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.

Menstrual blood-derived endometrial stem cell, a unique and promising alternative in the stem cell-based therapy for chemotherapy-induced premature ovarian insufficiency.

Chemotherapy can cause ovarian dysfunction and infertility since the ovary is extremely sensitive to chemotherapeutic drugs. Apart from the indispensable role of the ovary in the overall hormonal milieu, ovarian dysfunction also affects many other organ systems and functions including sexuality, bones, the cardiovascular system, and neurocognitive function. Although conventional hormone replacement therapy can partly relieve the adverse symptoms of premature ovarian insufficiency (POI), the treatment cannot fundamentally prevent deterioration of POI. Therefore, effective treatments to improve chemotherapy-induced POI are urgently needed, especially for patients desiring fertility preservation. Recently, mesenchymal stem cell (MSC)-based therapies have resulted in promising improvements in chemotherapy-induced ovary dysfunction by enhancing the anti-apoptotic capacity of ovarian cells, preventing ovarian follicular atresia, promoting angiogenesis and improving injured ovarian structure and the pregnancy rate. These improvements are mainly attributed to MSC-derived biological factors, functional RNAs, and even mitochondria, which are directly secreted or indirectly translocated with extracellular vesicles (microvesicles and exosomes) to repair ovarian dysfunction. Additionally, as a novel source of MSCs, menstrual blood-derived endometrial stem cells (MenSCs) have exhibited promising therapeutic effects in various diseases due to their comprehensive advantages, such as periodic and non-invasive sample collection, abundant sources, regular donation and autologous transplantation. Therefore, this review summarizes the efficacy of MSCs transplantation in improving chemotherapy-induced POI and analyzes the underlying mechanism, and further discusses the benefit and existing challenges in promoting the clinical application of MenSCs in chemotherapy-induced POI.

Efficiency and safety of acupuncture for women with premature ovarian insufficiency: study protocol for a randomized controlled trial.

Acupuncture has been widely used as an alternative and complementary therapy for premature ovarian insufficiency (POI) in China. However, research to date has not shown that acupuncture is effective for POI compared with hormone replacement therapy (HRT). We will conduct a randomized, controlled, and outcome assessor-blind trial to evaluate the efficacy and safety of acupuncture on POI. Seventy-six patients with POI will be randomly assigned to two groups. The treatment group will receive twenty-eight one-hour sessions of acupuncture treatments, and the control group will receive 12-week HRT. The whole study will consist of a 12-week treatment plan and a 12-week follow-up session. The primary outcome is measured by changes in serum anti-Müllerian hormone and follicle-stimulating hormone (FSH) levels at weeks 12 and 24. Secondary outcome measures include estradiol, luteinizing hormone (LH), LH/FSH ratio, Kupperman index, and menstrual condition. This trial is expected to clarify whether or not acupuncture is effective and safe for POI compared with HRT.

LIPUS combined with TFSC alleviates premature ovarian failure by promoting autophagy and inhibiting apoptosis.

OBJECTIVE: Premature ovarian failure (POF), also known as primary ovarian insufficiency, is a major cause of infertility in female worldwide. Excessive apoptosis and impaired autophagy in ovarian granulosa cells are the main pathological mechanisms of POF. The total flavonoids from semen cuscutae (TFSC) are often used in the treatment of gynecological endocrine disorders. In addition, low intensity pulsed ultrasound (LIPUS) is report as an effective method to improve ovarian function. This study aims to investigate the protective effect of POF by the combined use of TFSC and LIPUS. METHODS: POF rats model and granulosa cell model were successfully induced by tripterygium glycosides and cyclophosphamide, respectively. After that, model rats and cells received TFSC plus LIPUS administration. Then ovarian histomorphology, senescence, estrus cycle, and serum sex hormone levels were detected in rats. Ovarian tissue and granulosa cells autophagy and apoptosis levels were also assessed. RESULTS: Disturbed sex hormone levels, atrophied and senescent ovaries, and abnormal estrous cycle were found in POF rats. Meanwhile, cell autophagy was inhibited and cell apoptosis was activated in POF ovarian tissue and granulosa cells. However, TFSC combined with LIPUS improved these changes, and this combination treatment exhibited synergistic effects. The abnormal expression of the cell apoptosis-, autophagy-, and PI3K/AKT/mTOR signaling pathway-related proteins were also improved by combination treatment. CONCLUSION: The study found that the combination of TFSC and LIPUS can alleviate POF by modulating cell autophagy and apoptosis. The findings may provide a viable scientific basis for POF treatment.

[Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress].

OBJECTIVE: To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress. METHODS: Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot. RESULTS: Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01). CONCLUSION: Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.

[Effect of penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on expression of TRAIL and its receptors in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effect of penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on the expressions of death receptor pathway-related protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, as death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) in premature ovarian insufficiency (POI) rats, so as to explore its mechanisms underlying improvement of POI. METHODS: Forty female SD rats were randomly divided into blank control, model, penetrative needling and medication (estradiol valerate) groups, with 10 rats in each group. The POI model was established by intraperitoneal injection of cyclophosphamide (D1: 50 mg·kg-1·d-1, D2 to D15: 8 mg·kg-1·d-1, for a total of 15 d). After successful modeling, the rats in the penetrative needling group received penetrative needling of BL54 through ST28, with the needle retained for 30 min, once a day for a total of 4 weeks. Rats of the medication group received gavage of estradiol valerate (0.09 mg·kg-1·d-1) once daily for 4 weeks. After the intervention, the content of serum follicles of stimulation hormone (FSH),lateinizing hormone (LH),estradiol (E2) and vascular endothelial growth factor (VEGF) were assayed using enzyme-linked immunosorbent assay, and histopathological changes of ovarian tissue and the number of follicles were observed under light microscope after H.E. staining. The expression levels of TRAIL, DR4, DR5, DcR1, DcR2, and Fas-associated death domain (FADD) in ovarian tissues were detected using quantitative real-time PCR, and the immunoactivity of ovarian TRAIL, DR4 and DR5 detected using immunohistochemistry. The body weight and the damp weight of ovary were measured for calculating the ovarian coefficient. RESULTS: Compared with the blank control group, the E2 and VEGF contents, ovarian coefficient, and the number of the primary, secondary and sinus follicles were significantly decreased (P<0.01) in the model group, whereas FSH and LH contents, the atretic follicle number, TRAIL, DR4 and DR5 immunoactivity, and the expression levels of TRAIL, DR4, DR5 and FADD mRNAs considerably increased in the model group (P<0.01). In comparison with the model group, the decrease of the VEGF content, ovarian coefficient, and the number of the primary, secondary and sinus follicles, and the increase of the atretic follicle number, TRAIL, DR4 and DR5 immunoactivity, and expression levels of TRAIL, DR4, DR5 and FADD mRNAs were reversed in both penetrative needling and medication groups (P<0.01, P<0.05). The number of primary follicles was significantly more in the medication group than in the penetrative needling group (P<0.01). CONCLUSION: Penetrative needling of BL54 and ST28 can improve ovarian weight and promote follicular development in POI rats, which may be associated with its function in down-regulating the expression of pro-apoptotic proteins TRAIL, DR4, DR5 and FADD of the death receptor pathway to inhibit apoptosis of ovarian granulosa cells.

[Protective effect of moxibustion preconditioning in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effects of moxibustion preconditioning on ovarian function, fertility and ovarian granulosa cell apoptosis in rats with premature ovarian insufficiency (POI), so as to investigate its underlying mechanism in improving POI. METHODS: Forty-two female SD rats with two complete estrous cycles were randomly divided into control group, model group and pre-moxibustion group, with 14 rats in each group. The pre-moxibustion group was pretreated with mild moxibustion for 14 days before POI model establishment at 1) "Guanyuan" (CV4) and "Zhongwan" (CV12) and 2) bilateral "Shenshu" (BL23) as two sets of acupoints on alternate days, once each day, for 10 min each acupoint. After 14-day mild moxibustion intervention, 75 mg·kg-1·d-1 tripterygium glycoside tablet suspension was administered to rats in the pre-moxibustion group and the model group by gavage, for 14 consecutive days, while equivalent saline was given to rats in the control group in the same way. After modeling, the effect of moxibustion preconditioning on ovarian reserve function was evaluated by the estrous cycles, pregnancy rate and embryo number, morphological changes of ovaries, and serum sex hormone levels. TUNEL staining was used to detect the rate of granulosa cell apoptosis in ovaries. Immunohistochemistry and real time quantitative PCR were used to detect the relative expression of Caspase-3 and Caspase-9 proteins and mRNA levels in ovaries. RESULTS: Compared with the control group, the estrous cycles were disturbed; the pregnancy rate and number of embryos, the wet weight of ovary and ovarian index, the number of total follicles and different level of follicles, serum Estradiol (E2) and anti-mullerian hormone (AMH) levels were all significantly decreased (P<0.01,P<0.05), while the number of atretic follicles, serum follicule-stimulating hormone (FSH) and luteinizing hormone (LH) levels, the number of TUNEL-positive granulosa cells, the expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were significantly increased (P<0.01) in the model group. Compared with the model group, the disordered estrous cycles were improved; the pregnancy rate, the embryo numbers, the wet weight of ovary, and the total follicle number and primary follicle number, serum AMH level were significantly increased (P<0.01，P<0.05), while the number of atretic follicles, serum FSH level, the number of TUNEL-positive granulosa cells, expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were all significantly decreased (P<0.01,P<0.05) in the moxibustion group. CONCLUSION: Moxibustion preconditioning could improve ovarian function and improve fertility of POI rats, which may be associated with reducing the apoptosis of ovarian granulosa cells.

BCAA insufficiency leads to premature ovarian insufficiency via ceramide-induced elevation of ROS.

Premature ovarian insufficiency (POI) is a disease featured by early menopause before 40 years of age, accompanied by an elevation of follicle-stimulating hormone. Though POI affects many aspects of women's health, its major causes remain unknown. Many clinical studies have shown that POI patients are generally underweight, indicating a potential correlation between POI and metabolic disorders. To understand the pathogenesis of POI, we performed metabolomics analysis on serum and identified branch-chain amino acid (BCAA) insufficiency-related metabolic disorders in two independent cohorts from two clinics. A low BCAA diet phenotypically reproduced the metabolic, endocrine, ovarian, and reproductive changes of POI in young C57BL/6J mice. A mechanism study revealed that the BCAA insufficiency-induced POI is associated with abnormal activation of the ceramide-reactive oxygen species (ROS) axis and consequent impairment of ovarian granulosa cell function. Significantly, the dietary supplement of BCAA prevented the development of ROS-induced POI in female mice. The results of this pathogenic study will lead to the development of specific therapies for POI.

Moxibustion mitigates mitochondrial dysfunction and NLRP3 inflammatory activation in cyclophosphamide-induced premature ovarian insufficiency rats.

AIMS: This study aimed to investigate the protective effects of moxibustion on ovarian dysfunction in rats with cyclophosphamide (Cy)-induced premature ovarian insufficiency (POI). It also aimed at revealing its potential mechanisms and emphasizing its role in mitigating the mitochondrial dysfunction and NLRP3 inflammatory activation. MATERIALS AND METHODS: POI models were established by the intraperitoneal administration of Cy using female Sprague-Dawley rats. Moxibustion (BL23 or CV4, CV8) was used to treat POI models for fifteen days. Vaginal smears, enzyme-linked immunosorbent assay, hematoxylin-eosin, tunnel staining, flow cytometry analysis, immunohistochemistry staining, qRT-PCR, and western blotting were conducted to evaluate the ovarian function, mitochondrial dysfunction, and NLRP3 inflammatory activation in this study. KEY FINDINGS: Moxibustion could improve the disorder of the estrous cycles and reproductive hormone levels, promote follicular growth, reduce the number of atresia follicles, and alleviate the apoptosis of ovarian granulosa cells (GCs) in rats with POI. Furthermore, moxibustion mitigated the mitochondrial damage, reversed the elevated serum levels of IL-18 and IL-1ß, and decreased their protein expression in the ovaries of rats with POI. Moxibustion significantly inhibited the expression of the mRNAs and proteins of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase 1, and gasdermin D (GSDMD) in the ovaries of rats with POI. SIGNIFICANCE: These results supported that moxibustion may ameliorate Cy-induced POI by mitigating the mitochondrial dysfunction and NLRP3 inflammatory activation. Targeted treatment of mitochondrial damage and NLRP3 inflammatory activation may be a novel therapeutic strategy for POI.

Electroacupuncture attenuates ac4C modification of P16 mRNA in the ovarian granulosa cells of a mouse model premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is a type of pathological aging, which seriously interferes with the fertility of affected women. Electroacupuncture (EA) may have a beneficial effect; however, its mechanism of action is unknown. The purpose of this study was to determine the effect of EA on ovarian function in ovarian granulosa cells (OGCs) in a cyclophosphamide (CTX)-induced mouse model of POF. METHODS: Mice were divided into three groups: wild type (WT) group, CTX group and CTX + EA group. EA was administered under isoflurane anesthesia at CV4, ST36 and SP6 for 30 min every 2 days, 2-3 times per week for a total of 4 weeks. Effects of EA on ovarian weight and level of estrogen were examined. The mRNA and protein expression levels of cell cycle-associated proteins were detected and mRNA modifications were analyzed. RESULTS: EA significantly increased ovarian weight and reduced the proportion of atretic follicles in mice with CTX-induced POF (p < 0.05). EA increased the level of estrogen in the peripheral blood of mice and inhibited the modification of total mRNA N4-acetylcytidine (ac4C). A significant increase in the expression of P16 and N-acetyltransferase 10 (NAT10) and a significant decrease in the expression of Cyclin D (CCND1) and cyclin-dependent kinase 6 (CDK6) were observed in the OGCs of POF mice (p<0.05). After EA, P16 and NAT10 expression was decreased, and CCND1 and CDK6 expression was increased. Finally, EA reduced the ac4C modification of P16 mRNA-specific sites in the OGCs of POF mice. CONCLUSION: This study demonstrated that EA promoted the repair of the ovarian microenvironment by inhibiting the ac4C modification of P16 mRNA to decrease its stability and expression intensity, and by altering the activity of the P16/CDK6/CCND1 axis in OGCs.

Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress / 中国针灸

OBJECTIVE@#To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress.@*METHODS@#Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot.@*RESULTS@#Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01).@*CONCLUSION@#Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.

Study on the Mechanism of Action of Different Acupuncture Regimens on Premature Ovarian Failure Model Rats.

Objective: To study the mechanism of acupuncture on premature ovarian failure (POF) through the apoptosis pathway mediated by Bcl-2/Bax. Methods: POF rats were successfully obtained by cyclophosphamide. They were divided into five groups. After that, acupuncture was performed. The blank group and model group were not treated. The routine acupuncture group was acupuncture at Guanyuan, Sanyinjiao, Zhongji, and Guilai four points. The Neck-seven-acupuncture group was selected from Fengchi, Fengfu, Tianzhu, and Wangu four acupoints; the three-viscera simultaneous treatment group selected Guanyuan, Shenshu, Sanyinjiao, Taichong, and Baihui five points; and the data mining group selected Guanyuan and Sanyinjiao two points for 14 days of treatment. During the treatment, some rats were shed one after another due to the side effects of bone marrow suppression caused by mold-making. After treatment, serum estradiol (E2), follicle forming hormone (FSH), and luteinizing hormone (LH) were detected by radioimmunoassay, Bcl-2 and Bax proteins were analyzed by Western blot method, and Bcl-2 and Bax RNA were analyzed by PCR method. Results: Bcl-2 increased and Bax decreased in rats with premature ovarian failure treated with acupuncture. It shows that acupuncture can affect the secretion of ovarian-related hormones and the expression of apoptosis-related proteins, which is more significant in the conventional acupuncture point group. Conclusion: Acupuncture can inhibit the apoptosis of granulosa cells in ovarian tissue of rats with premature ovarian failure and improve ovarian function. The mechanism of its effect is to promote Bcl-2 gene expression and protein synthesis and inhibit Bax gene expression and protein synthesis. The conventional treatment group works best. This provides an experimental basis for the clinical use of acupuncture to intervene in the treatment of premature ovarian failure.

Advances in the cellular immunological pathogenesis and related treatment of primary ovarian insufficiency.

PURPOSE OF REVIEW: To describe the immunological mechanism in the pathogenesis of premature ovarian insufficiency (POI), summarize the alternations in humoral immunity and cellular immunity in patients with POI, and review some associated treatment methods based on these immunological changes. RECENT FINDINGS: Symptoms of patients with POI are similar to that of menopause, such as amenorrhea, anovulation, early depletion of the ovarian reserve, decreased estrogen, and elevated follicle-stimulating hormone (FSH) levels. Therefore, most previous research has focused on finding ovary-related antibodies. In recent years, advances in the field of immunology have increased our understanding of the pathogenic mechanism of POI, which includes changed levels of cytokines and immunological cells especially Tregs cells and Th17 cells. Based on these advances, many associatedtreatment methods including DHEA supplementation, Traditional Chinese Medicine, adoptive transfer of Treg cells and stem cell transplantation have become research hotspots. SUMMARY: This review focuses on the current literature regarding alterations in cellular immunity in patients with POI, as well as the role in the pathogenesis of POI, and summarizes treatment methods based on these immunological alternations, which have a great application potential in the clinical practice.

Preventive Electroacupuncture Alleviates Oxidative Stress and Inflammation via Keap1/Nrf2/HO-1 Pathway in Rats with Cyclophosphamide-Induced Premature Ovarian Insufficiency.

Electroacupuncture (EA) is a popular therapeutic therapy for premature ovarian insufficiency (POI). However, little has been known about the underlying processes of EA therapy. To investigate the benefit of EA and reveal the mechanism, thirty SD female rats were allocated into the control, model, sham, EA, and GnRHa groups at random. Vaginal smears were used to monitor the rats' estrous cycle. Serum liver and renal function (ALT, AST, BUN, and Cr), sex hormone (FSH, E2, and AMH), oxidative stress markers (SOD, GSH, and MDA), and inflammatory cytokine (IL6, IL1ß, and TNFα) levels were measured by enzyme-linked immunosorbent assay (ELISA). Their ovary morphology was observed by hematoxylin-eosin staining. Transmission electron microscope was used to remark the ultrastructure of the granulocytes. Protein and gene expressions of Keap1/Nrf2/HO-1 pathway were detected by western blot and RT-PCR. Compared with the model group, in the EA group, the levels of serum sex hormones recovered to normal levels. Moreover, it reduced oxidative stress in rats, as demonstrated by increased SOD and GSH levels and decreased MDA levels. Meanwhile, Keap1 mRNA and protein expression dropped considerably in the EA group, while the mRNA and protein expressions of Nrf2 and HO-1 increased. We found that preventive EA might rescue rats with CTX-induced ovarian dysfunction. The anti-inflammatory and antioxidative stress properties of EA, which elevated the Keap1/Nrf2/HO-1 signaling pathway, might be the underlying mechanism. Furthermore, as compared to GnRHa, electroacupuncture did not raise the burden of the liver (ALT and AST) or the kidney (BUN and Cr). Electroacupuncture has a meaningful impact and a sufficient level of safety, making it useful for therapeutic setting in POI.

Electro-Acupuncture Regulates Metabolic Disorders of the Liver and Kidney in Premature Ovarian Failure Mice.

As per the theory of traditional Chinese medicine (TCM), the liver and kidney dysfunction are important pathogenies for premature ovarian failure (POF). POF is a common gynecological disease that reduced the pregnancy rate. Electro-acupuncture (EA) is a useful non-pharmaceutical therapy that supposedly regulates the function of the liver and kidney in the treatment of POF with TCM. However, the underlying mechanism of EA in the treatment of POF has not been adequately studied through metabonomics with reference to the theory of TCM. Accordingly, we investigated the effect of EA on the liver and kidney metabolites in POF mice through metabolomics. POF mice were established via intraperitoneal injection of cisplatin. Both Sanyinjiao (SP6) and Guanyuan (CV4) were stimulated by EA for 3 weeks. The biological samples (including the serum and the ovary, liver, and kidney tissues) were evaluated by histopathology, molecular biology, and hydrogen-1 nuclear magnetic resonance (1HNMR)-based metabolomics to assess the efficacy of EA. 1HNMR data were analyzed by the orthogonal partial least squares discriminant analysis (OPLS-DA). The results revealed that EA was beneficial to ovarian function and the menstrual cycle of POF. Both the energy metabolism and neurotransmitter metabolism in the liver and kidney were regulated by EA. Notably, EA played an important role in regulating energy-related metabolism in the kidney, and the better effect of neurotransmitter-related metabolism in the liver was regulated by EA. These findings indicated that the ovarian functions could be improved and the metabolic disorder of the liver and kidney caused by POF could be regulated by EA. Our study results thus suggested that the EA therapy, based on the results for the liver and kidney, were related to POF in TCM, as preliminarily confirmed through metabolomics.

The Evaluation of Ovarian Function Recovery Following Treatment of Primary Ovarian Insufficiency: A Systematic Review.

Background: Primary ovarian insufficiency (POI) is gaining awareness as its prevalence increases and its effect on patients is extremely negative. To date, several therapies have been designed to treat POI, but the conclusions are conflicting, in part, due to inconsistent evaluation methods. Thus, we explore a multi-index of ovarian function assessment methods to evaluate the recovery of ovarian function after various therapies in order to evaluate effectiveness in a more comprehensive manner. Aim: The purpose of this review is to assess the effectiveness of various therapies to recover ovarian function in patients with POI. The primary outcome measures were anti-Müllerian hormone (AMH) levels, follicle stimulating hormone (FSH) levels, and antral follicle count (AFC). The secondary outcomes included the change of mean ovarian volume, menstruation recovery, and pregnancy rate. Methods: Our systematic searching including PubMed, Web of Science, Cochrane, and Embase databases was conducted to find all human clinical trial articles published from January 2000 to April 2021 and related to POI treatment, including the keywords: POI, AFC, and hormones. All prospective and retrospective studies exploring ovarian function recovery that include AFC, AMH levels, and FSH levels evolution throughout treatment were included. All patients included in the studies met the POI criteria described by the European Society for Human Reproductive Embryology (ESHRE) guideline. Results: Six studies were selected based on the criteria: one randomized controlled trial and five observational studies. Among them, two studies focused on the intraovarian platelet-rich plasma (PRP) infusion treatment, two studies focused on dehydroepiandrosterone (DHEA) supplements, one study focused on hormone replacement therapy (HRT), and one study focused on autologous adipose-derived stromal cells (ADSCs) treatment. There was insufficient scientific evidence that any approach could help ovarian function recovery in patients with POI because the ovarian function markers in each study had inconsistent changes with 26 patients (6.2%) reporting spontaneous pregnancy. Conclusion: Serum AMH levels, FSH levels, and AFC are sensitive indicators and reflect the evolution of ovarian function. Large randomized controlled trials are necessary, and the data on ovarian function should be collected comprehensively to evaluate the effectiveness of a variety of treatments.

Efficacy of needling acupoints of Guanyuan (CV4), Sanyinjiao (SP6), Zusanli (ST36), Pishu (BL20), Shenshu (BL23), Zigong (EX-CA1) on expression of p38 mitogen-activated protein kinase in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide.

OBJECTIVE: To investigate the efficacy of needling acupoins of Guanyuan (CV4), Sanyinjiao (SP6), Zusanli (ST36), Pishu (BL20), Shenshu (BL23), Zigong (EX-CA1) on the expression of p38 mitogen-activated protein kinase (p38MAPK) in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide, and to study the underlying mechanism. METHODS: Forty specific pathogen free female Sprague-Dawley rats were randomly divided into the blank group, the model group, the acupuncture group, the Western Medicine group and the Western Medicine combined with acupuncture group. Except the blank group, the rest of the rats were given with cyclophosphamide for 14 d to establish premature ovarian failure model. No intervention was conducted in the blank group and model group; the acupuncture group was given with acupuncture daily; the Western Medicine group was given with estradiol valerate (0.09 mg/kg) by intragastrical gavage daily; the combination group was given with acupuncture combined with estradiol valerate (0.09 mg/kg) daily. Each group was intervened in continuously for 14 d. After the last treatment, the levels of estradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected by enzyme-linked immunosorbent assay, then the ovarian tissue was dissected. Western blot was used to detect the expression of p38MAPK protein. RESULTS: Compared with the blank group, E2 in the serum of rats in the model group was significantly decreased (P < 0.05), FSH and LH were significantly increased (P < 0.05), and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly increased (P < 0.05). Compared with the model group, E2 in the serum of the acupuncture group, Western Medicine group and the combination group were significantly increased (P < 0.05), FSH and LH levels were significantly decreased (P < 0.05), and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly decreased (P < 0.05). However, there was no statistically significant difference between the Western Medicine group and the acupuncture group (P > 0.05). CONCLUSIONS: Acupuncture has the same effect as estrogen in interfering POF caused by cyclophosphamide, and its mechanism may be related to inhibiting the expression of p38MAPK protein in ovarian tissue and affecting the activation of p38MAPK signaling pathway.

[Acupuncture-moxibustion treatment by stages based on the theory of "transformation of yin and yang" for premature ovarian insufficiency].

OBJECTIVE: To compare the efficacy between acupuncture-moxibustion treatment by stages and femoston for premature ovarian insufficiency (POI). METHODS: A total of 66 patients with POI were randomly divided into an observation group (33 cases, 3 cases dropped off) and a control group (33 cases, 2 cases dropped off). The patients in the observation group, based on the theory of "transformation of yin and yang", were treated with acupuncture-moxibustion by stages in the postmenstrual period, ovulatory period, premenstrual period and menstrual period, once every other day, 3 times a week. The patients in the control group were treated with oral administration of femoston (estradiol tablets/estradiol and dydrogesterone tablets, 1 tablet per day). Both groups were treated for 3 menstrual cycles. The ovarian function (serum follicle-stimulating hormone [FSH], luteinizing hormone [LH], estradiol [E2], anti-mullerian hormone [AMH] and antral follicle count [AFC]) and perimenopausal symptoms [modified Kupperman index (KI) scale score] were observed before and after treatment, and the menstrual improvement of the two groups was compared. RESULTS: Compared before treatment, the serum levels of FSH and LH were decreased (P<0.01), the levels of E2 were increased (P<0.01) in the two groups after treatment; the serum level of AMH and AFC in the observation group were increased after treatment (P<0.01). After treatment, the serum level of AMH and AFC in the observation group were higher than those in the control group (P<0.05). After treatment, there was no significant difference in the menstrual return rate and menstrual regularity rate between the amenorrhea patients of the two groups (P>0.05). After treatment, the modified KI scale scores in the two groups were reduced (P<0.01), and the modified KI scale score in the observation group was lower than that in the control group (P<0.05). CONCLUSION: Acupuncture- moxibustion treatment by stages based on the theory of "transformation of yin and yang" has similar efficacy with femoston in improving serum sex hormone level and menstrual symptoms in patients with POI, and has advantages in improving serum AMH level, AFC and perimenopausal symptoms.

[WANG Yin¢s experience in treatment of primary ovarian insufficiency].

The clinical experience of professor WANG Yin in the treatment of primary ovarian insufficiency are summarized. Regarding acupoints selection, the sancai, named "heaven-earth-human being" method is adopted, focusing on the regulation of the middle jiao and shen (mind/spirit). Regarding needling technique, the elongated needle is used in preference. Besides, the bloodletting and cupping method with filiform-fire needle are innovated to achieve both the reinforcing and the reducing purposes as well as to eliminate stasis. The periodic therapy is applied to comply with the changes in the physiological cycle of gynecology. Acupuncture, Chinese herbal medicine, or the combination of them is selected in consideration of the concrete individual cases of primary ovarian insufficiency.

[Effect of regulating menstruation and promoting pregnancy acupuncture therapy on negative emotion in patients with premature ovarian insufficiency].

OBJECTIVE: To observe the influence of regulating menstruation and promoting pregnancy acupuncture therapy on negative emotions in patients with premature ovarian insufficiency (POI). METHODS: A total of 60 patients with POI were randomly divided into an acupuncture group and a western medication group, 30 cases in each group. The acupuncture group was treated with regulating menstruation and promoting pregnancy acupuncture therapy at Baihui (GV 20), Shenting (GV 24), Guanyuan (CV 4), Sanyinjiao (SP 6), Shenshu (BL 23), Ciliao (BL 32), etc. once a day, 5 times a week for 3 months. The western medication group was treated by oral administration of climen. The drug was given 1 tablet a day for 21 days and was stopped for 1 week as a course. The treatment was required 3 consecutive courses. The self-rating anxiety scale (SAS) score, modified Kupperman index (KI) score, agitated and depressive symptom scores in KI and serum level of follicle stimulating hormone (FSH) before and after treatment were compared between the two groups. RESULTS: After treatment, the SAS scores, KI scores and serum levels of FSH in the two groups and the scores of agitated and depressive symptom in the acupuncture group were lower than those before treatment (P<0.05), and the acupuncture group was lower than the western medication group (P<0.05). CONCLUSION: Regulating menstruation and promoting pregnancy acupuncture therapy can effectively improve the negative emotions of patients with POI and reduce serum level of FSH .