Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey

The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula-blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.     

Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes

BACKGROUND: Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS: Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS: The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS: The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.     

Study on the developing potentiality of mouse morula produced in vitro or in vivo after vitrification

Great horovement haS been made in the cryopreservation Of inalnlnalian elnbmp since wnttingham['] reported his Pioneering wold about successful cryOPreservationof mouse embryo. It results in the availability of cryOPreserVation of embryos of all stages[']. HOwever, cryopreserved embryos can not avoid the damages by ice crystal,which is a major cause of cen death in the freezing andthawing ProceduresL',']. SO it is necesseq to seareh for anew and more efficient method for embryO cryOPrese…     

Improved implantation rate after chemical removal of the zona pellucida

OBJECTIVE: To evaluate the implantation rate achieved after chemical removal of the zona pellucida from day 5 human in vitro-derived embryos. DESIGN: Prospective, randomized, controlled study. SETTING: A tertiary care infertility clinic. PATIENT(S): Two hundred fifty-seven patients undergoing IVF with transfer of morulas or blastocysts on day 5 after oocyte retrieval. All patients had had at least two previous implantation failures. INTERVENTION(S): Chemical removal of zona pellucida by using acidic Tyrode's solution vs. no removal (controls). MAIN OUTCOME MEASURES: Clinical pregnancy rate and implantation rate per transfer. RESULT(S): Embryos without zona pellucida implanted at nearly twice the rate of control embryos (15.7% vs. 27.5%). The pregnancy rate was also significantly higher in the zona pellucida-free group than the control group (31.0% vs. 46.1%). Removal of zona pellucida was most effective in embryos with delayed development, which reached the morula or early cavitating stage on day 5 of in vitro culture (implantation rate, 12.1% vs. 25.7%). CONCLUSION(S): Chemical removal of zona pellucida from day 5 in vitro cultured human embryos is an effective and safe method of significantly improving the implantation rate, especially of embryos with delayed development.     

Cryopreservation of human embryos at the morula stage and outcomes after transfer

OBJECTIVE: To evaluate the survival rate of human morula embryo freezing and the morphological alterations during freezing, during and after thawing, and their applications in embryo selection. DESIGN: Retrospective observational study. SETTING: Private infertility clinic. PATIENT(S): Consecutive patients under age 39 undergoing frozen morula embryo transfers from December 1999 to May 2003. INTERVENTION(S): Embryo freezing was performed at the morula stage. Embryo thaw and post-thaw ETs were conducted on the same day, which is equivalent to a day 4 ET. MAIN OUTCOME MEASURE(S): Morphological alterations during freezing and thawing and after thawing. Post-thaw embryo survival rates, transferable rates, pregnancy rates, and implantation rates. RESULT(S): Morula embryos showed reversed morphological alterations during the freezing process; these alterations were recovered during thawing or shortly after the thawing. Post-thaw survival rates showed no significant difference between any of the morula substages. However, embryos scored as grade 3, which represented good quality, had significantly higher post-thaw survival and transferable rates than grade 2 and 1 embryos. Patients who received at least one grade 3 embryo had significantly higher pregnancy rates, implantation rates, and ongoing/live birth rates than other groups. CONCLUSION(S): An acceptable survival rate can be achieved after cryopreservation of human morula embryos, and morphological alterations that occur during and shortly after an embryo thaw can be a feasible index for determining viable embryos.     

玻璃化冷冻法在人桑葚胚胎和卵裂期胚胎冻融周期中的临床研究

目的探讨玻璃化冷冻法对人桑葚胚和卵裂期胚胎解冻后的妊娠结局。方法回顾性分析河北医科大学第二医院生殖中心行冻融胚胎移植患者的解冻及妊娠情况,并根据冷冻方法和胚龄分为3组。A组为前期采用慢速程序法冷冻卵裂期胚胎的冻融移植周期,共215周期;B组为前期采用玻璃化法冷冻卵裂期胚胎的冻融移植周期,共208周期;C组为前期采用玻璃化法冷冻桑葚期胚胎的冻融移植周期,共159周期。比较A组和B组解冻后的胚胎存活率、存活胚胎的完整率、临床妊娠率、胚胎种植率和流产率;比较B组和C组解冻后的胚胎存活率、存活胚胎的完整率、临床妊娠率、胚胎种植率和流产率。结果 A组和B组比较,胚胎存活率和存活胚的完整率差异有统计学意义(P<0.01),胚胎种植率、临床妊娠率、流产率差异有统计学意义(P<0.05),临床妊娠率差异无统计学意义(P>0.05),而胚胎种植率、流产率差异有统计学意义(P<0.05)。B组和C组比较,存活胚完整率差异有统计学意义(P<0.01),胚胎种植率差异有统计学意义(P<0.05),胚胎存活率和临床妊娠率C组高于组B,但差异无统计学意义(P>0.05)。结论玻璃化冷冻法在冷冻人卵裂期胚胎由于具有较高的胚胎存活率和存活胚完整率而优于慢速程序化冷冻法。而桑葚期胚胎的玻璃化冷冻法在存活胚的完整率和胚胎种植率上优于卵裂期胚胎,可能是玻璃化法冷冻保存胚胎的较好时期。