction of the "λ" sign in late second trimester cannot exclude dichorionicity [41, 42]. Nonetheless, a false positive "λ" sign might also exist. This can be due to umbilical cord insertion into the intertwin membrane or because of the visualization of a hematoma presented along the insertion of the membrane. Another interesting reason that may lead to

the two opposing amnions "standing" at the base of the intertwin membrane [10].

**Figure 4.** Nondisjunction of chromosome Y.

#### *3.2.2. Number of distinct placentas*

It is logical that the visualization of two separate placental masses confirms dichorionicity as a single placenta identifies monochorionicity [4]. Careful ultrasound evaluation has to be done in order to define the presence of a single placenta or two placentas in abutment.

As the pattern above, monochorionic twins may form a bipartite placenta. This sonographic finding is visible in 3% of monochorionic twin pregnancies. As a result, two separated placental masses are present with two nearly equal-sized placental lobes, which can be totally separated or connected by chorion laeve. Things can be more complicated when each placental mass has its own umbilical cord connection. Bipartite placenta can be distinguished from the dichorionic placental masses by using color Doppler and identifying vascular anastomoses that are present between the two lobes. Thus, this leads to the conclusion that if an ostensibly dichorionic pregnancy is complicated with TTTS, the diagnosis of a monochorionic pregnancy with bipartite placenta has to be considered [32–35].
