| Part1Effects of modified vitrification media on the development potential of human vitrification-warmed in vitro matured oocytesObjective To certify the effects of modified vitrification media on the developmenta potential of human in-vitro matured oocytes.Methods Immature oocytes were collected from controlled ovarian stimulation cycles for ICSI treatment. All these immature oocytes were cultured for18-24hrs for maturation. Only matured oocytes were enrolled in this study. Gathered human in-vitro matured oocytes were randomly divided into control and three vitrified group.Group A was control group without vitrification treatment. Group B was vitrified with EG and DMSO. Group C was vitrified with EG and PROH. Group D was vitrified with EG, DMSO and PROH. The developmental potential was evaluated with survival rate, fertilization rate,2PN rate, cleavage rate of2PN, high-quality embryo rate, balstocyst rate and high-quality blastocyst rate.Result The vitrification process affected the development potential of these in-vitro matured oocytes. The fertilization rates, high-quality embryo rates, and balstocyst rates in the three vitrification groups were significantly lower than the control group (P<0.01).Though the survival rate (84.8%),2PN rate (46.7%), high-quality embryo rate (5.7%), balstocyst rate (7.6%) in Group D were higher than those of in Group B (77.2%,42.4%,2.2%,2.2%, respectively) and Group C (80.0%,40.0%,2.2%,3.3%, respectively), there was no significant difference (P>0.05). In three vitrifcaiton goups, high-quality blastocysts were got only in Group D (1.9%). Though high-quality blastocysts in Group D was lower than that of in Group A (6.7%), there was no significant difference (P>0.05).Conclusion The vitrification process reduced the development potential of the in-vitro matured oocytes. The modified vitrification media is effective for in-vitro matured oocytes vitrification through increasing the high-quality blastocyst rate.Part2Effects of modified vitrification media on the morphorlogy and ultrastructure of human vitrification-warmed in vitro matured oocytes.Objective To study the effects of modified vitrification media on the morphorlogy and ultrastructure of human in vitro matured oocytes.Methods Immature oocytes were collected from controlled ovarian stimulation cycles for ICSI treatment. All these immature oocytes were cultured for18-24hrs for maturation. Only matured oocytes were enrolled in this study. Gathered human in-vitro matured oocytes were randomly divided into control and two vitrified group. Matured oocytes in conventinonal vitrification group were vitrified with EG and DMSO. Modified vitrification group was vitrified with EG, DMSO and PROH. All the vitrified oocytes were warmed in1to5months. Control group was not given vitrification. The oocytes in control group and two vitrification groups were stained by Rhodamine-phalloidine and observed by laser confocal microscopy (LSCM). Some of the oocytes in the three groups were fixed and observed under transmission electron microscopy (TEM).Result There were a lot of vesicles in part of the warmed oocytes in the two vitrification groups, distributed under the membrane of the oocytes. The vesicles enclosed oocytes rate was18.7%in the modified vitrification group, which was lower significantly than the conventional group (66.7%)(P=0.000). There was no significant difference between modified vitrification group and conventional group in the rate of microfilament normal distribution (43.8%vs33.3%, P>0.05), but lower significantly than the control group (71.1%)(P<0.05). TEM results revealed that there were more vesicles in the cytoplasm in the two vitrification groups comparing with the control group. The morphology of mitochondria and vesicles were similar between the vitrification groups and control group. The distribution of the cortical granules in the vitrification-warmed oocytes was nearer to the periphery of the oocytes. Though the cortical granules extrusion was not observed, the number of the cortical granules was less in the vitrificaiton-warmed oocytesConclusion The modified vitrification protocol is useful for the recovery of morphorlogy and ultrastructure of the vitrification-warmed human in vitro matured oocytes compared with conventional vitrification protocol. Vitrificaiton process affected the number and distribution of cortical granules.Part3Effects of different vitrification media on the development potential of vitrification-warmed in vivo matured oocytes and clinical results Objective To study the effects of different vitrification media on the development potential of vitrification-warmed in vivo matured oocytes and clinical results.Methods This retrospective study was involved in all of the vitrification-warmed oocytes between2012January and2013December. All of the oocytes were vitrified in the last five years. Different vitrification media were used (MC media, KT media and modified media). After warming, the rates of oocyte survival, fertilization, high-quality embryo, blastulation, clinical pregnancy and implantation were recorded and analyzed. Also, the usage rate of every warmed oocyte was calculated. Data from self-oocytes warming and donor-oocytes warming were calculated separately.Result For the self-oocytes warming group, the oocytes survival rate of modified media (92.0%) was significantly higher than the MC media (88,2%) and KT media (77.3%)(P<0.05). High-quality embryo rate in modified media group (35.8%) was significantly higher than the other groups (29.0%vs28.3%)(P<0.05). There was no significant difference in the clinical pregnancy rate and implantation rate between MC, KT and modified media groups (37.2%vs30.2%vs39.6%and21.9%vs18.8%vs27.4%)(P>0.05). In spite of that, the number of embryo transfered in every cycle (1.89±0.59) was significantly lower in modified group compared with MC media group (2.28±0.83)(P=0.000). When it comes to oocytes usage efficiency, the high-quality embryo rate for every warmed oocyte was higher significantly than other two groups (P<0.01). Though the utilization of the oocyte was higher than the other two groups, there was no significant difference (P>0.05). Only MC media and KT media were used in the donor-oocyte cycles. There was no statistical difference in the rates of survival, fertilization, high-quality embryo, clinical pregnancy and implantation (P>0.05). Compared with data in the self-oocytes group for MC media and KT media, the clinical pregnancy rates were perfectly higher (50%and43.5%, respectively).Conclusion This clinical study showed that the modified vitrification media could get more high-quality embryos and higher clinical pregnancy rate. This modified media would be used effectively in clinical application since it is an efficient vitrification protocol. More subtle study should be carried out for a reliable conclusion. |
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