Xuan Gao

Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3, 2p21 and 9q33.3

Polycystic ovary syndrome (PCOS) is a common metabolic disorder in women. To identify causative genes, we conducted a genome-wide association study (GWAS) of PCOS in Han Chinese. The discovery set included 744 PCOS cases and 895 controls; subsequent replications involved two independent cohorts (2,840 PCOS cases and 5,012 controls from northern Han Chinese; 498 cases and 780 controls from southern and central Han Chinese). We identified strong evidence of associations between PCOS and three loci: 2p16.3 (rs13405728; combined P-value by meta-analysis Pmeta = 7.55 × 10−21, odds ratio (OR) 0.71); 2p21 (rs13429458, Pmeta = 1.73 × 10−23, OR 0.67); and 9q33.3 (rs2479106, Pmeta = 8.12 × 10−19, OR 1.34). These findings provide new insight into the pathogenesis of PCOS. Follow-up studies of the candidate genes in these regions are recommended.

Changes in the distribution of mitochondria before and after in vitro maturation of human oocytes and the effect of in vitro maturation on mitochondria distribution

OBJECTIVE To clarify the relationship between oocyte maturation and mitochondria distribution and assess the effects of in vitro maturation (IVM) on the distribution of mitochondria in human oocytes. DESIGN Prospective randomized trial. SETTING Hospital-based IVF center. PATIENT(S) One hundred fifty-eight patients undergoing intracytoplasmic sperm injection (ICSI) treatment for male factors or combined with oviduct infertility and fifteen patients undergoing controlled ovarian hyperstimulation followed by coitus or IUI. INTERVENTION(S) Of all the 284 immature oocytes, 140 were fixed directly. The others were prepared for IVM before they were fixed. All the 21 oocytes matured in vivo were fixed directly and stained for mitochondria. Both immature and mature oocytes were stained by Mito Tracker Green FM. The distribution of mitochondria was observed using a confocal laser scanning microscope. MAIN OUTCOME MEASURE(S) Mitochondrial distribution. RESULT(S) Three mitochondria distribution patterns were identified: peripheral, semiperipheral, and evenly diffused. A peripheral distribution of mitochondria was presented by 64.1% (50/78) of the germinal vesicle (GV) oocytes; 45.2% (28/62) of the meiosis I oocytes maintained the peripheral distribution; and 38.7% (24/62) presented a diffused status. After IVM, 75.5% (80/106) of the oocytes displayed an evenly diffused type of distribution. The mitochondria were more abundant in the inner cytoplasm than in the peripheral region in most of the oocytes matured in vivo. CONCLUSION(S) There are obvious changes in the distribution of mitochondria in human oocytes before and after maturation. Distribution of mitochondria in oocytes matured in vitro is slightly different from that of oocytes matured in vivo. The results may partially explain the reduced developmental potential of oocytes matured in vitro compared with those matured in vivo.

The role of male chromosomal polymorphism played in spermatogenesis and the outcome of IVF/ICSI-ET treatment

Chromosomal polymorphism has been reported to be associated with infertility, but its effect on IVF/ICSI-ET outcome is still controversial. To evaluate whether or not chromosomal polymorphism in men plays a role in spermatogenesis and the outcome of IVF/ICSI-ET, we retrospectively analysed 281 infertile couples. Measures included fertilization rate, implantation rate, pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, early miscarriage rate and preterm rate. Men with chromosomal polymorphism had significantly higher frequencies of severe oligozoospermia and azoospermia than those without (37.12% vs. 16.11%, p < 0.001; 27.27% vs. 10.74%, p < 0.001; respectively). Significantly, lower fertilization rate (68.02% vs. 78.00%, p < 0.001) and clinical pregnancy rate (45.00% vs. 66.67%, p = 0.031) were observed in polymorphism-carrying men with severe oligozoospermia compared with non-carriers with severe oligozoospermia. This suggests that chromosomal polymorphism has adverse effects on spermatogenesis, negatively influencing the outcome of IVF/ICSI-ET treatment. Polymorphic variations on the Y chromosome have been found to be the most prevalent polymorphism in infertile men, most frequently occurring in patients with severe oligozoospermia.

Spindle and chromosome changes of human MII oocytes during incubation after slow freezing/fast thawing procedures.

This experiment investigated the optimal time required for cryopreserved human oocytes to reform their spindles upon re-warming. Metaphase II oocytes were cryopreserved with a slow freezing method. Oocytes from each patient were randomly allocated into one of the 3 groups with different culture periods: 1, 3, or 5 hours, respectively, after thawing. Tubulin and chromosome configurations were visualized by confocal microscopy after immunostainings. By morphological assessment, 87.3% oocytes survived the process of freezing and thawing. Oocytes with normal spindle configuration increased significantly after 3 or 5 hours of incubation compared to those incubated for only 1 hour ( P < 0.05). There were no differences in the chromosome configurations among the treatment groups ( P > 0.05). This experiment demonstrated that cryopreserved human oocytes need a certain minimum period of incubation time (3 h) to recover their disrupted MII spindles and this information can be used in development of human IVF protocols with frozen oocytes.

STMN1 Promotes Progesterone Production Via StAR Up-regulation in Mouse Granulosa Cells

Stathmin 1 (STMN1) is a biomarker in several types of neoplasms. It plays an important role in cell cycle progression, mitosis, signal transduction and cell migration. In ovaries, STMN1 is predominantly expressed in granulosa cells (GCs). However, little is known about the role of STMN1 in ovary. In this study, we demonstrated that STMN1 is overexpressed in GCs in patients with polycystic ovary syndrome (PCOS). In mouse primary GCs, the overexpression of STMN1 stimulated progesterone production, whereas knockdown of STMN1 decreased progesterone production. We also found that STMN1 positively regulates the expression of Star (steroidogenic acute regulatory protein) and Cyp11a1 (cytochrome P450 family 11 subfamily A member 1). Promoter and ChIP assays indicated that STMN1 increased the transcriptional activity of Star and Cyp11a1 by binding to their promoter regions. The data suggest that STMN1 mediates the progesterone production by modulating the promoter activity of Star and Cyp11a1. Together, our findings provide novel insights into the molecular mechanisms of STMN1 in ovary GC steroidogenesis. A better understanding of this potential interaction between STMN1 and Star in progesterone biosynthesis in GCs will facilitate the discovery of new therapeutic targets in PCOS.

Nonsense mutation of EMX2 is potential causative for uterus didelphysis: first molecular explanation for isolated incomplete müllerian fusion

OBJECTIVE To investigate the association between human empty spiracles homeobox 2 gene (EMX2) and incomplete müllerian fusion (IMF). DESIGN Case-control study. SETTING University-based hospital. PATIENT(S) Cohort of 517 clinically well-characterized IMF cases and 563 control women. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) In cases and control women, direct sequencing of EMX2 exons and further functional studies; for functional studies, wild-type and mutant EMX2 expression plasmids constructed; human embryonic kidney cells (HEK293FT) transfected with empty vector, wild-type EMX2, mutant EMX2, and 1:1 combination (wild-type/mutant plasmids) with additional functional studies performed to clarify the deleterious effect of the novel mutation detected. RESULT(S) A novel nonsense mutation p.E142X was detected in one woman with a didelphic uterus (1 of 517, 0.19%). The results of Western blot analysis confirmed that the mutation caused a truncated protein as predicted, and functional studies proved that it resulted in a dominant negative effect. CONCLUSION(S) The novel nonsense mutation we detected-EMX2, p.E142X- resulted in a dominant negative effect. The functional data were exemplified in HEK293FT cells. This reinforced the likelihood that EMX2 contributed to the pathophysiology of IMF. Although it is uncommon (0.19%), EMX2 is the first gene identified that if perturbed may cause isolated IMF.

Allotransplantation of cryopreserved prepubertal mouse ovaries restored puberty and fertility without affecting methylation profile of Snrpn-DMR.

OBJECTIVE To evaluate the genetic safety of vitrification on the methylation imprints and the development and fertility potential of prepubertal mouse ovaries. DESIGN Experimental animal study. SETTING University-based fertility center. ANIMAL(S) Institute of Cancer Research (ICR) 10-day-old female mice, 10-week-old adult female mice, and 12-week-old adult male mice. INTERVENTION(S) Vitrification of juvenile mouse ovaries was performed using ED20 and EG5.5/30 solutions followed by retrieval of fresh and vitrified-warmed germinal vesicle (GV) oocytes for Snrpn differentially methylated regions (DMR) methylation analyses, collection of mature oocytes from superovulated ovarian grafts, in vitro fertility(IVF), and early embryonic development after heterotopic allotransplantation. MAIN OUTCOME MEASURE(S) Analysis of methylation status of Snrpn-DMR, percentage of fertilization, and blastocysts formation. RESULT(S) Methylation status of Snrpn-DMR from vitrified-warmed GV oocytes did not show significant alteration compared with that of controls, although a significant reduction of viable oocytes was observed. Puberty as well as endocrine function was restored, and no significant difference was shown in number of follicles, percentage of mice retaining fertility, and blastocyst formation among three groups. CONCLUSION(S) Our study proved that vitrification of prepubertal mouse ovaries did not alter the methylation profile of Snrpn-DMR and subsequent allotransplantation; IVF could restore the development and fertility potential.

Family-based analysis of eight susceptibility loci in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that is proposed to have a genetic basis. A recent genome-wide association study (GWAS) identified eight new risk loci that are independently associated with PCOS. To further validate the findings, a total of 321 case-parent trios (963 participants) who had a proband affected with PCOS were recruited for the family-based study. The transmission disequilibrium test (TDT) was used to analyze associations between PCOS and ten single nucleotide polymorphisms (SNPs) mapped to eight new susceptibility loci. Significant differences in transmission were observed for the SNPs rs2349415 (located in the FSHR gene, P = 0.0001) and rs3802457 (located in the C9orf3 gene, P = 0.0001), even after correction for multiple testing bias. The present data provides further evidence for an association between two susceptibility loci, 2p16.3 and 9q22.32, and PCOS. Follow-up functional studies on the FSHR and C9orf3 genes are required to understand their roles in PCOS development.

Selection of viable human spermatozoa with low levels of DNA fragmentation from an immotile population using density gradient centrifugation and magnetic‐activated cell sorting

We aimed to determine whether density gradient centrifugation and magnetic‐activated cell sorting (DGC‐MACS) could select viable spermatozoa, with lower levels of DNA fragmentation, from an immotile population. Analysis involved sixteen patients, each with a sperm count ≥107/mL. All samples were immotile despite exhibiting a live population >40%. Spermatozoa were prepared using DGC‐MACS and selected spermatozoa evaluated for membrane and DNA integrity using the hypo‐osmotic swelling (HOS) test, vital staining and the TUNEL test. The mean proportion of spermatozoa with an intact membrane in control, DGC and DGC‐MACS populations, was 52.5 ± 12.21%, 69.38 ± 7.87% and 81.81 ± 5.29%. The mean proportion of live spermatozoa in control, DGC and DGC‐MACS populations, was 65.88 ± 12.77%, 77.25 ± 7.39% and 85.81 ± 5.2%. DGC‐MACS reduced the within‐sample discrepancy between HOS test and vital stain results from 13.18% to 4.12%. The mean proportion of spermatozoa exhibiting DNA damage in control, DGC and DGC‐MACS populations, was 9.56 ± 3.39%, 5.25 ± 1.61% and 2.75 ± 1.13%. Finally, analysis showed that 71.23% of the DNA‐fragmented spermatozoa in unprocessed samples were removed following DGC‐MACS and that the addition of MACS to an existing DGC protocol reduced fragmented spermatozoa by a further 26.15% compared to DGC alone. Consequently, DGC‐MACS is a clinically viable method able to select viable spermatozoa with lower levels of DNA fragmentation from an immotile population.

Correlation of genetic results with testicular histology, hormones and sperm retrieval in nonobstructive azoospermia patients with testis biopsy

To investigate the frequency and types of genetic results in different testicular histology of patients with nonobstructive azoospermia (NOA), and correlated with hormones and sperm retrieval (SR), a retrospective study was conducted in 286 Chinese NOA patients who underwent testis biopsy and 100 age‐matched fertile men as the control group. Chromosome karyotype analyses were performed by the peripheral blood chromosome G‐band detection method. Screening of Y chromosome microdeletions of azoospermia factor (AZF) region was performed by polymerase chain reaction (PCR) amplification of 11 sequence‐tagged sites (STS). The serum levels of follicle‐stimulating hormone, luteinising hormone and testosterone (T) and the appearance of scrotal ultrasound were also obtained. In 286 cases of NOA, 14.3% were found to have chromosomal alterations. The incidence of chromosomal abnormality was 2.8%. Sex chromosomal abnormalities were seen in six cases (four cases of Klinefelters syndrome (47, XXY) and two cases of mosaics). The incidence of polymorphic chromosomal variants was 3% in the normal group and 11.5% in the NOA group. In total, 15.7% of NOA patients were found to have AZF microdeletions and AZF (c + d) was the most frequent one. The results of hormone and SR were found to be significantly different among all testicular histological types, whereas no significant differences were found when it comes to genetic alterations. It is concluded that the rate of cytogenetic alterations was high in NOA patients. So screening for chromosomal alterations and AZF microdeletions would add useful information for genetic counselling in NOA patients with testis biopsy and avoid vertical transmission of genetic defects by assisted reproductive technology.