Spontaneous pregnancy after thread-embedding therapy treatment with premature ovarian insufficiency after unilateral oophorectomy: a case report.

Premature ovarian insufficiency (POI) is a condition characterized by menstrual disturbance, subfertility, and estrogen deficiency symptoms. Women with POI have a small chance of natural conception, which may be even smaller when complicated with unilateral ovarian due to reduction of the ovarian follicular reserve. In China, acupuncture has been widely used to treat POI and POI-induced infertility, and studies have shown that acupuncture is helpful for improving ovarian function. Thread-embedding therapy is a method of acupuncture treatment development and extension, which can make the acupuncture effect last. In this article, we report a patient diagnosed with POI after unilateral oophorectomy (UO) who spontaneously conceived after thread-embedding therapy. Thread-embedding therapy may improve ovarian function in patients with POI, thereby providing a treatment strategy for infertility in patients with POI. This case report was written in accordance with the CARE guidelines.

Wine- and stir-frying processing of Cuscutae Semen enhance its ability to alleviate oxidative stress and apoptosis via the Keap 1-Nrf2/HO-1 and PI3K/AKT pathways in H2O2-challenged KGN human granulosa cell line.

BACKGROUND: Cuscutae Semen (CS) has been prescribed in traditional Chinese medicine (TCM) for millennia as an aging inhibitor, an anti-inflammatory agent, a pain reliever, and an aphrodisiac. Its three main forms include crude Cuscutae Semen (CCS), wine-processed CS (WCS), and stir-frying-processed CS (SFCS). Premature ovarian insufficiency (POI) is a globally occurring medical condition. The present work sought a highly efficacious multi-target therapeutic approach against POI with minimal side effects. Finally, it analyzed the relative differences among CCS, WCS and SFCS in terms of their therapeutic efficacy and modes of action against H2O2-challenged KGN human granulosa cell line. METHODS: In this study, ultrahigh-performance liquid chromatography (UPLC)-Q-ExactiveTM Orbitrap-mass spectrometry (MS), oxidative stress indices, reactive oxygen species (ROS), Mitochondrial membrane potential (MMP), real-time PCR, Western blotting, and molecular docking were used to investigate the protective effect of CCS, WCS and SFCS on KGN cells oxidative stress and apoptosis mechanisms. RESULTS: The results confirmed that pretreatment with CCS, WCS and SFCS reduced H2O2-induced oxidative damage, accompanied by declining ROS levels and malondialdehyde (MDA) accumulation in the KGN cells. CCS, WCS and SFCS upregulated the expression of antioxidative levels (GSH, GSH/GSSG ratio, SOD, T-AOC),mitochondrial membrane potential (MMP) and the relative mRNA(Nrf2, Keap1, NQO-1, HO-1, SOD-1, CAT). They inhibited apoptosis by upregulating Bcl-2, downregulating Bax, cleaved caspase-9, and cleaved caspase-3, and lowering the Bax/Bcl-2 ratio. They also exerted antioxidant efficacy by partially activating the PI3K/Akt and Keap1-Nrf2/HO-1 signaling pathways. CONCLUSIONS: The results of the present work demonstrated the inhibitory efficacy of CCS, WCS and SFCS against H2O2-induced oxidative stress and apoptosis in KGN cells and showed that the associated mechanisms included Keap1-Nrf2/HO-1 activation, P-PI3K upregulation, and P-Akt-mediated PI3K-Akt pathway induction.

Bioinformatic analysis of the role of immune checkpoint genes and immune infiltration in the pathogenesis and development of premature ovarian insufficiency.

PURPOSE: With advances in immunology, increasing evidence suggests that immunity is involved in premature ovarian insufficiency (POI) pathogenesis. This study investigated the roles of immune checkpoint genes and immune cell infiltration in POI pathogenesis and development. METHODS: The GSE39501 dataset and immune checkpoint genes were obtained from the Gene Expression Omnibus database and related literature. The two datasets were intersected to obtain immune checkpoint-related differentially expressed genes (ICRDEGs), which were analyzed using Gene Ontology and Kyoto Encyclopedia of Gene and Genomes enrichment analysis, weighted correlation network analysis, protein-protein interaction and related microRNAs, transcription factors, and RNA binding proteins. The immune cell infiltration of ICRDEGs was explored, and receiver operating characteristic curves were used to validate the diagnostic value of ICRDEGs in POI. RESULTS: We performed ICRDEG functional enrichment analysis and found that these genes were closely related to immune processes, such as T cell activation. Specifically, they are enriched in various biological processes and pathways, such as cell adhesion molecule and T cell receptor signaling pathways. Weighted correlation network analysis identified seven hub genes: Cd200, Cd274, Cd28, neurociliary protein-1, Cd276, Cd40lg, and Cd47. Furthermore, we identified 112 microRNAs, 17 RNA-binding proteins, and 101 transcription factors. Finally, immune infiltration analysis showed a clear positive correlation between hub genes and multiple immune cell types. CONCLUSION: Bioinformatic analysis identified seven potential ICRDEGs associated with POI, among which the immune checkpoint molecules CD200 and neurociliary protein-1 may be involved in the pathogenesis of POI.

Moxibustion ameliorates ovarian function in premature ovarian insufficiency rats by activating cAMP/PKA/CREB to promote steroidogenesis in ovarian granulosa cells.

Premature ovarian insufficiency (POI) presents a substantial challenge to women's physiological and psychological well-being. Hormone replacement therapy, as the preferred therapeutic approach, involves solely exogenous supplementation of estrogen. Moxibustion, a traditional Chinese external treatment, has been investigated in our previous studies. It not only improves hormone levels and clinical symptoms in POI patients but also safeguards ovarian reserve. This study aims to explore the regulatory mechanisms by which moxibustion modulates hormone levels and restores ovarian function in POI. A POI rat model was established using cyclophosphamide, and moxibustion treatment was applied at acupoints "CV4" and "SP6" for a total of four courses. Subsequently, ovaries from each group were subjected to transcriptome sequencing (Bulk RNA-seq). Target pathways and key genes were selected through enrichment analysis and GSVA scoring, with validation using various techniques including electron microscopy, ELISA, Western blot, and immunohistochemistry. The results demonstrated that moxibustion restored the estrous cycle in POI rats, improved sex hormone levels, reduced the number of atretic follicles, and increased the count of dominant follicles (P<0.05). Bulk RNA-seq analysis revealed that moxibustion downregulated pathways associated with ovarian dysfunction, infertility, and immune responses, upregulated pathways related to follicular development and ovarian steroidogenesis. Furthermore, our data confirmed that moxibustion significantly increased the number of ovarian granulosa cells (GCs) and upregulated the expression of proteins related to steroidogenesis in GCs, including FSHR, P450 arom, cAMP, PKA, and CREB (P<0.05), with no significant effect observed on proteins related to steroidogenesis in theca cells. These outcomes aligned with the RNA-seq results. In conclusion, these findings propose that moxibustion enhances steroidogenesis in GCs through the activation of the cAMP/PKA/CREB pathway, consequently improving impaired ovarian function in POI rats. This study provides robust evidence supporting moxibustion as a targeted intervention for treating POI by specifically regulating steroidogenesis in GCs.

Gonadotropin-Releasing Hormone (GnRH) Agonists Do Not Protect Ovarian Function in Patients Undergoing Stem Cell Transplants.

Introduction Medical indications for fertility preservation include any malignancy, chronic illness, or disease that would require gonadotoxic chemotherapy or radiation (conditioning regimens), which would impede a woman's ability to conceive in the future. Thus, any patient who plans to undergo a gonadotoxic regimen is advised to cryopreserve oocytes or embryos, which can be used in the future at the patient's convenience. Attempts have been made to suppress ovarian function with gonadotropin-releasing hormone agonists (GnRH-a) to induce ovarian quiescence and, thereby, theoretically limit the gonadotoxic impact on the follicular pool. We explored the use of leuprolide (a type of GnRH-a) in preventing primary ovarian insufficiency (POI) in a cohort study of patients who underwent hematopoietic stem cell transplants (HSCT) at the National Institutes of Health (NIH); since the conditioning regimens for HSCT include cyclophosphamide and other gonadotoxic therapies, we hypothesized that GnRH-a would be ineffective in preventing POI. Methods We assessed patients who underwent fertility preservation prior to their stem cell transplant, as their follicular-stimulating hormone (FSH) levels were evaluated prior to and post-chemotherapy. Twenty-nine patients who underwent hormonal evaluation prior to and post-chemotherapy were included. The control group did not receive GnRH-a prior to chemotherapy, while the treatment group did receive GnRH-a pre-chemotherapy. Results Our data revealed that 80% of the control group had menopausal levels post-chemotherapy, while 91% of the treatment group still had menopausal levels post-chemotherapy (p=0.33). Conclusions Thus, our hypothesis that GnRH-a is ineffective in reducing the risk of POI in a cohort of patients who receive conditioning regimens for HSCT was confirmed.

X chromosome rearrangement associated with premature ovarian insufficiency as diagnosed by molecular cytogenetic methods: a case report and review of the literature.

BACKGROUND: Premature ovarian insufficiency (POI) is a clinical condition characterized by ovarian dysfunction in women under 40. The etiology of most POI cases remains unidentified and is believed to be multifactorial, including factors such as autoimmunity, metabolism, infection, and genetics. POI exhibits significant genetic heterogeneity, and it can result from chromosomal abnormalities and monogenic defects. CASE PRESENTATION: The study participant, a 33-year-old woman, presented with a history of irregular menstruation that commenced two years ago, progressing to prolonged menstrual episodes and eventual cessation. The participant exhibits a rearrangement of the X chromosome, characterized by heterozygosity duplication on the long arm and heterozygosity deletion on the short arm by whole exome sequencing(WES) combined with cell chromosome detection. CONCLUSIONS: This study expands the spectrum of mutations associated with POI resulting from X chromosomal abnormalities. WES-Copy number variation analysis, in conjunction with chromosome karyotype analysis and other detection techniques, can provide a more comprehensive understanding of the genetic landscape underlying complex single or multi-system diseases.

Birth weight and premature ovarian insufficiency: a systematic review and meta-analysis.

OBJECTIVE: To comprehensively evaluate the effect of low birth weight on premature ovarian insufficiency. METHODS: We performed a systematic review of the literature by searching MEDLINE, EMBASE, Web of Science, Scopus, Wanfang and CNKI up to August 2023. All cohort and case-control studies that included birth weight as an exposure and premature ovarian insufficiency as an outcome were included in the analysis. Data were combined using inverse-variance weighted meta-analysis with fixed and random effects models and between-study heterogeneity evaluated. We evaluated risk of bias using the Newcastle Ottawa Scale and using Egger's method to test publication bias. All statistical analyses were performed with the use of R software. RESULTS: Five articles were included in the review. A total of 2,248,594 women were included, including 21,813 (1%) cases of premature ovarian insufficiency, 150,743 cases of low birth weight, and 220,703 cases of macrosomia. We found strong evidence that changed the results of the previous review that low birth weight is associated with an increased risk of premature ovarian insufficiency (OR = 1.15, 95%CI 1.09-1.22) in adulthood compared with normal birth weight. No effect of macrosomia on premature ovarian insufficiency was found. CONCLUSIONS: Our meta-analysis showed strong evidence of an association between low birth weight and premature ovarian insufficiency. We should reduce the occurrence of low birth weight by various methods to avoid the occurrence of premature ovarian insufficiency.

miR-21-5p-loaded bone mesenchymal stem cell-derived exosomes repair ovarian function in autoimmune premature ovarian insufficiency by targeting MSX1.

RESEARCH QUESTION: What is the effect of micro-RNA (miR)-21-5p-loaded bone marrow mesenchymal stem cell-derived exosomes (miR-21-Exo) on autoimmune premature ovarian insufficiency (POI)? DESIGN: The Cell Counting Kit 8 (CCK8) assay, fluorescence-activated cell sorting, western blotting, quantitative reverse transcriptase (qRT)-PCR and enzyme-linked immunosorbent assay (ELISA) verified the effect of miR-21-Exo on interferon-γ (IFN-γ)-induced KGN cells. qRT-PCR, western blotting and dual-luciferase reporter gene assays verified that miR-21-Exo mediated Msh homeobox 1 (MSX1) regulation of the Notch signalling pathway and that miR-21 interacted directly with MSX1. The effects of miR-21-Exo on the ovaries were verified by monitoring of the oestrous cycle, haematoxylin and eosin staining, follicle counts, ELISA, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), western blotting and qRT-PCR. RESULTS: The results showed that miR-21-Exo promoted IFN-γ-induced KGN cell proliferation and hormone synthesis, and inhibited apoptosis. Using dual-luciferase reporter gene assays, miR-21 and MSX1 were shown to have direct interactions. Moreover, the findings elucidated that miR-21-Exo inhibited cell apoptosis and promoted hormone synthesis by mediating MSX1 to regulate the Notch signalling pathway. miR-21-Exo restored the ovarian structure in a mouse model of autoimmune POI, promoted endocrine function and proliferation, and inhibited apoptosis and inflammation in vivo. CONCLUSIONS: This study demonstrates that miR-21-Exo regulates the MSX1-mediated Notch signalling pathway to inhibit granulosa cell apoptosis and improve hormone synthesis function, providing insight into a potential mechanism of molecular therapy for the treatment of autoimmune POI.

Exosomes derived from mesenchymal stem cells attenuate NLRP3-related pyroptosis in autoimmune premature ovarian insufficiency via the NF-κB pathway.

RESEARCH QUESTION: What is the effect of exosomes derived from bone marrow mesenchymal stem cells (MSC-Exos) on the pyroptosis and recovery of granulosa cells in autoimmune premature ovarian insufficiency (POI)? DESIGN: In vitro, KGN cells were exposed to interferon-gamma to simulate immune injury. Samples were collected after a 48 h incubation with MSC-Exos (30 µg/ml). The cell viability, secretion of oestrogen and expression of key molecules in pyroptosis and the nuclear factor kappa B (NF-κB) pathway were tested. In vivo, the BALB/c mouse model of autoimmune POI model induced by zona pellucida glycoprotein 3 was used. Fertility testing and sample collection were applied 4 weeks after the ovarian subcapsular injection of MSC-Exos (150 µg for each ovary). Hormone concentration measurements, follicle counting and pyroptotic pathway analyses were conducted for each group. RESULTS: In vitro, MSC-Exos significantly promoted the proliferation rate and secretion of oestrogen, while at the same time suppressing apoptosis and pyroptosis. In vivo, exosomal treatment normalized the irregular oestrous cycles, rescued the follicular loss and increased the pregnancy rate and number of offspring in POI mice. Elevated serum concentrations of oestrogen and anti-Müllerian hormone, as well as decreased concentrations of FSH and interleukin-1ß, were shown. Furthermore, MSC-Exos down-regulated the expression of the NLRP3/Casp1/GSDMD pathway and inhibited activation of the NF-κB pathway. CONCLUSIONS: These findings demonstrate for the first time that MSC-Exos exert a significant effect on restoring ovarian function in autoimmune POI in vivo and in vitro by suppressing the NLRP3/Casp1/GSDMD pathway and pyroptosis. The NF-κB pathway may contribute to the regulation of NLRP3-related pyroptosis.

Shifting the landscape: Dominant C-terminal rare missense FOXL2 variants in non-syndromic primary ovarian failure etiology.

Pathogenic germline variants in the FOXL2 gene are associated with Blepharophimosis, Ptosis, and Epicanthus Inversus syndrome (BPES) in humans, an autosomal dominant condition. Two forms of BPES have emerged: (i) type I (BPES-I), characterized by ocular signs and primary ovarian failure (POI), and (ii) type II (BPES-II) with no systemic associations. This study aimed to compare the distribution of FOXL2 variants in idiopathic POI/DOR (diminished ovarian reserve) and both types of BPES, and to determine the involvement of FOXL2 in non-syndromic forms of POI/DOR. We studied the whole coding region of the FOXL2 gene using next-generation sequencing in 1282 patients with non-syndromic POI/DOR. Each identified FOXL2 variant was compared to its frequency in the general population, considering ethnicity. Screening of the entire coding region of the FOXL2 gene allowed us to identify 10 different variants, including nine missense variants. Of the patients with POI/DOR, 14 (1%) carried a FOXL2 variant. Significantly, six out of nine missense variants (67%) were overrepresented in our POI/DOR cohort compared to the general or specific ethnic subgroups. Our findings strongly suggest that five rare missense variants, mainly located in the C-terminal region of FOXL2 are high-risk factors for non-syndromic POI/DOR, though FOXL2 gene implication accounts for approximately 0.54% of non-syndromic POI/DOR cases. These results support the implementation of routine genetic screening for patients with POI/DOR in clinical settings.

Pyrroloquinoline quinone promotes human mesenchymal stem cell-derived mitochondria to improve premature ovarian insufficiency in mice through the SIRT1/ATM/p53 pathway.

BACKGROUND: DNA damage and oxidative stress induced by chemotherapy are important factors in the onset of premature ovarian insufficiency (POI). Studies have shown that mitochondria derived from mesenchymal stem cells (MSC-Mito) are beneficial for age-related diseases, but their efficacy alone is limited. Pyrroloquinoline quinone (PQQ) is a potent antioxidant with significant antiaging and fertility enhancement effects. This study aimed to investigate the therapeutic effect of MSC-Mito in combination with PQQ on POI and the underlying mechanisms involved. METHODS: A POI animal model was established in C57BL/6J mice by cyclophosphamide and busulfan. The effects of MSC-Mito and PQQ administration on the estrous cycle, ovarian pathological damage, sex hormone secretion, and oxidative stress in mice were evaluated using methods such as vaginal smears and ELISAs. Western blotting and immunohistochemistry were used to assess the expression of SIRT1, PGC-1α, and ATM/p53 pathway proteins in ovarian tissues. A cell model was constructed using KGN cells treated with phosphoramide mustard to investigate DNA damage and apoptosis through comet assays and flow cytometry. SIRT1 siRNA was transfected into KGN cells to further explore the role of the SIRT1/ATM/p53 pathway in combination therapy with MSC-Mito and PQQ for POI. RESULTS: The combined treatment of MSC-Mito and PQQ significantly restored ovarian function and antioxidant capacity in mice with POI. This treatment also reduced the loss of follicles at various stages, improving the disrupted estrous cycle. In vitro experiments demonstrated that PQQ facilitated the proliferation of MitoTracker-labelled MSC-Mito, synergistically restoring mitochondrial function and inhibiting oxidative stress in combination with MSC-Mito. Both in vivo and in vitro, the combination of MSC-Mito and PQQ increased mitochondrial biogenesis mediated by SIRT1 and PGC-1α while inhibiting the activation of ATM and p53, consequently reducing DNA damage-mediated cell apoptosis. Furthermore, pretreatment of KGN cells with SIRT1 siRNA reversed nearly all the aforementioned changes induced by the combined treatment. CONCLUSIONS: Our research findings indicate that PQQ facilitates MSC-Mito proliferation and, in combination with MSC-Mito, ameliorates chemotherapy-induced POI through the SIRT1/ATM/p53 signaling pathway.

Bu-Shen-Ning-Xin decoction inhibits macrophage activation to ameliorate premature ovarian insufficiency-related osteoimmune disorder via FSH/FSHR pathway.

Limited studies are associated with premature ovarian insufficiency (POI)-related osteoimmune disorder currently. Bu-Shen-Ning-Xin decoction (BSNXD) displayed a favorable role in treating postmenopausal osteoporosis. However, its impact on the POI-related osteoimmune disorder remains unclear. The study primarily utilized animal experiments and network pharmacology to investigate the effects and underlying mechanisms of BSNXD on the POI-related osteoimmune disorder. First, a 4-vinylcyclohexene dioxide (VCD)-induced POI murine model was conducted to explore the therapeutical action of BSNXD. Second, we analyzed the active compounds of BSNXD and predicted their potential mechanisms for POI-related osteoimmune disorder via network pharmacology, further confirmed by molecular biology experiments. The results demonstrated that VCD exposure led to elevated follicle-stimulating hormone (FSH) levels, a 50% reduction in the primordial follicles, bone microstructure changes, and macrophage activation, indicating an osteoimmune disorder. BSNXD inhibited macrophage activation and osteoclast differentiation but did not affect serum FSH and estradiol levels in the VCD-induced POI model. Network pharmacology predicted the potential mechanisms of BSNXD against the POI-related osteoimmune disorder involving tumor necrosis factor α and MAPK signaling pathways, highlighting BSNXD regulated inflammation, hormone, and osteoclast differentiation. Further experiments identified BSNXD treatment suppressed macrophage activation via downregulating FSH receptor (FSHR) expression and inhibiting the phosphorylation of ERK and CCAAT enhancer binding proteins ß. In conclusion, BSNXD regulated POI-related osteoimmune disorder by suppressing the FSH/FSHR pathway to reduce macrophage activation and further inhibiting osteoclastogenesis.

Transcriptome analysis during 4-vinylcyclohexene diepoxide exposure-induced premature ovarian insufficiency in mice.

The occupational chemical 4-Vinylcyclohexene diepoxide (VCD) is a reproductively toxic environmental pollutant that causes follicular failure, leading to premature ovarian insufficiency (POI), which significantly impacts a woman's physical health and fertility. Investigating VCD's pathogenic mechanisms can offer insights for the prevention of ovarian impairment and the treatment of POI. This study established a mouse model of POI through intraperitoneal injection of VCD into female C57BL/6 mice for 15 days. The results were then compared with those of the control group, including a comparison of phenotypic characteristics and transcriptome differences, at two time points: day 15 and day 30. Through a comprehensive analysis of differentially expressed genes (DEGs), key genes were identified and validated some using RT-PCR. The results revealed significant impacts on sex hormone levels, follicle number, and the estrous cycle in VCD-induced POI mice on both day 15 and day 30. The DEGs and enrichment results obtained on day 15 were not as significant as those obtained on day 30. The results of this study provide a preliminary indication that steroid hormone synthesis, DNA damage repair, and impaired oocyte mitosis are pivotal in VCD-mediated ovarian dysfunction. This dysfunction may have been caused by VCD damage to the primordial follicular pool, impairing follicular development and aggravating ovarian damage over time, making it gradually difficult for the ovaries to perform their normal functions.

POI-associated EIF4ENIF1 mutations exhibit impaired translation regulation abilities.

Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P < 0.0001) lower translation efficiency (TE) than empty vector or GFP overexpression control group. Surprisingly, EIF4ENIF1 Q842P overexpression failed to repress global translation, showing an overall TE significantly higher than WT group. Overexpression R208H significantly (P < 0.0001) lowered the overall TE, whereas exhibiting a reduced translation inhibitory effect on high-TE genes (TE > 2 in GFP control group). Several fertility-associated genes, such as AMH in Q842P group and SERPINE1 and THBS1 in R208H group, was translationally up-regulated in mutant groups versus WT control, suggesting a potential mechanism of mutated EIF4ENIF1 causing POI via impaired translation repression. It is further proposed that T&T-seq can be a sensitive evaluation tool for the measurement of functional alteration by variants in many other translational regulator genes, not only EIF4ENIF1, helping to eliminate misinterpretation of clinical significance of genetic variants.

Primary Amenorrhea and Premature Ovarian Insufficiency.

This review focuses on primary amenorrhea and primary/premature ovarian insufficiency due to hypergonadotropic hypogonadism. Following a thoughtful, thorough evaluation, a diagnosis can usually be discerned. Pubertal induction and ongoing estrogen replacement therapy are often necessary. Shared decision-making involving the patient, family, and health-care team can empower the young person and family to successfully thrive with these chronic conditions.

The mechanisms of MicroRNA 21 in premature ovarian insufficiency mice with mesenchymal stem cells transplantation : The involved molecular and immunological mechanisms.

Umbilical cord-derived mesenchymal stem cell (UCMSC) transplantation has been deeply explored for premature ovarian insufficiency (POI) disease. However, the associated mechanism remains to be researched. To explore whether and how the microRNA 21 (miR-21) functions in POI mice with UCMSCs transplantation, the autoimmune-induced POI mice model was built up, transplanted with or without UCMSCs transfect with the LV-hsa-miR-21-5p/LV-hsa-miR-21-5p-inhibition, with the transfection efficiency analyzed by QRT-PCR. Mice hormone secretion and the anti-Zona pellucida antibody (AZPAb) levels were analyzed, the ovarian morphological changes and folliculogenesis were observed, and the ovarian apoptosis cells were detected to evaluate ovarian function. The expression and localization of the PTEN/Akt/FOXO3a signal pathway-related cytokines were analyzed in mice ovaries.Additionally, the spleen levels of CD8 + CD28-T cells were tested and qualified with its significant secretory factor, interleukin 10 (IL-10). We found that with the LV-hsa-miR-21-5p-inhibition-UCMSCs transplantation, the mice ovarian function can be hardly recovered than mice with LV-NC-UCMSCs transplantation, and the PTEN/Akt/FOXO3a signal pathway was activated. The expression levels of the CD8 + CD28-T cells were decreased, with the decreased levels of the IL-10 expression. In contrast, in mice with the LV-hsa-miR-21-5p-UCMSCs transplantation, the injured ovarian function can be reversed, and the PTEN/AKT/FOXO3a signal pathway was detected activated, with the increased levels of the CD8 + CD28-T cells, and the increased serum levels of IL-10. In conclusion, miR-21 improves the ovarian function recovery of POI mice with UCMSCs transplantation, and the mechanisms may be through suppressing the PTEN/AKT/FOXO3a signal pathway and up-regulating the circulating of the CD8 + CD28-T cells.

Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

A 14-year-old girl with premature ovarian insufficiency but with a positive pregnancy test.

OBJECTIVES: Childhood cancer survivors are at risk for premature ovarian insufficiency, especially after treatment with alkylating agents. The objective of this report is to highlight a case in which this phenomenon caused a false-positive pregnancy test. CASE PRESENTATION: A workup was performed in a 14-year-old girl with a positive pregnancy test. She was diagnosed with stage IV neuroblastoma of the left adrenal gland at the age of 4 years. She received extensive treatment, including alkylating agents, and had been diagnosed with premature ovarian insufficiency. An LH/hCG suppression test was performed using high dose 17 bèta-estradiol: hCG levels normalized. CONCLUSIONS: The pregnancy test was false-positive due to production of low amounts of hCG by the pituitary gland as a result of high LH concentrations following premature ovarian insufficiency. It may be helpful to perform the LH/hCG suppression test to prove pituitary origin of the hCG overproduction.

Screening of premature ovarian insufficiency associated genes in Hungarian patients with next generation sequencing.

BACKGROUND: Premature ovarian insuffiency (POI) is one of the main cause behind infertility. The genetic analysis of POI should be part of the clinical diagnostics, as several genes have been implicated in the genetic background of it. The aim of our study was to analyse the genetic background of POI in a Hungarian cohort. METHODS: The age of onset was between 15 and 39 years. All patients had the 46,XX karyotype and they were prescreened for the most frequent POI associated FMR1 premutation. To identify genetic alterations next-generation sequencing (NGS) of 31 genes which were previously associated to POI were carried out in 48 unrelated patients from Hungary. RESULTS: Monogenic defect was identified in 16.7% (8 of 48) and a potential genetic risk factor was found in 29.2% (14 of 48) and susceptible oligogenic effect was described in 12.5% (6 of 48) of women with POI using the customized targeted panel sequencing. The genetic analysis identified 8 heterozygous damaging and 4 potentially damaging variants in POI-associated genes. Further 10 potential genetic risk factors were detected in seven genes, from which EIF2B and GALT were the most frequent. These variants were related to 15 genes: AIRE, ATM, DACH2, DAZL, EIF2B2, EIF2B4, FMR1, GALT, GDF9, HS6ST2, LHCGR, NOBOX, POLG, USP9X and XPNPEP2. In six cases, two or three coexisting damaging mutations and risk variants were identified. CONCLUSIONS: POI is characterized by heterogenous phenotypic features with complex genetic background that contains increasing number of genes. Deleterious variants, which were detected in our cohort, related to gonadal development (oogenesis and folliculogenesis), meiosis and DNA repair, hormonal signaling, immune function, and metabolism which were previously associated with the POI phenotype. This is the first genetic epidemiology study targeting POI associated genes in Hungary. The frequency of variants in different POI associated genes were similar to the literature, except EIF2B and GALT. Both of these genes potential risk factor were detected which could influence the phenotype, although it is unlikely that they can be responsible for the development of the disease by themselves. Advances of sequencing technologies make it possible to aid diagnostics of POI Since individual patients show high phenotypic variance because of the complex network controlling human folliculogenesis. Comprehensive NGS screening by widening the scope to genes which were previously linked to infertility may facilitate more accurate, quicker and cheaper genetic diagnoses for POI. The investigation of patient's genotype could support clinical decision-making process and pave the way for future clinical trials and therapies.

Human umbilical cord mesenchymal stem cells alleviate chemotherapy-induced premature ovarian insufficiency mouse model by suppressing ferritinophagy-mediated ferroptosis in granulosa cells.

Primary ovarian insufficiency (POI) in younger women (under 40) manifests as irregular periods, high follicle-stimulating hormone (FSH), and low estradiol (E2), often triggered by chemotherapy. Though mesenchymal stem cell (MSC) therapy shows promise in treating POI, its exact mechanism remains unclear. This study reveals that human umbilical cord-derived MSCs (hUC-MSCs) can protect ovarian granulosa cells (GCs) from cyclophosphamide (CTX)-induced ferroptosis, a form of cell death driven by iron accumulation. CTX, commonly used to induce POI animal model, triggered ferroptosis in GCs, while hUC-MSCs treatment mitigated this effect, both in vivo and in vitro. Further investigations using ferroptosis and autophagy inhibitors suggest that hUC-MSCs act by suppressing ferroptosis in GCs. Interestingly, hUC-MSCs activate a protective antioxidant pathway in GCs via NRF2, a stress-response regulator. Overall, our findings suggest that hUC-MSCs improve ovarian function in CTX-induced POI by reducing ferroptosis in GCs. This study not only clarifies the mechanism behind the benefits of hUC-MSCs but also strengthens the case for their clinical use in treating POI. Additionally, it opens up a new avenue for protecting ovaries from chemotherapy-induced damage by regulating ferroptosis.