Non-identical monozygotic twins, intermediate twin types, zygosity testing, and the non-random nature of monozygotic twinning: A review


  • Geoffrey Machin

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    • 3931 Cherrilee Crescent, Victoria, British Columbia, Canada, V8N 1R7.
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    • Dr. Machin is a fetal/placental pathologist at Mount Sinai Hospital in Toronto, Ontario, Canada; and a part-time clinical Professor at the University of Toronto, Toronto, Ontario, Canada. He has published over 100 papers, many book chapters, and co-authored the Atlas of Multiple Pregnancy. His principal interests include twin pregnancy and pathology, perinatal pathology, genetics of Wilms tumor, and the etiology of hydrops fetalis. Currently, he resides with his wife of forty-four years; and enjoys choral singing, gardening, and spending time with his two sons and grandson.

  • How to cite this article: Machin G. 2009. Non-identical monozygotic twins, intermediate twin types, zygosity testing, and the non-random nature of monozygotic twinning: A review. Am J Med Genet Part C Semin Med Genet 151C:110–127.


Monozygotic twins (MZ) are rarely absolutely “identical.” This review discusses the types of genetic/epigenetic and prenatal environmental post-zygotic mechanisms that cause discordance within such twin pairs. Some of these mechanisms—ranging from heterokaryotypia to skewed X-chromosome inactivation—may cause extreme discordance, but these extremes are merely the more emphatic examples of discordance that, to some degree, underlies the majority of MZ twin pairs. Because of the entrenched misconception that MZ twins are necessarily identical, many MZ twin pairs are mistakenly designated as dizygotic (DZ). Clinical benefits to accurate zygosity determination include correct solid organ transplantation matching, if one twin requires donation for a non-genetically mediated disease; the opportunity of preventive management for disorders that do not manifest synchronously; and better counseling to parents regarding their individually unique, and often psychologically puzzling, twin offspring. In twin pairs with complex and confusing biological origins, more detailed zygosity testing may be required. For example, intermediate trigametic and tetragametic chimeric dizygotic twins are reviewed, some of whom are, nevertheless, monochorionic (MC). Because of inter-fetal vascular anastomoses in MC twins, genetic results from blood samples may not accurately reflect discordance in solid organs. Previously, it was thought that MZ twinning was some sort of embryological fluke. However, familial monozygotic twinning is more common than suggested by the literature. Seven new families are presented in an accompanying paper. Despite the difficulties and dangers of twin pregnancy (especially so for MC twins), human twinning persists, and continues to both challenge and fascinate parents, clinicians and geneticists. © 2009 Wiley-Liss, Inc.

“There are many events in the womb of time which will be delivered.” Shakespeare. Othello, act 1, sc.3, 1.


Pedigree analysis is used for tracking single-gene disorders in relatives of varying degrees. In most instances, the closest relatives to manifest the effects of similar chromosomal segregation and recombination during meiotic gametogenesis are related to the first degree: full siblings, DZ twins and parents/offspring pairs. Epigenetic mechanisms also play a role during meiosis, whereas chromosomal mosaicism is clearly a post-zygotic event.

But how are we to regard people who are “zeroth” degree relatives, that is, monozygotic (MZ) twins? Are such pairs compelled by their single zygotic genome to be phenotypically “identical”? This simplistic view remains a cornerstone for most parents of twins and even many health care professionals; however, it is steadily being eroded by recent refinements in our understanding of the mechanisms that modify gene structure and expression during meiosis and mitosis. MZ twin pairs are living human laboratories, and when they begin to diverge phenotypically, we can discover new ways in which the genome/epigenome profoundly changes post-zygotically. Some of these divergences occur in embryonic stem cells soon after zygosis [Fuentealba et al., 2008], perhaps in concert with events that underlie determination and differentiation of axes, organs, and tissues during embryogenesis. It is not clear whether any or most of these divergences actually stimulate the MZ twinning process. Other genetic/epigenetic divergences probably accumulate more slowly over the full lifetime of any MZ twin pair [Fraga et al., 2005]. In addition to genetic/epigenetic events within MZ twins that drive a wedge in their common zygotic phenotype, there are also significant prenatal, intra-uterine environmental considerations that especially apply to the majority of MZ twin pairs (about two-thirds) who are MC, that is, twins who are both connected to a truly single (not fused) placenta. The remaining one-third of MZ twins are dichorionic (DC), developing in separate sacs. Parents of like-sexed DC twins are frequently misinformed that their twins are necessarily dizygotic (DZ).

Many parents and professionals continue to use “MZ” and “identical” synonymously. As a result, the majority of parents of MZ twins think of their twins as DZ, because, on close and repeated inspection (and to preserve parental sanity), the twins reveal the ways that they differ in every respect. An alternative scenario commonly occurs in families whose MZ twins have been falsely labeled as DZ twins: naturally, the twins appear too uncomfortably alike to be DZ twins, and the startled parents may wonder if rare forms of twinning intermediate between MZ and DZ might provide an explanation for these uncanny similarities. Potentially harmful consequences of incorrect zygosity determination include failure to provide accurate genetic counseling to families; to initiate preventive medicine strategies (when one of an MZ pair presents phenotypically with disease before the other); and to offer optimal organ transplantation (the correct match and potential avoidance of immunosuppression in MZ twin recipients).

This paper reviews genetic/epigenetic and environmental mechanisms that ensure that no MZ twin pair is identical. Some striking examples of phenotypic discordance are well known (e.g., acardius), but they are generally thought to be rare and extreme. It may be more realistic to regard them as the tip of an iceberg, overlying a mass of genetic/epigenetic/phenotypic/environmental discordance that pervades MZ twinning in general. In fact, these epigenetic/genetic events may be more potent in phenotypic discordance than the supposed “environmental” influences that have previously been thought to drive the phenotypes of MZ (and DZ) twins apart [Singh et al., 2002a; Wong et al., 2005] (Fig. 1).

Figure 1.

Original “twin model” proposed that discordance in MZ and DZ twins could be separately ascribed to “genetic” and “environmental” influences on development. However, environmental factors have not been well delineated. An alternative model proposes that genetic and epigenetic factors largely account for discordance in MZ and DZ twin pairs. It is moot whether at least some of the epigenetic modification may in fact be driven by environmental exposure.

MZ twin pairs are usually discordant for major and regional malformations. Cerebral laterality is sometimes reversed within MZ twin pairs. The effect of MC placentation is a confounding prenatal environmental hazard. Methods of reliable zygosity diagnosis are not clear. Unusual forms of intermediate twins have recently been described, but they tend to have severely abnormal phenotypes, and so they do not readily explain MZ twin pairs who are similar but not identical. Finally, there is evidence that MZ twinning is not a random teratogenic event. Given the plethora of post-zygotic genetic/epigenetic and environmental events that modify the original zygotic genome, it is in fact surprising that many MZ twin pairs actually display close resemblance.

There is evidence that MZ twinning is not a random teratogenic event. Given the plethora of post-zygotic genetic/epigenetic and environmental events that modify the original zygotic genome, it is in fact surprising that many MZ twin pairs actually display close resemblance.

Regular readers of this journal will already be familiar with many of these facts, because numerous important papers of MZ twin discordance have been published in the American Journal of Medical Genetics.


A previous paper reviewed much of what was then known about genetic/epigenetic and prenatal environmental differences within MZ twin pairs [Machin, 1996]. The main facts then known included: unequal allocation of blastomeres to MZ twin inner cell masses, confined MZ twin chromosomal mosaicism, skewed X-inactivation, imprinting (e.g., in Beckwith–Wiedemann syndrome; BWS), lateral asymmetry, discordance for major malformations and the abnormal prenatal environment of MC MZ twins. In the subsequent dozen years, many publications have confirmed and expanded these ideas. The present paper updates the survey as comprehensively as possible.

Unequal Blastomere Allocation

There is evidence that the original allocation of stem cells to each twin may not always be scrupulously fair and equitable (larger clone sizes from fewer precursor cells in one twin) [Nance, 1990]. The evidence comes from female twin pairs in whom one twin has random X-inactivation whereas the other has skewed inactivation, presumably arising, by chance, from a smaller original set of stem cells. This could account for discordant fetal (and lifetime) growth, including diminished ability of the smaller embryo to acquire an adequate share of an MC placenta, and perhaps in rare and extreme cases resulting in acardius [Masuzaki et al., 2004] and parasitic conjoined twins. Growth discordance is a common reason for mistakenly thinking that MZ twins are DZ. Not only is the biological process of MZ twinning imagined to produce twins who look alike, but they are also expected to be of exactly the same size (cookie-cutter effects). In fact, growth-discordant MZ twins often have facial dissimilarity that makes them look unalike (Fig. 2).

Figure 2.

Evidence from skewed/random X-inactivation in some twin pairs indicates that the MZ twinning process can result in unequal allocation of founder cells with a cascade of events that include primary growth discordance, and could also be responsible for inability to acquire equal sharing of the MC placenta, as well as abnormal embryo development, for example, acardius, parasitic conjoined twin.

Genetic/Epigenetic Discordance

  • (1)Chromosomal mosaicism (Table I). Post-zygotic non-disjunction in one MZ twin (confined twin mosaicism) can lead to heterokaryotypia for all the known autosomal trisomies and for the gonosomes (chromosomes involved in sex determination) (Table I). In the latter context, a 47,XXY zygote can give rise to MZ twins who are 46,XY and 46,XX, with varying degrees of mosaicism in solid tissues and/or blood [Zech et al., 2008]. A more extreme example of non-identical MZ twins than sex discordance is hard to imagine!Various autosomal and gonosomal structural abnormalities can also lead to MZ twin mosaicism and phenotypic discordance. Post-zygotic non-disjunction may also lie behind many pairs with twin reversed arterial perfusion (acardia), resulting in a widely discordant phenotype between the pump and acardiac twin [Machin, 1996]. But such twins can only survive because they are MC, with an interchanged blood supply. Of six consecutive heterokaryotypic MC twin pairs, four involved the X chromosome and two involved autosomes [Lewi et al., 2006]. Dual amniocentesis was superior to villous sampling in prenatal diagnosis.
  • (2)Point mutation in single gene.A pair of MZ twins was reported [Kondo et al., 2002] who are discordant for van der Woude syndrome. The affected twin had a (post-zygotic) nonsense mutation in the IRF6 gene (IRF6, glu92ter), whereas the unaffected twin did not have the mutation. In contrast, a pair of MZ twins discordant for semilobar holoprosencephaly showed the same heterozygosity for a Ser362Leu in the SHH gene [Peng et al., 2007].
  • (3)Mutations in mitochondrial DNA (mtDNA).MZ twin brothers discordant for Leber hereditary optic neuropathy were heteroplasmic for a de novo 14484 mtDNA mutation [Biousse et al., 1997]. With current renewed interest in mitochondrial disorders in the public and clinical setting, mitochondrial DNA mutations have been currently investigated as modifiers that determine phenotypic variability for common Mendelian disorders. For example, in the case of neurofibromatosis type I, it has been postulated that MZ twin pair variability may be related to mtDNA heteroplasmy because of co-localization with the tumor suppressor protein neurofibromin. However, in four discordant twin pairs no evidence of heteroplasmy in mDNA was found [Detjen et al., 2007]. Likewise, MZ twins phenotypically discordant for adrenoleukodystrophy showed identical mtDNA [Wilichowski et al., 1998]. In MZ twin pairs discordant for obesity, transcript levels of genes involved in the oxidative phosphorylation pathway in adipose tissue were lower (P < 0.05) in the obese as compared to the non-obese co-twins [Mustelin et al., 2008].
Table I. Chromosomal Aneuploidy in MZ Twins Resulting in Phenotypic Discordance
Zygotic chromosomesTwin chromosomesPhenotypeRefs.
  • DPS, discordant phenotypic severity; n, normal; M, male; F, female; CH, cystic hygroma; TS, Turner syndrome.

  • a

    A complex series of events involving inheritance of different maternal chromosome 21 alleles.

Autosomal trisomyConcordant autosomal trisomy, e.g., trisomy 18DPSLee et al. 2004
Autosomal structural abnormalityConcordant structural abnormality:  
 dup(4q25-qter) del 1p36;DPSAngle et al. 2002
 dup(4q28.3-qter);DPSCelle et al. 2000
 del(18q22.3-qter) dup(18q12.1-qter);DPSCourtens et al. 1998
 Discordant structural anormality: 46,XX/46,XX,dup(11)(p12p15); presumed post-zygotic mutationDPSBourthoumieu et al. 2005
 46,XX/46,XX, der(15)t(11;15) (p12;p11.2), presumed post-zygoticDPSMarcus-Soekarman et al. 2004
Autosomal trisomyDiscordant trisomy, e.g., trisomy 13; trisomy 21 and trisomy 21/nTrisomy 13/n CH/naTaylor et al. 2008; Dahoun et al. 2008
EuploidDiscordant autosomal monosomy, e.g., 46,XY/45,XY,−21Trisomy 21/monosomy 21/normalCheng et al. 2006
Gonosomal aneuploidDiscordant gonosomal euploid, e.g., ?47,XXY->46,XY/46,XXNormal M4/F5Zech et al. 2008
Gonosomal euploidDiscordant gonosomal monosomy, e.g., 46,XY->46,XY/45,X; 46,XY->46,XY/45,X 46,XX->46,XX/45,X 46,XX->46,XX/45,XM/F, gonadal dysgenesis M/CHTho et al. 2007; Fernández-Martínez et al. 2007
  F/CH Mild TS/nGilbert et al. 2002; Su et al. 2006
Gonosomal structural abnormality46,X,del(X)(p11.1)/45,X; 46,X,i(Y)(p10)/45,X 47,X,idic(Y),idic(Y)/46,X,idic(Y)/45,XCH/n CH/M Hypospadic M/F with dysgenetic testesGentilin et al. 2008; Schmid et al. 2000; Nonomura et al. 2002

Epigenetic Phenomena

X-inactivation and imprinting are known to be discordant within some MZ twin pairs. Recent publications are summarized in Tables II and III. The frequency of discordant BWS in MZ twin pairs is well known. The majority of these pairs are female, and the affected twins show loss of methylation at imprinting center 2 (IC2) of 11p15.5 [Smith et al., 2006]. However, concordance and discordance is also reported in male MZ pairs. A discordant male pair showed paternal uniparental disomy for 11p15 in the affected twin [Smith et al., 2006]. Discordant trinucleotide expansion with discordant phenotype has been found in FRAX [Helderman-van den Enden et al., 1999]. The affected MZ male twin had the full mutation, whereas the unaffected twin was mosaic for the full mutation and the premutation. Another example is a pair of MZ twins who had the same 39 expansion of the CAG repeat in the HD gene, were discordant for clinical onset of Huntington disease by 7 years [Friedman et al., 2005].

Table II. Discordant Phenotypes With Unequal X-Inactivation in Female MZ Twin Pairs
Fabry, 231 Asp->AsnRedonnet-Vernhet et al. 1996
Lesch-Nyhan, IVS8 + 4 A->GDe Gregorio et al. 2005
Acardius, skewed in acardiusMasuzaki et al. 2004
FRAX, full mutationWillemsen et al. 2000
Hemophilia ABennett et al. 2008
Table III. Discordant Imprinting/DNA Methylation With Phenotypic Consequences in MZ Twin Pairs
DisorderAffected epigenotypeRefs.
  1. IC2, imprinting center 2; UPD, uniparental disomy; DRD2, dopamine D2 receptor gene; AXIN1, axis inhibitor 1; COMT, catechol-O-methyltransferase gene.

Beckwith–WiedemannFemale: loss of methylation of IC2, 11p15; male: paternal UPD, 11p15Smith et al. 2006
SchizophreniaNon-uniform methylation of DRD2Petronis et al. 2003
Bipolar depressionDiscordant X chromosome methylationRosa et al. 2008
Bipolar depressionUpregulated Wnt pathwayMatigian et al. 2007
Bipolar depressionAberrant DNA methylationKuratomi et al. 2008
Caudal duplicationIncreased methylation of AXIN1 in affected twinOates et al. 2006
Discordant birth weightDiscordant methylation of COMTMill et al. 2006

Repeats play a major role not just in clinical disorders but in zygosity testing. For example, a pair of MZ twins was diagnosed by analysis of many variable number tandem repeats/RFLP analysis from peripheral blood leucocytes. However, use of one VNTR (D183394) showed 260/264 repeats in one twin and 249/260 repeats in the other, thus raising the possibility of dizygosity [Keith and Machin, 1997]. This post-zygotic VNTR discordance calls into question the validity of zygosity testing based on a limited repertoire of VNTRs (see below, methods of zygosity testing).

Using gene chip methods, rare “errors” were reported in SNP analysis between MZ twins of a pair [Montgomery et al., 2005]. Even more confounding is the possibility that these could, in fact, be genuine post-zygotic events.

DNA copy number variations (CNVs) were found within 19 MZ twin pairs with either concordant or discordant phenotype [Bruder et al., 2008].

In a comprehensive survey of epigenetic phenomena [Fraga et al., 2005], age-dependent discordance within MZ twin pairs was found for X-inactivation, DNA methylation and histone acetylation in 35% of 40 twin pairs

In a comprehensive survey of epigenetic phenomena, age-dependent discordance within MZ twin pairs was found for X-inactivation, DNA methylation and histone acetylation in 35% of 40 twin pairs.

(Fig. 3). Competitive hybridization of DNA from pairs showed that discordances mostly occurred in telomeric regions and some gene-rich regions. The discordance presumably represents the emergence of heterogeneity in dividing tissue cells, and does not imply significant change in the “essential genome/epigenome” of the individual. Nevertheless, divergence between twins of an MZ pair is analogous to evolution of species from a common, extinct ancestor, in this case the ephemeral zygotic genome. Interestingly, discordances were greater in pairs who had spent significant periods of their lives apart from one another. This raises the question as to whether epigenetic changes might be caused by environmental discordance, thus squaring the circle of epigenetic versus environmental causation of phenotypic discordance in MZ twins.

Figure 3.

Differential methylation in MZ twin pairs [Fraga et al., 2005]. Degree of discordance in 5-year-old and 50-year-old twin pairs is mapped by comparative genomic hybridization of methylated DNA on chromosomes 1, 3, 12, and 17. Yellow signals seen in the 3-year-old pairs represent similar methylated DNA distribution. However, the 50-year-old twins show widespread discordance in DNA methylation (red = hypomethylation, green = hypermethylation). Figure is a direct reproduction of Figure 3 in Fraga et al. 2005.

Major and Regional Malformations

  • (1)Congenital heart defects. Discordance for presence and severity of cardiac malformation is the rule within MZ twin pairs [Goodship et al., 1995; Fryer, 1996; Hatchwell, 1996; Yamagishi et al., 1998; Vincent et al., 1999; Hillebrand et al., 2000; Lu et al., 2001; Singh et al., 2002b]. This is the case in both the context of 22q11 deletions and multifactorial heart disease.MC twin pairs may experience unequal blood flow through the developing cardiovascular system because of episodes of twin-to-twin transfusion (TTT) that may later manifest as the full TTT syndrome. In a series of 136 MC twin pairs, the prevalence of congenital heart malformation at birth was 3.8% versus 0.56% in singletons [Karatza et al., 2002]. The prevalence in TTT syndrome and in uncomplicated MC twins was 6.9% and 2.3% respectively. The lesions included hypoplastic ventricles and valvular stenoses. In a series of 165 MC twin pairs, 15 (9%) had structural heart defects [Manning and Archer, 2006]. The prevalence was 7% in MC,DA twins, but 4/7 (57%) in MC,MA twins. In 4 of the 15 pairs with heart defects, both twins were affected. In a series of 87 MC twin pairs, 11 (13%) had congenital heart disease [Hidaka et al., 2007]. In two cases, both twins were affected. There was only one pair with TTT syndrome, in which the donor had coarctation of the aorta. In a literature meta-analysis of the prevalence of heart defects among MC,DA twins, the frequency was higher in twins with TTT syndrome than without [Bahtiyar et al., 2007]. Atrial and ventricular septal defects and pulmonary stenosis were the common defects. Individual reports draw attention to flow anomalies in MC twins with and without TTT. In one pair [Young et al., 2001], the MC, monoamniotic (MA) twins were discordant for left heart hypoplasia (LHH); because of inter-twining of the cords, the cord of the unaffected twin was inadvertently occluded. In the other pair [del Río et al., 2005], the MC,MA twins were discordant for TTT and LHH. The cords were not entwined and selective cord occlusion was successful.Issues of selective termination in MC,MA twins are discussed below. Unless proven otherwise, congenital cardiac disease in MC twins is best regarded as a deformation secondary to mechanical (flow) events, and can be counseled accordingly.
  • (2)Neural tube defects (NTDs). Discordance is more common than concordance. A significant proportion of MC twins with NTDs are MA. Reports of discordant anencephalic MC,MA twin pairs [Kriplani et al., 1998; Lim et al., 2005; Middeldorp et al., 2008] include both expectant management and selective termination. It is not clear which management option is best, but it is known that anencephalic MC twins may die in utero [Vandecruys et al., 2006] with fetal demise of the normal co-twin in the majority of MC cases. A concordant pair of MC,MA anencephalic fetuses has been reported [Hansen and Donnenfeld, 1997]. A pair of discordant MZ twins with isolated occipital encephalocele has been reported [Djientcheu et al., 2006], whereas another pair were MC,MA and concordant [Ertunc et al., 2005].
  • (3)Body stalk anomaly. This has been described in four pairs of MZ twins; in two reports there was concordance, but no chorion status was reported [Shih et al., 1996; Aksoy et al., 2000]; two discordant pairs are also reported [Daskalakis and Nicolaides, 2002; Vidaeff et al., 2005], of which the latter were MC,MA.
  • (4)Caudal mesodermal defects. MZ twins concordant for omphalocele, exstrophy, imperforate anus, spinal defects (OEIS) have been reported [Lee et al., 1999]. In addition, MZ twins discordant for caudal duplication have been described [Kroes et al., 2002]. MC,MA twins discordant for urorectal septum malformation have been reported [Achiron et al., 2000]. MZ twins discordant for Mayer–Rokitansky–Kuster–Hauser syndrome have been described [Steinkampf et al., 2000]. A pair of MC,DA twins were discordant for posterior urethral valves [Sutherland, 1999]. Two more cases of MC twins discordant for lower urinary tract obstruction were reported [Sepulveda et al., 2005]. One pair were MC,DA and the other MC,MA. Pregnancies were managed by serial vesicocentesis and the normal co-twins survived. The “masking” effect of discordant urinary tract malformation in MC,MA twins continues to be reported. Oligohydramnios results in pulmonary hypoplasia, which is the lethal effect of urinary tract malformation when there is only one fetus per amniotic cavity. Urine production by the normal co-twin protects the affected MC,MA twin from developing Potter syndrome [Klinger et al., 1997; LiVoti et al., 1998; Perez-Brayfield et al., 1998]. Imperforate anus in discordant MZ twins has been reported [Kubiak and Upadhyay, 2005].

Other Examples of Discordance

  • (1)Discordance for sex, gender, gonadal function, sexual preference. A pair of 17-year-old MZ twins, raised as females, had 46,XY karyotypes. One twin had gonadal agenesis, whereas the other had pure gonadal dysgenesis with dysgerminoma [Chen et al., 2006]. A pair of MZ twins with discordant female/male phenotypes both had 46,XY karyotypes, and no mutation was found in the SRY conserved motif. The male had mixed gonadal dysgenesis and the female had pure gonadal dysgenesis [Somkuti et al., 2000]. Ten MZ pairs have been reported as discordant for premature ovarian failure, with inter-twin transplantation, resulting in pregnancies in eight cases [Silber et al., 2008]. Two MZ twin pairs have been reported who were discordant for female-to-male transsexualism [Segal, 2006]. In a study of seven pairs of MZ twins discordant for sexual orientation (lesbian/heterosexual), the lesbian twins had significantly lower 2nd/4th digit length ratios on both hands. In a control group of five lesbian/lesbian MZ twin pairs, no such difference was found [Hall and Love, 2003].
  • (2)Phenotypic discordance in miscellaneous syndromes and diseases. A wide variety of conditions has been described in which there are examples of phenotypic discordance within MZ twin pairs, including:Proteus syndrome, male [Biesecker et al., 1998; Brockmann et al., 2008]; tibial aplasia with ectrodactyly, female [Dayer et al., 2007]; lymphedema-distichiasis [Kumar et al., 2007]; microphthalmia, syndromic 3, male [Zenteno et al., 2006]; Melnick–Needles syndrome, female [Robertson et al., 2006]; Klippel–Feil syndrome, female [Toyoshima et al., 2006]; Klippel–Trenaunay syndrome, male [Hofer et al., 2005]; frontonasal dysplasia, five discordant pairs [Mohammed et al., 2004]; Alagille syndrome 1 [Kamath et al., 2002]; Leopard syndrome 1 [Rudolph et al., 2001]; Fryns syndrome [Vargas et al., 2000]; Joubert syndrome, female [Raynes et al., 1999]; orofaciodigital syndrome 1, female [Shotelersuk et al., 1999]; trichorhinophalangeal syndrome 1, female [Naselli et al., 1998]; Sotos syndrome [Brown et al., 1998]; Aicardi syndrome, female [Costa et al., 1997]; Say syndrome [Ashton-Prolla and Félix, 1997; Elçioğlu and Berry, 1997]; Schimmelpenning–Feuerstein–Mims syndrome [Schworm et al., 1996]; hereditary pancreatitis, three pairs [Amann et al., 2001; Munar-Qués et al., 1999; Ando et al., 2000; Holmgren et al., 2004]; van der Woude syndrome [Kondo et al., 2002]; Rett syndrome [Subramaniam et al., 1997].
  • (3)Cerebral and visceral organ laterality. Excluding the constraints of axial orientation in conjoined twins, MZ twin formation may occur after the original three axial planes have already been defined, at least in molecular terms. If this is so, the only tolerable plane of “splitting” would be sagittal, since this delivers cranial/caudal and dorsal/ventral components into each twin. Therefore, the twin-on-their-right would need to regenerate a new left side, and vice versa. This may be the basis for discordant cerebral laterality and handedness in MZ twin pairs. Analysis of cerebral dominance in language tasks by functional MRI (fMRI) in 25 MZ twin pairs is summarized in Table IV [Sommer et al., 2002]. Twelve pairs were concordantly right-handed (RH/RH) and 13 pairs were discordantly handed, that is, right-handed/non-right-handed (RH/NRH). Chorionicity was known in 20 pairs. There was an excess of RH/NRH pairs among the MC twins. Likewise, right cerebral dominance was only found in the RH/NRH MC pairs (and one of unknown chorionicity). As expected, the mean within-pair lateralization difference for language function was also highest in the RH/NRH MC pairs. RH/RH pairs had a female excess of 58%, whereas the RH/NRH group had a male excess of 62%. These data support the concept that axial determination is occurring at the same time as the “decision” for MC MZ twinning. In a study of 20 female MZ twin pairs discordant for handedness, the right-handers showed stronger lateralization than the left-handers, but chorionicity was not analyzed [Gurd et al., 2006]. In tests of discordant manual dexterity, no difference in prevalence of discordance was found between DZ and MZ twins, nor was there a chorion effect [Carlier et al., 1996]. However, other researchers have been unable to confirm discordant handedness in MZ twins [Derom et al., 1996]. This author knows of only one report in which MZ twins are discordant for visceral situs [Noone et al., 1999]. This occurred within the context of ciliary dyskinesia, in which absence of ciliary movement permits situs solitus and situs inversus in equal proportions. One female twin had situs solitus and the other situs inversus totalis. It is not clear whether the discordance occurred randomly, as a stochastic event, or whether the twinning process itself was also involved (see discussion above regarding sagittal plane of splitting).
  • (4)Neurodegenerative and mental disorders and brain imaging. MZ twin pairs show more frequent discordance for mental disorders than for any other kind of discordance listed in this summary paper. Careful analysis of brain structure and function in vivo and post-mortem may therefore offer valuable etiological clues to these disorders in the wider patient population.

    MZ twin pairs show more frequent discordance for mental disorders than for any other kind of discordance listed in this summary paper. Careful analysis of brain structure and function in vivo and post-mortem may therefore offer valuable etiological clues to these disorders in the wider patient population.

    A few selected examples are given here. In three MZ twin pairs with Alzheimer disease, there was discordance for age of onset (4–18 years), clinical characteristics and neuropathology [Brickell et al., 2007]. In MZ twin pairs discordant for obsessive–compulsive disorder, fMRI showed significant decreased brain activation during planning in the affected twins [den Braber et al., 2008]. Ten pairs of MZ twins discordant for risk of anxiety and depression were found to have reduced temporal lobe volumes in the affected twin [deGeus et al., 2007]. MZ twins discordant for Tourette syndrome show discordant D2 dopamine receptor binding in the head of the caudate nucleus [Wolf et al., 1996].
  • (5)Neoplastic diseases. In a family with multiple endocrine neoplasia type 1 (MEN1), MZ twins inherited from their mother a novel mutation in the MEN1 gene; one twin had hyperparathyroidism and an insulinoma, whereas the other twin was asymptomatic [Concolino et al., 2008]. In a pair of female MZ twins with WAGR syndrome, one twin developed a Wilms' tumor at the age of 19 months, whereas the other twin was tumor-free to the age of 6 years [Brémond-Gignac et al., 2005]. A pair of MZ twins were discordant for retinoblastoma. One twin had multifocal bilateral retinoblastomas, whereas the other twin was free of disease. No mutations were found in the retinoblastoma gene [Marcus et al., 1999]. Both concordance and discordance for common neoplasms (breast, colon, prostate, lung) are known in MZ twins. Concordance for neoplasia is one of the strongest arguments for testing when zygosity is in doubt (see below). Post-zygotic epigenetic variation within MZ twin pairs could account for the less than 100% concordance for neoplasm with known molecular basis, for example, BWS [Cooper et al., 2005].
  • (6)Disruptions. Reports continue to appear concerning discordance for disruptions, for example, Goldenhar syndrome [Verona et al., 2006], VACTERL association [Becker et al., 2005; Camacho et al., 2008] and vasculogenic fetal akinesia [Ho, 2000]. It seems likely that these anomalies (as well as flow-type congenital hearts defects) are caused by temporary hypotension/hypoperfusion of major territories as the result of twin transfusional events that do not develop into TTT. It has been suggested that discordance for several types of congenital brain anomalies (focal cortical dysgenesis, periventricular nodular heterotopias) might also in some cases be caused by vascular instability in MC twins [Sisodiya et al., 1999; Brodtkorb et al., 2000; Briellmann et al., 2001].
  • (7)Same intrauterine environment, MZ genome, discordance for congenital heart block. In two remarkable pairs [Cooley et al., 1997], the MZ twins were discordant for congenital complete heart block whose mothers had anti-Ro 52 and anti-Ro 60 antibodies.
  • (8)Masked discordance. The protective effect of a normal MA co-twin against the effects of oligohydramnios in a co-twin with anuria has already been discussed. The presence of inter-fetal vascular anastomoses in MC twin placentas can lead to subtle effects that mask twin discordance. There are reports of MZ twins discordant for thyroid dysgenesis. In fact, there are no known cases in which MZ twins are concordant for thyroid dysgenesis. In the presence of vascular anastomoses, the normal MC co-twin transfuses TSH and thyroid hormones into the thyroid dysgenetic co-twin, with protection from the prenatal onset of hypothyroidism and production of a false-positive newborn TSH screen result [Perry et al., 2002]. The dysgenetic twins have later onset neonatal hypothyroidism, with the potential for lasting effects. It has therefore been recommended that all same-sex twins should have a follow-up TSH screen at 14 days.Transfusion of bone marrow hematopoietic precursor cells has been implicated in childhood acute lymphoblastic leukemia in MZ twins [Maia et al., 2001; Mori et al., 2002]. There is modest concordance (level of 5%) in MZ twins, with protracted latency in some cases. However, retrospective analysis of newborn bloodspots reveals the presence of common leukemia fusion genes (e.g., TEL-AML1 and AML-ETO) in both twins.
  • (9)Special considerations for MA,MC MZ twins. The environment for MA MZ fetuses is particularly hostile because of the almost universal presence of braided cords, the presence of which is prenatally diagnostic of MA twinning, regardless of uncertainties over presence/absence of septal membranes, which permits secure prenatal diagnosis without recourse to dye studies. Selective termination is difficult, because definitive identification of the cord of the affected twin is required [Middeldorp et al., 2008]. MA twins show both concordance and discordance for major regional malformations. In a report of 12 pairs of MA twins diagnosed in the first trimester, 4 pairs were conjoined and 4 pairs were discordant for major malformation: kyphoscoliosis, anencephaly, body stalk defect, diaphragmatic hernia [Sebire et al., 2000]. Of the remaining four structurally normal pairs, three fetuses died of cord entanglement.
  • (10)Phenotypic discordance in single gene disorders. MZ twins with neurofibromatosis II have variable expression in terms of onset and location of tumors (e.g., intracranial tumors other than vestibular schwannomas) [Baser et al., 1996]. Similar considerations apply to MZ twins with tuberous sclerosis [Humphrey et al., 2004].
  • (11)Miscellaneous disorders. Concordance for Crohn disease was 63.6% and 3.6% respectively in MZ and DZ twins [Jess et al., 2005]. Forty-four percent of the twin pairs had NOD2/CARD15 mutations. Taking advantage of 11 MZ pairs discordant for rheumatoid arthritis (RA), cDNA derived from lymphoblastoid B cell lines was hybridized with a 20,000 element microarray chip [Haas et al., 2006]. The three most significantly over-expressed genes were laeverin (an enzyme with sequence homology to CD13), 11 beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (an angiogenic factor). The products of these genes, previously uncharacterized in RA, were expressed in RA synovial tissues. These examples show the potential for involving discordant MZ twins in the investigation of complex traits [Martin et al., 1997].
Table IV. Cerebral Dominance in MZ Twin Pairs Analyzed by Handedness and Chorionicity [Sommer et al., 2002]
HandednessTotalMC, n (%)DC, n (%)Not known, n (%)
RH/RH125 (42)4 (33)3 (25)
Mean lateralization index0.70.70.770.52
Mean within-pair lateralization difference0.120.090.0650.33
Female7 (58)4 (80)1 (25)2 (67)
Dominant right hemisphere0000
RH/NRH138 (62)3 (23)2 (15)
Mean lateralization index0.340.310.720.18
Mean within-pair lateralization difference0.350.530.060.14
Female5 (38)2 (25)1 (33)2 (100)
Dominant right hemisphere5 (38)4 (50)01 (50)


Because MZ twins are never “identical,” attempts have been made to explain twins who lie between the extremes of theoretical “MZ identity” and indubitably DZ twins who have the expected similarity of first degree relatives. Not all of these explanations have been satisfactory, and it remains true that, for the reasons discussed above, most MZ twins are best described as being too similar to be DZ. Furthermore, intermediate forms of twinning have usually been identified because they are MC, but are opposite-sexed and/or with significantly abnormal phenotypes; hence these intermediate forms are invoked to explain some twins pairs with less than complete “identity” in phenotypically normal same-sexed twins, most of whom, in reality, are nevertheless MZ. Four types of intermediate twinning have been described. The majority are chimeric in blood and involve division of products of oogenesis or of the early zygote such as to compromise the volume of oocytic cytoplasm generally regarded as optimal for normal early development; the chromosomal status of binovular ovarian follicles is not known. Alternatively, fusion of trophoblasts results in MC twinning although the twins are DZ. All are at least trigametic, always with different paternal haploid contributions, and sometimes with different maternal contributions also (i.e., true DZ twins).

  • (1)Polar body twinning. This is the most frequently cited hypothesis for similar but not “identical” twins, but it is more subtle than might at first appear. In terms of heterozygous maternal alleles, the first polar body is the completely reciprocal opposite of the secondary oocyte and ovum, because of chromosomal segregation, with or without recombination. The first polar body contains 23 whole chromosomes (46 chromatids) and is, in effect, diploid; any fertilization will result in a triploid twin fetus [Bieber et al., 1981]. Normally, the polar body degenerates, but fertilization of the polar body can lead to a twin pregnancy. A pair reported by Bieber et al. involved a triploid (necessarily MC), 69,XXX acardiac fetus and a normal diploid, 46,XY MC chimeric twin, both derived from the same primary oocyte. Even if a mechanism existed to haploidize the first polar body prior to fertilization, the resulting diploid twins would be less similar than DZ twins because of reciprocal allocation of heterozygous maternal alleles to the secondary oocyte/ovum and the first polar body. In the case of second polar body fertilization, recombination would have resulted in reciprocal dissimilarity for heterozygous alleles in the recombined segments such that the maternal contributions to the two zygotes would not be identical. However, the twins would probably have phenotypes intermediate between MZ and DZ, depending on the degree of dissimilarity of the paternal genomic contributions. No such twin pairs have so far been described.
  • (2)Parthenogenetic activation and cleavage of the ovum would result in identical maternal genomic contribution if both parthenogenetic components are fertilized. This is one possible explanation for a pair of opposite-sexed discordant chimeric twins, one of whom is a true hermaphrodite

    Parthenogenetic activation and cleavage of the ovum would result in identical maternal genomic contribution if both parthenogenetic components are fertilized. This is one possible explanation for a pair of opposite-sexed discordant chimeric twins, one of whom is a true hermaphrodite.

    [Souter et al., 2007] (Fig. 4). In the pair reported by Souter et al., 2007, the twins (spontaneously conceived, but of unknown chorionicity) were assigned opposite genders, and both twins had 46,XX and 46,XY cell lines in peripheral blood and skin fibroblasts, as well as XX, XY and X cells found by FISH in touch preparations from gonads of both twins. DNA markers and SNPs showed that both twins had inherited the same informative maternal alleles and SNPs (100%), and 52% of same paternal SNPs. In some informative loci, three different alleles were represented. The fact that 100% of the tested maternal alleles were held in common by both twins excludes second polar body twinning. The twinning process involved allocation of both cell lines into each chimeric twin. The female was a true hermaphrodite, with ambiguous external genitalia and bilateral ovotestes, whereas the male had normal external genitalia and testes. In this explanation, the two products of parthenogenetic cleavage of the ovum were fertilized by X- and Y-bearing spermatozoa, sharing only the expected 50% of the paternal genome. This was followed by aggregation of the cell lines, followed by twinning.
  • (3)Dispermic fertilization followed by diploidization of the triploid zygote [Golubovsky, 2003] is another possible explanation for the twins described by Souter et al. 2007. In this mechanism, two cell lines emerge from the dispermic triploid zygote; one cell line has extruded one male (X-bearing) pronucleus, whereas the other cell line has extruded the other male (Y-bearing) pronucleus. Aggregation of the cells is followed by chimeric twinning.
  • (4)MC dizygotic twin pairs with fusion of trophoblasts have been reported by several authors. These cases represent a particular type of chimerism, limited to blood and presumably placenta; but rather than the chimeric cell lines being incorporated into one body, the twinning process proceeded, with paradoxical MC placentation. All but one of the pairs was ascertained because they were opposite-sexed and/or had genital or other anomalies. Because of the manufacture of an MC placenta, most DZ twin pairs showed blood lymphocyte chimerism and/or blood group chimerism via inter-fetal anastomoses, but there was no chimerism in fetal solid tissues, implying that the cells destined for the inner cell masses did not aggregate and mix prior to twinning; when studied, even trophoblastic mixing was minimal [Souter et al., 2003]. The first such pair fully studied [Redline, 2003; Souter et al., 2003] (Fig. 5) resulted from in vitro fertilization (IVF) with three blastocysts implanted, one of which did not survive in early gestation. Ultrasound showed opposite sexed twins with MC placentation, later confirmed by pathology (with presence of inter-fetal vascular anastomoses). The twins were delivered at 36 weeks, a male weighing 2,114 g and a female weighing 2,183 g. External and internal genitalia were unambiguous. In situ hybridization of placental tissues for X and Y signals showed Y signals in solid placental tissues in the male zone and two X signals in the female zone of the placenta respectively. Amniotic cells were not chimeric. Peripheral blood lymphocyte studies at 3 months of age showed 46,XX/46,XY chimerism in both twins, with a predominance of 46,XY cells. Initial DNA studies were interpreted as MZ, because of the predominance of male cells in peripheral blood, but minor bands for female alleles were later discovered. DNA studies of skin fibroblasts showed pure dizygosity without evidence of chimerism. A similar twin pair was reported [Williams et al., 2004], conceived by IVF and intra-cytoplasmic sperm injection (ICSI) with assisted hatching and transfer of two embryos. The placenta was MC by ultrasound, confirmed by pathology. Delivery at 27 weeks for pregnancy-induced hypertension and severe fetal growth discordance resulted in the birth of a 654 g female with ambiguous genitalia, and a 1,146 g male. The female had a velamentous cord insertion. Ovaries were subsequently found in bilateral inguinal hernias. Peripheral blood lymphocyte karyotyping showed 46,XX/46,XY chimerism, with a majority of 46,XX cells in both twins. Microsatellite marker studies of buccal swab cells showed clear dizygosity without chimerism. FISH studies of buccal cells, skin fibroblasts and ovarian tissue likewise showed no chimerism.An earlier report [Kühl-Burmeister et al., 2000] an incomplete study documented blood chimerism in a dizygotic triplet pregnancy resulting from IVF with implantation of three embryos. Chorionicity was unknown. Born at 32 weeks, the female weighed 1,520 g, and the males 2,050 and 1,940 g. There were no genital abnormalities. Blood chimerism in the female was detected during presurgical blood typing (blood group A/AB). Further investigation showed the same blood chimerism in both male co-triplets. Using X- and Y-probes by in situ hybridization on peripheral blood lymphocytes, the female was 84% XY, and the males were 92% and 89% XY. No DNA single locus studies were done on buccal cells or fibroblasts from the males, but their serological markers were identical. It seems reasonable to assume that they were MZ because three embryos were implanted. Blood chimerism is best explained by monochorionicity of the MZ males and between the males and the female.A remarkable pair of non-chimeric MC DZ twins has been described [Yoon et al., 2005]. The twins were conceived by IVF/ICSI, and three embryos were transferred but only two gestational sacs were identified. Born at 33 weeks, one male twin weighed 2,660 g and had BWS (glossomegaly, omphalocele, and hemihypertrophy) and a 47,XYY peripheral blood karyotype. The co-twin was a phenotypically normal male, birth weight 2,221 g, with a 46,XY karyotype. Placentation was MC by pathology, but the status of anastomoses was not given. Dizygosity was confirmed by eight DNA single locus probes. There was no evidence of blood chimerism.Five pairs with blood chimerism in MC opposite-sexed Japanese twins have been reported [Miura and Niikawa, 2005]. Three pairs resulted from IVF, one from induced ovulation and artificial insemination and one from ICSI. All were MC by ultrasound, confirmed by pathology in three cases. One had anastomoses, and another pair (originally triplets with selective reduction) had some form of twin transfusion. Blood chimerism was diagnosed by karyotype in four and by ABO blood groups in one. Two tested pairs were negative for chimerism in skin fibroblasts.Alarmingly, a pair of spontaneously conceived DZ MC male twins has been reported [Shalev et al., 2006]. Amniocentesis for advanced maternal age showed 46,XY and 47,XY,+21 fetuses, which could plausibly be explained by MZ mosaicism. However, molecular studies showed dizygosity. There was cordocentesis blood chimerism in both twins.A pair of phenotypically normal MC (ultrasound and pathology) male twins, conceived by ovulation induction, were born at 34 weeks [Aoki et al., 2006]. Routine blood grouping showed blood group (AB/B) chimerism. Blood chimerism was confirmed in five of nine DNA microsatellite loci. These same probes showed pure dizygosity in hair root cells.Another IVF pregnancy with transfer of two embryos [Walker et al., 2007] resulted in MC male fetuses who were only diagnosed as DZ because buccal cells were analyzed as part of a research protocol. There was blood chimerism.A pair of DZ MC unlike-sexed twins developed TTT [Quintero et al., 2003]. Mode of conception was not stated. The male twin was the donor. Appropriate but occluded anastomoses were seen on pathology examination following laser therapy. 46,XX and 46,XY karyotypes were found in cultured fibroblasts. Presumably there was blood chimerism.A further pair of male/female MC DZ twins with blood chimerism has been described [Ginsberg et al., 2005]. The twins were conceived by ovulation induction, amniocentesis revealed 46,XX and 46,XY karyotypes.In summary (Table V), 13 pairs of MC DZ twins have so far been reported. One pair was conceived spontaneously; four pairs were like-sexed, and dizygosity was diagnosed because of phenotypic abnormalities or through research protocols that are not standard clinical practice in twin pregnancy. All had blood chimerism but no chimerism of solid fetal tissues. All were truly tetragametic and DZ. It remains to be seen whether any of these four mechanisms would be investigated in MC and DC like-sexed twins who were phenotypically normal, but whose degree of similarity was thought to lie intermediate between MZ and DZ. In a series of 110 consecutive MC twin pairs tested to confirm zygosity, all were like-sexed and none showed results suggesting chimerism in placental tissue tested with low stringency VNTR probes [Machin et al., 1995]. It seems that MC DZ twins are rare. The practical implication of MC DZ twins is that they are expected to be immunologically tolerant for transplantation, at least to some degree, and possibly not symmetrically tolerant

    The practical implication of MC DZ twins is that they are expected to be immunologically tolerant for transplantation, at least to some degree, and possibly not symmetrically tolerant.

    [Boklage, 2006].
Figure 4.

Diagrammatic representation of possible events leading to trigametic blood chimeric DZ twin pair [Souter et al., 2007]. The ovum cleaves by parthenogenesis, with products that are genetically identical. Each maternal product is fertilized, and, in this twin pair, X- and Y-bearing sperm are involved. Cells derived from the 46,XX and 46,XY zygotes aggregate during early proliferation, resulting in twins who differ in the ratio of constituent 46,XX and 46,XY cells. The high ratio of 46,XX to 46,XY cells in one twin results in true hermaphroditism. Figure is redrawn and modified from Figure 2A [Souter et al., 2007].

Figure 5.

Origin of DZ but MC twins in an IVF pregnancy [Redline, 2003; Souter et al., 2003]. A: DZ twins are considered always to be DC. B. MZ twins may be DC, MC, DA or MC,MA. C. However, the twin pair described by Souter et al. 2003 were DZ; there was no admixture of inner cell masses but trophoblasts aggregated to form an (acquired) MC placenta. The MC placenta resulted in blood chimerism for the male and female cell lines, but there was no chimerism in fixed cells derived from the inner cell masses. Figure reproduced with permission from Redline 2003.

Table V. Thirteen Reported Pairs of MC DZ Twins
  • BWS, Beckwith–Wiedemann syndrome; TESE, testicular sperm extraction.

  • a

    Diagnosed by research protocols.

Souter et al. 2003F, MIVF, 3 embryosRegional placental and blood chimerism by genotyping
Williams et al. 2004F, MIVF/ICSI/hatching, 2 embryosBlood chimerism by genotyping; ovaries in inguinal hernias
Kühl-Burmeister et al. 2000Triplets, F, M, MIVF, 3 embryosChorion not established. Blood group chimerism at presurgical blood grouping
Yoon et al. 2005M, 47,XXY with BWS; M, 46,XYIVF/ICSI, 3 embryosDZ results from investigation of BWS, 47,XXY

Miura and Niikawa 2005

, case 1
F, MIVFLeukocyte genotyping
Case 2F, MOvulation induction/AIMethodology not stated
Case 3F, M; triplets with reductionTESE/ICSITTT. Leukocyte genotyping
Case 4F, MIVFMethodology not stated
Case 5F, MIVFChimeric blood group
Shalev et al. 2006M, 47,XY,+21, M, 46,XYSpontaneousInvestigation of heterokaryotypia
Aoki et al. 2006M, MOvulation inductionBlood group chimerisma
Walker et al. 2007M, MIVF, 2 embryosBuccal cell genotypinga
Quintero et al. 2003F, MNot statedTTT, male donor. Presumed blood chimerism. Fibroblast genotyping.
Ginsberg et al. 2005F, MOvulation inductionDZ by amniocentesis


Whereas it is recognized that DZ twinning can be familial, it is usually assumed that MZ twinning is not familial and that the etiology of the process is obscure. In reality, there are two factors that negate this view. First, MZ twinning can be familial [Harvey et al., 1977; Shapiro et al., 1978; Hamamy et al., 2004; Machin, 2009]. Inheritance appears autosomal dominant in many cases. Some new examples are illustrated and discussed in the accompanying paper [Machin, 2009]. Genotyping in these families might uncover candidate genes for MZ twinning that have, for example, more obvious roles in cell adhesion. Familial MC and DC MZ twins may be inherited via different genes.

Second, the prevalence of spontaneous MZ twinning (unlike DZ twinning) appears to be constant by geography and ethnicity, at around 3.5/1,000 births [Bomsel-Helmreich and Al Mufti, 2005]. MZ twinning is an intrinsic feature of human reproduction. Because of the many disadvantages of MC placentation [Barigye et al., 2005], it is not clear what the evolutionary advantage of MZ twinning might be. It may merely be a phenocopy of DZ twinning, whose advantage would be a more rapid recovery per completed pregnancy following population loss through war, famine or disease. After all, Mengele's interest in twins was spurred by the belief that the Lebensraum could best be quickly repopulated by increased twin birth [Nyiszli, 2001].

Third, the female excess in MZ twinning increases towards the “extremes” of MZ twinning. Thus, the female excess in least in DC MZ twins and greatest in MC,MA twins. Fully 75% of conjoined twins are female. There is an excess of females among MZ triplets. Females predominate among MZ twins discordant for BWS. These facts might merely indicate an increased male fetal vulnerability, or may indicate differences in the rate of early development in males and females, but they are quite striking among MZ twins.


Although this review has sought to modify the over-simplistic concept of “identical” twins, the fact remains that MZ twins are concordant to a great degree for traits, disorders and diseases that may develop metachronously; preventive and early therapy can be considered. For instance, it is valid to consider that MZ twin women have four breasts when calculating cancer risks [Peto and Mack, 2000]. The risk for a breast cancer patient of developing (non-metastatic) new primary cancer in the opposite breast is about 0.8% per annum, whereas the risk that an MZ twin of that breast cancer patient will develop breast cancer is 1.3% per annum, that is, 0.7% per breast. In other words, the four breasts are equally susceptible to cancer development. This offers opportunities for preventive medical (tamoxifen) or surgical therapy. Similar considerations apply to several other cancers.

As previously mentioned, MZ twins can often act as solid organ transplant recipient/donor pairs without possibility of transplant rejection [St Clair et al., 1998; Silber et al., 2008]. To some degree, this may apply also to DZ MC twins [Boklage, 2006].

Many workers in the field of twin medical services and research feel that all twin pairs have the right to know their zygosity. My experience is that many MZ twin pairs are designated as DZ because of zealous expectations that MZ twins should be “identical.” This can have adverse results in the clinical situations described above, especially in transplantation

Many workers in the field of twin medical services and research feel that all twin pairs have the right to know their zygosity. My experience is that many MZ twin pairs are designated as DZ because of zealous expectations that MZ twins should be “identical.” This can have adverse results in the clinical situations described above, especially in transplantation.

[St Clair et al., 1998].

In view of the above discussion, it is clear that determination of zygosity is not as simple as was once thought. Nor is zygosity testing widely available. A number of laboratories offer testing based on buccal cell samples, using 8 or so variable number tandem repeats (VNTRs). But MZ twins can be quite dissimilar via a number of mechanisms: genetic, epigenetic and prenatal environmental; chimeric MC DZ twins must also be considered. When using any method for zygosity testing, DZ twinning is usually diagnosed when a genotypic difference is detected, but MZ twinning can only be diagnosed statistically by failing to detect genotypic difference after testing to a reasonable degree of exhaustiveness, compatible with reasonable time and cost. There is no positive “litmus” test for MZ twinning. However, post-zygotic mutation at loci and sequences used for genotyping may result in designation of MZ twins as DZ. MC status is more like a screening tool, which should be implemented in practice, rather than a safely diagnostic modality of MZ twinning. Chorionicity determination, like any screen, clearly has false positives and negatives. For example, it is clearly wrong when the twins are unlike-sexed and have been shown to be chimeric.

Using placentation, for present purposes, seems reasonable to continue to designate spontaneously conceived MC twins as MZ; but DZ MC chimerism should be considered if the twins clearly have or develop a DZ phenotype, especially when reproduction has been artificial. One-third of MZ twins are DC, so like-sexed DC twins should not be assumed to be DZ.

In genotyping, the most common method is to extract markers from buccal samples by PCR, and analyze eight variable microsatellite loci. Any discordant results are thought to indicate DZ twins. However, post-zygotic mutations in repeat number can complicate this issue [Keith and Machin, 1997]. At present, it is not known how many VNTR single locus discordances are tolerable within MZ twinning, and much more needs to be discovered before genotypic criteria for zygosity diagnosis can be standardized. In doubtful cases, testing can be repeated using low stringency methods, although these are not available from small DNA samples. Misleading results can be obtained from blood DNA samples in MC twins. For prenatal diagnosis, double amniocentesis is preferable to chorionic villus sampling. Again, cordocentesis samples can be misleading in MC twins. In testing MZ twins discordant for suspected genetic and developmental disorders, blood mosaicism can compromise results unless it is certain that the twins were DC.