Embryo Implantation Period Research Articles

Mechanisms of LPS–induced epithelial mesenchymal transition in bEECs

High-concentrate diets cause subacute ruminal acidosis, resulting in increased blood lipopolysaccharide (LPS) levels in cows. We found that the peak LPS in cows fed with high-concentrate diets coincides the period of embryo implantation in a large-scale dairy farm. As epithelial-mesenchymal transition (EMT) should be tightly regulated during normal embryo implantation in cows, we speculated that increased LPS may cause abnormal EMT, thereby inhibiting embryo implantation in cows. To confirm that elevated LPS levels induce abnormal EMT in cows, we treated bovine endometrial epithelial cells (bEECs) with LPS for 48 h and analyzed the protein levels of ZEB1, a major EMT-related transcription factor, which is positively regulated by the TGFβ/SMAD3 pathway. In addition, we analyzed the changes in expression of three EMT-related genes (E-cadherin, N-cadherin, and Vimentin), and examined the morphology and migratory ability of the cells. The results showed that elevated LPS levels increased protein expression of ZEB1, vimentin, and N-cadherin, and reduced that of E-cadherin. Elevated LPS also increased bEECs migration rate, and induced the cells to acquire a mesenchymal phenotype. Furthermore, benzyl butyl phthalate (BBP)-induced ZEB1 overexpression significantly decreased E-cadherin levels and increased N-cadherin levels. As LPS treatment also decreased the expression of Bta-miR-200b, we further found that Bta-miR-200b targets to the 3′UTR of ZEB1 through the confirmation of dual-luciferase reporter system. And the increased level of Bta-miR-200b by mimic enhanced the expression of E-cadherin and yet inhibited the expression of N-cadherin in protein, which exactly opposite to the results induced by LPS. In conclusion, LPS induced EMT in bEECs by upregulating ZEB1, while Bta-miR-200b could inhibit the occurrence of EMT by binding ZEB1 3′UTR. These results provide a new insight for low reproductive rate of dairy cows under the background of high-concentrate diets.

Proteomic profiles and the function of RBP4 in endometrium during embryo implantation phases in pigs

BackgroundEndometrial receptivity plays a vital role in the success of embryo implantation. However, the temporal proteomic profile of porcine endometrium during embryo implantation is still unclear.ResultsIn this study, the abundance of proteins in endometrium on days 9, 10, 11, 12, 13, 14, 15 and 18 of pregnancy (D9, 10, 11, 12, 13, 14, 15 and 18) was profiled via iTRAQ technology. The results showed that 25, 55, 103, 91, 100, 120, 149 proteins were up-regulated, and 24, 70, 169, 159, 164, 161, 198 proteins were down-regulated in porcine endometrium on D10, 11, 12, 13, 14, 15 and 18 compared with that on D9, respectively. Among these differentially abundance proteins (DAPs), Multiple Reaction Monitoring (MRM) results indicated that S100A9, S100A12, HRG and IFI6 were differentially abundance in endometrial during embryo implantation period. Bioinformatics analysis showed that the proteins differentially expressed in the 7 comparisons were involved in important processes and pathways related to immunization, endometrial remodeling, which have a vital effect on embryonic implantation.ConclusionOur results reveal that retinol binding protein 4 (RBP4) could regulate the cell proliferation, migration and apoptosis of endometrial epithelial cells and endometrial stromal cells to affect embryo implantation. This research also provides resources for studies of proteins in endometrium during early pregnancy.

Extracellular vesicles derived from endometrial epithelial cells deliver exogenous miR-92b-3p to affect the function of embryonic trophoblast cells via targeting TSC1 and DKK3

BackgroundExtracellular vesicles (EVs) could mediate embryo-maternal communication to affect embryo implantation by delivering biology information, including microRNA (miRNA), protein, lipid. Our previous research shows that miR-92b-3p was differentially expressed in EVs of uterine flushing fluids during the embryo implantation period. However, the role of miR-92b-3p from EVs in embryo implantation remains elusive.Materials and methodsEVs were isolated from porcine endometrial epithelial cells (EECs) by ultracentrifugation. MiR-92b-3p mimics and EVs were used to regulate the expression of miR-92b-3p in porcine trophoblast cells (PTr2 cells). Cell proliferation, migration and adhesion analyses were used to observe the phenotype. RT-qPCR, western blot and dual-luciferase reporter assay were used to assess the targets of miR-92b-3p.ResultsIn this study, EVs derived from porcine EECs were identified and could be taken up by PTr2 cells. We found that the EVs derived from EECs transfected with miR-92b-3p mimic (EVs-miR-92b-3p) significantly promoted the proliferation, migration and adhesion of PTr2 cells. We verified that Tuberous sclerosis complex subunit (TSC1) and Dickkopf 3 (DKK3) were the target genes of miR-92b-3p. Moreover, our study showed that miR-92b-3p plays a vital role in PTr2 cells via targeting TSC1 and DKK3. Furthermore, the 3'UTR vectors of TSC1 and DKK3 can rescue the effect of miR-92b-3p on PTr2 cells.ConclusionsTaken together, this study reveals a novel mechanism that EVs derived from porcine EECs treated with miR-92b-3p crosstalk with trophoblasts by targeting TSC1 and DKK3, leading to an enhanced ability for implantation.

P–619 Oral dydrogesterone (OD) versus micronized vaginal progesterone (MVP) for luteal phase support (LPS) in IVF/ICSI: a double blind, cross-over, pharmacokinetic study

Abstract Study question How does the blood pharmacokinetic (PK) profile of OD/MVP differ after the first and last administration dose when used as LPS for fresh embryo transfer? Summary answer: The PK profile differed strongly between both LPS administration strategies with a more rapid absorption, metabolism and clearance of OD in comparison with MVP. What is known already Adequate LPS is crucial to achieve a successful pregnancy following ovarian stimulation (OS) and fresh embryo transfer. OD has been proven to be non-inferior compared to MVP in two phase III clinical trials. Additionally, a combined individual participant data and aggregate data meta-analysis showed an odds ratio in favor of OD for live birth. Little information is available on the PK of LPS strategies, leaving an important field unexplored. Individualization of LPS has recently gained more interest and insight into the PK of progestogens is essential to correctly interpret the potential impact of circulating hormone levels on reproductive outcomes. Study design, size, duration Twenty oocyte donors underwent two OS cycles followed by one week of LPS (OD or MVP) in a randomized, cross-over, double blind, double dummy fashion. As both dydrogesterone (D) and 20αdihydrodydrogesterone (DHD) are progestogenic, D, DHD and progesterone (P) plasma levels were established using a validated liquid chromatography tandem mass spectrometry assay in each cycle, on the 1st (single dose PK) and 8th day (multiple dose PK) of LPS (9 and 12 harvesting time-points, respectively). Participants/materials, setting, methods All oocyte donors were <35 years, had regular menstrual cycles, no intra-uterine contraceptive device, AMH within normal range and BMI ≤ 29 kg/m2. OS was performed in a GnRH antagonist protocol followed by dual triggering (1000U hCG + 0.2mg triptorelin) as soon as ≥ 3 follicles of 20mm were present. Following oocyte retrieval, subjects initiated LPS consisting of MVP 200 mg (Utrogestan®) or OD 10 mg (Duphaston®), both three times daily. Main results and the role of chance The mean (±SD) age of the subjects was 27.4 (± 3.8) years and BMI was 24.0 (±3.2) kg/m2. The mean (±SD) number of oocytes retrieved was 19.7 ±10. No adverse events were reported during the intake of the study medication. The PK results are best estimates as sampling was reduced compared to a formal PK study. Following the intake of the first dose of OD, the observed maximal plasma concentrations (Cmax) for D and DHD were 2.9 and 77 ng/ml (single dose). The Cmax for D and DHD was reached after 1.5 and 1.6 hours (=Tmax), respectively. On the 8th day of LPS the first administration of that day gave rise to a Cmax of 3.6 and 88 ng/ml for D and DHD (multiple dose). For both, the observed Tmax was 1.5 hours. Following the intake of the first dose of MVP, the Cmax for P was 16 ng/mL with a Tmax of 4.2 hours. On the 8th day of LPS the first administration of that day showed a Cmax for P of 21 ng/mL with a Tmax of 7.3 hours. Although low, the role of chance could be influenced by the relatively low sampling numbers and frequency. Limitations, reasons for caution Peripheral concentrations do not necessarily reflect the steroidogenic effect on endometrial progesterone receptors. Extrapolation to clinical practice is therefore difficult, however, molecular analyses of endometrial tissue harvested within this study protocol are underway to investigate further pharmacodynamics and the progestogenic impact on endometrial receptivity during the embryo implantation period. Wider implications of the findings: This is the first study comparing OD/MVP pharmacokinetics in IVF/ICSI. Results suggest administration frequency to be as important as dose, definitely for OD, showing a rapid absorption/clearance. More studies are needed to investigate blood levels in relation to time of LPS administration, especially in (artificially prepared) FET and LPS individualization. Trial registration number EUDRACT 2018–000105–23

Interferon-τ regulates the expression and function of bovine leukocyte antigen by downregulating bta-miR-204.

IFN-τ is a pregnancy recognition factor that regulates embryo implantation in ruminants. IFN-τ has been suggested to be involved in the expression of microRNA (miRNA/miR) and bovine leukocyte antigen (BoLA), which is an analog of the human major histocompatibility complex class I. However, little is known about whether the miRNAs are involved in the expression of BoLA in ruminants. The present study firstly verified that bta-miR-204 was downregulated and that BoLA was upregulated in the uterine tissues of dairy cows during early pregnancy. Subsequently, luciferase reporter assays, reverse transcription-quantitative PCR and western blot analysis were used to validate BoLA as the target gene of bta-miR-204. Moreover, BoLA was markedly upregulated and bta-miR-204 was downregulated in bovine endometrial epithelial cells (bEECs) treated with IFN-τ. In addition, the results indicated that when the expression level of BoLA was increased by IFN-τ, the expression level of programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) was also increased. Furthermore, when BoLA was silenced in bEECs by small interfering RNA, the expression of PD-L1 and PD-L2 was not affected by IFN-τ. The expression level of PD-L1 and PD-L2 was also increased in the uterine tissues of pregnant dairy cattle. In conclusion, IFN-τ may function by suppressing the expression of bta-miR-204 to increase the expression of BoLA during the embryo implantation period in cattle. IFN-τ may induce PD-L1 and PD-L2 transcription by regulating BoLA, which may influence the T cell immune response, thereby regulating pregnant cattle immunization.

ASSOCIATION BETWEEN THE PERCENTAGE OF p16-POSITIVE SENESCENT CELLS AND THE EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS IN HUMAN ENDOMETRIUM DURING THE MID-LUTEAL PHASE

Progesterone and estrogen secretion and the expression of their receptors are strongly connected with the cyclic changes in endometrium and its receptivity. We previously reported that the frequency of occurrence of p16-positive senescent cells in human endometrium appear to be also associated with endometrial receptivity and successful embryo implantation. The objective of this study was to analyze the relationship between the percentage of senescent cells and estrogen and progesterone receptors expression in human endometrium during the mid-luteal phase of the menstrual cycle. Prospective observational study. This study includes 383 women who had an endometrial biopsy during the mid-lutheal phase (7 days after LH surge) of the natural cycles. Tissue sections were stained immunohistochemically for ER, PR and p16 using the following monoclonal antibodies: progesterone receptor (PR) (NCL-PGR-312, Leica Microsystems), 1:100 dilution; estrogen receptor (ER) (NCL-L-ER-6F11, Leica Microsystems), 1:100 dilution; p16 ink4a antibody (MAD-000690QD–7, Master Diagnostica), ready-to-use. The percentage of positively stained p16 cells and the levels of ER/PR expression were evaluated by Image-J software (NIH, Maryland, USA) in multiple endometrial sections. Statistical analysis was performed by Spearman’s correlation test using SPSS v.21 (IBM Corp., Armonk, NY, USA). No correlation was found between the frequencies of occurrence of senescent p16-positive stromal, luminal and glandular epithelial cells and PR (p>0.05). In contrast, the glandular expression of ER showed low, but a significant correlation with the percentage of p16+ glandular epithelial cells (R=0.34; p=0.03). In conclusion, we found that the increased cell senescence in endometrial glands during the mid-luteal phase is associated with increased expression of glandular ER in the mid-luteal phase of the cycle. These results suggest an estrogen related change of endometrial senescent cells during the period of embryo implantation.

Regulation of Integrin β3 Protein Secretion on Implantation Embryo of Mouse (Mus musculus L.) Induced by Oil Atsiri of Purple Nutsedge Tubers (Cyperus rotundus L.)

Aims: This study aims to determine the regulation of β3 integrin protein in the period of embryo implantation after being induced by oil atsiri of purple nutsedge tubers.
 Study Design: The study employed a completely randomised design using four concentration levels repeated six times.
 Place and Duration of Study: Organic Chemistry Laboratory and Zoology Laboratory of Faculty of Mathematics and Natural Sciences University of Lampung, between June 2015 and April 2016.
 Methodology: This study used mouse as experimental animals with 24 female mouse, then mated with male mouse with a ratio (1 female: 1 male). After the vaginal plug was found in female mouse, then the essential oil of purple nutsedge tubers was induced orally (force-fed) according to the treatment dose. Each 6 female mice were grouped as a control group, and 3 treatment groups, consisting of controls (C) = 0.4 ml aquabides, treatment 1 (T1) = 4.5 mg/40g body weight, treatment 2 (T2) = 45 mg/40g body weight, and treatment 3 (T3) = 135 mg/40g body weight. After the one way Anova test, Ho is rejected, meaning there are significant differences and the analysis is continued with multiple comparison tests, in this case the Least Significant Difference (LSD) test is used.
 Results: The results shown that the induction of essential oils of purple nutsedge tubers can reduce the level of β3 integrin protein in uterus mouse significantly.
 Conclusion: It is suggested essential oil of purple nutsedge tubers has potential decrease and can reduce the level of β3 integrin protein in uterus mouse during the period of embryo implantation.

Unique and independent role of the GABAB1 subunit in embryo implantation and uterine decidualization in mice

Embryo implantation and decidualization are crucial for successful pregnancy, which include multiple genes and signaling pathways, while the precise mechanism regarding embryo implantation and decidualization has yet to be explored. The GABA which activates GABAA or GABAB receptors has been found playing an important role in early pregnancy. Here we seek to investigate whether GABAB receptors participate in embryo implantation in mice. This study first characterized the spatiotemporal expression pattern of GABAB receptors in the uterus during the peri-implantation period and found that GABAB1 expression was drastically upregulated in stromal cells on days 4–6, a period of embryo implantation and early stages of decidualization. Embryo delayed implantation and oil-induced decidualization models were further used to confirm that the GABAB1 was associated with embryo implantation and decidualization. We also found estrogen or progesterone had no directly effect on expression of GABAB1 in ovariectomized model. Because we were unable to detect significant GABAB2 which couples with GABAB1 to form whole GABAB receptors, and the agonist and antagonist of whole GABAB receptors had weak effect on the proliferation and differentiation of stromal cells as well, we excluded the possibility whole GABAB receptors function, and concluded it should be non-classical signals of GABAB1 involving in embryo implantation and decidualization. Future studies should focus on investigating the roles and mechanisms of GABAB1 during embryo implantation and decidualization.

MicroRNA-30a-3p regulates epithelial-mesenchymal transition to affect embryo implantation by targeting Snai2†.

To study the potential role of miR-30a-3p in embryo implantation and explore underlying mechanisms. We first established normal pregnancy, pseudopregnancy, delayed implantation, and artificial decidualization mouse models. Next, we detected miR-30a-3p expression profiles of these models with real-time reverse transcription PCR(qRT-PCR), then predicted potential target genes through a dual-luciferase assay. Immunofluorescence-fluorescence in situ hybridization co-located miR-30a-3p and target genes. We then examined the effect of miR-30a-3p on embryo implantation in vivo and in vitro. Wound healing and transwell assays were employed to explore possible miR-30a-3p effects on epithelial-mesenchymal transition (EMT), before molecules related to the latter process were examined with qRT-PCR. MiR-30a-3p expression decreased significantly on embryo implantation day, compared with the peri-implantation period (P<0.05). Identified target gene Snai2 expression increased significantly during implantation (P<0.05). In vivo and in vitro analysis showed that up-regulation of miR-30a-3p by agomir and mimics resulted in decreased implantation sites and embryo implantation rate. Transfection of miR-30a-3p mimics to HEC-1-b cells decreased expression of Snai2 and mesenchymal markers (Vimentin and N-cadherin). Furthermore, wound healing area decreased, as did migration and invasion capacity. MiR-30a-3p is down-regulated in the embryo implantation period and might have some effect on embryo implantation by acting as a suppressor of EMT through targeting Snai2.

Investigation of Eph-ephrin A1 in the regulation of embryo implantation in sows.

Eph A1 and ephrin A1 (Eph-ephrin A1) is a key receptor-ligand pair of Eph-ephrin system, which plays important roles in the migration and adhesion of cells, tissue morphogenesis and vasculogenesis in mammals. In order to investigate the regulation of Eph-ephrin A1 during porcine embryo implantation, the expressions of mRNA and protein of Eph-ephrin A1 were detected in different reproductive tissues from twelve sows during embryo implantation period on pregnancy day 13, 18 and 24, respectively. Functions of Eph-ephrin A1 on the migration and adhesion of porcine endometrial epithelial cells were analysed by RNA interference (RNAi), transwell migration assays and MTT assays. Results showed that mRNA levels of Eph-ephrin A1 were highly expressed in endometrial attachment site when compared to other reproductive tissues (p<0.05) and were peaked on pregnancy day 18 during embryo implantation (p<0.05). Protein levels of Eph-ephrin A1 were highly expressed in endometrial attachment site and were peaked on pregnancy day 18 (p<0.05). Eph-ephrin A1 proteins were located in endometrial luminal epithelium, stroma of attachment site and inter-attachment site during embryo implantation, and the protein levels were higher during implantation compared to pre-implantation or post-implantation. Furthermore, silencing ephrin A1 gene significantly reduced the migration and adhesion capacity of porcine endometrial epithelial cells. These findings suggest that the Eph-ephrin A1 protein likely targets endometrial attachment site to enhance the migration and adhesion of porcine endometrial epithelial cells around pregnancy day 18 during pregnancy in sows.

MicroRNA Dysregulation and Steroid Hormone Receptor Expression in Uterine Tissues of Rats with Endometriosis during the Implantation Window.

Background:Estrogen receptor (ER) and progesterone receptor (PR) are involved in endometriosis, but the involvement of microRNAs (miRNAs) is unknown. The aim of the study was to explore the correlation between miRNA and ER/PR in uterine tissues of rats with endometriosis during the implantation window.Methods:Twenty female Sprague-Dawley rats were randomized in three groups: endometriosis (n = 7), fat tissue control (n = 6), and normal (n = 7) groups. The female rats were mated and sacrificed on day 5 (implantation). Uterine tissues were obtained for hematoxylin-eosin staining, immunohistochemistry, and miRNA expression. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the expression of rno-miR-29c-3p, rno-miR-34c-5p, rno-miR-141-5p, rno-miR-24-1-5p, and rno-miR-490-5p.Results:The 475 miRNAs were found to differentially express between the endometriosis and normal control groups, with 127 being upregulated and 348 being downregulated. Expression of five miRNAs (rno-miR-29c-3p, rno-miR-34c-5p, rno-miR-141-5p, rno-miR-24-1-5p, and rno-miR-490-5p) were validated by RT-PCR and found to be differentially expressed among the three groups. Expression of ER and PR proteins (immunohistochemistry) in the glandular epithelium and endometrial stroma was significantly different among the three groups (all P < 0.05). Five miRNAs were involved in pathways probably taking part in implantation and fertility.Conclusions:The results suggested that miRNAs, ER, and PR could play important roles in the embryo implantation period of rats with endometriosis. These miRNAs might play a role in endometrial receptivity in endometriosis.

Low serum progesterone on the day of embryo transfer is associated with a diminished ongoing pregnancy rate in oocyte donation cycles after artificial endometrial preparation: a prospective study.

Is there a relationship between serum progesterone (P) and endometrial volume on the day of embryo transfer (ET) with ongoing pregnancy rate (OPR) in artificial endometrium preparation cycles? Patients with serum P < 9.2 ng/ml on the day of ET had a significantly lower OPR but endometrial volume was not related with OPR. A window of optimal serum P levels during the embryo implantation period has been described in artificial endometrium preparation cycles. A very low endometrial volume is related to poor reproductive outcome. Prospective cohort study with 244 patients who underwent ET in an oocyte donation cycle after an artificial endometrial preparation cycle with estradiol valerate and vaginal micronized progesterone (400 mg/12 h). The study period went from 22 February 2016 to 25 October 2016 (8 months). Sample size was calculated to detect a 20% difference in OPR (35-55%) between two groups according to serum P levels in a two-sided test (80% statistical power, 95% confidence interval (CI)). Patients undergoing their first/second oocyte donation cycle, aged <50, BMI < 30 kg/m2, triple layer endometrium >6.5 mm and 1-2 good quality transferred blastocysts. A private infertility centre. Serum P determination and 3D ultrasound of uterine cavity were performed on the day of ET. Endometrial volume measurements were taken using a virtual organ computer-aided analysis (VOCAL™) system. The primary endpoint was OPR beyond pregnancy week 12. About 211 of the 244 recruited patients fulfilled all the inclusion/exclusion criteria. Mean serum P on the day of embryo transfer was 12.7 ± 5.4 ng/ml (Centiles 25, 9.2; 50, 11.8; 75,15.8). OPRs according to serum P quartiles were: Q1: 32.7%; Q2: 49.1%; Q3: 58.5%; Q4: 50.9%. The OPR of Q1 was significantly lower than Q2-Q4: 32.7% versus 52.8%; P = 0.016; RR (95% CI): 0.62 (0.41-0.94). The mean endometrial volume was 3.4 ± 1.9 ml. Serum P on the day of ET did not correlate with endometrial volume. A logistic regression analysis, adjusted for all the potential confounders, showed that OPR significantly lowered between women with serum P < 9.2 ng/ml versus ≥9.2 ng/ml (OR: 0.297; 95%CI: 0.113-0.779); P = 0.013. The ROC curve showed a significant predictive value of serum P levels on the day of ET for OPR, with an AUC (95%CI) = 0.59 (0.51-0.67). Only the women with normal uterine cavity, appropriate endometrial thickness and good quality blastocysts transfer were included. Extrapolation to an unselected population or to other routes and/or doses of administering P needs to be validated. The role of endometrial volume could not be fully defined as very few patients presented a very low volume. The present study suggests a minimum threshold of serum P values on the day of ET that needs to be reached in artificial endometrial preparation cycles to optimize outcome. No upper threshold could be defined. None. NCT02696694.

Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.

MicroRNA-429 functions as a regulator of epithelial-mesenchymal transition by targeting Pcdh8 during murine embryo implantation.

What is the role of miR-429 in murine embryo implantation? miR-429 functions as a suppressor of epithelial-mesenchymal transition (EMT) during the process of embryo implantation by reverse regulation of Pcdh8. MicroRNAs (miRNAs) may serve as promising regulators of embryo implantation. miR-429 was recently found to be down-regulated during embryo implantation period in a microarray analysis. The expression profile of miR-429 was clarified in a series of models, and the target gene was confirmed. The in vivo and in vitro effect of miR-429 on embryo implantation was examined. Pregnancy was produced by natural mating between female C57BL6/J mice and male mice, and a series of models, including pseudopregnancy, delayed implantation and artificial decidualization, were established. The expression profile of miR-429 during the embryo implantation period was clarified in these models. Candidate target genes of miR-429 were predicted by bioinformatic analysis and tested by luciferase activity assay. The in vivo effects of miR-429 on embryo implantation were also examined. The in vitro effects of miR-429 on EMT were studied by examining migratory and invasive capacities by transwell assay and expression profiles of cadherin family members by western blotting and qRT-PCR. The expression profile of miR-429 in animal models suggested its down-regulation should be dependent on the presence and status of blastocysts and on endometrial decidualization. The luciferase activity assay showed that Pcdh8, a member of cadherin gene family, was the target gene of miR-429, and miR-429 suppressed the expression of Pcdh8 mRNA and protein. Gain-of-function of miR-429 in vivo resulted in a significant reduction of the number of implantation sites, but had little effect on fertilization. Up-regulation of miR-429 in vitro led to suppression of mesenchymal marker genes Vim, Cdh2, Zeb1 and Zeb2, and activation of epithelial marker gene Cdh1, resulting in suppression of the migratory and invasive capacities of cells. miR-429 also partially abrogated TGF-beta-induced EMT. The dysregulated expression profiles of EMT markers during embryo implantation period could be partially reversed by gain-of-function of miR-429 in vivo. The association of miR-429 with other members of the miR-200 family in embryo implantation remains to be determined. The relationship between miR-429 and the cadherin family needs more intensive description and the detailed mechanism of miR-429 in regulating the cadherin family needs to be elucidated. Our findings indicate that miR-429 plays a major role in embryo implantation as a suppressor of EMT by targeting Pcdh8. This information could contribute to a better understanding of the mechanisms involved in the miRNA-mediated regulation of embryo implantation, and subsequently improve treatments for infertility. The findings are consistent with that from previous research of the other members in miR-200 family in embryo implantation and in the EMT. This study was supported by the Natural Science Foundation of China (Grant number: 81170592), and Special Fund from National Excellent Doctoral Dissertation (Grant number: 201079). There was no conflict of interest.

Effect of anti-PMSG on distribution of estrogen receptor alpha and progesterone receptor in mouse ovary, oviduct and uterus.

It is well established that estrogen and progesterone are critical endogenous hormones that are essential for implantation and pregnancy in females. However, the distribution of estrogen receptor α (ERα) and progesterone receptor (PR) in female reproductive tracts is elusive. Herein, we report that after serial treatments with pregnant mare's serum gonadotrophin (PMSG) with or without anti-PMSG (AP), mice could regulate the distribution of ERα and PR in the murine ovary, oviduct and uterus and the level of estradiol in serum. ERα and PR regulation by PMSG and anti-PMSG was estrous cycle-dependent and critical for promoting the embryo-implantation period. Furthermore, our results suggested that AP-42 h treatment is more effective than the other treatments. In contrast, other treatment groups also affected the distribution of ERα and PR in mouse reproductive tracts. Thus, we found that anti-PMSG has the potential to restore the distribution of ERα and PR, which could effectively reduce the negative impact of residual estrogen caused by the normal superovulation effect of PMSG in mice.

PARP1 during embryo implantation and its upregulation by oestradiol in mice

Pregnancy requires successful implantation of an embryo, which occurs during a restricted period defined as 'receptivity of the endometrium' and is influenced by the ovarian steroids progesterone and oestradiol. The role of poly(ADP-ribose)polymerase-1 (PARP1) in apoptosis is well established. However, it is also involved in cell differentiation, proliferation and tissue remodelling. Previous studies have described the presence of PARP in the uterus, but its exact role in embryo implantation is not yet elucidated. Hence, in this study, we studied the expression of PARP1 in the uterus during embryo implantation and decidualisation, and its regulation by ovarian steroids. Our results show upregulation of the native form of PARP1 (∼116 kDa) in the cytosolic and nuclear compartments of implantation and non-implantation sites at day 5 (0500 h), followed by downregulation at day 5 (1000 h), during the embryo implantation period. The transcript level of Parp1 was also augmented during day 5 (0500 h). Inhibition of PARP1 activity by the drug EB-47 decreased the number of embryo implantation sites and blastocysts at day 5 (1000 h). Further, cleavage of native PARP1 was due to the activity of caspase-3 during the peri-implantation stage (day 5 (0500 h)), and is also required for embryo implantation, as inhibition of its activity compromised blastocyst implantation. The native (∼116 kDa) and cleaved (∼89 kDa) forms of PARP1 were both elevated during decidualisation of the uterus. Furthermore, the expression level of PARP1 in the uterus was found to be under the control of the hormone oestrogen. Our results clearly demonstrate that PARP1 participates in the process of embryo implantation.

Progesterone Promotes HOXa-10 Expression in Mouse During Embryo Implantation Period

Progesterone Promotes HOXa-10 Expression in Mouse During Embryo Implantation Period

The expression of specific genes and their predicated micro-RNA (miRNA) in human endometrium during the window of embryo implantation

Using microarray we identified a number of differently expressed genes and microRNAs (miRNAs), non-protein-coding small RNAs that regulate gene expression through mRNA degradation, in human endometrium. This study was designed to validate the expression of 23 of these genes functionally identified as inflammatory mediator, transcription factors, tissue turnover, cell growth etc. and their related miRNAs in the endometrium during the period of embryo implantation.

Expression of Adiponectin Receptors and Its Possible Implication in the Human Endometrium

Adiponectin, a pleiotropic cytokine, exerts its effects via the specific receptors AdipoR1 and AdipoR2. Whereas circulating adiponectin concentrations decrease in women with endometriosis and endometrial cancer, possible effects of adiponectin and the presence of the receptors in the endometrium have not been determined. In this study, we examined the expression of adiponectin receptors AdipoR1 and AdipoR2 in the human endometrium and assessed effects of adiponectin in endometrial cells. Expression of AdipoR1 and AdipoR2 in endometrial tissues was evaluated by real-time quantitative PCR, in situ hybridization, and Western blotting. The effects of adiponectin on phosphorylation of AMP-activated protein kinase, a regulator of energy homeostasis, in cultured endometrial stromal cells (ESCs) and epithelial cells (EECs) were studied by Western blotting. The effects of adiponectin on IL-1beta-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein 1 from cultured ESCs were determined using specific ELISAs. The expression of AdipoR1 and AdipoR2 was detected in the endometrium. The expression of both genes was increased in the midluteal phase, the period of embryo implantation. In situ hybridization revealed that both AdipoR1 and AdipoR2 appeared to be equally expressed in the epithelial cells and in the stromal cells. Adiponectin increased phosphorylation of AMP-activated protein kinase in ESCs and EECs. Adiponectin decreased IL-1beta-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein 1 from ESCs. These findings suggest that adiponectin exerts energy-homeostatic and antiinflammatory effects in the endometrium, and these effects might be relevant to pathological and physiological endometrium-related events such as implantation and endometriosis.

Localization of connective tissue growth factor during the period of embryo implantation in the mouse.

A role for connective tissue growth factor (CTGF) in reproductive function has been suggested from recent studies in the pig. To extend these findings, we have analyzed the immunohistochemical localization of CTGF during the estrous cycle and early pregnancy in mice. During the diestrous and early proestrous stages, CTGF was localized at high levels to both luminal and glandular uterine epithelial cells and at much lower levels in the stroma or myometrium. Epithelial expression of CTGF was considerably reduced at estrus. On Days 1.5-3.5 of pregnancy, CTGF was localized mainly to the uterine epithelial cells, which showed a substantially reduced level of CTGF on Day 4.5. On Days 5.5 and 6.5, CTGF was present at high levels in uterine decidual cells. CTGF was detected in the trophectoderm and inner cell mass of the preimplantation embryo on Day 4.5 and became preferentially localized to embryonic endoderm and mesoderm on Days 5.5-6.5. Multiple mass forms of CTGF (Mr 14 000-38 000) were present in endometrial extracts and uterine luminal flushings. Collectively, these data support a role for CTGF in uterine cell growth, migration, adhesion, and extracellular matrix production during the estrous cycle and early pregnancy, as well as in early development of the embryo.