Yunxia Cao

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

OBJECTIVEnTo compare the survival, fertilization, early embryonic development, and meiotic spindle assembly and chromosome alignment in frozen-thawed human oocytes after slow-freezing and vitrification.nnnDESIGNnA randomized study.nnnSETTINGnA university-affiliated assisted reproductive center.nnnPATIENT(S)nDonated extra eggs from women undergoing assisted reproduction treatment.nnnINTERVENTION(S)nA total of 605 mature oocytes were divided into a slow-freezing group and a vitrification group for cryopreservation.nnnMAIN OUTCOME MEASURE(S)nAfter 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.nnnRESULT(S)nThe 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%).nnnCONCLUSION(S)nVitrification 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.

Biallelic SUN5 Mutations Cause Autosomal-Recessive Acephalic Spermatozoa Syndrome

Acephalic spermatozoa syndrome is a rare and severe form of teratozoospermia characterized by a predominance of headless spermatozoa in the ejaculate. Family clustering and consanguinity suggest a genetic origin; however, causative mutations have yet to be identified. We performed whole-exome sequencing in two unrelated infertile men and subsequent variant filtering identified one homozygous (c.824C>T [p.Thr275Met]) and one compound heterozygous (c.1006C>T [p.Arg356Cys] and c.485T>A [p.Met162Lys]) SUN5 (also named TSARG4) variants. Sanger sequencing of SUN5 in 15 additional unrelated infertile men revealed four compound heterozygous (c.381delA [p.Val128Serfs∗7] and c.824C>T [p.Thr275Met]; c.381delA [p.Val128Serfs∗7] and c.781G>A [p.Val261Met]; c.216G>A [p.Trp72∗] and c.1043A>T [p.Asn348Ile]; c.425+1G>A/c.1043A>T [p.Asn348Ile]) and two homozygous (c.851C>G [p.Ser284∗]; c.350G>A [p.Gly114Arg]) variants in six individuals. These 10 SUN5 variants were found in 8 of 17 unrelated men, explaining the genetic defect in 47.06% of the affected individuals in our cohort. These variants were absent in 100 fertile population-matched control individuals. SUN5 variants lead to absent, significantly reduced, or truncated SUN5, and certain variants altered SUN5 distribution in the head-tail junction of the sperm. In summary, these results demonstrate that biallelic SUN5 mutations cause male infertility due to autosomal-recessive acephalic spermatozoa syndrome.

Association between CYP19 gene SNP rs2414096 Polymorphism and polycystic ovary syndrome in Chinese women

BackgroundSeveral studies have reported the association of the SNP rs2414096 in the CYP19 gene with hyperandrogenism, which is one of the clinical manifestations of polycystic ovary syndrome (PCOS). These studies suggest that SNP rs2414096 may be involved in the etiopathogenisis of PCOS. To investigate whetherthe CYP19 gene SNP rs2414096 polymorphism is associated with the susceptibility to PCOS, we designed a case-controlled association study including 684 individuals.MethodsA case-controlled association study including 684 individuals (386 PCOS patients and 298 controls) was performed to assess the association of SNP rs2414096 with PCOS. Genotyping of SNP rs2414096 was conducted by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that was performed on genomic DNA isolated from blood leucocytes. Results were analyzed in respect to clinical test results.ResultsThe genotypic distributions of rs2414096 (GG, AG, AA) in the CYP19 gene (GG, AG, AA) in women with PCOS (0.363, 0.474, 0.163, respectively) were significantly different from that in controls (0.242, 0.500, 0.258, respectively) (P = 0.001). E2/T was different between the AA and GG genotypes. Age at menarche (AAM) and FSH were also significantly different among the GG, AG, and AA genotypes in women with PCOS (P = 0.0391 and 0.0118, respectively). No differences were observed in body mass index (BMI) and other serum hormone concentrations among the three genotypes, either in the PCOS patients or controls.ConclusionsOur data suggest that SNP rs2414096 in the CYP19 gene is associated with susceptibility to PCOS.

No association of the insulin gene VNTR polymorphism with polycystic ovary syndrome in a Han Chinese population

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disorder associated with an increased risk of type II diabetes mellitus. The results of previous research about the association of the VNTR polymorphism in 5-prime flanking region of the insulin (INS) gene with PCOS have been inconsistent. The present study was to investigate the association of the INS-VNTR polymorphism with PCOS in a Han Chinese population.MethodsThe -23/HphI polymorphism as a surrogate marker of the INS-VNTR length polymorphism was genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 216 PCOS patients and 192 non-PCOS women as a control group. Allelic and genotypic frequencies were compared between patients and controls, and these results were analyzed in respect to clinical test data.ResultsNo significant differences were observed between the cases and controls groups either in allele (P = 0.996) or genotype (P = 0.802) frequencies of INS-VNTR polymorphism; Regarding anthropometric data and hormone levels, there were no significant differences between INS-VNTR genotypes in the PCOS group, as well as in the non-PCOS group.ConclusionThe present study demonstrated for the first time that the INS-VNTR polymorphism is not a key risk factor for sporadic PCOS in the Han Chinese women. Further studies are needed to give a global view of this polymorphism in pathogenesis of PCOS in a large-scale sample, family-based association design or well-defined subgroups of PCOS.

Polymorphisms of TCF7L2 and HHEX genes in Chinese women with polycystic ovary syndrome

PurposeThis study was to evaluate whether polymorphisms of TCF7L2 (rs7903146) and HHEX (rs1111875) genes responsible for insulin secretion are associated with the polycystic ovary syndrome (PCOS) in Chinese people.Methods326 PCOS patients and 290 healthy individuals as controls were studied. Blood samples were obtained for DNA analyses and hormone measurements. Genotyping of the TCF7L2 (rs7903146) and HHEX (rs1111875) genes was carried out by the polymerase chain reaction–restriction fragment length polymorphism method.ResultsWe did not find statistically significant differences in the distribution of the TCF7L2 rs7903146 and HHEX rs1111875 polymorphisms between the Chinese women with PCOS and the controls. Levels of hormones such as insulin, FSH, LH, LH/FSH, P, T and E2 were also similar between the different genotypes of the genes TCF7L2 and HHEX, respectively, which was confirmed within either the PCOS subjects or controls.ConclusionsThere was no association of either of the two variants, rs7903146 of TCF7L2 and rs1111875 of HHEX, with the occurrence of PCOS in the Chinese population.

Association between polymorphisms of the CYP11A1 gene and polycystic ovary syndrome in Chinese women

The cholesterol side chain cleavage enzyme (CYP11A1) gene plays an important part in the synthesis of sex hormones and has been reported to be involved in the pathogenesis of polycystic ovary syndrome. A case–control study including 314 PCOS patients and 314 controls was conducted to assess the association of the SNPs rs4077582 and rs11632698 in CYP11A1 with PCOS using the polymerase chain reaction-restriction fragment length polymorphism method. Thereafter, 100 DNA samples were re-genotyped by direct sequencing for confirmation. The genotypic distribution of rs4077582 in women with PCOS differed from that in controls (Pxa0=xa00.002). No such distributional difference was found in rs11632698 (Pxa0=xa00.912). Data from our previous study of these two SNPs in another population including 290 PCOS patients and 344 controls was combined with the current data. Combined analysis (a total of 1262 participants, including 604 PCOS patients and 658 control women) showed a much more significant difference in the genotypic distribution of rs4077582 between PCOS and controls (Pxa0<xa00.001). The T allele was more prevalent in PCOS patients (Odds ratioxa0=xa01.314; 95xa0% CI 1.122–1.540). The testosterone levels among the three genotypes for rs4077582 were different in the control group, as were the LH levels and the LH/FSH ratio. Therefore, SNP rs4077582 in CYP11A1 is strongly associated with susceptibility to PCOS and may alter the testosterone levels by the regulation of LH in different genotypes. No association was observed in rs11632698.

SNP rs2470152 in CYP19 is correlated to aromatase activity in Chinese polycystic ovary syndrome patients

CYP19 encodes aromatase, a key enzyme essential for estrogen biosynthesis. Single nucleotide polymorphism (SNP) rs2470152 in CYP19 is associated with serum estradiol (E2) level and the E2/T (estradiol/testosterone) ratio. A case‑control study including 661 individuals [364 polycystic ovary syndrome (PCOS) patients and 297 controls] was conducted to assess the association of SNP rs2470152 with PCOS. The subjects were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. Hormone levels were analyzed among various genotypes. The genotypic distributions of rs2470152 did not differ in PCOS patients when compared to the controls. However, differences in the E2/T ratio were detected, exhibiting a lower ratio in the heterozygous TC genotype in PCOS patients (p=0.01036) and controls (p=0.000). Testosterone levels also differed between the three genotypes of PCOS patients (p=0.00625), with a higher level in the TC genotype. Therefore, rs2470152 in CYP19 was not a major etiological factor for PCOS; however, the heterozygous TC genotype may inhibit aromatase activity, resulting in hyperandrogenism, particularly in PCOS patients.

Polymorphic CAG repeat in the androgen receptor gene in polycystic ovary syndrome patients

Human androgen receptor (AR) contains a highly polymorphic polyglutamine tract encoded by CAG repeats [(CAG)n] in exon 1 of the AR gene. The CAG repeats, ranging from 11 to 38, have been reported to be inversely correlated with AR activity. A case-control study involving 261 polycystic ovary syndrome (PCOS) patients and 278 healthy controls was conducted. Fluorescently labeled DNA fragments containing (CAG)n were obtained by PCR and genotyped via capillary electrophoresis. AR (CAG)n ranges were 6, 12-28 in PCOS cases and 9, 10, 12-32 in controls. In the PCOS group, a higher frequency of short (CAG)n alleles was found compared with that of controls (P=0.007). Similarly, CAG biallelic mean distributions also showed statistical difference between the two groups (P=0.025). In conclusion, shorter alleles of the (CAG)n in exon 1 of the AR gene enhanced the susceptibility to PCOS, either by upregulating AR activity or by causing hyperandrogenism.

Essential role for SUN5 in anchoring sperm head to the tail.

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5−/− mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility.

Effects of vitrification cryopreservation on follicular morphology and stress relaxation behaviors of human ovarian tissues: sucrose versus trehalose as the non-permeable protective agent

STUDY QUESTIONnIs sucrose more effective than trehalose in human ovarian tissue cryopreservation?nnnSUMMARY ANSWERnThe effect of sucrose as a cryoprotective agent (CPA) was not significantly different from that of trehalose in human ovarian tissue cryopreservation.nnnWHAT IS KNOWN ALREADYnSugars have the ability to keep the cell membrane intact and can decrease the toxicity of CPAs. Sucrose is the most commonly used non-permeable CPA, while trehalose is rarely used in human ovarian tissue cryopreservation. Although various methods are utilized to evaluate the efficiency of human ovarian tissue cryopreservation, few studies have evaluated the effect of cryopreservation from the viewpoint of biomechanics.nnnSTUDY DESIGN, SIZE, DURATIONnA total of 15 ovarian tissue samples were collected from 15 patients (20-41 years old) with benign ovarian tumors or malignancies, and each was dissected into six slices. Two slices were taken as the fresh control group. The remaining four slices were vitrified using different vitrification protocols. After warming, samples in each group were either fixed for histological evaluation or destined for stress relaxation test.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnThe CPA solutions for the control and vitrified groups were composed of EDS and EDT (E, ethylene glycol; D, dimethylsulphoxide; S, sucrose; T, trehalose). The stress relaxation experiments were carried out at room temperature using a dynamic mechanical analyzer. Ovarian tissue samples were assessed for both their morphology and viscoelasticity. Stress relaxation data (SRD) were calculated as a percentage, representing the ability to maintain the initial stress after stretching. The percentage of morphologically normal follicles was compared between groups, which was represented by morphologic preservation ratio.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe morphologic preservation ratio of the primordial follicles in the fresh control group (87.58%) was higher than that in group S (72.33%) (P = 0.000) and group T (79.56%) (P = 0.002). Although not statistically significant, compared with the S group, vitrification with T suggested a trend toward a higher morphologic preservation ratio of the primordial follicles. The SRD in the fresh control group (0.6433 ± 0.7233) was significantly different from that in group S (0.5200 ± 0.8331, P = 0.000) or in group T (0.5667 ± 0.6415, P = 0.000). However, no significant difference was found between groups S and T.nnnLIMITATIONS, REASONS FOR CAUTIONnExperimental samples were directly exposed to the air, which will result in a discrepancy in the viscoelastic properties between experimental tissues and in vivo tissues.nnnWIDER IMPLICATIONS OF THE FINDINGSnOur study suggested a trend toward a higher morphologic preservation ratio of the primordial follicles after vitrification in trehalose compared with sucrose, which may provide a basis for further optimizing human ovarian tissue vitrification. In addition, it was possible to evaluate the effect of ovarian tissue cryopreservation from a biomechanics perspective.nnnSTUDY FUNDING/COMPETING INTERESTSnThis study was supported by the grants from the Medical Scientific Research Subject, Health Ministry of Anhui Province (2010B014) and National Basic Research Program of China (973 Program) (2012CB944704), and the National Natural Science Foundation of China (Nos. 51276179 and 51476160). The authors declare that there is no conflict of interests regarding the publication of this original paper.