Genome-wide association study meta-analysis of dizygotic twinning illuminates genetic regulation of female fecundity.

STUDY QUESTION: Which genetic factors regulate female propensity for giving birth to spontaneous dizygotic (DZ) twins? SUMMARY ANSWER: We identified four new loci, GNRH1, FSHR, ZFPM1, and IPO8, in addition to previously identified loci, FSHB and SMAD3. WHAT IS KNOWN ALREADY: The propensity to give birth to DZ twins runs in families. Earlier, we reported that FSHB and SMAD3 as associated with DZ twinning and female fertility measures. STUDY DESIGN, SIZE, DURATION: We conducted a genome-wide association meta-analysis (GWAMA) of mothers of spontaneous dizygotic (DZ) twins (8265 cases, 264 567 controls) and of independent DZ twin offspring (26 252 cases, 417 433 controls). PARTICIPANTS/MATERIALS, SETTING, METHODS: Over 700 000 mothers of DZ twins, twin individuals and singletons from large cohorts in Australia/New Zealand, Europe, and the USA were carefully screened to exclude twins born after use of ARTs. Genetic association analyses by cohort were followed by meta-analysis, phenome wide association studies (PheWAS), in silico and in vivo annotations, and Zebrafish functional validation. MAIN RESULTS AND THE ROLE OF CHANCE: This study enlarges the sample size considerably from previous efforts, finding four genome-wide significant loci, including two novel signals and a further two novel genes that are implicated by gene level enrichment analyses. The novel loci, GNRH1 and FSHR, have well-established roles in female reproduction whereas ZFPM1 and IPO8 have not previously been implicated in female fertility. We found significant genetic correlations with multiple aspects of female reproduction and body size as well as evidence for significant selection against DZ twinning during human evolution. The 26 top single nucleotide polymorphisms (SNPs) from our GWAMA in European-origin participants weakly predicted the crude twinning rates in 47 non-European populations (r = 0.23 between risk score and population prevalence, s.e. 0.11, 1-tail P = 0.058) indicating that genome-wide association studies (GWAS) are needed in African and Asian populations to explore the causes of their respectively high and low DZ twinning rates. In vivo functional tests in zebrafish for IPO8 validated its essential role in female, but not male, fertility. In most regions, risk SNPs linked to known expression quantitative trait loci (eQTLs). Top SNPs were associated with in vivo reproductive hormone levels with the top pathways including hormone ligand binding receptors and the ovulation cycle. LARGE SCALE DATA: The full DZT GWAS summary statistics will made available after publication through the GWAS catalog (https://www.ebi.ac.uk/gwas/). LIMITATIONS, REASONS FOR CAUTION: Our study only included European ancestry cohorts. Inclusion of data from Africa (with the highest twining rate) and Asia (with the lowest rate) would illuminate further the biology of twinning and female fertility. WIDER IMPLICATIONS OF THE FINDINGS: About one in 40 babies born in the world is a twin and there is much speculation on why twinning runs in families. We hope our results will inform investigations of ovarian response in new and existing ARTs and the causes of female infertility. STUDY FUNDING/COMPETING INTEREST(S): Support for the Netherlands Twin Register came from the Netherlands Organization for Scientific Research (NWO) and The Netherlands Organization for Health Research and Development (ZonMW) grants, 904-61-193, 480-04-004, 400-05-717, Addiction-31160008, 911-09-032, Biobanking and Biomolecular Resources Research Infrastructure (BBMRI.NL, 184.021.007), Royal Netherlands Academy of Science Professor Award (PAH/6635) to DIB, European Research Council (ERC-230374), Rutgers University Cell and DNA Repository (NIMH U24 MH068457-06), the Avera Institute, Sioux Falls, South Dakota (USA) and the National Institutes of Health (NIH R01 HD042157-01A1) and the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health and Grand Opportunity grants 1RC2 MH089951. The QIMR Berghofer Medical Research Institute (QIMR) study was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485, 552498, 1050208, 1075175). L.Y. is funded by Australian Research Council (Grant number DE200100425). The Minnesota Center for Twin and Family Research (MCTFR) was supported in part by USPHS Grants from the National Institute on Alcohol Abuse and Alcoholism (AA09367 and AA11886) and the National Institute on Drug Abuse (DA05147, DA13240, and DA024417). The Women's Genome Health Study (WGHS) was funded by the National Heart, Lung, and Blood Institute (HL043851 and HL080467) and the National Cancer Institute (CA047988 and UM1CA182913), with support for genotyping provided by Amgen. Data collection in the Finnish Twin Registry has been supported by the Wellcome Trust Sanger Institute, the Broad Institute, ENGAGE-European Network for Genetic and Genomic Epidemiology, FP7-HEALTH-F4-2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA-12502, AA-00145, AA-09203, AA15416, and K02AA018755) and the Academy of Finland (grants 100499, 205585, 118555, 141054, 264146, 308248, 312073 and 336823 to J. Kaprio). TwinsUK is funded by the Wellcome Trust, Medical Research Council, Versus Arthritis, European Union Horizon 2020, Chronic Disease Research Foundation (CDRF), Zoe Ltd and the National Institute for Health Research (NIHR) Clinical Research Network (CRN) and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. For NESDA, funding was obtained from the Netherlands Organization for Scientific Research (Geestkracht program grant 10000-1002), the Center for Medical Systems Biology (CSMB, NVVO Genomics), Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL), VU University's Institutes for Health and Care Research (EMGO+) and Neuroscience Campus Amsterdam, University Medical Center Groningen, Leiden University Medical Center, National Institutes of Health (NIH, ROI D0042157-01A, MH081802, Grand Opportunity grants 1 RC2 Ml-1089951 and IRC2 MH089995). Part of the genotyping and analyses were funded by the Genetic Association Information Network (GAIN) of the Foundation for the National Institutes of Health. Computing was supported by BiG Grid, the Dutch e-Science Grid, which is financially supported by NWO. Work in the Del Bene lab was supported by the Programme Investissements d'Avenir IHU FOReSIGHT (ANR-18-IAHU-01). C.R. was supported by an EU Horizon 2020 Marie Sklodowska-Curie Action fellowship (H2020-MSCA-IF-2014 #661527). H.S. and K.S. are employees of deCODE Genetics/Amgen. The other authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

Trends in Dizygotic and Monozygotic Spontaneous Twin Births During the Period 2007-2017 in Lombardy, Northern Italy: A Population-Based Study.

In this study, we analyzed the estimated frequency of monozygotic (MZ) and dizygotic (DZ) spontaneous twins in Lombardy during the period 2007-2017. This is a population-based study using the regional healthcare utilization databases of the Lombardy Region. The total number of spontaneous twin deliveries, in separate strata of like and unlike sex, was obtained. Moreover, estimates of DZ and MZ twin births were calculated using Weinberg's method. The standardized rates (SRs), adjusted for maternal age, of DZ and MZ twin births were computed according to calendar period. The twinning rates were calculated among strata of parity and maternal age. Finally, DZ:MZ ratio was calculated. Among the 734,278 spontaneous deliveries, 9176 (12.5 out of 1000 births) couples of twins were identified. In the three periods considered (i.e. 2007-2010, 2011-2014 and 2015-2017), no trend in the SRs of MZ twins was observed, respectively 0.41 (95% CI [0.40, 0.43]), 0.43 (95% CI [0.42, 0.45]) and 0.43 (95% CI[0.42, 0.45]). Differently, a slightly decreasing trend was observed in DZ twins SRs, respectively 0.87 (95% CI [0.84, 0.89]), 0.81 (95% CI [0.79, 0.83]), and 0.78 (95% CI [0.76, 0.80]). As concerns parity and maternal age, the rate of DZ twin births was consistently higher in nulliparae women aged 35 years or more. In our cohort, despite the increase of maternal age, a decline of spontaneous twin births emerged, especially due to the downward trend of DZ twins.

On the origin of zygosity and chorionicity in twinning: evidence from human in vitro fertilization.

Assisted reproduction is presumed to increase monozygotic twin rates, with the possible contribution of laboratory and medical interventions. Monozygotic dichorionic gestations are supposed to originate from the splitting of an embryo during the first four days of development, before blastocyst formation. Single embryo transfers could result in dichorionic pregnancies, currently explained by embryo splitting as described in the worldwide used medical textbooks, or concomitant conception. However, such splitting has never been observed in human in vitro fertilization, and downregulated frozen cycles could also produce multiple gestations. Several models of the possible origins of dichorionicity have been suggested. However, some possible underlying mechanisms observed from assisted reproduction seem to have been overlooked. In this review, we aimed to document the current knowledge, criticize the accepted dogma, and propose new insights into the origin of zygosity and chorionicity.

Fertility in men with hypospadias: A nationwide register-based study using dizygotic twinning rates as an indicator of semen quality.

BACKGROUND: It is not known if impaired fertility in men with hypospadias is caused by decreased semen quality or other factors. Semen quality in men born with hypospadias may be impaired due to effects of androgens or testicular dysgenesis but has been very little studied. OBJECTIVES: To study semen quality in men with hypospadias using dizygotic twinning rates as an epidemiological indicator. We further aimed to study men treated for cryptorchidism, given a hypothesized mutual etiology for decreased semen quality. MATERIALS AND METHODS: We conducted a population-based study using national Swedish registers. A total of 4,363,165 births between 1964 and 2013 were included. The association between hypospadias and cryptorchidism, and fathering dizygotic multiple births was estimated using logistic regression and presented as odds ratios. The main analyses excluded births conceived using assisted reproductive technology (ART). RESULTS: We identified a total of 5317 births with fathers with hypospadias, including 26 dizygotic births conceived unassisted. No significant association was found between hypospadias and dizygotic twinning (OR 1.10, 0.75-1.61). We estimated a significantly increased odds for dizygotic multiple births in men treated for cryptorchidism (OR 1.35, 1.01-1.81) which was decreased after exclusion of ART, but the estimate was not significant (OR 0.75, 0.48-1.18). DISCUSSION: Using dizygotic twinning rates as an indicator of semen quality, we did not find any difference between fathers with hypospadias and controls. Due to sample size, we could not analyze phenotypes separately and can therefore not exclude impaired semen quality in severe hypospadias. We could not demonstrate any association between dizygotic twinning and cryptorchidism. Men treated for cryptorchidism were more likely than controls to use ART to conceive. CONCLUSION: Men with hypospadias who conceived without ART were not shown to have impaired semen quality using dizygotic twinning as an epidemiological indicator.

Perinatal outcomes in dichorionic diamniotic twins with multifetal pregnancy reduction versus expectant management: A systematic review and meta-analysis.

BACKGROUND: Published findings on perinatal outcomes of multifetal pregnancy reduction (MPR) of dichorionic diamniotic (DCDA) twin pregnancy to singleton are controversial. We performed a meta-analysis to appraise the effects of MPR of DCDA twin pregnancy versus expectant management on perinatal outcomes. METHODS: Four electronic databases were searched from their inception to June 15, 2019, to identify publications that appraised MPR before 15 weeks of gestation. Studies reporting perinatal outcomes of both MPR of DCDA twin pregnancy to singleton and expectant management were considered. The relative risks (RRs) and mean differences with 95% confidence intervals (CIs) were pooled using a random-effects model. RESULTS: Six studies involving 7398 participants showed that MPR of DCDA twin pregnancy to singleton was associated with a lower risk of preterm birth (5 studies with 7297 participants; RR: 0.30, 95% CI: 0.22-0.40; P < .001) and higher birth weight (4 studies with 5763 participants; mean differences: 548.10 g, 95% CI: 424.04-672.15; P < .001) than expectant management; there was no difference in the occurrence of miscarriages (5 studies with 7355 participants; RR: 1.57, 95% CI: 0.90-2.75; P = .11). Sensitivity analysis showed that all the results were stable and reliable, with the omission of 2 studies with serious risk of bias. CONCLUSION: Compared to expectant management, MPR of DCDA twin pregnancy to singleton prevents preterm birth and low birth weight, without increasing the risk of miscarriages. Regarding perinatal morbidity related to preterm birth, MPR can be reserved as a remediation measure to improve the perinatal outcomes of DCDA twin pregnancies.

An age-dependent ovulatory strategy explains the evolution of dizygotic twinning in humans.

Dizygotic twinning, the simultaneous birth of siblings when multiple ova are released, is an evolutionary paradox. Twin-bearing mothers often have elevated fitness, but despite twinning being heritable, twin births occur only at low frequencies in human populations. We resolve this paradox by showing that twinning and non-twinning are not competing strategies; instead, dizygotic twinning is the outcome of an adaptive conditional ovulatory strategy of switching from single to double ovulation with increasing age. This conditional strategy, when coupled with the well-known decline in fertility as women age, maximizes reproductive success and explains the increase and subsequent decrease in the twinning rate with maternal age that is observed across human populations. We show that the most successful ovulatory strategy would be to always double ovulate as an insurance against early fetal loss, but to never bear twins. This finding supports the hypothesis that twinning is a by-product of selection for double ovulation rather than selection for twinning.

The Genetics of Human DZ Twinning.

In the course of twin studies whose main focus was elucidation of genetic and environmental factors on behavioral traits, many twin researchers became aware of the strong tendency for dizygotic (DZ) twinning to run in families. Over four decades, Nick Martin and others initiated hormone and ultrasound studies, performed segregation and pedigree analyses, tested candidate genes, carried out linkage projects in sister pairs and formed large collaborations to illuminate the genetics of DZ twinning by genome-wide association studies and meta-analysis. This article summarizes the early work on hormone and genetic studies and describes the meta-analyses that have at last met with success in finding the first genes that predispose to DZ twinning, which also appear to influence many other female reproductive traits.

Placental T2* estimated by magnetic resonance imaging and fetal weight estimated by ultrasound in the prediction of birthweight differences in dichorionic twin pairs.

INTRODUCTION: Intertwin birthweight (BW) difference is associated with an increased risk of adverse outcome. Ultrasound estimated fetal weight (EFW) is the current method to predict intertwin BW difference, however, the sensitivity is poor. Therefore, new methods are needed. Placental T2* estimated by magnetic resonance imaging (MRI) provides non-invasive information about the placental function. This study aimed to investigate placental T2* difference as a new predictor of BW difference, and to compare it to the EFW. METHODS: We included 25 dichorionic twin pairs at 19-38 weeks' gestation. Placental T2* was obtained by MRI and EFW by ultrasound. Correlations between each predictor and BW difference were examined by simple linear regression, and the combined model was analyzed by multiple linear regression and likelihood ratio test. RESULTS: Strong positive correlations were demonstrated between intertwin differences in placental T2* and BW (r = 0.80, p < 0.005), and EFW and BW (r = 0.64, p < 0.005). Placental T2* difference was a strong independent predictor of BW difference (p < 0.001), and the combined model performed better than each predictor alone (p < 0.0001). DISCUSSION: This pilot study demonstrates that placental T2* difference may be a predictor of intertwin BW difference irrespectively of fetal size. The clinical potential of this method deserves further investigation in a larger clinical study.

Associations Between Features of Placental Morphology and Birth Weight in Dichorionic Twins.

Low birth weight is associated with perinatal and long-term morbidity and mortality, and may be a result of abnormal placental development and function. In studies of singletons, associations have been reported between features of placental morphology and birth weight. Evaluating similar associations within twin pairs offers a unique opportunity to control for key confounders shared within a twin pair, including gestational age, parental characteristics, and intrauterine environment. Data from 3 studies in the United States that were completed from 2012 to 2013, 2006 to 2008, and 1959 to 1966 were used in our analysis of 208 sets of dichorionic twins with unfused placentas. We used linear regression to model difference in birth weight within a twin pair as a function of differences in placental characteristics (i.e., thickness, 2-dimensional surface area, intraplacental difference in diameter). After controlling for sex discordance, a 75.3- cm2 difference in placental surface area, which reflects the interquartile range, was associated with a difference in birth weight of 142.1 g (95% confidence interval (CI): 62.9, 221.3). The magnitude of the association also may be larger for same-sex male pairs than same-sex female pairs (males: 265.8 g, 95% CI: 60.8, 470.8; females: 133.0 g, 95% CI: 15.7, 250.3). Strong associations between surface area and birth weight are consistent with reported results for singleton pregnancies.