The tendency to conceive spontaneous dizygotic (DZ) twins is a complex trait with important contributions from both environmental factors and genetic disposition. Twins are relatively common and occur on average 13 times per 1000 maternities, though the twinning frequency varies over time and geographic location. This variation is mostly attributed to the differences in DZ twinning rate, since the monozygotic twinning rate is relatively constant. DZ twinning is in part under genetic control, with mothers of DZ twins reporting significantly more female family members with DZ twins than mothers of monozygotic twins. Maternal factors such as genetic history, advanced age and increased parity are known to increase the risk of DZ twins. Recent research confirmed that taller mothers and mothers with a high body mass index (30>) are at greater risk of DZ twinning. Seasonality, smoking, oral contraceptive use and folic acid show less convincing associations with twinning. Genetic analysis is beginning to identify genes contributing to the variation in twinning. Mutations in one of these genes (growth differentiation factor 9) are significantly more frequent in mothers of DZ twins. However, the mutations are rare and only account for a small part of the genetic contribution for twinning.

In 2004 the Netherlands Twin Register (NTR) started a large scale biological sample collection in twin families to create a resource for genetic studies on health, lifestyle and personality. Between January 2004 and July 2008, adult participants from NTR research projects were invited into the study. During a home visit between 7:00 and 10:00 am, fasting blood and morning urine samples were collected. Fertile women were bled on day 2–4 of the menstrual cycle, or in their pill-free week. Biological samples were collected for DNA isolation, gene expression studies, creation of cell lines and for biomarker assessment. At the time of blood sampling, additional phenotypic information concerning health, medication use, body composition and smoking was collected. Of the participants contacted, 69% participated. Blood and urine samples were collected in 9,530 participants (63% female, average age 44.4 (SD 15.5) years) from 3,477 families. Lipid profile, glucose, insulin, HbA1c, haematology, CRP, fibrinogen, liver enzymes and creatinine have been assessed. Longitudinal survey data on health, personality and lifestyle are currently available for 90% of all participants. Genome-wide SNP data are available for 3,524 participants, with additional genotyping ongoing. The NTR biobank, combined with the extensive phenotypic information available within the NTR, provides a valuable resource for the study of genetic determinants of individual differences in mental and physical health. It offers opportunities for DNA-based and gene expression studies as well as for future metabolomic and proteomic projects.

Context:Genes from the ovarian bone morphogenetic signaling pathway (GDF9 and BMP15) are critical for normal human fertility. We previously identified a deletion mutation in GDF9 in sisters with spontaneous dizygotic (DZ) twins, but the prevalence of rare GDF9 variants in twinning families is unknown. Objective:The objective was to evaluate the frequency of rare variants in GDF9 in families with a history of DZ twinning. Design and Subjects:We recruited 3450 individuals from 915 DZ twinning families (1693 mothers of twins) and 1512 controls of Caucasian origin. One mother of DZ twins was selected from 279 of the 915 families, and a DNA sample was screened for rare variants in GDF9 using denaturant HPLC. Variants were confirmed by DNA sequencing and genotyped in the entire sample by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results:We found two novel insertion/deletions (c.392-393insT, c.1268-1269delAA) and four missense alterations in the GDF9 sequence in mothers of twins. Two of the missense variants (c.307CϾT, p.Pro103Ser and c.362CϾT, p.Thr121Leu) were located in the proregion of GDF9 and two (c.1121CϾT, p.Pro374Leu and c.1360CϾT, p.Arg454Cys) in the mature protein region. For each variant, the frequencies were higher in cases compared with controls. The proportion of mothers of DZ twins carrying any variant (4.12%) was significantly higher (P Ͻ 0.0001) than the proportion of carriers in controls (2.29%). Conclusion:We describe new variants in the GDF9 gene that are significantly more common in mothers of DZ twins than controls, suggesting that rare GDF9 variants contribute to the likelihood of DZ twinning. . Women with a history of DZ twinning have an increased incidence of multiple follicle growth (4, 5), and in other mammals, members of the TGF␤ superfamily, growth differentiation factor-9 (GDF9), and bone morphogenetic protein-15 (BMP15) play crucial roles in determining follicle growth and ovulation rate (6).These genes are critical for human fertility. A dominantnegative mutation in BMP15 in Italian sisters resulted in ovarian dysgenesis (7), and rare mutations in both GDF9 and BMP15 contribute to premature ovarian failure (POF) (8 -10). We previously reported a heterozygous loss-of-function mutation in the pro-region of GDF9 in two sisters with spontaneous DZ twins (11), and rare mutations in GDF9 may contribute to an increased frequency of twins in some families. We therefore conducted a screen for GDF9 variants in mothers of spontaneous DZ twins and assessed the frequency of the variants in DZ twinning families. Subjects and MethodsStudy subjects were recruited from 915 families with a history of DZ twinning [755 families from Australia and New Zealand and 160 families from The Netherlands] with 3450 individuals available for genotyping. We recruited families with two or more sisters who gave birth to spontaneous DZ twins (12) and also families with a single case where at least one third-degree female relative had DZ twins. Samples were also obtained fr...

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We studied twinning and fertility indices in mothers with spontaneous monozygotic (MZ) and dizygotic (DZ) twins and in mothers who conceived their twins after the use of assisted reproduction techniques (ART). Participants in this study consisted of 8,222 and 5,505 women with spontaneous DZ and MZ offspring and 4,164 and 250 women with ART DZ and MZ twin pairs, respectively. Women were compared with respect to the number of sibs and offspring, the presence of other relatives with twins and the time it took to conceive the twins. We also compared familial twinning between a younger and an older age group.Women with spontaneous DZ twins more often reported female relatives with twins than those with spontaneous MZ twins. The proportion of DZ versus MZ twin offspring in relatives was also larger in women with spontaneous DZ offspring than in women with MZ offspring. The first group of women reported a shorter time to conceive. Women with ART twins had fewer sibs and offspring and less often reported relatives with twins. We did not observe that DZ twinning was more familial in women who had their twins before age 36 years compared to older women.Familial DZ twinning is clearly present in mothers of spontaneous DZ twins. The mechanisms underlying spontaneous and non-spontaneous DZ twinning are different and fertility treatment should be taken into account in any study of twinning. Twinning is not more familial in women who have their twins at a younger age.