Honghua Xiaoyao tablet combined with estradiol improves ovarian function in D-galactose-induced aging mice by reducing apoptosis and affecting the release of reproductive hormones: an in vivo study.

Context: It is very necessary to delay ovarian aging and prevent age-related health problems. The active ingredient in Honghua Xiaoyao tablet (HHXYT) has the effects of anti-oxidation, anti-inflammation, immune regulation and so on. Objective: To explore the effect and mechanism of Honghua Xiaoyao tablet on aging model mice. Materials and methods: The aging model was established by intraperitoneal injection of D-galactose in model mice. The mice in the HHXYT-L,M,H group were given 0.3 g/kg, 0.6 g/kg and 1.2 g/kg Honghua Xiaoyao tablet suspension respectively, and the HHXYT-M + E2 group was given 0.6 g/kg HHXYT +0.13 mg/kg estradiol valerate for 30 days. In this study, ELISA, HE, Western blot, IH and TUNEL were used. Results: HHXYT + E2 can improve the gonadal index, estrous cycle of aging mice. In HHXYT-M + E2 group, the level of FSH and LH decreased, while E2 and AMH increased significantly. The number of growing follicles in HHXYT-M + E2 group increased, which was better than that of HHXYT alone. Western blot results showed that HHXYT-M + E2 group decreased the expression of Bax, cleaved-Parp, cleaved-Casp-3 and CytC molecules and increased the expression of Bcl-2 in ovarian tissue. FSHR expression decreased in model group and increased in HHXYT group. TUNEL staining showed that the number of apoptotic cells in HHXYT group was reduced, and the HHXYT-M + E2 group was the most significantly. Discussion and conclusion: HHXYT can improve the level of sex hormones and increase the number of growing follicles in aging mice. HHXYT-M + E2 group has the best effect, and its mechanism may be related to reducing ovarian granulosa cell apoptosis.

Dingkun Pill modulate ovarian function in chemotherapy-induced premature ovarian insufficiency mice by regulating PTEN/PI3K/AKT/FOXO3a signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Dingkun Pill (DKP) is a traditional Chinese medicine that has been shown to have beneficial effects on reproductive function. However, the specific mechanism underlying its effect on POI is not well understood. AIM OF THE STUDY: To investigate the effect of different doses of Dingkun Pill on ovarian function in cyclophosphamide (CTX)-induced premature ovarian insufficiency (POI) mice and to explore its molecular mechanism through PTEN/PI3K/AKT/FOXO3a signaling pathway. This study will provide valuable insights into the potential clinical application of Dingkun Pill for the treatment of POI. MATERIALS AND METHODS: Fifty female ICR mice were randomly divided into normal control (NC) group, model control (MC) group, and Dingkun Pill low, medium, high dose (DKP-L, M, H) groups. Mice were injected with CTX to construct the POI model. Mice in the DKP-L, M, and H groups were given 0.9 g/kg, 1.8 g/kg, and 3.6 g/kg of Dingkun Pill suspension for 21 days, respectively. Mice in the NC and MC groups were given the same amount of normal saline by gavage. Changes in body weight, estrous cycle and gonadal index were observed in each group of mice. Serum levels of FSH, LH, E2 and AMH were detected by ELISA. Hematoxylin-eosin (HE) staining observed the changes of ovarian pathological morphology and follicle counts at all levels. qRT-PCR was used to measure the levels of the PTEN and FOXO3a genes in ovarian tissue. The expression of PTEN/PI3K/AKT/FOXO3a signaling pathway related proteins were detected by Western-blot and immunohistochemistry (IHC). RESULTS: In POI mice, Dingkun Pill increased body weight, promoted the recovery of estrous cycle, increased ovarian index, and improved pathological morphology of the ovaries. The FSH level decreased in the medium dose group (P < 0.05), the LH level reduced significantly in the medium and high dose groups (P < 0.01), and the E2 level in the high dose group increased (P < 0.05). There was no significant difference in AMH levels across all dose groups. The number of growing follicles improved at all levels in the low and medium dose groups, but declined significantly in the high dose group. However, the number of corpus luteum increased significantly in the high dose group (P < 0.001), and the atretic follicles in the three dose groups decreased. Results from qRT-PCR, Western-blot and IHC showed that the moderate dose of Dingkun Pill suppressed the levels of the p-PI3K and p-AKT proteins by upregulating the expression of PTEN in the ovarian tissues of POI mice, thereby inhibiting the expression of the key protein p-FOXO3a. However, the inhibitory effect of the higher dose may be less than that of the lower and intermediate dose groups. CONCLUSIONS: The Dingkun Pill modulated hormonal levels, promoted follicle growth and induced ovulation in mice with CTX-induced POI, with better results in the low and moderate dose groups. Its mechanism may be related to the regulation of the PTEN/PI3K/AKT/FOXO3a signaling pathway.

miRNA-122-5p in POI ovarian-derived exosomes promotes granulosa cell apoptosis by regulating BCL9.

This study is to explore the therapeutic effect and potential mechanisms of exosomal microRNAs (miRNAs) derived from the ovaries with primary ovarian insufficiency (POI). The POI mouse model was established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan. The apoptosis of granulosa cells (GCs) incubated with exosomes extracted from ovarian tissues of control and POI groups was analyzed by flow cytometry. Then, high-throughput sequencing was performed to detect the difference of miRNAs profile in ovarian tissue-derived exosomes between the control and POI mice. The effect of differential miRNA on the apoptosis of CTX-induced ovarian GCs was analyzed by flow cytometry. The results showed that POI mouse model was successfully established. Exosomes extracted from ovarian of normal and POI group have different effects on apoptosis of GCs induced by CTX. miRNA-seq found that exosomal miR-122-5p in POI group increased significantly. miR-122-5p as the dominant miRNA targeting BCL9 was significantly upregulated in ovarian tissues of chemotherapy-induced POI group. Exosomes derived from the ovaries in the control group and miR-122-5p inhibitor group attenuated the apoptosis of primary cultured ovarian GCs. In conclusion, exosomal miR-122-5p promoted the apoptosis of ovarian GCs by targeting BCL9, suggested that miR-122-5p may function as a potential target to restore ovarian function.

MicroRNA-200 and microRNA-30 family as prognostic molecular signatures in ovarian cancer: A meta-analysis.

BACKGROUND: MicroRNAs (miRs) play a vital role in the occurrence, development, and progression of human cancers, but its role in the prognosis of ovarian cancer is unclear. METHODS: We performed a meta-analysis by searching PubMed, Embase, and Web of Science databases for eligible studies. The pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were used to explore the association between miRs expression and overall survival (OS) and progression-free survival (PFS) on ovarian cancer patients. We also used Kaplan-Meier to analyze the relationship between miRs and OS in OncoLnc dataset. RESULTS: A total of 15 records were included into the meta-analysis. The expression level of miR-200 family showed significant association with OS (HR = 0.78, 95% CI: 0.64-0.94) and insignificant association with PFS (HR = 0.72, 95% CI: 0.50-1.03). Subgroup analysis revealed that an increased expression level of miR-200c was associated with better OS (HR = 0.59, 95% CI: 0.45-0.74). An increased expression level of miR-200a, miR-200c, and miR-141 was associated with better PFS (miR-200a, HR = 0.59, 95% CI: 0.42-0.75; miR-200c, HR = 0.50, 95% CI: 0.14-0.87, miR-141, HR = 0.38, 95% CI: 0.12-0.63). Similarly, higher expression of miR-30 family was associated with elevated OS/PFS for ovarian cancer (OS, HR = 0.43, 95% CI: 0.13-0.74; PFS, HR = 0.76, 95% CI: 0.64-0.87). The OncoLnc dataset presented that elevated expression level of miR-30d-5p was associated with better OS (n = 470, P = .0197). CONCLUSION: The meta-analysis reveals that miR-200 family and miR-30 family could be promising prognostic biomarkers of ovarian cancer.

Melatonin inhibits 17ß-estradiol-induced migration, invasion and epithelial-mesenchymal transition in normal and endometriotic endometrial epithelial cells.

BACKGROUND: Melatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear. Here, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic endometrial epithelial cells and explored the pathway that might be involved. METHODS: This hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the expression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by CCK8 assays. RESULTS: Compared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of Notch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by specific inhibitor blocked 17ß-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells. CONCLUSIONS: Our data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present in endometriotic endometrium. Melatonin may block 17ß-estradiol-induced migration, invasion and EMT in normal and endometriotic epithelial cells by upregulating Numb expression and decreasing the activity of the Notch signaling pathway.

Identifying risk factors for cesarean scar pregnancy: a retrospective study of 79 cases.

OBJECTIVES: To explore the possible risk factors for cesarean scar pregnancy (CSP), the incidence of which is increasing rapidly in China. MATERIAL AND METHODS: 79 patients with CSP and 69 non-CSP expectant mothers with at least 1 previous cesarean section were employed in the study. The obstetric histories of the participants were collected and analyzed using Chi square test. RESULTS: We found that 77.2% CSP patients had ≥ 3 pregnancies and only 36.2% women had ≥ 3 pregnacies in non-CSP group. During the previous cesarean delivery, 21.5% of CSP patients had entered the first stage of labor, which was 43.5% in non-CSP group (P < 0.05). Cephalopelvic disproportion occurred in 51.9% of CSP patients, which was significantly higher than that (23.2%) in non-CSP group (P < 0.01). 11.4% of CSP patients had undergone cesarean section due to breech and shoulder presentation in the past, which was only 1.4% in non-CSP group. However, no significance was noted (P > 0.05). We did not find significant differences between the CSP and non-CSP patients in maternal age, multiple cesarean sections, gestational age, emergency or elective caesarean section. CONCLUSIONS: Multiple pregnancies, absence of the first stage of labor, and cephalopelvic disproportion might be the risk factors for the occurrence of CSP.

Dydrogesterone has no effect on uterine fibroids when used to prevent miscarriage in pregnant women with uterine fibroids.

OBJECTIVES: To analyse the effect of dydrogesterone use during pregnancy on uterine fibroids, pregnancy complications, and pregnancy outcome. MATERIAL AND METHODS: In all, 372 pregnant women with uterine fibroids who were treated at the Affiliated Provincial Hospital of Shandong University were included in this study. Thirty-three of these women received dydrogesterone and constituted the treatment group, and the 27 women who were found to have uterine fibroids during the first trimester but did not receive intervention to prevent miscarriage composed the control group. The changes in uterine fibroids before and after pregnancy and the pregnancy complications were recorded; immunohistochemistry was used to detect the expression of progesterone receptor (PR) and proliferation- and apoptosis-related proteins in the uterine fibroid tissue. RESULTS: No significant difference was observed in the change in uterine fibroid volume during pregnancy between the treatment group and the control group (p > 0.05). The percentage of uterine fibroids with red degeneration was lower in the treatment group than in the control group, but the difference was not statistically significant. No significant difference was observed in newborn weight, height, Apgar score, threatened miscarriage, or premature birth, among other characteristics, between the two groups (p > 0.05). Immunohistochemistry showed no significant difference in the expression of PR, cyclinD1, insulin-like growth factor (IGF1), or B-cell lymphoma 2 (Bcl2) between the two groups. CONCLUSIONS: The use of dydrogesterone during pregnancy has no significant effect on uterine fibroids, pregnancy progression, or pregnancy outcomes in pregnant patients with uterine fibroids.

Removal of Hg(II) from aqueous solution using sodium humate as heavy metal capturing agent.

An environmental friendly and economic natural biopolymer-sodium humate (HA-Na) was used to capture Hg(II) from aqueous solutions, and the trapped Hg(II) (HA-Na-Hg) was then removed by aluminium coagulation. The best Hg(II) capturing performance (90.60%) was observed under the following conditions: initial pH of 7.0, coagulation pH of 6.0, HA-Na dosage of 5.0 g L-1, Al2(SO4)3.18H2O dosage of 4.0 g L-1, initial Hg(II) concentration of 50 mg L-1 and capturing time of 30 min. The HA-Na compositions with the molecular weight beyond 70 kDa showed the most intense affinity toward Hg(II). The results showed that the reaction equilibrium was achieved within 10 min (pH 7.0), and could be well fitted by the pseudo-second-order kinetics model. The capturing process could be well described by the Langmuir isotherm model and the maximum capturing capacity of Hg(II) was high up to 9.80 mg g-1 at 298 K (pH 7.0). The Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis showed that the redox reaction between Hg(II) and HA-Na and the coordination reaction of carboxyl and hydroxy groups of HA-Na with Hg(II) were responsible for Hg(II) removal. The successive regeneration experiment showed that the capturing efficiency of humates for Hg(II) was maintained at about 51% after five capture-regeneration recycles.

Large moderately-differentiated ovarian Sertoli-Leydig cell tumor in a 13-year-old female: A case report.

Sertoli-Leydig cell tumor of the ovary, also known as androblastoma, is a rare neoplasm from the group of sex cord-stromal tumors of the ovary. The tumor accounts for <0.5% of all primary ovarian neoplasms. The clinical signs and symptoms of Sertoli-Leydig cell tumors can be associated with either hormonal production or the presence of a mass-occupying lesion. In the current study, a 13-year-old female was diagnosed with a stage Ic ovarian Sertoli-Leydig cell tumor following abdominal pain and distension. One month after a right oophorectomy, the follow-up magnetic resonance imaging scan was negative for residual or recurrent tumor. The overall 5-year survival rate for moderately-differentiated (grade 2) and poorly-differentiated (grade 3) Sertoli-Leydig cell tumors is 80%, and long-term follow-up is therefore highly advised in this patient.

The feasibility of establishing classification system for ovarian function.

The occurrence, development, and decline of ovarian function are the foundation in women's whole life stages, which reflect the process beginning from embryo formation to the aging. Correct assessment of ovarian function is significant for evaluating the potential reproductive ability and predicting the age of menopause, as well as providing both individualized and proper treatment and preventive care based on physiological characteristics of women in different phases. Ovarian reserve (OR) is used to predict the potential fertility of women by evaluating the follicles and the quantity and quality of eggs. Currently, there are multiple indexes used to evaluate ovarian reserve, including anti-Millerian hormone (AMH), follicle-stimulating hormone (FSH), estradiol (E2), inhibin B, antral follicle count (AFC), etc. Although some scholars combine multiple indexes to evaluate the ovarian function, these indexes are far less accurate, detailed, and comprehensive. To find an ideal method for evaluation of ovarian reserve is the hotspot in research of reproductive endocrine. The present authors, for the first time, put forward a classification system of ovarian reserve function after summarizing numerous cases. It can both accurately and effectively evaluate the ovarian function quantitatively. It is of great help in making clinical decisions and of great significance in future development.

Mutation screening of HOXA7 and HOXA9 genes in Chinese women with Müllerian duct abnormalities.

HOXA genes in groups 7-13 have been proven to play a role in determining positional identity along the genitalia axis. The aim of the present study was to explore the relationship between HOXA7 and HOXA9 mutations and Müllerian duct abnormalities (MDA). One hundred and ninety-two Chinese patients with MDA abnormalities and 192 healthy controls were recruited. All coding regions of HOXA7 and HOXA9 were amplified and sequenced directly. Rs2301721 and rs2301720 in HOXA7, rs35355140 and rs7810502 in HOXA9 were identified in patients with MDA and controls. One rare single nucleotide polymorphism rs189587233 in 3' UTR of HOXA9 gene was detected in one patient with didelphic uterus and absent in the 192 controls. This polymorphism, however, is known to exist in the normal Chinese population. Our results indicated that variants in the HOXA7 and HOXA9 genes were not common in Chinese women with Müllerian duct abnormalities.

Sohlh2 inhibits ovarian cancer cell proliferation by upregulation of p21 and downregulation of cyclin D1.

Spermatogenesis and oogenesis basic helix-loop-helix (bHLH) transcription factor 2 (Sohlh2) functions as a bhlh transcription factor to regulate mouse germ cell differentiation. Our previous data showed that Sohlh2 was highly expressed in human normal tissues, but low level of Sohlh2 was observed in many cancer cell lines, suggesting a possible role of Sohlh2 in tumorigenesis. In this study, we examined this possibility by using immunohistochemistry, MTT, 5-bromo-2-deoxyuridine, clonogenic assay and tumor xenograft techniques. Our results showed that the expression of Sohlh2 was decreased in epithelial ovarian carcinoma (EOC) tissues compared with benign ovarian tumors and ovarian tumors with low malignant potential. Forced expression of Sohlh2 led to a significant reduction in cancer cell proliferation in vitro and tumorigenesis in nude mice. Conversely, silencing of Sohlh2 enhanced ovarian cancer cell proliferation. Furthermore, Sohlh2 had opposite effects on its two direct targets p21 and cyclin D1: overexpression of Sohlh2 upregulated p21 but downregulated cyclin D1 expression. p21 knockdown could reverse the effects of Sohlh2 overexpression on inhibiting cell proliferation, and cyclin D1 knockdown could reverse the effects of Sohlh2 ablation on promoting cell proliferation. Thus, our data indicate that Sohlh2 likely functions as a tumor suppressor in EOCs, which is achieved by inducing p21 expression but repressing cyclin D1 expression.

Mutational analysis of SKP2 and P27 in Chinese Han women with premature ovarian failure.

P27 and SKP2, a major regulator of P27, play a crucial role in ovarian function in mice. Both P27-deficient and SKP2-deficient female mice develop premature ovarian failure (POF). The coding regions of SKP2 and P27 were examined in 200 Chinese women with POF and 200 control volunteers. This study is the first to investigate SKP2 in POF. No plausible pathogenic mutations were detected. The results suggest that mutations in SKP2 and P27 are not common in Chinese Han women with POF.

Effects of BMP4/SMAD signaling pathway on mouse primordial follicle growth and survival via up-regulation of Sohlh2 and c-kit.

Bone morphogenetic protein 4 (BMP4) is essential for the development of primordial follicles, although its underlying mechanism remains largely unknown. By using cultured ovaries, the effects of BMP4 and the potential signal transduction pathways were investigated. Ovaries from 3-day-old female mouse pups were maintained in organ culture in the absence (control) or presence of BMP4 (100 ng/ml). At different culture time, the effects of BMP4 on primordial follicle growth and survival were assayed by follicle count and TUNEL labeling. The expression of phospho-SMAD1/5/8, Sohlh2, and c-kit were measured by immunohistochemistry, RT-PCR, and Western blotting. Immunohistochemistry was also performed to determine the expression pattern of BMP4, pSMAD1/5/8, Sohlh2, and c-kit in vivo during ovarian development. The results showed treatments of ovaries with BMP4 resulted in a significant (P < 0.05) increase on the primordial-to-primary follicle transition. The oocytes of primordial follicles treated with BMP4 were also less likely to undergo apoptosis. BMP4 enhanced the phosphorylation of SMAD1/5/8 and up-regulated the expression of Sohlh2 and c-kit in primordial follicles. During ovarian development in vivo, Sohlh2, and c-kit exhibited similar expression patterns to BMP4 and pSMAD1/5/8 in primordial follicles. The present studies suggest that BMP4/SMAD signaling pathway initiate primordial follicle growth and prevented oocyte apoptosis via up-regulation of Sohlh2 and c-kit.

LHX1 mutation screening in 96 patients with müllerian duct abnormalities.

OBJECTIVE: To investigate whether LHX1 gene mutations exist in Han Chinese patients with müllerian duct abnormalities (MDAs). DESIGN: Mutation screening. SETTING: University hospital. PATIENT(S): Ninety-six MDA patients and 105 control subjects from a Han Chinese population. The parents of the patients carrying the genetic variation were also screened. INTERVENTION(S): Gene sequencing. MAIN OUTCOME MEASURE(S): Karyotype, LHX1 gene sequencing. RESULT(S): We found no significant mutation in coding regions of LHX1. However, there is a new rare polymorphism of LHX1 gene, c.1070-1081del, found in 1 out of 77 incomplete müllerian fusion patients and 1 out of 105 control individuals in the Han Chinese population (thus affecting ∼1% of Han Chinese). CONCLUSION(S): No causative perturbation was identified in the LHX1 gene. Mutations in the coding regions of LHX1 may not be a common genetic etiologic factor involved in Han Chinese MDA patients.

Variants of the WNT7A gene in Chinese patients with müllerian duct abnormalities.

OBJECTIVE: To search for WNT7A gene mutations in a cohort of 191 Chinese Han patients with müllerian duct abnormalities (MDAs). DESIGN: Phenotypic and mutational study. SETTING: University hospital. PATIENT(S): A total of 191 Chinese Han patients with MDAs and 192 healthy control individuals. INTERVENTION(S): Genomic DNA extracted from blood samples, all coding regions amplified by polymerase chain reaction (PCR) then directly sequenced to screen variants. MAIN OUTCOME MEASURE(S): Not applicable. RESULT(S): The sequence analysis revealed one novel synonymous variant and three known single-nucleotide polymorphisms (SNPs). CONCLUSION(S): The results indicate that mutations in the coding sequence of WNT7A are not responsible for müllerian duct abnormalities in the Chinese population.

Granular cell tumor from a 7-year swelling of the vulva: a case report.

Granular cell tumor (GCT) is a rare neoplastic process of the skin and soft tissue. About 10% of GCTs are found in the vulva. A 27-year-old client presented to the outpatient service of our hospital with painless edematous lesions on both sides of labia majora and minora. After a thorough preoperative preparation, the patient underwent complete excision of the mass under general anesthesia. Pathologic examination and immunohistochemical study supported the diagnosis of GCT.

Successful pregnancies with uterine leiomyomas and myomectomy at the time of caesarean section.

OBJECTIVE To ascertain the impact of uterine leiomyomas on pregnancy outcome, and to determine the effectiveness of myomectomy at the time of caesarean delivery. METHODS A retrospective study was conducted on pregnant women with uterine leiomyomas. Clinical information including the course of the pregnancy, mode of delivery, pathology findings, and postpartum course were extracted from medical records and analysed for statistical significance. RESULTS There were 50 pregnancies associated with uterine leiomyomas. During pregnancy, the leiomyomas grew >2 cm in 46% of cases. Only 6% were affected by symptoms of red degeneration. There were 2% mid trimester inductions of labour, 6% vaginal deliveries, and 92% caesarean sections. Of the 46 caesarean sections, 47% were due to obstetrical factors and 89% of patients underwent myomectomy at the time of caesarean delivery. The mean blood loss from myomectomy at the time of caesarean section was 260 ml (200-700 ml), and 5% of patients who underwent myomectomy were transfused. CONCLUSIONS It is possible to carry a pregnancy successfully to term when the pregnancy is complicated by uterine leiomyomas. When caesarean delivery is needed, myomectomy can be performed at the time of caesarean section routinely without significant complications.