Hormone replacement therapy in women with iatrogenic premature ovarian insufficiency after radiotherapy for cervical cancer: A retrospective cohort and survey study.

OBJECTIVES: This study assessed the uptake of hormone replacement therapy (HRT) in cervical cancer patients with iatrogenic menopause. Survival in relation to HRT use was assessed via a retrospective chart study, and the severity of menopausal symptoms, motivations and barriers to starting HRT were examined via questionnaires. STUDY DESIGN: In total, 293 women under the age of 51 and treated with radiotherapy for cervical cancer between 2010 and 2020 were identified. Medical records were searched for information on HRT use. If still living, women were sent digital questionnaires addressing menopausal symptoms, quality of life (QoL) and potential barriers and motivations concerning HRT use. Univariable data were analysed using Mann-Whitney U, chi-square, and Fisher's exact test, while logistical regression was used to analyse relationships between certain variables and use of HRT and survival. RESULTS: Overall HRT uptake was 78.1 %, which was related to age and Charlson Comorbidity Index, but independent of the duration of radiotherapy or FIGO stage. Overall survival was higher for HRT users (χ2(1) = 4.3, p = 0.038). Questionnaires were sent to 193 patients and 100 completed it (response rate 51.8 %). Main reasons for HRT use were relief of hot flushes and improvement in QoL. For women below age 51, QoL was indeed higher for current HRT users than for non-HRT users (EQ-index 0.8 vs. 0.7, p = 0.008). CONCLUSIONS: HRT prescription rate was inversely correlated with age. Survival was not negatively affected by HRT use. It is important to stress the benefits of HRT and address women's fears regarding its use.

Evaluation of the therapeutic effect of very small stem cells from peripheral blood on the treatment of Premature Ovarian Failure: A pilot study.

OBJECTIVE: Premature ovary failure (POF) is a severe health condition with multiple negative outcomes, which deteriorate a patient's life. The current study aimed to evaluate the therapeutic effect of mesenchymal stem cells (MSCs) derived from peripheral blood in the treatment of women with the POF background. METHODS: The current study was a pilot study carried-out on women younger than 40 with premature ovarian failure. Study participants underwent 4-months cell therapy using Mesenchymal stem cells extracted from peripheral bloods. Serum level of Follicle-stimulating hormone (FSH), Estradiol (E2), Anti-mullerian hormone (AMH), and Antral follicle count (AFC) were the main investigated outcomes that were assessed at baseline, month two and month four of the very small stem cell intervention. RESULTS: Average serum level of FSH was 45.0 (12.1) mIU/mL at baseline and continually decreased during the study and reached 33.2 (12.4) mIU/mL in the fourth month. The average AMH level was 0.10 ng/mL prior to the intervention and increased to 0.13 ng/mL in the 2nd month and 0.15 ng/mL in the fourth month. The level E2 was 85.7 (23.6) pg/ml on average at baseline, while the average E2 reduced to 77.2 (25.6) pg/ml in the fourth month. Average number of AFC was 2.0 (0.8) at baseline. We observed a gradual increase in the second month (Mean AFC=2.2) and after four months it increased to 3.1 (1.8) as the highest menstrual restoration and pregnancy was observed in 10% of our study participants. CONCLUSIONS: MSCs could significantly improve hormone secretion in women with POF. Implantation of MSCs in women with POF background was associated with an increase in AMH and AFC, while it downed the serum level of E2 and FSH. MSCs could also lead to menstrual restoration and pregnancy in women with POF.

Endometrial stem cells alleviate cisplatin-induced ferroptosis of granulosa cells by regulating Nrf2 expression.

BACKGROUND: Premature ovarian failure (POF) caused by cisplatin is a severe and intractable sequela for young women with cancer who received chemotherapy. Cisplatin causes the dysfunction of granulosa cells and mainly leads to but is not limited to its apoptosis and autophagy. Ferroptosis has been also reported to participate, while little is known about it. Our previous experiment has demonstrated that endometrial stem cells (EnSCs) can repair cisplatin-injured granulosa cells. However, it is still unclear whether EnSCs can play a repair role by acting on ferroptosis. METHODS: Western blotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were applied to detect the expression levels of ferroptosis-related genes. CCK-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assays were used to evaluate cell viability. Transmission electron microscopy (TEM) was performed to detect ferroptosis in morphology. And the extent of ferroptosis was assessed by ROS, GPx, GSSG and MDA indicators. In vivo, ovarian morphology was presented by HE staining and the protein expression in ovarian tissue was detected by immunohistochemistry. RESULTS: Our results showed that ferroptosis could occur in cisplatin-injured granulosa cells. Ferroptosis inhibitor ferrostatin-1 (Fer-1) and EnSCs partly restored cell viability and mitigated the damage of cisplatin to granulosa cells by inhibiting ferroptosis. Moreover, the repair potential of EnSCs can be markedly blocked by ML385. CONCLUSION: Our study demonstrated that cisplatin could induce ferroptosis in granulosa cells, while EnSCs could inhibit ferroptosis and thus exert repair effects on the cisplatin-induced injury model both in vivo and in vitro. Meanwhile, Nrf2 was validated to participate in this regulatory process and played an essential role.

Granulosa Cells-Related MicroRNAs in Ovarian Diseases: Mechanism, Facts and Perspectives.

MicroRNAs (miRNAs) are a class of short single-stranded, noncoding RNAs that affect the translation of mRNAs by imperfectly binding to homologous 3'UTRs. Research on miRNAs in ovarian diseases is constantly expanding because miRNAs are powerful regulators of gene expression and cellular processes and are promising biomarkers. miRNA mimics, miRNA inhibitors and molecules targeting miRNAs (antimiRs) have shown promise as novel therapeutic agents in preclinical development. Granulosa cells (GCs) are supporting cells for developing oocytes in the ovary. GCs regulate female reproductive health by producing sex hormones and LH receptors. Increasing research has reported the relevance of miRNAs in GC pathophysiology. With in-depth studies of disease mechanisms, there are an increasing number of studies on the biomolecular pathways of miRNAs in gynecology and endocrinology. In the present review, we summarize the different functions of GC-related microRNAs in various ovarian disorders, such as polycystic ovary syndrome, premature ovarian insufficiency, premature ovarian failure and ovarian granulosa cell tumors.

Transplantation of the LRP1high subpopulation of human umbilical cord-derived mesenchymal stem cells improves ovarian function in mice with premature ovarian failure and aged mice.

BACKGROUND: Premature ovarian failure (POF) has a profound impact on female reproductive and psychological health. In recent years, the transplantation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) has demonstrated unprecedented potential in the treatment of POF. However, the heterogeneity of human UC-MSCs remains a challenge for their large-scale clinical application. Therefore, it is imperative to identify specific subpopulations within UC-MSCs that possess the capability to improve ovarian function, with the aim of reducing the uncertainty arising from the heterogeneity while achieving more effective treatment of POF. METHODS: 10 × Genomics was performed to investigate the heterogeneity of human UC-MSCs. We used LRP1 as a marker and distinguished the potential therapeutic subpopulation by flow cytometry, and determined its secretory functions. Unsorted UC-MSCs, LRP1high and LRP1low subpopulation was transplanted under the ovarian capsules of aged mice and CTX-induced POF mice, and therapeutic effects was evaluated by assessing hormone levels, estrous cycles, follicle counts, and embryo numbers. RNA sequencing on mouse oocytes and granulosa cells after transplantation was performed to explore the mechanism of LRP1high subpopulation on mouse oocytes and granulosa cells. RESULTS: We identified three distinct functional subtypes, including mesenchymal stem cells, multilymphoid progenitor cells and trophoblasts. Additionally, we identified the LRP1high subpopulation, which improved ovarian function in aged and POF mice. We elucidated the unique secretory functions of the LRP1high subpopulation, capable of secreting various chemokines, cytokines, and growth factors. Furthermore, LRP1 plays a crucial role in regulating the ovarian microenvironment, including tissue repair and extracellular matrix remodeling. Consistent with its functions, the transcriptomes of oocytes and granulosa cells after transplantation revealed that the LRP1high subpopulation improves ovarian function by modulating the extracellular matrix of oocytes, NAD metabolism, and mitochondrial function in granulosa cells. CONCLUSION: Through exploration of the heterogeneity of UC-MSCs, we identified the LRP1high subpopulation capable of improving ovarian function in aged and POF mice by secreting various factors and remodeling the extracellular matrix. This study provides new insights into the targeted exploration of human UC-MSCs in the precise treatment of POF.

FTO attenuates the cytotoxicity of cisplatin in KGN granulosa cell-like tumour cells by regulating the Hippo/YAP1 signalling pathway.

Premature ovarian failure (POF) is a devastating condition for women under 40 years old. Chemotherapy, especially the use of cisplatin, has been demonstrated to promote the apoptosis of granulosa cells in primary and secondary follicles, leading to POF. Our previous studies demonstrated that fat mass- and obesity-associated (FTO) plays an essential role in protecting granulosa cells from cisplatin-induced cytotoxicity. Various studies have suggested that the Hippo/YAP signalling pathway plays a significant role in regulating cell apoptosis and proliferation. Additionally, YAP1 is the main downstream target of the Hippo signalling pathway and is negatively regulated by the Hippo signalling pathway. However, whether the Hippo/YAP signalling pathway is involved in the protective effect of FTO on granulosa cells has not been determined. In this study, we found that after cisplatin treatment, the apoptosis of granulosa cells increased in a concentration-dependent manner, accompanied by the downregulation of FTO and YAP1. Furthermore, overexpression of FTO decreased cisplatin-induced granulosa cell apoptosis, inhibited the Hippo/YAP kinase cascade-induced phosphorylation of YAP1, and promoted the entry of YAP1 into the nucleus. The downstream targets of YAP1 (CTGF, CYR61, and ANKRD1) were also increased. Si-RNA-mediated downregulation of FTO promoted cisplatin-induced granulosa cell apoptosis, activated the Hippo/YAP kinase cascade, and inhibited the YAP1 entry into the nucleus. These effects were completely reversed by the small molecule inhibitor of YAP1-verteporfin (VP). Taken together, these data suggested that FTO-YAP1 plays a positive role in regulating the proliferation of injured granulosa cells induced by cisplatin.

Inhibition of p38 MAPK/NF-κB p65 signaling pathway activity by rare ginsenosides ameliorates cyclophosphamide-induced premature ovarian failure and KGN cell injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C. A. Mey., one of the most used herbs in the world, shows effective treatment in reproductive injury. Recent studies have proven that the processed product, red ginseng, which is more active than ginseng itself. Therefore, it is speculated that its main functional component, rare ginsenosides (heat-transformed saponin, HTS), may be effective in treating premature ovarian failure (POF), but its efficacy has not yet been experimentally confirmed. AIM OF THE STUDY: To evaluate whether HTS could attenuate cyclophosphamide-induced inflammation and oxidative damage in POF model rats and the human granulosa-like KGN cell line and protect granulosa cell proliferation. MATERIAL AND METHODS: HTS were isolated from ginsenosides and high performance liquid chromatography (HPLC) analysis was used to analyze the HTS components. Cyclophosphamide (CP) was used to establish a POF rat model and KGN cell injury model. Reactive oxygen species (ROS) and antioxidant enzyme production was determined using specific assays, while inflammatory cytokine secretion was measured by enzyme-linked immunosorbent assay (ELISA). The proliferative function of granulosa cells was assessed using high-content screening and immunohistochemistry to determine the Ki67 protein level. Protein expression in ovarian tissues and KGN cells was analyzed by Western blotting, quantitative real-time PCR (qRT-PCR) was used to determine the transcriptional changes in ovarian tissues and KGN cells. RESULTS: In CP-treated POF model rats, HTS significantly decreased malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels, increased glutathione oxidase (GSH) levels, and upregulated Ki67 expression in ovarian granulosa cells. In addition, HTS significantly increased cell survival and Ki67 expression levels in CP-treated cells, and superoxide dismutase (SOD) levels were significantly increased. HTS significantly downregulated IL-6, TNF-α, and interleukin-1ß (IL-1ß) mRNA expression and significantly inhibited nuclear factor kappa-B p65 (NF-κB p65) and p38 mitogen activated protein kinase (p38 MAPK) phosphorylation in POF model rats and KGN cells. Moreover, NF-κB p65 and p38 MAPK levels were significantly increased in ovarian granulosa cells. p65 and p38 protein and gene expression was significantly downregulated. CONCLUSION: HTS ameliorated CP-induced POF and human granulosa cell injury, possibly by inhibiting inflammation and oxidative damage mediated by the p38 MAPK/NF-κB p65 signaling pathway.

HNF1A gene mutations and premature ovarian failure (POF): evidence from a clinical paradigm combining MODY 3 and POF.

Premature ovarian failure (POF) defines the occurrence of ovarian failure prior to the age of 40. It occurs in one out of 100 women but is very rare before age 20 (1:10,000). Maturity-onset diabetes of the young (MODY), caused by mutations in the HNF1A gene, is also a rare disorder; all types of MODY account for 1-2% of adult diabetic cases. These two rare nosologic entities coexisted in an adolescent girl evaluated for delayed puberty. Although this combination could represent a chance association, an interrelation might exist. We examined HNF1A expression in human fetal and adult ovaries by immunohistochemistry using a polyclonal HNF1A antibody. HNF1A protein was expressed in both the fetal and adult human ovaries. Based on these findings, we hypothesize that HNF1A participates in ovarian organogenesis and/or function and that mutations in the HNF1A gene might represent another molecular defect causing POF, possibly in combination with other genetic factors. The study underlines the importance of rare clinical paradigms in leading the way to elucidation of the pathogenetic mechanisms of rare diseases.

Intraovarian platelet-rich plasma injection and IVF outcomes in patients with poor ovarian response: a double-blind randomized controlled trial.

STUDY QUESTION: Does platelet-rich plasma (PRP) intraovarian injection increase the number of retrieved oocytes in successive ovarian punctions among patients with poor ovarian reserve (POR)? SUMMARY ANSWER: The injection of PRP increases the number of retrieved oocytes without increasing the quality of developed blastocysts. WHAT IS KNOWN ALREADY: Management of women with reduced ovarian response to stimulation is one of the significant challenges in reproductive medicine. Recently, PRP treatment has been proposed as an adjunct in assisted reproduction technology, with controversial results. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial included 60 patients with POR stratified according to the POSEIDON classification groups 3 and 4. It was conducted to explore the efficacy and safety of intraovarian PRP injection. Patients were proposed to undergo three consecutive ovarian stimulations to accumulate oocytes and were randomized to receive either PRP or placebo during their first oocyte retrieval. Randomization was performed using computer-generated randomization codes. Double blinding was ensured so that neither the participant nor the investigators knew of the treatment allotted. All patients underwent three ovarian stimulations and egg retrieval procedures. ICSI was performed after a third ovarian puncture. The primary endpoint was the number of mature oocytes retrieved after PRP or placebo injection in successive ovarian punctures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixty women (30-42 years) fulfilling inclusion criteria were randomized in equal proportions to the treatment or control groups. MAIN RESULTS AND THE ROLE OF CHANCE: The baseline demographic and clinical characteristics [age, BMI, anti-Müllerian hormone (AMH) levels] were comparable between the groups. Regarding the primary endpoint, the cumulative number (mean ± SEM) of retrieved mature oocytes was slightly higher in the treatment group: 10.45 ± 0.41 versus 8.91 ± 0.39 in the control group, respectively (95% CI of the difference 0.42-2.66; P = 0,008). The number of mature oocytes obtained among all patients increased in successive egg retrievals: 2.61 ± 0.33 (mean ± SEM) in punction 1 (P1), 3.85 ± 0.42 in P2, and 4.73 ± 0.44 in P3. However, the increase was higher among patients receiving the assessed PRP treatment. In P2, the number of retrieved mature oocytes was 4.18 ± 0.58 versus 3.27 ± 0.61 in controls (95% CI of the difference: -0.30 to 2.12; P = 0.138) and in P3, 5.27 ± 0.73 versus 4.15 ± 0.45 (95% CI of the difference: 0.12-2.12; P = 0.029). The mean ± SEM number of developed and biopsied blastocysts was 2.43 ± 0.60 in the control group and 1.90 ± 0.32 in the treatment group, respectively (P = 0.449). The mean number of euploid blastocysts was 0.81 ± 0.24 and 0.81 ± 0.25 in the control and treatment groups, respectively (P = 1.000). The percentages of patients with euploid blastocysts were 53.33% (16 out of 30) and 43.33% (13 out of 30) for patients in the control and treatment groups, respectively (Fisher's exact test P = 0.606). The overall pregnancy rate per ITT was 43% (26 out of 60 patients). However, the percentage of clinical pregnancies was higher in the control group (18 out of 30, 60%) than in the treatment group (8 out of 30, 27%) (P = 0.018). There was also a trend toward poorer outcomes in the treatment group when considering full-term pregnancies (P = 0.170). There were no differences between control and treatment groups regarding type of delivery, and sex of newborns. LIMITATIONS, REASONS FOR CAUTION: The mechanism of the potential beneficial effect of PRP injection on the number of retrieved oocytes is unknown. Either delivered platelet factors or a mechanical effect could be implicated. Further studies will be needed to confirm or refute the data presented in this trial and to specify the exact mechanism of action, if any, of PRP preparations. WIDER IMPLICATIONS OF THE FINDINGS: The increasing number of women with a poor response to ovarian stimulation supports the exploration of new areas of research to know the potential benefits of therapies capable of increasing the number of oocytes available for fertilization and improving the quality of developed blastocysts. An increase in the retrieved oocytes in both arms of the trial suggests that, beyond the release of growth factor from platelets, a mechanical effect can play a role. However, neither improvement in euploid blastocyst development nor pregnancy rates have been demonstrated. STUDY FUNDING/COMPETING INTEREST(S): This trial was supported by Basque Government and included in HAZITEK program, framed in the new Euskadi 2030 Science and Technology Plan (PCTI 2030). These aids are co-financed by the European Regional Development Fund (FEDER). The study funders had no role in the study design, implementation, analysis, manuscript preparation, or decision to submit this article for publication. No competing interests are declared by all the authors. TRIAL REGISTRATION NUMBER: Clinical Trial Number EudraCT 2020-000247-32. TRIAL REGISTRATION DATE: 3 November 2020. DATE OF FIRST PATIENT'S ENROLLMENT: 16 January 2021.

Ameliorative effect of salidroside on the cyclophosphamide-induced premature ovarian failure in a rat model.

BACKGROUND: Oxidative stress injury is an important pathological factor of premature ovarian failure (POF). Salidroside, extracted from the Chinese herb-Rhodiola rosea, has advantages in antioxidant characteristics. However, their therapeutic efficacy and mechanisms in POF have not been explored. PURPOSE: This study aims to assess the therapeutic effects of salidroside in chemotherapy-induced ovarian failure rats. METHODS: A POF rat model was established by injection of cyclophosphamide, followed by treatment with salidroside. The therapeutic effect of salidroside was evaluated based on hormone levels, follicle count, and reproductive ability. Oxidative stress injury was assessed by the detection of SOD enzyme activity and MDA levels. Differential gene expression of Keap1, Nrf2, HMOX1, NQO1, AMH, BMP15, and GDF9, were identified by qRT­PCR. The protein expression of Keap1, Nrf2, P53, and Bcl-2 were detected by western blot. RESULTS: Salidroside treatment markedly restored FSH, E2, and AMH hormone secretion levels, reduced follicular atresia, and increased antral follicle numbers in POF rats. In addition, salidroside improves fertility in POF rats, activates the Nrf2 signaling pathway, and reduces the level of oxidative stress. The recovery function of high dose salidroside (50 mg/kg) in a reproductive assay was significantly improved than that of lower dose salidroside (25 mg/kg). Meanwhile, the safety evaluation of salidroside treatment in rats showed that salidroside was safe for POF rats at doses of 25-50 mg/kg. CONCLUSIONS: Salidroside therapy improved premature ovarian failure significantly through antioxidant function and activating Nrf2 signaling.

Current Status and Future Prospects of Stem Cell Therapy for Infertile Patients with Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI), also known as premature menopause or premature ovarian failure, signifies the partial or complete loss of ovarian endocrine function and fertility before 40 years of age. This condition affects approximately 1% of women of childbearing age. Although 5-10% of patients may conceive naturally, conventional infertility treatments, including assisted reproductive technology, often prove ineffective for the majority. For infertile patients with POI, oocyte donation or adoption exist, although a prevalent desire persists among them to have biological children. Stem cells, which are characterized by their undifferentiated nature, self-renewal capability, and potential to differentiate into various cell types, have emerged as promising avenues for treating POI. Stem cell therapy can potentially reverse the diminished ovarian endocrine function and restore fertility. Beyond direct POI therapy, stem cells show promise in supplementary applications such as ovarian tissue cryopreservation and tissue engineering. However, technological and ethical challenges hinder the widespread clinical application of stem cells. This review examines the current landscape of stem cell therapy for POI, underscoring the importance of comprehensive assessments that acknowledge the diversity of cell types and functions. Additionally, this review scrutinizes the limitations and prospects associated with the clinical implementation of stem cell treatments for POI.

Gonadotropin upregulates intraovarian calpains-1 and -2 during ovarian follicular recruitment in the SD rat model.

Calpain role has been shown in the cumulus cell-oocyte complexes and, corpus luteum. We investigated the association of calpains-1 and -2 in ovarian folliculogenesis using the Sprague-Dawley (SD) rat model and steroidogenesis in the human granulosa cells (hGCs). We induced PCOS in 42-day-old SD rats by letrozole oral gavage for 21 days. Premature ovarian failure (POF) was induced in 21-day-old SD rats by 4-vinylcyclohexene diepoxide (VCD). Ovulation and ovarian hyperstimulatory (OHS) syndrome were induced by pregnant mare gonadotropin (PMSG) + human chorionic gonadotropin (hCG) treatments in 21 days SD rats, respectively. Steroidogenesis is stimulated in human granulosa cells (hGCs) by forskolin and the response of 17-beta-estradiol (E2) on calpains expression was checked in hGCs. The protein expression by immunoblotting and activity by biochemical assay of calpains-1 and -2 showed an oscillating pattern in the ovarian cycle. PMSG-induced follicular recruitment showed upregulation of calpains-1 and -2, but with no change during ovarian function cessation (POF). Upregulated calpain-2 expression and calpain activity was found in the hCG +PMSG-induced ovulation. Letrozole-induced PCOS showed downregulation of calpain-1, but upregulation of calpain-2. PMSG+hCG-induced OHS led to the upregulation of calpain-1. Letrozole and metformin separately increased the expression level of calpains-1 and -2 in the hGCs during luteinization. In conclusion, the expression levels of calpains -1 and -2 are increased with ovarian follicular recruitment by PMSG and calpain-1 is decreased in the PCOS condition, and letrozole and metformin upregulate the expression of calpains-1 and -2 during luteinization in the hGCs possibly via E2 action.

Efficacy of natural products on premature ovarian failure: a systematic review and meta-analysis of preclinical studies.

OBJECTIVE: This study aims to investigate the effects of natural products on animal models of premature ovarian failure (POF). METHODS: We conducted comprehensive literature searches and identified relevant studies that examined the protective effects of natural products on experimental POF. We extracted quantitative data on various aspects such as follicular development, ovarian function, physical indicators, oxidative stress markers, inflammatory factors, and protein changes. The data was analyzed using random-effects meta-analyses, calculating pooled standardized mean differences and 95% confidence intervals. Heterogeneity was assessed using the I2 statistic, and bias was estimated using the SYRCLE tool. RESULTS: Among the 879 reviewed records, 25 articles met our inclusion criteria. These findings demonstrate that treatment with different phytochemicals and marine natural products (flavonoids, phenols, peptides, and alkaloids, etc.) significantly improved various aspects of ovarian function compared to control groups. The treatment led to an increase in follicle count at different stages, elevated levels of key hormones, and a decrease in atretic follicles and hormone levels associated with POF. This therapy also reduced oxidative stress (specifically polyphenols, resveratrol) and apoptotic cell death (particularly flavonoids, chrysin) in ovarian granulosa cells, although it showed no significant impact on inflammatory responses. The certainty of evidence supporting these findings ranged from low to moderate. CONCLUSIONS: Phytochemicals and marine natural product therapy (explicitly flavonoids, phenols, peptides, and alkaloids) has shown potential in enhancing folliculogenesis and improving ovarian function in animal models of POF. These findings provide promising strategies to protect ovarian reserve and reproductive health. Targeting oxidative stress and apoptosis pathways may be the underlying mechanism.

L-carnitine fails to rescue chemotherapy injured ovaries by epigenetic changes of transcription factors.

Cyclophosphamide (CP), as an anti-cancer drug, is frequently used to treat various types of cancer. A decreased number of ovarian follicles impaired normal ovarian function, and subsequent premature ovarian failure (POF) presented as a side effect of cyclophosphamide usage. These events may eventually affect the fertility rate of individuals. The present study showed the effect of cyclophosphamide on ovarian reserves and the protective effect of L-carnitine (LC) as an antioxidant to prevent POF. To design the study, six to eight-week-old NMRI female mice were divided into three groups: control, cyclophosphamide (CP), and cyclophosphamide + L-carnitine (CP + LC). Mice received drugs intraperitoneally (IP) for 21 days. In the following 24 h after the last injection, both ovaries were used to evaluate the expression of Sohlh1 and Lhx8 genes by Real-time PCR. Furthermore, the alteration of Lhx8 promoter methylation was examined by Methylation-sensitive high-resolution melting analysis (MS-HRM). The present data showed the negative effect of CP on regulator genes of oogenesis including Sohlh1 and Lhx8. In addition, an examination of the epigenetic status of the Lhx8 gene showed a change in promoter methylation of this gene following cyclophosphamide injection. Although, L-carnitine is an effective antioxidant in relieving oxidative stress caused by cyclophosphamide and its damage, in the present study, however, the use of L-carnitine failed to protect the ovaries from changes caused by CP injection. So, using cyclophosphamide can alter the expression of folliculogenesis genes through its effects on epigenetic changes and may cause POF. The results of the present study showed that L-carnitine consumption can't protect the ovaries against the adverse effects of CP.

Zishen Yutai pills restore fertility in premature ovarian failure through regulating arachidonic acid metabolism and the ATK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The Zishen Yutai pills (ZYP), a Chinese medicinal formulation derived from the Qing Dynasty prescription "Shou Tai pills", have been documented to exhibit beneficial effects in clinical observations treating premature ovarian failure (POF). However, the anti-POF effects and its comprehensive systemic mechanism have not yet been clarified. AIM OF THE REVIEW: Therapeutic effects and systemic mechanism of ZYP in POF were evaluated. MATERIALS AND METHODS: After pulverization, sieving, and stirring, ZYP was administered intragastrically to cisplatin-induced POF mice at a dose of 1.95 mg/kg/d for 14 days. The anti-POF effects of ZYP were investigated by assessing the number of ovarian follicles at different developmental stages, as well as measuring serum estradiol (E2) levels and ovarian-expressed anti-Müllerian hormone (AMH). Reproductive performance and offspring health were evaluated to predict fertility restoration. Furthermore, a combination of proteomic and metabolomic profiling was employed to elucidate the underlying molecular mechanism of ZYP in treating POF. Western blot (WB) analyses and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to explore the mechanisms through which ZYP exerted its anti-POF effects. RESULTS: We have demonstrated that oral administration of ZYP reversed the reduction in follicles at different developmental stages and stimulated the expressions of serum E2 and ovarian-expressed AMH in a cisplatin-induced POF model. Additionally, ZYP ameliorated follicle apoptosis in ovaries affected by cisplatin-induced POF. Furthermore, treatment with ZYP restored the quantity and quality of oocytes, as well as enhanced fertility. Our results revealed 62 differentially expressed proteins (DEPs) through proteomic analyses and identified 26 differentially expressed metabolites (DEMs) through metabolomic analyses. Both DEPs and DEMs were highly enriched in the arachidonic acid (AA) metabolism pathway. ZYP treatment effectively upregulated the protein and mRNA expression of critical targets in AA metabolism and the AKT pathway, including CYP17α1, HSD3ß1, LHR, STAR, and AKT, in cisplatin-induced POF mice. CONCLUSIONS: These results indicated that ZYP exerted protective effects against POF and restored fertility from cisplatin-induced apoptosis. ZYP could be a satisfying alternative treating POF.

Protective effect of Luffa cylindrica fermentation liquid on cyclophosphamide-induced premature ovarian failure in female mice by attenuating oxidative stress, inflammation and apoptosis.

Premature ovarian failure (POF) is a leading cause of women's infertility without effective treatment. The purpose of this study was to investigate the protective effects of Luffa cylindrica fermentation liquid (LF) on cyclophosphamide (CTX) -induced POF in mice and to preliminarily investigate the underlying mechanisms. Thirty-two Balb/c mice were divided into four groups randomly. One group served as the control, while the other three received CTX injections to establish POF models. A 14-day gavage of either 5 or 10 µL/g LF was administered to two LF pretreatment groups. To analyze the effects of LF, the ovarian index, follicle number, the levels of serum sex hormones, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), inflammatory factors, and apoptosis of the ovarian cells were measured. The effects of LF pretreatment on the expression of TLR4/NF-κB and apoptosis pathways were also evaluated. We found that LF pretreatment increased the ovarian index and the number of primordial and antral follicles while decreasing those of atretic follicles. LF pretreatment also increased the serum levels of estradiol (E2) and anti-Müllerian hormone (AMH), while decreasing those of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Furthermore, LF pretreatment increased the levels of SOD and GSH in the ovaries, while decreasing those of MDA, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). LF administration reduced the amount of TUNEL+ ovarian cells and the levels of TLR4 and NF-κB P65 protein expression. In conclusion, LF has antioxidant, anti-inflammatory as well as anti-apoptotic effects against CTX-induced POF, and the inhibition of TLR4/NF-κB and apoptosis pathways may be involved in its mechanisms.

Danggui Shaoyao San protects cyclophosphamide-induced premature ovarian failure by inhibiting apoptosis and oxidative stress through the regulation of the SIRT1/p53 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: It has been reported that apoptosis and oxidative stress are related to cyclophosphamide (CYC)-induced premature ovarian failure (POF). Therefore, anti-apoptotic and anti-oxidative stress treatments exhibit therapeutic efficacy in CYC-induced POF. Danggui Shaoyao San (DSS), which has been extensively used to treat gynecologic diseases, is found to inhibit apoptosis and reduce oxidative stress. However, the roles of DSS in regulating apoptosis and oxidative stress during CYC-induced POF, and its associated mechanisms are still unknown. AIM OF THE STUDY: This work aimed to investigate the roles and mechanisms of DSS in inhibiting apoptosis and oxidative stress in CYC-induced POF. MATERIALS AND METHODS: CYC (75 mg/kg) was intraperitoneally injected in mice to construct the POF mouse model for in vivo study. Thereafter, alterations of body weight, ovary morphology and estrous cycle were monitored to assess the ovarian protective properties of DSS. Serum LH and E2 levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was employed for examining ovarian pathological morphology and quantifying follicles in various stages. Meanwhile, TUNEL staining and apoptosis-related proteins were adopted for evaluating apoptosis. Oxidative stress was measured by the levels of ROS, MDA, and 4-HNE. Western blot (WB) assay was performed to detect proteins related to the SIRT1/p53 pathway. KGN cells were used for in vitro experiment. TBHP stimulation was carried out for establishing the oxidative stress-induced apoptosis cell model. Furthermore, MTT assay was employed for evaluating the protection of DSS from TBHP-induced oxidative stress. The anti-apoptotic ability of DSS was evaluated by hoechst/PI staining, JC-1 staining, and apoptosis-related proteins. Additionally, the anti-oxidative stress ability of DSS was measured by detecting the levels of ROS, MDA, and 4-HNE. Proteins related to SIRT1/p53 signaling pathway were also measured using WB and immunofluorescence (IF) staining. Besides, SIRT1 expression was suppressed by EX527 to further investigate the role of SIRT1 in the effects of DSS against apoptosis and oxidative stress. RESULTS: In the in vivo experiment, DSS dose-dependently exerted its anti-apoptotic, anti-oxidative stress, and ovarian protective effects. In addition, apoptosis, apoptosis-related protein and oxidative stress levels were inhibited by DSS treatment. DSS treatment up-regulated SIRT1 and down-regulated p53 expression. From in vitro experiment, it was found that DSS treatment protected KGN cells from TBHP-induced oxidative stress injury. Besides, DSS administration suppressed the apoptosis ratio, apoptosis-related protein levels, mitochondrial membrane potential damage, and oxidative stress. SIRT1 suppression by EX527 abolished the anti-apoptotic, anti-oxidative stress, and ovarian protective effects, as discovered from in vivo and in vitro experiments. CONCLUSIONS: DSS exerts the anti-apoptotic, anti-oxidative stress, and ovarian protective effects in POF mice, and suppresses the apoptosis and oxidative stress of KGN cells through activating SIRT1 and suppressing p53 pathway.

Microfluidic Encapsulation of Exosomes Derived from Lipopolysaccharide-Treated Mesenchymal Stem Cells in Hyaluronic Acid Methacryloyl to Restore Ovarian Function in Mice.

Premature ovarian failure (POF) features an upward incidence nowadays, and the human umbilical cord mesenchymal stem cells (hUC-MSCs)-derived exosomes (MSC-Exos) have shown applied values in the recovery of ovarian function. Here, a novel exosome-encapsulated microcarrier prepared by microfluidic technology for ovarian repair after chemotherapy damage is presented. The exosomes derived from lipopolysaccharide (LPS)-preconditioned hUC-MSCs are encapsulated with hyaluronic acid methacryloyl (HAMA) via microfluidic electrospray, which is named HAMA/MSC-Exos. Attributing to the biocompatibility and semipermeable property of HAMA, the encapsulated exosomes show great viability and controllable release behavior from HAMA. It is demonstrated that in situ transplantation of HAMA/MSC-Exos can rescue ovarian functions of cyclophosphamide-induced ovarian failure in mice by increasing ovarian volume, improving the number of antral follicles and restoring fertility. It is believed that the transplantation of HAMA/MSC-Exos will provide a new concept for the treatment of POF in clinical practice.

Comprehensive analysis of lncRNA-miRNA-mRNA ceRNA network and key genes in granulosa cells of patients with biochemical primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is a common condition leading to the pathological decline of ovarian function in women of reproductive age, resulting in amenorrhea, hypogonadism, and infertility. Biochemical premature ovarian insufficiency (bPOI) is an intermediate stage in the pathogenesis of POI in which the fertility of patients has been reduced. Previous studies suggest that granulosa cells (GCs) play an essential role in the pathogenesis of POI, but their pathogenetic mechanisms remain unclear. To further explore the potential pathophysiological mechanisms of GCs in POI, we constructed a molecular long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) network using GC expression data collected from biochemical premature ovarian failure (bPOI) patients in the GEO database. We discovered that the GCs of bPOI patients had differential expression of 131 mRNAs, 191 lncRNAs, and 28 miRNAs. By systematic network analysis, we identified six key genes, including SRSF1, PDIA5, NEURL1B, UNK, CELF2, and CFL2, and five hub miRNAs, namely hsa-miR-27a-3p, hsa-miR-24-3p, hsa-miR-22-3p, hsa-miR-129-5p, and hsa-miR-17-5p, and the results suggest that the expression of these key genes may be regulated by two hub miRNAs, hsa-miR-27a-3p and hsa-miR-17-5p. Additionally, a POI model in vitro was created to confirm the expression of a few important genes. In this study, we discovered a unique lncRNA-miRNA-mRNA network based on the ceRNA mechanism in bPOI for the first time, and we screened important associated molecules, providing a partial theoretical foundation to better understand the pathogenesis of POI.

Mechanism Exploration of Environmental Pollutants on Premature Ovarian Insufficiency: a Systematic Review and Meta-analysis.

As a public health problem, premature ovarian insufficiency leads to infertility or sub-fertility. In addition to premature ovarian insufficiency (POI) increases the lifetime risk of bone fragility, cardiovascular disease, and cognitive impairment. To investigate the effects of environmental pollutants on the occurrence of POI and explore its mechanism, we conducted a computer search for articles published in electronic databases by December 13, 2022. Three reviewers independently examined all included studies and scored the qualities of included studies using the Newcastle-Ottawa Scale criteria. In this meta-analysis, eight clinical studies as well as ten preclinical findings showed a pooled OR of 2.331 and 95% CI of 1.968-2.760. This confirms that environmental pollutants, including POPs, heavy metals, PAEs, PAHs, cosmetic and pharmaceutical products, and cigarette smoke, are indeed significant risk factors for POI. In addition, it is demonstrated from the results of this study that signaling pathway of calcium and PI3K Akt and Xpnpep2, Col1, Col3, Col4, Cx43, Egr3, Tff1, and Ptgs2 genes may all be involved in the process. Environmental pollutants, including POPs, heavy metals, PAEs, PAHs, cosmetic and pharmaceutical products, and cigarette smoke, are indeed significant risk factors for POI.