Idiopathic Primary Ovarian Insufficiency Research Articles

Does the Serum Vaspin Level Have a Role in the Diagnosis of Primary Ovarian Insufficiency?

OBJECTIVE: This study was designed to compare the serum vaspin levels between patients with primary ovarian insufficiency (POI) and healthy fertile women. STUDY DESIGN: Sixty-nine patients diagnosed with idiopathic POI and 70 age-matched healthy fertile women were included in this case-control study. General gynecological and physical examination findings and serum basal hormone levels were recorded. Vaspin levels in serum samples were determined using the Enyzme-Linked ImmunoSorbent Assay (ELISA) method. RESULTS: The mean age of the patients with POI was 31.9±5.4 years, while it was 31.2±3.8 years in the control group. There were no statistically significant differences in body mass index, gravidity, parity, and cigarette consumption between the groups. However, the mean follicle-stimulating hormone level in POI patients was 55.4±25.1 IU/L, significantly higher than the 6.2±1.8 IU/L observed in the control group. Luteinizing hormone levels were also significantly higher in POI patients, while estradiol and anti-Mullerian hormone values were significantly lower. Furthermore, the mean serum vaspin level in POI patients was 0.79±0.56 ng/mL, compared to 1.08±0.64 ng/mL in the control group (p<0.001). CONCLUSIONS: Serum vaspin levels were found to be decreased in women with POI. Therefore, monitoring the serum vaspin levels in women with POI may help in the early identification of patients with POI.

Selected Genetic Factors Associated with Primary Ovarian Insufficiency

Primary ovarian insufficiency (POI) is a heterogeneous disease resulting from non-functional ovaries in women before the age of 40. It is characterized by primary amenorrhea or secondary amenorrhea. As regards its etiology, although many POI cases are idiopathic, menopausal age is a heritable trait and genetic factors play an important role in all POI cases with known causes, accounting for approximately 20% to 25% of cases. This paper reviews the selected genetic causes implicated in POI and examines their pathogenic mechanisms to show the crucial role of genetic effects on POI. The genetic factors that can be found in POI cases include chromosomal abnormalities (e.g., X chromosomal aneuploidies, structural X chromosomal abnormalities, X-autosome translocations, and autosomal variations), single gene mutations (e.g., newborn ovary homeobox gene (NOBOX), folliculogenesis specific bHLH transcription factor (FIGLA), follicle-stimulating hormone receptor (FSHR), forkhead box L2 (FOXL2), bone morphogenetic protein 15 (BMP15), etc., as well as defects in mitochondrial functions and non-coding RNAs (small ncRNAs and long ncRNAs). These findings are beneficial for doctors to diagnose idiopathic POI cases and predict the risk of POI in women.

Prolidase as a marker of fibrogenesis in idiopathic primary ovarian insufficiency

ObjectivesTo evaluate the serumlevel of prolidase,which isa marker of fibrogenic activity, in women with idiopathic primary ovarian insufficiency (POI). Study DesignThis is a prospective case-control study. Serum prolidase level was compared between the study group including 68 women with POI and control group including 65 normally menstruating women. Serum proline and hydroxyproline levels were also compared. Correlation analyses were performed between the prolidase level and POI related parameters including estradiol (E), follicle stimulating hormone (FSH), anti-mullerian hormone (AMH) levels, and presence of POI family history. ResultsSerum prolidase and proline level were significantly increased in women with the diagnosis of POI compared to the control group (1082.57 (147.53) vs 981.13 (223.26) U/L, 233.30 (83.16) vs 218.94 (82.59) µmol/L, respectively). Prolidase level found to have significant correlations with AMH, E, FSH levels, and presence of POI family history (r = −0.49, p = 0.001; r = −0.39, p = 0.001; r = 0.42, p = 0.001; r = 0.22, p = 0.01; respectively). In receiver operating characteristics analysis, prolidase was shown to be a discriminative factor for POI at 1031.14 U/L cut-off value with 75 % sensitivity and 65 % specificity. Thearea under curve was 0.71 [(95 % CI: 0.62–0.79), p = 0.001]. ConclusionThe current study revealed increased prolidase level in women withPOI. Serum prolidase level was also negatively correlated with the serum AMH level. Considering the present findings,prolidase may be a candidate molecule in assessment of POI cases.

Approach to Evaluation and Comprehensive Care for Primary Ovarian Insufficiency

Idiopathic primary ovarian insufficiency (POI) is a reproductive endocrine disorder defined as the loss of ovarian function before age 40. The condition is characterized by infertility, estrogen deficiency, and premature menopause. POI has significant long-term physical and psychosocial sequelae. Because POI is underrecognized and challenging to diagnose, patients often experience a “diagnostic odyssey” that contributes to impaired health-related quality of life. This review provides a state-of-the-science overview of POI, approach to diagnosis and management, and interprofessional aspects of care. We highlight unanswered questions and future directions and underscore the nurse practitioner’s role in providing holistic care for women with POI.

Premature menopause and autoimmune primary ovarian insufficiency in two international multi-center cohorts.

ObjectiveTo investigate markers of premature menopause (<40 years) and specifically the prevalence of autoimmune primary ovarian insufficiency (POI) in European women.DesignPostmenopausal women were categorized according to age at menopause and self-reported reason for menopause in a cross-sectional analysis of 6870 women.MethodsVariables associated with the timing of menopause and hormone measurements of 17β-estradiol and follicle-stimulating hormone were explored using multivariable logistic regression analysis. Specific immunoprecipitating assays of steroidogenic autoantibodies against 21-hydroxylase (21-OH), side-chain cleavage enzyme (anti-SCC) and 17alpha-hydroxylase (17 OH), as well as NACHT leucine-rich-repeat protein 5 were used to identify women with likely autoimmune POI.ResultsPremature menopause was identified in 2.8% of women, and these women had higher frequencies of nulliparity (37.4% vs 19.7%), obesity (28.7% vs 21.4%), osteoporosis (17.1% vs 11.6%), hormone replacement therapy (59.1% vs 36.9%) and never smokers (60.1% vs 50.9%) (P < 0.05), compared to women with menopause ≥40 years. Iatrogenic causes were found in 91 (47%) and non-ovarian causes in 27 (14%) women, while 77 (39%) women were classified as POI of unknown cause, resulting in a 1.1% prevalence of idiopathic POI. After adjustments nulliparity was the only variable significantly associated with POI (odds ratio 2.46; 95% CI 1.63–3.42). Based on the presence of autoantibodies against 21 OH and SCC, 4.5% of POI cases were of likely autoimmune origin.ConclusionIdiopathic POI affects 1.1% of all women and almost half of the women with premature menopause. Autoimmunity explains 4.5% of these cases judged by positive steroidogenic autoantibodies.

FMR1 gene CGG repeat distribution among the three individual cohorts with intellectual disability, autism, and primary ovarian insufficiency from Tamil Nadu, Southern India.

Fragile X syndrome is the most common genetic cause of intellectual disability (ID) and is also well known to have a role in primary ovarian insufficiency (POI) and fragile X-associated tremor ataxia syndrome (FXTAS) that expresses across generations. The objective was to compare the CGG repeat variants in FMR1 gene among three correlating cohorts of ID, autism and idiopathic POI. Thirty-six patients with ID, 12 with autism spectrum disorder (ASD) and 13 females with idiopathic POI were screened for FMR1 CGG repeat size by fluorescent methylation-specific PCR and GeneScan analysis, irrespective of Hagerman checklist clinical scores. Among 29 males and seven females, 11 FMR1 allelic variants ranging from 21 to >200 CGG repeats were observed. Three (CF2-3, 39-5, 44-2) out of 29 males had full mutation alleles accounting for a 10.34% incidence of FXS among idiopathic ID males. One of them was a mosaic for CGG repeats with both premutation and full mutation alleles. The frequency of fragile X syndrome is high among patients with idiopathic ID; they also had a high score for the clinical check list. A cascade testing that begins with checklist evaluation prior to DNA analysis will be cost-effective for establishing early diagnosis in South India. With the huge disease burden, there is a need for the establishment of more molecular diagnostics and self-help groups for fragile X syndrome.

Idiopathic primary ovarian insufficiency is associated with anogenital distance, a marker for prenatal environment

ObjectiveIn a significant number of women diagnosed with primary ovarian insufficiency (POI), the underlying cause cannot be found. On the other hand, the ovarian reserve set prenatally has been shown to be affected by intrauterine nutrition, environmental and hormonal factors. We aimed to evaluate the relationship between anogenital distance (AGD), which is considered as a biomarker for prenatal hormonal environment, and idiopathic POI in adolescents and young women. Study designThis case control study was conducted between September 2018 and December 2019. The study group consisted of patients between the ages of 15–30, who developed POI following menarche without any identified genetic or iatrogenic cause. Controls were patients of the same age range who were having regular menstrual periods and were not diagnosed with polycystic ovary syndrome or endometriosis. Patients were excluded from the study if they were pregnant, had vaginal delivery or genital surgery. Distance between the anterior clitoral surface and the upper verge of the anus (AGDAC), and between the posterior fourchette and the upper verge of the anus (AGDAF) were measured in all subjects. ResultsIn total, 37 POI patients and 44 controls were included in the study. Two groups were similar for demographic parameters such as age and body mass index (BMI). Shorter AGDAC and AGDAF measurements were found to be associated with idiopathic POI. ConclusionOur results provide the first evidence of a strong association between shorter AGD measurements and the presence of idiopathic POI suggesting that prenatal environment may have role in the development of POI.

Association between the FMR1 CGG repeat lengths and the severity of idiopathic primary ovarian insufficiency: a meta analysis

Aim Reports on the association of the CGG repeat length in the FMR1 gene with the severity of idiopathic POI are inconclusive. Therefore, a meta analysis was performed to investigate the relationship between the expansion of repeat CGG and idiopathic POI risk. Methods Up to January 2019, 18 case-control or cohort studies involving 3022 idiopathic POI patients and 8461 controls were included for meta analysis. Results Thirteen studies, including 2047 cases and 6912 controls, met our criteria for the assessment of the premutation and intermediate repeat length in patients patients with POI. Compared with controls, FMR1 gene premutation is significantly associated with POI (OR = 8.13; 95% CI: 4.35–15.19; p < .00001), whereas there was no significant correlation between intermediate repeat length and POI (OR = 0.86; 95% CI: 0.62–1.18; p = .34). Six studies, representing 975 patients and 1749 controls, were eligible for evaluation of the premutation and intermediate repeat length in diminished ovarian reserve (DOR). The association between premutation and DOR was significant (OR=14.87; 95% CI:5.20-42.52; p < 0.00001), while no significant correlation of intermediate size to DOR was found in the case-control comparison (OR = 0.98; 95% CI: 0.65–1.47; p = 0.93). Conclusion There is a close association between premutation of the FMR1 gene and increased susceptibility to idiopathic POI of each stage and no correlation between intermediate repeat length of the FMR1 gene and the severity of idiopathic POI.

Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.

Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI. To investigate the genetic etiology of idiopathic POI. We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology. A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles. Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.

Primary Ovarian Insufficiency and Adolescent Vaccination.

Published case series have suggested a potential association between human papillomavirus (HPV) vaccination and primary ovarian insufficiency (POI). We describe POI incidence and estimate POI risk after HPV; tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis, adsorbed (Tdap); inactivated influenza (II); and meningococcal conjugate (MenACWY) vaccination. We searched Kaiser Permanente Northwest electronic health records for outpatient diagnoses suggestive of POI in female patients aged 11 to 34 years between 2006 and 2014. We reviewed and adjudicated the medical record to confirm diagnoses and estimate symptom onset dates. We excluded cases with known causes and calculated the incidence of idiopathic POI. We estimated risk by calculating hazard ratios and 95% confidence intervals (CIs). From a cohort of 199 078 female patients, we identified 120 with diagnoses suggestive of POI. After adjudication and exclusion of 26 POI cases with known causes, we confirmed 46 idiopathic POI cases. POI incidence was low in 11- to 14-year-olds (0.87 per 1 000 000 person-months) and increased with age. One confirmed case patient received the HPV vaccine 23 months before the first clinical evaluation for delayed menarche. The adjusted hazard ratio was 0.30 (95% CI: 0.07-1.36) after HPV, 0.88 (95% CI: 0.37-2.10) after Tdap, 1.42 (95% CI: 0.59-3.41) after II, and 0.94 (95% CI: 0.27-3.23) after MenACWY vaccination. We did not find a statistically significant elevated risk of POI after HPV, Tdap, II, or MenACWY vaccination in this population-based retrospective cohort study. These findings should lessen concern about POI risk after adolescent vaccination.

"Evaluation of four genes associated with primary ovarian insufficiency in a cohort of Mexican women".

Primary ovarian insufficiency (POI) is a clinical condition observed in women younger than 40years of age, characterized by amenorrhea, hypoestrogenism, high levels of follicle-stimulating hormone (FSH), and infertility. Mutations in some master regulators of the development, maturation, and maintenance of ovarian follicles such as BMP15, FSHR, FOXL2, and GDF9 have been suggested as etiological factors in the development of POI. The aim of this study, the first in the Mexican population, is to evaluate the presence of mutations or polymorphisms in these four candidate genes. In a sample of 20 Mexican patients with idiopathic POI, we looked for and analyzed genetic variants in BMP15, FSHR, FOXL2, and GDF9 genes. We observed two polymorphisms: a coding change, c.919G>A (p.Ala307Thr), in the FSHR gene and a synonymous variant, c.447C>T (p.Thr149Thr), in the GDF9 gene. These two variants have been reported previously as polymorphisms (rs6165 and rs254286, respectively). We observed no significant difference associated with POI in the patients when compared with a healthy control group (p > 0.05). Also, no exonic variants were found for the genes BMP15 and FOXL2 in the individuals tested. The lack of association of the evaluated genes in this sample of Mexican women is consistent with the complex genetic etiology of POI that is observed across cohorts studied thus far.

Chromosomal instability in women with primary ovarian insufficiency.

What is the prevalence of somatic chromosomal instability among women with idiopathic primary ovarian insufficiency (POI)? A subset of women with idiopathic POI may have functional impairment in DNA repair leading to chromosomal instability in their soma. The formation and repair of DNA double-strand breaks during meiotic recombination are fundamental processes of gametogenesis. Oocytes with compromised DNA integrity are susceptible to apoptosis which could trigger premature ovarian aging and accelerated wastage of the human follicle reserve. Genomewide association studies, as well as whole exome sequencing, have implicated multiple genes involved in DNA damage repair. However, the prevalence of defective DNA damage repair in the soma of women with POI is unknown. In total, 46 women with POI and 15 family members were evaluated for excessive mitomycin-C (MMC)-induced chromosome breakage. Healthy fertile females (n = 20) and two lymphoblastoid cell lines served as negative and as positive controls, respectively. We performed a pilot functional study utilizing MMC to assess chromosomal instability in the peripheral blood of participants. A high-resolution array comparative genomic hybridization (aCGH) was performed on 16 POI patients to identify copy number variations (CNVs) for a set of 341 targeted genes implicated in DNA repair. Array CGH revealed three POI patients (3/16, 18.8%) with pathogenic CNVs. Excessive chromosomal breakage suggestive of a constitutional deficiency in DNA repair was detected in one POI patient with the 16p12.3 duplication. In two patients with negative chromosome breakage analysis, aCGH detected a Xq28 deletion comprising the Centrin EF-hand Protein 2 (CETN2) and HAUS Augmin Like Complex Subunit 7 (HAUS7) genes essential for meiotic DNA repair, and a duplication in the 3p22.2 region comprising a part of the ATPase domain of the MutL Homolog 1 (MLH1) gene. Peripheral lymphocytes, used as a surrogate tissue to quantify induced chromosome damage, may not be representative of all the affected tissues. Another limitation pertains to the MMC assay which detects homologous repair pathway defects and does not test deficiencies in other DNA repair pathways. Our results provide evidence for functional impairment of DNA repair in idiopathic POI, which may predispose the patients to other DNA repair-related conditions such as accelerated aging and/or cancer susceptibility. Funding was provided by the National Institute of Child Health and Human Development. There were no competing interests to declare.

Sequence variants of KHDRBS1 as high penetrance susceptibility risks for primary ovarian insufficiency by mis-regulating mRNA alternative splicing.

Does a novel heterozygous KHDRBS1 variant, identified using whole-exome sequencing (WES) in two patients with primary ovarian insufficiency (POI) in a pedigree, cause defects in mRNA alternative splicing? The heterozygous variant of KHDRBS1 was confirmed to cause defects in alternative splicing of many genes involved in DNA replication and repair. Studies in mice revealed that Khdrbs1 deficient females are subfertile, which manifests as delayed sexual maturity and significantly reduced numbers of secondary and pre-antral follicles. No mutation of KHDRBS1, however, has been reported in patients with POI. This genetic and functional study used WES to find putative mutations in a POI pedigree. Altogether, 215 idiopathic POI patients and 400 healthy controls were screened for KHDRBS1 mutations. Two POI patients were subjected to WES to identify sequence variants. Mutational analysis of the KHDRBS1 gene in 215 idiopathic POI patients and 400 healthy controls were performed. RNA-sequencing was carried out to find the mis-regulation of gene expression due to KHDRBS1 mutation. Bioinformatics was used to analyze the change in alternative splicing events. We identified a heterozygous mutation (c.460A > G, p.M154V) in KHDRBS1 in two patients. Further mutational analysis of 215 idiopathic POI patients with the KHDRBS1 gene found one heterozygous mutation (c.263C > T, p.P88L). We failed to find these two mutations in 400 healthy control women. Using RNA-sequencing, we found that the KGN cells expressing the M154V KHDRBS1 mutant had different expression of 66 genes compared with wild-type (WT) cells. Furthermore, 145 genes were alternatively spliced in M154V cells, and these genes were enriched for DNA replication and repair function, revealing a potential underlying mechanism of the pathology that leads to POI. Although the in vitro assays demonstrated the effect of the KHDRBS1 variant on alternative splicing, further studies are needed to validate the in vivo effects on germ cell and follicle development. This finding provides researchers and clinicians a better understanding of the etiology and molecular mechanism of POI. This study was supported by the Ministry of Science and Technology of China (2012CB944704; 2012CB966702), National Research Institute for Family Planning (2017GJZ05), the National Natural Science Foundation of China (31171429) and Beijing Advanced Innovation Center for Structural Biology. The authors declare no conflict of interest.

The significance of polymorphism and expression of oestrogen metabolism-related genes in Chinese women with premature ovarian insufficiency

The aim of this study was to investigate whether polymorphism and expression of CYP17, CYP1A1, COMT and SULT1A1 affected the risk of idiopathic primary ovarian insufficiency (POI) in Chinese women. DNA sequencing and real-time PCR were used to detect these genes in 132 cases of idiopathic POI and 132 normal women. A significant increase in the C allele of CYP17 (rs743572) polymorphism was observed in women with POI compared with controls (PFDR = 0.046). A significant decrease was observed in the C allele of CYP1A1 (rs4646903) in women with POI compared with controls (PFDR = 0.004). The A allele of COMT (rs4680) polymorphism was more frequent in women with POI compared with controls (PFDR = 0.029). The genotypic frequency of SULT1A1 (rs9282861) was not significantly different between the two groups. For the relative expression of CYP17 and COMT were statistically significant (both PFDR = 0.066), with false discovery rate controlled at 0.1. No significant difference was observed in the RNA levels of CYP1A1 and SULT1A1 between the two groups. The frequency of expression of the CYP17 T/C variant tended to be higher and the A allele of COMT polymorphism together with down-regulation of its mRNA expression may be more frequent in Chinese women with idiopathic POI.

Identification of patients with primary ovarian insufficiency caused by autoimmunity

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.

Idiopathic Primary Ovarian Insufficiency in Adolescents

Idiopathic Primary Ovarian Insufficiency in Adolescents

TBP2 gene may not be associated with primary ovarian insufficiency

Objective: It has previously been reported that TATA-binding protein 2 (TBP2) knockout female mice are sterile. Our objective was to assess the association between the TBP2 gene and primary ovarian insufficiency (POI) in a Chinese population.Methods: A cohort of 60 POI patients matched with 60 fertile controls was recruited by Nanjing Medical University. There were no interventions. The complete TBP2 exon regions were analyzed by direct sequencing in all POI patients and controls.Results: A known single nucleotide polymorphism (rs8019270) was identified in both POI and control groups. There was no difference in the genotype distribution or allelic frequencies between the control group and the POI group. No plausible pathogenic mutations were identified.Conclusion: Our study indicates that the TBP2 gene is not responsible for idiopathic POI in the Chinese Han population.

Identification of Multiple Gene Mutations Accounts for a new Genetic Architecture of Primary Ovarian Insufficiency.

Idiopathic primary ovarian insufficiency (POI) is a major cause of amenorrhea and infertility. POI affects 1% of women before age 40 years, and several genetic causes have been reported. To date, POI has been considered a monogenic disorder. The aim of this study was to identify novel gene variations and to investigate if individuals with POI harbor mutation in multiple loci. One hundred well-phenotyped POI patients were systematically screened for variants in 19 known POI loci (and potential candidate genes) using next-generation sequencing. At least one rare protein-altering gene variant was identified in 19 patients, including missense mutations in new candidate genes, namely SMC1β and REC8 (involved in the cohesin complex) and LHX8, a gene encoding a transcription factor. Novel or recurrent deleterious mutations were also detected in the known POI candidate genes NOBOX, FOXL2, SOHLH1, FIGLA, GDF9, BMP15, and GALT. Seven patients harbor mutations in two loci, and this digenicity seems to influence the age of symptom onset. Genetic anomalies in women with POI are more frequent than previously believed. Digenic findings in several cases suggest that POI is not a purely monogenic disorder and points to a role of digenicity. The genotype-phenotype correlations in some kindreds suggest that a synergistic effect of several mutations may underlie the POI phenotype.

A dominant negative mutation at the ATP binding domain of AMHR2 is associated with a defective anti-Müllerian hormone signaling pathway.

Does a heterozygous mutation in AMHR2, identified in whole-exome sequencings (WES) of patients with primary ovarian insufficiency (POI), cause a defect in anti-Müllerian hormone (AMH) signaling? The I209N mutation at the adenosine triphosphatebinding domain of AMHR2 exerts dominant negative defects in the AMH signaling pathway. Previous studies have demonstrated the associations of several sequence variants in AMH or AMHR2 with POI, but no functional assay has been performed to verify whether there was any defect on AMH signaling. Ninety-six unrelated female Chinese Han patients were diagnosed with idiopathic POI and subjected to WES. In silico analysis was done for the sequence variants followed by molecular assays to examine the functional effects of the sequence variants in human granulosa cells. In silico analysis, immunostaining, Western analysis, genome-wide expression analysis, quantitatively polymerase chain reaction were applied to the characterization of the sequence variants. We identified one novel heterozygous missense variant, p.Ala17Glu (A17E), in AMHR2. Subsequently, A17E and two independently reported missense variants, p.Ile209Asn (I209N) and p.Leu354Phe (L354F), were evaluated for effects on the AMH signaling pathway. In silico analysis predicted that all three variants may be deleterious. However, only one variant, I209N, showed severe defects in transducing the AMH signal as well as impaired SMAD1/5/8 phosphorylation. Furthermore, using genome-wide gene expression analysis, we identified genes whose expression was affected by the mutation, these included genes previously reported to participate in AMH signaling as well as newly identified genes. They are EMILIN2, FAM155A, GATA2, HES5, ID1, ID2, RLTPR, SMAD7, CBL, MALAT1 and SMARCA2. None. Although the in vitro assays demonstrated the causative effect of I209N on AMH signaling, further studies need to validate its long-term effects on folliculogenesis and POI. These results will aid both researchers and clinicians in understanding the molecular pathology of AMH signaling and POI to develop diagnostic assays or therapeutics approaches. Research funding is provided by the Ministry of Science and Technology of China [2012CB944704; 2012CB966702], and the National Natural Science Foundation of China [Grant number: 31171429]. The authors declare no conflict of interest.

Increased Incidence of Mitochondrial Cytochrome C Oxidase 1 Gene Mutations in Patients with Primary Ovarian Insufficiency.

Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is defined as more than six months of cessation of menses before the age of 40 years, with two serum follicle stimulating hormone (FSH) levels (at least 1 month apart) falling in the menopause range. The cause of POI remains undetermined in the majority of cases, although some studies have reported increased levels of reactive oxygen species (ROS) in idiopathic POF. The role of mitochondrial DNA in the pathogenesis of POI has not been studied extensively. This aim of this study was to uncover underlying mitochondrial genetic defects in patients with POI. The entire region of the mitochondrial genome was amplified in subjects with idiopathic POI (n=63) and age-matched healthy female controls (n=63) using nine pair sets of primers, followed by screening of the mitochondrial genome using an Illumina MiSeq. We identified a total of 96 non-synonymous mitochondrial variations in POI patients and 93 non-synonymous variations in control subjects. Of these, 21 (9 in POI and 12 in control) non-synonymous variations had not been reported previously. Eight mitochondrial cytochrome coxidase 1 (MT-CO1) missense variants were identified in POI patients, whereas only four missense mutations were observed in controls. A high incidence of MT-CO1 missense variants were identified in POI patients compared with controls, and the difference between the groups was statistically significant (13/63 vs. 5/63, p=0.042). Our results show that patients with primary ovarian insufficiency exhibit an increased incidence of mitochondrial cytochrome c oxidase 1 gene mutations, suggesting that MT-CO1 gene mutation may be causal in POI.