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OBSTETRICS A review of the mechanisms and evidence for typical and atypical twinning Helen C. McNamara, MB, BS; Stefan C. Kane, MB, BS; Jeffrey M. Craig, PhD; Roger V. Short, ScD; Mark P. Umstad, MD

mirror-image , polar body twins, The mechanisms responsible for twinning and disorders of gestations have been the complete hydatidiform mole with coex- subject of considerable interest by physicians and scientists, and cases of atypical istent twin, vanishing twins, fetus papy- twinning have called for a reexamination of the fundamental theories invoked to explain raceus, , superfetation, and twin gestations. This article presents a review of the literature focusing on twinning and superfecundation. A detailed discussion atypical twinning with an emphasis on the phenomena of chimeric twins, phenotypically of , conjoined discordant monozygotic twins, mirror-image twins, polar body twins, complete hydati- twins, and twin-reversed arterial perfu- diform mole with a coexistent twin, vanishing twins, fetus papyraceus, fetus in fetu, sion sequence is beyond the scope of superfetation, and superfecundation. The traditional models attributing monozygotic this review and has been presented twinning to a fission event, and more recent models describing monozygotic twinning as elsewhere.1,2 a fusion event, are critically reviewed. Ethical restrictions on scientific experimentation with human and the rarity of cases of atypical twinning have limited opportu- Traditional models of twinning nities to elucidate the exact mechanisms by which these phenomena occur. Refinements Traditionally it has been thought that in the modeling of early embryonic development in twin pregnancies may have significant dizygotic twins result from fertilization clinical implications. The article includes a series of figures to illustrate the phenomena of 2 distinct ova by 2 separate sperma- described. tozoa, whereas monozygotic twins are the product of a single ovum and Key words: assisted reproductive technologies, chimeric twins, complete hydatidiform that subsequently divide to form 2 mole with coexistent twin, dizygotic, fetus in fetu, fetus papyraceus, mirror-image twins, embryos.1 monozygotic, twins, phenotypically discordant monozygotic twins, polar body twins, Widely accepted models of mono- superfecundation, superfetation, zygotic twinning are based on the un- proven hypothesis of postzygotic division of the conceptus (Figure 1). In this idespread use of assisted repro- Traditional models of twinning pertain- model, the number of fetuses, , W ductive technologies (ART) in- ing to a fission event have been con- and amnions are determined by the volving the manipulation of the natural tested. New models involving fusion of timing of the splitting (Table 1). mechanisms of fertilization and im- fetal membranes have been proposed. Proposed triggers for splitting include plantation has been accompanied by an This article presents a review of the postzygotic gene , abnormal- increasing number of reports of atypical literature regarding the typical and ities in surface proteins, and abnor- twinning. Cases of atypical twinning atypical twinning, highlighting the phe- malities in the formation of the zona have inspired a reexamination of the nomena of chimeric twins, phenotypi- pellucida.3 The incidence of mono- fundamental theories of twinning. cally discordant monozygotic twins, zygotic twins is increased 2- to 5-fold in ART pregnancies,3 which might be pre- disposed to splitting because of From the Department of Maternal-Fetal Medicine (Dr McNamara, Dr Kane and Dr Umstad), Royal Womens Hospital, Department of Obstetrics and Gynaecology (Dr Kane and Dr Umstad), and handling, media, and microinjection or Faculty of Medicine, Dentistry and Health Sciences (Dr Short), University of , and because of the intrinsic abnormalities Murdoch Childrens Research Institute (Dr Craig), Royal Children’s Hospital, Melbourne, Victoria, associated with infertility.4-6 . It has conventionally been asserted Received March 2, 2015; revised Oct. 28, 2015; accepted Oct. 29, 2015. that monochorionicity confirms mono- J.M.C. is supported by grants from the Australian National Health and Medical Research Council , opposite-sex twins confirm (grants 1011070 and 1083779), the Financial Markets Foundation for Children (grant 032-2007), and dizygosity, and same-sex dichorionic ’ ’ the Murdoch Children s Research Institute, which is funded by the Victorian Government s twins remain of uncertain zygosity until Operational Infrastructure Support Program. S.C.K. is supported by a postgraduate scholarship from 7 the Australian National Health and Medical Research Council. postnatal evaluation occurs. The authors report no conflict of interest. New models of twinning Corresponding author: Mark P. Umstad, MD. [email protected] In 2013 Herranz8 argued that the 0002-9378/$36.00 ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2015.10.930 hitherto-unchallenged hypothesis of

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FIGURE 1 The traditional model of twinning

Dizygotic twins are the product of 2 distinct fertilization events, resulting in dichorionic diamniotic twins with each conceptus developing to become a genetically distinct individual. Monozygotic twins result from postzygotic splitting of the product of a single fertilization event. Splitting on days 1e3 (up to the morula stage) results in dichorionic diamniotic twins, on days 3e8 (during which hatching occurs) in monochorionic diamniotic twins, on days 8e13 in monochorionic monoamniotic twins, and if no split has occurred by day 13, in (not shown). In this diagram, 2 of the 3 oocyte-derived polar bodies are shown at the stage. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. postzygotic splitting lacked scientific Herranz8 offered an alternative theory Denker9 opposed Herranz’s argu- proof. He argued that factors that initiate of twinning based on the following 2 ment, emphasizing that a lack of cleavage have not been specified, that principles: (1) monozygotic twinning oc- evidence may stem from ethical limita- coexistenceofseparateembryoswithina curs at the first cleavage division of the tions on scientific experimentation with single zona pellucida is unlikely, that zygote and (2) subsequent chorionicity human embryos. He highlighted that postzygotic splitting becomes more un- and amnionicity is determined by the data regarding twinning mechanisms in likely with the passage of time, and that degree offusion of embryonicmembranes animals, differences in the nature of the splitting has never been observed in vitro. within the zona pellucida (Figure 2). zona pellucida in vivo and in vitro, and

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may lead to erro- TABLE 1 neous conclusions.36 Chorionicity and amnionicity by time of zygote splitting Zygosity Twins Time of split Chorions Amnions Fetal mass Phenotypically discordant Dizygotic DC DA No split 2 2 2 monozygotic twins Phenotypic discordance in monozygotic Monozygotic DC DA Days 1e32 2 2 twins commonly occurs as a conse- Monozygotic MC DA Days 3e81 2 2 quence of epigenetic, mitochondrial, Monozygotic MC MA Days 8e13 1 1 2 and genetic discordance.1,37,38 Epige- Monozygotic Conjoined After day 13 1 1 1 netics has been implicated as a mediator of stochastic and twin-specific environ- DA, diamniotic; DC, dichorionic; MA, monoamniotic; MC, monochorionic. mental factors.39-41 Genetic differences McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. within monozygotic pairs must arise de novo soon after zygotic cleavage if they are found in multiple somatic the developmental potential of cell line- twins with confined hematological and/ tissues and later in development if they ages after fertilization were not dis- or tissue chimerism have been reported are .42 Such differences can be cussed. Denker9 concluded that both the (Appendix 1).7,12,16-29 single base pair mutations42,43 or copy traditional fission model and Herranz’s Concerns have been raised that number variation44,45 or involve whole fusion model were unsubstantiated. chimeric twins might exhibit reproduc- .46 Further examination of twinning tive dysfunction analogous to that of the On a -scale, the frequency of processes in typical and atypical twins bovine .30 Early follow-up epigenetic differences within mono- might confirm which, if either, of these studies described normal genitalia, go- zygotic pairs is likely to be high.39,40 Less models is more accurate. nads and endocrinological function in is known about the frequency of genetic gender-discordant chimeric twins to a discordance in monozygotic twins, Atypical twinning maximum of 18 months of age. How- although it is likely to be low.47,48 A review of the evidence for atypical ever, in 2013 Choi et al26 reported a case However, more genetic variation might twinning provides insights into the of MCDZ twins complicated by death in occur outside coding regions.49 Little is mechanisms of twinning and challenges utero of the female twin and severe known about the frequency of mito- aspects of traditional models of gonadal failure in the male cotwin. The chondrial discordance in twins.50 twinning. authors concluded that close observa- tion of chimeric infants is necessary to Mirror-image twins Chimeric twins ensure that gonadal failure/dysfunction Mirror-image twinning has been A is a single contain- is identified and appropriately managed. described in monozygotic twins with ing 2 populations of genetically distinct The mechanisms underlying human phenotypic features that are asymmet- cells originating from 2 different twin chimerism and monochorionic rical. As many as 25% of monozygotic .1 Chimerism in humans was dizygotic twin pregnancies remain twins may have mirror-image features.51 initially observed in studies of the ABO incompletely defined.18 Theories pro- Mirror asymmetries observed include blood group.10 Blood group chimerism posed are outlined in Table 2 and direction of occipital hair whorl, dental was demonstrated via in depicted in Figure 3.7,14,17,19,25,27,31-35 patterns, unilateral eye and ear defects, 197611 and is now considered com- Nevertheless, it is clear that the dogma cleft lip and palate, bony abnormalities, mon10,12 and persistent.13 of monochorionicity being synonymous and tumor patterns (Appendix 2).51-62 Chimerism has since been described with monozygosity is no longer Mirror-image central nervous system in twins with monochorionic dizygotic appropriate. abnormalities, including optic glioma, (MCDZ) placentation. Early reports of Assumptions regarding the antenatal colpocephaly, and arachnoid cysts,63-66 MCDZ twins were discounted because diagnosis of zygosity on the basis of and cases of mirror-image later- of the absence of formal placental his- sonographic features24 may be unreliable, ality in heterotaxy syndromes67,68 have topathology14 or confirmatory genetic with important implications for antenatal been described. testing.15 risk stratification,16 screening, and diag- It has been suggested that higher- In 2003 Souter et al15 reported the nosis. Failure to diagnose MCDZ twins order cerebral functions including first confirmed case of sex-discordant might have long-term consequences.26 dominant handedness,69 eye domi- MCDZ twins born to a 48 year old Individuals with blood or tissue chime- nance,52 and cerebral lateralization for woman following in vitro fertilization rism might be at increased risk in the language and mental rotation tasks70 (IVF). Cytogenetic analyses demon- context of transfusion or trans- also exhibit mirror asymmetries. How- strated chimerism in peripheral blood plantation,22 and modeling of epigenetic ever, Derom et al69 demonstrated that leukocytes. A further 20 cases of MCDZ and genetic factors of disease in although left-handedness may be more

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FIGURE 2 An alternative model of monozygotic twinning

In this model, splitting occurs at the postzygotic 2 cell stage, with each cell forming a distinct individual. If twin hatch from the zona pellucida together, dichorionic diamniotic twins will result. If the 2 trophectoderms fuse before hatching and the inner cells masses are separated within the shared trophectoderm, monochorionic diamniotic twins will result. If the inner cell masses are fused and separated later, monochorionic monoamniotic twins will result. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016.

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TABLE 2 Theories proposed to explain chimeric and MCDZ twinning Hypothesis Evidence 1 Placental anastomoses allowing early transfer of In cases of chimeric twins affected by TTTS, recipient twins are signifi- genetic material cantly more chimeric than donor twins.7,25 Tissue chimerism might be due to migration and subsequent ectopic differentiation of chimeric hematopoietic stem cells.27 Chimerism has been demonstrated to persist after selective laser photocoagulation of placental anastomoses.25,31 2 Fusion of elements of 2 genetically distinct zygotes Chorions might fuse in early pregnancy with subsequent degeneration of intervening tissue.14 might fuse preimplantation. Fusion of preimplantation em- bryos has been achieved in vitro.34 MCDZ twinning is more common in ART pregnancies. Handling with disruption of the zona pellucida, and multiple embryo transfer with spatial proximity of embryos, might predispose to fusion.17,19 3 Fertilization of a binovular follicle Binovular follicles have been observed in women undergoing ovulation induction with gonadotropins.33 Fertilization of a binovular follicle has been achieved in vitro, but pro- gression to a viable pregnancy has not been observed.150-152 ART, assisted reproductive technologies; binovular follicles, follicles in which 2 oocytes exist within a single zona pellucida; MCDZ, monochorionic dizygotoic; TTTS, twin-to-twin transfusion syndrome. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016.

common in twins, there is no evidence monochorionic twin pregnancy with a ascribing previously reported cases to to suggest that discordant handedness normal male (XY karyotype) and an chimerism, and instead suggested em- represents mirror imaging. These con- acardiac female (triploid XXX karyo- bryonic fusion as an alternative but clusions were supported by large- type). Cytogenetic studies suggested a poorly understood possibility.77 scale data from Australia and The diploid contribution from the mother in .71,72 the acardiac twin. Human leukocyte Complete hydatidiform mole with According to traditional models of antigen (HLA) typing suggested dis- coexistent twin twinning, mirror-imaging results from permic fertilization. Thus, it was pro- A multiple pregnancy with a complete late zygotic splitting at days 9-12, posed that independent fertilizations of hydatidiform mole (CHM) and a coex- just prior to the formation of conjoined a haploid ovum and its diploid first polar isting live fetus (CLF) is characterized by twins (Table 1).1,60 Cases of heterotaxy body had occurred. The authors hy- the presence of a fetus with normal syndrome have been likened to cases pothesized that the proximity of the karyotype, anatomy, and placentation of conjoined twins in whom the close ovum and its first polar body allowed the alongside a molar component with no proximity of the body axes gives development of distinct inner cell masses identifiable fetal parts, a with rise to organ laterality of one twin within a common . A fusion diploid paternal chromosomes, and the affecting that of the other.73 Neverthe- mechanism for twinning was deemed characteristic sonographic and histo- less, little evidence exists to support these unlikely.76 logical features of a CHM (Figure 5). hypotheses.67,69,74 In contrast, Fisk et al77 performed CHM-CLF is rare, with a reported inci- cytogenetic analyses on the tissues of dence of 1 in 22,000 to 1 in 100,000 Polar body twins 9 twin pregnancies affected by twin- pregnancies.78,79 A polar body is a small, cellular by- reversed arterial perfusion sequence. All Three conditions require differentia- product of the meiotic division of an twin pairs were monochorionic and tion from CHM-CLF. First, a singleton oocyte. Apoptosis usually occurs within discordant for the acardiac anomaly. pregnancy with a partial hydatidiform 17-24 hours of formation, and the Deoxyribonucleic acid (DNA) finger- mole may occur in which the fetus has resulting fragments remain within the printing revealed monozygosity. The triploidy resulting from dispermic zona pellucida.75 calculated likelihood of fertilization of fertilization of a haploid normal oocyte. It has been hypothesized that fertil- an ovum and its first or second polar Second, a twin pregnancy may occur ization of an ovum and its first or second body in any twin pair and in all twin with a normal twin in one sac and a polar body by 2 distinct sperm may pairs was less than 3.6% and 0.0003%, partial mole in the other sac. Third, result in polar body twinning (Figure 4). respectively. The authors disputed the mesenchymal dysplasia may occur and In 1981 Bieber et al76 described a existence of polar body twinning, is associated with an enlarged cystic

176 American Journal of Obstetrics & Gynecology FEBRUARY 2016 ajog.org Obstetrics Expert Reviews placenta, fetal growth restriction, and, FIGURE 3 occasionally, fetal death.80 Models of chimeric twinning Historically, most CHM-CLF preg- nancies have been terminated. More recently it has become apparent that the prognosis is not as poor as previously thought. The live birth rate varies from 21% to 40%.81,82 Complications include vaginal bleeding, hyperemesis grav- idarum, thyrotoxicosis, early-onset se- vere preeclampsia, and fetal death. Outcomes in higher-order multiple pregnancies remain poor.83,84 Conflicting evidence exists regarding rates of persistent gestational tropho- blastic disease in CHM-CLF compared with CHM alone. Sebire et al82 showed a rate of 19% for CHM-CLF compared with 16% for CHM alone. Massardier et al81 showed a rate of 50% for CHM- CLF compared with 14% for CHM alone. The risk of gestational tropho- blastic disease is independent of gesta- tion and whether the pregnancy is terminated or is allowed to continue.85 Although fetal loss is the most likely outcome for CHM-CLF, continuing the pregnancy is possible as long as maternal complications are manageable and the pregnancy is closely monitored.

Vanishing twins Vanishing twin syndrome (VTS) refers to multiple pregnancies affected by the < Hypothesis 1 (not shown) accords with the traditional model of monochorionic diamniotic twinning (Figure 1) in which placental anasto- moses may result in intertwin transfer of blood cells with subsequent blood cell chimerism. Such blood cells might subsequently infiltrate saliva. Hypothesis 2 follows the traditional model of dizygotic twinning up to the hatching stage. If 2 hatched blastocysts are in close proximity, as with the use of assisted reproductive technolo- gies, trophectoderm fusion may occur. Hypoth- esis 3 involves fertilization of a binovular follicle in which 2 oocytes exist within a single zona pellucida. In each hypothesis, fusion might also occur after implantation. In rare cases, cells from the may be transferred between twins, resulting in some degree of somatic chimerism (not shown). McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016.

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cortical sclerosis, microcephaly, and FIGURE 4 FIGURE 5 multicystic encephalomalacia have been Polar body twinning Complete hydatidiform mole 100 reported. Initially it was suggested with coexistent twin that VTS might increase the risk of ce- rebral palsy for survivors.101 Subsequent studies revealed no statistically signifi- cant increase in cerebral palsy or adverse neurological sequelae.94,102 Longer-term follow-up has suggested a possible increased risk of cerebral impairment at 1 year of age, but again, findings did not reach statistical significance.100 Further- more, survivors of VTS are deemed less psychologically vulnerable by their par- A transabdominal ultrasound scan of a dichor- ents to a maximum age of 11 years.103 ionic diamniotic twin pregnancy at 12 weeks’ VTS may have an impact on prenatal gestation demonstrating a normal fetus (left screening and diagnosis. Studies evalu- panel ) and a complete hydatidform mole (right ating serum markers in pregnancies panel ). affected by VTS have reported incon- McNamara. Typical and atypical twinning. Am J Obstet sistent results, perhaps because of dif- Gynecol 2016. ferences in mean gestational age at sampling and the interval between sampling and fetal loss. Recent evidence diagnosed on ultrasound prior to 15 suggests the presence of a vanishing weeks’ gestation frequently gave rise to twin is associated with a 21% increase the delivery of singleton infants.86 After in pregnancy-associated plasma protein the introduction of routine first- A(P ¼ .0026), a 10% increase in trimester ultrasound, VTS was increas- alpha-fetoprotein (P < .0001), and a ingly observed.87-90 13% increase in dimeric inhibin A VTS is thought to be underreported in (P ¼ .0470).104 spontaneous twin pregnancies.91 Fetal VTS might also affect the interpreta- reduction may occur prior to recogni- tion of noninvasive prenatal testing tion of pregnancy, with up to 80% (NIPT) using cell-free fetal DNA. Cases occurring prior to 9 weeks’ gestation of misdiagnosis of fetal sex using NIPT (Figure 6).92 Most cases are asymptom- may be due to VTS with subsequent atic but can be accompanied by vaginal persistence of sex se- bleeding.93 Alternatively, VTS is well quences from the vanishing twin.105,106 characterized in ART pregnancies, with In 2015 a large-scale evaluation of re- reported rates ranging from 10.4% to sults of NIPT in 30,795 consecutive cases 18.8%.35,92,94 identified 130 cases with additional fetal If an oocyte and one of its polar bodies are each Conflicting evidence exists regarding haplotypes, 76 of which could be clini- fertilized by a different sperm (illustrated in red potential adverse effects of a vanishing cally correlated. VTS was evident in and blue), 2 zygotes within a single zona pellucida twin on the remaining pregnancy. Pin- 42.1% of these 76 cases. Fetal haplotypes may result. If these 2 products of fertilization fuse borg et al94,95 described the outcomes of remained detectable via NIPT for at the blastocyst stage, monochorionic diamniotic 642 survivors of VTS. When compared up to 8 weeks after the fetal loss.107 It twins (shown) or monochorionic monoamniotic with singletons, survivors were found to has been concluded that early ultrasound twins (not shown) may result. If the first polar be at an increased risk of small for monitoring and careful pretest and body, from the first meiotic division, is fertilized, gestational age, low and very low birth- posttest counseling regarding NIPT are the twin will be triploid. If one of the second polar weight, and preterm birth. The degree of essential. bodies is fertilized, the twin will be diploid. risk was inversely proportional to the 94,95 McNamara. Typical and atypical twinning. Am J Obstet timing of fetal loss. Further studies Fetus papyraceus Gynecol 2016. have demonstrated inconsistent results Fetus papyraceus refers to a fetus in a (Appendix 3).35,96-99 multiple pregnancy that dies in utero and spontaneous loss of an embryo or fetus Concerns have been raised regarding then appears as a compressed, mummi- in the first trimester. In 1976 it was the impact of VTS on neuro- fied mass at delivery (Figure 7).108 Fetal observed that twin pregnancies developmental outcomes. Cases of focal death is thought to occur between 12 and

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FIGURE 6 FIGURE 8 Fetuses continuing at 11 weeks’ gestation Computed tomography scan of the bony outline of a fetus in fetu

A 64 slice computed tomography scan of the bony outline of a fetus in fetu presenting as an Number of fetuses continuing at 11 weeks’ gestation following the initial diagnosis of 2, 3, or 4 abdominal mass in a 2 month old child is shown. Reproduced, with permission, from Gangopadhyay AN, Srivas- gestational sacs or embryos early in the first trimester. tava A, Srivastava P, Gupta DK, Sharma, SP, Kumar V. Twin fetus Adapted from Rodrı´guez-Gonza´lez et al.92 in fetu in a child: a case report and review of the literature. J Med Case Rep 2010;4:96-102 (http://openi.nlm.nih.gov/ McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. detailedresult.php?img¼2852393_1752-1947-4-96-3&query¼ fetus in fetu&req¼4&npos¼4; licence reference, http://openi. nlm.nih.gov/faq.php#copyright). ’ McNamara. Typical and atypical twinning. Am J Obstet 20 weeks gestation. The incidence is 1 in Fetus papyraceus has been reported in Gynecol 2016. 200 twin pregnancies and 1 in 12,000 both monochorionic109,111,112 and pregnancies overall.109 dichorionic109,113 twin pregnancies and In most cases, fetus papyraceus is of higher-order multiple pregnancies.114 Historically, fetus in fetu was consid- no consequence to the surviving preg- There are no associations with age or ered to represent a well-formed mature nancy. However, associations with apla- parity, but there is a trend toward . However, in contrast to the sia cutis congenital110-114 intestinal increased frequency with mono- disorganized tissues derived from un- atresia, gastroschisis, and cardiac and chorionicity and velamentous cord controlled pluripotent cell replication in pulmonary anomalies have been insertion.109 teratomata, fetus in fetu is characterized described.109,110 Many of the abnormalities associated by the presence of vertebrae with with fetus papyraceus can be attributed appropriately organized limbs and or- to thromboembolic events following the gans (Figure 9).118 Serology and molec- 108 FIGURE 7 death of a monochorionic twin. ular genetic testing have indicated that Fetus papyraceus However, shared vascular anastomoses fetus in fetu represents a monozygotic, alone are not a complete explanation monochorionic diamniotic twin gesta- because the condition is seen in dichor- tion.119,120 Persistent anastomoses of the ionic twins.113 In dichorionic twins, fetal vitelline circulation lead to the absorp- death might be due to placental ischemia tion of one twin inside the other during leading to fetus papyraceus and conse- the ventral folding of the trilaminar quences for the cotwin.108 embryonic disc.121 Nevertheless, an association between Fetus in fetu/parasitic twins teratomata and fetus in fetu has been Fetus in fetu refers to 1 or more partially observed.122 Both are commonly located formed fetuses situated entirely within in the retroperitoneum123 and are his- the body of another normally formed topathologically similar.124 Cases of fetus (Figure 8). First described in teratoma and fetus in fetu occurring in Reproduced, with permission, from CC BY-SA 3.0 Bobjgalindo/ 115,116 Wikimedia (http://commons.wikimedia.org/wiki/File:Fetus_ approximately 1800, this event has the same individual have been re- papyraceus.JPG; licence reference, http://creativecommons. been estimated to occur once in every ported.125,126 Fetus in fetu has been org/licenses/by-sa/3.0/). 500,000 births.117 Fetus in fetu remains described as part of a parasitic contin- McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. rare despite the advent of ART, with uum including conjoined twins, acardiac fewer than 200 cases documented. twins, and teratomata.127

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the incidental identification of an discordance because of placental insuf- FIGURE 9 abdominal mass in a neonate or in- ficiency, infection, congenital anomalies, Pathological specimen of fetus in 131 fant. Advances in fetal sonography or twin-to-twin transfusion syndrome is fetu 141 have led to increased prenatal diag- not uncommon in twins. Second, in- nosis.120,132,133 Unlike teratomata, terval delivery might contribute to the fetuses in fetu do not demonstrate ma- subsequent appearance of twins with lignant potential but may cause signifi- different gestational ages. Third, in cases cant mass effect, necessitating surgical in which there is a misdiagnosis of a removal (Figure 10).126,134 singleton pregnancy, multiple pregnancy on further imaging might be attributed Superfetation to superfetation rather than to sono- Superfetation refers to fertilization and graphic diagnostic error.140 implantation of a second conception In contrast, with the advent of ART, during pregnancy (Figure 11). Early cases it has been recognized that natural bar- Pathological specimen of fetus in fetu demon- of suspected superfetation were reported riers to superfetation can be overcome. in the context of growth discordance. In Cases of superfetation resulting from strates 2 miniature fetuses joined by a cord-like 135 structure. 1989 Bhat et al reported a case of ART with or without additional sponta- Reproduced, with permission, from Gangopadhyay AN, Srivas- dichorionic diamniotic twins delivered at neous conception have been reported tava A, Srivastava P, Gupta DK, Sharma, SP, Kumar V. Twin fetus 36 weeks’ gestation with discordant (Appendix 4).142,143 in fetu in a child: a case report and review of the literature. 141 J Med Case Rep 2010;4:96-102 (http://openi.nlm.nih.gov/ birthweights and the subsequent death of In 2005 Harrison et al attempted to detailedresult.php?img¼2852393_1752-1947-4-96-3&query¼ fi fi fetus in fetu&req¼4&npos¼4; licence reference, http://openi. the second twin. Given the signi cantly con rm superfetation in a triplet preg- nlm.nih.gov/faq.php#copyright). different Dubowitz scores,136 superfeta- nancy by estimating the gestational age McNamara. Typical and atypical twinning. Am J Obstet tion was presumed. postnatally using neurosonography and Gynecol 2016. In 2003 Singhal et al137 reported a case ophthalmic evaluation. The authors of twins born to a mother with raised the possibility that superfetation pseudodidelphys. At the time of pre- can be determined conclusively.141 sentation, discordant estimated gesta- Further research is necessary to defini- Fetuses in fetu have been identified tional age was determined. In the tively prove ordisprove this phenomenon. in the mediastinum, scrotum, mouth, absence of Doppler parameters suggest- and .123,128,129 Usually there is 1 fetal ing intrauterine growth restriction, su- Superfecundation mass, but cases of up to 11 fetuses perfetation was presumed. The demise Superfecundation refers to the fertiliza- in fetu have been reported.130 The diag- of the second twin was confirmed at tion of 2 oocytes via separate instances of nosis is commonly made following 32 weeks’ gestation, whereas the first coital or artificial insemination during twin was live born at 35 weeks’ the polyovulatory period (Figure 11). gestation.137 Heteropaternal superfecundation occurs FIGURE 10 The plausibility of the phenomenon after coitus with multiple partners. 138 The fetus in fetu mass enveloped of superfetation has been questioned. Monopaternal superfecundation occurs in a sac at the time of surgery Whereas ovulation has been reported after coitus with one partner on to occur in the first trimester of multiple occasions. The latter is prob- pregnancy,139 the up-regulation of ably more common and not frequently the hypothalamic-pituitary-ovarian axis detected.142 by luteal and then placental proges- In 1978 Terasaki et al144 described a terone typically suppresses ovulation. case of suspected heteropaternal super- Progesterone-induced changes in cervi- fecundation on the basis of HLA typing. cal mucus may limit successful fertiliza- In 1982 heteropaternal superfecunda- tion. The presence of a gestational sac in tion was again suspected in a pair of the uterus may limit a successful im- twins born with different skin colors.145 plantation. Therefore, it has been argued Additional cases have been described that spontaneous superfetation is in the context of paternity disputes 140 146 Reproduced, with permission, from Gangopadhyay AN, Srivas- unlikely. (Appendix 5). tava A, Srivastava P, Gupta DK, Sharma, SP, Kumar V. Twin fetus in fetu in a child: a case report and review of the literature. Three alternative explanations for More recently, superfecundation has J Med Case Rep 2010;4:96-102 (http://openi.nlm.nih.gov/ detailedresult.php?img¼2852393_1752-1947-4-96-3&query¼ described cases of superfetation have been associated with ART. In 2001 fetus in fetu&req¼4&npos¼4; licence reference: http://openi. been proposed. First, pregnancies Amsalem et al142 reported spontaneous nlm.nih.gov/faq.php#copyright). complicated by growth discordance may monopaternal superfecundation in a McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. give rise to the appearance of twins 25 year old woman undergoing IVF of differing gestational ages. Growth for secondary infertility. After an

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uncomplicated transfer of 2 embryos, 5 complications unique to multiple gesta- FIGURE 11 embryos were detected on ultrasound. tions; utilizing cell-free DNA for aneu- Superfetation and Following multifetal pregnancy reduc- screening in multiple superfecundation tion, male twins were born at term. pregnancies; interpreting twin data to Subsequent cytogenetic testing determine the relative contributions of confirmed monopaternity and suggested genetic, epigenetic, and environmental that cleavage/duplication was unlikely. factors to various phenotypic outcomes; In 2005 Peigné et al143 reported a case and reducing the incidence of sponta- wherein monopaternal superfecunda- neous and assisted multiple pregnancies. tion was observed following multiple An examination of the anomalies of the intrauterine inseminations and IVF cy- placenta and umbilical cord in twin cles. Again, a selective fetal reduction gestations has been recently published resulted in the delivery of live-born and provides an excellent review.149 twins.143 Further research, including series of Evaluation of large parentage data- extended cytogenetic analyses, is needed bases has led to a reported overall to refine our understanding of early frequency of heteropaternal superfe- embryonic development in twin cundation of 2.4%.147 It has been pro- pregnancies. - posed that 1 in 400 twin pairs born to married white women in the United States are the result of heteropaternal REFERENCES superfecundation. Monopaternal super- 1. fecundation is thought to be more Hall J. Twinning. Lancet 2003;362:735-43. 2. Weber M, Sebire N. Genetics and develop- common, with an estimated prevalence mental pathology of twinning. Semin Fetal of 1:12 dizygotic twins born to mothers Neonatal Med 2010;15:313-8. in the United Kingdom.148 The rate 3. Kilby M, Baker P, Critchley H, Field D. of superfecundation depends on com- Consensus views arising from the 50th study munity rates of coital frequency, group: multiple pregnancy. London: RCOG Press; 2006:283-6. polyovulation, and subsequent DNA 4. Derom C, Vlietinck R, Derom R, Van den detection. The reported incidence may Berghe H, Thiery M. Increased monozygotic be increasing because of increased pa- twinning rate after ovulation induction. Lancet ternity testing.146 1987;1:1236-8. 5. Given that superfecundation may Alikani M, Noyes N, Cohen J, Rosenwaks Z. Monozygotic twinning in the human is associ- occur with ART, women should be ated with the zona pellucida architecture. Hum advised to consider avoiding intercourse Reprod 1994;9:1318-21. after embryo transfer to reduce the risk 6. Blickstein I, Verhoeven HC, Keith LG. Zygotic of subsequent higher-order multiple splitting after assisted reproduction. N Engl J pregnancy and/or ectopic pregnancy.142 Med 1999;340:738-9. 7. Umstad MP, Short RV, Wilson M, Craig JM. Chimaeric twins: why monochorionicity does not Superfetation may result when 2 embryos are Conclusion guarantee monozygosity. Aust N Z J Obstet produced at different time points, resulting in Twinning is a complex and multifacto- Gynaecol 2012;52:305-7. asynchronous development in utero. At birth, rial phenomenon and elements of the 8. Herranz G. The timing of monozygotic twin- this may result in apparent growth discordance twinning process remain incompletely ning: a criticism of the common model. Zygote 2013:1-14. of dichorionic diamniotic twins. Superfecunda- understood. A conventional model of 9. Denker HW. Comment on G. Herranz: The tion may result when the fertilization of 2 oocytes monozygotic twinning is based on timing of monozygotic twinning: a criticism of the via separate instances of coital or artificial fission events in the developing embryo. common model. Zygote (2013). Zygote 2013: insemination occur during a single polyovulatory This model lacks definitive evidence 1-3. 10. period. Because superfecundation is in many and is challenged by cases of atypical van Dijk BA, Boomsma DI, de Man AJM. Blood group chimerism in human multiple births ways similar to superfetation, both are illustrated twinning. is not rare. Am J Med Genet 1996;61:264-8. by the same figure. Fertilization in superfecun- An alternative model proposes that 11. Robinson E, North D, Horsfield G, Kelly F. dation may be monopaternal or heteropaternal embryonic fusion events underlie A case of twin chimerism. J Med Genet 1976;13: and both are illustrated by red and blue sperm. monozygotic twinning. However, sup- 528-30. 12. Viëtor HE, Hamel BC, van Bree SP, et al. McNamara. Typical and atypical twinning. Am J Obstet porting evidence is similarly limited. Gynecol 2016. Immunological tolerance in an HLA non-identical Elucidating the precise mechanisms by chimeric twin. Hum Immunol 2000;61:190-2. which twinning occurs will have signifi- 13. Sudik R, Jakubiczka S, Nawroth F, cant implications for managing Gilberg E, Wieacker P. Chimerism in a fertile

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184 American Journal of Obstetrics & Gynecology FEBRUARY 2016 ajog.org APPENDIX 1 Cases of monochorionic dizygotic twins Gestation Placental Studies Maternal ART status Antenatal delivery histology Twin 1 Twin 2 Blood Tissue Genitalia Follow-up Nylander et al,14 1970, 1 35 y old Spontaneous — — MCDA Male Female — — Examination (N) — western Nigeria Multi anastomoses 2490 g 1760 g present Vie¨tor et al,12 2000, 2 29 y old — Spontaneous 34.5 — Male Female Yes No Examination (N) — The Netherlands G1P0 reduction triplet (skin Ultrasound (N) fibroblast) Quintero et al,16 2003, 3 28 y old — TTTS — MCDA FDIU FDIU Yes — Examination (N) NA United States G1P0 Anastomoses Male Female 3 lasered Souter et al,15 2003, 4 48 y old IVF with donor — 37 MCDA Male Female Yes No (skin Examination (N) 5mo United States G1P0 eggs; 3 ET Anastomoses 2114 g 2183 g fibroblast) Ultrasound (N) present Williams et al,17 2004, 5 38 y old Ovarian Severe 28 MCDA Male Female Yes No (ovarian Twin 2 clitoromegaly; 7 wks United States 0111 stimulation, IVF, preeclampsia Emergency Twin 2 2114 g 654 g biopsy) ultrasound: ICSI CD velamentous not seen insertion of the cord Ginsberg et al,18 2005, 6 35 y old Ovarian stimulation Preterm labor 22 MCDA Male Female Yes — Limited autopsy: Expired United States (clomiphene) normal internal and d14

ERAY2016 FEBRUARY external genitalia Miura and Niikawa,19 7 — IVF, ET — — MC Male Female Yes No Examination (N) — 2005, Japan 8 — FSH ovarian — — MC Male Female — — Examination (N) — stimulation, IUI mrcnJunlo Obstetrics of Journal American 9 — TESE, ICSI Spontaneous — MC Male Female Yes No — —

fetal reduction Obstetrics 10 — IVF, ET — — — Male Female — — — — 11 — IVF, ET — — — Male Female Yes — — — Yoon et al,20 2005, 12 34 y old IVF, ICSI, 3 ET — 33.5 MCDA Male Male Yes — Examination (N) —

United States G1P0 2660 g 2221 g Reviews Expert XXY Aoki et al,21 2006, 13 27 y old Ovarian stimulation —34MCDA Male Male Yes No (hair root —3mo

& Japan G1P0 (clomiphene) Elective CD Anastomoses 2002 g 2132 g cells)

Gynecology present Walker et al,22 2007, 14 30 y old IVF, 2 ET — 36 MCDA Male Male Yes No (buccal Examination (N) 15 mo Australia G2P1 Elective CD Not sent for 2395 g 1960 g cells) Ultrasound (N) histology 185 McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. (continued) xetReviews Expert 186 APPENDIX 1 mrcnJunlo Obstetrics of Journal American Cases of monochorionic dizygotic twins (continued) Gestation Placental Studies Maternal ART status Antenatal delivery histology Twin 1 Twin 2 Blood Tissue Genitalia Follow-up Ekelund et al,23 2008, 15 38 y old ICSI, assisted TTTS 32 MCDA Male Female Yes No (buccal Examination (N) 6mo Denmark Multi hatching, 3 ET Elective CD 2265 g 1550 g cells) Ultrasound (N; 1 identifiable ovary)

Hackmon et al,24 2009, 16 32 y old Spontaneous — 37 MCDA — — Yes No (buccal Examination (N) 18 mo Obstetrics

& United States G3P2 NVD cells) Ultrasound (N)

Gynecology Assaf et al,25 2010, 17 28 y old ICSI, 2 ET TTTS 37þ4 MCDA Female Male Yes No (buccal Examination (N) 9 mo United States G1P0 Elective CD of 3487 g 2892 g cells) vascular communications ERAY2016 FEBRUARY Hawcutt et al,32 2011, 18 IVF, 2ET Preterm labor 25þ1 — Male Female Yes No (Buccal Examination (N) 10 mo United Kingdom Emergency 710 g 740 g cells) Ultrasound (N) CD Umstad et al,7 2012, 19 36 y old Spontaneous TTTS 36 MCDA Male Male Yes No (Buccal Examination (N) 14 mo Australia Elective CD 2490 g 2310 g cells) Choi et al,26 2013, 20 31 y old IVF, assisted — 33.8 MCDA Male FDIU Yes No (Skin Twin 1: Twin 1: South Korea 0020 hatching, 3 ET CD 2160 g Female fibroblast) undescended 39 mo 2335 g left testis Fumoto et al,27 2014, 21 32 y old IVF, 2 ET — 35 — Female Male Yes Yes (Buccal Examination (N) 7mo Japan Elective CD 1880 g 2548 g cells) Ultrasound (N) Lee et al,28 2014, 22 — IVF-ET — — — Female Male Yes No Hydrocele in 23 mo Korea male twin Lee et al,29 2014, 23 34 y old IVF, 2ET — 38þ0 MCDA Female Male Yes No (skin Examination (N) 23 mo Korea G1P0 NVD 2490 g 2340 g fibroblast) Ultrasound (N) ART, assisted reproductive technologies; CD, cesarean delivery; ET, embryo transfer; FDIU, fetal death in utero; FSH, follicle stimulating hormone; ICSI, intracytoplasmic sperm injection; IUI, intrauterine insemination; IVF, in vitro fertilization; MC, monochorionic; MCDA, monochorionic diamniotic; Multi, multiparous; N, normal; NA, not applicable; NVD, normal vaginal delivery; TTTS, twin-to-twin transfusion syndrome; US, ultrasound. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. ajog.org ajog.org APPENDIX 2 Cases of mirror-image twins Studies Maternal ART status Antenatal Delivery Twin 1 Twin 2 Zygosity Twin 1 Twin 2 Evaluation Rife,52 1933, 1 Dominant handedness, United States direction of hair whorl, eye dominance Letts and Shokeir,153 2 — — No family history, 36 Female Female Nixon’s discriminant, — — Coxa vara 1975, Canada uncomplicated NVD 1979 g 1097 g probability 0.9695 MZ; chromosome studies Springer and 3 — — — — Female Female HLA antigens were 52 y 52 y Breast ductal carcinoma in Searleman,51 1978, similar; considered situ United States MZ Novak, 1981,56 439y — Spontaneous 35 Female Female Placenta PN PN Laryngotracheosophageal United States old G10 preterm labor CD 2210 g 1940 g examined ¼ MCDA cleft, displacement of heart, unilateral pulmonary hypoplasia Nigro et al,64 1991, 5 — Clomid Spontaneous 33þ4 Male Male Identical 7 y, 9 y 7 y, 9 y Colpocephaly (enlarged United States preterm labor CD 1616 g 1758 g occipital horns of the lateral ventricles), dominant handedness Williams and Kapila,154 6 — — — — Male Male — 5 y 5 y Posterior urethral valves with 1993, United Kingdom unilateral hydronephrosis

ERAY2016 FEBRUARY and bladder diverticulum Morison et al,62 1994, 7 — — — — Male Male — 60 y 62 y Lung adenocarcinoma United States Sperber et al,53 1994, 8 G2P1 — Nonconsanguineous, — Female Female Peripheral blood 4 y 4 y Dental fusion (mandibular Canada no family history DNA analysis ¼ MZ lateral incisor and canine mrcnJunlo Obstetrics of Journal American fusion) Obstetrics Karaca et al,57 1995, 9 30 y old — Consanguineous, NVD — — Monozygotic 6 mo 6 mo Cleft lip and palate Turkey G5P4 uncomplicated Satoh et al,58 1995, 10 — — Nonconsanguineous, NVD, Female Female ABO blood typing PN, 5 y PN, 5 y First and second branchial Japan uncomplicated term HLA typing, DNA arch syndrome, spinal

typing ¼ MZ scoliosis, unilateral cleft lip/ Reviews Expert palate Sommer et al,70 1999, 11 — — Uncomplicated No Female Female Genotyping ¼ MZ — — Cerebral lateralization for

& The Netherlands trauma 3300 g 3500 g language mental rotation Gynecology Okamoto et al,155 12 — — — 38 2360 g 2500 g Monozygotic 20 20 Myopic anisometropia 2001, Japan Male Male months months

McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. (continued) 187 xetReviews Expert 188 APPENDIX 2 mrcnJunlo Obstetrics of Journal American Cases of mirror-image twins (continued) Studies Maternal ART status Antenatal Delivery Twin 1 Twin 2 Zygosity Twin 1 Twin 2 Evaluation Morini et al, 2002,156 13 33 y old — Prenatal diagnosis NVD Male Male Placenta examined ¼ PN PN Posterior urethral valves, hydronephrosis MCDA, ABO typing bilateral vesicoureteric reflux, hydroureter, nonfunctioning , cyst Dirani et al,54 2006, 14 — — No family history 3 wks 1500 g 2000 g Standard 55 y 55 y Congenital esotropia Australia preterm genotyping ¼ MZ Obstetrics

& Hwang et al,68 2006, 15 26 y old — No family history, 38 2580 g 2934 g DNA testing ¼ MZ PN PN Asplenia syndrome Gynecology Taiwan uncomplicated CD Male Male Helland and Hester,65 16 24 y old — Uncomplicated 40 2900 g 3370 g Placenta examined ¼ 12 y 12 y Arachnoid cysts in 2007, Norway NVD Female Female MC cerebellopontine angle

ERAY2016 FEBRUARY Goto et al,61 2008, 17 — — — — Male Male Monozygotic 10 y 11 y Solitary bone cysts of the Japan humerus, handedness, hair whorl direction Thacker et al,67 2009, 18 37 y old — US cardiac defect 34 Female Female Monozygotic PN PN Heterotaxy syndrome United States G2P3 El CD 1720 g 2000 g Zhou et al,66 2011, 19 24 y old — Uncomplicated 35 1700 g 2300 g Placenta examined ¼ 14 mo 14 mo Arachnoid cysts in temporal China CD Male Male MC lobe Riess et al,59 2012, 20 G2P1 — US cardiac defect; 32þ6 1660 g 1945 g DNA testing PN PN Kabuki syndrome, mirror Germany Karyotype NAD CD Male Male (peripheral blood cleft lip and palate leukocytes) ¼ MZ Wang et al,60 2012 21 — — — — Male Male Genetic testing ¼ MZ 11 mo 17 mo Trigger thumb United States with > 99% CI Hu et al,55 2014 22 — — — — Female Female DNA testing ¼ MZ 10 y 10 y External ear deformity: China microtia ART, assisted reproductive technologies; CD, cesarean delivery; DNA, deoxyribonucleic acid; HLA, human leukocyte antigen; MCDA, monochorionic diamniotic; MZ, monozygotic; NVD, normal vaginal delivery; PN, postnatal; US, ultrasound. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. ajog.org ajog.org APPENDIX 3 Vanishing twins: outcomes of survivors of a vanishing twin vs primary singletons Mean GA LBW VLBW Adverse Studies Years ART status Case, n Control, n (delivery) < 2500 g < 1500 g SGA CP neurology Dickey et al,96 2002, 1 1976e2000 Infertility 140 4683 15.7 vs 4.5a United States treatment P ¼ .32 Chasen et al,97 2006, 2 2003e2005 IVF 55 168 39.4 vs 39.1 14.5 vs 9.6a United States P ¼ .44 P ¼ .32 La Sala et al,35 2006, 3 1992e2004 IVF with or 84 602 38.2 vs 38.3 OR 1.2 OR 2.7 (0.4e18.9) Italy without ICSI P > .05 (0.6e2.8) Shebl et al,98 2008, 4 1999e2005 IVF with or 46 92 39.0 vs. 39.3 26.1% vs 12.0% 4.3% vs 1.1% 32.6% vs 16.3% Austria without ICSI P ¼ .69 P ¼ .036a P ¼ .22 P ¼ .029a Almog et al,99 2010, 5 1999e2007 IVF with or 57 171 35.1 vs. 38.2 33.3% vs 11.7% 3.5% vs 0.6% 14.0% vs 17.5% Israel without ICSI P ¼ .001b P ¼ .0001b NS NS Pinborg et al, 2005,94 6 1995e2001 IVF with or 642 5237 OR 1.7 (1.2e2.2) OR 2.1 (1.3e3.6) OR 1.50 (1.03e2.20)b OR 1.9 (0.7e5.2) 4.7% vs and 2007,95 Denmark without ICSI 4.1 % P ¼ .5 Anand et al,100 2007, 7 1999e2001 Mixed 22 103 RR 6.1 United Kingdom (1.5e8.3) P ¼ .03b ART, assisted reproductive technologies; CP, cerebral palsy; GA, gestational age; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; LBW, low birthweight; NS, not statistically significant; OR, odds ratio; RR, relative risk; SGA, small for gestational age; VLBW, very low birthweight. a b

ERAY2016 FEBRUARY Birthweight less than 10th percentile; Statistically significant. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. mrcnJunlo Obstetrics of Journal American Obstetrics xetReviews Expert & Gynecology 189 xetReviews Expert 190 APPENDIX 4 mrcnJunlo Obstetrics of Journal American Cases of superfetation Maternal ART status Antenatal Progress Delivery Twin 1 Twin 2 Evaluation Bhat et al,135 1 Gravida 5 — Admitted 36/40 — Spontaneous NVD Female 2100 g Male 1500 g Presumed superfetation on 1989, at 36/40 Dubowitz 36/40 Dubowitz 30/40 the basis of different estimated gestational age (Dubowitz score) without evidence of IUGR Kobayashi et al157 2 33 y old Spontaneous Presented with Second pregnancy — — — Presumed superfetation

1996, Japan G1P0 abdominal pain; identified with Obstetrics

& E/O left anterior increase in hCG;

Gynecology broad ligament estimated 4/40 Serial hCG monitoring Tuppen et al,158 1999, 3 38 y old Spontaneous Vaginal bleeding 7/40 US 7/40: 4 wks Spontaneous NVD Female 3288 g Male 2324 g Presumed superfetation on the United Kingdom G3P2 growth discordance Dubowitz 39/40 Dubowitz 35/40 basis of different estimated ERAY2016 FEBRUARY gestational age (Dubowitz score) Singhal et al,137 4 20 y old — US 28/40: 5 wks US 32/40: FDIU Emergency CD Female 2200 g Female 660 g Presumed superfetation on the 2002, India G1P0 growth discordance; twin 2 Confirmed (macerated basis of different estimated no IUGR; bicornuate pseudodidelphys ) gestational age (Dubowitz score) uterus uterus without evidence of IUGR ART, assisted reproductive technologies; CD, cesarean delivery; E/O, excision of; FDIU, fetal death in utero; hCG, human chorionic gonadotropin; IUGR; intrauterine growth restriction; NVD, normal vaginal delivery; US, ultrasound. McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. ajog.org ajog.org APPENDIX 5 Cases of superfecundation Maternal ART status Antenatal Progress Delivery Twin 1 Twin 2 Evaluation Context Archer,159 1810 1 One white twin Racially different One mulatto twin in appearance Terasaki et al,144 1978 2 HLA typing Paternity suit Harris et al,145 1982 3 One white twin Racially different One black twin in appearance Wenk et al,147 1991, 4 HLA typing Paternity suit United States Verma et al,160 1992 5 Chromosome heteromorphic Paternity suit markers Lu et al,161 1994 6 HLA typing Paternity suit Conventional marker typing 7 HLA typing Paternity suit Conventional marker typing Girela et al,146 1997, 8 Female Female PCR typing of loci HLA Paternity suit Spain 2100 g 1750 g Restricted fragment length polymorphism Amsalem et al, 2001,142 925y IVF with ICSI; US Day 27: Selective fetal 37 Male Male DNA analysis of 3 embryos Antenatal care; IVF Israel old 2 ET (7 cell/8 5 embryos reduction 3 Elective CD 2390 g 2170 g (amniotic fluid), live-born cell stage) twins (cord blood), ; ERAY2016 FEBRUARY genetically distinct Peigne´ et al, 2011,143 10 31 y 3 IUI; 2 IVF; US 4/40 Selective fetal 31 Female Male DNA analysis of 3 embryos, Antenatal care; IVF France old G0 2 ET second cycle 5 embryos reduction 3 Elective CD 780 g 1240 g live-born twins, parents; genetically distinct

mrcnJunlo Obstetrics of Journal American ART, assisted reproductive technologies; CD, cesarean delivery; DNA, deoxyribonucleic acid; ET, embryo transfer; HLA, human leukocyte antigen; ICSI, intracytoplasmic sperm injection; IUI, intrauterine injection; IVF, in vitro fertilization; NVD, normal vaginal delivery;

US, ultrasound. Obstetrics McNamara. Typical and atypical twinning. Am J Obstet Gynecol 2016. xetReviews Expert & Gynecology 191