This study primarily investigates the effect of Liuwei Dihuang Pills on the activation and proliferation of female germline stem cells(FGSCs) in the ovaries and cortex of mice with premature ovarian failure(POF), and how it improves ovarian function. ICR mice were randomly divided into the control group, model group, Liuwei Dihuang Pills group, Liuwei Dihuang Pills double-dose group, and estradiol valerate group. A mouse model of POF was established by intraperitoneal injection of cyclophosphamide. After successful modeling, the mice were treated with Liuwei Dihuang Pills or estradiol valerate for 28 days. Vaginal smears were prepared to observe the estrous cycle and body weight. After the last administration, mice were sacrificed and sampled. Serum levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-Müllerian hormone(AMH) were measured by enzyme-linked immunosorbent assay(ELISA). Hematoxylin-eosin(HE) staining was used to observe ovarian morphology and to count follicles at all stages to evaluate ovarian function. Immunohistochemistry was used to detect the expression of mouse vasa homolog(MVH), a marker of ovarian FGSCs. Immunofluorescence staining, using co-labeling of MVH and proliferating cell nuclear antigen(PCNA), was used to detect the expression and localization of specific markers of FGSCs. Western blot was employed to assess the protein expression of MVH, octamer-binding transcription factor 4(Oct4), and PCNA in the ovaries. The results showed that compared with the control group, the model group exhibited disordered estrous cycles, decreased ovarian index, increased atretic follicles, and a reduced number of follicles at all stages. FSH and LH levels were significantly elevated, while AMH and E_2 levels were significantly reduced, indicating the success of the model. After treatment with Liuwei Dihuang Pills or estradiol valerate, hormone levels improved, the number of atretic follicles decreased, and the number of follicles at all stages increased. MVH marker protein and PCNA proliferative protein expression in ovarian tissue also increased. These results suggest that Liuwei Dihuang Pills regulate estrous cycles and hormone disorders in POF mice, promote the proliferation of FGSCs, improve follicular development in POF mice, and enhance ovarian function.
Animals ; Female ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Cell Proliferation/drug effects* ; Mice, Inbred ICR ; Ovary/cytology* ; Primary Ovarian Insufficiency/genetics* ; Follicle Stimulating Hormone/metabolism* ; Humans ; Anti-Mullerian Hormone/blood* ; Antineoplastic Agents/adverse effects* ; Luteinizing Hormone/metabolism* ; Cyclophosphamide/adverse effects*

In this study, we explored the pharmacological effects of Siwu Decoction in treating premature ovarian insufficiency(POI) and its molecular mechanism based on the mitophagy pathway modulated and mediated by estrogen receptor(ER) subtypes. Female Balb/c mice were divided into a control group, model group, as well as high-dose and low-dose groups of Siwu Decoction. The POI mice model was constructed by intraperitoneal injection of cisplatin. The high-dose and low-dose groups of Siwu Decoction were administered intragastrically with Siwu Decoction each day for 14 days. During this period, we monitored the estrous cycle and body weight of the mice and calculated the ovarian index. The morphology of the ovaries was detected by hematoxylin-eosin(HE) staining, and the number of primordial follicles was counted. The apoptosis of the ovarian tissue was detected by TUNEL staining. The expression levels of anti-Müllerian hormone(AMH), apoptosis-associated and mitophagy-associated proteins, ER subtypes, and the expression levels of key proteins of its mediated molecular pathways were detected by Western blot and immunohistochemistry. KGN cells were divided into a control group, model group, Siwu Decoction group, and gene silencing group. The apoptosis model was induced by H_2O_2, and PTEN-induced putative kinase 1(PINK1) gene silencing was induced by siRNA transfection. The Siwu Decoction group and gene silencing group were added to the medium containing Siwu Decoction. Cell viability was detected by CCK-8 assay. Cell senescence was detected by senescence-associated-β-galactosidase. The expression levels of apoptosis-associated and mitophagy-associated proteins were detected by Western blot. The results of in vivo experiments showed that compared with the model group, the mice in the high-dose and low-dose groups of Siwu Decoction significantly recovered the rhythm of the estrous cycle, and the levels of ovarian index, number of primordial follicles, and expression of AMH, representative indexes of ovarian function, were significantly higher, suggesting that the level of ovarian function was significantly improved. The expression levels of the apoptosis-related proteins, cytochrome C(Cyt C), cysteinyl aspartate specific proteinase 3(caspase 3), B-cell lymphoma-2(Bcl-2)-associated X(Bax), and mitophagy-associated indicator(Beclin 1) were significantly decreased, and the expression levels of Bcl-2 was significantly elevated. The positive area of TUNEL was significantly reduced, suggesting that the apoptosis level of the ovaries was significantly reduced. The expression levels of PINK1, Parkin, and sequestosome 1(p62) were significantly reduced, suggesting that the level of ovarian mitophagy was significantly down-regulated. The expression levels of ERα and ERβ were significantly elevated, and the ratio of ERα/ERβ was significantly reduced. The expression levels of key proteins in the pathway, phosphoinositide 3-kinase(PI3K) and protein kinase B(Akt), were significantly reduced, suggesting that the regulation of ER subtypes and the mediation of PI3K/Akt pathway were the key mechanisms. In vitro experiments showed that compared with the model group, the proportion of senescent cells in the Siwu Decoction group was significantly reduced. Cyt C, caspase 3, Beclin 1, Parkin, and p62 were significantly reduced, which was in line with in vivo experimental results. The proportion of senescent cells and the expression level of the above proteins were further significantly reduced after PINK1 silencing. It can be seen that Siwu Decoction can regulate the expression level and proportion of ER subtypes in KGN cells, then mediate the PI3K/Akt pathway to inhibit excessive mitophagy and apoptosis, and exert therapeutic effects of POI.
Animals ; Female ; Drugs, Chinese Herbal/administration & dosage* ; Mitophagy/drug effects* ; Primary Ovarian Insufficiency/physiopathology* ; Mice ; Mice, Inbred BALB C ; Humans ; Receptors, Estrogen/genetics* ; Apoptosis/drug effects* ; Ovary/metabolism* ; Signal Transduction/drug effects* ; Anti-Mullerian Hormone/genetics*

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

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*

Chitooligosaccharide-zinc (COS·Zn) is a powerful anti-oxidant and anti-aging scavenger, whose anti-oxidative ability immensely exceeds vitamin C. Therefore, this study was aimed to investigate the protective effects of COS·Zn against premature ovarian failure (POF) and potential mechanisms. Female KM adult mice were divided into the following groups: a treatment group (150 mg·kg
Animals ; Chitosan ; Female ; Humans ; Mice ; NF-E2-Related Factor 2/genetics* ; Nuclear Proteins ; Oligosaccharides ; Primary Ovarian Insufficiency/drug therapy* ; Signal Transduction ; Zinc

OBJECTIVE: To explore the clinical and genetic features of an adult female with premature ovarian failure (POF) and mosaic ring chromosome 13. METHODS: The patient was subjected to G-banding karyotyping and chromosomal microarray analysis (CMA). RESULTS: The patient was diagnosed as POF and had a karyotype of 46,XX,r(13)(p13q34)[86]/45,XX,-13[9]/46,XX,r(13;13)[5]. CMA analysis has failed to detect any deletion in the long arm of chromosome 13. Literature review suggested that the ring chromosome 13 may have clinical symptoms similar to those of sex chromosome abnormalities. CONCLUSION A case of mosaic ring chromosome 13 and POF has been reported. Mosaic ring chromosome 13 may lead to symptoms of POF similar to sex chromosomal abnormalities.
Adult ; Chromosome Aberrations ; Female ; Humans ; Karyotype ; Karyotyping ; Phenotype ; Primary Ovarian Insufficiency ; genetics

OBJECTIVETo analyze partial deletion of the long arm of X chromosome in a family and explore the mechanism underlying its phenotypes.

METHODSG-banding technique was employed to analyze the karyotypes of the subjects, and fluorescence in situ hybridization (FISH) was used to analyze their X chromosomes with Xpter, Xqter and WCPX probes.

RESULTSThe karyotypes of the proband, her mother and her fetus were all 46,X,del(X)(q24). Combined FISH and karyotyping analysis suggested that the proband and her fetus both carried a Xq24q27.3 deletion.

CONCLUSIONThe Xq24q27.3 deletion carried by the family is closely related with premature ovarian failure but not with short stature, gonadal dysgenesis and primary amenorrhea.

Adult ; Chromosome Banding ; Chromosome Deletion ; Chromosomes, Human, X ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Primary Ovarian Insufficiency ; genetics