miR-4433a-3p promotes granulosa cell apoptosis by targeting peroxisome proliferator-activated receptor alpha and inducing immune cell infiltration in polycystic ovarian syndrome.

BACKGROUND: Granulosa cell (GC) proliferation and apoptosis are critical events of the ovum energy supply, which lead to follicular growth retardation or atresia, and various ovulatory obstacles, eventually resulting in the development of ovarian disorders such as polycystic ovarian syndrome (PCOS). Apoptosis and dysregulated miRNA expression in GCs are manifestations of PCOS. miR-4433a-3p has been reported to be involved in apoptosis. However, there is no study reporting the roles of miR-4433a-3p in GC apoptosis and PCOS progression. METHODS: miR-4433a-3p and peroxisome proliferator-activated receptor alpha (PPAR-α) levels in GCs of PCOS patients or in tissues of a PCOS rat model were examined by quantitative polymerase chain reaction and immunohistochemistry. Bioinformatics analyses and luciferase assays were used to examine the association between miR-4433a-3p and PPAR-α, as well as PPAR-α and immune cell infiltration, in PCOS patients. RESULTS: miR-4433a-3p expression in GCs of PCOS patients was increased. miR-4433a-3p overexpression inhibited the growth of the human granulosa-like tumor cell line (KGN) and promoted apoptosis, while co-treatment with PPAR-α and miR-4433a-3p mimic rescued miR-4433a-3p-induced apoptosis. PPAR-α was a direct target of miR-4433a-3p and its expression was decreased in PCOS patients. PPAR-α expression was also positively correlated with the infiltration of activated CD4+ T cells, eosinophils, B cells, gamma delta T cells, macrophages, and mast cells, but negatively correlated with the infiltration of activated CD8+ T cells, CD56+ bright natural killer cells, immature dendritic cells, monocytes, plasmacytoid dendritic cells, neutrophils, and type 1 T helper cells in PCOS patients. CONCLUSION: The miR-4433a-3p/PPAR-α/immune cell infiltration axis may function as a novel cascade to alter GC apoptosis in PCOS.

Frozen versus fresh single blastocyst transfer in ovulatory women: a multicentre, randomised controlled trial.

BACKGROUND: Elective single embryo transfer (eSET) has been increasingly advocated, but concerns about the lower pregnancy rate after reducing the number of embryos transferred have encouraged transfer of multiple embryos. Extended embryo culture combined with electively freezing all embryos and undertaking a deferred frozen embryo transfer might increase pregnancy rate after eSET. We aimed to establish whether elective frozen single blastocyst transfer improved singleton livebirth rate compared with fresh single blastocyst transfer. METHODS: This multicentre, non-blinded, randomised controlled trial was undertaken in 21 academic fertility centres in China. 1650 women with regular menstrual cycles undergoing their first cycle of in-vitro fertilisation were enrolled from Aug 1, 2016, to June 3, 2017. Eligible women were randomly assigned to either fresh or frozen single blastocyst transfer. The randomisation sequence was computer generated, with block sizes of two, four, or six, stratified by study site. For those assigned to frozen blastocyst transfer, all blastocysts were cryopreserved and a delayed frozen-thawed single blastocyst transfer was done. The primary outcome was singleton livebirth rate. Analysis was by intention to treat. This trial is registered at the Chinese Clinical Trial Registry, number ChiCTR-IOR-14005405. FINDINGS: 825 women were assigned to each group and included in analyses. Frozen single blastocyst transfer resulted in higher rates of singleton livebirth than did fresh single blastocyst transfer (416 [50%] vs 329 [40%]; relative risk [RR] 1·26, 95% CI 1·14-1·41, p<0·0001). The risks of moderate or severe ovarian hyperstimulation syndrome (four of 825 [0·5%] in frozen single blastocyst transfer vs nine of 825 [1·1%] in fresh single blastocyst transfer; p=0·16), pregnancy loss (134 of 583 [23·0%] vs 124 of 481 [25·8%]; p=0·29), other obstetric complications, and neonatal morbidity were similar between the two groups. Frozen single blastocyst transfer was associated with a higher risk of pre-eclampsia (16 of 512 [3·1%] vs four of 401 [1·0%]; RR 3·13, 95% CI 1·06-9·30, p=0·029). INTERPRETATION: Frozen single blastocyst transfer resulted in a higher singleton livebirth rate than did fresh single blastocyst transfer in ovulatory women with good prognosis. The increased risk of pre-eclampsia after frozen blastocyst transfer warrants further studies. FUNDING: The National Key Research and Development Program of China.

[Influence of male age on the pregnancy outcomes of IVF-ET].

OBJECTIVE: To investigate the influence of male age on the pregnancy outcomes of in vitro fertilization-embryo transfer (IVF-ET). METHODS: We retrospectively analyzed 7,533 cycles of IVF-ET performed between January 1, 2009 and October 31, 2013. We divided the samples into three groups according to the female age (< 30, 30-34, and 35-38 yr), each again subdivided into six groups based on the male age (< 30, 30-32, 33-35, 36-38, 39-41, and ≥ 42 yr). We compared the rates of implantation, pregnancy, miscarriage, and live birth among different age groups. RESULTS: There were no statistically significant differences in basal E2, FSH, endometrium thickness on the day of hCG administration, number of oocytes retrieved, and days of embryo transfer among different male age groups (P > 0.05). The implantation rate showed an age-dependent decrease in the < 30, 30-32, 33-35, 36-38, 39-41, and ≥ 42 yr male groups, 41.1, 42.0, 39.5, 31.3, 40.7, and 48.6% among the women aged < 30 years (P < 0.05), 40.3, 36.4, 35.1, 35.3, 29.4, and 37.3% among the women aged 30-34 years (P < 0.05), and 48.2, 17.8, 25.3, 23.5, 22.1, and 23.8% among the women aged 35-38 years (P < 0.05). The miscarriage rate was significantly higher in the ≥ 39 yr than in the 30-32 and 33-35 yr male age groups among the women aged 30-34 years (P < 0.05), but showed no remarkable differences among the other male age groups in the women aged < 30 and 35-38 years (P > 0.05). No statistically significant differences were observed in the rates of pregnancy and live birth among different male age groups (P > 0.05). CONCLUSION: Male age has some influence on the rates of implantation and miscarriage but not on the rates of pregnancy and live birth in IVF-ET.

Chemerin isoform analysis in human biofluids using an LC/MRM-MS-based targeted proteomics approach with stable isotope-labeled standard.

Targeted proteomics has advantages over earlier conventional technologies for protein detection. We developed and validated an LC/MRM-MS-based targeted proteomic method combined with immunoaffinity precipitation for the enrichment and detection of low abundance chemerin isoforms in human biofluids. After tryptic digestion, each chemerin isoform was characterized by isoform-specific peptides, and the absolute quantification was achieved by using stable isotope-labeled peptides as internal standards. In serum, follicular fluid and synovial fluid, a total of 6 chemerin isoforms were identified and quantified, among which a novel natural isoform 153Q was discovered for the first time. The relative content of the six chemerin isoforms in human serum was 157S ≫ 156F ≫ 158K > 154F ≥ 155A > 153Q in the ratio of 25:17:5:2.5:2.2:1, respectively. The absolute contents were in the range of 88-3.5 ng/mL. This distribution remained consistent among the 3 biofluids analyzed. Total chemerin were found to be increased in both polycystic ovary syndrome (serum and follicular fluid) and rheumatoid arthritis (serum) patients. However, chemerin isoform analysis revealed that only 156F & 157S were increased in the former, while 155A, 156F & 157S were increased in the latter. This demonstrates the potential of this method in detailed characterization of changes in chemerin isoforms that may be of clinical relevance.

Insulin Reduces Reaction of Follicular Granulosa Cells to FSH Stimulation in Women With Obesity-Related Infertility During IVF.

CONTEXT: Women with obesity usually need larger doses of FSH for ovarian stimulation, resulting in poor outcomes; however, the mechanism is still unclear. OBJECTIVE: To investigate the molecular regulation of FSH receptor (FSHR) expression associated with obesity. DESIGN: Case-control study to improve in vitro fertilization (IVF) outcomes. PATIENTS: Women with obesity (82) and women who were overweight (457) undergoing IVF and 1790 age-matched controls with normal weight from our reproductive medicine center. MAIN OUTCOME MEASURES: FSHR expression was decreased in parallel with body mass index (BMI), whereas the estradiol (E2) level on the human chorionic gonadotropin (hCG) trigger day was significantly lower. RESULTS: FSHR expression in human granulosa cells (hGCs), both mRNA (P = 0.02) and protein (P = 0.001) levels, was decreased in women who were overweight or obese. Both insulin (P < 0.001) and glucose (P = 0.0017) levels were positively correlated with BMI in fasting blood and follicle fluids (FFs) but not with FFs leptin level. We treated human granulosa-like tumor cells (KGN) cells with insulin; E2 production was compromised; the level of phosphorylated (p)-protein kinase B (p-Akt2) decreased, whereas p-glycogen synthase kinase 3 (GSK3) increased; and there were similar changes in hGCs from women with obesity. Stimulated hGCs from women with obesity with compound 21 (CP21), an inhibitor of GSK3ß, resulted in upregulated ß-catenin activation and increased FSHR expression. CP21 also increased the expression of insulin receptor substrate 1 and phosphatidylinositol 3-kinase (PI3K), as well as p-Akt2. CONCLUSIONS: Women with obesity in IVF were associated with reduced FSHR expression and E2 production caused by a dysfunctional insulin pathway. Decreased FSHR expression in hGCs from women with obesity and insulin-treated KGN cells could be rescued by an inhibitor of GSK3ß, which might be a potential target for the improvement of the impaired FSH-stimulation response in women with obesity.

Na7CrCuW11O39.16H2O induces apoptosis in human ovarian cancer SKOV3 cells through the p38 signaling pathway.

Ovarian carcinoma is a common malignant disease worldwide with a poor therapeutic response. The present study investigated the effects of Na7CrCuW11O39.16H2O (CrCuW11) on ovarian cancer cell growth and investigated the mechanisms underlying its actions. The effects of CrCuW11 on cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, acridine orange/ethidium bromide staining and electron microscopy in human ovarian cancer SKOV3 cells. The expression of bcl-2-like protein 4 (Bax), B-cell lymphoma 2 (Bcl-2), cytochrome c, phosphorylated (p)-p38 and p38 was determined by western blot analysis. Caspase-3 activity was measured by caspase-3 activity kit. CrCuW11 concentrations of 1.87×10-3 mol. l-1 at 12 h reduced viability induced apoptosis in SKOV3 cells in a concentration-and time-dependent manner. Forced expression of CrCuW11 upregulated the expression of certain proteins (Bax, cytochrome c, and p-p38), and downregulated Bcl-2 protein expression. Furthermore, CrCuW11 also enhanced caspase-3 activity. The p38 inhibitor SB203580 was able to inhibit the activity of CrCuW11. Caspase-3 and p38 signaling pathways were associated with CrCuW11-regulated multiple targets involved in SKOV3 cell proliferation. Therefore, the results of the present study indicated that CrCuW11 may be used as a novel clinical drug for the treatment of ovarian cancer.

Transcription factor­microRNA synergistic regulatory network revealing the mechanism of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common type of endocrine disorder, affecting 5­11% of women of reproductive age worldwide. Transcription factors (TFs) and microRNAs are considered to have crucial roles in the developmental process of several diseases and have synergistic regulatory actions. However, the effects of TFs and microRNAs, and the patterns of their cooperation in the synergistic regulatory network of PCOS, remain to be elucidated. The present study aimed to determine the possible mechanism of PCOS, based on a TF­microRNA synergistic regulatory network. Initially, the differentially expressed genes (DEGs) in PCOS were identified using microarray data of the GSE34526 dataset. Subsequently, the TFs and microRNAs which regulated the DEGs of PCOS were identified, and a PCOS­associated TF­microRNA synergistic regulatory network was constructed. This network included 195 DEGs, 136 TFs and 283 microRNAs, and the DEGs were regulated by TFs and microRNAs. Based on topological and functional enrichment analyses, SP1, mir­355­5p and JUN were identified as potentially crucial regulators in the development of PCOS and in characterizing the regulatory mechanism. In conclusion, the TF­microRNA synergistic regulatory network constructed in the present study provides novel insight on the molecular mechanism of PCOS in the form of synergistic regulated model.

A subpathway-based method of drug reposition for polycystic ovary syndrome.

The need for development of new therapeutic agents for polycystic ovary syndrome (PCOS) is urgent due to general lack of efficient and specialized drugs currently available. We aimed to explore the metabolic mechanism of PCOS and inferred drug reposition for PCOS by a subpathway-based method. Using the GSE34526 microarray data from the Gene Expression Omnibus database, we first identified the differentially expressed genes (DEGs) between PCOS and normal samples. Then, we identified 13 significantly enriched metabolic subpathways that may be involved in the development of PCOS. Finally, by an integrated analysis of PCOS-involved subpathways and drug-affected subpathways, we identified 54 novel small molecular drugs capable to target the PCOS-involved subpathways. We also mapped the DEGs of PCOS and a potential novel drug (alprostadil) into purine metabolism pathway to illustrate the potentially active mechanism of alprostadil on PCOS. Candidate agents identified by our approach may provide insights into a novel therapy approach for PCOS.

Arrested human embryos are more likely to have abnormal chromosomes than developing embryos from women of advanced maternal age.

BACKGROUND: Aneuploidy is one of the major factors that result in low efficiency in human infertility treatment by in vitro fertilization (IVF). The development of DNA microarray technology allows for aneuploidy screening by analyzing all 23 pairs of chromosomes in human embryos. All chromosome screening for aneuploidy is more accurate than partial chromosome screening, as errors can occur in any chromosome. Currently, chromosome screening for aneuploidy is performed in developing embryos, mainly blastocysts. It has not been performed in arrested embryos and/or compared between developing embryos and arrested embryos from the same IVF cycle. METHODS: The present study was designed to examine all chromosomes in blastocysts and arrested embryos from the same cycle in patients of advanced maternal ages. Embryos were produced by routine IVF procedures. A total of 90 embryos (45 blastocysts and 45 arrested embryos) from 17 patients were biopsied and analyzed by the Agilent DNA array platform. RESULTS: It was found that 50% of the embryos developed to blastocyst stage; however, only 15.6% of the embryos (both blastocyst and arrested) were euploid, and most (84.4%) of the embryos had chromosomal abnormalities. Further analysis indicated that 28.9% of blastocysts were euploid and 71.1% were aneuploid. By contrast, only one (2.2%) arrested embryo was euploid while others (97.8%) were aneuploid. The prevalence of multiple chromosomal abnormalities in the aneuploid embryos was also higher in the arrested embryos than in the blastocysts. CONCLUSIONS: These results indicate that high proportions of human embryos from patients of advanced maternal age are aneuploid, and the arrested embryos are more likely to have abnormal chromosomes than developing embryos.

[Observation on the efficacy of acupuncture at key acupoints combined with rehabilitation therapy for spasmodic hemiplegia after cerebral infarction].

OBJECTIVE: To assess the clinical efficacy of acupuncture at key acupoints combined with the routine rehabilitation training of limb function on spasmodic hemiplegia after cerebral infarction. METHODS: Eighty-six cases were randomized into an acupuncture combined with rehabilitation group (group A, 44 cases) and a rehabilitation group (group B, 42 cases). In group A, the key acupoints were selected from head, face, chest, abdomen, shoulder, back, hands, feet and ankles such as Cuanzhu (BL 2), Danzhong (CV 17), Jianyu (LI 15) and Yanglao (SI 16) were stimulated with acupuncture. In combination, the routine limb rehabilitation training was applied, once every day. In group B, the routine limb rehabilitation training was applied alone. In both groups, 10 treatments made one session and 2 sessions were required totally. Before and after treatment, Fugl-Meyer scale and functional independent measurement (FIM) scale were adopted to assess the limb motor level and the activity of daily life in the patients respectively. The modified Ashworth scale was used to assess the effect of anti-spasm. RESULTS: The total effective rate of anti-spasm was 90.9% (40/44) in the group A, which was superior to 73.8% (32/42) in the group B (P < 0.05). After 2 sessions of treatment, Fugl-Meyer score and FIM score were all improved in both groups (all P < 0.05) and the results in the group A were better than those in the group B (all P < 0.05). Additionally, the improvement of FIM score after the 1st session of treatment in the group A was better as compared with the group B, indicating the significant difference (P < 0.05). CONCLUSION: Acupuncture at key acupoints combined with rehabilitation therapy effectively relieves the spasmodic condition of the patients with post-stroke spasmodic hemiplegia, improves the limb function and the life activity of the patients.

Augmentation of transgenic expression by ultrasound­mediated liposome microbubble destruction.

Non-invasive, efficient and tissue-specific transgenic technologies could be valuable in gene therapy. Although non-viral carriers may be safer and cheaper, they have a much lower transfection efficiency than viral gene carriers. The present study was designed to test the transgenic expression and safety of red fluorescent protein (RFP) in HeLa cells in vitro and in transplanted tumors of nude mice in vivo under ultrasound-mediated liposome microbubble destruction (UMLMD) conditions. Plasmids containing RFP were gently mixed with liposome microbubbles (LMs). The mixture was added to HeLa cells or injected into BALB/c mice by the tail vein under various ultrasound exposure and LM parameters, and then the transfection efficiencies were examined. The results in vivo and in vitro demonstrated that, following a comparison of the plasmid group, the ultrasound + plasmid group and the LM + plasmid group, UMLMD significantly increased the transgenic expression (P<0.01) without causing any apparent detrimental effect. From the study, we concluded that UMLMD could be a non-invasive, effective and promising non-viral technique for gene therapy and transgenic research.

Optimization and apoptosis induction by RNAi with UTMD technology in vitro.

Apoptosis induction by short hairpin RNA (shRNA) expression vectors may be an efficient and promising strategy for cancer gene therapy. Ultrasound-targeted microbubble destruction (UTMD) is an appealing technique; however, there few data are available to demonstrate the feasibility and to optimize the methodology for this technology. The aim of this study was to optimize this technique and to elucidate the effects on gene inhibition and apoptosis induction in vitro. Human cervical cancer cell lines were obtained and cultured.shRNA vectors were constructed, and the UTMD technique was examined to determine whether or not it was suitable for shRNA transfection into cells. Cells were then examined using flow cytometry. The results revealed that the optimal irradiation parameters obtained higher transfection efficiency and did not affect the integrity of plasmid DNA. We concluded that survivin downregulation with shRNA expression vectors, mediated by the optimal UTMD parameters, markedly induced cell apoptosis and caused cell cycle arrest, laying a foundation for further investigation of this cancer therapy.

[Effects of patient age, number and quality of embryo transferred on the outcome of in vitro fertilization-embryo transfer].

OBJECTIVE: To study the effect of patient age, the number and quality of embryo transferred on pregnancy outcome of in vitro fertilization-embryo transfer (IVF-ETs). METHODS: A retrospective study was performed in infertile women who underwent a total of 1800 cycles of IVF-ET and intracytoplasmic sperm injection (ICSI). The patients were divided into three age groups, namely<30 years group, 30-35 years group, and>or=35 years group. The clinical pregnancy rate and the multiple pregnancy rate were compared when 1, 2 or 3 embryos and 0, 1, 2 or 3 good-quality embryos were transferred. RESULTS: In patients<30 years, no significant differences was found in the clinical pregnancy rate between 1, 2 and 3 embryos transfer groups; 2 and 3 good-quality embryos transfer resulted in similar pregnancy rate, which was significantly higher than that resulted from 0 and 1 good-quality embryo transfer. Multiple pregnancy was not found in 1 embryo transfer group. In patients aged 30-35 years, the pregnancy rate showed no significant differences not only between 1 and 2 embryos transfer groups, but also between 2 and 3 good-quality embryos transfer groups; multiple embryo transfer led to significantly increased multiple pregnancy rate. In patients aged>or=35 years, the transfer of 1, 2 and 3 embryos resulted in similar pregnancy rate; transfer of 3 good-quality embryos had obviously higher pregnancy rate than 0, 1 and 2 good-quality embryos transfer groups. Increased numbers of embryos and good-quality embryos transferred were both associated with increased multiple pregnancy rate. CONCLUSION: One good-quality embryo transfer in patients<30 years and 2 good-quality embryos transfer in patients>or=30 years can obtain ideal pregnancy rate and reduce the incidence of multiple pregnancy. For patients aged>or=35 years, transfer of only good-quality embryo is recommended.

[In vitro maturation of human oocytes from pregnant late term: a new source of oocytes].

OBJECTIVE: To investigate the in vitro maturation of human oocytes (IVM) from pregnant late term, natural cycles and Gn stimulating cycles and the effect of granulose cells on IVM from pregnant late term. METHODS: A total of 1086 immature oocytes were obtained including 633 oocyte-cumulus complexes (OCCs) and 453 denuded oocytes (DOs). OCCs were divided into pregnant late term group, natural cycle group and IVM group, and DOs were divided into pregnant late term group, natural cycle group and controlled ovarian hyperstimulation (COH) group. All the oocytes were matured in IVM culture system and fertilized by ICSI. The embryos were cultured to blastocyst stage except that those in IVM group were transferred into the uterus. The main outcomes were assessed including maturation rate (MR), fertilization rate (FR), cleavage rate (CR), and blastulation rate (BR) (natural cycle group, pregnant late term group and COH group) and pregnancy rate per transfer cycle (PR) of IVM group. RESULTS: MR of OCCs in pregnant late term group, natural cycle group and COH group was 74.3%, 76.9% and 82.2%, respectively, showing statistical difference between pregnant late term cycle group and IVM group. No statistical difference was observed in FR, CR or BR between the three groups. For IVM cycle group, clinical pregnancy rate of 20% per aspiration was achieved. For DOs, MR of COH group (86.0%) was significantly higher than that of the natural cycle group (72.5%) and pregnant late term group (72.7%) (P<0.01). FR, CR and BR showed no statistical difference among the 3 groups. No difference was found in MR, FR, CR and BR between OCCs group and DOs group from pregnant late term. CONCLUSIONS: The oocytes from pregnant late term have the same development potential as those from natural cycles or Gn stimulating cycles in vitro, and provide a new source of donor oocytes. Granulose cells do not affect the IVM from pregnant late term.