The effects of assisted reproduction on the trends and zygosity of multiple births in England and Wales 1974-99.

Assisted reproductive techniques have led to an increase in the proportion of maternities that are multiple. Though predominantly dizygotic, they are at greater risk of monozygotic division than those spontaneously conceived. England and Wales data 1974-99 on stillbirths and livebirths were analysed for 4 periods: 1974-80 (pre-assisted reproduction; 1982-8; 1989-91 (pre-redefinition of stillbirth); 1993-9 (post-redefinition of stillbirth). For twin data, Weinberg's rule was applied to estimate the proportions that were mono- (MZ) and dizygotic (DZ). Compared with the period before assisted reproduction, the most recent period shows an increase in twin maternities of 3.81 per 1,000 comprised of 3.22 (95% CI 3.10 to 3.33; p < 0.0001) DZ and 0.60 (95% CI 0.51 to 0.68; p < 0.0001) MZ twins. It is estimated that 15.7% of assisted reproduction twins are MZ. Higher order multiple births showed an increase of 3.06 (95% CI 2.85 to 3.29; p < 0.0001) per 10,000 maternities. Stillbirth rates in MZ twins are of the same order of magnitude as those in higher order multiple births but higher than those in DZ twins. The improvement in stillbirth rates over the 26 year study period is of the same order magnitude in singletons, DZ and MZ twins and higher order multiples. Assisted reproduction has led to a significant increase in the proportion of MZ twins. These are at high risk of fetal death and this needs to be considered when local stillbirth and perinatal mortality rates are used in auditing obstetric services.     

Monthly trend and seasonal variation in twinning rate in Japan, 1975–1994

We examined birth records from Japanese statistics, 1975–1994, to investigate the seasonality of twin births. We could identify 198 924 pairs of twins (97.9% of all the registered twin records) and estimated the numbers of mono- and dizygotic twin pairs. The seasonal index of the twinning rate for each month was calculated by dividing the crude rate by the estimated trend value for the month. There were significant variations in the seasonal index for overall, dizygotic and monozygotic twinning rates. Peak months with values more than 3% higher than expected were July and October–December for dizygotic twins, and April and June for monozygotic twins; these seasonalities were statistically significant by analysis of variance and the patterns were similar in recent years, with a sharp increase in the total twinning rate. When observed year-by-year, however, there were years that did not show these typical seasonalities. It is suggested that the mechanisms for probable seasonal variations in twinning rates are different for dizygotic and monozygotic twin pregnancies, and that factors involved in these variations are not effective every year. Received: 2 September 1998 / Revised: 6 May 1999 / Accepted: 26 May 1999     

Twin births to mothers who are twins: a registry based study.

OBJECTIVES: To estimate the risk of having twin infants for mothers who are twins; to investigate the genetic influence on twinning. DESIGN: Retrospective study of multiple births in two nationwide registries. SETTING: Sweden. SUBJECTS: Multiple births among 31,586 deliveries between 1973 and 1991 to women who were twins. MAIN OUTCOME MEASURES: Numbers of monozygotic and dizygotic twin births expected and estimated. RESULTS: Women who are dizygotic twins have a moderately increased risk of having twins (relative risk 1.30, 95% confidence interval 1.14 to 1.49) which seems to be completely the result of dizygotic twinning. When a mother is a monozygotic twin, her risk of having twins of the same sex is significantly increased (1.47; 1.10 to 1.97). This is the result of an excess of monozygotic twins (39 pairs estimated, 18 expected). CONCLUSIONS: Women who are twins have an increased risk of giving birth to twins. Genetic components of monozygotic and dizygotic twinning seem to be independent.     

Twinning rates in a rural area of Bangladesh

In this study we investigate the incidence of twin births over a period of 16 years in a rural area of Bangladesh using data from the Demographic Surveillance System of the International Centre for Diarrhoeal Disease Research. Over the study period twinning rates fluctuated between 7.8 and 11.2 per 1000 live births. The twinning rate was strongly correlated with maternal age; the rate for mothers over 35 years of age was about 3 times higher than for mothers younger than 20 years. The variation in twinning rate with maternal age is due to the variation in dizygotic twinning; the rate of monozygotic twinning is almost constant for all ages. Twinning rates were higher in the treatment area than in the comparison area after controlling for maternal age and parity. The rates were lower for monozygotic twinning and higher for dizygotic twinning in the treatment area than in the comparison area. Seasonality was observed for both twins and singletons, but the peak for twinning precedes that for singleton births by more than a month.     

Twinning and heteropaternity in chimpanzees (Pan troglodytes)

Unlike monozygotic (MZ) twins, dizygotic (DZ) twins develop from separate ova. The resulting twins can have different sires if the fertilizing sperm comes from different males. Routine paternity testing of a pair of same-sexed chimpanzee twins born to a female housed with two males indicated that the twins were sired by two different males. DNA typing of 22 short-tandem repeat (STR) loci demonstrated that these twins were not MZ twins but heteropaternal DZ twins. Reproductive data from 1926-2002 at five domestic chimpanzee colonies, including 52 twins and two triplets in 1,865 maternities, were used to estimate total twinning rates and the MZ and DZ components. The average chimpanzee MZ twinning rate (0.43%) equaled the average human MZ rate (0.48%). However, the chimpanzee DZ twinning rate (2.36%) was over twice the human average, and higher than all but the fertility-enhanced human populations of Nigeria. Similarly high twinning rates among African chimpanzees indicated that these estimates were not artifacts of captivity. Log-linear analyses of maternal and paternal effects on recurrent twinning indicated that females who twinned previously had recurrence risks five times greater than average, while evidence for a paternal twinning effect was weak. Chimpanzee twinning rates appear to be elevated relative to corresponding estimated human rates, making twinning and possibly heteropaternity more important features of chimpanzee reproductive biology than previously recognized.     

Twinning rate in spontaneous abortions.

An unselected series of spontaneous abortions and their mothers were karyotyped with Q-bands to obtain a frequency of twin conceptions lost during the first trimester. Among 661 spontaneous abortions, 15 twin pairs were identified including two sets of conjoined twins. Analysis of Q-band variants permitted the exclusion of cases with two cell lines that could be attributed to maternal contamination or mosaicism. The twinning rate among spontaneous abortions was 1/44 compared with 1/103 live births and stillbirths in the Ontario population. If Weinberg's differential method is applied to these data, the frequency would be as high as 1/30 under the assumption that the incidence of monozygotic twins among abortions is the same as that for live births.     

Maternal smoking and twinning.

In order to investigate a possible association between maternal smoking during pregnancy and twinning, information on 1,096,330 single births and 12,342 twin births in 1983-95 was obtained from the Swedish Medical Birth Registry (MBR). All odds ratios (OR) were estimated after stratification for year of birth and maternal age, parity, and educational level. Smoking women, compared with non-smoking women, were at increased risk of having dizygotic (DZ) twins, but the risk increase was only evident among multiparas. A strong association between previous involuntary childlessness and dizygotic (DZ) twinning (especially in primiparas) was found. The strongest association between maternal smoking and DZ twinning was found among multiparas without any history of involuntary childlessness (OR: 1.35, 95%CI:1.22-1.49), whereas among women who had experienced involuntary childlessness, the opposite was seen (OR: 0.82, 95%CI:0.66-1.00, no difference between parity strata). Weinberg's differential method was used to estimate the number of monozygotic (MZ) twins, and a method of estimating stratified ORs among mothers of MZ twins was presented. No association was found between MZ twinning and maternal smoking (OR: 0.96, 95%CI:0.86-1.07), and no confounding by parity or previous involuntary childlessness was indicated. Several non-causal explanations to the positive association between DZ twinning and maternal smoking among multiparas were discussed, but homogeneity over strata indicated that maternal smoking may be a true risk factor for double ovulation.     

Zygosity misclassification of twins at birth in Japan.

Though twinning rates have been rapidly increasing in Japan, the problem of zygosity misclassification at birth has been paid little attention. By analyzing four independent samples, the authors found that at a constant rate about 25-30% of monozygotic twins were misclassified as dizygotic twins at birth. This percentage is in very good accordance with that of monozygotic twins having dizygous placenta. Generally the obstetricians informed twins' parents about their children's zygosity. The number of placentas, as informed by obstetricians, was very strongly associated with zygosity. Concluding, even now many monozygotic twins in Japan may be misclassified as dizygotic at birth by obstetricians based solely on the number of placenta.     

Glucose and insulin metabolism in twins: influence of zygosity and birth weight.

Several epidemiological and metabolic studies have demonstrated an impact of the intrauterine environment on the development of disease in adult life, including Type 2 diabetes and glucose intolerance. Our finding of lower birth weights among monozygotic diabetic twins compared to their non-diabetic genetically identical co-twins confirms this association and, furthermore, eliminates the possibility that the association could be explained solely by common genes leading to both impaired intrauterine growth and increased risk of Type 2 diabetes. Due to an often shared placenta monozygotic twins may experience a more adverse intrauterine environment compared to dizygotic twins and may therefore be more prone to develop various metabolic abnormalities. Our findings of a higher glucose and insulin profile after oral glucose ingestion, and recently lower insulin-stimulated glucose uptake--indicating glucose intolerance and insulin resistance--among monozygotic compared to dizygotic twins may to some extent question the validity of classical twin studies in diabetes research where equal environmental influences in monozygotic and dizygotic twins is assumed. The potential role of an adverse intrauterine environment in causing Type 2 diabetes in humans, may to some degree alter our conception of the twin model in diabetes research including the interpretation of aetiological conclusions reached in previous classical twin studies of diabetes. However, our present knowledge is far too insufficient to discard the results from classical twin studies concerning the relative role of genes versus environment for the development of diabetes and its metabolic effects.     

X inactivation as a source of behavioural differences in monozygotic female twins.

Although members of monozygotic twin pairs are identical in genome sequence, they may differ in patterns of gene expression. One early and irreversible process affecting gene expression, which can create differences within pairs of female monozygotic twins, is X inactivation - one twin can express mainly paternally-received genes on the X chromosome while the other twin expresses mainly maternally-received genes. It follows that non-identical X chromosome expression may cause female monozygotic twins to correlate less strongly than male monozygotic twins on complex behavioural traits affected by X-linked loci. We tested this hypothesis using data from around 4000 same-sex twin pairs on 9 social, behavioural and cognitive measures at ages 2, 3 and 4. Consistent with our hypothesis, monozygotic males were generally more similar than monozygotic females. Three of four significant differences were in traits showing higher correlations in males than females, and these traits - prosocial behaviour, peer problems, and verbal ability - have all been proposed previously in the literature as being influenced by genes on the X chromosome. Interestingly, dizygotic twins showed the reverse pattern of correlations for similar variables, which is also consistent with the X inactivation hypothesis; taken together, then, our monozygotic and dizygotic results suggest the presence of quantitative trait loci on the X chromosome.     

The potential use of artificially produced monozygotic twins for comparative experiments

The uniformity of twins has been examined by assembling estimates of the intraclass correlation coefficient (rho(I)) available in the literature for a variety of parameters studied in cattle monozygotic twins and human dizygotic and monozygotic twins. The values of rho(I) vary considerably between parameters. In human monozygotic twins rho(I) is always larger compared to that found in dizygotic twins. There is insufficient evidence to determine whether artificial monozygotic twins are more uniform than natural monozygotic twins. A new measure of twin uniformity, given by T (3) = 1 (1-rho (I)) , is introduced. In practice 2T(3) gives the number of animals chosen at random that one member of a twin pair can replace without loss of statistical efficiency. A useful class of experimental designs for the exploitation of twin uniformity is incomplete block designs. These designs are defined by (v, k, b), where v is the number of treatments to be compared, k = 2, and b is the number of twin pairs. Each design has an associated efficiency (E). Provided rho(I)>1-E, an incomplete block design will be advantageous. In general, when only a few twin pairs are available, this relation will only hold for monozygotic and not dizygotic twins. Suitable arrangements of treatment comparisons for designs (3,2,8), (4,2,9), (5,2,10), (6,2,11) are presented.     

Does higher concordance in monozygotic twins than in dizygotic twins suggest a genetic component?

It is widely regarded that twins can be used as a natural experiment to subject hypotheses to empirical testing regarding the contributions of genetic factors to phenotypic variability in human traits, especially behavioral traits. In genetic epidemiology, a higher concordance rate in monozygotic (MZ) twins than in dizygotic (DZ) twins is often taken as prima facie evidence for a genetic component. While twins studies have been used to estimate the contributions of genetic factors to phenotypic variability in human traits, the corresponding methodology that allows the estimation entails several crucial assumptions. The most critical is that MZ and DZ twins are equally similar environmentally. Although MZ twins are genetically more similar than DZ twins, they are often environmentally more similar. This paper demonstrates that, even in the complete absence of any genetic factor and of any biases, the greater environmental similarity alone in MZ twins can result in higher concordance rate in MZ twins than in DZ twins. This is especially true when there are multiple environmental factors, which may have multiple exposure levels and/or interact strongly, although each of them may be of low risk. This may serve as a sobering antidote to the uncritical reliance on twin studies without examining the validity of the underlying assumptions.     

No paternal effect on monozygotic twinning in the Swedish Twin Registry.

Previous research has provided evidence for a genetic effect in monozygotic twinning, indicated by an increased risk for monozygotic women to have monozygotic offspring. However, since the biological mechanism for this trait is unknown, it is not clear if there exists a paternal inheritance. In this study we investigated twin pregnancies in offspring born in 1941-1996 to male twins in the Swedish Twin Registry and population controls born in 1926-1980. In total 4,225,331 offspring, of which 89,286 were twins, were studied. There was neither an increase in the probability for monozygotic men to have like-sexed twin offspring risk ratio (RR = 0.95; 95% CI = 0.77-1.13) nor an increase in the estimated number of monozygotic twin births. Thus, there is no evidence for a paternal effect on monozygotic twinning, suggesting that the gene(s) increasing the liability for division of the embryo are expressed in the mother and not in the fertilised egg.     

A twin study of dental dimension. I. Discordance,asymmetry, and mirror imagery

Bilateral tooth measurements in twins were partitioned into three orthogonal contrasts, each associated with one degree of freedom, to estimate three parameters: discordance, asymmetry, and mirror imagery. The probability levels of the within-pair variance ratios were used to test for significance of these estimates. The results provided strong evidences for the existence of significant genetic determinants of almost all of the individual tooth dimensions, but little or no evidence for a genetic basis of asymmetry. The analysis gave no indication that monozygotic twinning was associated with an increased degree of either fluctuating asymmetry or mirror imagery, when compared to dizygotic twins. The data on monozygotic twins further suggested that for most variables examined, the increment of environmental discordance resulting from the twinning phenomena was greater than the developmental noise that caused asymmetry within individual cotwins.     

Twins and the paradox of dental-age estimations: A caution for researchers and clinicians

The biological age difference among twins is frequently an issue in studies of genetic influence on various dental features, particularly dental development. The timing of dental development is a crucial issue also for many clinicians and researchers. The aim of this study was therefore to verify within groups of twins how dental development differs, by applying Demirjian's method, Mincer's charts of development of third molars and two of Cameriere's methods for dental age estimation, which are among the most popular methods both in the clinical and the forensic scenario. The sample consisted of 64 twin pairs: 21 monozygotic, 30 dizygotic same-sex and 13 dizygotic opposite-sex with an age range between 5.8 and 22.6 years. Dental age was determined from radiographs using the mentioned methods. Results showed that dental age of monozygotic twins is not identical even if they share all their genes. The mean intra-pair difference of monozygotic pairs was low and similar to the difference in dizygotic same-sex twins; the maximum difference between monozygotic twins, however, was surprisingly large (nearly two years). This should lead to some circumspection in the interpretation of systematic estimations of dental age both in the clinical and forensic scenario.     

Multiple birth and Down's disease]

The frequency of twinning among newborns with Down's syndrome (2,11+/-0,6%)was significantly higher than in the general populaltion (0,73+/-0,3%). The increase in the rate of multiple births of children with trisomy-21 occurred due to almost three-fold excess in the frequency of dizygotic (discordant) twin pairs over the expected level. The increase in the frequency of dizygotic twins with Down's syndrome was explained by the combined effect of two independent factors: the increase in probability of dizgotic twins natality and the enhanced rate of children birth with trisomy-21, which depended on the increase in mother's age.     

The role of the placenta in variability of fetal exposure to cocaine and cannabinoids: a twin study.

There is wide variability in the reported adverse fetal effects of cocaine and cannabinoids. The causes of this variability are largely unknown. We hypothesized that variability in placental handling of drugs affect fetal exposure. We used twin pregnancies as a paradigm to address the role of the placenta in this variability. We analyzed hair or meconium samples taken from dizygotic and monozygotic twins exposed in utero to illicit drugs. Out of 12 pairs, 5 had negative levels in both twins, and seven pairs of twins had chemical evidence of fetal exposure to cocaine (n = 5) or cannabinoids (n = 2). The one known monozygotic pair of twins had almost identical levels of cocaine. In contrast, the six dizygotic pairs had large disparities in either cocaine or cannabinoid concentrations. In three of these six dizygotic pairs, levels of cocaine (n = 2) or canabinoids (n = 1) were undetectable in one twin while positive in the other. Given that twins are theoretically exposed to similar maternal drug levels, our findings suggest that the placenta may have a major role in modulating the amounts of drug reaching the fetus.     

Zygosity diagnosis in the absence of genotypic data: an approach using latent class analysis.

For zygosity diagnosis in the absence of genotypic data, or in the recruitment phase of a twin study where only single twins from same-sex pairs are being screened, or to provide a test for sample duplication leading to the false identification of a dizygotic pair as monozygotic, the appropriate analysis of respondents' answers to questions about zygosity is critical. Using data from a young adult Australian twin cohort (N = 2094 complete pairs and 519 singleton twins from same-sex pairs with complete responses to all zygosity items), we show that application of latent class analysis (LCA), fitting a 2-class model, yields results that show good concordance with traditional methods of zygosity diagnosis, but with certain important advantages. These include the ability, in many cases, to assign zygosity with specified probability on the basis of responses of a single informant (advantageous when one zygosity type is being oversampled); and the ability to quantify the probability of misassignment of zygosity, allowing prioritization of cases for genotyping as well as identification of cases of probable laboratory error. Out of 242 twins (from 121 like-sex pairs) where genotypic data were available for zygosity confirmation, only a single case was identified of incorrect zygosity assignment by the latent class algorithm. Zygosity assignment for that single case was identified by the LCA as uncertain (probability of being a monozygotic twin only 76%), and the co-twin's responses clearly identified the pair as dizygotic (probability of being dizygotic 100%). In the absence of genotypic data, or as a safeguard against sample duplication, application of LCA for zygosity assignment or confirmation is strongly recommended.     

Observed frequency of monozygotic twinning in Holstein dairy cattle

Bonnier's equation is used to mathematically estimate the frequency of monozygotic (MZ) twinning in epidemiologic studies of twinning in dairy cattle; however, no empirical determination of MZ twinning has been reported in the literature. Our objectives were to empirically determine the frequency of MZ twinning in lactating Holstein cows and to compare this result with published estimates predicted using Bonnier's equation. Ear biopsies were collected from 107 sets of Holstein twins from six Wisconsin dairies resulting in 40 opposite-sex twins, 29 same-sex male twins, and 38 same-sex female twins. To empirically determine the frequency of MZ twinning, DNA extracted from ear biopsies collected from the 67 same-sex twins was PCR amplified using primers for a minimum of 5 polymorphic microsatellite DNA markers. Opposite-sex twins were classified as dizygotic (DZ) as well as same-sex twins differing in at least one microsatellite DNA marker. Same-sex twins were classified as MZ when all genotypes for a minimum of five markers were identical. Of the 67 same-sex twins, 62 were classified as DZ and 5 MZ resulting in a MZ twinning frequency of 7.5% of same-sex twins and 4.7% of all twins. The estimated frequency of MZ twinning in this population of twin calves using Bonnier's equation was 39.5% of same-sex twins and 24.7% of all twins. We concluded that MZ twinning occurred infrequently in Holstein cattle and perhaps less frequently than that reported in studies using Bonnier's equation to estimate MZ twinning.