Kun Qian

Hsa-miR-222 Is Involved in Differentiation of Endometrial Stromal Cells in Vitro

Decidualization is a critical step during embryo implantation and characterized by the differentiation of endometrial stromal cells (ESCs) into decidual cells. Because miRNAs are important determinants of cellular fate specification, in this study, the miRNA expression in ESCs during in vitro decidualization was profiled by using a microarray. Significance analysis of microarrays revealed that 49 miRNA genes were differently (>2-fold) expressed between the noninduced ESCs and induced ESCs with a false discovery rate of 0. The expression variance of hsa-miR-222, 221, 143, 101, 30d, 30c, 181b, 27b, 29b, 507, and 23a was validated by using quantitative PCR (P < 0.05). Based on microRNA (miRNA) and mRNA expression variance and predicted target genes of miRNAs, a bioinformatic model of miRNAs controlling ESCs differentiation was formulated. Finally, we proved that down-regulation of has-miR-222 could decrease the number of cells in S phase during ESCs differentiation (P < 0.05). Antisense oligonucleotides of has-miR-222 could increase reporter gene expression by targeting the 3 untranslated regions of CDKN1C/p57kip2 mRNAs as well as increase CDKN1C/p57kip2 protein levels (P < 0.05). In conclusion, our results suggest that a subset of miRNAs play a key role in gene reprogramming during ESCs decidualization and that hsa-miR-222 participates in ESC differentiation by regulating ESCs terminally withdrawing from the cell cycle.

Is frozen embryo transfer cycle associated with a significantly lower incidence of ectopic pregnancy? An analysis of more than 30,000 cycles

OBJECTIVEnTo analyze the incidence of ectopic pregnancy (EP) in fresh compared with frozen-thawed cycles.nnnDESIGNnRetrospective cohort study.nnnSETTINGnTeaching hospital.nnnPATIENT(S)nThirty-one thousand nine hundred twenty-five women undergoing inxa0vitro fertilization-embryo transfer (IVF-ET) from January 2006 to Decemberxa02013.nnnINTERVENTION(S)nFresh IVF-ET compared with frozen-thawed ET (FET).nnnMAIN OUTCOME MEASURE(S)nIncidence of EP with fresh IVF-ET compared with frozen-thawed ET cycles, clinical pregnancy rate, and rate of EP per clinical pregnancy.nnnRESULT(S)nFor the fresh IVF cycles, 19,173 patients underwent oocyte retrieval; 15,042 had an ET, 6,431 of these patients (42.7%) had a clinical pregnancy, and among these 297 (1.97%) appeared to have an EP. The group of patients undergoing frozen-thawed ET (12,752 patients) included 12,255; there were 5,564 pregnancies (45.4%) and 124 ectopic implants (1.01%). The incidence of an EP per clinical pregnancy was 4.62% for the fresh transfer group compared with 2.22% for the frozen-thawed cycle group; this difference was statistically significant. In addition, the fresh ET cycles had the highest risk of EP, followed by day-3 embryo FET cycles; blastocyst FET cycles had the lowest risk of EP, and the differences were all statistically significant.nnnCONCLUSION(S)nFrozen-thawed ET cycles were associated with a statistically significantly lower risk of EP when compared with fresh cycles. These findings are consistent with ovarian stimulation being associated with an increased risk of EP.

Administration of calcitonin promotes blastocyst implantation in mice by up-regulating integrin β3 expression in endometrial epithelial cells

STUDY QUESTIONnDoes exogenous calcitonin improve the efficiency of implantation in mice by increasing uterine receptivity?nnnSUMMARY ANSWERnThe administration of calcitonin could improve the efficiency of implantation by increasing the expression of several receptivity-related genes in endometrial epithelial cells (EECs).nnnWHAT IS KNOWN ALREADYnCalcitonin is one of the biomarkers of uterine receptivity, which is transiently produced in the uterine epithelia during the period of implantation both in humans and mouse.nnnSTUDY DESIGN, SIZE, DURATIONnHormone-replaced mice were used for in vivo experiments. To evaluate the effect of calcitonin on uterine receptivity, the expression of endometrial genes was analyzed 36 h after i.p. injection of 0.5 IU calcitonin in a treatment group versus saline in the control. To evaluate the effect of calcitonin on implantation efficiency in vivo, two groups received 0.5 IU or 2 IU calcitonin (i.p.) 24 h before embryo transfer, and a control group received saline (i.p.) (n = 18 mice per group). Implantation sites were counted 7 days after embryo transfer. The RL95-2 human endometrial carcinoma cell line was used to study the mechanisms underlying the effect of calcitonin on gene expression in the endometria. Using an in vitro model of endometrium-trophoblast interaction, established with RL95-2 cells and JAR (human choriocarcinoma cell line) trophoblast, endometrial receptivity was evaluated by comparing attachment and outgrowth of JAR spheroids in control and treatment groups.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnUterine receptivity in ovariectomized mice was induced by injection of estradiol and progesterone. Expression of eight genes in murine endometrium and RL95-2 cells was analyzed by real-time RT-PCR, western blot, immunohistochemical analysis, flow cytometry and enzyme-linked immunosorbent assay. We tested the effects of a protein kinase C inhibitor, matrigel and an antibody against integrin αvβ3 using RL95-2 cells and performed attachment and outgrowth assays using the in vitro model of endometrium-trophoblast interaction. Implantation efficiency was evaluated by counting the implantation sites after embryo transfer.nnnMAIN RESULTS AND THE ROLE OF CHANCEnCalcitonin up-regulated αvβ3 in RL95 cells, which in turn resulted in increased levels of the leukemia inhibitory factor (LIF) and heparin binding-epidermal growth factor (HB-EGF) mRNA (both P < 0.01 versus control) and protein (both P < 0.05 versus control). The attachment and expansion of JAR spheroids was promoted by pretreatment of EECs with calcitonin (P < 0.05 versus control) together with significantly increased expression of αvβ3, LIF and HB-EGF. Moreover, the injection of calcitonin in the preimplantation phase increased the total number of implantation sites in treatment groups (55 in control versus 78 and 85 in 0.5 and 2 IU groups, respectively). Compared with the control group (3.11 ± 2.14), the average number of implantation sites in the 2 IU calcitonin treatment group increased (4.72 ± 1.87, P = 0.022).nnnLIMITATIONS, REASONS FOR CAUTIONnExperiments were performed in mice and human cell lines but not in primary cultures of human endometrial cells.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe findings presented here have important implications, in that calcitonin administration (currently used for treatment of hypercalcemia or osteoporosis) may have clinical benefits in assisted reproduction programs, by facilitating endometrial receptivity and embryo implantation. However, further studies are required to confirm these findings.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis work was supported by National Science Foundation of China (No. 81170619). There are no financial or commercial conflicts in this study.

Bioinformatics and microarray analysis of microRNA expression profiles of murine embryonic stem cells, neural stem cells induced from ESCs and isolated from E8·5 mouse neural tube

Abstract To better understand whether microRNAs (miRNAs) are involved in the self-renewal of stem cells and fate determination of neural stem cells and to identify the miRNA expression patterns of different neural stem cells (NSC) in vitro and in vivo, we examined miRNA expression profiles of murine embryonic stem cells (ESC), NSC induced from ESC and isolated from E8·5 mouse neural tube (E8·5-NSC) using microarray technique. It was found that a total of 40 miRNAs had similar expression level in all the three cells [false discovery rate (FDR)=0, fold change <3·0]. Moreover, q-PCR showed that some members of miR-106b and miR-17–92 families were expressed in the ESC, NSC induced from ESC (ESC-NSC) and hematopoietic stem cells (HSC). Bioinformatical analysis showed that stemness genes (p21/CDKN1A, p57/CDKN1C and PTEN) were putative targets of miR-106b and miR-17–92 families. A total of 95 miRNAs were found to experience significant change (FDR=0, fold change >5·0) when the ESC differentiated into NSC. On the basis of miRNA, mRNA expression variance and predicted target genes of miRNA, we formulated a bioinformatical model for miRNA control of ESC-NSC differentiation. Then, the miRNA expression pattern was compared between NSC obtained in vitro and in vivo, and it was found that only 8% of miRNAs were different between the two NSCs. This study suggested that miR-106b and miR-17–92 families may promote the renewal of stem cells by targeting PTEN, p21/CDKN1A and p57/CDKN1C. Some miRNAs may play a key role in gene re-programming during ESC-NSC differentiation, and a substantial homogeneity exists between NSCs derived in vitro and those in vivo.

Myeloid ecotropic viral integration site 1 (MEIS) 1 involvement in embryonic implantation

BACKGROUND The HOXA10 homeobox gene controls embryonic uterine development and adult endometrial receptivity. The three-amino-acid loop extension (TALE) family homeobox genes like myeloid ecotropic viral integration site 1 (MEIS) provide enhanced target gene activation and specificity in HOX-regulated cellular processes by acting as HOX cofactors. METHODS AND RESULTS Analysis of an Affymetrix data set in the public domain showed high expression of MEIS1 in human endometrium. MEIS1 expression was confirmed during the human menstrual cycle by RT–PCR and in situ hybridization and was increased during the secretory compared with proliferative phase of the cycle (P = 0.0001), the time of implantation. To assess the importance of maternal Meis1 expression in a mouse model, the uteri of Day 2 pregnant mice were injected with Meis1 over-expression or small interfering RNA (siRNA) constructs. Blocking Meis1 expression by siRNA before implantation significantly reduced average implantation rates (P = 0.00001). Increased or decreased Meis1 expression significantly increased or decreased the expression of integrin β3, a transcriptional target of HOXA10 and an important factor in early embryo-endometrium interactions (P = 0.006). Manipulating Meis1 expression before implantation also dramatically affected the number of pinopodes, uterine endometrial epithelial projections that develop at the time of endometrial receptivity. CONCLUSIONS The results suggest that in mouse, meis1 contributes to regulating endometrial development during the menstrual cycle and establishing the conditions necessary for implantation.

Neonatal outcomes after early rescue intracytoplasmic sperm injection: an analysis of a 5-year period

OBJECTIVEnTo evaluate the safety and efficacy of early rescue intracytoplasmic sperm injection (ICSI).nnnDESIGNnRetrospective cohort study.nnnSETTINGnTeaching hospital.nnnPATIENT(S)nThere were 13,232 ovarian stimulation cycles (IVF, n = 9,631; ICSI, n = 2,871; early rescue ICSI, n = 730) that resulted in the delivery of 5,001 babies (IVF, n = 3,670; ICSI, n = 1,095; early rescue ICSI, n = 246) from August 2008 to August 2013.nnnINTERVENTION(S)nEarly rescue ICSI.nnnMAIN OUTCOME MEASURE(S)nClinical pregnancy rates, neonatal outcomes, and congenital birth defects were analyzed.nnnRESULT(S)nThe early rescue ICSI cycles did not seem to have a negative effect on the clinical pregnancy rate (43.42%) when IVF cycles (45.33%) were compared with ICSI cycles (44.39%). In the early rescue ICSI group, a total of 254 clinical pregnancies were achieved: 197 (33.67%) live births, 38 (6.49%) miscarriages, 2 (0.79%) induced abortions, 3 (1.18%) fetal deaths, and 4 pregnancies (1.57%) without completion at follow-up. Overall, the multiple gestations, the delivery method, mean gestational age, preterm deliveries, mean birth weight, and rate of congenital birth defects of the early rescue ICSI group were similar to those in the conventional IVF and ICSI groups.nnnCONCLUSION(S)nEarly rescue ICSI had similar clinical pregnancy rates when compared with conventional IVF and ICSI, in addition to the delivery of healthy children. The clinical evidence from the early rescue ICSI group did not show an elevated rate of malformations. Early rescue ICSI seems to be a safe alternative method for individuals with total fertilization failure or near total fertilization failure when compared with conventional IVF treatment.

Regulation of myeloid ecotropic viral integration site 1 and its expression in normal and abnormal endometrium

OBJECTIVEnTo identify the expression profile and sex steroid regulation pattern of myeloid ecotropic viral integration site 1 (MEIS1) in endometrium.nnnDESIGNnMolecular studies in human and animal tissue.nnnSETTINGnReproductive medicine center of a university hospital.nnnPATIENT(S) AND ANIMAL(S)nWomen with normal menstrual cycles for male infertility and female infertility with endometriosis. Sexually mature female mice (Kunming White strain).nnnINTERVENTION(S)nPrimary cultured endometrial stromal cells, Ishikawa cells, and oophorectomized mice were treated with sex steroid.nnnMAIN OUTCOME MEASURE(S)nMEIS1 expression in the human endometrium during the menstrual cycle, mouse uterus during the peri-implantation period of pregnancy, and eutopic endometrium from patients with endometriosis was analyzed by immunohistochemistry staining and western blot. In addition, MEIS1 expression in response to sex steroid was examined both inxa0vitro and inxa0vivo by immunohistochemistry staining and western blot.nnnRESULT(S)nMEIS1 expression was markedly increased in endometrium during the implantation period, and in decidualizing stromal cells in human endometrium and murine uterus. Steroid hormones increased MEIS1 expression in primary cultured endometrial stromal cells, Ishikawa cells, and endometrium of oophorectomized mice. The effects of estrogen and progesterone were more marked in oophorectomized mice and were additive. MEIS1 expression was significantly lower in eutopic endometrium compared with normal endometrium in the midsecretory stage.nnnCONCLUSION(S)nMEIS1 is likely a key mediator between sex steroid and genes for uterine receptivity. Diminished endometrium MEIS1 expression may contribute to implantation failure in endometriosis.

CDKN1C (P57): one of the determinants of human endometrial stromal cell decidualization

Abstract Decidualization is regulated by crosstalk of progesterone and the cAMP pathway. It involves extensive reprogramming of gene expression and includes a wide range of functions. To investigate how cell cycle regulatory genes drive the human endometrial stromal cell (ESC) exit cell cycle and enter differentiation, primary cultured ESC was treated with 8-Br-cAMP and MPA and cell cycle distribution was investigated by flow cytometry. High-throughput cell cycle regulatory gene expression was also studied by microarray. To validate the results of microarray chip, immunohistochemistry and semi-quantitative method of optical density were used to analyze the expression of cell cycle regulator proteins in proliferative phase of endometrium (n = 6) and early pregnancy decidua (n = 6). In addition, we selected cyclin-dependent kinase inhibitor 1c (CDKN1C, also known as P57) and cyclin-dependent kinase inhibitor 2b (CDKN2B, also known as P15) in order to study their role in the process of decidualization by the RNAi method. ESC was arrested at G0/G1 checkpoints during decidualization. Cell cycle regulatory genes P57 and P15 were upregulated, while cyclin D1 (CCND1), cyclin-dependent kinase 2 (CDK2), and cell division cycle protein 2 homolog (CDC2) were downregulated during ESC differentiation both in vitro and vivo. P57 siRNA impaired ESC decidualization and caused differentmorphological and ultrastructural changes as well as a relatively low secretion of prolactin, but P15 siRNA had no effects. We concluded that P15, CCND1, CDK2, and CDC2 may participate in ESC withdraw from the cell cycle and go into differentiation both in vitro and in vivo. P57 is one of the key determinants of ESC differentiation due to its effect on the cell cycle distribution, but its association with the decidua-specific transcription factor needs further investigation. Summary Sentence Cell cycle regulatory genes P57 and P15 are upregulated, while Cyclin D1, CDK2, and CDC2 are downregulated during the ESC differentiation and P57 is one of the key regulators that modulates ESC differentiation by controlling cell cycle distribution.

A retrospective analysis of ovarian stimulation with letrozole in women undergoing artificial insemination by donor

The aim of this retrospective study was to determine the clinical pregnancy rate in women undergoing letrozole ovarian stimulation and artificial insemination by donor (AID). Between 2012 and 2015, 130 natural cycles, 939 letrozole cycles and 130 letrozole plus gonadotrophin cycles were conducted. Letrozole cycles were divided into three groups according to LH concentration on the day of HCG administration (LH <10 mIU/ml and follicle size ≥18u2009cm; LH ≤10 to <20 mIU/ml; and LH ≥20 mIU/ml). Pregnancy rates were 17.3%, 22.4% and 26.8%, respectively (P = 0.012). In women given 10 mIU/ml LH or more, logistic regression identified oestradiol (OR 1.002, 95% CI, 1.000 to 1.004, P = 0.029) and leading follicle size (OR 0.861, 95% CI, 0.772 to 0.960, P = 0.007) as significant predictive factors of pregnancy rate; the higher the oestradiol and the smaller the follicles, the better the pregnancy rate. The pregnancy rate was significantly higher in the letrozole plus gonadotrophin group than the letrozole group (P = 0.04). Better pregnancy rates can be achieved if LH surge occurs before HCG administration, especially with higher oestradiol and lower follicle size; treatment with letrozole plus gonadotrophin was significantly more effective than letrozole alone in AID.