The Research On The Safety And Efficiency Of Human Oocyte Vitrification

Mature oocyte cryopreservation is currently the best technic for female fertility preservation.It has irreplaceable advantages in the field of assisted reproductive technology,and extends the research field of human gametes and embryo development potential.Oocyte cryopreservation is more likely to associate with damage during the cryopreservation procedure due to its special structure characteristics.The damage of cell structure,spindle,plasma membrane and zona pellucida could affect the fertilization and embryo developmental potential after warming.Oocyte cryopreservation technology showed little promising progress in the early days,only in the past ten years,it has a rapid development due to the vitrification technic and application and intracytoplasmic sperm injection(ICSI).Oocyte cryopreservation has not been used as routine procedure for ART,for more clinically sample and further research are needed to prove its safety.The principle of vitrification is to increase the speed of temperature and concentration of Cory-protectant agent,transfer the inter and outer cellular from fluid to solid as a non-crystal state,forming a solid-glassed state,keep the molecular and ionic distribution inside the cellular fluid.Vitrification has been proved to be more efficient to oocyte compared with slow cooling,but the high concentration of cryoprotectant could be poison to the cell,the key factors of vitrification is the cooling rate is quickly enough so that the cryoprotectant do not harm to the cell.The aim of this study is focus on the oocyte morphologically analysis,the time it needs to digest the zona pellucida,ROS level,spindle structure and the expression of HIFOO after vitrification-warming procedure from morphology,cellular and molecular perspective.Compare the developmental potential of fresh and vitrified human oocyte after warming procedure and recruit CNV-SEQ test to the blastocyst for vitrified oocyte to confirm the safety of cryopreservation on genome perspective.Finally,we analysis the clinically data,use a-CGH to detect the safety of the generation of oocyte vitrification family,and its effect on further safety and effective issue of oocyte vitrification,provide the improvement and clinically application in the future.Part 1 The effect of vitrification on the function and mechanism of oocyteAim: To investigate the effect of vitrification on the function and mechanism of oocyte.Method: Collect the routine unfertilized oocyte for IVF cycle,record the morphology parameter change before and after vitrification,including diameter,perivitelline space,zona pellucida and polar body.The unfertilized oocytes were random divided into fresh and vitrification group.Compare the time Tyroid acid digest cumulus oocyte,plasma ROS level and H1 FOO expression.IVM the immature oocyte from IVF cycle,culture them to MII oocyte,and then observe the spindle,divided group into A,B,C group according to the image.Compare the morphology change before and after vitrification using immune-fluorescent dying to observe spindle structure using confocal microscope.Result:(1)There were totally 236 oocytes,201 oocytes survived after vitrification.(85.17% survival rate),there were significantly differences on oocyte diameter and perivitelline space between two groups 0 hour after warming,no difference on the parameter of zona pellucida and polar body.No significant difference on morphology parameter of oocyte 2 hours after vitrification.(2)The zona pellucida of 50 oocytes were digest on each group,the vitrified group need more time to digest the zona pellucida(p<0.05).(3)188 unfertilized oocytes were recruit to detect the ROS level,ROS were lower for 0hr after warming group(49 oocytes)compare with unvitrified group(42 oocytes).There were 51 oocytes in 2hrs after warming group,the ROS level were higher than unvitrified group.And 46 oocytes in 4hrs after warming group,slightly higher than unvitrified group,no significantly difference between them.(4)The H1 FOO expression were detected for all104 oocytes,50 for unvitrified group and 54 for 2 hrs after warming group,the H1 FOO expression were lower in vitrified group compare with unvitrified group(p<0.05).(5)195 oocyte were survived after vitrification for totally 230 IVM oocytes,the survival rate for vitrification was significantly different according to the type of spindle(p<0.05).No difference between type A and unvitrified oocyte.(6)There were totally 16 MII oocytes after IVF and only 2 had spindle malformation,30 oocytes for vitrified group and 4 of them were abnormal(13.3% abnormal rate).So vitrification had no effect on spindle structure.Conclusion: Vitrification had negative effect on oocyte zona pellucida,ROS level 2 hrs after warming and H1 FOO gene expression.No effect on morphology and spindle of oocyte.Part ? The effect of vitrification on oocyte developmental potential and blastocyst CNV-Seq.Aim: Compare the developmental potential for vitrified oocyte,and detect the safety issue of blastocyst from the genome perspective.Method: IVM the immature oocyte from IVF cycle to MII oocyte,random divided into fresh and vitrified group,then ICSI and culture the oocytes,compare the fertilization rate,cleavage rate and development potential.And CNV-SEQ test were recruit for available blastocyst from vitrified group.Result: 313 MII oocyte were obtained after IVM,106 oocytes were detect do ICSI and culture.207 oocytes were vitrified before ICSI and culture,175 were survived after warming(84.54% survival rate).No significant difference on fertilization rate,cleavage rate.The obtaining embryo and blastocysts are significantly lower in vitrified group compare with fresh group(p<0.05).The number of more than 5 blastomere D3 embryo were lower in vitrified group(p<0.05).No difference on fragmentation rate over 50% embryo for two groups.The results for CNV-Seq test from blastocysts obtaining from vitrified group showed only one blastocyst of 8 blastocytes successfully examinated were abnormal(12.5% abnormal rate).Although two blastocyst of 5 blastocytes obtaining from fresh oocytes successfully examinated wereabnormal(40% abnormal rate).Conclusion: Oocyte vitrification had no effect on fertilization rate,but affect the embryo developmental potential due to the developmental delay.It is safe for the blastocyst from genome sequence result.Part ? The research on clinical application and the safety of generation for oocyte vitrification.Aim: To investigate the clinical data of oocyte vitrification and the safety of generation using genome test.Method: Record the oocyte vitrification data from 2011 to 2013 in our clinic,and run a-CGH test to the birth baby from two families to detect the safety of oocyte vitrification.Result: There are totally 82 oocyte vitrification cycles,55 cycle were no available sperm during the OPU day,23 partial oocyte vitrification,and 4 cycle for fertility preservation.There are totally 623 MII oocytes,the average oocyte vitrification number is 7.6.And 45 cycle(338 oocytes)were warming,the survival rate was 84.19%,fertilization rate was 81.23%,the cleavage rate was 92.44%,D3 available embryo rate was 39.54%,28 cycle embryos weretransferred,the pregnancy rate was 25%(7 cycles).The age of no available sperm group were higher than partial group(p<0.05),it had lower available embryo rate(p<0.05),no significant difference on pregnancy rate due to the small sample(19.05% vs 42.86%,p>0.05).The survived oocytes and cycles with available embryos from patient lower than 35 were higher than patient over 35,no significant difference on pregnancy rate due to the small sample(27.27% vs 16.67%).From the a-CGH result,it indicated there is no new gene mutation after oocyte vitrification.Conclusion: Oocyte vitrification had beneficial for those patients couldn't provide sperm during the OPU day and patients with large number of oocytes,although it is not efficient for patient over 35,but the safety of oocyte vitrification generation was preliminary proved.