The Research On The In-vitro Maturation And Vitrification Of The Immature Oocytes From Superovulation Cycles And Natural Cycles

Part 1 The in-vitro maturation of immature oocytes from superovulation cycles and natural cyclesObjective:the traditional assisted reproductive techniques (ART), the patients, undergoing controlled ovarian stimulation cycle, could obtain a large number of oocytes. There were about 15% immature oocytes in these oocytes. The immature oocytes were abandoned due to unable to be fertilized. In this study, the immature oocytes from controlled ovarian stimulation cycles and nature cycles were matured in vitro. After IVM, matured oocytes were inseminated by ICSI. Then the fertilization rate, embryo development and pregnancy rate were analyzed. The results help us to evaluate the clinical application value of these immature oocytes, and improve the utilization rate of oocytes.Methods:1. Objects:248 immature oocytes in superovulation group from 64 patients, undergoing the ART treatment in the Assisted Reproductive Medical Center, NanFang hospital, Southern Medical University, between 2010 and 2014, and 45 immature oocytes from natural cycles were selected with in vitro maturation treatment.2. Methods:The patients were started on a long protocol in the luteal phase of the last cycle for down-regulation. At 14 or 15 days after GnRH agonist administration, their serum levels of E2, P and LH, as well as transvaginal ultrasound were examined. Ovarian stimulation was commenced with gonadotrophin (Gn) after pituitary down-regulation. The doses of Gn were adjusted according to the outcome of transvaginal ultrasound and their serum levels. When there were more than 2 follicles whose diameter were more than 18 mm,10000 IU hCG was injected at the night. Thirty-six hours later, oocytes were collected under the guide of transvaginal ultrasound. The oocytes were incubated 4-6 hours at 37℃,6%CO2,95%humidity. Then the CCs were removed by mechanical pipetting. The collected immature oocytes were cultured in G-IVF plus medium 24-30 hours at 37℃,6%CO2,95% humidity. After culture, the mature oocytes were fertilized by ICSI and cultured in G-l plus medium 16-18 hours at 37℃,6%CO2,95% humidity. The conditions of fertilization were observed after 16-18 hours. The embryos with 2PN were cultured in G-l plus medium and the quality of embryo were assessed in 24 hours and 48 hours. The best embryos were transferred at day 3 and the others were frozen. Serum β-HCG was tested on day 12 after the embryo transfer. If the outcome was positive, the sac was confirmed by ultrasound on 4 weeks after embryo transfer, which was considered as a clinical pregnancy. Then the maturation rate, fertilization rate, cleavage rate, high quality embryo rate and clinical pregnancy rate were compared between the two groups.Results:The average age of women was similar in superovulation cycle group (34.48±5.7) and in natural cycle group (36.29±7.2) (P>0.05). There were 248 immature oocytes in superovulation cycle group and 45 in natural cycle group. After IVM,163 oocytes had matured in superovulation cycle group and 43 in natural cycle group. The maturation rate in the natural cycle group was significantly higher than that of superovulation cycle group (P<0.05). The fertilization rate was 64.42% in superovulation cycle group and 76.74% in natural cycle group. The cleavage rate was 89.89% in superovulation cycle group and 100% in natural cycle group. The high quality embryo rate was 17.50% in superovulation cycle group and 33.33% in natural cycle group. There was no significant difference between the two groups in the fertilization rate, cleavage rate and high quality embryo rate (P>0.05). However, clinical pregnancy rate in the natural cycle group were significantly higher than that of superovulation cycle group (P<0.05). The clinical pregnancy rate was 0% in superovulation cycle group and 26.32% in natural cycle group.Conclusion:1. Immature oocytes collected from natural cycle could reach MII and high quality embryo could be obtained after ICSI. There is a better clinical application value.2. The immature oocytes collected from superovulation cycle could reach MII, and high quality embryo could obtained after ICSI.Part 2 The Vitrification of immature oocytes from superovulation cyclesObjective:In the traditional assisted reproductive techniques (ART), the patients, undergoing controlled ovarian stimulation cycle, could obtain a large number of oocytes. There were about 15% immature oocytes in these oocytes. The immature oocytes were abandoned due to unable to be fertilized. In this study, the immature oocytes obtained from ICSI cycle were matured in vitro. After IVM, matured oocytes were cryopreserved by vitrification and thawed. Then the oocytes were inseminated by ICSI. The fertilization rate, embryo development were evaluated. Fertilization, embryo development of in-vitro matured oocytes which were cryopreserved and thawed by vitrification were investigated. To detect the reuse of immature oocytes.Methods:1.Objects:There were 92 immature oocytes in vitrification group and 248 immature oocytes in superovulation group from patients, undergoing the ART treatment in the Assisted Reproductive Medical Center, NanFang hospital, Southern Medical University, between 2010 and 2014, were selected to study.2. Methods:The immature oocytes of vitrification group were cultured in G2-plus medium 24-30 hours at 37℃,6%CO2,95% humidity. After culture, the mature oocytes were cryopreserved and thawed.1) vitrification:1-2 oocytes(no more than 3) were put into ES medium for 5-15 minutes, then the oocytes were taken into VS medium. Suck and blow oocytes 3 times or more in different position of VS medium. The oocytes were put on tip of the Cryotop and put into liquid nitrogen quickly. The Cryotop were preserved in liquid nitrogen canister.2) thawing:The Cryotop were taken out from liquid nitrogen canister and put into TS medium. Find the oocytes under a dissecting microscope. The oocytes were stayed in TS medium for 1 minute, then put them into DS medium for 3 minutes. After 3 minutes, the oocytes were put into WS1 medium for 5 minutes and WS2 medium for 5 minutes. Last, the oocytes were cultured in G-IVF plus medium at 37℃,6%CO2,95% humidity.3) The oocytes were cultured for 2 hours and then fertilized by ICSI.Results:The average age of women was 34.48±5.7 in IVM group and 32.00±4.2 in vitrification group, and there was no significant difference (P>0.05).248 immature oocytes were in IVM group and 92 in vitrification group. After IVM,163 immature oocytes were matured in IVM group and 78 in vitrification group. The mature rate was significant difference between the two groups. The vitrification group contained 51 GV and 41 MⅠ. After IVM,38 immature oocytes were matured in GV group and 40 in MⅠ group. The difference of mature rate had statistical significance (P<0.05). 35 mature oocytes were cryopreserved and 30 oocytes were survival after thawing in GV group.43 mature oocytes were cryopreserved and 33 oocytes were survival after thawing in MⅠ group. The survival rate between GV group and MⅠ group was no significant difference. After ICSI, The fertilization rate was 64.42% in IVM group and 77.78% in vitrification group. The cleavage rate was 89.89% in IVM group and 91.11% in vitrification group. The high quality embryo rate was 17.50% in IVM group and 21.95% in vitrification group. There was no significant difference between the two groups in the fertilization rate, cleavage rate and high quality embryo rate (P>0.05). The fertilization rate, cleavage rate and high quality embryo rate were no significant difference between GV group and MI group too(P>0.05). The fertilization rate was 73.33% in GV group and 81.82% in MⅠ group. The cleavage rate was 90.90% in GV group and 91.30% in MⅠ group. The high quality embryo rate was 15.00% in GV group and 28.57% in MⅠ group.Conclusion:1. The immature oocytes could reach MⅡ, which were cultured in G2-plus medium.2. The mature oocytes which were cryopreserved by vitrification and thawed could develop high quality embryo after ICSI.