Premature Ovarian Insufficiency Patients Research Articles

Effects of herbal medicine on ovulation in premature ovarian insufficiency patients during in vitro fertilization

Introduction: Premature ovarian insufficiency (POI) is a condition in which menopause occurs before the age of 40 and is known to be a relatively common disease that occurs in 1% of all women. The only way for patients diagnosed with POI to become pregnant is through oocyte donation, in which fertilized ovums are transplanted by receiving oocytes from donors.4 Can POI patients increase their chances of normal ovulation? POI patients were more likely to observe and collect follicles larger than the average 15 mm diameter when herbal medicine was administered. Methods: This study is a retrospective cohort study conducted on POI patients undergoing in vitro fertilization (IVF) who visited our hospital from 2020 to 2022. We would like to report the response rate to gonadotropin-releasing hormone (GnRH) during IVF in the hyperovulation induction low response group after herbal medicine treatment. Statistical analysis was performed using IBM’s SPSS Statistics to verify significance with a P-value<0.05. Results: In recovering ovarian POI patient's ovarian function, herbal medicine treatment increased the response rate to GnRH (the number of oocytes collected) during IVF, follicle-stimulating hormone (FSH) and cortisol levels decreased, and estradiol (E2) and luteinizing hormone (LH) levels increased. Conclusions: It was confirmed that herbal medicine treatment can increase the probability of normal ovulation in patients with idiopathic POI with high cortisol levels. Until now, it has been known that ovarian function cannot be recovered once irreversibly decreased, but by increasing the probability of ovulation through herbal medicine treatment, it is significant that it may be possible to suppress ovarian aging or preserve fertility.

MCM9 compound heterozygosity in an adolescent with premature ovarian insufficiency.

Delayed puberty in girls is often related to late maturation but is occasionally the first sign of premature ovarian insufficiency (POI). POI is a condition that affects ovarian function and fertility, and its etiology is unknown in most cases. Genetic factors have recently been identified in 20-25% of women with POI, involving genes that regulate various aspects of ovarian development and maintenance. We report a case of delayed puberty due to POI in an adolescent from a non-consanguineous family who carried two variants in the MCM9 gene. MCM9 is essential for DNA replication and repair, and its dysfunction can lead to chromosomal instability and ovarian failure. Our case highlights the importance of targeted gene panel analysis, particularly in POI patients with negative autoimmunity screening, and evidence of ovarian or uterine dysgenesis on pelvic imaging. Delayed puberty in girls is often self-limiting, but it can also indicate underlying conditions with lifelong implications, such as premature ovarian insufficiency (POI). Patients with POI, negative autoimmune screening, a normal karyotype, and no FMR premutation should undergo further genetic testing, preferably through targeted gene panels. Compound heterozygous variants in MCM9 can cause POI, presenting with delayed puberty and primary amenorrhea in girls without a consanguineous family.

Association of FOXL2 and ERCC6 variants with premature ovarian insufficiency and their potential use in clinical IVF guidance

Premature ovarian insufficiency (POI) is the main cause of infertility in women. Some cases of POI are thought to be caused by genetic defects and the clinical outcomes of these patients are unknown. Here, we performed whole-exome sequencing of the peripheral blood of a cohort of 55 subjects with POI and identified one heterozygous missense variant in FOXL2 (c.1045G>C; p.Arg349Gly) and two heterozygous missense variants in ERCC6 (c.379G>A; p.Val127Ile and c.4223A>C; p.Glu1408 Ala) in four POI patients. All of these heterozygous mutations were predicted to be deleterious and were parentally inherited from their heterozygous fathers. The mRNA and protein expression of FOXL2 and ERCC6 were absent or decreased in the patients. The patients carrying the variants of FOXL2 (c.1045G>C; p.Arg349Gly) and ERCC6 (c.379G>A; p.Val127Ile) failed to conceive in two and four assisted reproductive cycles, respectively. Another patient and her sister carrying the ERCC6 (c.4223A>C; p.Glu1408 Ala) variant achieved good clinical outcomes after assisted reproductive therapy. Our findings support the possible roles of FOXL2 and ERCC6 in POI and might contribute to the genetic counseling of POI patients.

Assessment of left ventricular myocardial systolic dysfunction in premature ovarian insufficiency patients using echocardiographic layer-specific myocardial strain imaging

BackgroundDue to the lack of oestrogen, premature ovarian insufficiency (POI) is an independent risk factor for ischaemic heart disease and overall cardiovascular disease. This study aimed to apply layer-specific myocardial strain for early quantitative evaluation of subclinical left ventricular myocardial systolic function changes in patients with POI.MethodsForty-eight newly diagnosed, untreated patients with POI (POI group) and fifty healthy female subjects matched for age, height and weight (control group) were enrolled. Standard transthoracic echocardiography was used to measure conventional parameters and layer-specific strain parameters.The layer-specific strain parameters included subendomyocardial global longitudinal strain (GLSendo), mid-layer myocardial global longitudinal strain (GLSmid), subepimyocardial global longitudinal strain (GLSepi), subendomyocardial global circumferential strain (GCSendo), mid-layer myocardial global circumferential strain (GCSmid), and subepimyocardial global circumferential strain (GCSepi). ResultsThere were no significant differences in age, body mass index (BMI), blood pressure, or left ventricular ejection fraction (LVEF) between the two groups. The end-diastolic interventricular septal thickness (IVST) was greater in the POI group (8.29 ± 1.32 vs. 7.66 ± 0.82, P = 0.008), and the POI group had lower E, E/A, and lateral e′ (all P < 0.05). As for systolic functions,the POI group had lower GLSendo, GLSmid, GLSepi, GCSendo, GCSmid, and GCSepi (all P < 0.05).The intraobserver and interobserver coefficients of GLSendo, GLSmid, GLSepi, GCSendo, GCSmid, and GCSepi were greater than 0.900. ConclusionsPOI patients with normal LVEF may suffer from subclinical left ventricular myocardial systolic dysfunction. Echocardiography of layer-specific myocardial strain could more sensitively detect subclinical impairment of left ventricular systolic function in POI patients.Graphical abstract

Metabolic Risk in Patients with a Diminished Ovarian Reserve and Premature Ovarian Insufficiency.

Objective: Diminished ovarian reserve (DOR) and premature ovarian insufficiency (POI) represent conditions of different severity, characterized by an earlier-than-expected decrease in ovarian activity. The present study aims to compare metabolic disturbances between women with DOR and patients with POI from a different origin. Materials and methods: A total of 226 women (28 healthy women; 77 individuals with DOR, and 121 patients with POI/36 with Turner syndrome [TS] and 85 with non-TS POI/) have been studied retrospectively. Data concerning anthropometric indices, and metabolic parameters were collected. Results: Patients with DOR, non-TS POI, and TS had increased blood pressure and liver enzymes, pronounced insulin resistance, and worse lipid profiles than controls (p < 0.008 for all). TS patients had significantly higher ASAT, GGT, and TSH levels compared to non-TS POI and DOR individuals. The prevalence of type 2 diabetes tended to be higher in TS women compared to other groups. The prevalence of previously diagnosed polycystic ovarian syndrome was lower in the non-TS POI patients than in the DOR patients (p = 0.005). Conclusions: patients with decreased ovarian function suffer from insulin resistance, abnormal lipid profile, and subtle hepatic disturbances, irrespective of the severity of the condition and the presence of chromosomal aberrations.

Bone mineral density: Comparison between women under hormone replacement therapy with Turner syndrome or idiopathic premature ovarian insufficiency

ContextTurner syndrome (TS) is characterized by short stature and premature ovarian insufficiency (POI). The main long-term complication of POI is osteoporosis, which can be prevented by hormone replacement therapy (HRT). ObjectiveThe objective of our study was to compare initial bone mineral density (BMD) and progression between TS and idiopathic POI patients under HRT. MethodsA single-center retrospective study was conducted between 1998 and 2018. All women had undergone at least two bone densitometry assessments at least 2 years apart. ResultsSixty-eight TS patients and 67 idiopathic POI patients were included. Mean age at initial assessment was 27 years (IQR, 21–35.5 years) in TS patients and 31.5 years (IQR, 23–37 years) in idiopathic POI patients (P=0.1). Lumbar and femoral neck BMD were lower in the TS group than in the idiopathic POI group (respectively 0.89g/cm2 versus 0.95g/cm2, P=0.03; 0.70g/cm2 versus 0.77g/cm2, P<0.0001). Mosaic karyotype was associated with better BMD in TS patients while history of growth hormone treatment had no impact on BMD. Over time, a significant gain in vertebral BMD was observed in TS patients versus a loss of BMD in idiopathic POI patients (P=0.0009). ConclusionTS patients had a lower BMD at baseline than idiopathic POI patients, at both spinal and femoral levels. Over time, on HRT, a significant gain in vertebral BMD was observed in patients with TS, compared with a loss of BMD in patients with idiopathic POI. We hypothesized that earlier initiation and longer duration of HRT played an important role in this finding. Long-term prospective follow-up to assess the incidence of fractures in TS would be useful.

Chromosome Segregation-1-like Gene Participates in Ferroptosis in Human Ovarian Granulosa Cells via Nucleocytoplasmic Transport.

Premature ovarian insufficiency (POI) is defined as the depletion of ovarian function before the age of 40 years. The global prevalence of POI is 3.5%. To date, genetic factors account for 23.5% of the etiology of POI. Herein, a previously uncharacterized pathogenic homozygous variant of the chromosome segregation-1-like gene (CSE1L) was identified in POI patients via targeted panel sequencing. It is reported that dysregulated iron metabolism is involved in many reproductive endocrine disorders; however, its precise role in POI remains obscure. In this study, we identified CSE1L as a potential candidate gene that plays an important role in maintaining iron homeostasis. Deficiency of CSE1L led to ferroptosis in human granulosa cells, which was confirmed by transmission electron microscopy. Mechanistically, coimmunoprecipitation identified the direct interaction between CSE1L and FoxO1. Inhibition of CSE1L led to the excessive accumulation of FoxO1 in the nucleus via nucleocytoplasmic transport. Then, FoxO1 bound to the promoter region of NCOA4 and promoted its transcription, which was verified by a chromatin immunoprecipitation assay. Moreover, inhibition of CSE1L in cumulus cell monolayer could impede oocyte maturation, which might be associated with oxidative stress. Consequently, our study first revealed that CSE1L participated in ferroptosis in human ovarian granulosa cells via nucleocytoplasmic transportation, which might be helpful in revealing the molecular mechanism of CSE1L in the development of POI. Importantly, these findings might provide new insights into the application of ferroptosis inhibitors in the treatment of POI.

P-353 Perfluorooctanoic acid (PFOA) impacts murine ovarian follicle development through apoptotic signaling pathways and exhibits transgenerational effects

Abstract Study question How does Perfluorooctanoic Acid (PFOA) exposure impact ovarian function and the physiological and reproductive health of subsequent offspring? Summary answer PFOA exposure leads to ovarian dysfunction, characterized by follicular depletion and hormonal imbalances, and adversely affects the reproductive health of offspring. What is known already Mass spectrometry reveals higher perfluorinated compound levels in premature ovarian failure patients compared to healthy individuals. PFOA, an environmental contaminant, is known for its endocrine-disrupting effects, but its specific impact on ovarian function and transgenerational health remains less explored. This study addresses the gap by examining PFOA’s direct ovarian effects and its consequential impacts on offspring, using both in vivo and in vitro models. Study design, size, duration This study employs mouse models and ovarian granulosa cells to assess PFOA’s impact. Mice were administered PFOA (50mg/Kg) and evaluated for ovarian function and offspring health. Ovarian tissue underwent histological analysis and hormone assays. RNA transcriptome sequencing identified activated apoptotic pathways post-PFOA exposure. Offspring physiological and reproductive parameters were also examined. Parallel in vitro experiments with ovarian granulosa cells at 50uM PFOA concentration provided insights into cellular apoptosis. Participants/materials, setting, methods Clinically, we collected serum samples from approximately 100 Premature Ovarian Insufficiency (POI) patients and 100 healthy individuals, analyzed using mass spectrometry. In murine experiments, we simulated human environmental exposure to PFOA (particularly in high-exposure professions) using a 50mg/Kg dose administered via gavage. Transcriptome sequencing analyzed potential signaling pathways in both experimental and control mice. Offspring underwent physiological metabolic assessments and fertility evaluations, including fertilization and blastocyst rates. Main results and the role of chance Mass spectrometry results from human samples revealed significantly higher levels of perfluorinated compounds in POI patients compared to healthy controls, suggesting a potential link between PFOA exposure and ovarian dysfunction. In murine models, PFOA administration at 50mg/Kg resulted in notable ovarian changes. Histological analysis showed a reduction in follicle counts and abnormalities in AMH and FSH levels, indicative of premature ovarian failure. Transcriptome sequencing of these mice highlighted the activation of apoptotic pathways, especially the PI3K/Akt/mTOR signaling, post-PFOA exposure. This suggests a mechanistic pathway through which PFOA may induce ovarian dysfunction. Furthermore, offspring of PFOA-exposed mice displayed significant reproductive health challenges. They exhibited irregularities in estrous cycles and a decrease in oocyte fertilization rates, pointing to potential transgenerational effects of PFOA exposure. These findings in murine models align with the elevated perfluorinated compound levels observed in human POI patients, reinforcing the hypothesis of PFOA’s detrimental impact on ovarian health and subsequent generational fertility. The consistency of results across human clinical data and animal models strengthens the argument for a causal relationship between PFOA exposure and reproductive health risks. Limitations, reasons for caution While these findings highlight PFOA’s detrimental effects on ovarian function and offspring health, translating results from animal models to humans necessitates caution. The specific mechanisms and potential variability in human response to environmental PFOA exposure require further investigation. Wider implications of the findings This research underscores the far-reaching impacts of environmental contaminants like PFOA on reproductive health. It emphasizes the need for stringent regulation of such pollutants and further studies to understand their long-term effects on human health, particularly regarding fertility and intergenerational wellbeing. Trial registration number not applicable

P-791 The 4-step ASCOT technique modifies the plasma proteome of patients with premature ovarian insufficiency to resemble that of normoresponder women

Abstract Study question Does the 4-step ASCOT technique induce any modification in the plasma proteomic profile of women with premature ovarian insufficiency (POI)? Summary answer The 4-step ASCOT technique elicits substantial changes in plasma proteome of POI patients, leading to a more similar composition to that observed in normoresponder women. What is known already Nowadays, treatment of POI women is still challenging and different approaches have been proposed to improve their reproductive potential, including stem cells (SC) and platelet-rich plasma (PRP). Previous studies showed that plasma treatments containing a combination of both SC-secreted and platelet-enclosed factors are the most promising regenerative treatment in mouse and human ovarian tissues. Therefore, we designed the 4-step ASCOT technique, consisting in: 1) SC mobilization from bone marrow to peripheral blood to enrich plasma with SC secreted factors, 2) Platelet concentration, 3) platelet activation to deliver additional growth factors and 4) direct ovarian injection. Study design, size, duration Experimental study with plasma samples of 10 POI women undergoing the 4-step ASCOT reactivation technique (NCT04475744) and 5 normoresponder fertile women. Peripheral plasma samples from POI women were collected at recruitment (Pre) and three months after SC-PRP ovarian injection (Post). Proteomic profiles of Pre and Post plasma samples were compared between them and with the normoresponder samples. Participants/materials, setting, methods Proteins were isolated from all plasmas and quantified by the data-independent acquisition parallel accumulation serial fragmentation (diaPASEF) approach. Principal component analysis was performed to compare the plasma proteomic profile of pre- and post-treatment samples, applying the vip function to determine which proteins explained the differences between both time-points (vip&amp;gt;1.5). Then, ElasticNet and Limma methods were employed to determine the statistically significant changes and functional analysis performed to establish the enriched KEGG pathways (false discovery rate&amp;lt;0.05). Main results and the role of chance Plasma proteome of POI patients at recruitment exhibited a distinctly different proteomic pattern from that of normoresponder women. However, principal component analysis revealed a clear distinction between plasma samples obtained from POI patients at recruitment and three months after the 4-step ASCOT technique (principal component 1 (PC1) accounting for 20% of explained variance, and PC2 for a 15.4%). Differences between both time-points were mainly determined by 56 proteins involved in processes with a key role in ovarian function, such as complement and coagulation cascades, platelet activation, Hippo pathway, cell cycle, oocyte meiosis and PI3K-Akt signaling pathway. Of these, fifteen proteins exhibited significantly differential expression between both time points, including the age-related APOF protein and the GC vitamin D binding protein, which was the most prominently changed protein after the treatment. Interestingly, the 4-step ASCOT technique allowed the plasma proteome of patients with POI to reach a similar profile to that observed in normoresponder women. In fact, a detailed analysis uncovered 72 differentially expressed proteins between pre-treatment and normoresponder samples, with only 28 in post-treatment vs. normoresponders. Moreover, the magnitude of expression changes, when compared to normoresponder women, was more significant in pre-treatment than in post-treatment samples. Limitations, reasons for caution Further research will be necessary to determine the extent of the regenerative effects of the identified differentially expressed proteins within the ovaries, and their direct implications in ovarian aging and function. Wider implications of the findings Our findings highlight that the 4-step ASCOT technique induces changes in plasma proteome of POI patients, reaching similar values to normoresponders. These changes, that remain for few months, might contribute to ovarian reactivation, being the stepping stone to design more effective and personalized treatments for patients with impaired ovarian reserves. Trial registration number NCT04475744

Genetic Variants in KNDy Pathway Lack Association with Premature Ovarian Insufficiency in Mexican Women: A Sequencing-Based Cohort Study.

Previous studies have demonstrated the essential role of the Kisspeptin/Neurokinin B/Dynorphin A (KNDy) pathway in female reproductive biology by regulating the activity of the hypothalamic-pituitary-gonadal axis. Identified loss-of-function mutations in these genes are linked to various reproductive disorders. This study investigated genetic disorders linked to mutations in the KNDy genes related to premature ovarian insufficiency (POI). A cohort of 14 Mexican POI patients underwent genetic screening using PCR-SSCP and Sanger sequencing, assessing the genetic variations' impact on protein function thereafter using multiple in silico tools. The PCR excluded extensive deletions, insertions, and duplications, while SSCP detected five genetic variants. Variations occurred in the KISS1 (c.58G>A and c.242C>G), KISS1R (c.1091A>T), PDYN (c.600C>T), and OPRK1 (c.36G>T) genes, whereas no genetic anomalies were found in NK3/NK3R genes. Each single-nucleotide variant underwent genotyping using PCR-SSCP in 100 POI-free subjects. Their allelic frequencies paralleled the patient group. These observations indicate that allelic variations in the KNDy genes may not contribute to POI etiology. Hence, screening for mutations in KNDy genes should not be a part of the diagnostic protocol for POI.

Successful Pregnancy and Delivery after Premature Ovarian Insufficiency Combined with Undifferentiated Connective Tissue Disease: A Case Report

Background:: Premature ovarian insufficiency (POI) is extremely rare in the early stage of undifferentiated connective tissue disease. Patients with POI find it difficult to achieve successful pregnancy and delivery. Case Presentation:: A 27-year-old female visited an outpatient department for premature ovarian insufficiency (POI) and infertility. She had regular menstrual periods since she was 14 years old and had no history of systemic disease. Laboratory tests showed low estrogen (15 ng/L, range 19.6-144.2 ng/L), elevated follicle-stimulating hormone (34 U/L), low anti-Mullerian hormone (0.1 μg/L), normal prolactin (11.48 ng/mL), and thyroid stimulating hormone (TSH) levels (0.97 mU/L). She demonstrated smaller bilateral ovarian volume and positivity to antinuclear and antiphospholipid antibodies. After the failure of conventional drug therapy and in vitro fertilization, the patient became pregnant naturally after treatment with glucocorticoids. Conclusion:: Immunosuppression could help improve ovarian function and pregnancy outcomes in POI patients, but the therapeutic mechanisms are not clear and should be elucidated with more clinical studies.

Introducing a value-based healthcare approach for women with premature ovarian insufficiency (POI): Recommendations for patient-centered outcomes in clinical practice

ObjectiveTo establish a set of clinician and patient-reported outcome measures (PROMs) and present the initial findings of a value-based healthcare (VBHC) program in patients with premature ovarian insufficiency (POI). MethodsEmploying a four-phase approach, we identified the most crucial domains for patients with POI and translated these into PROMs. Prior to each visit, patients completed questionnaires to evaluate: depression (BDI-II), menopausal symptoms (GCS), work ability (WAS) and infertility (FertiQoL). During the visits, cardiovascular health assessments and dual-energy X-ray absorptiometry (DEXA) scans to measure bone mineral density were performed. Data at intake is presented, and comparisons are drawn between women using and those not using hormone replacement therapy (HRT). Patient-reported experience measures (PREMs) were evaluated by a questionnaire. ResultsA total of 267 POI patients completed the PROM questionnaires, of whom 58.1 % were using HRT at intake. Over half of the patients (53.5 %), had a BDI-II score of 14 or higher, indicating mild to severe depression. The mean total GCS score was 20.9 (SD 11.3). The median work ability score was 7.5 (IQR 6.0–8.0) and the mean FertiQoL score 63.9 (SD 15.7). Additionally, 22.7 % of patients presented with hypertension, 6.2 % with hypercholesterolemia, and almost 50 % had low bone mass. Patients rated the VBHC program with a mean of 8.6 (SD 1.2). ConclusionsThese findings underscore the necessity of a multidisciplinary VBHC program incorporating standardized screening and psychological treatment. We advocate for the implementation of patient-centered outcomes for clinical practice, which have been found to be highly relevant by patients with POI.

Novel compound heterozygous variants in FANCI cause premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common reproductive aging disorder due to a dramatic decline of ovarian function before 40 years of age. Accumulating evidence reveals that genetic defects, particularly those related to DNA damage response, are a crucial contributing factor to POI. We have demonstrated that the functional Fanconi anemia (FA) pathway maintains the rapid proliferation of primordial germ cells to establish a sufficient reproductive reserve by counteracting replication stress, but the clinical implications of this function in human ovarian function remain to be established. Here, we screened the FANCI gene, which encodes a key component for FA pathway activation, in our whole-exome sequencing database of 1030 patients with idiopathic POI, and identified two pairs of novel compound heterozygous variants, c.[97C > T];[1865C > T] and c.[158-2A > G];[c.959A > G], in two POI patients, respectively. The missense variants did not alter FANCI protein expression and nuclear localization, apart from the variant c.158-2A > G causing abnormal splicing and leading to a truncated mutant p.(S54Pfs*5). Furthermore, the four variants all diminished FANCD2 ubiquitination levels and increased DNA damage under replication stress, suggesting that the FANCI variants impaired FA pathway activation and replication stress response. This study first links replication stress response defects with the pathogenesis of human POI, providing a new insight into the essential roles of the FA genes in ovarian function.

α-Ketoglutarate Improves Ovarian Reserve Function in Primary Ovarian Insufficiency by Inhibiting NLRP3-Mediated Pyroptosis of Granulosa Cells.

Premature ovarian insufficiency (POI) is a common female infertility problem, with its pathogenesis remains unknown. The NOD-like receptor family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis has been proposed as a possible mechanism in POI. This study investigates the therapeutic effect of α-ketoglutarate (AKG) on ovarian reserve function in POI rats and further explores the potential molecular mechanisms. POI rats are caused by administration of cyclophosphamide (CTX) to determine whether AKG has a protective effect. AKG treatment increases the ovarian index, maintains both serum hormone levels and follicle number, and improves the ovarian reserve function in POI rats, as evidence by increased the level of lactate and the expression of rate-limiting enzymes of glycolysis in the ovaries, additionally reduced the expression of NLRP3, Gasdermin D (GSDMD), Caspase-1, Interleukin-18 (IL-18), and Interleukin-1 beta (IL-1β). In vitro, KGN cells are treated with LPS and nigericin to mimic pyroptosis, then treated with AKG and MCC950. AKG inhibits inflammatory and pyroptosis factors such as NLRP3, restores the glycolysis process in vitro, meanwhile inhibition of NLRP3 has the same effect. AKG ameliorates CTX-induced POI by inhibiting NLRP3-mediated pyroptosis, which provides a new therapeutic strategy and drug target for clinical POI patients.

Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-β (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-β1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Resveratrol-βcd inhibited premature ovarian insufficiency progression by regulating granulosa cell autophagy

BackgroundThe ovarian environment of premature ovarian insufficiency (POI) patients exhibits immune dysregulation, which leads to excessive secretion of numerous proinflammatory cytokines that affect ovarian function. An abnormal level of macrophage polarization directly or indirectly inhibits the differentiation of ovarian granulosa cells and steroid hormone production, ultimately leading to POI. Resveratrol, as a health supplement, has been widely recognized for its safety. There is a substantial amount of evidence indicating that resveratrol and its analogs possess significant immune-regulatory functions. It has also been reported that resveratrol can effectively inhibit the progression of POI. However, the underlying immunological and molecular mechanisms through which resveratrol inhibits the progression of POI are still unclear.ResultsOur preliminary reports have shown that resveratrol-βcd, the beta-cyclodextrin complex of resveratrol, significantly enhances the stability of resveratrol. Resveratrol-βcd could regulate the dysfunctional immune status of macrophages and T cells in the tumor microenvironment. In this study, we treated busulfan and cyclophosphamide (B/C)-treated mice, which were used as a POI model, with resveratrol-βcd. After resveratrol-βcd treatment, the levels of IL-6 in the ovaries were significantly increased, and the progression of POI was suppressed. IL-6 activated granulosa cells (GCs) through soluble IL-6R (sIL-6R), promoting autophagy in GCs. Resveratrol-βcd and IL-6 had a synergistic effect on enhancing autophagy in GCs and promoting E2 secretion.ConclusionsWe partially elucidated the immune mechanism by which resveratrol inhibits the progression of POI and the autophagy-regulating function of GCs. This provides a theoretical basis for using resveratrol to prevent POI in future studies and clinical guidance.

Melatonin regulates autophagy in granulosa cells from patients with premature ovarian insufficiency via activating Foxo3a.

Premature ovarian insufficiency (POI) is a diverse form of female infertility characterized by a decline in ovarian function before the age of 40. Melatonin (MT) is a potential clinical treatment for restoring or safeguarding ovarian function in POI. However, the specific therapeutic mechanism underlying this effect remains unclear. To address this, we conducted experiments using human granulosa cells (GCs) from both POI and normal patients. We examined the expression levels of autophagy-related genes and proteins in GCs through qRT-PCR and western blot analysis. Autophagy flux was monitored in GCs infected with GFP-LC3-adenovirus, and the regulatory function of MT in autophagy was investigated. Additionally, we employed pharmacological intervention of autophagy using 3-Methyladenine (3-MA) and RNA interference of Forkhead box O-3A (FOXO3A) to elucidate the mechanism of MT in the autophagy process. Compared to GCs from normal patients, GCs from POI patients exhibited irregular morphology, decreased proliferation, increased apoptosis, and elevated ROS levels. The expression of autophagy-related genes was downregulated in POI GCs, resulting in reduced autophagic activity. Furthermore, MT levels were decreased in POI GCs, but exogenous MT effectively activated autophagy. Mechanistically, melatonin treatment downregulated FOXO3A expression and induced phosphorylation in POI GCs. Importantly, silencing FOXO3A abolished the protective effect of melatonin on GCs. These findings indicate that autophagy is downregulated in POI GCs, accompanied by a deficiency in MT. Moreover, we demonstrated that supplementing MT can rescue autophagy levels and enhance GC viability through the activation of FOXO3A signaling. Thus, MT-FOXO3A may serve as a potential therapeutic target for POI treatment.

Aberrantly High FBXO31 Impairs Oocyte Quality in Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI), which is defined as loss of ovarian function that occurs before the age of 40, causes menstrual disturbances, infertility, and diverse health problems in females. Despite the limited understanding of the molecular basis underlying POI pathology, we had previously demonstrated that the cooperation of miR-106a and FBXO31 plays a pivotal role in diminished ovarian reserve (DOR), with FBXO31 serving as a putative target of miR-106a. In this study, we found that FBXO31 is aberrantly expressed in granulosa cells of POI patients, leading to accumulated reactive oxygen species (ROS) and cell apoptosis via the p53/ROS pathway. Furthermore, our results demonstrated that high levels of FBXO31 in mouse ovaries impair oocyte quality. Our study revealed that FBXO31 may serve as a novel indicator and play a significant role in the etiology of POI.

Single-cell RNA-seq identified novel genes involved in primordial follicle formation.

The number of primordial follicles (PFs) in mammals determines the ovarian reserve, and impairment of primordial follicle formation (PFF) will cause premature ovarian insufficiency (POI). By analyzing public single-cell RNA sequencing performed during PFF on mice and human ovaries, we identified novel functional genes and novel ligand-receptor interaction during PFF. Based on immunofluorescence and in vitro ovarian culture, we confirmed mechanisms of genes and ligand-receptor interaction in PFF. We also applied whole exome sequencing (WES) in 93 cases with POI and whole genome sequencing (WGS) in 465 controls. Variants in POI patients were further investigated by in silico analysis and functional verification. We revealed ANXA7 (annexin A7) and GTF2F1 (general transcription factor IIF subunit 1) in germ cells to be novel potentially genes in promoting PFF. Ligand Mdk (midkine) in germ cells and its receptor Sdc1 (syndecan 1) in granulosa cells are novel interaction crucial for PFF. Based on immunofluorescence, we confirmed significant up-regulation of ANXA7 in PFs compared with germline cysts, and uniform expression of GTF2F1, MDK and SDC1 during PFF, in 25 weeks human fetal ovary. In vitro investigation indicated that Anxa7 and Gtf2f1 are vital for mice PFF by regulating Jak/Stat3 and Jnk signaling pathways, respectively. Ligand-receptor (Mdk-Sdc1) are crucial for PFF by regulating Pi3k-akt signaling pathway. Two heterozygous variants in GTF2F1, and one heterozygous variants in SDC1 were identified in cases, but no variant were identified in controls. The protein level of GTF2F1 or SDC1 in POI cases are significantly lower than that of controls, indicating the pathogenic effects of the two genes on ovarian function were dosage dependent. Our study identified novel genes and novel ligand-receptor interaction during PFF, and further expanding the genetic architecture of POI.

Homozygous missense variant in MEIOSIN causes premature ovarian insufficiency.

Are variants of genes involved in meiosis initiation responsible for premature ovarian insufficiency (POI)? A MEIOSIN variant participates in the pathogenesis of human POI by impairing meiosis due to insufficient transcriptional activation of essential meiotic genes. Meiosis is the key event for the establishment of the ovarian reserve, and several gene defects impairing meiotic homologous recombination have been found to contribute to the pathogenesis of POI. Although STRA8 and MEIOISN variants have been found to associate with POI in a recent study, the condition of other meiosis initiation genes is unknown and direct evidence of variants participating in the pathogenesis of POI is still lacking. This was a retrospective genetic study. An in-house whole exome sequencing (WES) database of 1030 idiopathic POI patients was screened for variations of meiosis initiation genes. Homozygous or compound heterozygous variations of genes involved in meiosis initiation were screened in the in-house WES database. The pathogenicity of the variation was verified by in vitro experiments, including protein structure prediction and dual-luciferase reporter assay. The effect of the variant on ovarian function and meiosis was demonstrated through histological analyses in a point mutation mouse model. One homozygous variant in MEIOSIN (c.1735C>T, p.R579W) and one in STRA8 (c.258 + 1G>A), which initiates meiosis via the retinoic acid-dependent pathway, were identified in a patient with idiopathic POI respectively. The STRA8 variation has been reported in the recently published work. For the MEIOSIN variation, the dual-luciferase reporter assay revealed that the variant adversely affected the transcriptional function of MEIOSIN in upregulating meiotic genes. Furthermore, knock-in mice with the homologous mutation confirmed that the variation impacted the meiotic prophase I program and accelerated oocyte depletion. Moreover, the variant p.R579W localizing in the high-mobility group (HMG) box domain disrupted the nuclear localization of the MEIOSIN protein but was dispensable for the cell-cycle switch of oocytes, suggesting a unique role of the MEIOSIN HMG box domain in meiosis initiation. Further studies are needed to explore the role of other meiosis initiation genes in the pathogenesis of POI. The MEIOSIN variant was verified to cause POI by impaired transcriptional regulation of meiotic genes and was inherited by a recessive mode. The function of HMG box domain in MEIOSIN protein was also expanded by this study. Although causative variations in meiotic initiation genes are rare in POI, our study confirmed the pathogenicity of a MEIOSIN variant and elucidated another mechanism of human infertility. This work was supported by the National Key Research & Developmental Program of China (2022YFC2703800, 2022YFC2703000), National Natural Science Foundation for Distinguished Young Scholars (82125014), National Natural Science Foundation of China (32070847, 32170867, 82071609), Basic Science Center Program of NSFC (31988101), Natural Science Foundation of Shandong Province for Grand Basic Projects (ZR2021ZD33), Natural Science Foundation of Shandong Province for Excellent Young Scholars (ZR2022YQ69), Taishan Scholars Program for Young Experts of Shandong Province (tsqn202211371), and Qilu Young Scholars Program of Shandong University. The authors declare no conflict of interest. N/A.