MicroRNAs in POI, DOR and POR.

PURPOSE: Premature ovarian insufficiency (POI) is a clinical syndrome defined by loss of ovarian activity before the age of 40 years. However, the etiology of approximately 90% patients remains unknown. Diminished ovarian reserve (DOR) and poor ovarian response (POR) are related to POI in clinic. The main purpose of this review was to evaluate the roles of microRNAs (miRNAs) in the pathogenesis and therapeutic potential for POI, DOR and POR. METHODS: A literature search was conducted using six databases (PubMed, EMBASE, Web of Science, Cochrane Library, CNKI and Wangfang Data) to obtain relevant studies. RESULTS: This review enlightens expression profiles and functional studies of miRNAs in ovarian insufficiency in animal models and humans. Functional studies emphasized the role of miRNAs in steroidogenesis, granulosa cell proliferation/apoptosis, autophagy and follicular development by regulating target genes in specific pathways, such as the PI3K/AKT/mTOR, TGFß, MAPK and Hippo pathways. Differentially expressed circulating miRNAs provided novel biomarkers for diagnosis and prediction, such as miR-22-3p and miR-21. Moreover, exosomes derived from stem cells restored ovarian function through miRNAs in chemotherapy-induced POI models. CONCLUSION: Differential miRNA expression profiles in patients and animal models uncovered the underlying mechanisms and biomarkers of ovarian insufficiency. Exosomal miRNAs can restore ovarian function against chemotherapy-induced POI, which needs further investigation to develop novel preventive and therapeutic strategies in clinical practice.

Caulis Sargentodoxae Prescription Plays a Therapeutic Role with Decreased Inflammatory Cytokines in Peritoneal Fluid in the Rat Endometriosis Model.

Caulis Sargentodoxae prescription has been confirmed by the gynecological clinical observation to be effective in the treatment of endometriosis (EMs), and inflammatory cytokines were involved in EMs. In this paper, animal experiments were designed to explain anti-inflammatory mechanisms of Caulis Sargentodoxae prescription on endometriosis. The EMs model was established by autoplastic transplantation, and rats were randomly divided into seven groups: normal control group, model group, ovariectomized group, gestrinone (Western medicine) group, Caulis Sargentodoxae prescription (Chinese medicine) group, celecoxib (inhibitor) group, and combination (Chinese medicine + inhibitor) group. After oral administration for 21 days, the growth inhibitory rates of the ectopic endometria in treatment groups were evaluated, and the levels of inflammatory cytokines in the serum and peritoneal fluid were determined by ELISA, as well as the expression of prostaglandin endoperoxide synthase 2 (PTGS2) in the ectopic endometrial tissues was detected by real-time polymerase chain reaction, immunohistochemistry, and western blotting. The growth inhibitory rates of the ectopic endometria were significantly higher in the Caulis Sargentodoxae prescription group and gestrinone group, in comparison with the model group (p < 0.05). In the Caulis Sargentodoxae prescription group, the levels of inflammatory cytokines including IL-1, IL-2, IL-6, TNF-α, and PGE2 were all reduced in the serum and peritoneal fluid (p < 0.05). In addition, the specific expression of PTGS2 in the ectopic endometrial tissues significantly decreased in the Caulis Sargentodoxae prescription group and PTGS2 inhibitor celecoxib group both at mRNA and protein levels, but in the steroid hormone drug gestrinone group not at the mRNA level. So, Caulis Sargentodoxae prescription has a reliable therapeutic effect on the EMs by its comprehensive anti-inflammatory roles, possibly in a way different from gestrinone.

Gene UCHL1 expresses specifically in mouse uterine decidual cells in response to estrogen.

UCHL1 is expressed specifically in the brain and gonads of almost all studied model organisms including Drosophila, zebrafish, amphibians, and mammals, suggesting a high degree of evolutionary conservation in its structure and function. Although UCHL1 has been involved in spermatogenesis in mice, its specific expression in mammal placenta remains elusive. Our previous work has revealed that UCHL1 is highly expressed in oocytes, and has been involved in mouse ovarian follicular development. Here, we further examined UCHL1 expression change in endometria during early natural pregnancy, with different stages of the estrous cycle and pseudopregnancy as control. The UCHL1 gene deletion model showed that UCHL1 protein is associated with endometrial development, and its deletion leads to infertility. Notably, we demonstrate evidence showing the distinct expression pattern of UCHL1: weak expression over the uterine endometria, strong expression in decidualized stromal cells at the implantation site with a peak at pregnancy D6, and a shift with primary decidualization to secondary decidualized zones. Using the delayed implantation, the delayed implantation activation, and the artificial decidualization models, we have demonstrated that strong expression of UCHL1 occurred in response to decidualization and estrogen stimulation. These observations suggest that during the early proliferation and differentiation of mouse uterine decidua, UCHL1 expression is up-regulated, and formed an unique intracellular distribution mode. Therefore, we proposed that UCHL1 is involved in decidualization, and possibly in response to estrogen regulation.

Sequence variants in FSHR and CYP19A1 genes and the ovarian response to controlled ovarian stimulation.

OBJECTIVE: To examine whether sequence variants within the FSHR and CYP19A1 genes are related to the ovarian response to controlled ovarian stimulation (COS). DESIGN: Genetic association study using both single-gene and combined analyses of women with sequence variants undergoing in vitro fertilization treatment. SETTING: Academic research institute hospital. PATIENT(S): Seven hundred and five women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (FSH). INTERVENTION(S): Peripheral blood extraction, DNA purification, and FSHR c.919G>A (rs6165, p.Thr307Ala) and CYP19A1 c.*19C>T (rs10046) sequence variants analyses. MAIN OUTCOME MEASURE(S): Single-gene statistical analysis and combined statistical analysis with the SPSS17.0 software; FSHR c.919G>A and CYP19A1 c.*19C>T sequence variant genotypes and clinical parameters related to the COS response as oocyte retrieval and hormone levels, doses of exogenous FSH. RESULT(S): Women with genotype Ala/Ala at FSHR position 307 had higher basal levels of FSH and were more likely to have a low ovarian response compared with other genotypes. Women with genotype TT at CYP19A1 yielded fewer oocytes after ovarian stimulation. The combined analysis of these two sequence variants revealed that these two single-nucleotide variants have a synergistic effect in conferring the risk of a low ovarian response. CONCLUSION(S): Our results support an association of sequence variants in the genes that participate in estrogen synthesis, notably the FSHR and CYP19A1 genes, with the outcome of COS.

Decreased NDRG1 expression is associated with pregnancy loss in mice and attenuates the in vitro decidualization of endometrial stromal cells.

Embryo implantation is an essential step for a successful pregnancy, and any defect in this process can lead to a range of pregnancy pathologies. The objective of this study was to explore the role of N-myc downregulated gene 1 (NDRG1) in embryo implantation. It was found that uterine NDRG1 expression has a dynamic pattern during the estrous cycle in nonpregnant mice and that uterine NDRG1 expression was elevated during the implantation process in pregnant mice. The distinct accumulation of NDRG1 protein signals was observed in the primary decidual zone adjacent to the implanting embryo during early pregnancy. Furthermore, uterine NDRG1 expression could be induced by activated implantation or artificial decidualization in mice. Decreased uterine NDRG1 expression was associated with pregnancy loss in mice and was associated with recurrent miscarriages in humans. The in vitro decidualization of both mouse and human endometrial stromal cells (ESCs) was accompanied by increased NDRG1 expression and downregulated NDRG1 expression in ESCs effectively inhibited decidualization. Collectively, these data suggest that NDRG1 plays an important role in decidualization during the implantation process, and the abnormal expression of NDRG1 may be involved in pregnancy loss.

The ovarian estrogen synthesis function was impaired in Y123F mouse and partly restored by exogenous FSH supplement.

BACKGROUND: LepR tyrosine site mutation mice (Y123F) exhibit decreased serum E2 levels, immature reproductive organs, infertility as well as metabolic abnormalities. Although the actions of leptin and lepR in the control of reproductive function are thought to be exerted mainly via the hypothalamic-pituitary-gonadal axis, relatively less is known regarding their local effects on the peripheral ovary, especially on steroid hormone synthesis. Meanwhile, whether the decreased fertility of Y123F mouse could be restored by gonadotropin has not been clear yet. METHODS: The serum levels of E2, P4, FSH, LH, T and leptin of Y123F and WT mice at the age of 12 weeks were measured by enzyme-linked immunosorbent assay. Immunohistochemistry was used to compare the distribution of hormone synthases (STAR, CYP11A1, CYP19A1, HSD17B7) and FSHR in adult mouse ovaries of two genotypes. Western blot and real-time PCR were used to detect the expression levels of four ovarian hormone synthases and JAK2-STAT3 / STAT5 signaling pathway in 4 and 12 weeks old mice, as well as the effects of exogenous hFSH stimulation on hormone synthases and FSHR. RESULTS: Compared with WT mice, the serum levels of FSH, LH and E2 in 12-week-old Y123F mice were significantly decreased; T and leptin levels were significantly increased; but there was no significant difference of serum P4 levels. STAR, CYP11A1, HSD17B7 expression levels and the phosphorylation levels of JAK2 and STAT3 were significantly decreased in adult Y123F mice, while the expression of CYP19A1 and phospho-STAT5 were significantly increased. No significant differences were found between 4-week-old Y123F and WT mice. After exogenous hFSH stimulation, E2 levels and expression of CYP19A1 and HSD17B7 were significantly higher than that in the non-stimulated state, but significant differences still existed between Y123F and WT genotype mice under the same condition. CONCLUSIONS: Abnormal sex hormone levels of Y123F mice were due to not only decreased gonadotropin levels in the central nervous system, but also ovarian hormone synthase abnormalities in the peripheral gonads. Both FSH signaling pathway and JAK2-STAT3/STAT5 signaling pathway were involved in regulation of ovarian hormone synthases expression. Exogenous FSH just partly improved the blood E2 levels and ovarian hormone synthase expression.

[Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion].

The purpose of this study was to investigate the effects of ubiquitin C-terminal hydrolase-L1 (UCHL1) on non-small cell lung cancer cell line A549. UCHL1 gene knockout A549 cell line was constructed by CRISPR-CAS9 gene editing technique. The mRNA and protein levels of UCHL1 were examined by RT-PCR and Western blot, respectively. Cell proliferation and cycles were analyzed by CCK-8 method and flow cytometry, respectively. The sensitivity of A549 cells to cisplatin was detected by CCK-8 method. Migration ability of A549 cells was detected by scratch assay and Transwell test, and p-Erk expression level was assessed by Western blot. The results showed that UCHL1 gene knockout A549 cells were successfully constructed by CRISPR-CAS9 gene editing technique. After UCHL1 gene knockout, there was no significant change in cell proliferation and cell cycle ratios in A549 cells. UCHL1 gene knockout A549 cells exhibited decreased sensitivity to cisplatin and migration activity, as well as increased p-Erk expression level. These results suggest that the loss of UCHL1 gene function may reduce the sensitivity and migration ability of A549 cells, and this effect may be related to the activation of Erk1/2 signaling pathway.

Novel mutations in genes encoding subcortical maternal complex proteins may cause human embryonic developmental arrest.

Successful human reproduction initiates from normal gamete formation, fertilization and early embryonic development. Abnormalities in any of these steps will lead to infertility. Many infertile patients undergo several failures of IVF and intracytoplasmic sperm injection (ICSI) cycles, and embryonic developmental arrest is a common phenotype in cases of recurrent failure of IVF/ICSI attempts. However, the genetic basis for this phenotype is poorly understood. The subcortical maternal complex (SCMC) genes play important roles during embryonic development, and using whole-exome sequencing novel biallelic mutations in the SCMC genes TLE6, PADI6 and KHDC3L were identified in four patients with embryonic developmental arrest. A mutation in TLE6 was found in a patient with cleaved embryos that arrested on day 3 and failed to form blastocysts. Two patients with embryos that arrested at the cleavage stage had mutations in PADI6, and a mutation in KHDC3L was found in a patient with embryos arrested at the morula stage. No mutations were identified in these genes in an additional 80 patients. These findings provide further evidence for the important roles of TLE6, PADI6 and KHDC3L in embryonic development. This work lays the foundation for the genetic diagnosis of patients with recurrent IVF/ICSI failure.

Increased Expression of NDRG3 in Mouse Uterus During Embryo Implantation and in Mouse Endometrial Stromal Cells During In Vitro Decidualization.

Decidualization is an indispensable event in the embryo implantation process, but its underlying molecular mechanisms remain elusive. In this study, we showed that in mice, the uterine expression of N-myc downstream-regulated gene 3 (NDRG3), a member of the α/ß hydrolase superfamily, was induced by estradiol and progesterone. During the embryo implantation process, uterine Ndrg3 expression was remarkably upregulated, and its expression level at implantation sites (IS) was significantly higher than that at inter-IS. Increased uterine expression of Ndrg3 was associated with artificial decidualization and the activation of delayed implantation. The in vitro decidualization of mouse endometrial stromal cells (ESCs) induced by estradiol and progesterone was also accompanied by increased Ndrg3 expression, and downregulated Ndrg3 expression in ESCs effectively inhibited decidualization. miR-290b-5p was identified as an upstream regulator of Ndrg3, and the uterine expression level of miR-290b-5p was decreased during the implantation process. Furthermore, overexpression of miR-290b-5p in mouse ESCs inhibited their in vitro decidualization. Taken together, these data suggested that Ndrg3 might play an important role in embryo implantation by regulating decidualization potentially via the estrogen/progesterone/miR-290b-5p pathway.

LEPR gene polymorphism and plasma soluble leptin receptor levels are associated with polycystic ovary syndrome in Han Chinese women.

AIM: To investigate the association of LEPR polymorphisms and plasma leptin and soluble leptin receptor (sOB-R) levels with polycystic ovary syndrome (PCOS) in Chinese women. PATIENTS & METHODS: LEPR Lys109Arg (rs1137100) and Gln223Arg (rs1137101) polymorphisms of PCOS patients and the controls were genotyped. Plasma leptin and sOB-R levels of two groups were measured. RESULTS: The genotypic distributions of Lys109Arg (rs1137100) differed between the PCOS and control groups. Plasma sOB-R levels increased significantly in PCOS patients and were associated with PCOS independent of BMI. Furthermore, luteinizing hormone, triglyceride and fasting blood glucose correlated significantly to PCOS patients' sOB-R levels. CONCLUSION: LEPR Lys109Arg (rs1137100) was associated with PCOS susceptibility and genotype AA was deduced to be a protective factor for PCOS; sOB-R levels might be recognized as a new indicator for the severity of PCOS.

Inhibition of the Binding between RGS2 and ß-Tubulin Interferes with Spindle Formation and Chromosome Segregation during Mouse Oocyte Maturation In Vitro.

RGS2 is a negative regulator of G protein signaling that contains a GTPase-activating domain and a ß-tubulin binding region. This study aimed to determine the localization and function of RGS2 during mouse oocyte maturation in vitro. Immunofluorescent staining revealed that RGS2 was widely expressed in the cytoplasm with a greater abundance on both meiotic spindles and first/second polar bodies from the fully-grown germinal vesicle (GV) stage to the MII stages. Co-expression of RGS2 and ß-tubulin could also be detected in the spindle and polar body of mouse oocytes at the MI, AI, and MII stages. Inhibition of the binding site between RGS2 and ß-tubulin was accomplished by injecting anti-RGS2 antibody into GV-stage oocytes, which could result in oocytes arrest at the MI or AI stage during in vitro maturation, but it did not affect germinal vesicle breakdown. Moreover, injecting anti-RGS2 antibody into oocytes resulted in a significant reduction in the rate of first polar body extrusion and abnormal spindle formation. Additionally, levels of phosphorylated MEK1/2 were significantly reduced in anti-RGS2 antibody injected oocytes compared with control oocytes. These findings suggest that RGS2 might play a critical role in mouse oocyte meiotic maturation by affecting ß-tubulin polymerization and chromosome segregation.

Mutations in TUBB8 cause a multiplicity of phenotypes in human oocytes and early embryos.

BACKGROUND: TUBB8 is a primate-specific ß-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. METHODS AND RESULTS: Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. CONCLUSIONS: Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).

Uterine Expression of NDRG4 Is Induced by Estrogen and Up-Regulated during Embryo Implantation Process in Mice.

Embryo implantation is an essential step for the establishment of pregnancy and dynamically regulated by estrogen and progesterone. NDRG4 (N-myc down-regulated gene 4) is a tumor suppressor that participates in cell survival, tumor invasion and angiogenesis. The objective of this study was to preliminarily explore the role of NDRG4 in embryo implantation. By immunohistochemistry (IHC) and quantitive RT-PCR (qRT-PCR), we found that uterine expression of NDRG4 was increased along with puberal development, and its expression in adult females reached the peak at the estrus stage during the estrus cycle. Furthermore, uterine NDRG4 expression was significantly induced by the treatment of estradiol (E2) both in pre-puberty females and ovariectomized adult females. Uterine expression pattern of NDRG4 during the peri-implantation period in mice was determined by IHC, qRT-PCR and Western blot. It was observed that NDRG4 expression was up-regulated during the implantation process, and its expression level at the implantation sites was significantly higher than that at the inter-implantation sites. Meanwhile, an increased expression in NDRG4 was associated with artificial decidualization as well as the activation of delayed implantation. By qRT-PCR and Western blot, we found that the in vitro decidualization of endometrial stromal cells (ESCs) was accompanied by up-regulation of NDRG4 expression, whereas knockdown of its expression in these cells by siRNA inhibited the decidualization process. In addition, Western blot analysis showed that NDRG4 protein expression was decreased in human villus tissues of recurrent miscarriage (RM) patients compared to normal pregnant women. Collectively, these data suggested that uterine NDRG4 expression could be induced by estrogen, and NDRG4 might play an important role during early pregnancy.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other ß-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one ß-tubulin polypeptide (α/ß-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed ß-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/ß-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).

The Influence of LepR Tyrosine Site Mutations on Mouse Ovary Development and Related Gene Expression Changes.

Leptin exerts many biological functions, such as in metabolism and reproduction, through binding to and activating the leptin receptor, LepRb, which is expressed in many regions of the brain. To better understand the roles of LepR downstream signaling pathways, Y123F mice, which expressed mutant leptin receptors with phenylalanine (F) substituted for three tyrosines (Y) (Tyr985, Tyr1077 and Tyr1138), were generated. The body weight and abdominal fat deposits of Y123F homozygous mice (HOM) were higher than those of wild-type mice (WT). HOM ovaries were atrophic and the follicles developed abnormally; however, the HOM ovaries did not exhibit polycystic phenotypes. Moreover, Y123F HOM adults had no estrous cycle and the blood estrogen concentration remained stable at a low level below detection limit of 5 pg/ml. LepR expression in HOM ovaries was higher than in WT ovaries. Using cDNA Microarrays, the mRNA expressions of 41 genes were increased, and 100 were decreased in HOM vs. WT ovaries, and many signaling pathways were evaluated to be involved significantly. The expressions of 19 genes were validated by real-time quantitative PCR, most of which were consistent with the microarray results. Thus, Y123F HOM mice were suggested as a new animal model of PCOS for research that mainly emphasizes metabolic disorders and anovulation, but not the polycystic phenotype. Meanwhile, using the model, we found that JAK-STAT and hormone biosynthesis pathways were involved in the follicular development and ovulation disorders caused by LepR deficiency in ovaries, although we could not exclude indirect actions from the brain.

Effect of high ovarian response on the expression of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in peri-implantation endometrium in IVF women.

OBJECTIVE: To investigate the effect of ovarian stimulation on the expression of EG-VEGF mRNA and protein in peri-implantation endometrium in women undergoing IVF and its relation with endometrial receptivity (ER). DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENTS: Eighteen women in stimulated cycles (SC) as study subjects and 18 women in natural cycles (NC) as controls. Women in SC group were classified with two subgroups, high ovarian response (SC1, n=9) with peak serum E2>5,000 pg/mL and moderate ovarian response (SC2, n=9) with peak serum E2 1,000-5,000 pg/mL. INTERVENTION(S): Endometrial biopsies were collected 6 days after ovulation in NC or after oocyte retrieval in SC. MAIN OUTCOME MEASURE(S): Endometrium histological dating was observed with HE staining. EG-VEGF mRNA expression levels determined by real-time polymerase chain reaction analysis, and protein levels by immunohistochemistry. RESULTS: All endometrial samples were in the secretory phase. The endometrial development in SC1 was 1 to 2 days advanced to NC, and with dyssynchrony between glandular and stromal tissue. Immunohistochemistry analysis showed that EG-VEGF protein was predominantly expressed in the glandular epithelial cells and endothelial cells of vessels, and also presented in the stroma. The image analysis confirmed that both the gland and stroma of endometrium in SC1 had a significantly lower EG-VEGF protein expression than that in SC2 and NC endometrium. Moreover, EG-VEGF mRNA levels were significantly lower in SC1 than in NC. Both EG-VEGF protein and mRNA levels had no significant difference between SC2 and NC. CONCLUSION: Decreased expression of EG-VEGF in the peri-implantation is associated with high ovarian response, which may account for the impaired ER and lower implantation rate in IVF cycles.

Aberrant Placental Villus Expression of miR-486-3p and miR-3074-5p in Recurrent Miscarriage Patients and Uterine Expression of These MicroRNAs during Early Pregnancy in Mice.

MicroRNAs (miRNAs) are critical regulators of gene expression; they have emerged as new players in the pathophysiology of reproductive disorders such as preeclampsia and recurrent miscarriage (RM). In this study, miRNA expression profiles were determined by deep sequencing analysis in placental villi obtained from women with RM and with gestational age-matched normal pregnancy (NP). A total of 69 miRNAs were found to be aberrantly expressed in RM. Five of these human (Homo sapiens) miRNAs (hsa-miRNAs), including hsa-miR-3074-5p, -486-3p, -1269b, -6765-3p and -144-3p, were validated by qRT-PCR in 12 RM and 10 NP placental villi. Hsa-miR-3074-5p expression was significantly higher, while hsa-miR-486-3p expression was significantly lower, in RM compared to NP. Subsequently, the expression of mouse (Mus musculus) miR-486-3p (mmu-miR-486-3p) and mmu-miR-3074-5p, which are identical to hsa-miR-486-3p and hsa-miR-3074-5p, respectively, were examined by qRT-PCR in the uterus during early pregnancy. Mmu-miR-486-3p expression was significantly increased during the peri-implantation period, and the levels were significantly higher at implantation than at non-implantation sites. In contrast, the expression of mmu-miR-3074-5p was markedly decreased at implantation compared to non-implantation sites. These data suggest that miR-486-3p and miR-3074-5p may be involved in embryo implantation and that their aberrant expression is associated with RM. Larger studies are warranted to follow up this pilot study. © 2015 S. Karger AG, Basel.

Altered microRNAs expression profiling in cumulus cells from patients with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age, and oocyte developmental competence is altered in patients with PCOS. In recent years microRNAs (miRNAs) have emerged as important regulators of gene expression, the aim of the study was to study miRNAs expression patterns of cumulus cells from PCOS patients. METHODS: The study included 20 patients undergoing in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI): 10 diagnosed with PCOS and 10 matching controls. We used deep sequencing technology to identify the miRNAs differentially expressed in the cumulus cells of PCOS. RESULTS: There were 17 differentially expressed miRNAs in PCOS cumulus cells, including 10 miRNAs increase and 7 miRNAs decrease. These miRNAs were predicted to target a large set of genes with different functions, including Wnt- and MAPK- signaling pathways, oocyte meiosis, progesterone-mediated oocyte maturation and cell cycle. Unsupervised hierarchical clustering analysis demonstrated that there was a specific miRNAs expression pattern in PCOS cumulus cells. CONCLUSION: We found that the miRNAs expression profile was different in cumulus cells isolated from PCOS patients compared with control. This study provided new evidence for understanding the pathogenesis of PCOS.

Deficiency of monoclonal non-specific suppressor factor beta (MNSFB) promotes pregnancy loss in mice.

Maternal immune tolerance to the semi-allogenic fetus is required for successful pregnancy in mammals. Monoclonal nonspecific suppressor factor beta (MNSFB) is an immunosuppressive factor present in uterine epithelial and stromal cells, as well as in macrophages and T cells. Although the functional neutralization of MNSFB using specific antibodies against it lead to failed embryo implantation in mice, the exact role of MNSFB at the fetal-maternal interface remains unclear. The present study generated conditional heterozygous Mnsfb-deficient (Mnsfb(+/) (-) ) mice using the LoxP/Cre system. Western-blot analyses showed that uterine MNSFB protein in Mnsfb(+/-) mice was remarkably down-regulated compared to that in the wild-type (Mnsfb(+/+) ) mice. The litter size of female Mnsfb(+/-) mice was significantly reduced, which corresponded to developmental failure of embryos beyond Day 11 of pregnancy. The expression level of MNSFB protein was also lower in the failing compared to the normal embryos. An aberrant interaction between the embryos of Day-4 pregnant wild-type mice and endometrial stromal cells of female Mnsfb(+/-) mice was observed in vitro. The uterine Day-5 abundance of P53, BAX, and BCL-G in pregnant Mnsfb(+/-) mice was significantly decreased compared to that of wild-type mice, whereas the expression of P27 and tumor necrosis factor alpha (TNFA) was elevated. By comparison, the levels of MNSFB and BAX proteins in human decidual tissues obtained from recurrent spontaneous miscarriage patients were significantly reduced compared to those obtained from legal medial abortion, highlighting the involvement of MNSFB in the pathogenesis of recurrent spontaneous miscarriage. Together, these results demonstrated that a deficiency in MNSFb is associated with pregnancy loss, probably through reduced P53 and/or increased TNFA production at the fetal-maternal interface.