*3.1.2. Number of amniotic sacs within the chorionic cavity*

Identification of the number of amniotic sacs present in a single gestational sac helps define amnionicity in a monochorionic pregnancy. Prior to the 10th week of gestation, the amnions grow outward from the embryonic disk and at that age are not big enough to contact each other and create the intertwin septum [4]. As a result, separate and distinct amnions indicate a diamniotic twin pregnancy (**Pictures 2a**, **b** and **3a**, **b**). The evaluation of the amnion should be done diligently via transvaginal ultrasound since the intertwin membrane is extremely thin and it may be invisible via transabdominal ultrasound. Even when the separate amnions cannot be visualized via the transvaginal ultrasound, their absence can be confirmed by demonstrating umbilical cord enlargement by using pulsed wave Doppler and identifying two distinct heart rates [3]. In addition, the impossible visualization of the intertwin membrane may be technical: if the membrane is parallel to the ultrasound beam or because the ultrasound gain is low, the membrane may be hard to evaluate. This problem can be solved by changing the angle of insonation and increasing gain facilitates visualization [5]. Another way to confirm amnionicity, wherever there is any doubt about the presence of the intertwin membrane, is to suggest a small chain of repeat scans [4].

However, is evaluation of intertwin membrane always that simple? There are two rare yet important situations that may lead to a false diagnosis of monoamniotic twins. The first case

is when the monochorionic-diamniotic twins are complicated with twin-to-twin transfusion syndrome (TTTS) the donor twin has severe oligohydramnios or anhydramnios, and the intertwin membrane collapses resulting in wrapping the donor twin. The collapse of the membrane can be overtaken if we evaluate extremely carefully the wrapping membrane around the limbs of the donor twin. A possible rupture of the intertwine membrane is another case that may lead to "pseudo-MA" twins. Rupture of the membrane may occur spontaneously, but more often is a complication of invasive *in utero* procedures. Discontinuity of the membrane and cord enlargement can be visualized on the ultrasound scan. Other facts helping

**Picture 2.** (a) 3D imaging of dichorionic diamniotic pregnancy at 6 weeks of gestation. (b) 3D imaging of dichorionic

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diamniotic pregnancy at 6 weeks of gestation.

**Picture 1.** Dichorionic diamniotic pregnancy at 5 weeks of gestation. The two separate gestational sacs with one yolk sac each are visible and a thick septum separates them.

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gestational sacs implies a dichorionic pregnancy (**Picture 1**) [18]. The number of gestational sacs is the parameter with the highest accuracy to define chorionicity which is extremely close

Identification of the number of amniotic sacs present in a single gestational sac helps define amnionicity in a monochorionic pregnancy. Prior to the 10th week of gestation, the amnions grow outward from the embryonic disk and at that age are not big enough to contact each other and create the intertwin septum [4]. As a result, separate and distinct amnions indicate a diamniotic twin pregnancy (**Pictures 2a**, **b** and **3a**, **b**). The evaluation of the amnion should be done diligently via transvaginal ultrasound since the intertwin membrane is extremely thin and it may be invisible via transabdominal ultrasound. Even when the separate amnions cannot be visualized via the transvaginal ultrasound, their absence can be confirmed by demonstrating umbilical cord enlargement by using pulsed wave Doppler and identifying two distinct heart rates [3]. In addition, the impossible visualization of the intertwin membrane may be technical: if the membrane is parallel to the ultrasound beam or because the ultrasound gain is low, the membrane may be hard to evaluate. This problem can be solved by changing the angle of insonation and increasing gain facilitates visualization [5]. Another way to confirm amnionicity, wherever there is any doubt about the presence of the intertwin

However, is evaluation of intertwin membrane always that simple? There are two rare yet important situations that may lead to a false diagnosis of monoamniotic twins. The first case

**Picture 1.** Dichorionic diamniotic pregnancy at 5 weeks of gestation. The two separate gestational sacs with one yolk sac

to 100% [16].

66 Multiple Pregnancy - New Challenges

*3.1.2. Number of amniotic sacs within the chorionic cavity*

membrane, is to suggest a small chain of repeat scans [4].

each are visible and a thick septum separates them.

**Picture 2.** (a) 3D imaging of dichorionic diamniotic pregnancy at 6 weeks of gestation. (b) 3D imaging of dichorionic diamniotic pregnancy at 6 weeks of gestation.

is when the monochorionic-diamniotic twins are complicated with twin-to-twin transfusion syndrome (TTTS) the donor twin has severe oligohydramnios or anhydramnios, and the intertwin membrane collapses resulting in wrapping the donor twin. The collapse of the membrane can be overtaken if we evaluate extremely carefully the wrapping membrane around the limbs of the donor twin. A possible rupture of the intertwine membrane is another case that may lead to "pseudo-MA" twins. Rupture of the membrane may occur spontaneously, but more often is a complication of invasive *in utero* procedures. Discontinuity of the membrane and cord enlargement can be visualized on the ultrasound scan. Other facts helping

*3.1.3. Number of yolk sacs*

**3.2. 10th–14th week of gestation**

*3.2.1. Sex discordance*

characteristics and (4) chorionic peak sign—'λ' sign.

twins, leading to a false diagnosis of dichorionic twins.

cells having the abnormal karyotype (**Figure 2**) [19, 20].

SOX9 which inhibits the expression of SRY gene [23, 24].

Over the past few years, there is an uncertainty regarding the relation between the number of yolk sacs and amnionicity. If there are two yolk sacs present in the extraembryonic coelom, the pregnancy will be regarded as diamniotic. However, a single yolk sac cannot set the definitive diagnosis of a monoamniotic pregnancy. This is well-established since it is known that the differentiation of a yolk sac and an amnion occur very close to each other in time, around 6–8 days after fertilization [5]. If a single yolk sac is detected, a repeat first trimester scan is undertaken, or a refer to a tertiary center with advanced experience in multiple pregnancies can be helpful [3, 4].

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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

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

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

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

**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.

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

#### *3.1.3. Number of yolk sacs*

Over the past few years, there is an uncertainty regarding the relation between the number of yolk sacs and amnionicity. If there are two yolk sacs present in the extraembryonic coelom, the pregnancy will be regarded as diamniotic. However, a single yolk sac cannot set the definitive diagnosis of a monoamniotic pregnancy. This is well-established since it is known that the differentiation of a yolk sac and an amnion occur very close to each other in time, around 6–8 days after fertilization [5]. If a single yolk sac is detected, a repeat first trimester scan is undertaken, or a refer to a tertiary center with advanced experience in multiple pregnancies can be helpful [3, 4].
