Female ; Humans ; Consensus ; Menopause, Premature ; Primary Ovarian Insufficiency/therapy*

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P<0.01), and elevated the level of FSH and LH(P<0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P<0.05), and lowered the levels of FSH and LH(P<0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.
Humans ; Mice ; Female ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; bcl-2-Associated X Protein ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/metabolism* ; Saline Solution/therapeutic use* ; Signal Transduction ; Granulosa Cells ; Primary Ovarian Insufficiency/drug therapy* ; Follicle Stimulating Hormone/therapeutic use* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis

A 13-year and 6-month-old girl attended the Hunan Children's Hospital due to delayed menarche. The laboratory test results indicated increased follicle-stimulating hormone and luteinizing hormone, decreased anti-Mullerian hormone, and pelvic ultrasound showed a cord-like uterus and absence of bilateral ovaries. Her 11-year and 5-month-old younger sister had the same laboratory and imaging findings, and both girls were diagnosed with primary ovarian insufficiency. Whole exome sequencing and Sanger sequencing confirmed that the proband and her sister carried heterozygous variants of HROB gene c.718C>T (p.Arg240*) and c.1351C>T (p.Arg451*), which were inherited from their parents respectively and consistent with autosomal recessive inheritance. Oral estradiol valerate at an initial dose of 0.125 mg/d was given to the proband, and the secondary sexual characteristics began to develop after 6 months.
Humans ; Female ; Child ; Infant ; Primary Ovarian Insufficiency/genetics* ; Luteinizing Hormone ; Estradiol

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.
Humans ; Rats ; Female ; Animals ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; Tumor Suppressor Protein p53/genetics* ; Apoptosis Regulatory Proteins ; Acupuncture Therapy ; Primary Ovarian Insufficiency/therapy* ; Apoptosis ; RNA, Messenger ; Oxidative Stress ; Acupuncture Points

Healthy birth and child development are the prerequisite for improving the overall quality of the population. However, premature ovarian failure(POF) threatens the reproductive health of women. The incidence of this disease has been on the rise, and it tends to occur in the young. The causes are complex, involving genetics, autoimmune, infectious and iatrogenic factors, but most of the causes remain unclear. At the moment, hormone replacement therapy and assisted reproductive technology are the main clinical approaches. According to traditional Chinese medicine(TCM), kidney deficiency and blood stasis are one of the major causes of POF, and TCM with the effects of tonifying kidney and activating blood has a definite effect. Through clinical trials, TCM prescriptions for POF have excellent therapeutic effect as a result of multi-target regulation and slight toxicity. In particular, they have no obvious side effects. A large number of studies have shown that the kidney-tonifying and blood-activating TCM can regulate the neuroendocrine function of hypothalamic-pituitary-ovarian axis, improve ovarian hemodynamics and microcirculation, reduce the apoptosis of granulosa cells, alleviate oxidative stress injury, and modulate immunologic balance. The mechanism is that it regulates the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt), vascular endothelial growth factor(VEGF), transforming growth factor(TGF)-β/Smads, nuclear factor E2-related factor 2(Nrf2)/antioxidant response element(ARE), and nuclear factor-kappa B(NF-κB) signaling pathways. This article summarized the pathological mechanisms of tonifying kidney and activating blood TCM in the prevention and treatment of POF and explored the biological basis of its multi-pathway and multi-target characteristics in the treatment of this disease. As a result, this study is expected to serve as a reference for the treatment of POF with the tonifying kidney and activating blood therapy.
Child ; Humans ; Female ; Primary Ovarian Insufficiency/drug therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Vascular Endothelial Growth Factor A ; Medicine, Chinese Traditional ; NF-kappa B ; Kidney

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.
Female ; Humans ; Mice ; Animals ; Primary Ovarian Insufficiency/chemically induced* ; Proteomics ; Signal Transduction ; Glycosides/adverse effects*

This study aims to explore the molecular mechanism of Lycium barbarum polysaccharides(LBP) in alleviating premature ovarian failure(POF) in mice via the 5'-adenosine monophosphate activated protein kinase(AMPK)/silent information regulator 1(Sirt1) signaling pathway. The POF mouse model was established by D-galactose(D-gal) injection at the back. Six groups were set up, including a normal control group, a model group, a LBP group, a 3-MA(autophagy inhibitor 3-methyladenine) group, an AMPK inhibitor group, and a LBPAMPK inhibitor group, with 15 mice in each group. After 28 continuous days of administration, enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of sex hormones [estradiol(E_2), luteinizing hormone(LH), and follicle-stimulating hormone(FSH)] in serum. The ovarian mass coefficient was measured. Senescence-associated β-Galactosidase(SA-β-Gal) staining and hematoxylin-eosin(HE) staining were performed for observing the state of ovarian senescence and the morphological changes of the ovary. Immunohistochemical method was used to measure the expression of the autophagy marker LC3-Ⅱ in ovarian tissue. Western blot was employed to measure the expression levels of the senescence marker p16~(INK4 a), autophagy markers(LC3-Ⅱ and Beclin-1), the autophagy substrate p62, lysosome-associated membrane protein 2(LAMP2), and the proteins in the AMPK/Sirt1 pathway and mammalian target of rapamycin complex 1(mTORC1)/UNC-51-like kinase 1 Ser757 site(Ulk1 Ser757) pathway. Compared with the normal control group, the modeling of POF decreased the ovarian granulosa cells and follicles, led to the ovarian aging and severe sex hormone secretion disorders, weakened ovarian autophagy activity, and down-regulated the expression of proteins in the AMPK/Sirt1 pathway(P<0.05). Compared with the model group, the treatment with LBP increased ovarian granulosa cells and follicles, alleviated aging and sex hormone disorders, increased autophagy activity, and activated the AMPK/Sirt1 pathway(P<0.05). Both 3-MA and AMPK inhibitor can inhibit autophagy and aggravate ovarian damage and aging in mice. AMPK inhibitor can partially attenuate the role of LBP in promoting autophagy activation and alleviating aging and ovarian tissue damage(P<0.05). LBP can alleviate the symptoms of POF induced by D-gal by promoting the activation of AMPK/Sirt1 pathway.
Animals ; Female ; Humans ; Mice ; AMP-Activated Protein Kinases/metabolism* ; Autophagy/drug effects* ; Follicle Stimulating Hormone/blood* ; Lycium/chemistry* ; Polysaccharides/therapeutic use* ; Primary Ovarian Insufficiency/drug therapy* ; Sirtuin 1/metabolism*

OBJECTIVE: To investigate the clinical significance of miRNA-146, OX-LDL and ROS in patients with primary ovarian insufficiency (POI). METHODS: 100 patients with POI were prospectively collected and 100 women with normal ovarian function were randomly selected as control group. Serum miRNA-146 expression level was detected by qRT-PCR and serum OX-LDL and ROS expression levels were detected by ELISA. Ovarian granulosa cells of mouse were transfected with miRNA-146 mimics or inhibitors, and then treated with OX-LDL. Cell viability, colony forming ability, apoptosis rate and toll like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) of pathway proteins were evaluated respectively. RESULTS: Compared with control group, the expression level of miRNA-146 in POI group was significantly lower, the expression level of OX-LDL and ROS were significantly higher, and the ovarian volume and peak systolic blood flow velocity of ovarian artery were significantly decreased in POI group. Upregulation of miRNA-146 expression had a protective effect on OX-LDL injured ovarian granulosa cells, as evidenced by increased ovarian granulosa cell viability and colony number, reduced apoptosis, and downregulation of TLR4/NF-κB expression. CONCLUSION miRNA-146 can target downstream TLR4/NF-κB signaling pathway affects oxidative stress and inflammatory response of POI induced by OX-LDL and ROS, and is expected to become a biomarker for early prediction of POI and a new target for treatment.
Humans ; Female ; Mice ; Animals ; Toll-Like Receptor 4/metabolism* ; NF-kappa B/metabolism* ; MicroRNAs/metabolism* ; Reactive Oxygen Species/pharmacology* ; Primary Ovarian Insufficiency/genetics* ; Apoptosis/genetics*

OBJECTIVE: To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which Fkbp38 gene deletion causes premature ovarian insufficiency (POI). METHODS: The Cre-loxp system was used to construct oocyte-specific Fkbp38 knockout transgenic mice. The genotype of the transgenic mice was identified using PCR, and the expression of FKBP38 in the oocytes was verified. The numbers of primordial follicles, primary follicles, secondary follicles and antral follicles in Fkbp38 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of Fkbp38 deletion on follicular development. The fertility and serum sex hormone levels of the mice were determined by reproduction experiments and ELISA to assess ovarian function. Ovarian granulosa cell apoptosis of the mice was assessed using TUNEL assay. The activity of the downstream target protein of phosphorylated ribosomal S6 (PS6) of mTOR signaling pathway was detected, and the expressions of BCL-2 and BAX proteins were determined using immunofluorescence assay for assessing oocyte development in the mice. RESULTS: The oocyte-specific Fkbp38 knockout transgenic mouse model was successfully constructed, which showed decreased fertility, disordered sex hormone levels, and significantly reduced primordial follicles, primary follicles and secondary follicles in the ovary (P < 0.05), demonstrating POI-like changes. Compared with the control mice, oocyte-specific Fkbp38 knockout caused activation of the mTOR signaling pathway, significantly increased apoptosis of the granulosa cells, and obviously increased the BAX/BCL- 2 ratio by increasing BAX expression and reducing BCL-2 expression in the oocytes (P < 0.05). CONCLUSION FKBP38 plays an important role in follicle development, and Fkbp38 gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.
Female ; Humans ; Mice ; Animals ; Primary Ovarian Insufficiency/genetics* ; Apoptosis ; Signal Transduction ; Proto-Oncogene Proteins c-bcl-2 ; Granulosa Cells ; Mice, Transgenic ; Mice, Knockout ; TOR Serine-Threonine Kinases

OBJECTIVE: To explore the correlation between Fragile X mental retardation gene-1 (FMR1) gene CGG repeats with diminished ovarian reserve (DOR). METHODS: For 214 females diagnosed with DOR, DNA was extracted from peripheral blood samples. FMR1 gene CGG repeats were determined by PCR and capillary electrophoresis. RESULTS: Three DOR patients were found to carry FMR1 premutations, and one patient was found to carry gray zone FMR1 repeats. After genetic counseling, one patient and the sister of another patient, both carrying FMR1 permutations, conceived naturally. Prenatal diagnosis showed that both fetuses have carried FMR1 permutations. CONCLUSION FMR1 gene permutation may be associated with DOR. Determination of FMR1 gene CGG repeats in DOR patients can provide a basis for genetic counseling and guidance for reproduction.
Female ; Fragile X Mental Retardation Protein/metabolism* ; Fragile X Syndrome/genetics* ; Humans ; Ovarian Diseases ; Ovarian Reserve/genetics* ; Primary Ovarian Insufficiency/genetics* ; Trinucleotide Repeats/genetics*