By Jeffrey M Craig, Kate Murphy, Tessa L Cutler, Monica Rankin, Jane Denton, Deborah Schofield, Margaret Otlowski, Mark P Umstad, Susan Carrick
- Monozygotic (MZ) twins are genetically identical, while dizygotic (DZ) twins are as genetically similar as non-twin siblings.
- Accurate knowledge of zygosity is important for medical, social, financial and ethical reasons, but incorrect assumptions mean twins are often misclassified.
- Chorionicity, sex and genetic zygosity testing can be used to differentiate between MZ and DZ twins.
- Medical professionals should receive more education about twin zygosity; parents should be informed of their twins’ chorionicity and encouraged to undertake zygosity testing; twins and their families should be educated about twin zygosity, its testing and its implications.
- The cost of zygosity testing should be reduced, and parents of twins should receive higher family payments to offset costs and increase testing rates.
Zygosity traditionally refers to the degree of genetic similarity between DNA sequences inherited from each parent. For twins, zygosity refers to the degree of genetic similarity within each pair; monozygotic (identical) twins have the same genes, whereas dizygotic twins differ genetically as much as any pair of siblings. The same applies to higher-order multiple births. Hence, in this paper, for ease of reading, the word “twins” refers to all multiples.
Accurate knowledge of zygosity is important for a range of medical reasons, including the estimation of disease risk and tissue compatibility. It is also important for social reasons, including defining social relationships, personal identity, determining the likelihood of further twin pregnancies, and avoiding embarrassment or emotional stress over uncertainty about twins’ genetic identities. Yet, despite medical health advancements, incorrect information on whether twins are monozygotic or dizygotic is still given to twins and their parents.
In this article we focus on zygosity as an important research, community and public health issue. We look at types of twins and what we know about zygosity. We review historical and contemporary data and relevant commentary. We also look at the reasons why zygosity testing is so important – medically, socially, financially and ethically – and at the options for improving our understanding of zygosity. We conclude with recommendations for universal, affordable, targeted testing and recording of zygosity in multiple births. We also make recommendations for increased awareness and education, particularly within the healthcare setting and for dialogue with government.
Suggested citation: Craig, J.M., K. Murphy, T.L. Cutler, M. Rankin, J. Denton, D. Schofield, M. Otlowski, M.P. Umstad, and S. Carrick, The importance of zygosity knowledge for twins and science, Conversations in Twins Research, Twins Research Australia, Melbourne, 2019, https://www.twins.org.au/2019/02/14/importance-of-zygosity-knowledge-for-twins-and-science/
Monozygotic (MZ) twins result from the splitting of a single embryo early in gestation and are genetically the same [1, 2]. Dizygotic (DZ) twins result from two separate fertilisation events and are as genetically similar as non-twin siblings. In some cases, zygosity can be determined from information about the sex and chorionicity (number of placentas; see below for more details) of twins. In other cases, only a genetic test for zygosity can provide accurate information.
We  and other researchers [4, 5] have found that a substantial proportion of parents and twins are misinformed about their zygosity status, and that this misinformation may come from parents or medical professionals. This misinformation is often based on incorrect assumptions, such as the belief that only DZ twins can have two placentas/chorions, and that twins can only be MZ if they are physically and behaviourally identical.
The determination of zygosity at birth of all same-sex twins has been recommended by geneticists and epidemiologists since the early 1990s [6-9]. This recommendation was made because the information is useful in determining organ transplant compatibility, for research into the biology and pathology of MZ twinning, and for the investigation of discordance and concordance for many genetic diseases and disorders with multifactorial inheritance. Knowledge of zygosity also provides twins and their families with understanding of their identity and relationships. Additionally, increased education for health professionals and the community will help ensure that twins have the same opportunity to participate in research as non-twins; to do so requires clarity about their zygosity.
In this article we explain the most accurate methods for determining zygosity and examine the most common misconceptions that lead to inaccurate zygosity determination. The value of zygosity knowledge for twins and for science is explored in detail and recommendations on how to achieve more accurate zygosity knowledge are made.
There are two main types of twins (Figure 1). DZ (fraternal) twins result from two separate fertilisations. Approximately half are the same sex. Each twin has its own placenta, which is continuous with the chorion (outer membrane) and its own amnion (inner sac) with rare exceptions, and each pair is as genetically similar as any pair of siblings. MZ (identical) twins result from the splitting of a single embryo early in gestation. Our current understanding of MZ twins indicates that approximately one-third of MZ twins have separate placentas. Approximately two-thirds of MZ twins share a single placenta despite maintaining their own amnion, umbilical cord and share of the placental mass. Almost all MZ twins are the same sex and genetically identical. Sharing a placenta results in vascular (blood vessel) connections in all monochorionic twin pairs, with one fifth of these exchanging significant amounts of blood, which may cause injury and possibly death to both twins if left untreated.
|Figure 1. The formation of the main types of twins
Three parameters are used to differentiate between MZ and DZ twins: chorionicity, sex and genetic zygosity testing (Figure 2) [1, 10, 11]. Blood type may also be useful as differing blood types indicates dizygosity. Chorionicity is most accurately determined by the thickness of the membrane between the twins, most accurately before 14 weeks gestation (Figure 3) . In dichorionic twins a thick, lambda-shaped () membrane separates the twins. In monochorionic twins this membrane is much thinner and joins the placenta to form a T shape. Ultrasounds taken later in gestation are less reliable due to the increased crowding of twins in the uterus.
|Figure 2. Decision tree for determining zygosity in twins
|Figure 3. Determination of chorionicity at birth
Physical examination of the inter-twin membranes at birth should also be used to determine chorionicity (Figure 3). This will provide confirmation of early ultrasound data and determination of chorionicity in twins without early ultrasound information. Again, dichorionic membranes are thick, opaque and can be pulled apart, whereas monochorionic membranes are thin and semi-transparent.
When sex and chorionicity are known, we can label different-sex twins as DZ and same-sex monochorionic twins as MZ (with rare exceptions in cases of chromosomal abnormalities) (Figure 2). When blood type is known, we can label twins with different blood types as DZ. If chorionicity and blood type are unknown then a test is required to accurately determine zygosity. Same-sex dichorionic twin pairs with the same blood type also require a test to determine their zygosity.
Zygosity tests usually involve the analysis of 12–16 highly variable genetic markers (DNA “fingerprints”), which are identical in MZ twins and 40–60% identical in DZ twins. All that is needed is a cheek swab, saliva or blood sample, which can be collected by a medical professional or by parents or twins, then sent though the post. The test can be performed quickly and easily at any age, including newborns. In Australia, parents of twins and adult twins who are members of Twins Research Australia currently pay AU$120 for a zygosity test (https://www.twins.org.au/twins-and-families/zygosity-testing).
The steps in Figure 2 can be used to accurately determine twin zygosity in almost all pairs. However, our studies have found that misclassification of twins occurs in up to one third of pairs due to incorrect advice from medical professionals or the incorrect assumptions of parents or twins themselves . The most common incorrect assumptions are that (1) all dichorionic twin pairs are DZ and (2) genetically-identical MZ twins must have identical phenotypes.
All DZ twin pairs are dichorionic; however, a third of MZ twin pairs are also dichorionic. The belief that all dichorionic twins are DZ can lead to a third of MZ twins being incorrectly categorised as DZ. In addition, the number of chorions can be difficult to determine from ultrasounds. Dichorionic twin pairs may have fused placentas which can be mistaken for a single placenta without careful examination at birth. This can lead to misclassification of DZ pairs as MZ.
Monozygotic twins usually look and behave more similarly than DZ twins due to their greater genetic similarity. However, MZ twins are often not physically and behaviourally identical due to differences in the environments they encounter from conception onwards. Following the splitting of the embryo, with each MZ foetus having the same genes, uterine environmental factors can cause changes to the way the genes work, which can make the twins different. Ninety-nine per cent of twins have their own amnion, umbilical cord and (share of the) placenta (Figure 1), all of which can differ morphologically within pairs. These factors means that many twin pairs begin to experience different environments even when they are in the womb. MZ twins and their parents report that they are acutely aware of any differences in physical appearance (e.g., height), medical conditions (e.g., allergy discordance) and even behaviour (e.g., sporting interests) between the twins and that these differences lead them to believe the twins could be DZ. Clearly, accurate testing and reporting of twin chorionicity and zygosity would conclusively identify whether a set of twins is identical or fraternal.
The importance of zygosity knowledge
Accurate knowledge of zygosity is important to both twins and science. Medical, personal, financial, scientific, legal and ethical reasons support greater testing and reporting of twin zygosity [9, 13, 14].
MZ pairs are perfectly compatible organ donors. This means they require much less post-transplant immunosuppression than DZ twins and have better chances of long-term survival.
As nearly all diseases have at least some genetic component to their origin, the diagnosis of a disease in one twin means the co-twin is at increased risk of that disease, more so for MZ pairs than DZ pairs. Genetic sequence data will almost always be the same for MZ co-twins, so the implications of this for the co-twin should be considered if testing is undertaken. Knowledge of a genetic disorder manifested in only one of a pair of identical twins is likely to lead to early detection in the second twin, thus leading to a greater chance of successful prevention or treatment.
There are psychological benefits for parents and twins in knowing whether they are MZ or DZ. In the event that one or both twins die before or soon after birth, it is vital that parents and/or the surviving twin have this fundamental information about twin zygosity as it bears upon immediate bereavement response, the long-term identity of the surviving twin, and future family planning.
Accurate determination of zygosity is important postnatally for estimation of the likelihood of the mother or close female relatives giving birth to further sets of twins. This is because (with rare exceptions) only DZ twins can run in families, mainly through the maternal line.
Knowledge of zygosity also aids in understanding the physical and behavioural differences and similarities between twins.
Increased understanding of zygosity helps define social relationships and helps define twins as individuals. As noted above, it helps determine the likelihood of further twin gestations in the family (which is increased only for mothers of DZ pairs). It also helps avoid embarrassment over uncertainty when asked about by zygosity by family, friends, teachers and strangers.
Knowledge of zygosity can provide peace of mind and positive emotional responses for twins and their families. Some twins experience significant emotional stress if they discover later in life that their belief about their zygosity is incorrect. Knowledge of zygosity is also a prerequisite for twin research, and incorrect assumptions mean many twins believe they are unable to participate .
Knowledge of a genetic disorder manifested in one identical twin is likely to lead to early detection in the other, thus improving health outcomes and reducing costs for treatment and management compared to detection at a later stage of disease. Costs incurred for zygosity testing would be outweighed, in the case of a suspected genetic disorder, by the savings from genetic testing of the second twin after a diagnosis in the first (this is only applicable to MZ twins).
Inaccurate knowledge of zygosity affects the results and findings of medical research involving twins. Accurate knowledge of zygosity also saves both time and expense for researchers and participants as additional testing is not required.
The International Council for Multiple Birth Organisations and the International Society of Twin Studies, in their Declaration of Rights (http://icombo.org/wp-content/uploads/2010/11/Declaration-of-Rights-2014.pdf), state that:
- parents have a right to expect accurate recording of placentation, determination of chorionicity and amnionicity via ultrasound, and the diagnosis of zygosity of same sex multiples at birth;
- older, same sex multiples of undetermined zygosity have a right to testing to ascertain their zygosity; and
- zygosity should be respected as any other human trait and deserves the same privacy rules.
In its Universal Declaration on the Human Genome and Human Rights, UNESCO supports the right to know one’s genetic identity/information (www.ohchr.org/EN/ProfessionalInterest/Pages/HumanGenomeAndHumanRights.aspx).
Respect for the individual recognises the importance of the concept of identity for a person, which is important for wellbeing and for avoiding harms, including those affecting human dignity, freedom and human rights, resulting from misinformation.
Knowledge of zygosity at birth also avoids any erroneous assumptions from the outset, including those that would result in damaging psychological or emotional impact if zygosity assumptions are proven incorrect, such as if twins had assumed incorrectly that they were identical. Certainty can be very reassuring, especially for parents . For these reasons and those listed above, we argue that not offering twin zygosity testing at birth is unethical.
Arguments against testing
It has been suggested that it would be inappropriate to undertake zygosity testing on a baby or child, one of the arguments being that it removes the individual’s ability to decide whether they want to have this information [157, 16]. This argument seems to misconceive the nature of zygosity testing and the practical circumstances of same-sex twins. Zygosity testing is quite distinct from other forms of genetic testing of children that raise legitimate ethical concerns. Most notably, it is not a test for a gene mutation or predisposition to a disease but rather to determine whether twins are MZ or DZ. It therefore cannot be compared with the weight of knowledge about predisposition to a genetic disease, particularly for a late-onset condition such as Huntington’s disease for which it is always preferable for individuals to decide for themselves whether to undertake genetic testing. Nor can it realistically be suggested that zygosity testing invades privacy, given the practical reality that parents of twins and the twins themselves, once they are old enough, will frequently be asked whether they are identical twins, so this issue will inevitably by at the forefront, irrespective of whether zygosity testing is undertaken at birth.
Importantly, we are not recommending mandatory zygosity testing for twins. We acknowledge that some twins may not wish to know, and that cases in which only one twin in the pair seeks such information could raise ethical implications for testing. Counselling options are available in such instances. Similarly, in the case of infants/children, some parents may prefer not to know.
The impact of zygosity knowledge on twins
In one study, twins who received zygosity test results were asked why it was important for them to know their zygosity . Some of the responses are below.
For health reasons, I feel it was necessary to know so if one was to have a disease etc. we would pay closer attention to the other twin. (Mother of MZ twins aged two years, previously unsure of their zygosity)
[It meant] the world! I felt close to my sister and was always really curious to know if we were actually identical or not so finally finding out was like settling a piece of unknown family history. It was fabulous. (MZ twin aged 25 years, previously believed she was a DZ twin)
Assurance of total compatibility and similarity for medical reasons. (MZ twin aged 40, previously guessed he was MZ)
We take part in twin research and don’t know if we are identical or non-identical. (MZ twins aged 39, previously guessed they were DZ)
The importance of twins in research
The study of twins helps us disentangle genetic from environmental influences on human traits and conditions, because identical twins share all of their genetic variation while non-identical twins share around half. In relation to health conditions that are affected to a high degree by genetics, identical twins will be much more alike than non-identical twins.
Conversely, if a health condition is largely affected by the family environment (e.g., living in dire poverty), then both MZ and DZ twins will be similarly affected. In addition, if a condition is brought about by an aspect of the environment that twins do not share, for example if only one twin smokes, then the disease in twins may not be any more alike than in any two people picked at random from the population.
Research involving twins (that uses the uniqueness of twinning) has been conducted for decades . This ongoing research has led to the conclusion that for almost all behavioural and health conditions, twins are representative of the population as a whole [18, 19]. The research has proved invaluable in helping to separate the effects of genes and environment on variation in human characteristics, behaviours, and susceptibility to diseases. We argue that increasing the accuracy of zygosity reporting will increase the accuracy and validity of twin research.
Furthermore, this research, combined with advances in technology, represents a powerful approach to identifying and understanding the molecular pathways that underlie complex human traits. Twin research has become even more valuable in recent times due to the ability to control genes and the shared environment through study design, and the rapid expansion of the number of twin registries worldwide [17, 20].
Based on the information presented above, we make 9 recommendations.
- Increase education about twin zygosity, its testing and its implications, for medical professionals, including obstetricians, neonatologists, midwives, maternal child health nurses, paediatricians and ultrasonographers..
- Obstetricians should give all parents of newborn twins a written report of their twins’ chorionicity and its implications for zygosity. If their twins are the same sex and dichorionic and they wish to know the zygosity, they should be provided with enough information to make an informed decision about whether to perform the test.
- Universal zygosity testing of same-sex twins should be encouraged as early in life as possible as standard practice. Parents of same-sex twins and their families should always be advised that if they wish to know the pair’s zygosity, the only way to be certain, except when twins have different blood groups, is to have a zygosity test.
- From birth, all children should be recorded as a singleton or a twin in medical records. This way, if a single twin sees a medical professional, issues related to risk to the co-twin can be raised. Such information is also essential for data linkage and twin research.
- Medical professionals should ask twins and parents of twins whether they are identical or fraternal and asked about how they know. Those who are unsure, or who have made an incorrect assumption, should be encouraged to have a zygosity test. They should receive accessible and accurate information about how to take the test and the costs involved.
- Increase education for twins and their families about twin zygosity, its testing and its implications. This would help to dispel incorrect assumptions, encourage them to ask for further advice from medical professionals when needed, and enable them to make more informed decisions about zygosity testing. In addition, families of twins should be provided with details of any local or national twin registries and multiple birth organisations for reasons of peer support and contribution to research.
- With the help of government and genetic testing companies, decrease the burden of the cost of zygosity testing for individuals. Currently in Australia, 1.5% of all births are twins and the most commonly accessed zygosity test costs $120. The annual cost of zygosity testing all same-sex dichorionic twins born in Australia would be around AUD230,000, or AUD345,000 for all same-sex twins irrespective of chorionicity. These are modest costs given the potential significance of this knowledge.
- Undertake a study to improve our understanding of the health benefits and cost effectiveness of zygosity testing to provide evidence for funding of zygosity testing through the public health system.
- Raise family payments to families with twins in recognition of the elevated cost of raising them (estimated at five times the cost of singletons in the United States ). This approach would be consistent, in Australia, with the multiple birth allowance for triplets and quads (http://guides.dss.gov.au/family-assistance-guide/1/2/16). This would give families greater ability to undertake zygosity testing if they choose.
We have detailed how twin zygosity is determined and why this knowledge is important for medical, personal, financial, scientific, legal and ethical reasons. We argue that incorrect assumptions about zygosity act as barriers to optimal health and wellbeing in twins and to scientific research. We strongly recommend engagement with all stakeholders – twins, medical and healthcare professionals, government and the community at large – to increase education about zygosity. We recommend that chorionicity be determined at birth and reported to all parents of twins, who should also receive information about zygosity testing if required.
This publication was made possible by a Centre of Research Excellence Grant (1079102) from the National Health and Medical Research Council of Australia. JMC is supported by grants from the Australian National Health and Medical Research Council (grant numbers 1011070 and 1083779), the Financial Markets Foundation For Children (grant number 032-2007), and the Murdoch Childrens Research Institute, which is funded by the Victorian Government’s Operational Infrastructure Support Program. We thank Lucas Calais-Ferreira for comments on the manuscript.
About the authors
Associate Professor Jeffrey Craig works in the Centre for Molecular and Medical Research, at the Deakin University School of Medicine.
Tessa L Cutler is the Research Coordinator and Liaison at Twins Research Australia, Melbourne School of Population and Global Health, University of Melbourne.
Kate Murphy is the Deputy Director of Twins Research Australia at The Melbourne School of Global Population Health, The University of Melbourne.
Monica Rankin is the Chairperson of the International Council of Multiple Birth Organisations.
Jane Denton is the Director of the Multiple Births Foundation, Queen Charlotte’s & Chelsea Hospital, London.
Deborah Schofield is Professor and Chair of Health Economics, University of Sydney.
Margaret Otlowski is Dean & Head of School, Faculty of Law, University of Tasmania.
Mark P Umstad is Director of Maternity Services, Royal Women’s Hospital, Melbourne.
Susan Carrick is the Deputy Director of Twins Research Australia, based at The Charles Perkins Centre’s Twin Research Node, The Charles Perkins Centre, University Of Sydney.
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Figure 1. The formation of the main types of twins
DZ twins result from two separate fertilisation events. Each twin has its own placenta which is continuous with the chorion and its own amnion (inner sac), and they are referred to as “dichorionic diamniotic”. MZ twins result from the splitting of a single embryo early in gestation; the timing of splitting determines the type of MZ twin. Approximately one-third of MZ twins split early and are dichorionic diamniotic. Approximately two-thirds of MZ twins split later and share a single placenta despite maintaining their own amnion, umbilical cord and share of the placental mass. They are referred to as “monochorionic diamniotic”. Approximately 1% of twins split even later and share a single chorion and amnion. They are referred to as “monochorionic monoamniotic”. Taken from  with permission.
Figure 2. Decision tree for determining zygosity in twins
*Number of placentas should be determined by ultrasound prior to 14 weeks’ gestation and confirmed at birth by physical examination of membranes .
Figure 3. Determination of chorionicity at birth
Fused dichorionic and monochorionic placentas are compared at the macroscopic (a) and microscopic (b) levels. In fused dichorionic placentas, twins are separated by two amnions (dark blue) and two fused chorions (red). When rubbed between thumb and fingers, the dividing membranes can be separated into two semi-transparent amnions and a thicker layer of fused chorions. The fused chorionic membranes are continuous with the placentas, resulting in a slight ridge on the placental surface (*) corresponding to the lambda sign previously observed on ultrasound scans (). In monochorionic placentas, twins are separated by two amnions only. When rubbed between thumb and fingers, the dividing membranes are thinner and semi-transparent. The placental surface is flat and the placental junction between the two twins corresponds to the T sign previously observed on ultrasound scans.