1. Altered Sex Ratio and Increased Monozygotic Twinning Following Day 6 Blastocyst Transfers in Human IVF-ET Ellen H. Roots, L. Keith Smith and M. Janelle Odom Dorsett The Centre for Reproductive Medicine, Lubbock, TX Introduction Blastocyst culture with Day 5 transfers has been reported to increase the male/female sex ratio of babies born following IVF-ET (1) and to increase the rate of monozygotic twinning (2). Our laboratory has recently demonstrated that Day 6 blastocyst transfers can increase the implantation, pregnancy and live birth rates compared to Day 5 blastocyst transfers (3). We now report the effects of Day 6 blastocyst transfers on the male/female sex ratio, the rate of monozygotic twinning and the live birth weights in babies born following IVF- ET. Objective The objective of this study was to determine if Day 6 blastocyst transfers altered the male/female sex ratio, the rate of monozygotic twinning and the live birth weights in babies born following IVF-ET. Design A five-year retrospective analysis of all IVF cases resulting in live births following Day 5 or Day 6 blastocyst transfers from April 15, 1998 to July 15, 2003. Materials/Methods Oocyte fertilization, embryo culture and blastocyst transfers were performed using S1/S2 or G1.2/G2.2 sequential culture media (Vitrolife). Blastocyst transfers were performed on Day 5 or Day 6. IVF-ICSI was performed on ~10% of the cases. Oocyte donors were utilized in ~5% of the cases. All birth information was obtained directly from the patients (verbal or written). The normal male/female sex ratio was defined as 51% male/49% female = 1.04. The normal rate of monozygotic twinning was defined as 0.42%/delivery (4). Statistical analyses of the data were performed using Chi square or Student t-tests. Results Table 1: Sex Ratio of Babies Born Following IVF-ET TransferTotalSex DayBirthsMalesFemalesRatio ___________________________________________________________ Day 2/317088 (51.8%)82 (48.2%)1.07 Day 55636 (64.3%)20 (35.7%)1.80 Day 6192128 (66.7%)64 (33.3%)2.00 *** *** Results differ from the normal male/female sex ratio 51%/49% = 1.04 at P < 0.005 Chi square. Are new embryo culture techniques altering the male/female sex ratio of babies born following IVF-ET? Monozygotic twins as seen on vaginal ultrasound at 9 weeks post-conception. References 1. Menezo YJR, Chouteau J, Torello MJ, Girard A, Veiga A. Birth weight and sex ratio after transfer at the blastocyst stage in humans. Fertil Steril 1999;72:221-224. 2. Milki AA, Jun SH, Hinckley MD, Behr B, Giudice LC, Westphal LM. Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer. Fertil Steril 2003;79:503-506. 3. Smith K, Roots E, Odom Dorsett J. Day 6 blastocyst transfers can increase implantation, pregnancy and live birth compared to Day 5 blastocyst transfers. Embryologists’ Newsletter 2003;6:1-9. 4. Bulmer MG. The biology of twinning in man. Oxford:Clarendon Press, 1970. Table 3: Birth Weights of Babies Born Following Day 5 or Day 6 Blastocyst Transfers PregnancyTransferTotalBirth TypeDayBirthsWeight (grams) ________________________________________________________ SingletonDay 5183124 ± 576 Day 6753182 ± 548 TwinDay 5202078 ± 635 a Day 6932090 ± 737 a TripletDay 591514 ± 238 b Day 6121534 ± 496 b Birth weights are expressed as the mean ± one standard deviation (grams). a Results differ from singleton births at P < 0.05 Student t-test. b Results differ from singleton births at P < 0.01 Student t-test. Table 2: Monozygotic Twinning in Day 5 vs. Day 6 Blastocyst Transfers TransferTotalMonozygotic DayDeliveriesTwinsPercentP-Value ____________________________________________________________ Day 54012.5%NS Day 616453.0%< 0.05 Results compared to the normal rate of monozygotic twinning (0.42%) by Chi square. Day 6 blastocyst transfers also significantly increased the probability of monozygotic twinning. The mechanism for this increase is currently unknown. Day 6 blastocyst transfers did not significantly alter birth weights when compared to Day 5 transfers in singleton, twin or triplet births. Conclusions Patients should be counseled as to the increased male/female sex ratio, as well as the increased probability of monozygotic twinning associated with blastocyst culture. Discussion Day 6 blastocyst transfers significantly increased the male/female sex ratio in babies born following IVF-ET. Since the Day 2/3 data does not demonstrate this altered ratio, one possible explanation is that small developmental rate differences exist between male and female embryos that become apparent when in vitro culture is extended to 6 days. Another possibility is that extended culture conditions in vitro may simply favor survival of the male embryos. Results are expressed as the % of monozygotic twins/ delivery. *** Results differ from the natural rate of monozygotic twinning at P < 0.05 Chi square. *** Results differ from the normal male/female sex ratio 51%/49% = 1.04 at P < 0.005 Chi square.