Rare spontaneous monochorionic dizygotic twins: a case report and a systematic review

Monochorionic dizygotic (MCDZ) twins are a rare condition, mostly related to assisted reproductive technology (ART) [1]. Here we present a case of spontaneous monochorionic dizygotic twins with isolated levocardia and a normal cardiac structure, situs inversus abdominalis, discordant for abdominal anomalies, with only one twin affected by biliary atresia with splenic malformation syndrome (BASM).

We reviewed all cases of spontaneous monochorionic dizygotic twins reported in literature, highlighting the clinical features, the obstetrical implications and the challenges related to this unusual and still not fully known twinning event.

A 27-year-old woman, gravida 2, para 0, with no history of previous disease and no family history of congenital anomalies, smoker and with a BMI of 18.3 received a diagnosis of spontaneous twin pregnancy at 12 weeks. The assessment of chorionicity in this gestational age was however hampered by the presence of an hematoma measuring 44 × 38 mm separating the two membranes, thus preventing the identification of the “lambda” or “T” sign.

The anatomy scan at 20 weeks revealed two female fetuses, both presenting isolated levocardia (IL) with normal heart and situs inversus abdominalis with a left-sided liver and right-sided stomach and spleen (Fig. 1). Noteworthy, in one fetus (A) the gallbladder was not visible.

An amniocentesis was performed, with conventional cytogenetic evaluation indicating normal karyotype 46XX for both fetuses. No genetic anomaly was reported with the Chromosomal Microarray Analysis (CMA). Zygosity was assessed by microsatellite analysis; as shown in Table 1, twins share only a fraction of paternal and maternal alleles, indicating dizygosity.

At 36 weeks one fetus was diagnosed to be growth restricted, having an abdominal circumference and an estimated fetal weight less than 3rd centile. At 37 weeks and 1 day, the pulsatility index (PI) of the umbilical artery of the growth restricted fetus, with an estimated fetal weight of 2171 g (below the 3rd centile, according to Hadlock growth chart), was 0.89 (corresponding to 46° centile), while the PI of the middle cerebral artery was 1.45 (26° centile). By contrast, the other twin had an estimated fetal weight of 2521 g with normal Doppler parameters. At 37 weeks and 3 days the mother underwent a scheduled cesarean delivery. The birthweight of the two female neonates were 2430 g and 2185 g.

The histological analysis of the placenta confirmed the monochorionicity of the twin pregnancy, revealing the juxtaposition of an amnion on each surface of the dividing membrane (Fig. 2: Hematoxillin-Eosin image obtained using ECLPISE Ni-U equipment, with 10 × magnification, acquired through DS-Fi3 Nikon Digital Camera). The chorion was not visible between the two amnion surfaces, thus excluding the rare occurrence of partially fused placentas [2, 3]. Postnatally, by using DNA extracted from blood samples, the molecular analysis confirmed the dizygosity of the twins, as already previously determined through amniocentesis.

Moreover, the two babies turned out to have the same situs anomaly, with IL and situs inversus, thus confirming the prenatal diagnosis. In order to identify possible genetic causes of such a concordant abnormal phenotype, the twins were subjected to clinical exome analysis with evaluation of 17 genes known to be associated to situs inversus. However, according to the variant classification ACMG (The American College of Medical Genetics and Genomics), no pathogenic or probably pathogenetic variants have been identified. By opening the analysis to the whole clinical exome (4490 genes), the twins did not share pathogenic or probably pathogenetic variants. In addition, the twin whose gallbladder was not visualized prenatally, developed jaundice with acholic stool in her neonatal period.

The complete abdominal scan performed at 20 days of extrauterine life showed a left sided, damage-free liver with regular size (lateral diameter of 6,3 cm), a reversed relation between superior mesenteric vein and artery and right-placed inferior vena cava in relation to the aorta. These findings perfectly fit with abdominalis situs inversus with isolated levocardia. The scan revealed for the first time a shriveled gallbladder, which led to the diagnosis of biliary atresia type IV associated with splenic malformations syndrome (Fig. 3). The clinical exome analysis was unable to identify the genetic cause of these abdominal abnormalities.

The twin with BASM at 38 days of extrauterine life underwent Kasai portoenterostomy, second-hand appendectomy and Ladd bridle dissection. Later, during the fifth month of extrauterine life, the baby was diagnosed to have a subclinical acute cholangitis, which was treated with continuous infusion of piperacillin tazobactam during hospitalization. Unfortunately, the baby had recurrent cholangitis during the whole first year of extrauterine life, and these are still occurring.

A systematic review was conducted using Pubmed, Scopus, OVID, and Cochrane Library electronic databases. The citations were identified with the use of a combination of the following key words: “monochorionic dizygotic twins”; “monochorionic dizygotic chimerism”; “monochorionic dizygotic freemartinism”; “spontaneous monochorionic dizygotic twins”; “spontaneous monochorionic heterosexual twins” from the inception of each database through January 2022. Only articles in English language were selected, while no restrictions for geographic location were applied (Supplementary material).

Overall 14 cases of spontaneously conceived and 4 cases of monochorionic dizygotic twins conceived after ovulation induction were identified. All available articles were case reports. A description of each case is provided in two tables: Table 2 describes case reports on spontaneously conceived monochorionic dizygotic twins [4‐17], while Table 3 describes case reports on monochorionic dizygotic twins conceived after ovulation induction [18‐21]. The mean maternal age at delivery in the group of spontaneously conceived monochorionic dizygotic twins was 31.08 weeks (SD ± 4.69). The sonographic assessment of chorionicity was established in the first trimester in 11 cases [5, 6, 8‐16]. Follow-up scans revealed the following complications during pregnancy: 2 cases of Twin-to-Twin Transfusion syndrome (TTTS) [5, 14],  1 case of Twin Reversed Arterial Perfusion sequence (TRAP) [7], 1 case of Twin Anemia Polycythemia sequence (TAPS) [13] and 1 case of discordant growth pattern [10]. Sex discordance between twins in monochorionic pregnancy was reported in 4 cases [12, 13, 15, 16]. Amniocentesis was performed to validate dizygosity in 2 cases [12, 15], while in 1 case it was done to exclude trisomy 21 [6]. Out of 14 cases reported, 1 case underwent miscarriage after laser procedure performed at 18 weeks gestation for TTTS [5], 1 case underwent a voluntary termination of pregnancy because of the diagnosis of trisomy 21 in one twin [6], 6 cases had a cesarean delivery which occurred at a mean gestational age of 35.83 weeks (SD ± 2.31) [9, 11, 12, 14‐16], 3 cases had a spontaneous vaginal delivery at a mean gestational age of 35 weeks (SD ± 2.64) [7, 8, 10] and 3 cases did not report the delivery mode [4, 13, 17]. A monochorionic pregnancy was confirmed at gross examination in all cases. Dizygosity was demonstrated by the phenotypical evidence of sex discordance between twins in 2 cases [4, 13] and it was proven by cytogenetic analysis showing sex discordant karyotypes between twins in 3 cases [5, 7, 8]. Chimerism was found to be confined to blood in 6 cases [6, 8, 10, 14, 16, 17], to be present in a non-shared tissue in 3 cases [9, 12, 15] and it was detected in blood as well as in tissue in 1 case [11]. The mean maternal age at delivery in the group of monochorionic dizygotic twins conceived after ovulation induction was 31.00 weeks (SD ± 3.36) [18‐21]. The sonographic assessment of chorionicity was determined in the first trimester in all cases. No typical monochorionic complications were detected during the follow up scans. However, TAPS was found in one case at delivery [21]. Sex discordance between twins in monochorionic pregnancy was reported in 3 cases [18, 20, 21]. Invasive prenatal diagnosis aiming at validating dizygosity was performed in only one case [18]. Preterm delivery occurred in all cases; twins were delivered through cesarean section in 2 cases [19, 20]. A monochorionic pregnancy was confirmed at gross examination in all cases. Chimerism was found to be confined to blood in 2 cases [19, 20], to be present in a non-shared tissue in 1 case [18], and it was detected in blood as well as in tissue in 1 case [21]. In this review, we present a case of spontaneous monochorionic dizygotic twins with isolated levocardia and a normal cardiac structure, situs inversus abdominalis, discordant for abdominal anomalies, with only one twin affected by BASM. Moreover, we reviewed 14 cases of spontaneously conceived MCDZ twins [4‐17]. Monochorionicity has been traditionally considered to be a guarantee of monozygosity. A growing body of evidence has now demonstrated that monochorionic twins could be dizygotic. A very rare phenomenon of “sesquizygosis” has been described in the literature [12]. In this case, the twins share the same haploid genome from one parent and, therefore, are intermediate between mono and dizygotic twinning. The microsatellite analysis of our twins excluded sesquizygosis (Table 1). A previous systematic review on this issue showed that assisted reproductive technology is the major responsible for the origin of this unusual way of twinning [1]. Several hypotheses have been advanced to explain a monochorionc dizygotic twin pregnancy: the fusion of the trophoblasts from two different embryos before implantation [22], the presence of binovular follicles where a single zona pellucida includes two distinct oocytes leading to close contact between embryos [19] and the penetration of an oocyte and second polar body surrounded by one zona pellucida by more than one sperm [23]. The chance of cell fusion seems to be small in a natural pregnancy, but not impossible to occur as proven by the number of cases found in our systematic research. Based on this review, this type of twinning is burdened by the same risk of pregnancy complications found in MCMZ pregnancies, i.e. TTTS, TRAP, TAPS and selective Fetal Growth Restriction (sFGR). These findings suggest the importance of a correct diagnosis of chorionicity in the first trimester of pregnancy, in order to timely and adequately manage possible complications. Chorionicity should be determined before 14 weeks of gestation, examining the dividing membrane carefully. In dichorionic diamniotic (DCDA) twin pregnancy, the twins are separated by a thick layer of fused chorionic membranes with two thin amniotic layers, one on each side (the so-called “full lambda” sign), while in monochorionic diamniotic (MCDA) pregnancy only two thin amniotic layers separate the two fetuses (the T-sign). In this case report the ultrasonographic examination of these signs was prevented by the presence of a large hematoma at the site of insertion of the amniotic membrane into the placenta. Since 3% of monochorionic pregnancies have two placental masses (also defined “bipartite placenta” [24]) on ultrasound and dichorionic placentae are commonly appearing as a single mass, the reliability of the number of placental masses at ultrasound is questionable. Therefore, we did not consider this feature for the diagnosis of chorionicity. Interestingly, bipartite placenta seems to have some relevant clinical implications. A recent study found bipartite placenta in five MCDA pregnancies and showed that this occurrence was associated with a higher rate of complications, such as TTTS and sFGR and might impair prenatal surgical interventions [24]. The diagnosis of monochorionicity is even more important in the context described in our case report, since sex discordance of twins could lead to the automatic assumption of dichorionicity. Chimerism is characterized by cells originating from more than one genetically distinct zygote. Chimerism was found in most reviewed cases. Blood confined chimerism is likely to be consequent to the blood sharing between the dizygotic twins via the unique placenta. It has been theorized that the “outer cell mass” of the two distinct dizygotic embryos undergo fusion with the development of a single chorion and anastomoses. However, in 21.42% of cases tissue chimerism was found, which is more difficult to explain [9, 11, 15]. It is unknown whether chimerism has clinical consequences. Bogdanova et al. reported a possible case of human freemartinism in a female twin with aplasia of the uterus [25]. According to the Author the lack of Mullerian structures in this female was caused by her exposition to the effect of the Mullerian inhibiting substance transferred from the male twin via the common placenta in early pregnancy. Recently, Peters et al. investigated whether there is a prevalence of male microchimerism in women with Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome [26]. However, their observational case–control study, involving 95 women with MRKH syndrome and 99 control women, showed that the prevalence of male microchimerism was significantly higher in the control group than in the MRKH group, thus rejecting the initial hypothesis. We compared our case report with the cases of spontaneously conceived MCDZ twins present in our systematic review. Molecular analysis performed on tissue and blood samples of the two female twins confirmed dizygosity. What is peculiar of our case report, is the very rare anomaly shared by both twins, though in a different way. Both have IL and situs inversus abdominalis, but only one twin is affected by biliary atresia with splenic malformation syndrome. No genetic cause of these abnormalities was identified by clinical exome analysis; but this should not rule out genetic determinants of the phenotypic abnormalities. There are indeed several distinct methodological features able to explain the non-identification of genetic causes. Among them, the fact that the clinical exome evaluates only genes known to be associated to human diseases or the possibility that the putative causative variant might be located in control regions of gene expression (promoter or enhancers, for example), not analyzed by the exome approach. Biliary atresia is recognized as a key feature in two distinct types of syndromes: the Cat-eye syndrome (CES) and the Biliary Atresia Splenic Malformation (BASM) syndrome. The first one is determined by aneuploidy of chromosome 22 and patients affected typically have coloboma, cardiac anomalies and anorectal malformations. On the other hand, BASM is characterized by a constellation of visceral anomalies in different combinations. Patients with BASM could have polysplenia, asplenia or double spleen, situs inversus with and without malrotation; preduodenal portal vein, a complete absence of intrahepatic vena cava and cardiac anomalies. According to some researchers [27, 28] the association between BA and laterality defects of the abdominal viscera may suggest a defect in the embryonic development to explain the etiology of BASM. The bile duct development begins at 4 gestational weeks and ends at about 13 weeks; in the same period laterality defects, such as development of left–right axis reversal (GA 2–3 weeks), splenic malformations (GA 3–6 weeks), preduodenal portal vein (GA 4–8 weeks), and interrupted vena cava (GA 6–8 weeks) are thought to occur [27]. A recent metanalysis, aiming at analyzing the characteristics of biliary atresia in twins, found that 97% of twins were discordant for the anomaly. In more than half of the cases twins were monozygotic, thus indicating that zygosity is not the main causative factor of the onset of the disease [29]. However, even assuming the role of epigenetic factors in the pathogenesis of BA, our case report still remains a fascinating enigma. It is hard to find an explanation of why two monochorionic dizygotic sex concordant twins should share a very rare laterality anomaly of the abdominal viscera, sparing one twin from developing biliary atresia. The main strength of this study is the singularity and originality of our case report, where two MCDZ twins share the same malformation in a slight different way, with important clinical consequences on one twin. Moreover, we did a comprehensive systematic review on MCDZ pregnancies naturally conceived, which is the first in the literature to the best of our knowledge. It is interesting to note that when spontaneously conceived MCDZ pregnancies were affected by a malformation, this was present in one twin only, according to our review. Our case report is therefore unique. However, some limitations should be recognized too. First of all, we did not perform the whole exome sequencing, thus preventing the possibility to identify some variants located in control region of gene expression. In addition we did not investigate the occurrence of chimerism, either in blood and in other different tissues.

In conclusion, spontaneously conceived MC-DZ twins are a rare condition, with only 14 cases described in literature. The evidence so far suggests the importance to timely diagnose the chorionicity, in order to adequately manage the typical complications associated with monochorionicity. Furthermore, the clinician should keep in mind that monochorionicity does not always correspond to monozygosity. Rarely, MC twins could be DZ, even in naturally conceived pregnancies, as shown in this comprehensive review. It is still unclear how this unusual type of twinning can occur in spontaneous conception. The mystery deepens considering the peculiarity of our case report, where the two MCDZ twins share a very rare anomaly, for which no genetic cause has been found through clinical exome analysis.