Cytogenetic diagnosis of patients with suspected premature ovarian failure in Manaus, Brazil

Premature ovarian insufficiency: step-by-step genetics bring new insights

Chapter 18 - Premature ovarian insufficiency

The Fanconi anemia pathway is a key pathway involved in the repair of deoxyribonucleic acidinterstrand crosslinking damage, which chiefly includes the following four modules: lesion recognition, Fanconi anemia core complex recruitment, FANCD2-FANCI complex monoubiquitination, and downstream events (nucleolytic incision, translesion synthesis, and homologous recombination). Mutations or deletions of multiple Fanconi anemia genes in this pathway can damage the interstrand crosslinking repair pathway and disrupt primordial germ cell development and oocyte meiosis, thereby leading to abnormal follicular development. Premature ovarian insufficiency is a gynecological clinical syndrome characterized by amenorrhea and decreased fertility due to decreased oocyte pool, accelerated follicle atresia, and loss of ovarian function in women <40years old. Furthermore, in recent years, several studies have detected mutations in the Fanconi anemia gene in patients with premature ovarian insufficiency. In addition, some patients with Fanconi anemia exhibit symptoms of premature ovarian insufficiency and infertility. The Fanconi anemia pathway and premature ovarian insufficiency are closely associated.

Study question Is it possible to identify a genetic cause of familial premature ovarian failure (POF) with whole-exome sequencing (WES)? Summary answer Whole-exome sequencing is the most efficient strategy to identify probably pathogenic mutations in different genes in pathologies of polygenic etiology such as premature ovarian failure. What is known already Premature ovarian failure is the loss of ovarian function before the age of 40, and it is a common cause of infertility in women. This pathology has a heterogeneous etiology. Some chromosomal and genetic alterations have been described, and could explain approximately 20% of cases. However, in most patients the origin remains unknown. Recent studies with next-generation sequencing (NGS) have identified new variants in candidate genes related with premature ovarian insufficiency (POI) or premature ovarian failure (POF). These genes are not only involved in processes such as folliculogenesis, but also with DNA damage repair, homologous recombination, and meiosis. Study design, size, duration Fourteen women, from 7 families, affected by idiopathic POF were included in the study from October 2019 to September 2020. Seven POF patients were recruited when they came to our clinic to undergo assisted reproductive treatment. In the anamnesis, it was found that they had relatives with a diagnosis of POF, who were also recruited for the study. The inclusion criteria were amenorrhea before 38 years old and analytical and ultrasound signs of ovarian failure. Participants/materials, setting, methods WES was performed using TrusightOne (Illumina®). Sequenced data were aligned through BWA tool and GATK algorithm was used for SNVs/InDel identification. VCF files were annotated using Variant Interpreter software. Only the variants shared by each family were extracted for analysis and these criteria were followed: (1) Exonic/splicing variants in genes related with POF or involved in biological ovarian functions (2) Variants with minor allele frequency (MAF) ≤0.05 and (3) having potentially moderate/strong functional effects. Main results and the role of chance Seventy-nine variants possibly related with the POF phenotype were identified in the seven families. All these variants had a minor allele frequency (MAF) ≤0.05 in the gnomAD database and 1000 genomes project. Among these candidate variants, two were nonsense, six splice region, one frameshift, two inframe deletion and 68 missense. Thirty-two of the missense variants were predicted to have deleterious effects by minimum two of the four in silico algorithms used (SIFT, PolyPhen–2, MutationTaster and PROVEAN). All variants were heterozygous, and all the families carried three or more candidate variants. Altogether, 43 probably damaging genetic variants were identified in 39 genes expressed in the ovary and related with POF/POI or linked to ovarian physiology. We have described genes that have never been associated to POF pathology, however they may be involved in key biological processes for ovarian function. Moreover, some of these genes were found in two families, for example DDX11, VWF, PIWIL3 and HSD3B1. DDX11 may function at the interface of replication-coupled DNA repair and sister chromatid cohesion. VWF gene is suggested to be associated with follicular atresia in previous studies. PIWIL3 functions in development and maintenance of germline stem cells, and HSD3B1 is implicated in ovarian steroidogenesis. Limitations, reasons for caution Whole-exome sequencing has some limitations: does not cover noncoding regions of the genome, it also cannot detect large rearrangements, copy-number variants (large deletions/duplications), mosaic mutations, mutations in repetitive or high GC rich regions and mutations in genes with corresponding pseudogenes or other highly homologous sequences. Wider implications of the findings: WES has previously shown to be an efficient tool to identify genes as cause of POF, and has demonstrated the polygenic etiology. Although some studies have focused on it, and many genes are identified, this study proposes new candidate genes and variants, having potentially moderate/strong functional effects, associated with POF. Trial registration number Not applicable

Premature ovarian insufficiency (POI), formerly known as premature menopause, is characterized by the loss of ovarian function in women under 40 years of age. The clinical manifestations of this condition arise due to estrogen deficiency and may include symptoms such as amenorrhea, oligomenorrhea, vasomotor instability (hot flashes, night sweats), sleep disturbances, vulvovaginal atrophy, changes in urinary frequency, dyspareunia, decreased libido, and general weakness. This article will discuss the autoimmune origin of POI and its relationship with other autoimmune diseases. According to statistics, from 4 to 30% of cases of POI are caused by autoimmune processes. POI often occurs with other autoimmune conditions, such as Addison’s disease, hypothyroidism, Graves’ disease, Hashimoto’s thyroiditis, and diabetes mellitus, although it can also occur in isolation. The pathogenesis of POI is associated with hypoestrogenism and the loss of residual follicles, which leads to menstrual cycle disorders, infertility and a decrease in quality of life. To avoid the long-term effects of estrogen deficiency, hormone replacement therapy is recommended. Studying the causative factors of POI, the relationship of this condition with other autoimmune diseases and analyzing existing methods of early diagnosis will ensure more effective management of such patients and increase the chances of improving their fertility.

BackgroundThe loss of ovarian function in women, referred to as premature ovarian insufficiency (POI), is associated with a series of concomitant diseases. POI is genetically heterogeneous, and in most cases, the etiology is unknown.MethodsWhole-exome sequencing (WES) was performed on DNA samples obtained from patients with POI, and Sanger sequencing was used to validate the detected potentially pathogenic variants. An in silico analysis was carried out to predict the pathogenicity of the variants.ResultsWe recruited 24 patients with POI and identified variants in POI-related genes in 14 patients, including bi-allelic mutations in DNAH6, HFM1, EIF2B2, BNC, and LRPPRC and heterozygous variants in BNC1, EIF2B4, FOXL2, MCM9, FANCA, ATM, EIF2B3, and GHR. No variants in the above genes were detected in the WES data obtained from 29 women in a control group without POI. Determining a clear genetic etiology could significantly increase patient compliance with appropriate intervention strategies.ConclusionsOur study confirmed that POI is a genetically heterogeneous condition and that whole-exome sequencing is a powerful tool for determining its genetic etiology. The results of this study will aid researchers and clinicians in genetic counseling and suggests the potential of WES for the detection of POI and thus early interventions for patients with POI.

New candidate gene POU5F1 associated with premature ovarian failure in Chinese patients

Terapias innovadoras en pacientes con baja reserva ovárica e insuficiencia ovárica primaria

Premature ovarian insufficiency (POI) is a clinical syndrome resulting from loss of ovarian function before the age of 40. It is a state of hypergonadotropic hypogonadism, characterised by amenorrhoea or oligomenorrhoea, with low ovarian sex hormones (oestrogen deficiency) and elevated pituitary gonadotrophins. POI with primary amenorrhoea may occur as a result of chromosomal and genetic abnormalities, such as Turner syndrome, Fragile X, or autosomal gene defects; secondary amenorrhoea may be iatrogenic after the surgical removal of the ovaries, radiotherapy, or chemotherapy. Other causes include autoimmune diseases, viral infections, and environmental factors; in most cases, POI is idiopathic. Appropriate replacement of sex hormones in women with POI may facilitate the achievement of near normal uterine development. However, the optimal effective hormone therapy (HT) regimen to maximise the reproductive potential for women with POI remains unclear. To investigate the effectiveness and safety of different hormonal regimens on uterine and endometrial development in women with POI. We searched the Cochrane Gynaecology and Fertility (CGF) Group trials register, CENTRAL, MEDLINE, Embase, PsycINFO, CINAHL, and two trials registers in September 2021. We also checked references of included studies, and contacted study authors to identify additional studies. We included randomised controlled trials (RCTs) investigating the effect of various hormonal preparations on the uterine development of women diagnosed with POI. We used standard methodological procedures recommended by Cochrane. The primary review outcome was uterine volume; secondary outcomes were endometrial thickness, endometrial histology, uterine perfusion, reproductive outcomes, and any reported adverse events. We included three studies (52 participants analysed in total) investigating the role of various hormonal preparations in three different contexts, which deemed meta-analysis unfeasible. We found very low-certainty evidence; the main limitation was very serious imprecision due to small sample size. Conjugated oral oestrogens versus transdermal 17ß-oestradiol We are uncertain of the effect of conjugated oral oestrogens compared to transdermal 17ß-oestradiol (mean difference (MD) -18.2 (mL), 95% confidence interval (CI) -23.18 to -13.22; 1 RCT, N = 12; very low-certainty evidence) on uterine volume, measured after 12 months of treatment. The study reported no other relevant outcomes (including adverse events). Low versus high 17ß-oestradiol dose We are uncertain of the effect of a lower dose of 17ß-oestradiol compared to a higher dose of 17ß-oestradiol on uterine volume after three or five years of treatment, or adverse events (1 RCT, N = 20; very low-certainty evidence). The study reported no other relevant outcomes. Oral versus vaginal administration of oestradiol and dydrogesterone We are uncertain of the effect of an oral or vaginal administration route on uterine volume and endometrial thickness after 14 or 21 days of administration (1 RCT, N = 20; very low-certainty evidence). The study reported no other relevant outcomes (including adverse events). No clear conclusions can be drawn in this systematic review, due to the very low-certainty of the evidence. There is a need for pragmatic, well designed, randomised controlled trials, with adequate power to detect differences between various HT regimens on uterine growth, endometrial development, and pregnancy outcomes following the transfer of donated gametes or embryos in women diagnosed with POI.

Background: Premature ovarian insufficiency (POI) is a condition defined by loss of ovarian activity before the age of 40 years, accompanied by menopausal symptoms. It is marked by amenorrhea or oligomenorrhea, absence of ovulation, elevated gonadotropins, and low estradiol levels. POI can be classified into spontaneous non-iatrogenic and iatrogenic forms. The prevalence of non-iatrogenic POI in the population ranges from 1% to 3.5%. The X chromosome’s Numerical and structural abnormalities are key etiological factors. Objective: To determine the clinical peculiarities and types of POI caused by X chromosome anomalies. Material and Methods: The study included 26 patients aged 16 to 24 with non-iatrogenic spontaneous POI. Of these, 12 patients were diagnosed with numerical or structural abnormalities of the X chromosome based on clinical, laboratory, and instrumental studies. In some cases, x-chromosome abnormalities were detected using G-banding in peripheral blood lymphocyte cultures; fluorescence in situ hybridization (FISH) and molecular cytogenetic methods were employed. Results: Among the 26 patients with non-iatrogenic spontaneous POI, four were diagnosed with Turner syndrome. These patients exhibited severe growth retardation, somatic anomalies typical of Turner syndrome, and sexual development according to Tanner’s scheme (Ma0PaAx0Me0), including markedly elevated gonadotropins and decreased estradiol levels, By US – streak gonads and uterus, without follicles. Two patients were identified with a 45, X karyotype, while another had a 46, X, i(Xq) karyotype. Additionally, a mosaic karyotype with a 45, X cell line was detected in 5 patients (4 with 45, X/46, XX and 1 with 45, X/47, XXX). Structural abnormalities of the X chromosome, specifically deletions, were found in 2 patients. Patients with mosaicism and structural abnormalities of the X chromosome exhibited mild growth retardation and premature ovarian failure, which was characterized by primary amenorrhea, hypergonadotropinemia, and a significantly reduced number of antral follicles. Only one patient with a 45, X /47, XXX mosaic karyotype experienced secondary amenorrhea and spontaneous puberty. One patient with tetrasomy X (karyotype 48, XXXX) and POI presented with tall stature, mental retardation, secondary amenorrhea, hypergonadotropinemia, and a few follicles in the ovaries. To initiate puberty, all patients were treated with monotherapy using natural estrogen analogs, followed by replacement therapy with estrogen-gestagens until the age of natural menopause. Conclusion: • For all patients with non-iatrogenic premature ovarian insufficiency, despite the absence of subjective signs of estrogen deficiency and the characteristic signs of Turner’s syndrome, karyotyping is recommended. • Oocyte donation is the optimal method to achieve fertility in patients with premature ovarian insufficiency and numerical and structural anomalies of the X chromosome. • In patients with X chromosome anomalies at an early stage of diagnosis of chromosomal anomalies and in cases of an acceptable number of ovarian follicles, cryopreservation of reproductive materials may be considered with the using genetic testing of the embryo.

Emergency IVF versus ovarian tissue cryopreservation: decision making in fertility preservation for female cancer patients

BackgroundPremature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear.MethodsWe measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose.ResultsUSP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo.ConclusionsUpregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.

Study question Can multiple intraovarian injections of platelet-rich plasma (PRP) enhance ovarian function and fertility outcomes in a mouse model of premature ovarian insufficiency (POI)? Summary answer A double administration of PRP ameliorates the deleterious effects of chemotherapy on ovarian fibrosis and embryo development in mice. What is known already PRP has emerged as an alternative approach to restore fertility in women with POI, a condition characterized by the loss of ovarian function before the age of 40. Previous studies suggest that PRP therapy has the potential to enhance ovarian tissue regeneration and follicular activation in damaged ovaries. However, the clinical efficacy and optimal protocols for PRP preparation and administration remain uncertain. Further research is needed to establish effective treatment regimens. Therefore, this study aims to evaluate the potential benefits of repeated PRP injections, providing sustained stimulation across multiple menstrual cycles. Study design, size, duration POI condition was induced in thirteen 8-week-old NOD/SCID female mice, while 4 untreated mice were used as a reference (Young-Healthy). Thirteen days after POI induction, mice were randomized to receive 1 or 2 intraovarian injections of PRP (POI PRP-1X, n = 4 and POI PRP-2X, n = 5 respectively) or 2 injections of PBS (POI-Sham, n = 4), with a 13-day interval between injections. Twelve days after treatments, mice underwent controlled ovarian stimulation (COS) and euthanasia to evaluate ovarian function. Participants/materials, setting, methods POI was induced by intraperitoneal administration of cyclophosphamide and busulphan (120 mg/kg, 12 mg/kg). For intraovarian injections, 10 µl of either PBS or activated PRP from normoresponder patients (1801-FIVI-003-AP) was injected into each ovary. Following COS and euthanasia, ovaries were collected and fixed to evaluate ovarian fibrosis (Picrosirius Red staining) and vascularization (Isolectin-B4 inmunostaining). Retrieved oocytes underwent in vitro fertilization (IVF) with C57BL/6 sperm, and embryos were cultured to assess fertilization rate and embryo development. Main results and the role of chance Chemotherapy administration successfully reproduced the POI condition in mice as evidenced by the signs of ovarian damage observed in the POI-Sham group. Specifically, the ovarian/body mass ratio (POI-Sham: 0.3±0.1, Young-Healthy: 0.7±0.1; p &amp;lt; 0.001) and the total number of ovulated oocytes (POI-Sham= 1.6±1.8, Young-Healthy: 25.0±13.7; p = 0.001) were significantly reduced in POI-Sham mice compared with Young-Healthy females. The POI-Sham group also exhibited higher levels of ovarian fibrosis (POI-Sham: 14.8±3.5%, Young-Healthy: 4.9±0.6%; p = 0.005), although no differences were found in ovarian vascularization. Single and double PRP intraovarian injections did not improve ovarian mass or oocyte yield in POI mice. However, both PRP regimens were able to reduce ovarian fibrosis, being this decrease statistically significant in the 1PRP dose group (POI PRP-1X: 7.2±0.9%, POI PRP-2X: 10.9±3.7% vs. POI-Sham: 14.8±3.5%; p = 0.008 and p = 0.061 respectively). Chemotherapy did not affect fertilization rates, but the POI-Sham group showed impaired embryo development, with reduced blastocyst formation (POI-Sham: 81.8%, Young-Healthy: 98.3%; p = 0.002) and hatching rates (POI-Sham: 63.6%, Young-Healthy: 92.5%, p = 0.035) compared to controls. Notably, these negative effects were not observed in those POI mice treated with two PRP injections (POI PRP-2X: 91.3%, 91.3% respectively; p=NS vs. Young-Healthy). Limitations, reasons for caution This study was conducted in a mouse model of POI, requiring additional studies with human samples to confirm this results and assess their clinical application. Wider implications of the findings PRP treatment mitigates some of the deleterious effects induced by chemotherapy in mice, and a double PRP injection seems to improve the benefits of this treatment in embryo development. However, further research is still needed to clarify the therapeutic potential of PRP to promote ovarian function and restore female fertility. Trial registration number No

Berberine protects cyclophosphamide and busulfan-induced premature ovarian insufficiency in mouse model

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure