NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis.

BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.

Increased ApoE Expression in Follicular Fluid and the ApoE Genotype Are Associated With Endometriosis in Chinese Women.

More than 10% of women suffer from endometriosis (EMT) during their reproductive years. EMT can cause pain and infertility and requires further study from multiple perspectives. Previous reports have indicated that an increase inapolipoprotein E (ApoE) may be associated with a lower number of retrieved mature oocytes in older women, and an association between ApoE and spontaneous pregnancy loss may exist in patients with EMT. The purpose of this study was to investigate the existence of an increase in ApoE in follicular fluid (FF) and the possible relationship between ApoE and EMT in Chinese women. In the current study, 217 Chinese women (111 control subjects and 106 EMT patients) were included. The ApoE genotypes were identified by Sanger sequencing. We found that ApoE expression in FF was higher in patients with EMT than in the control group. In addition, a significant difference in ApoE4 carriers (ϵ3/ϵ4, ϵ4/ϵ4) was found between the control subjects and the patients with EMT. Furthermore, a nonparametric test revealed significant differences in the numbers of blastocysts and high-quality blastocysts, but not the hormone levels of FSH, LH, and E2, between the two groups. We also established a multifactor (BMI, high-quality blastocysts, and ϵ4) prediction model with good sensitivity for identifying patients who may suffer from EMT. Our results demonstrate that ApoE expression in FF is increased in EMT, the ApoE-ϵ4 allele is significantly linked to EMT, and a combined analysis of three factors (BMI, high-quality blastocysts, and ϵ4) could be used as a predictor of EMT.

Downregulation of LINC01508 contributes to cisplatin resistance in ovarian cancer via the regulation of the Hippo-YAP pathway.

BACKGROUND: Some long non-coding RNAs (lncRNAs) have been found to contribute to cisplatin resistance. Here, we identified a novel lncRNA that was downregulated in cisplatin-resistant to ovarian cancer (OC) cells and aimed to examine the contribution of LINC01508 to cisplatin resistance in OC cells. METHODS: Differences in the lncRNA expression profile between OV2008 and C13K cells were assessed by lncRNA expression microarray. The expression of LINC01508 in ovarian epithelial cells, four OC cells, and OC, benign ovary tumor and normal ovary, cisplatin-resistant and non-resistant OC specimens were evaluated by quantitative real-time polymerase chain reaction (qPCR). The role of LINC01508 in OC cisplatin-resistant was evaluated by cell counting kit-8 (CCK-8), flow cytometry, colony formation, wound healing, Transwell, and tumor growth inhibition study in vivo. The clinical associations of LINC01508 in OC were evaluated using correlation analysis. The effects of verteporfin (VP) on cisplatin were explored to reveal the function of the hippo-YAP pathway on the cisplatin tolerance of C13K. RESULTS: LINC01508 was downregulated in cisplatin-resistant OC cells and platinum-resistant OC tissue (p<0.01). LINC01508 downregulation was correlated with tumor size, residual tumor, and platinum resistance. The overexpression of LINC01508 improves in vitro and in vivo sensitivity to cisplatin while predicts the poor overall survival which need further follow-up research. The increased level of LINC01508 could suppress the cisplatin resistance of OC cells through the inhibition of the hippo-YAP pathway. CONCLUSIONS: The study proposes that dysregulation of LINC01508 expression results in resistance of OC to cisplatin through the inhibition of the hippo-YAP pathway.

Melatonin maintains mitochondrial membrane potential and decreases excessive intracellular Ca2+ levels in immature human oocytes.

AIMS: Previous reports have demonstrated that melatonin exists in multiple extrapineal sites, and higher amounts of melatonin are present in human follicular fluid than in serum, which indicates that it might play key roles in human oocyte maturation and subsequent embryonic development. Melatonin has been shown to be a potent antioxidant and might be beneficial to human oocytes during in vitro maturation (IVM). However, the underlying mechanisms of melatonin action during IVM have not been thoroughly investigated. MAIN METHODS: Immunofluorescence staining, western blotting, and ELISA were applied to investigate whether melatoninergic components are expressed in the cultured human ovarian cumulus cells. TMRE staining and Fluo-4 AM staining were performed to detect the mitochondrial membrane potential and intracellular Ca2+ levels of immature human oocytes respectively. KEY FINDINGS: First, cultured human ovary cumulus cells synthesized melatonin in vitro, and it expressed serotonin (the precursor of melatonin) and the two key enzymes, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Additionally, the results suggest that melatonin maintains the mitochondrial membrane potential and decrease excessive Ca2+ levels in immature human oocytes during IVM. SIGNIFICANCE: In conclusion, we provide evidence that the melatoninergic components were expressed in cultured human ovarian cumulus cells, and melatonin might reduce oxidative stress of human oocytes by ameliorating mitochondrial function. In view of the significant clinical value that immature human oocytes have in assisted reproductive technology (ART), our findings highlight a potential treatment strategy of using melatonin to improve mitochondrial function and to enhance the quality of human oocytes during IVM.

Prevalence and risk factors of monochorionic diamniotic twinning after assisted reproduction: A six-year experience base on a large cohort of pregnancies.

OBJECTIVE: To characterize the incidence and risk factors for monochorionic diamniotic(MC-DA) twinning after assisted reproductive technologies (ART). DESIGN: Retrospective population-based study. SETTING: The study was conducted in China; Department of Reproductive Medicine Center at The First Affiliated Hospital of Anhui Medical University. POPULATION: A cohort of 8860 clinical pregnancies after embryo transfer (ET) carried out in our center between 2011 and 2016 were retrospectively analyzed for the incidence of MC-DA twinning. METHODS: Logistic regression was used to model the effect on the incidence of MC-DA twinning after ART. Different clinical data (maternal age) and laboratory procedures (type of ET (fresh versus frozen), insemination (IVF or ICSI)), embryo stage at time of ET (cleavage or blastocyst)) on the incidence of MC-DA twinning were evaluated. MAIN OUTCOME MEASURES: Monochorionic-diamniotic pregnancies were identified when more than one fetal poles was visualized in one gestational sac via trans-vaginal ultrasound at early first-trimester (7 to 8 weeks). RESULTS: The overall MC-DA twinning rate was 2.55% (226/8860). Eighty one MC-DA twinnings occurred in the fresh cycles and 145 in the frozen cycles (2.67% vs. 2.49%). MC-DA twinning incidence showed no significant difference whether ICSI was performed or not (2.79% vs. 2.44%). The MZT that resulted from single embryo transfer (SET) cycles (1.99%) was slightly lower than multiple embryo transfer cycles (2.61%),but with non-significance. However, women <35 years displayed a significant higher rate (2.81%) than women ≥35 years old (1.16%). Blastocyst transfer was associated with a significantly increase in MC-DA twinning incidence than cleavage-stage embryos transfer (2.79% VS 2.02%, P = 0.008). In the results of logistic regression analysis, blastocyst transfer is a major risk factor of MZT in the fresh cycles (P = 0.044), while maternal age plays a more important role in the frozen cycles (P = 0.004). CONCLUSIONS: There is an elevated prevalence of MC-DA twinning after ART. Both maternal age and blastocyst transfer are risk factors of monozygotic pregnancy independently. Blastocyst transfer is a major risk factor of MC-DA twinning in the fresh cycles, while maternal age plays a more important role in the frozen cycles.

Diagnostic Significance of Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Complex 1 in Preeclampsia.

OBJECTIVE: To examine the association of phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (m-TORC1) with preeclampsia (PE) and to explore their diagnostic significance in PE. METHODS: A total of 153 singleton pregnant women were enrolled into our study, among which there were 97 patients with PE (mild PE [MPE]: n = 51; severe PE [SPE]: n = 46) and 56 healthy pregnant women (normal controls, NCs). Enzyme-linked immunosorbent assay (ELISA) and Western blot were used in this study. Moreover, a receiver-operating characteristic (ROC) curve was used to estimate the diagnostic significance. RESULTS: After adjustment for confounding factors, at 24 to 28 gestational weeks, the serum levels of PI3K and m-TORC1 were both higher in the MPE and the SPE groups compared to those in the NC group (all P < .001). The serum levels of PI3K were positively correlated with the serum levels of m-TORC1 in both the NC and the PE groups at both 15 to 21 and 24 to 28 gestational weeks (both P < .001). Multivariable linear regression indicated that both PI3K and m-TORC1 were positively correlated with the systolic pressure (both P < .001). At 24 to 28 gestational weeks, there remained relatively high sensitivity and specificity when the serum levels of PI3K and m-TORC1 were used to diagnose PE (both P < .001). A Western blot assay found that there were significant differences in the PI3K and m-TORC1 protein expression among the 3 groups (all P < .001). CONCLUSION: The serum levels of PI3K and m-TORC1 might have the potential to diagnose PE, while PI3K and m-TORC1 fail to predict PE during early pregnancy.

YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells.

OBJECTIVE: To identify the role of YAP in cisplatin resistance in human ovarian cancer cells and in the regulation of autophagy in these cancer cells. MATERIALS AND METHODS: The cisplatin-sensitive OV2008 parental cell line and its cisplatin-resistant variant C13K were cultured. RNA interference was used to knock down the YAP gene. Accumulation of GFP-LC3 puncta was performed by fluorescence microscopy. The formation of autophagosomes was observed by transmission electron microscopy. Drug sensitivity was examined using CCK-8 assay, while apoptosis, the level of intracellular rhodamine 123 and lysosomal acidification were analyzed by fluorescence-activated cell sorting. Acid phosphatase activity was measured using an acid phosphatase-assay kit. Real-time polymerase chain reaction, Western blotting, and immunofluorescence detection were used to detect the protein and messenger RNA expression of YAP, YAP target genes, CCND1, cleaved PARP, and caspase 3, Atg-3 and -5, and the LC3B protein. RESULTS: YAP signaling may regulate cisplatin resistance in ovarian cancer cells by augmenting cellular autophagic flux. After knockdown of YAP-sensitized C13K cells to cisplatin by inducing a decrease in autophagy, YAP led to an increase in autophagy via enhancement of autolysosome degradation. CONCLUSION: YAP-mediated autophagy may play a protective role in cisplatin-resistant human ovarian cancer cells. Therefore, YAP-mediated autophagy should be explored as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

Molecular mechanism of increased sensitivity of cisplatin to ovarian cancer by inhibition of microRNA-23a expression.

OBJECTIVE: The aim of this study is to investigate the sensitivity change and the preliminary mechanism of ovarian cancer cells on the resistant to chemotherapeutic drugs by inhibiting miR-23a expression. METHODS: The ovarian cancer cell lines A2780 was administrated with antagomir-23a and platinum, and then the cell proliferation inhibition rate was determined by MTT assay. The cell cycle distribution was detected by flow cytometric analysis. The apoptotic morphological changes were analyzed by Hoechst33258 staining. The glycoprotein P-gp expression changes were detected by Western blot analysis. RESULTS: The cell proliferation inhibition rate increased significantly after the administration of miR-23a and platinum (P<0.01). The middle concentration of drug efficacy IC50 in experimental group decreased by 83.76% compared with that in control group, which was 17.89 µmol/L vs 110.18 µmol/L (P<0.01). The cell lines A2780 were arrested in G0/G1 phase and apoptosis rate kept increasing (P<0.05). The cell nuclei stained by Hoechst33258 were obviously enhanced and demonstrated apoptosis morphology, such as condense, pyknosis. Compared with control group, the levels of P-gp protein expression in experimental group decreased along with the increase of the cisplatin concentration (P<0.05). CONCLUSION: The inhibition of miR-23a expression could significantly increase the sensitivity of cisplatin towards tumor cells, and it was probably because the negative regulatory factors of miR-23a target genes was released, and as a result, the expression of P-gp protein was inhibited.

Association of genetic variants in SOHLH1 and SOHLH2 with non-obstructive azoospermia risk in the Chinese population.

OBJECTIVE: Spermatogenesis and oogenesis specific basic helix-loop-helix 1 (SOHLH1) and spermatogenesis and oogenesis specific basic helix-loop-helix 2 (SOHLH2) play essential roles for both spermatogenesis and oogenesis. The aim of this study was to evaluate the association of SOHLH1 and SOHLH2 single nucleotide polymorphisms (SNPs) with non-obstructive azoospermia (NOA) in the Chinese population. STUDY DESIGN: In this study, we assessed 7 single nucleotide polymorphisms (SNPs) of SOHLH1 and SOHLH2 with Sequenom iplex technology in 361 NOA cases and 368 fertile controls. RESULTS: We found that the SNPs rs1328626 and rs6563386 of SOHLH2 were significantly associated with NOA risk, of which, a protective effect of minor allele T of rs1328626 on NOA (P = 0.038, odds ratio [OR] = 0.799, 95% confidence interval [CI] = 0.645-0.988) and a significantly increased risk of the SNP rs6563386 with the minor allele G to NOA (P = 0.029, OR = 1.402, 95% CI = 1.034-1.9) were observed, respectively. Our data indicated that the haplotype GC of the variants rs1328626 and rs6563386 conferred a significantly increased risk of NOA (P = 0.031, OR = 1.397, 95% CI = 1.031-1.895). Moreover, we found the genotype distribution of rs1328641 was significantly associated with testes volume in the NOA patients (P = 0.022). CONCLUSIONS: The polymorphisms rs1328626 and rs6563386 of the SOHLH2 gene would be the genetic risk factors for NOA in the Chinese population. The SNP rs1328641 might influence testes development in the NOA patients.

CREM variants rs4934540 and rs2295415 conferred susceptibility to nonobstructive azoospermia risk in the Chinese population.

To evaluate the association of variants related to spermatogenesis with susceptibility to Chinese idiopathic nonobstructive azoospermia (NOA), seventeen tag single-nucleotide polymorphisms (SNPs) in CREM, ACT, KIF17b, and SPAG8 were analyzed in 361 NOA patients and 368 controls by Sequenom iplex technology. The results showed that two CREM SNPs, rs4934540 and rs22954152, were significantly associated with NOA and played protective roles against the disease (P value with Bonferroni correction = 0.00017, odds ratio [OR] = 0.624 and P = 0.012, OR = 0.686, respectively). Haplotype analysis of CREM gene variants suggested that haplotype CGTG of the SNPs, rs4934540, rs2295415, rs11592356, and rs1148247, exhibited significant protective effect against the occurrence of NOA (P = 0.001, OR = 0.659). The haplotype TATG conferred a significantly increased risk of NOA (P = 0.011, OR = 1.317). Furthermore, making use of quantitative RT-PCR, we demonstrated that relative mRNA expression of CREM in NOA patients with maturation arrest was only one-third of that in the controls with normal spermatogenesis (P < 0.0001). Our findings indicated that the polymorphisms of CREM gene were associated with NOA in the Chinese population and low CREM expression might be involved in the pathogenesis of spermatogenesis maturation arrest.

[Association between recurrent miscarriages and insulin resistance: a meta analysis].

OBJECTIVE: To evaluate the association between recurrent miscarriages and insulin resistance. METHODS: The case-control studies on the association between recurrent spontaneous abortion and insulin resistance from June 1996 to April 2012 were collected from Medline, Elsevier, Chinese Journal Full-text Database, Chinese Biological Medicine Database, data base of Wanfang, Springer link and EMBASE. RevMan 5.1 software was used for Meta analysis. RESULTS: According to the included criteria, 7 clinical trials were finally selected. Total 467 cases with recurrent pregnancy loss were enrolled in study group, while 413 women with no history of abnormal pregnancies were enrolled in control group. No significant difference was found in average age and body mass index between the two groups (P > 0.05). Meta analysis results showed that the level of fasting glucose was no statistical difference between study group and control group (WMD = 2.27, 95%CI: -1.11 to 5.65, P > 0.05); fasting insulin level was higher 2.05 mU/L in study group than that of in control group, the difference was statistically significant (WMD = 2.05, 95%CI: 1.03 to 3.08, P < 0.01). Case number of study group on Homa-insulin resistance > 4.5 was more than that of control group (OR = 3.36, 95%CI: 1.72 to 6.57, P < 0.01). Case number of study group on glucose/insulin ratio < 4.5 was more than that of the control group, statistical difference was found (OR = 3.37, 95%CI: 1.90 to 5.99, P < 0.01). CONCLUSION: Insulin resistance is associated with the susceptibility to recurrent miscarriages, and it may contribute to the occurrence of recurrent miscarriages.

[Polymorphism of CYP11A1 gene in Chinese patients with polycystic ovarian syndrome].

OBJECTIVE: To investigate the association between polymorphism of cytochrome P450 subfamily XIA polypeptide 1 (CYP11A1) gene and polycystic ovarian syndrome (PCOS) in Chinese population. METHODS: From May 2005 to Dec. 2008, 290 PCOS cases treated in the First affiliated hospital of Anhui Medical University matched with 344 reproductive women as controls were enrolled in this study. Genotypes of 7 tagging single nucleotide polymorphisms (tSNP, rs12438594, rs4077582, rs9806234, rs16968477, rs4887139, rs1843090, rs11632698) covering CYP11A1 gene (r(2) > or = 0.8) and 3 microsatellite markers (D15S1547, D16S520, D15S1546) were chosed from the phase II database of Han population in HapMap data. Genotype and frequency of allele were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and haplotype of gene polymorphism were analyzed in 290 PCOS cases and 344 controls. RESULTS: Among 7 tSNPs and 3 microsatellite markers, the frequency of rs4077582, D15S1547, D15S1546 and rs11632698 between two groups reached statistical difference (P = 0.010, 0.044, 0.018 and 0.026). The allele frequency of rs4077582, rs4887139, rs1843090, D15S1547 and D16S520 showed significant difference between two groups (P = 0.002, 0.048, 0.030, 0.001 and 0.024). Among 5 haplotype of CYP11A1 (ACGCA13/6/9AG, ACGTA16/6/11AA, GCACG12/8/8AA, GTACA14/4/7GG, GTGCA13/6/7AG), the frequency of GTACA14/4/7GG and ACGCA13/6/9AG were 7.8% (45/580) and 25.3% (147/580) in PCOS group and 11.9% and 19.6% in control group, which showed statistical difference (P < 0.05). There was no significant difference in the level of serum androgen at difference genotype from rs4077582 between two groups (P > 0.05). CONCLUSION: The polymorphism of CYP11A1 gene was associated with PCOS, however, the relationship between gene sequence covered by tSNP/microsatellite markers and hyperandrogenism of PCOS should be further investigated.

Cryopreservation of immature and in-vitro matured human oocytes by vitrification.

The objective of this study was to determine the efficacy of vitrification of human oocytes before and after in-vitro maturation (IVM). The immature oocytes recovered (n = 472) were divided into two groups: (i) immature oocytes (n = 219) vitrified at the germinal vesicle (GV) stage; and (ii) immature GV-stage oocytes (n = 253) that were firstly matured in vitro (MII-stage oocytes; n = 178), then vitrified (n = 79). The remaining oocytes (n = 99), which were not vitrified, were processed as controls. After warming, the oocyte survival, maturation and fertilization rates, as well as embryonic development, were compared. The results showed no significant difference between the survival rates of the oocytes vitrified at GV stage and those vitrified at MII stage (85.4% versus 86.1%). However, oocyte maturation rates were significantly reduced (P < 0.05) when oocytes were vitrified at immature GV stage followed by IVM (50.8%) in comparison with the control group (70.4%). Following insemination by intracytoplasmic sperm injection, there was no difference in the fertilization (62.1% versus 58.8%), cleavage (69.5% versus 67.5%) and blastocyst development (0.0% versus 0.0%) rates between these two groups. However, these results were significantly lower (P < 0.05) than those achieved in the control group. This suggests that better results can be achieved by vitrifying mature oocytes rather than immature oocytes.

[Effect of double activation on the development of frozen-thawed oocytes after intracytoplasmic sperm injection].

OBJECTIVE: To investigate the influence on developmental potential of frozen-thawed rabbit oocytes with double assisted activation followed by intracytoplasmic sperm injection (ICSI). METHODS: A total of rabbit oocytes were collected and thawed after vitrification cryopreservation. Among all oocytes were cultured for 1 hour followed by ICSI. 156 Survived oocytes were divided into 5 groups randomly. I0634 single activation: 30 oocytes were added with calcium ionomycin (I0634) at 5 micromol/L for 5 minutes; SrCl(2) single activation: 26 oocytes were added with strontium chloride at 10 mmol/L for 10 minutes; I0634 double activation: 33 oocytes were activated by I0634 twice; SrCl(2) double activation: 28 oocytes were activated by strontium chloride twice. CONTROL GROUP: 39 oocytes were not added with any activators. The rate of fertilization, cleavage and blastocysts formation were observed and compared between various groups. RESULT: The rates of fertilization, cleavage and blastocysts formation were in group of SrCl(2) single activation were higher than those of I0634 single activation group without statistical difference (54% vs.33%, 27% vs. 17%, 8% vs. 3%, P < 0.05). However, those above rates in double activation by I0634 were higher significantly than those of single I0634 activation (82% vs. 33%, 55% vs. 17%, 15% vs. 3%, P < 0.05). The rates of fertilization (61%) was higher and the rate of cleavage (21%) and blastocysts formation (7%) were lower in group of SrCl(2) double activation in comparison with group of SrCl(2) single activation without reaching statistical difference (P < 0.05). Notably, the rates of fertilization, cleavage and blastocysts formation in I0634 double activation group were higher than those in group of SrCl(2) double activation with statistical difference (82% vs. 61%, 55% vs. 21%, 15% vs. 7%, P < 0.05). CONCLUSION: It might enhance the potential of fertilization of oocytes and early embryo development treated by double activation following ICSI, however, those activated oocytes demonstrate rapid cleavage.

Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification.

OBJECTIVE: To compare the survival, fertilization, early embryonic development, and meiotic spindle assembly and chromosome alignment in frozen-thawed human oocytes after slow-freezing and vitrification. DESIGN: A randomized study. SETTING: A university-affiliated assisted reproductive center. PATIENT(S): Donated extra eggs from women undergoing assisted reproduction treatment. INTERVENTION(S): A total of 605 mature oocytes were divided into a slow-freezing group and a vitrification group for cryopreservation. MAIN OUTCOME MEASURE(S): After frozen-thawing, the oocyte survival rate, spindle assembly, and chromosome alignment were compared. The surviving oocytes were inseminated by intracytoplasmic sperm injection, and the rate of fertilization and embryo development were also compared in two groups. RESULT(S): The oocyte survival rate was statistically significantly lower in the slow-freezing group (75 out of 123, 61.0%) than the vitrification group (268 out of 292, 91.8%). The fertilization rate was the same for both groups, but the cleavage rate of zygotes was statistically significantly different between two groups: (slow-freezing, 25/46 (54.4%) versus vitrification, 142 out of 182 (78.0%). There was a considerable difference in the percentage of high-quality embryos between slow-freezing and vitrification groups: 6 out of 25 (24.0%) versus 60 out of 142 (42.3%), respectively. The percentage of blastocyst development was statistically significantly higher in the vitrification group (47 out of 60, 33.1%) than in the slow-freezing group (3 out of 25, 12.0%). There was a much higher percentage of oocyte abnormalities in terms of spindle assembly and chromosome alignment in the slow-freezing group (25 out of 64, 39.1%) compared with the vitrification group (11 out of 62, 17.7%). CONCLUSION(S): Vitrification is superior to the slow-freezing method, leading to improved oocyte survival rate, fertilization, and embryonic development in vitro. These results may be related to vitrified human oocytes incurring less damage to spindle integrity and chromosome alignment.

[Effect of culture time on the spindle and chromosome configurations of human thawed oocytes with slow-cooling and raid-thawing protocol].

OBJECTIVE: To examine the metaphase II spindle and chromosome configurations of human oocytes cultured for different times after thawing. METHODS: Using slow-cooling and raid-thawing protocol combined with 0.3 mol/L sucrose and 1.5 mol/L 1, 2-propanedio 1 (1, 2-PROH) to cryoprotect human mature oocytes (n = 102), the 64 survival oocytes without abnormal zona pellucida and cytoskeletal were randomly assigned to three groups after thawing: group A: culture 1 hour (n = 20), group B: culture 3 hour (n = 22), group C: culture 5 hours (n = 22), the fresh oocytes served as control group (n = 18). Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II spindle and chromosome. RESULTS: (1) The normal spindle rates of groups A, B and C were 10% (2/20), 46% (10/22) and 41% (9/22) respectively, significantly decreased compared with control group (83%, 15/18; P < 0.05). The rates of absent spindle in group A (45%, 9/20) was significantly higher than control group (6%, 1/18; P < 0.01). Also, the rates of absent spindle in group A was higher than groups B (14%, 3/20) and C (14%, 3/20; P < 0.05). However, no significant differences were observed in groups B and C (P > 0.05). (2) A significant increase in abnormal chromosome rate was observed in group A (30%, 0; 6/20) compared to groups B (68%, 15/22), C (64%,14/22) and control group (78%, 14/18; P < 0.05). No differences in chromosome morphology were observed in groups B, C and control group (P > 0.05). CONCLUSIONS: The cryoprotectant protocol leads to a deleterious effect on the organization of the meiotic spindle and chromosome at MII stage. The 3 - 5 hours post-thawing incubation could permit restoration of the meiotic spindles and chromosome.