| Objective: To explore the effect of low oxygen（5%O2）and individual protocol onin-vitro maturation of immature human oocyte from ICSI cycle.Methods: Totally556immature human oocytes were collected in ICSI cycles，and wererandomly divided into two groups: conventional group（20%O2）,319immatureoocytes for IVM; Low oxygen group（5%O2）,X237immature oocytes. All the immatureoocytes in the two groups were cultured in vitro, and then in-vitro matured oocytes wereperformed with ICSI,1618h later, the status of fertilization of the injected oocyteswere identified on the basis of the appearance of the second polar body and formation ofpronuclear, subsequently, normally-fertilized oocytes were picked up and cultured toblastocyst stage, at the same time, observing and recording the rates of maturation, fertilization, cleavage, D3high-quality embryos, blastocyst and high-quality blastocysts.According to in-vitro maturation protocol of immature oocytes, conventional group（20%O2）and low oxygen（5%O2）group were also randomly divided into two groups,respectively：conventional IVM group, all of the immature oocytes were placed intoIVM medium to cultured in37℃,6%CO2,20%O2and100%humidity condition,36hlater, the oocytes developing to metaphase II（MII）stage were conducted with ICSI;Individualized IVM group, all of the oocytes in this group were cultured for4h in thesame condition as the former, then observing the appearance of the first polar bodyevery two hours under the inverted microscope, picking up the oocyte with the firstpolar body and placing them into fertilization medium for a further4hours of in-vitrocuture,4h later, ICSI and embryo culture was conducted. The remaining immatureoocytes repeated the previous procedure until the longest36hours of in-vitromaturation time. In the process of the experiment, the rates of maturation, fertilization,cleavage, D3high-quality embryos, blastocyst and high-quality blastocysts wererecorded. Furthermore, All of the formed high-quality blastocysts were analysed for8pairs of chromosomes：13，15，16，18，21，22，X and Y through two rounds of FISH.In the first round of FISH,13,21,22,16and18pairs chromosomes were analyzed witha corresponding probe marked with red, green, gold, aqua and blue fluorescent; In thesecond round of FISH,15, X and Y chromosomes were diagnosed, and thecorresponding probes were respectively marked with green, orange and aquafluorescent,.Results: conventional group（20%O2）：319GV+MI oocytes were collected，258oocyteswere matured with IVM，227matured oocytes were aquired and fertilized with ICSI，1622PN embryos were obtained，in which153oocytes were cleavaged，formed53good quality embryos （D3），37blastocysts including9high-quality blastocysts wereobtained. Comparison between the two groups showed no significantly difference inmaturation rate, fertilization rate,2PN rate,2PN cleavage rate and high-quality blastocyst rate（P＞0.05）. However, D3-good quality embryos rate and blastocyst rateshowed highly statistical difference（P＜0.01，X2ï¼15.13; P＜0.01，X2ï¼8.65）. Underthe condition ofX20%O2,conventional IVM group contained112oocytes,94oocytesmatured after IVM，84oocytes fertilized after ICSI，containedX2PN embryos76，inwhich702PN embryos cleavaged，10D3-good quality embryos，6blastocysts wereformed, no high-quality blastocysts were obtained. Individualized IVM group contained207oocytes,164oocytes matured after IVM，143oocytes fertilized after ICSI，125embryos（including2PN），in which1142PN embryos cleavaged，20D3-good qualityembryos，16blastocysts were formed which included4high-quality blastocysts.Comparison between the two groups, there was no significantly difference in maturationrate, fertilization rate,2PN rate,2PN cleavage rate and high-quality blastocyst rate（P＞0.05），D3-good quality embryos formation rate and blastocyst formation rate showedhighly statistically difference（P＜0.01，X2ï¼18.51; P＜0.01，X2ï¼13.78）.13high-quality blastocyst were obtained，all of them were fixed successfully for thefollowing FISH diagnosis,9high-quality blastocysts in the low oxygen group（5%O2）,4showed aneuploid and aneuploid rate was44.4%; For4high-quality blastocysts in theconventional group（20%O2）,2showed abnormal after FISH diagnosis and abnormalrate50%. No statistical difference in abnormal embryonic rate was found between thetwo groups（P＞0.05）.Conclusion Low oxygen（5%O2）is better to improve in-vitro maturation of immatureoocyte, fertilization and the development of the formed embryos; individualized IVMprotocol improves the development of the resulting embryos; high-quality blastocystscan be obtained from the immature oocytes followed by IVM，ICSI and early embryoculture. In a word，in the controlled ovarian hyperstimulation cycle，immature oocytesfrom ICSI cycle can achieve a better IVM result through an individualized IVMprotocol in a low oxygen（5%O2） of in-vitro culture condition. i. The style of Cryopreservation of human immature oocytes followed by in vitromaturation: slow-freezingVs.Vitrification.Objective: To explore the survival rate, maturation rate, the fertilization rate andembryonic development of immature human oocytes from ICSI cycle afterslow-freezing and vitrification style.Methods: A total of454immature oocytes [germinal vesicle（GV）and metaphase I（MI）stages] were collected and randomly divided into a slow-freezing group [1.5mol/L-1,2-propanediol（PROH）+0.2mol/l sucrose] and vitrification group [20%PROH+20%ethylene glycol（EG）+0.5mol/l sucrose].Results: The vitrification protocol yielded a better survival rate than the slow freezingprotocol at each maturation stage assessed. Regardless of the maturation stage（GV+MI）, the slow freezing protocol had a significantly lower survival rate than thevitrification protocol（P＜0.001）. In addition, a significantly difference was found inthe survival rates between GV and MI oocytes totally in two groups（90.1Vs.64.7%,respectively;P＜0.01）. We also found that the maturation rates of GV and MI oocytesfrom the slow freezing and vitrification groups were16.7Vs.24.4%and50.8Vs55.4%,respectively. The GV oocytes had significantly lower viability than MI oocytes after36h of in vitro maturation（21.2Vs54.0%, respectively;P＜0.01）totally in two groups.In addition, the GV and MI oocytes in the slow freezing group had a markedly lowermaturation rate than those in the vitrification group（33.6Vs.43.1%, respectively）, butno statistical difference was found between the two groups（P＞0.05）. For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, whilea19.0%（4/21）fertilization rate was obtained in the vitrification group. For theMI-matured oocytes, fertilization rates in the slow-freezing and vitrification group were36%and61.1%, respectively, but no significant difference was found between the twogroups（P＞0.05）. In the GV vitrification group, no embryo formed; however, for MIoocytes, in slow-freezing group,12oocytes were fertilized, but only two cleavageembryos had been got and were subsequently blocked at the2-cell stage. In vitrificationgroup, a total of22embryos were obtained from MI oocytes, five embryos developed tothe blastocyst stage.Conclusions: Vitrification is superior to the slow-freezing method in terms of thesurvival and developmental rates for the cryopreservation of human immature oocytes.In addition, GV oocytes appeared to be more tolarete than MI oocytes to the lowtemperature and cryopreservation cryoprotectant.ii. Effect of ethanol activation/application of trehalose on the developmental potential offrozen-thawed immature human oocytes from ICSI cycleObjective: To observe the survival rate, maturation rate, the fertilization rate andembryonic development and the aneuploid rate of the immature oocytes after ethanolactivation/application of trehalose.Methods: All the immature oocytes were randomly divided into three groups：thesucrose virification group，274immature oocytes were frozen and thawed using mediumcontaining sucrose as cryoprotectantï¼›the trehalose virification group，245immatureoocytes were used trehalose; the control group（fresh group），291fresh immatureoocytes were directly conducted with IVM culture. all of the in-vitro matured oocytesfrom the three groups were used with ICSI fertilization, after ICSI, these oocytes inthree groups were further divided into six subgroups according to using ethanolactivation or not: no-activation sucrose virification group and sucrose virification groupwith activation, no-activation trehalose virification group and trehalose virification group with activation, no-activation fresh group and fresh group with activation. Therates of maturation, fertilization, cleavage, D3high-quality embryos, blastocysts andhigh-quality blastocysts were recorded. Furthermore, All of the formed high-qualityblastocysts were analysed with8chromosomes：13，15，16，18，21，22，X and Y throughtwo rounds of FISH. In the first round of FISH,13,21,22,16and18chromosomeswere analyzed with a corresponding probe marked with red, green, gold, aqua and bluefluorescent; in the second round of FISH,15, X and Y chromosomes were diagnosed,and the corresponding probes were respectively marked with green, orange and aquafluorescent.Results: In the no activation fresh group（GV+MI）：112oocytes were cultrued withIVM，94matured oocytes was obtained,76of them were fertilized，70of themcleavaged，10D3-good quality embryos and6blastocyst were obtained，but nohigh-quality blastocyst formed. In fresh group with activation（GV+MI）：179oocyteswere cultured with IVM and152matured, after ICSI, the injected oocytes wereexposed to7%ethanol for6minutes, subsequently cultured,130fertilized,124cleavaged, and39D3high-quality embryos,25blastocysts and14high-qualityblastocysts were obtained. In the no-activation sucrose virification grou（pGV+MI）:128immature oocytes were obtained.110survived after thawing,100matured after IVM,82fertilized after ICSI,48cleavaged，no high-quality embryos and blastocysts formed;In the sucrose virification group with activation（GV+MI）：146immature oocytes wereobtained，124survived after thawing,104matured after IVM,86fertilized,56cleavaged,16high-quality embryos and8blastocysts including4high-qualityblastocysts formed. Comparison between the fresh group with activation andno-activation, no statistical difference was found in maturation rate, fertilization rateand cleavage rate（P＞0.05）, and a significant statistical difference for high-qualityembryos rate （31.5%Vs14.3%，P＜0.01，X2ï¼6.98）, blastocysts formation andhigh-quality blastocysts rate （20.2%Vs8.6%，P＜0.05，X2ï¼4.48ï¼›56.0%Vs0，P ＜0.05，X2ï¼6.13）. Comparison between the no-activation vitrification group and withactivation, there was a significant statistically difference in high-quality embryos andblastocysts formation rate （28.6%Vs0%，P＜0.01，X2ï¼16.21ï¼›14.3%Vs0，P＜0.01，X2ï¼7.43）, and4high-quality blastocysts were obtained in the activation group.Comparison between the fresh group with activation and vitrification group withactivation,no significant statistically difference was found for maturation, fertilization,high-quality embryo, blastocyst and high-quality blastocyst rates（P＞0.05）, butcleavage rate in the fresh group （95.4%）was significantly higher than vitrificationgroup（65.1%）（P＜0.01，X2ï¼34.14）. Comparison between the no-activation freshgroup and no-activation vitrification group, cleavage rate, high-quality embryo rate andblastocyst rate in fresh group were significantly higher than those in the vitrificationgroup respectively（92.1%Vs58.5%;14.3%Vs0;8.6%Vs0）（P＜0.01，X2ï¼23.51ï¼›P＜0.01，X2ï¼7.49ï¼›P＜0.05，X2ï¼4.33）. In the trehalose virification group, immatureoocytes was125, the survived108were obtained after thawing,98matured after IVM,82fertilized after ICSI,45cleavaged, and5D3high-quality embryos,3blastocystswere obtained, but no high-quality blastocyst. In the trehalose virification group withactivation, totally120immature oocytes，100survived after thawing,87matured afterIVM,70fertilized after ICSI,38cleavaged, subsequently cultured,13D3high-qualityembryos,10blastocysts including6high-quality blastocysts were obtained. Comparisonbetween trehalose virification group and sucrose virification group, no statisticallydifference was founded for survival rate, maturation rate, fertilization rate, cleavage rate,high-quality embryo rate, blastocyst rate and high-quality blastocyst rate （P＞0.05）.Also, the parameters in trehalose virification group with activation and sucrosevirification group with activation O2no difference（P＞0.05）.Totally24high-quality blastocyst were obtained in fresh group with activation and virificationgroup with activation. All of them were fixed successfully for the following FISHdiagnosis,14high-quality blastocyst in fresh group,5abnormal embryos were founded （abnormal rate35.7%）; for4high-quality blastocysts from the sucrose virificationgroup with activation,3abnormal embryos were founded （the aneuploid rate75%）;6embryos in the trehalose virification group with activation,3O2aneuploidy（aneuploid rate50%）. no difference was found in aneuploid rate between the freshgroup and virification group（P＞0.05）.Conclusions oocytes suffer damage in the freezing-thawing process and reduce theformation of embryos; Artificial activation can not improve the formation of embryos，but their development; Trehalose as impermeable protectant appears not to improve thefreezing–thawing effect. Objective: Aim to analyse freezing-thawing damage by using co-focal microscopy toobserve ultramicrostructure of frozen-thawed matured oocytes derived from immatureoocytes from ICSI cycle.Methods:62immature oocytes with IVM and51matured,42of them with a normalshape were picked up and randomly divided into two groups: IVM-sucrose group，20oocytes were observed using co-focal microscopy after thawing with vitrificationmedium containing sucrose to analyze the structure change of the spindles. IVM-trehalose group，22oocytes using vitrification medium containing trehalose. The controlgroup，20MII oocytes with normal shape were collected from IVF cycles（patients hadsigned informed consent and agreed to oocyte donation）,2of them were directly fixedand then observed the configuration of their spindles, for the other18oocytes，9of themwere frozen and thawed using vitrification medium containing sucrose, and the other9using trehalose, subsequently, all the survived oocytes were fixed to be observed the configuration of their spindles through co-focal microscope.Results: A total of42mature oocytes was frozen,,20of them were used withvitrification medium containing sucrose, after thawing,17survived （IVM-sucrosegroup）, the other22with trehalose medium and19survived after thawing（IVM-trehalose group）. In IVF-sucrose and IVF-trehalose groups, respectively8survived. There was no significantly difference for surviving rate among four groups（P＞0.05）. all of the survived oocytes were included in the four groups were fixed. InIVM-sucrose group,10were successfully fixed,11in IVM-trehalose group,6IVF-sucrose group and7IVF-trehalose group had been fixed seccessfully. The oocyteswere observed under co-focal microscope, the normal morphology both inchromosomes and spindle morphology of the oocytes were l3（30.0%）,4（36.4%）,4（66.7%） and5（71.4%）, respectively. There were no significantly difference innormal morphology rate of spindle and chromosomes among four groups （P＞0.05）.Conclusions Application of trehalose as an impermeable protectant during vitrificationof human MII oocytes can achieve an acceptible results. In the freezing-thawing process,both in-vivo and in-vitro mature oocytes suffer serious damage, but in-vivo matureoocyte appears to be more tolerate to cryopreservation. |
