1. HATCHING OF BLASTOCYSTS IN VITRO ON DAY 6 OF EMBRYO CULTURE IS CORRELATED WITH INCREASED PREGNANCY, IMPLANTATION AND LIVE BIRTH RATES IN HUMAN IVF-ET L.K. Smith, E.H. Roots and M.J. Odom Dorsett The Centre for Reproductive Medicine, Lubbock, TX Introduction Recent advances in ART have made it possible to routinely culture human embryos to the blastocyst stage. Blastocyst culture and transfer has been reported to increase pregnancy and implantation rates and to reduce the risk of higher order multiple pregnancies in human IVF-ET (1-4). Blastocyst transfers are performed primarily on Day 5 of embryo culture, with the most advanced embryos presenting as expanded blastocysts. Recent studies in our laboratory suggest that Day 6 blastocyst transfers increase pregnancy, implantation and live birth rates by increasing the efficacy of embryo selection. The most advanced embryos present as hatched zona-free (HZF) blastocysts or hatching blastocysts on Day 6. We have investigated what effect the hatching of blastocysts in vitro has on pregnancy, implantation and live birth rates in IVF-ET. Objective The objective of this study was to determine if the hatching of blastocysts in vitro was correlated with pregnancy, implantation and live birth rates in IVF-ET. Design A three-year retrospective analysis of all IVF-ET cases with Day 6 blastocyst transfers from January 1, 1999 to December 31, 2001. Materials/Methods Blastocyst culture was performed on all IVF-ET cases. Oocytes were co-incubated with 200,000 motile sperm/mL in 100 µL drops of S1 or G1.2 media (Vitrolife) under mineral oil (Squibb). All cultures were performed in a 37ºC incubator with 5% CO 2. Denuding and fertilization checks were performed at 18 hrs. Embryos were washed and moved to 30-100 µL drops of S1 or G1.2 media under mineral oil and cultured until Day 3. The embryos were then washed and transferred to 30-100 µL drops of S2 or G2.2 media under mineral oil and cultured until Day 6. Blastocyst transfers were performed on Day 6 with an 18 cm Wallace transfer catheter. Statistical analysis of the data was performed using Chi square. Results Blastocyst Classification Criteria: Early Blastocyst Blastocoele formation has begun. Cavitation is < 100%. Expanding Blastocyst Cavitation is 100%. Blastocyst expansion has begun. Hatching Blastocyst Blastocyst has herniated the zona. Hatched Zona-Free (HZF) Blastocyst Blastocyst has completely evacuated the zona. Blastocysts were classified as describe above. Results are expressed as a percent of the total blastocysts on Day 6. Table 1 : Effect of Blastocyst Hatching In Vitro on Pregnancy, Implantation and Live Birth Rates Blastocyst Stage:EarlyExpandingHatchingHZF BlastocystBlastocystBlastocystBlastocyst ------------------------------------------------------------------------------------------------------- # Transfers 41 42 71 33 Positive HCG/Transfer 26.8% * 61.9% 83.1% * 97.0% * Positive US/Transfer 19.5% * 42.9% 69.0% * 75.8% * Live Birth/Transfer 19.5% * 40.5%67.6% * 72.7% * % Implantation 13.7% * 34.7% 51.0% * 61.8% * Transfers were classified based on the developmental stage of the best embryo at transfer on Day 6. * Results differ from control (Expanding Blastocyst) at p < 0.01 Chi square. Conclusions The hatching of blastocysts in vitro was correlated with an increase in pregnancy, implantation and live birth rates in IVF-ET cases with Day 6 blastocyst transfers. These results suggest that the hatching of blastocysts in vitro may be used as a positive selection criterion for embryo transfers. IVF-ET cases that present  1 HZF or hatching blastocysts on Day 6 have implantation rates that make them excellent candidates for single embryo transfers. References 1. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998;69:84-88. 2. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434-3440. 3. Behr B, Pool TB, Milki AA, Moore D, Gebhardt J, Dasig D. Preliminary clinical experience with human blastocyst development in vitro without co-culture. Hum Reprod 1999;14:454-457. 4. Marek D, Langley M, Gardner DK, Confer N, Doody KM, Doody KJ. Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program. Fertil Steril 1999;72:1035-1040.