The incidence of monozygotic twinning (MZT) is greatly increased among IVF patients compared to the general population (0.7-13% vs. 0.45%, respectively), but the reason for this remains unclear1.To further investigate this association, we sought to ascertain whether MZT events occur in clusters and, if so, to explore possible explanations for this clustering. Retrospective cohort study. Using an established and validated IVF database from a single large urban IVF center, consecutive fresh IVF cycles (autologous and oocyte donation) resulting in a viable clinical pregnancy (confirmation of a gestational sac(s) and presence of a fetal pole with a heartbeat(s) on ultrasound) from Jan 2002 to Dec 2013 were retrospectively reviewed. The incidence of MZT overall and separately for each 6-month interval was calculated as the total number of MZT events divided by the number of viable clinical pregnancies. 6-month intervals with a MZT incidence rate >2 standard deviations (SD) higher than the overall rate were regarded as a high risk interval. Logistic regression modeling was then used to adjust for both time and non-time related risk factors for MZT events within our cohort (Table 1). Over the 12 year study period, 25,502 fresh IVF cycles were performed, resulting in 8,598 clinical pregnancies. Of these, 95 cycles (1.1%) resulted in MZ twins. Median patient age (±SD) in the MZT cohort was 35.4 years (±4.4). The % of MZT was >2SD higher than the overall % of MZT in 4 of the 24 6-month intervals. PGD, extended embryo culture (≥4 days), and more recent cycle (2005 or later) were independent risk factors for MZT. Conversely, increasing numbers of embryos transferred appeared to decrease the risk of MZT. Use of multivariable logistic regression modeling to control for risk factors for MZT did not correct for this clustering effect, with both high-risk interval (clustering) and extended embryo culture remaining significant after adjustment (Table 1.) This study supports our hypothesis that MZ occurs in clusters, and that this clustering effect could not be explained by demographics and cycle characteristics alone. Our study would suggest that external factors both clinical (such as type of stimulation) and laboratory (type or lots of cultured media, days in culture) may be involved.Tabled 1Table 1. Risk Factors for MZT events among 8598 fresh IVF cycles from Jan 2002 through Dec 2013.MZT (N=95)Non-MZT (n=8503)Un-adjustedp-valueAdjusted OR (95% CI) and p-valueHigh-risk 6-month interval35.8% (34)15.5% (1321)<.00012.37 (1.53, 3.66) p=.0001Embryo Biopsy12.6% (12)3.5% (298)<.00011.63 (0.82, 3.23) p=.16Assisted Hatching7.4% (7)11.1% (940)0.2537--Donor cycle8.4% (8)8.7% (743)0.9133--ICSI31.6% (30)38.5% (3277)0.1655--Year 2005 or later86.3% (82)72.9% (6197)0.00331.56 (0.84, 2.91)p=0.16Extended Culture45.3% (43)18.1% (1541)<.00012.68 (1.66, 4.33) p<.0001Oocyte provider >35 years46.3% (44)48.6% (4136)0.6519--Age at cycle start35.4 ±4.435.9 ±4.60.3753-->2 embryos transferred20.0% (19)31.7% (2692)0.0150.75 (0.45, 1.40) p=0.42 Open table in a new tab