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Primordial follicle activation is fundamental for folliculogenesis and for the maintenance of fertility. An effective therapeutic strategy for patients with premature ovarian insufficiency (POI) is to promote the activation of residual primordial follicles. The secretome of human umbilical cord mesenchymal stromal cells (hUC-MSC-sec) contains several components that might promote the activation of primordial follicles. In the present study, we revealed that treatment with the hUC-MSC-sec significantly increased the proportion of activated primordial follicles in mouse ovaries both in vitro and in vivo. The activating effects of hUC-MSC-sec on primordial follicles were attributed to the activation of the PI3K-AKT signaling pathway by hepatocyte growth factor (HGF). While the effect of the hUC-MSC-sec was attenuated by the neutralizing antibodies against HGF, application of exogenous HGF alone also promoted the activation of primordial follicles. Furthermore, we demonstrated that HGF promoted the expression of KITL in granulosa cells by binding with the HGF receptor c-Met, thereby increasing the activity of the PI3K-AKT signaling pathway to activate primordial follicles. Taken together, our findings demonstrate that hUC-MSC-sec promotes primordial follicle activation through the functional component HGF to increase the PI3K-AKT signaling activity, highlighting the application of the hUC-MSC-sec or HGF for the treatment of POI patients. Graphical abstract

Premature ovarian insufficiency (POI) is one of the key aspects of ovarian infertility. Due to early cession of ovarian function, POI imposes great challenges on the physiological and psychological health of women and becomes a common cause of female infertility. In the worldwide, there has been a special outpouring of concern for about 4 million reproductive-aged women suffering from POI in China. Driven by advances in new technologies and efforts invested by Chinses researchers, understanding about POI has constantly been progressing over the past decade. Here, we comprehensively summarize and review the landmark development and achievements from POI studies in China spanning 2011–2020, which aims to provide key insights from bench to bedside.

Immune dysregulation has long been proposed as a component of premature ovarian insufficiency (POI), but the underlying mediators and mechanisms remain largely unknown. Here we showed that patients with POI had augmented T helper 1 (TH1) responses and regulatory T (Treg) cell deficiency in both the periphery and the ovary compared to the control women. The increased ratio of TH1:Treg cells was strongly correlated with the severity of POI. In mouse models of POI, the increased infiltration of TH1 cells in the ovary resulted in follicle atresia and ovarian insufficiency, which could be prevented and reversed by Treg cells. Importantly, interferon (IFN) ‐γ and tumor necrosis factor (TNF) ‐α cooperatively promoted the apoptosis of granulosa cells and suppressed their steroidogenesis by modulating CTGF and CYP19A1. We have thus revealed a previously unrecognized Treg cell deficiency‐mediated TH1 response in the pathogenesis of POI, which should have implications for therapeutic interventions in patients with POI., The Treg cells deficiency with decreased number and impaired suppression function mediate augmented TH1 responses in premature ovarian insufficiency (POI). The increased TH1 proinflammatory cytokines IFN‐γ and TNF‐α impair steroidogenesis by targeting CYP19A1 and promote apoptosis of granulosa cells partially by down‐regulation of CTGF via JAK‐STAT1 and NF‐κB activation, hence contribute to follicle atresia, ovarian dysfunction and premature insufficiency.

Premature ovarian insufficiency (POI) is highly heterogeneous in genetic etiology. Yet identifying causative genes has been challenging with candidate gene approaches. Recent approaches using next generation sequencing (NGS), especially whole exome sequencing (WES), in large POI pedigrees have identified new causatives and proposed relevant candidates, mainly enriched in DNA damage repair, homologous recombination, and meiosis. In the near future, NGS or whole genome sequencing will help better define genes involved in intricate regulatory networks. The research into miRNA and age at menopause represents an emerging field that will help unveil the molecular mechanisms underlying pathogenesis of POI. Shedding light on the genetic architecture is important in interpreting pathogenesis of POI, and will facilitate risk prediction for POI.

Context Premature ovarian insufficiency (POI) is characterized by cessation of menstruation before 40 years of age and elevated serum level of FSH (>25 IU/L). Recent studies have found a few causative genes responsible for POI enriched in meiotic recombination and DNA damage repair pathways. Objective To investigate the role of variations in homologous recombination genes played in POI pathogenesis. Methods The whole exome sequencing was performed in 50 POI patients with primary amenorrhea. Functional characterizations of the novel variants were carried out in budding yeast and human cell line. Results We identified 8 missense variants in 7 homologous recombination genes, including EXO1, RAD51, RMI1, MSH5, MSH2, MSH6, and MLH1. The mutation p.Thr52Ser in EXO1 impaired the meiotic process of budding yeast and p.Glu68Gly in RAD51-altered protein localization in human cells, both of them impaired the efficiency of homologous recombination repair for DNA double-stranded breaks in human cells. Conclusions Our study first linked the variants of EXO1 and RAD51 with POI and further highlighted the role of DNA repair genes in ovarian dysgenesis.

The genetic etiology of premature ovarian insufficiency (POI) has been well established to date, however, the role of long noncoding RNAs (lncRNAs) in POI is largely unknown. In this study, we identified a down-expressed lncRNA HCP5 in granulosa cells (GCs) from biochemical POI (bPOI) patients, which impaired DNA damage repair and promoted apoptosis of GCs. Mechanistically, we discovered that HCP5 stabilized the interaction between YB1 and its partner ILF2, which could mediate YB1 transferring into the nucleus of GCs. HCP5 silencing affected the localization of YB1 into nucleus and reduced the binding of YB1 to the promoter of MSH5 gene, thereby diminishing MSH5 expression. Taken together, we identified that the decreased expression of HCP5 in bPOI contributed to dysfunctional GCs by regulating MSH5 transcription and DNA damage repair via the interaction with YB1, providing a novel epigenetic mechanism for POI pathogenesis.

Meiosis is a germ cell-specific division that is indispensable for the generation of haploid gametes. However, the regulatory mechanisms of meiotic initiation remain elusive. Here, we report that the Wdr62 (WD40-repeat protein 62) is involved in meiotic initiation as a permissive factor rather than an instructive factor. Knock-out of this gene in a mouse model resulted in female meiotic initiation defects. Further studies demonstrated that Wdr62 is required for RA-induced Stra8 expression via the activation of JNK signaling, and the defects in meiotic initiation from Wdr62-deficient female mice could be partially rescued by JNK1 overexpression in germ cells. More importantly, two novel mutations of the WDR62 gene were detected in patients with premature ovarian insufficiency (POI), and these mutations played dominant-negative roles in regulating Stra8 expression. Hence, this study revealed that Wdr62 is involved in female meiotic initiation via activating JNK signaling, which displays a novel mechanism for regulating meiotic initiation, and mutation of WDR62 is one of the potential etiologies of POI in humans.

Accumulating evidence has indicated that the genes involved in meiosis are highly correlated with ovarian function. Pumilio 1 (PUM1) is a RNA-binding protein which is involved in the meiotic process. It has been reported that the Pum1 knockout female mice displayed subfertility due to the decrease in primordial follicle pool. The aim of our study is to investigate whether variants of the PUM1 gene are responsible for primary ovarian insufficiency (POI) in Chinese women. We analyzed coding sequence and untranslated regions of the PUM1 gene in 196 Han Chinese women with non-syndromic POI and 192 controls. Seven novel variants were identified, but one of them was synonymous and six were intronic. Besides, seven known single-nucleotide polymorphisms (SNPs) were found, and there were no significant differences in genotype and allele frequencies of the SNPs between patients and controls. The results suggest that the variants in PUM1 may not contribute to POI in Han Chinese women. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-017-1110-4) contains supplementary material, which is available to authorized users.

Premature ovarian insufficiency (POI) imposes great challenges on women’s fertility and lifelong health. POI is highly heterogeneous and encompasses occult, biochemical, and overt stages. MicroRNAs (miRNAs) are negative regulators of gene expression, whose roles in physiology and diseases like cancers and neurological disorders have been recognized, but little is known about the miRNAs profile and functional relevance in biochemical POI (bPOI). In this study, the expression of miRNAs and mRNAs in granulosa cells (GCs) of bPOI women was determined by two microarrays, respectively. MiR-379-5p, PARP1, and XRCC6 were differentially expressed in GCs of bPOI as revealed by microarrays. Subsequently, functional studies demonstrated that miR-379-5p overexpression inhibited granulosa cell proliferation and attenuated DNA repair efficiency. Furthermore, both PARP1 and XRCC6 showed lower levels in GCs from patients with bPOI and were identified as executives of miR-379-5p. Therefore, our data first uncovered potentially pathogenic miR-379-5p and two novel targets PARP1 and XRCC6 in bPOI, which corroborated the significance of DNA repair for POI, and brought up an epigenetic explanation for the disease.

Context Premature ovarian insufficiency (POI) is highly heterogeneous, both in phenotype and etiology. They are not yet clearly stated and correlated. Objective To characterize clinical presentations of a large, well-phenotyped cohort of women with POI, and correlate phenotypes with etiologies to draw a comprehensive clinical picture of POI. Design, patients, interventions, and main outcome measures In this retrospective study, a total of 955 Chinese women with overt POI between 2006 and 2015 were systemically evaluated and analyzed. The phenotypic features, including menstrual characteristics, hormone profiles, ovarian ultrasonography/biopsy, pregnancy/family history, and genetic/autoimmune/iatrogenic etiologies were assessed and further compared within different subgroups. Results Among 955 women with POI, 85.97% presented with secondary amenorrhea (SA) and 14.03% with primary amenorrhea (PA). PA represented the most severe ovarian dysfunction and more chromosomal aberrations than SA. The decline of ovarian function in patients with SA progressed quickly. They had shortened reproductive periods (approximately 10 years) and developed amenorrhea within 1 to 2 years after menstrual irregularity. The ovaries were invisible or small, and the presence of follicles (28.43%) was correlated with other good reproductive indicators. Familial patients (12.25%) manifested better ovarian status and fewer chromosomal aberrations than sporadic patients. The etiologies consisted of genetic (13.15%), autoimmune (12.04%), and iatrogenic (7.29%), approximately 68% remaining idiopathic. There were significant differences among different etiologies, with the genetic group representing the most severe phenotype. Conclusion Our results regarding distinct phenotypic characteristics and association with different etiologies further confirmed the high heterogeneity of POI. Additional longitudinal clinical studies and pathogenesis research are warranted.

Primary ovarian insufficiency (POI) is a genetically heterogeneous disorder that occurs in familial or sporadic fashion. Through whole exome sequencing in a Chinese pedigree with POI, we identified a novel homozygous missense mutation (ENST00000375755: c.1459G > T, p.D487Y) in the MSH5 gene in two sisters with POI. The homologous mutation in mice resulted in atrophic ovaries without oocytes, and in vitro functional study revealed that mutant MSH5 impaired DNA homologous recombination repair. From sanger sequencing of MSH5 in 200 sporadic POI patients, we identified three heterozygous mutations (ENST00000375755: c.1057C > A, p.L353M; c.1459G > T, p.D487Y and c.2107 A > G, p.I703V). Considering the heterozygous p.D487Y carrier in the POI pedigree was fertile, the causality of the three heterozygous mutations in POI need more evidence. Our studies confirmed that perturbation of genes involved in DNA damage repair could lead to non-syndromic POI. The underlying mechanism-inability to repair DNA damage-will receive increasing attention with respect to POI.

To evaluate the effect of ovarian biopsy and scratch on the resumption of ovarian function in women with premature ovarian insufficiency (POI), the follicle development and pregnancy outcome were retrospectively analyzed in women with POI after ovarian biopsy/scratch. Eighty patients with POI with secondary amenorrhea were accepted the laparoscopic surgery, and then hormone replacement treatment for 6 months was applied and in vitro fertilization and embryo transfer was suggested. No significant difference in clinical characteristics was observed before and after ovarian biopsy/scratch ( P > .05). After the ovarian surgery, 11 (13.75%) patients presented with ovarian function resumption spontaneously or with human menopausal gonadotropin stimulation. Three metaphases II oocytes were retrieved in 10 patients. Two embryos were formed and freshly transferred followed by a healthy singleton delivery in 1 (1.25%) patient. Patients with follicle development had higher baseline estradiol compared to those without (78.20 [18.53-161.08] pg/mL vs 15.70 [8.40-43.80] pg/mL, P < .01). The technique of ovarian biopsy/scratch could promote follicle development in vivo, suggesting it could bring promising benefits for some women with POI. However, future improvement in efficiency and practice criteria is warranted.

Global transcriptional activity increases as oocytes grow and is silenced in fully grown oocytes. Thus, the chromatin configuration varies during oocyte growth, but the molecular mechanisms regulating these changes remain to be clarified. Here, we studied a susceptibility gene of polycystic ovary syndrome (PCOS), RPS26, which is a ribosomal protein-encoding gene that is highly expressed in the ovary, but the functions of which remain unknown. Specific knockout of Rps26 in mouse oocytes resulted in retarded follicle development from pre-antral follicles to antral follicles, while the chromatin configurations of the oocytes were arrested at the transition from the non-surrounded nucleolus (NSN) to surrounded nucleolus (SN)-type. As a consequence, all oocytes died by postnatal day 84 resulting in premature ovarian failure (POF). Loss of Rps26 in oocytes led to decreased mRNA transcription and low levels of histone trimethylation on H3K4/H3K9 and DNA methylation at 5-cytosine, high levels of which are required for oocytes to transform from NSN to SN-type. Low protein levels of oocyte-derived growth differentiation factor 9, bone morphogenetic protein 15, and the oocyte-granulosa cell gap junction protein connexin 37 inhibited oocyte growth and retarded follicle development. The disruption of the phosphoinositide 3-kinase/protein kinase B/Forkhead box O-3a pathway contributed to oocyte death and follicle atresia. These results provide genetic clues for the clinical diagnosis of POF, especially in PCOS patients without treatment.

HELQ , a member of DNA repair gene family, is an enzyme required for DNA strands cross-links repair and closely related to age at natural menopause. It also possesses a critical role in the germ cell maintenance, and loss of HELQ gene leads to subfertility. The aim of the present study was to investigate whether mutations in HELQ contribute to premature ovarian failure (POF) in Chinese women. A cohort of 192 patients with POF was enrolled. All exons and exon–intron boundaries of genomic DNA were amplified and sequenced. Six known single-nucleotide polymorphisms were identified in both POF and control groups, including rs1494961, rs13141136, rs7665103, rs11099600, rs2047210 and rs12645412. No mutation was identified. Our study indicates for the first time that mutations in the coding sequence of the HELQ gene may not be responsible for premature ovarian failure in Chinese Han population.

BACKGROUND Primary ovarian insufficiency (POI) is characterized by marked heterogeneity, but with a significant genetic contribution. Identifying exact causative genes has been challenging, with many discoveries not replicated. It is timely to take stock of the field, outlining the progress made, framing the controversies and anticipating future directions in elucidating the genetics of POI. METHODS A search for original articles published up to May 2015 was performed using PubMed and Google Scholar, identifying studies on the genetic etiology of POI. Studies were included if chromosomal analysis, candidate gene screening and a genome-wide study were conducted. Articles identified were restricted to English language full-text papers. RESULTS Chromosomal abnormalities have long been recognized as a frequent cause of POI, with a currently estimated prevalence of 10–13%. Using the traditional karyotype methodology, monosomy X, mosaicism, X chromosome deletions and rearrangements, X-autosome translocations, and isochromosomes have been detected. Based on candidate gene studies, single gene perturbations unequivocally having a deleterious effect in at least one population include Bone morphogenetic protein 15 (BMP15), Progesterone receptor membrane component 1 (PGRMC1), and Fragile X mental retardation 1 (FMR1) premutation on the X chromosome; Growth differentiation factor 9 (GDF9), Folliculogenesis specific bHLH transcription factor (FIGLA), Newborn ovary homeobox gene (NOBOX), Nuclear receptor subfamily 5, group A, member 1 (NR5A1) and Nanos homolog 3 (NANOS3) seem likely as well, but mostly being found in no more than 1–2% of a single population studied. Whole genome approaches have utilized genome-wide association studies (GWAS) to reveal loci not predicted on the basis of a candidate gene, but it remains difficult to locate causative genes and susceptible loci were not always replicated. Cytogenomic methods (array CGH) have identified other regions of interest but studies have not shown consistent results, the resolution of arrays has varied and replication is uncommon. Whole-exome sequencing in non-syndromic POI kindreds has only recently begun, revealing mutations in the Stromal antigen 3 (STAG3), Synaptonemal complex central element 1 (SYCE1), minichromosome maintenance complex component 8 and 9 (MCM8, MCM9) and ATP-dependent DNA helicase homolog (HFM1) genes. Given the slow progress in candidate-gene analysis and relatively small sample sizes available for GWAS, family-based whole exome and whole genome sequencing appear to be the most promising approaches for detecting potential genes responsible for POI. CONCLUSION Taken together, the cytogenetic, cytogenomic (array CGH) and exome sequencing approaches have revealed a genetic causation in ∼20–25% of POI cases. Uncovering the remainder of the causative genes will be facilitated not only by whole genome approaches involving larger cohorts in multiple populations but also incorporating environmental exposures and exploring signaling pathways in intragenic and intergenic regions that point to perturbations in regulatory genes and networks.

In this retrospective study, the relationship between demographic characteristics, past medical history, general lifestyle habits and susceptibility of premature ovarian failure (POF) in Han Chinese population was investigated. Five hundred and fifty-three patients with POF and 400 women with normal ovarian function were recruited. A questionnaire was designed to gather information from responders. Logistic regression was carried out to calculate odds ratios (OR), 95% confidence intervals (95% CI) and P-values. History of pelvic surgery, mumps, having relatives with menstrual abnormalities and exposure to chemical agents were significantly associated with increased risk of POF (OR 5.53 [2.15 to 14.23]; 3.26 [2.38 to 4.47]; 28.12 [8.84 to 89.46]; 4.47 [2.09 to 9.58]). Vegetarian diet, tea and mineral water consumption reduced the risk of POF (OR 0.27 [0.19 to 0.37]; 0.04 [0.03 to 0.07]; 0.63 [0.47 to 0.85], respectively). Heredity, pelvic surgery, mumps and exposure to chemical agents were identified as risk factors for POF, whereas vegetarian diet, tea consumption and mineral water drinking were protective. Therefore, genetic consultation could help those women whose relatives manifested an early or premature menopause to avoid the consequences of possible premature ovarian function cessation. Avoidance of exposure to endocrine disrupters and flavonoids intake should be considered.

The gene PGRMC1 is highly expressed in the granulose and luteal cells of rodent and primate ovaries. Its role in anti-apoptosis and regulating cell-cycle progression suggests a role in regulating follicle growth. The hypothesis is supported by the study in mice and studies in Sweden. In this study, the coding exons of PGRMC1 were sequenced among 196 Chinese women with premature ovarian failure (POF) and 200 controls, and one novel missense mutation was identified (C.556C>T, p. Pro186Ser) in the POF group and one novel SNP (C.533C>T, p. Trh177Ile) was identified in both groups. The mutation is not considered causative because protein prediction did not indicate a deleterious effect. It is concluded that coding mutations of PGRMC1 do not seem to be a common cause of the disease in Han Chinese women. Future studies in larger cohorts from other ethnic groups are necessary to establish the role of PGRMC1 in POF.

Objective To identify the potential FSHR mutation in a Chinese woman with primary ovarian insufficiency (POI). Design Genetic and functional studies. Setting University-based reproductive medicine center. Patient(s) A POI patient, her family members, and another 192 control women with regular menstruation. Intervention(s) Ovarian biopsy was performed in the patient. Sanger sequencing was carried out for the patient, her sister, and parents. The novel variant identified was further confirmed with the use of control subjects. Main Outcome Measure(s) Sanger sequencing and genotype analysis to identify the potential variant of the FSHR gene; hematoxylin and eosin staining of the ovarian section to observe the follicular development; Western blotting and immunofluorescence to detect FSH receptor (FSHR) expression; and cyclic adenosine monophosphate (cAMP) assay to monitor FSH-induced signaling. Result(s) Histologic examination of the ovaries in the patient revealed follicular development up to the early antral stage. Mutational screening and genotype analysis of the FSHR gene identified a novel homozygous mutation c.175C>T (p.R59X) in exon 2, which was inherited in the autosomal recessive mode from her heterozygous parents but was absent in her sister and the 192 control women. Functional studies demonstrated that in vitro the nonsense mutation caused the loss of full-length FSHR expression and that p.R59X mutant showed no response to FSH stimulation in the cAMP level. Conclusion(s) The mutation p.R59X in FSHR is causative for POI by means of arresting folliculogenesis.

Objective To identify whether variants found in a large Han Chinese cohort – 8q22.3 SNPs rs3847153 and rs3108910; and one SNP each in HK3 (rs2278493), ESR1 (rs2234693) and BRSK1 (rs12611091) – are associated with premature ovarian failure (POF) in a different ethnic group (Serbian). Design Case–control genetic association study in 197 Serbian POF cases and 552 matched controls. Results None of the SNPs found associated with POF in Chinese cohort were found to be associated in the Serbian sample. Conclusions In contrast to Han Chinese, no association was found between POF in Serbian women and any of the four tested loci: 8q22.3, HK3, ESR1 and BRSK1. This indicates that ethnically distinct populations may show differences in gene-regulating pathways and genes causing POF.

Autoimmune pathogenesis is responsible for a subset of primary ovarian insufficiency (POI) cases. The significance of autoantibodies for POI, however, remains unclear. A total of 250 women with idiopathic POI and 256 age-matched healthy women were enrolled. The presence in serum of adrenal cortex autoantibody (AAA), detected by indirect immunofluorescence and non-organ-specific antibodies, including antinuclear antibody, anti-cardiolipin antibody, and anti-double stranded DNA antibody, detected by enzyme-linked immunosorbent assay, was compared. Ovarian biopsy was carried out for histology assessment. Adrenal function was followed-up in 15 women with POI who were positive for AAA. Higher frequency of positive AAA was observed in women with POI (19.2%) compared with controls (5.9%, P < 0.01). No difference in anti-cardiolipin antibody, antinuclear antibody and anti-double stranded DNA antibody was found between the two groups. Ovarian biopsies in 13 women with POI (six AAA positive and seven negative) showed atrophic ovaries devoid of follicles. One out of fifteen women positive for AAA had symptoms of adrenal insufficiency 3 years after POI diagnosis. Significantly higher positive frequency of AAA in POI patients suggests the role of autoimmune disturbance in pathogenesis. Therefore, AAA may serve as a biomarker for ovarian autoimmunity.

Insights into common genetic susceptibility between primary ovarian insufficiency (POI) and natural or early menopause have delivered an innovative way of assessing the genetic mechanisms involved in POI. PRIM1 plays a crucial role in DNA replication by synthesizing RNA primers for Okazaki fragments. It is closely associated with age at natural menopause, early menopause and POI in European women. In this study, we aimed to investigate whether mutations in PRIM1 contribute to POI in Chinese women. All exons and exon-intron boundaries of PRIM1 gene were sequenced in 192 Han Chinese women with non-syndromic POI. No plausible mutations were identified. The results suggest that the perturbations in PRIM1 gene are not a common explanation for POI in Chinese women.

Objective To explore the potential damaging effect of chronic pelvic inflammation on ovarian reserve. Design Case-control study. Patients A total of 122 women with bilateral tubal occlusion, diagnosed by hysterosalipingography (HSG) and 217 women with normal fallopians were recruited. Measurements Serum anti-Mullerian hormone (AMH), basic follicle-stimulating hormone (FSH), luteining hormone (LH), estradiol (E2), and testosterone (T) were measured; and antral follicle counts (AFCs) were recorded. Results Significantly lower level of AMH was observed in women with bilateral tubal occlusion compared to control group [2.62 (2.95) ng/ml vs. 3.37 (3.11) ng/ml, P = 0.03], and the difference remained after adjustment of BMI (Padjust = 0.04). However, no statistical difference was found in the levels of FSH [7.00 (2.16) IU/L vs. 6.74 (2.30) IU/L], LH [4.18 (1.52) IU/L vs. 4.63 (2.52) IU/L], E2 [35.95 (20.40) pg/ml vs. 34.90 (17.85) pg/ml], T [25.07±11.46 ng/dl vs. 24.84±12.75 ng/dl], and AFC [6.00 (4.00) vs. 7.00 (4.00)] between two groups (p>0.05). Conclusions Women with bilateral tubal occlusion showed decreased AMH level, suggesting that chronic pelvic inflammation may diminish ovarian reserve. More caution should be paid when evaluating the detriment of PID on female fertility.

STUDY QUESTION Are telomere length and telomerase activity associated with biochemical primary ovarian insufficiency (POI)? SUMMARY ANSWER Shortened telomere length and diminished telomerase activity were associated with biochemical POI. WHAT IS KNOWN ALREADY POI is a result of pathological reproductive aging and encompasses occult, biochemical and overt stages. Studies have indicated telomere length as a biomarker for biological aging. STUDY DESIGN, SIZE, DURATION A total of 120 patients with biochemical POI and 279 control women were recruited by the Center for Reproductive Medicine of Shandong University. PARTICIPANTS/MATERIALS, SETTING, METHODS Telomere length in peripheral blood leukocytes (LTL) and granulosa cells (GTL) was measured using a modified Quantitative Polymerase Chain Reaction technique. The relative telomerase activity (RTA) in granulosa cells was detected using a modified quantitative-telomeric repeat amplification protocol assay. MAIN RESULTS AND THE ROLE OF CHANCE After adjusting for age, patients with biochemical POI (n = 120) exhibited significantly shorter LTLs (0.75 ± 0.09 vs 1.79 ± 0.12, P < 0.001; OR = 0.54, 95% CI = 0.43-0.68) and GTLs (0.78 ± 0.09 vs 1.90 ± 0.23, P < 0.001; OR = 0.54, 95% CI = 0.41-0.70) than the controls (n = 279 for LTLs; n = 90 for GTLs). Significantly diminished RTAs in granulosa cells were detected in patients with biochemical POI (n = 31) compared with the controls (n = 38) (1.57 ± 0.59 vs 4.63 ± 0.93, P = 0.025; OR = 0.84, 95% CI = 0.72-0.98). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The cross-sectional nature of this study might have its limit in telomere length as well as telomerase activity along with the progressing decline in ovarian function. WIDER IMPLICATIONS OF THE FINDINGS These findings suggest that telomere length and telomerase activity may be considered as indicators for progression of ovarian decline. STUDY FUNDING/COMPETING INTERESTS This research was supported by the National Basic Research Program of China (973 Program) (2012CB944700), Science research foundation item of no-earnings health vocation (201402004) and the National Natural Science Foundation of China (31471352, 81270662, 81471509, 81300461, 81522018). The authors have no potential conflict of interest to declare.

Exonuclease 1 (EXO1) is required for both DNA repair and meiosis. Inactivation of EXO1 gene in mice leads to infertility. This study aimed to investigate whether variants in the EXO1 gene contribute to human premature ovarian failure (POF). The coding region of EXO1 was sequenced in 186 Han Chinese patients with non-syndromic POF. No plausible mutation was detected. The results suggest that mutations in the coding region of EXO1 may not be responsible for POF in Han Chinese women.

Premature ovarian failure (POF) is a heterogeneous disease. Though dozens of candidate genes have been identified for the genetic etiology of POF, it is largely unexplained in majority of patients. Recently, Wt1+/R394W mice was found to present POF-like phenotype, which indicates that WT1 might be a plausible candidate gene for non-syndromic POF. The coding region of WT1 gene was screened in 384 patients with POF and 6 novel variations were identified, including two missense mutations (p. Pro126Ser in exon1 and p. Arg370His in exon7) and four intronic variants (c.647-27C > T, c.647-13G > C, c.647-13G > A in intron1 and c.950 + 14T > C in intron 4). In vitro experiments showed that both mutant p. Pro126Ser and p. Arg370His repressed the expression of Amh and Cdh1 and induced the expression of Fshr and Cyp19 in mRNA level (P AMH, FSHR, CYP19 and CDH1) are required for granular cells (GCs) proliferation, differentiation and oocyte-GCs interaction. The novel mutant p. P126S and p. R370H in the WT1 gene potentially impaired GCs differentiation and oocyte-GCs interaction, which might result in loss of follicles prematurely. Therefore, WT1 is a plausible causal gene for POF.

Objective To determine whether plasma microRNAs (miRNAs) are differentially expressed between women with and without premature ovarian failure (POF), and to uncover the association of miRNAs with risk of POF. Design Microarray with real-time polymerase chain reaction validation. Setting University hospital. Patient(s) A total of 140 individuals with premature ovarian failure (POF) and 140 age- and body mass index–matched control subjects of Han Chinese ancestry. Intervention(s) None. Main Outcome Measure(s) Relative miRNA expression levels in plasma of POF and control group. Result(s) Fifty-one differentially expressed miRNAs were identified by chip-based discovery stage between ten patients with POF and ten control subjects, among which nine miRNAs (let-7b-5p, let-7c, miR-15b-5p, miR-22-3p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-151a-5p, and miR-151b) were selected and validated. The relative expression level of miR-22-3p was significantly down-regulated in POF compared with control subjects. MiR-22-3p yielded a receiver operating characteristic (ROC) curve area of 0.668 (95% confidence interval 0.602–0.733) in discriminating POF from controls. In addition, logistic binary regression analysis and linear regression analysis showed the miR-22-3p to be a protective factor for POF (odds ratio 0.766, 95% CI 0.643–0.912) and negatively associated with serum FSH. Furthermore, bioinformatics analysis indicated that the target function of miR-22-3p was involved in apoptosis, endocytosis, and tumorigenesis. Conclusion(s) Mir-22-3p showed a lower expression level in POF and was modestly effective in distinguishing POF from control subjects. The decreased expression of miR-22-3p in plasma of POF may reflect the diminished ovarian reserve and be a consequence of the pathologic process of POF.

BACKGROUND In premature ovarian failure (POF), cessation of menstruation occurs before the expected age of menopause. Approximately 1% of women are affected. FMR1 premutation was reported to be responsible for up to 3.3%-6.7% of sporadic POF and 13% of familial cases in Caucasians, while the data was absent in Chinese population. Therefore, the impact of FMR1 CGG repeat on ovarian reserve is needed to be investigated in large Chinese cohort. METHODS The number of FMR1 CGG repeat was determined in 379 Han Chinese women with well-defined 46, XX non-syndromic sporadic POF and 402 controls. The age of menopause onset in respect to CGG repeats was further analyzed. RESULTS The frequency of FMR1 premutation in Han Chinese POF was only 0.5% (2/379), although it was higher than that in matched controls (0%, 0/402), it was much lower than that reported in Caucasian with POF (3.3%-6.7%). The prevalence of intermediate FMR1 (41-54) was not increased significantly in sporadic POF than that in controls (2.9% vs. 1.7%, P = 0.343). However, POF patients more often carried a single additional CGG repeat in a single allele than did fertile women (allele-1: 29.7 vs. 28.8, P

Objective To investigate whether mutations in the minichromosome maintenance complex component 8 (MCM8) gene were present in 192 patients with sporadic primary ovarian insufficiency (POI). Design Retrospective case-control cohort study. Setting University-based reproductive medicine center. Patient(s) A total of 192 patients with sporadic POI and 312 control women with regular menstruation (192 age-matched women and 120 women >45 years old). Intervention(s) Sanger sequencing was performed in patients with sporadic POI, and potentially pathogenic variants were confirmed in matched controls. DNA damage was induced by mitomycinC (MMC) treatment, and DNA repair capacity was evaluated by histone H2AX phosphorylation level. Main Outcome Measure(s) Sanger sequencing for MCM8 was performed in 192 patients with sporadic POI, and functional experiments were performed to explore the deleterious effects of mutations identified. Result(s) Two novel missense variants in MCM8 , c. A950T (p. H317L), and c. A1802G (p. H601R), were identified in two patients with POI but absent in 312 controls (the upper 90% confidence limit for the proportion 2/192 is 2.24%). The HeLa cells overexpressing mutant p. H317L and p. H601R showed higher sensitivity to MMC compared with wild type. Furthermore, mutant p. H317L showed decreased repair capacity after MMC treatment with much more histone H2AX phosphorylation remaining after 2 hours of recovery. Conclusion(s) Our result suggests novel mutations p. H317L and p. H601R in the MCM8 gene are potentially causative for POI by dysfunctional DNA repair.

We screened growth differentiation factor 9 coding regions for mutations in a Chinese sample of 100 women with premature ovarian failure and discovered four novel single-nucleotide polymorphisms: c.436C>T (p.Arg146Cys), c.588A>C (silent), c.712A>G (p.Thr238Ala), and c.1283G>C (p.Ser428Thr). Nonsynonymous single-nucleotide polymorphisms c.436C>T and c.1283G>C were also detected in the control population. The c.712A>G perturbation results in a missense mutation (p.Thr238Ala) and was not present in any of 96 controls. Substitution of the hydrophobic amino acid residue alanine for hydrophilic threonine may disrupt growth differentiation factor 9 function.

Many genetic association studies have been performed to investigate disorders of female reproduction, such as polycystic ovary syndrome, premature ovarian failure and endometriosis. These disorders typically manifest heterogeneously, and their pathogeneses are influenced by polygenic and environmental factors. Researchers evaluating these genetic associations have chosen candidate genes related to hormone action, steroid biosynthesis, inflammatory cytokines and autoimmune factors. Several of these genes have yielded statistically significant associations with female reproductive disorders; however, few associations have been robust and reproducible. Whole-genome association studies generate more reliable and unbiased results and represent a breakthrough in genetic studies of female reproduction. Nevertheless, to date only a very small fraction of the overall heritability has been identified and so further studies are needed.

BACKGROUND: The etiology of most premature ovarian failure (POF) cases is usually elusive. Although genetic causes clearly exist and a likely susceptible region of 8q22.3 has been discovered, no predominant explanation exists for POF. More recently, evidences have indicated that mutations in NR5A1 gene could be causative for POF. We therefore screened for mutations in the NR5A1 gene in a large cohort of Chinese women with non-syndromic POF. METHODS: Mutation screening of NR5A1 gene was performed in 400 Han Chinese women with well-defined 46,XX idiopathic non-syndromic POF and 400 controls. Subsequently, functional characterization of the novel mutation identified was evaluated in vitro. RESULTS: A novel heterozygous missense mutation [c.13T>G (p.Tyr5Asp)] in NR5A1 was identified in 1 of 384 patients (0.26%). This mutation impaired transcriptional activation on Amh, Inhibin-a, Cyp11a1 and Cyp19a1 gene, as shown by transactivation assays. However, no dominant negative effect was observed, nor was there impact on protein expression and nuclear localization. CONCLUSIONS: This novel mutation p.Tyr5Asp, in a novel non-domain region, is presumed to result in haploinsufficiency. Irrespectively, perturbation in NR5A1 is not a common explanation for POF in Chinese.

P27 and SKP2, a major regulator of P27, play a crucial role in ovarian function in mice. Both P27-deficient and SKP2-deficient female mice develop premature ovarian failure (POF). The coding regions of SKP2 and P27 were examined in 200 Chinese women with POF and 200 control volunteers. This study is the first to investigate SKP2 in POF. No plausible pathogenic mutations were detected. The results suggest that mutations in SKP2 and P27 are not common in Chinese Han women with POF. P27 and Skp2, a major regulator of P27, play a crucial role in ovarian function in mice. Both P27-deficient and Skp2-deficient female mice develop premature ovarian failure (POF). The coding region of SKP2 and P27 were examined in 200 Chinese women with POF and 200 control volunteers. One known single-nucleotide polymorphism (SNP), rs6175530 in exon 7 of SKP2 , and one known SNP, rs1690837 in exon 1 of P27 , were identified. The present study is the first to discover variants occurring in SKP2 in association with POF. The results suggest that mutations in SKP2 and P27 are not common in Chinese Han women with POF.

The coding region of the disrupted meiotic cDNA gene ( DMC1 ) was examined in 192 Chinese women with premature ovarian failure. Two known single-nucleotide polymorphisms, c.8632C>T in intron 4 and c.32377G>C in intron 10, were identified. The results suggest that mutations in the coding sequence of DMC1 are not associated with premature ovarian failure in Chinese women. The coding region of the disrupted meiotic cDNA gene ( DMC1 ) was examined in 192 Chinese women with premature ovarian failure. Premature ovarian failure is defined as secondary amenorrhoea, infertility, oestrogen deficiency, and elevated gonadotrophin concentration in women younger than 40 years. DMC1 is a meiosis-specific gene, encoding a protein essential for meiotic homologous recombination. It participates in the formation of synaptonemal complexes and repair of double-strand breaks at recombination hotspots. Two known single-nucleotide polymorphisms, c.8632C>T in intron 4 and c.32377G>C in intron 10, were identified. No additional single-nucleotide polymorphisms or mutations were found. The results suggested mutations in the coding sequence of DMC1 are not associated with premature ovarian failure in Chinese women.

Background This retrospective cohort study was to determine the frequency and types of chromosomal abnormalities in Han Chinese women with well-documented premature ovarian failure (POF). Methods Karyotype analysis and correlation to phenotypes were performed on 531 Chinese patients with proven POF (FSH > 40 mIU/ml) attending four reproductive centers in China. G-banded metaphase chromosomes were prepared and analyzed, with mosaicism excluded by counting up to 100 cells from lymphocytes. Results Chromosomal abnormalities were present in 64 of 531 (12.1%) POF cases, of which 32 were X-structural aberrations (7 mosaic): 15 del(Xq), 2 del(Xp), 11 isochromosomes [6 i(Xp); 5 i(Xq)], 1 ring chromosome (mosaic), 1 inversion (mosaic), 1 isodicentric chromosome and 1 complex arrangement. Nine non-mosaic X-autosome translocations were detected, all but 1 involving Xq. Aneuploidy without a structurally abnormal X was found in 19 cases: 7 non-mosaic 45,X, 9 45,X mosaicisms and 3 47,XXX (1 mosaic with 46,XX line). Karyotypic abnormalities were more frequent in patients with primary amenorrhea (15/70, 21.4%) than those with secondary amenorrhea (49/461, 10.6%; P = 0.01). 45,X and 45,X/46,XX mosaicism were the complements most frequently associated with primary amenorrhea (46.7%). Two of the three cases with 46,XY or 45,X/46,XY karyotype presented with 'secondary amenorrhea'. One balanced autosomal Robertsonian translocation was also detected. Conclusions The overall prevalence of chromosomal abnormalities was 12.1% in this first large scale report of chromosomal aberrations in Chinese women with POF. In one of the largest samples of women with POF reported from any population, the prevalence of X-structural abnormalities, X-autosome translocations and X aneuploidy confirms the essential role X chromosomal abnormalities play in POF.

Premature ovarian failure (POF) is a complex heritable disorder known to be caused by chromosomal abnormalities and to date a limited number of known mutations, often autosomal. We sought to identify additional genetic loci associated with POF by performing the first large-scale genome-wide association study (GWAS). GWAS, using Affymetrix SNP 6.0 chip, was conducted in an initial discovery set of 391 well-documented (follicle-stimulating hormone >40 IU/ml) Chinese Han POF patients, compared with 895 unrelated Chinese female controls. A replication study on the most significant loci was then performed in an independent set of 400 cases and 800 controls. Suggestive significant associations were observed at 8q22.3. Replication of eight single-nucleotide polymorphisms (SNPs) (rs10464815, rs10808365, rs3847152, rs3847153, rs3847154, rs3843552, rs10955242, rs3843555) (P ≤ 3.86 × 10(-6)) was confirmed in verification sets. No specific candidate gene was found in the immediate region of 8q22.3. This GWAS, involving by far the largest sample of POF cases accumulated to date, revealed heretofore unrecognized association between POF and a novel genetic locus or region of unknown nature on 8q22.3. We speculate existence of a long-distance regulatory region that has relevance to the control of ovarian differentiation or oogenesis. Given failure to find association with any of the other autosomal regions known to harbor genes causing ovarian failure, our findings also underscore the likelihood of considerable genetic and etiologic heterogeneity in POF and the need for additional approaches like whole-genome sequencing.

The newborn ovary homeobox gene (NOBOX) is an oocyte-specific homeobox gene that plays a critical role in early folliculogenesis, thus representing an attractive candidate gene for nonsyndromic ovarian failure. We investigated whether perturbation in the homeodomain region of NOBOX was present in Chinese women with premature ovarian failure (POF). We sequenced 200 Chinese patients with POF, and discovered only two known single nucleotide polymorphisms: in intron 6 (c.1154+11 T>C and c.1155-22 G>A); neither offers plausible explanations for POF. Failing to find causative mutation contrasts with our previous study in a caucasian sample, in which we found plausible homeobox mutation in 1 of 96 POF cases. Mutations in the homeobox domain of NOBOX are not common explanations for POF in Chinese women.

Premature Ovarian Failure (POF) is a genetically heterogenous disorder that leads to hypergonadotropic ovarian failure and infertility. We screened 100 Chinese women with POF for mutations in the oocyte-specific gene FIGLA and identified three variants in four women: missense mutation c.11C → A (p.A4E) was found in two women; deletion c. 15–36 del (p.G6fsX66), resulting in a frameshift that leads to haploinsufficiency, was found in one woman; and deletion c.419–421 delACA (p.140 delN) was found in one. Functional analyses by the yeast two-hybrid assay demonstrated that the p.140 delN mutation disrupted FIGLA binding to the TCF3 helix-loop-helix (HLH) domain. Our findings show that a subset of Chinese women with sporadic, premature ovarian failure harbor mutations in FIGLA.

Infertility is one of important issues in patients with premature ovarian failure (POF) during their reproductive life. This study would analyze the features of infertility women with POF.138 Chinese infertility women with POF were accrued from the women who visited the Reproductive Medical Center in Shandong Provincal Hospital of Shandong University between May 2003 and November 2006.This study analyzed the clinical, etiology features and karyotype of 138 patients, then evaluates the effect of the therapy.It showed that most patients' physical situation were suitable for assisted reproductive therapy. The infertility women with POF had high rate of abnormal karyotype (11/65, 16.92%) and lower severe autoimmune disorders rate (2/138,1.45%). Some of them demonstrated family aggregation. FSH was dropped apparently after estrogen-progesterone therapy (p0.05). The spontaneous pregnancy incidence of the women was 2.17% (3/138).The infertility women with POF were suitable for assisted reproductive therapy and donor oocyte could be an alternate way. Their high chromosomal abnormality rate showed the importance of genetic screening. And estrogen-progesterone therapy was beneficial for improving their endocrine environment.

The bone morphogenetic protein-15 (BMP-15) gene is thought to be one of the important candidate genes involved in premature ovarian failure (POF). However, to date, there has been no consensus on the relationship between mutations in BMP-15 and ovarian dysfunction.To analyse mutations in the BMP-15 gene in Chinese women with POF.We sequenced the BMP-15 gene protein coding region of 92 patients and 76 healthy controls that cycle regularly.No mutations were found in the BMP-15 gene protein coding region. There was no difference in the incidence of single nucleotide polymorphism (SNP) rs 17003221 (CT) in exon 2 (p0.05), or SNP rs (3810682CG: ss16336587) in the putative promoter region of exon 1, between the two groups. However, the allele gene frequency in SNP rs (3810682CG: ss16336587) was C (97.92%) and G (2.08%), respectively, in Chinese women, which is different from other races.Our findings indicate that mutations in BMP-15 exons, or changes in BMP-15 pro-peptide, are rare in Chinese women with POF. In addition, our data suggest that the 2 SNPs are not related to POF in Chinese women, while providing evidence for SNP variation between different races.

The LHX8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian oogenesis. The authors investigated whether nucleotide changes were present in the LHX8 gene of Caucasian women with premature ovarian failure (POF), as compared with control women. When the authors sequenced 95 Caucasian women with POF, they discovered two novel single-nucleotide polymorphisms (SNPs) in intron 3 (c.769+10G>T) and 3′ untranslated region (c.1787A>G) of the LHX8 gene. These polymorphisms also were found in controls (n = 94), at frequencies that were not statistically different from those in POF women. Mutations in the LHX8 exons are uncommon in Caucasian women with POF.

Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurring before the age of 40 years. Genetic etiology is responsible for perhaps 25% of cases, but most cases are sporadic and unexplained. In this study, through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in the fusion gene CSB-PGBD3. Subsequently functional studies suggest that mutated CSB-PGBD3 fusion protein was impaired in response to DNA damage, as indicated by delayed or absent recruitment to damaged sites. Our data provide the first evidence that mutations in the CSB-PGBD3 fusion protein can cause human disease, even in the presence of functional CSB, thus potentially explaining conservation of the fusion protein for 43 My since marmoset. The localization of the CSB-PGBD3 fusion protein to UVA-induced nuclear DNA repair foci further suggests that the CSB-PGBD3 fusion protein, like many other proteins that can cause POF, modulates or participates in DNA repair., Author Summary Through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in CSB-PGBD3. Our functional studies implicate CSB-PGBD3, a gene which has previously shown association with DNA repair and Cockayne syndrome has a potential role in maintenance of ovarian function. This study also provides evidence for a pivotal role of DNA repair in POF.

Objective To investigate whether gene variants of SOHLH1 exist in Chinese and Serbian patients with primary ovarian insufficiency (POI). Design Case-control genetic study. Setting University hospitals. Patient(s) A total of 364 Han Chinese and 197 Serbian women with nonsyndromic POI and ethnically matched controls. Intervention(s) None. Main Outcome Measure(s) SOHLH1 gene sequencing. Result(s) We found 10 novel heterozygous variants in our cohorts of 561 women with POI but none in the 600 ethnically matched controls. Statistical and bioinformatic analyses indicated that three of the eight variants in Chinese POI cases are potentially disease causing. They comprise two missense variants (p.Ser317Phe and p.Glu376Lys) that might each change activity of the SOHLH1 protein as a transcription factor and one variant (c.*118C>T) located in the 3′ untranslated region of the SOHLH1 gene, which might generate a new binding site for the microRNA hsa-miR-888-5p. Of the two variants in the Serbian POI cases, both were synonymous, and no missense variant was identified. The allele frequencies of some known single-nucleotide polymorphisms were statistically significantly different between patients and controls in both the Chinese and Serbian groups. Conclusion(s) Our results suggest that SOHLH1 may be regarded as a new candidate gene for POI.

NANOS3 encodes an RNA-binding protein and has a conserved function in germ cell development. Our objective was to investigate whether mutations in NANOS3 were present in Chinese and Caucasian women with premature ovarian failure. A known synonymous single-nucleotide polymorphism (rs 2016163) in exon 1 was identified through sequencing 80 Chinese and 88 Caucasian women with premature ovarian failure. No additional single-nucleotide polymorphisms or mutations were found in exons encoding for NANOS3. Our findings suggest that mutations in NANOS3 exons are rare in both Chinese and Caucasian women with premature ovarian failure.

Objective To determine whether variants in the SOHLH2 gene contribute to human premature ovarian failure (POF) in different ethnicities. Design Case-control genetic study. Setting University hospitals. Patient(s) Chinese (364 cases) and Serbian (197 cases) women with nonsyndromic POF and ethnically matched controls. Intervention(s) None. Main Outcome Measure(s) Variation analysis of the SOHLH2 gene. Result(s) Eleven novel heterozygous variants were identified in cohorts of POF but were absent in matched controls. These included the nonsynonymous variants p.Glu79Lys (n = 2 cases), p.Glu105Gly, and p.Thr321Pro, which were found among four Chinese POF cases, and p.Leu120Phe (n = 3 cases) and p.Leu204Phe, which were found among four Serbian women. Protein alignments reveal that p.Glu79Lys and p.Glu105Gly involve amino acids highly conserved among mammals, both of which are predicted to be deleterious. The c.-210G>T found in the Chinese POF cohort lies in the core promoter region, which is enriched with transcription factor binding sites and CpG islands. In the Serbian cohort, the variant most likely to have a deleterious effect is c.530+6T>G, which is predicted to affect RNA splicing and result in nonsense mediated decay of transcripts. The other variants are less likely to be deleterious. Disturbing the expression, transactivation or homo-/ heterodimerization of the SOHLH2 protein could result in ovarian failure. Overall, four of the 11 novel variants seem plausible explanations for POF; the other seven variants are less likely but cannot be categorically excluded. Conclusion(s) Our identification of novel variants in the SOHLH2 gene, in women with POF of both Chinese and Serbian origin, strongly suggests an important role for SOHLH2 in human POF etiology.