RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

Creation of a rabbit model for intrauterine adhesions using electrothermal injury / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The pathogenesis and therapeutic treatment of intrauterine adhesions (IUAs) remain unsolved, highlighting the need for stable and effective experimental animal models. In this study, uterine electrocoagulation of twenty-one female New Zealand White rabbits was carried out to establish an IUA model. As rabbits have two completely separate uterine horns, each rabbit had its own internal control: one uterine horn was given an electrothermal injury (Group A, n=21), and the contralateral uterine horn received no treatment and served as the control (Group B, n=21). The endometrial morphology, number of endometrial glands, area of endometrial fibrosis, and number of implanted fetuses were compared between the two groups. In Group A, the numbers of endometrial glands on Days 7 and 14 and the number of implanted fetuses were significantly lower than those in Group B (P<0.05, P<0.05, and P<0.01, respectively), while the ratio of the area with endometrial stromal fibrosis to the total endometrial area was significantly increased (P<0.01). These results suggest that this method of electrothermal injury is effective for the establishment of a rabbit IUA model between 7 and 14 d after surgery.

Comparison of the Developmental Potential and Clinical Results of In Vivo Matured Oocytes Cryopreserved with Different Vitrification Media / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Oocyte vitrification is widely used throughout the world, but its clinical efficacy is inconsistent and depends on the vitrification media. This study compared the developmental potential and clinical results of in vivo matured oocytes cryopreserved with different vitrification media.</p><p><b>METHODS</b>This retrospective study involved vitrified-warmed oocytes at one in vitro fertilization laboratory. Vitrification media kits comprised the MC kit (ethylene glycol [EG] plus 1,2-propanediol [PROH]), the KT kit (EG plus dimethyl sulphoxide [DMSO]), and the Modified kit (EG plus DMSO and PROH kit). Rates of oocyte survival and subsequent developmental potential were recorded and analyzed. The t-test and the Chi-square test were used to evaluate each method's efficacy.</p><p><b>RESULTS</b>Oocyte survival rate was significantly higher for the Modified kit (92.0%) than for the MC kit (88.2%) (P < 0.05) and the KT kit (77.3%) (P < 0.001). The rate of high-quality embryo development in the Modified kit group (35.8%) was significantly higher than in the MC kit group (29.0%) and the KT kit group (28.3%) (P < 0.001). No significant differences were observed in the clinical pregnancy and implantation rates among the MC, KT, and Modified kit groups (37.2% vs. 30.2% vs. 39.6%; 21.9% vs. 18.8% vs. 27.4%, respectively) (P > 0.05). The high-quality embryo rate per warmed oocyte was significantly higher (23.4%) in the Modified kit group than in the other groups (P < 0.001). The embryo utilization and live birth rates per warmed oocyte were the highest in the Modified kit group, but not significantly (P > 0.05).</p><p><b>CONCLUSIONS</b>Modified vitrification media are efficient for oocyte vitrification and, with further verification, may be able to replace commercially available media in future clinical applications.</p>

High levels of testosterone inhibit ovarian follicle development by repressing the FSH signaling pathway / 华中科技大学学报（医学）（英德文版）

The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.

Application of calcium ionophore A23187 in ICSI for globozoospermia: A report of 2 cases and review of the literature / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the pathogenesis of globozoospermia, fertilization ability of round-headed sperm, and the application value of assisted oocyte activation in intracytoplasmic sperm injection (ICSI) for the wives of glohozoospermia men.</p><p><b>METHODS</b>We collected oocytes from the wives of 2 globozoospermia patients and randomly divided them into two groups after ICSI to receive calcium ionophore A23187-activation and conventional treatment, respectively. We reviewed the relevant literature published at home and abroad, and discussed the etiology of globozoospermia, fertilization ability of round-headed sperm, and treatment options for this disease.</p><p><b>RESULTS</b>Quality embryos were obtained in the A23187-activation group while no fertilized oocytes, oocyte cleavage, quality embryos, or blastular formation were found in the conventional treatment group. Both women achieved pregnancy and gave birth to healthy neonates after transfer of the quality embryos from the A23187-activation group.</p><p><b>CONCLUSION</b>Calcium ionophore A23187 can be applied to ICSI for the wives of globozoospermia men and bring about desirable clinical outcomes. Meanwhile, attention should be paid to its safety.</p>

Elective single blastocyst transfer is more suitable for normal responders than for high responders / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Embryo quality and receptivity of the endometrium are two factors that determine the results of in vitro fertilization/intra-cytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET). There is no consensus of the optimal transfer strategy for normal responders or high responders. The current study aimed to find the optimal transfer strategy for different subgroups of patients.</p><p><b>METHODS</b>From April 2010 to December 2010, patients who meet the following criteria were included in this study; primary infertility, female age ≤ 35 years, FSH level on female cycle day 2 - 3 ≤ 12 mIU/ml, at least six good quality embryos available on day three. The clinical outcomes using different transfer strategies between normal responders and high responders were reviewed and compared.</p><p><b>RESULTS</b>For the normal responders, the clinical pregnancy rate of day three double-embryo transfer (DET) was comparable to that of day five elective single blastocyst transfer (eSBT), 64.04% vs. 60.33% (P > 0.05). For the high responders, the clinical pregnancy rate of day five eSBT was significantly lower than that of day three DET, 43.35% vs. 57.21% (P < 0.05). For the high responders, the rates of clinical pregnancy and implantation in frozen-thawed embryo transfer (FET) cycles were notably higher than in eSBT cycles (64.56% vs. 43.35% and 62.11% vs. 43.35% respectively) (P < 0.05).</p><p><b>CONCLUSIONS</b>For normal responders, eSBT might be an applicable strategy to reduce multiple pregnancy rates while maintaining acceptable overall pregnancy rates. And in order to reduce multiple pregnancies and increase the chance of pregnancy of high responders, FET may be a preferable strategy.</p>

Research update of Cullin protein in reproductive system / 中国医学科学院学报

Culling protein is a member of Cullin-Ring-based E3-ligases ( CRLs family) , which belong to E3 ubiquitin ligases. Cullin plays diverse and essential roles in many biological processes through mediating the ubiquitination of target proteins. This article summarizes the potential functions of Culling proteins in gamete genesis and maturation, embryo development, and reproductive related disorders.

Twin delivery of a 46,XY gonadal dysgenetic woman following vitrified oocytes donation / 中华医学杂志(英文版)

A 46,XY gonadal dysgenetic woman gave birth to two healthy girls following vitrified oocytes donation. The loss of SRY gene was considered as the cause of this patient. Although similar cases have been reported about pregnancies of 46,XY pure gonadal dysgenetic women, successful delivery from vitrified oocytes has been hardly reported yet. Oocytes vitrification technique provides a beneficial way by saving superfluous oocytes from the pregnancy patients to these women who need.

Effects of abnormal structure of sperm chromatin on the outcome of in vitro fertilization and embryo transfer / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the effects of sperm chromatin structure abnormalities on the outcome of in vitro fertilization and embryo transfer (IVF-ET).</p><p><b>METHODS</b>Sperm DNA fragmentation and chromatin packaging defects were assessed in 136 couples undergoing IVF-ET because of infertility. The relationship between sperm DNA fragmentation, chromatin packaging defects and fertilization rate and clinical pregnancy was evaluated.</p><p><b>RESULTS</b>Both sperm DNA fragmentation and chromatin packaging defect had a negative correlation with fertilization rate (r=-0.198, P<0.05, and r=-0.389, P<0.01, respectively). Both parameters were higher in couples who failed to achieve pregnancy than those who achieved clinical pregnancy (10.74% vs. 5.40%, P<0.01 and 23.58% vs. 11.83%, P<0.01, respectively).</p><p><b>CONCLUSION</b>Abnormality of sperm chromatin structure is one of the reasons for IVF-ET failure. Examination of sperm chromatin structure is helpful in predicting the risk of IVF-ET failure and optimizing treatment of infertility.</p>

Clinical application of oocyte vitrification in failed testicular sperm extraction cycles: report of 8 cases / 中华男科学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical application value of oocyte vitrification in failed testicular sperm extraction cycles in non-obstructive azoospermia (NOA) patients.</p><p><b>METHODS</b>We retrospectively analyzed the clinical data of 8 women undergoing oocyte frozen-thawing cycles by vitrification because of failed testicular sperm extraction from their NOA husbands and no banked donor sperm on the day of oocyte retrieval. The oocytes were cryopreserved by vitrification with cryotop and thawed 2 months later. The surviving metaphase II (MII) oocytes were injected with the banked donor sperm of the same blood type as the husbands by intracytoplasmic sperm injection (ICSI) for fertilization. The rates of oocyte survival, fertilization, cleavage, good embryos and pregnancy were evaluated.</p><p><b>RESULTS</b>Sixty oocytes were vitrified and 47 (78.3%) survived after thawing, of which 41 MII oocytes underwent ICSI and 33 (80.5%) of them were fertilized. The rates of cleavage and good embryos were 81.8% (27/33) and 59.3% (16/27) respectively. Fifteen of the embryos were transferred to the 8 patients, with 1.9 +/- 0.8 per cycle, of which 5 (33.3%) were confirmed by ultrasound to have been implanted and 5 resulted in clinical pregnancy (62.5%), all singleton without miscarriage. Three normal boys and 1 normal girl were already born, with the pregnancy time of (39 + 4 +/- 0.4) wk and newborn body weight of (3787.5 +/- 513.7) g, respectively.</p><p><b>CONCLUSION</b>Vitrification of oocytes in failed testicular sperm extraction cycles is a promising technique for preserving female fertility, which, with ICSI of banked donor sperm, may result in satisfactory clinical outcomes.</p>

Use of L-carnitine before percutaneous epididymal sperm aspiration-intracytoplasmic sperm injection for obstructive azoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the use of L-carnitine before percutaneous epididymal sperm aspiration-intracytoplasmic sperm injection (PESA-ICSI) in the treatment of obstructive azoospermia.</p><p><b>METHODS</b>Seventy-nine cases of obstructive azoospermia treated in our center from Sep 2008 to Aug 2009 were divided into an L-carnitine (n = 43) and a control group (n = 36), the former given oral L-carnitine at 1 g bid for 3 months before PESA-ICSI, while the latter left untreated. Comparisons were made between the two groups in the number of retrieved oocytes and fertilized oocytes as well as the number and rate of good embryos.</p><p><b>RESULTS</b>There were no significant differences between the two groups in the number of retrieved oocytes and fertilized oocytes. But the number and rate of good embryos were significantly higher in the L-carnitine than in the control group (P < 0.05).</p><p><b>CONCLUSION</b>Three-month oral medication of L-carnitine before PESA-ICSI can raise the number and rate of good embryos in obstructive azoospermia patients and therefore benefit the therapeutic outcome.</p>

Effects of different proportions of cryoprotectant to seminal plasma on the motility of post-thaw human sperm / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of different proportions of cryoprotectant to seminal plasma on the motility of post-thaw human sperm.</p><p><b>METHODS</b>Different proportions of cryoprotectant to seminal plasma (1:1 and 1:3) were used for freezing sperm, and the forward movement and total motility rates of the frozen-thawed sperm were compared.</p><p><b>RESULTS</b>The forward movement and total motility rates were (58.60 +/- 5.57)% and (66.17 +/- 5.24)% before cryopreservation. The 1:1 proportion achieved post-thaw forward movement and total motility rates of (40.53 +/- 8.97)% and (51.23 +/- 9. 30)%, while the 1:3 (44.7 +/- 8.67)% and (51.50 +/- 7.40)%, respectively. Significantly decreased sperm motility was observed after cryopreservation (P < 0.05). Statistically significant differences were found in the forward movement but not in the total motility of the frozen-thawed sperm between the two proportions.</p><p><b>CONCLUSION</b>Cryopreservation causes obvious damage to human sperm. Higher proportion of cryoprotectant to seminal plasma (1:3) can improve the forward movement of post-thaw sperm as compared with the lower one (1:1).</p>

Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality.</p><p><b>METHODS</b>Two early apoptotic changes in the semen of 56 men were assessed using Annexin V (AN)/propidium iodide (PI) staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential (MMP). The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay.</p><p><b>RESULTS</b>The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN(-)/PI(-) spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL (+) spermatozoa. As for the AN(-)/PI(+) fraction, we found an opposite result in comparison to AN(-)/PI(-) spermatozoa. The level of early apoptotic AN(+)/PI(+) spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN+/PI+ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL (+) spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL (+) spermatozoa.</p><p><b>CONCLUSION</b>Although early apoptotic AN+/PI(-) spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa (especially, the percentage of AN(-)/PI(-) spermatozoa), and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization.</p>

Effects of two different cryoprotectants on the motility of post-thaw human sperm / 中华男科学杂志

<p><b>OBJECTIVE</b>To compare the effects of the cryoprotectant containing glucose and that containing sucrose on the motility of post-thaw human sperm.</p><p><b>METHODS</b>The cryoprotectant containing glucose and that containing sucrose were applied to 50 semen samples and the motility of the post-thaw human sperm was compared before and after cryopreservation and between the study groups.</p><p><b>RESULTS</b>The forward motility and total motility of the sperm were (58.4 +/- 5.7)% and (63.4 +/- 6.1)% before cryopreservation, (43.8 +/- 7.6)% and (48.4 +/- 7.6)% after thawing with the cryoprotectant containing glucose, and(42.6 +/- 8.9)% and (48.0 +/- 8.5)% after thawing with the cryoprotectant containing sucrose. Decreased sperm motility was observed after cryopreservation, with statistic significance (P < 0.01). There was no significant difference in the forward and total motility of the post-thaw sperm between the two cryoprotectants.</p><p><b>CONCLUSION</b>Cryopreservation inflicts obvious damage on sperm. Sucrose is a feasible sperm cryoprotectant.</p>

Cloning and expression of human interleukin-26 in Escherichia coli / 生物工程学报

To clone human interleukin-26 (hIL-26) and express it in E. coli efficiently. Two pairs of primers were synthesized according to the hIL-26 gene reported on GenBank. The hIL-26 gene was cloned by nest PCR following the first round RT-PCR from human peripherial blood monocytes total RNA, and then the PCR product was cloned into pMD18-T vector. Colony PCR, restriction analysis and sequence analysis showed that the gene cloned was the same as the reported hIL-26. The recombinant was cut with BamHI and EcoR I to obtain the hIL-26 fragment, and then the fragment was inserted into pBV220 which was cut with the same enzymes. The recombinant expression vector was induced to express hIL-26 at 42 degrees C, SDS-PAGE analysis showed that the recombinant protein accounted for up to 20% of the whole protein of E. coli, and the protein was also confirmed by Western blotting. Purity of the protein was found to be above 90% after purified with molecular sieve. After renaturalized with glutathione buffer, the promoting effect of it on the production of IFN-y in PBMC was detected by RT-PCR. A recombinant bacterial strain for expressing hIL-26 with biological activity was constructed successfully.

Sex chromosome analysis and IGF-II expression on activated human unfertilized oocytes after ICSI with calcium ionophore A23187 and puromycin / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the sex chromosomes and the expression of insulin-like growth factor-II (IGF-II) on activated human unfertilized oocytes after intracytoplasmic sperm injection(ICSI) with calcium ionophore A23187 and puromycin.</p><p><b>METHODS</b>All 95 discarded oocyes that showed no evidence of fertilization at 16-18 h after in vitro maturation and intracytoplasmic sperm injection cycles (IVM-ICSI)/conventional ICSI were exposed to calcium ionophore A23187 (5 micromol/L) for 5 min and then were incubated with puromycin (10 microg/mL) for 4 h. After activation, the oocytes were cultured in vitro for 3-5 days. The sex chromosome analysis was performed by dual color fluorescence in situ hybridization. The expression of IGF-II on the activated embryos, normal embryos, and parthenotes was examined.</p><p><b>RESULTS</b>The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 h after ICSI. The activated rate, cleavage rate, and quality of activated embryos of the IVM-ICSI group were similar to those of ICSI group, respectively. Sex chromosome analysis indicated that 8 male and 5 female embryos had been derived from two pronucleus and a second polar body. The expression of IGF-II on activated embryos and normal embryos was high and similar, which was much stronger than that of parthenotes.</p><p><b>CONCLUSION</b>The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 h after ICSI. Moreover, the unfertilized oocytes activated by calcium ionophore A23187 and puromycin had normal sex chromosomes and expression of IGF-II like the normal embryos. These suggest that oocyte activation may be considered as a remedial measure in the presence of total or nearly total fertilization failure in ICSI.</p>

Study on the (TAAAA)n repeat polymorphism in sex hormone-binding globulin gene and the SHBG serum levels in putative association with the glucose metabolic status of Chinese patients suffering from polycystic ovarian syndrome in Shandong province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship of (TAAAA)n repeat polymorphism in the promoter of the sex hormone-binding globulin (SHBG) gene and SHBG serum levels to the glucose metabolic status of Chinese polycystic ovary syndrome (PCOS) patients in Shandong province.</p><p><b>METHODS</b>GeneScan method was used to detect and identify (TAAAA)n repeat number (alleles) and genotypes for 156 controls and 157 patients who were divided into normal glucose tolerance without hyperinsulinemia (NIR group) and with hyperinsulinemia (HI group) and abnormal glucose metabolic (AGM) group according to the results of oral glucose test and insulin resistant test; IRMA was used to measure serum SHBG for part of them.</p><p><b>RESULTS</b>Five alleles containing (TAAAA) 6-10 repeats and 14 genotypes including 6/6, 6/7, 6/8, 6/9, 6/10, 7/7, 7/8, 7/9, 7/10, 8/8, 8/9, 8/10, 9/9, 9/10 repeats genotypes were present in the subjects. Genotype distribution of 6/10 repeats genotype is lower in PCOS than that in control, and 8/9 repeats genotype vice versa (P < 0.01); among PCOS subgroups, the eight repeat genotypes in NIR group is more frequent than that in HI group (P < 0.01), and 7/9 genotype distribution in AGM group is higher than that in NIR group and HI group(P < 0.05-0.01). The serum SHBG levels in homozygous genotype groups exhibit a sequence of 8/8 > 9/9 > 6/6, 7/7 repeats and the fall of serum SHBG trend is in reversed relation with the increase in body mass index (BMI), Homa-IR, and blood pressure. Serum SHBG levels in AGM exhibit a sequence of HI group < NIR group < control but show no statistical difference between both groups.</p><p><b>CONCLUSION</b>This study reveals that the repeat number, alleles, genotypes and their distributions in Chinese women are very different from these in foreigners. Some special genotypes and low serum SHBG levels may be associated with PCOS and its glucose metabolic status; some special genotypes may influence Chinese serum SHBG and need more studies, but both SHBG gene polymorphism genotype and serum SHBG are not good indicators to find out the PCOS individual at high risk.</p>

Clinical analysis of multiple pregnancy reduction / 中华男科学杂志

<p><b>OBJECTIVE</b>To analyse the effect of the reduction of multiple pregnancy through transvaginal ultrasonic monitoring on the pregnancy outcome.</p><p><b>METHODS</b>Eighty-four cases were divided into two groups according to whether they had vaginal hemorrhage before operation. And the pregnancy outcomes were analyzed.</p><p><b>RESULTS</b>The abortion rate and preterm birth rate of the vaginal hemorrhage group were higher, and the difference was statistically significant.</p><p><b>CONCLUSIONS</b>The reduction of multiple pregnancy through transvaginal ultrasonic monitoring is a safe operative method. But it is only a remedial treatment for multiple pregnancy, and how to prevent multiple pregnancy is of more practical value.</p>

Comparison of vitrification and slow-freezing of human day 3 cleavage stage embryos:postvitrification development and pregnancy outcomes / 中华妇产科杂志

Objective To compare the effects of vitrification with slow-freezing on the developmental ability of day 3 cleavage stage embryos.Methods Patients who had no less than 4 high quality embryos were included in this study.These embryos were cryopreserved using the methods of vitrification or slow-freezing.In the eryopreserved embryo transfer cycles,the embryos which were cryopreserved using one of the methods were chosen randomly.The developmental ability of embryos was compared between these two groups.Results A total of 80 patients were included in this study with 160 embryos.In the group of slow-freezing,73(91%)embryos were survived and achieved 15(38%)clinical pregnancies.Among these,3 were twins and the implantation rate was 25%(18/73).In the group of vitrification,71(89%)embryos were survived and achieved 19(48%)clinical pregnancies.Among these, 9 were twins and the implantation rate was 39%(28/71),which was significantly higher than the slow- freezing group(P

Spindle and chromosome configuration of human in vitro matured oocytes after slow freezing-fast thawing / 中华妇产科杂志

Objective To evaluate the development of immature oocytes after freezing-thawing by conventional cryopreservation method for mature oocytes.Methods Immature oocytes were collected from stimulated ovaries of intracytoplasmic sperm injection(ICSI)cycles.Immature oocytes were in vitro matured directly or after slow freezing-fast thawing and immunostained for tubulin and chromatin and at last visualized by confocal microscopy.Results No statistical difference was found in maturity rate between freezing groups and the controls.There was a statistically significant increase in abnormalities of chromosome(23.7% vs. 50%)and spindle(28.9% vs.53.9%)in the GV freezing group compared with the GV control(P