Vitrification solution without sucrose for cryopreservation in mouse blastocysts / 대한생식의학회지

OBJECTIVE: This study was designed to investigate the survival rate of vitrified mouse blastocysts depending on the presence or absence of sucrose in vitrification solution. METHODS: Mouse two-cell embryos were collected and cultured to blastocysts. Two vitrification solutions were prepared. The control solution was composed of 25% glycerol, 25% ethylene glycol, and 0.5 M sucrose (G25E250.5S) containing 2.5 mL glycerol, 2.5 mL ethylene glycol, 2 mL SSS, and 0.855 g sucrose in 5 mL PB1. The experimental solution was composed of 25% glycerol and 25% ethylene glycol (G25E25) and contained 2.5 mL glycerol and 2.5 mL ethylene glycol in 5 mL PB1. Artificial shrinkage was conducted by aspirating the blastocoelic fluid using an ICSI pipette. To examine the effect of sucrose in the vitrification solution on the survival rate of mouse blastocysts, the shrunken-equilibrated blastocysts were rehydrated or vitrified after being exposed to one of the two vitrification solutions. After exposure and the vitrification-thawing process, the re-expansion rate and hatching rate were evaluated after 6 hours of in vitro culture. RESULTS: The re-expansion rate of mouse blastocysts exposed to vitrification solution with and without sucrose were not different in the experimental solution (without sucrose) (98%) and the control solution (with sucrose) (92%) (p>0.05). The hatching rate was higher in the experimental solution (95%) than in the control solution (88%), but did not differ across two treatments (p>0.05). The re-expansion rate of mouse blastocysts vitrified in the control solution was 92% and 94%, respectively (p>0.05), and the hatching rate was higher in the experimental solution (90%) than in the control solution (74%) (p<0.05). CONCLUSION: Sucrose need not be added in vitrification solution for freezing of artificially shrunken mouse blastocysts.

Preliminary clinical outcome of novel strategy for the maximization of cumulative pregnancy rates per retrieval in normal responders / 대한생식의학회지

OBJECTIVE: We devised a novel strategy, a GnRH antagonist protocol with a GnRH agonist trigger followed by frozen-thawed blastocyst transfers with long zona dissection (LZD). The purpose of this study was to investigate the clinical outcomes of this new strategy according to age. METHODS: Ninety women aged less than 35 (group A) and 32 women aged 35 to 39 (group B) underwent the GnRH antagonist protocol with a GnRH agonist trigger in order to obtain many oocytes and prevent early-onset ovarian hyperstimulation syndrome (OHSS). All oocytes were cultured to the blastocyst stage and all blastocysts grade 3BB or better were cryopreserved. Embryo transfers were only performed in freeze-thaw cycles to prevent late-onset OHSS and to overcome embryo-endometrium dyssynchrony. LZD was performed just after thawing to improve hatching and implantation rates. RESULTS: The average numbers of retrieved oocytes and blastocysts grade 3BB or better were 12.8+/-5.5 and 4.4+/-2.6 in group A and 10.9+/-7.4 and 2.5+/-2.2 in group B, respectively, and OHSS did not occur in any of the women. Implantation rates were 46.7% in group A and 39.3% in group B. Cumulative clinical pregnancy rates per retrieval were 77.8% in group A and 62.5% in group B. Cumulative ongoing pregnancy rates per retrieval were 71.1% in group A and 53.1% in group B. CONCLUSION: GnRH antagonist protocol with GnRH agonist trigger followed by frozen-thawed blastocyst transfers with LZD can generate many blastocysts without OHSS and maximize cumulative pregnancy rates per retrieval. This strategy is more effective in young women aged less than 35 than in women aged 35 to 39.

Comparison of pregnancy and implantation rates in fresh embryo transfer (ET) and frozen-thawed ET cycles in infertile women with polycystic ovarian syndrome / 대한산부인과학회지

OBJECTIVE: Aim of this study is to evaluate the pregnancy and implantation rates in fresh-embryo transfer (ET) and frozen-thawed ET cycles in women with polycystic ovarian syndrome (PCOS). METHODS: PCOS was diagnosed by the Rotterdam criteria. In 4 cases of 72 stimulation cycles, ET was not conducted due to severe ovarian hyperstimulation syndrome (OHSS). Sixty eight cycles of fresh-ET and 40 cycles of frozen-thawed ET were included in this retrospective study. Age, gravidity, body mass index, infertility duration were compared between two groups. Number of embryos transferred, implantation rate, clinical pregnancy rate and multiple pregnancy rate were compared between two groups by using chi-square test and student's t-test. RESULTS: Number of embryos transferred showed significant difference between two groups. Fresh-ET group was 4.7 and frozen-thawed ET group was 2.8 (P<0.001). However, overall clinical outcomes with fresh-ET and frozen-thawed ET cycles were similar. Implantation rates were 8.3% vs 11.5%, clinical pregnancy rates were 27.9% vs 25.0% and multiple pregnancy rates were 36.8% vs 20.0%. CONCLUSION: Although more number of embryos were transferred in fresh-ET cycles, the clinical outcomes were similar between fresh-ET and frozen-thawed ET cycles. It may be due to decreased uterine receptivity in fresh-ET cycles. Frozen-thawed ET may be used as alternative plan for cases of severe OHSS and decreased uterine receptivity expected.

Effect of the decidualization of human endometrial cells on the hatching rates of the vitrified and the fresh mouse blastocysts / 대한산부인과학회지

OBJECTIVE: To compare the hatching rates of the vitrified and the fresh mouse blastocysts co-cultured with decidualized or non decdualized human endometrial cells and to confirm the necessity of assisted hatching in the vitrified mouse blastocyst. METHODS: Stromal and epitherial cells isolated from human endometrial tissue were co-cultured and decidualized with TGF-beta 1 and progesterone. The vitrified and the fresh mouse blastocysts were co-cultured with human decidualized or non-decidualized endometrial cells, respectively and the hatching rates were investigated. RESULTS: Epithelial and stromal cells isolated from endometrial tissue were cultured seperately for 24 hours and stained by immunohistochemical staining for cytokeratin (epithelial cells) or vimentin (stromal cells). The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. The co-cultured human stromal and epitherial cells were decidualized by administration of TGF-beta 1 and progesterone. The hatching rates of the fresh and the vitrified mouse blastocysts co-cultured with decidualized human endometrial cells were 89% and 31%, respectively. The hatching rates of the fresh and the vitrified mouse blastocysts co-cultured with non-decidualized human endometrial cells were 82% and 27%, respectively. The hatching rates were significantly higher in fresh mouse blastocysts than in vitrified mouse blastocysts regardless of decidualization of human endometrial cells (P<0.05). CONCLUSION: The hatching rate was significantly higher in fresh mouse blastocysts than in vitrified mouse blastocysts. This results showed that the cryopreservation procedure caused the zona hardening of mouse blastocyst and the decidualization of endometrial cells did not affect to hatching rate of the vitrified mouse blastocysts. We confirmed that assisted hatching was necessary for improving the hatching rate of cryopreserved mouse blastocysts.