Inhibited Embryo Implantation Research Articles

Friend leukemia integration 1 overexpression decreases endometrial receptivity and induces embryo implantation failure by promoting PART1 transcription in the endometrial epithelial cells.

In vitro fertilization-embryo transfer (IVF-ET) is a crucial assisted reproductive technology for treating infertility. However, recurrent implantation failure (RIF), a significant challenge in IVF-ET success, remains unresolved. This study aimed to explore the role and mechanism of FLI1 in endometrial receptivity and RIF. Differential endometrial cell proportions between patients with RIF and control subjects were assessed using single-cell RNA sequencing (scRNA-seq) analysis. The chromatin accessibility of FLI1 in the luteal endometrial tissue of patients with RIF and control subjects was examined using the single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). FLI1 mRNA and protein levels were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Cell viability and migration were examined via cell counting kit (CCK)-8 and scratch healing assays. Epithelial-mesenchymal transition markers were analyzed using western blotting. Mechanisms underlying FLI1's regulation of PART1 transcription and expression in endometrial epithelial cells were explored using chromatin immunoprecipitation and dual-luciferase reporter assays. Adeno-associated virus (AAV) carrying epithelial cell-specific FLI1/PART1 overexpression sequences was uterinely injected in mice to assess FLI1/PART1 effects. scRNA-seq revealed diminished endometrial epithelial cell proportions in RIF patients. Meanwhile, scATAC-seq indicated enhanced chromatin accessibility of FLI1 in these cells. FLI1 exhibited specific expression in RIF patients' endometrial epithelial cells. Specific FLI1 overexpression inhibited embryo implantation, while knockdown enhanced it. Pregnant mice injected with AAV encoding FLI1 overexpression had significantly lower implantation than AAV-negative controls. FLI1 binding to PART1 promoter heightened PART1 transcription and expression in endometrial epithelial cells. Rescue experiments illustrated FLI1's role in embryo implantation by boosting PART1 expression. PART1 was notably elevated in RIF patients' luteal endometrial tissue and non-receptive endometrial epithelial cells (HEC-1-A). Specific PART1 overexpression dampened embryo implantation, whereas knockdown promoted it. Pregnant mice injected with AAV encoding PART1 had lower implantation than negative controls. PART1 knockdown mitigated FLI1's inhibitory impact on HEC-1-A cell viability and migration. FLI1 overexpression in the endometrial epithelial cells of patients with RIF inhibited embryo implantation by binding to the PART1 promoter region to promote PART1 expression. These findings can aid in the development of novel therapeutic targets for RIF.

Uterine issues in infertile queens: Nine cases

This prospective case series investigated potential uterine causes of infertility in queens. Purebred queens with infertility (failure to conceive, embryonic death, or failure to maintain pregnancy and produce viable kittens), but no other reproductive disorders were examined approximately 1–8 weeks before mating (Visit 1), 21 days after mating (Visit 2), and 45 days after mating (Visit 3) if pregnant at Visit 2. Investigations included vaginal cytology and bacteriology, urine bacteriology, and ultrasonography. At Visit 2 or 3, uterine biopsy or ovariohysterectomy was performed for histology. Of nine eligible queens, seven were non-pregnant by ultrasound at Visit 2 and two had lost pregnancies by Visit 3. Ovulation was confirmed by serum progesterone concentration in all queens. Ultrasonic appearance of the ovaries and uterus was compatible with a healthy status except for one queen with signs of cystic endometrial hyperplasia (CEH) and pyometra, a follicular cyst in another, and fetal resorptions in two queens. Six cats had histologic lesions of endometrial hyperplasia, including CEH (n=1). Only one cat had no histologic uterine lesions. Bacteria were cultured from vaginal samples in seven queens at Visit 1, (two were non-evaluable), and in five of seven queens sampled at Visit 2. Uterine cultures were negative except for the cat with pyometra. All urine cultures were negative. In summary, the most frequent pathology observed in these infertile queens was histologic endometrial hyperplasia, which can potentially inhibit embryo implantation and healthy placental development. This suggests that uterine disease might contribute substantially to infertility in purebred queens.

Effect of miR-143-3p from Extracellular Vesicles of Porcine Uterine Luminal Fluid on Porcine Trophoblast Cells

Extracellular vesicles (EVs) in uterine luminal fluid (ULF) can reportedly affect the proliferation and migration function of porcine trophoblast cells (PTr2 cells) by mediating the maternal-fetal exchange of information. miR-143-3p is considered a crucial miRNA in early pregnancy in mammals; however, little is currently known about how it regulates the function of PTr2 cells. This study aimed to investigate the effects of ssc-miR-143-3p in ULF-EVs on the function of PTr2 cells during porcine embryo implantation. The uptake of ULF-EVs by PTr2 cells was confirmed, which significantly increased the expression of ssc-miR-143-3p. Ssc-miR-143-3p was found to facilitate the proliferation and migration of PTr2 cells in the CCK-8, EdU and wound-closure assays, while the opposite findings were observed after the knockdown of ssc-miR-143-3p. Bioinformatics analysis and the luciferase reporter assay showed that glycerol-3 phosphate dehydrogenase 2 (GDP2) was directly targeted by miR-143-3p. Inhibition of miR-143-3p was validated in mice to inhibit embryo implantation. In summary, ssc-miR-143-3p in ULF-EVs affects the proliferation and migration of PTr2 cells by mediating GPD2, thereby affecting embryo implantation.

FHL1 mediates HOXA10 deacetylation via SIRT2 to enhance blastocyst-epithelial adhesion

Recurrent implantation failure (RIF) is a rather thorny problem in the clinical practice of assisted reproductive technology. Due to the complex aetiology of RIF, its pathogenesis is far from fully understood, and there is no effective treatment available. Here, We explored the regulatory mechanism of the four half-domains of LIM domain 1 (FHL1), which is significantly downregulated in the endometrium of RIF patients, in blastocyst-epithelial adhesion. Indeed, FHL1 expression was dramatically increased in normal female mid-secretory endometrial epithelial cells and was abnormally reduced in RIF patients. Furthermore, FHL1 overexpression promoted blastocyst-epithelial adhesion, and interfering with FHL1 expression in the mouse uterus significantly inhibited embryo implantation. Mechanistically, FHL1 did not regulate HOXA10 mRNA expression but increased HOXA10 protein stability and activated HOXA10, thereby promoting its regulation of downstream gene expression and the β3 integrin/FAK pathway. Meanwhile, FHL1 regulates HOXA10 function by increasing HOXA10 deacetylation through enhanced binding of HOXA10 and SIRT2. SIRT2-specific inhibitors can significantly inhibit this effect. In the endometrial epithelial cells of RIF patients, the correlation between FHL1 and HOXA10 and its downstream target genes has also been verified. Finally, our data indicated FHL1 is a regulatory molecule that promotes blastocyst-epithelial adhesion. Altogether, downstream dysfunction due to aberrant FHL1 expression is an important molecular basis for embryo implantation failure in patients with RIF and to provide new potential therapeutic targets.

SIRT1 suppresses in vitro decidualization of human endometrial stromal cells through the downregulation of forkhead box O1 expression

SIRT1 regulates survival, DNA repair, and metabolism in human cells and has pleiotropic effects on age-related diseases through either deacetylating target proteins or inhibiting gene transcription. Forkhead box O1 (FOXO1) is one of the most important transcription factors during decidualization. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) are well-known FOXO1-dependent genes in decidualizing cells. To determine whether SIRT1 plays a role in decidualization, we investigated morphological changes in cells following artificially stimulated decidualization and expression levels of PRL, IGFBP1, and FOXO1 in the immortalized non-neoplastic human endometrial stromal cell line T HESCs. SIRT1 expression decreased in the decidualization condition and SIRT1 inhibited morphological changes caused by decidualization of T HESCs. SIRT1 suppressed PRL, IGFBP1, and FOXO1 expression; inhibited FOXO1, PRL, and IGFBP1 promoter activity; and decreased histone protein acetylation of the FOXO1 promoter. We found that FOXO1 expression increased in the secretory phase compared with the proliferative phase, whereas SIRT1 expression decreased in the secretory phase in the human endometrium. We also revealed that SIRT1 may inhibit embryo implantation according to the blastocyst-like spheroid implantation assay. Collectively, these results indicate that SIRT1 suppresses decidualization of human endometrial stromal cells by inhibiting FOXO1 expression.

Steroidal but not embryonic regulation of mucin 1 expression in bovine endometrium.

In cow herd management, inadequate embryo implantation leads to pregnancy loss and causes severe economic losses. Thus, it is crucial to understand the molecular mechanisms underlying endometrial receptivity and subsequent embryo implantation. Transmembrane glycocalyx mucin 1 (MUC1) has a large and highly glycosylated extracellular domain known to inhibit embryo implantation via steric hindrance. The role of MUC1 in the bovine endometrium remains to be explored. Herein, we used simple but reliable in vivo and in vitro experiments to investigate the expression and regulation of MUC1 in the bovine endometrium. MUC1 gene expression was analyzed in endometrial epithelial cells collected by the cytobrush technique using reverse transcription-quantitative polymerase chain reaction. MUC1 protein expression was evaluated by immunohistochemical analysis of endometrial samples collected from slaughtered cows. We used an in vitro cell culture model to study the regulation of MUC1 expression by treating cells with sex steroidal hormones or co-culturing cells with a blastocyst. The results revealed that MUC1 was expressed and localized to the apical surface of luminal epithelial cells in the bovine endometrium. MUC1 expression disappeared during the luteal phase of the estrous cycle and during pregnancy. 17β-estradiol induced MUC1 expression, whereas progesterone inhibited its increase and co-culturing with blastocysts did not affect the expression. A long postpartum interval is a known risk factor for reduced fertility, and MUC1 expression was higher in this compromised condition. Our results demonstrated the MUC1 regulation by steroid hormones in bovine endometrium for embryo implantation, and we observed a negative correlation between MUC1 expression and fertility.

MiR-183 impeded embryo implantation by regulating Hbegf and Lamc1 in mouse uterus

Embryo implantation plays a decisive role in pregnancy. While in the process of implantation, microRNA (miRNA) is an important regulatory factor in the post transcriptional level. However, the role of many miRNAs in embryo implantation remained unknown. In this study, microRNA-183 (miR-183) was found differentially expressed in mouse uterus during implantation. In vivo treatment of miR-183 agomir in the uterine horn before implantation could eliminate the number of implantation site. The localization of miR-183 in mouse uteri gradually changed from epithelial to stromal layer in early pregnancy. Mice implantation models demonstrated that the decrease of miR-183 was mainly caused by maternal factors. Loss and gain function of miR-183 in endometrial cell lines showed that miR-183 could inhibit cell migration, invasion and apoptosis. MiR-183 could inhibit embryo implantation by binding Heparin-Binding EGF-like growth factor (Hbegf) and Laminin gamma one (Lamc1), which were key genes in embryo apposition and penetration. All these evidences indicate that miR-183 plays an important role during embryo implantation. This study provides new insights into the functions of miR-183 during embryo implantation and the development of contraceptive drugs in early pregnancy.

Calpain7 impairs embryo implantation by downregulating \u03b23-integrin expression via degradation of HOXA10

Endometriosis (ENDO) is a common gynecological disease that causes infertility in many women. Previous studies noted that the dysregulation of Homeo box A10 (HOXA10) in the endometrium of women with ENDO was involved in the failure of embryo implantation. However, the mechanism by which HOXA10 expression is reduced in women with ENDO is still poorly understood. Here we found that a member of the calcium (Ca2+)-dependent cysteine protease family calpain7 (CAPN7), negatively correlated with HOXA10, was highly expressed in the endometrium of infertile women with ENDO and was significantly downregulated during the window of embryo implantation in mice. Overexpression of CAPN7 in Ishikawa cells or in the uterus of mice inhibited embryo implantation in vitro and in vivo. In the current study, we identified a sequence rich in proline, glutamic acid, serine, and threonine (PEST sequence) that enhanced the Ca2+-dependent degradation of HOXA10 by CAPN7. Furthermore, the interaction between HOXA10 and CAPN7 repressed the transcriptional activity and protein stability of HOXA10. In contrast, the administration of the calpain inhibitor ALLN reversed the CAPN7-induced HOXA10 degradation. Moreover, truncation of the PEST motif in HOXA10 abolished its CAPN7-dependent proteolysis. These studies reveal a novel pattern of HOXA10 regulation via PEST sequence-mediated calpain proteolysis that was demonstrated to be reversed by a calpain inhibitor. Thus, the inhibition of CAPN7-induced HOXA10 degradation may represent a novel potential therapeutic method to improve impaired embryo implantation in women with ENDO.

Novel function of pregnancy-associated plasma protein A: promotes endometrium receptivity by up-regulating N-fucosylation

Glycosylation of uterine endometrial cells plays important roles to determine their receptive function to blastocysts. Trophoblast-derived pregnancy-associated plasma protein A (PAPPA) is specifically elevated in pregnant women serum, and is known to promote trophoblast cell proliferation and adhesion. However, the relationship between PAPPA and endometrium receptivity, as well as the regulation of N-fucosylation remains unclear. We found that rhPAPPA and PAPPA in the serum samples from pregnant women or conditioned medium of trophoblast cells promoted endometrium receptivity in vitro. Moreover, rhPAPPA increased α1,2-, α1,3- and α1,6-fucosylation levels by up-regulating N-fucosyltransferases FUT1, FUT4 and FUT8 expression, respectively, through IGF-1R/PI3K/Akt signaling pathway in human endometrial cells. Additionally, α1,2-, α1,3- and α1,6-fucosylation of integrin αVβ3, a critical endometrium receptivity biomarker, was up-regulated by PAPPA, thereby enhanced its adhesive functions. Furthermore, PAPPA blockage with antibody inhibited embryo implantation in vivo, mouse embryo adhesion and spreading in vitro, as well as N-fucosylation level of the endometrium in pregnant mice. In summary, this study suggests that PAPPA is essential to maintain a receptive endometrium by up-regulating N-fucosylation, which is a potential useful biomarker to evaluate the receptive functions of the endometrium.

Inhibition of Endometrial Tiam1/Rac1 Signals Induced by miR-22 Up-Regulation Leads to the Failure of Embryo Implantation During the Implantation Window in Pregnant Mice.

This study assessed first the impact of endometrial Tiam1/Rac1 signals and microRNA-22 (miR-22) on embryo implantation in mice, and then the expression of the above three genes in the endometrium during the embryo implantation window in the natural menstrual cycle in women with repeated implantation failure (RIF) after in vitro fertilization treatment. Four hundred fifty-two Kun-ming female mice and 200 women (70 infertility patients with RIF, 130 women as controls) were entered into this study. Endometrial Tiam1/Rac1 signals and miR-22 expression were studied in clinical and mouse samples and serum estrogen (E2) and progesterone (P) were analyzed in clinical subjects. A pregnant mouse model based on an endometrial miR-22 and Tiam1 mRNA expression trend of patients with RIF was constructed and then the embryo implantation numbers were analyzed, and an ovariectomized mouse model was used to assess correlations of expression of these three genes with E2 and P. The results showed that during the embryo implantation window in the natural menstrual cycle, endometrial miR-22 was significantly higher whereas Tiam1/Rac1 signals were notably lower in patients with RIF than in controls, and the P:E2 ratio was statistically lower in the RIF group. Tiam1/Rac1 signal down-regulation and miR-22 up-regulation contributed to the inhibition of embryo implantation in mice. We also found a suppressive effect of miR-22 up-regulation on Tiam1/Rac1 signal expression, and reciprocal regulation of E2 and P for these three genes' expression in mice. In conclusion, miR-22 up-regulation and Tiam1/Rac1 signal down-regulation inhibited embryo implantation in mice; this mechanism may be partially due to the suppressive effect of miR-22 on Tiam1 expression, and is regulated to some extent by serum E2 and P. Our findings provide evidence that endometrial Tiam1/Rac1 signal down-regulation along with miR-22 up-regulation during embryo implantation window in the natural menstrual cycle may be one of the reasons for the failure of embryo implantation in patients with RIF.

Effects of bisphenol-A on blastocyst development and implantation

To determine the effects of bisphenol-A (BPA) on blastocyst development and implantation. According to completely randomized grouping method, 90 pregnant mice were divided into 100, 300, and 600 mg/(kg·d)BPA groups and control group. BPA-treated pregnant mice were orally administered with BPA at concentrations of 100, 300 and 600 mg/(kg·d) from day 0.5 to day 3.5 of their pregnancy. Blastocyst implantation and development were studied. In the 300 mg/(kg·d) BPA group, the number of implantation sites and implantation rate were significantly decreased. In the 600 mg/(kg·d) group, no implantation sites were observed among pregnant mice and BPA inhibited embryo implantation. Blastocyst development on day 4 was examined, and findings showed that the development rate and total numbers of blastocysts in BPA treatment groups had no significant difference from the control group. However, BPA at 300 and 600 mg/(kg·d) significantly reduced blastocyst hatching rate and dramatically increased the number of blastocyst apoptotic cells when compared with those in the control group. BPA at a high concentration damages the blastocyst development before implantation and inhibits embryo implantation.

Inhibition of embryo implantation in mice through vaginal administration of a proprotein convertase 6 inhibitor

Uterine proprotein convertase 6 (PC6) plays a critical role in embryo implantation in both mice and women. It was hypothesized that inhibiting uterine PC6 could prevent pregnancy. Vaginal administration of a PC6 inhibitor presents the ideal route for local drug delivery. A peptide-based PC6 inhibitor, C-30k-PEG Poly R that was previously shown to have properties of increased vaginal absorption and penetration was tested for its contraceptive potential in mice following vaginal administration. The study demonstrated that this approach could inhibit embryo implantation in some mice (24% completely and 47% partially inhibited).

Small Molecule Proprotein Convertase Inhibitors for Inhibition of Embryo Implantation

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound's lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.

Activated EGFR stimulates MUC1 expression in human uterine and pancreatic cancer cell lines

MUC1 is a large cell surface mucin glycoprotein that plays diverse roles in both normal and tumor cell biology. These roles include mucosal hydration and protection, inhibition of embryo implantation, protection of tumor cells from the immune system and reduction of cytotoxic drug uptake. Similarly, the EGFR family of cell surface receptors drives many normal developmental processes as well as various aspects of tumor growth and gene expression. EGFR family members have been demonstrated to form complexes with MUC1 in various cellular contexts. Nonetheless, the role that EGFR activation plays in modulating MUC1 levels has not been considered. In this study, we demonstrate that activated EGFR drives high level MUC1 expression in multiple cell lines of uterine adenocarcinoma and pancreatic cancer origins. In some cells, addition of exogenous EGFR ligands (EGF or HB-EGF) elevates MUC1 levels while addition of the EGFR tyrosine kinase inhibitor, AG1478, reduces MUC1 levels. The thiazolidinedione, rosiglitazone, previously shown to reduce progesterone-stimulated MUC1 expression, also blocks EGFR ligand-driven MUC1 expression. This activity was observed at relatively high rosiglitazone concentrations (above 10 µM) and appeared to be largely PPARγ independent indicating a novel utility of this drug to reduce mucin-expression in various tumor settings. Collectively, these data demonstrate that: (1) activation of EGFR stimulates MUC1 expression in multiple cellular contexts and (2) it may be possible to develop useful interventions to reduce MUC1 expression as a complementary strategy for tumor therapy.

Postcoital administration of asoprisnil inhibited embryo implantation and disturbed ultrastructure of endometrium in implantation window in mice

Asoprisnil, a member of the selective progesterone receptor modulators, exerts high progesterone receptor selectivity, endometrial targeted advantages and significant anti-implantation effect in rats. The purpose of this study was to confirm the anti-implantation effect of asoprisil, investigate the ultrastructural changes of the peri-implantation endometrium in mice and explore the effect of asoprisnil on endometrial receptivity and its targeted contraceptive proficiency. Post-coitus mice were administered with different dosages (0.2, 0.1, 0.05 mg·g(-1)·day(-1)) of asoprisnil from day 1 of pregnancy to day 3. Then 3 animals in each group were killed on day 5 of pregnancy, and uteri were collected to examine the ultrastructural changes of endometria under a transmission electron microscope (TEM). A total of 80 animals were sacrificed on day 8 of pregnancy, and the uterine horns were examined for the presence or absence of nidation sites and the number of implantation embryos. The results showed that the implantation rate and the average number of implantation embryos in asoprisnil groups were statistically significantly decreased as compared with the vehicle control group (P<0.05). The TEM results revealed that, in vehicle control group, the tight junction between the luminal epithelia cells was short and straight, the gap was wide; the luminal epithelia cells were covered with plenty of short, clavate and neatly arranged microvilli; the endometril stromal cells were large with plenty of cytoplasm, and showed significant decidual change; there was more than one nucleus in stromal cells, and the karyotheca was integrity. In low dosage and high dosage asoprisnil groups, the tight junction was longer and more curve than in the vehicle control group; microvilli were uneven and asymmetrically distributed in luminal epithelia; the stromal cells were small and the decidual change was not significant; there were karyopyknosis and karyolysis in stromal cells; there were abnormal thick-wall vessels in the endometrium. It was suggested that asoprisnil changed the ultrastructure of the endometrium in implantation window, disturbed the endometrial receptivity and finally resulted in embryo implantation failure.

209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL–1 β-oestradiol + 10 µg mL–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P &lt; 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P &lt; 0.03), and blastocyst (1.0- v. 2.1-fold; P &lt; 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.

Arginine enhances embryo implantation in rats through PI3K/PKB/mTOR/NO signaling pathway during early pregnancy

Our previous study has demonstrated that dietary arginine supplementation during early pregnancy enhanced embryo implantation in rats. However, the mechanism was not clear. The objective of this study was to determine the mechanism that arginine enhanced embryo implantation during early pregnancy. Rats were fed the basal diets supplemented with 1.3% (wt:wt) L-arginine-HCl or 2.2% (wt:wt) L-alanine (isonitrogenous control) once pregnancy. On d4 of pregnancy, rats were given intrauterine injection of L-NG-nitro arginine methyl ester (L-NAME, nitric oxide synthase inhibitor), α-difluoromethylornithine (DFMO, polyamine synthesis inhibitor), wortmannin (PI3K inhibitor), or rapamycin (mTOR inhibitor). On d7 of pregnancy, rats were killed. Intrauterine injection of L-NAME decreased the implantation sites, while dietary arginine supplementation increased the implantation sites. Intrauterine injection of DFMO decreased the pregnancy rate, which was reversed by dietary arginine supplementation. Intrauterine injection of rapamycin or wortmannin inhibited embryo implantation. However, dietary arginine supplementation did not reverse this inhibition. Western blot analysis revealed that the expression of uterine p-PKB and p-S6K1 was greater in rats fed the arginine-supplemented diet in the presence of L-NAME treatment compared with rats fed the control diet. In the presence of DFMO treatment, the expression of uterine iNOS and eNOS was significantly enhanced in the arginine group compared with the control group. Similarly, intrauterine injection of wortmannin or rapamycin decreased the expression of uterine iNOS and eNOS, which was enhanced by dietary arginine supplementation. These data indicated that dietary arginine supplementation during early pregnancy could enhance embryo implantation through stimulation of PI3K/PKB/mTOR/NO signaling pathway.

Immunosuppressive Factor MNSFβRegulates Cytokine Secretion by Mouse Lymphocytes and Is Involved in Interactions between the Mouse Embryo and Endometrial CellsIn Vitro

Immune tolerance at the fetomaternal interface must be established during the processes of implantation and pregnancy. Monoclonal nonspecific suppressor factor beta (MNSFβ) is a secreted protein that possesses antigen-nonspecific immune-suppressive function. It was previously reported that intrauterine immunoneutralization of MNSFβsignificantly inhibited embryo implantation in mice. In the present study, MNSFβprotein expression was up- or downregulated by overexpression or RNA interference, respectively, in HCC-94 cells and the culture supernatants used to determine effects of MNSFβon the secretion of IL-4 and TNFαfrom mouse lymphocytes as detected by ELISA. A coculture model of mouse embryos and endometrial stromal cells was also utilized to determine the effects of a specific anti-MNSFβantibody on hatching and growth of embryosin vitro. The results show that MNSFβinduced secretion of IL-4 and inhibited secretion of TNFαfrom mouse lymphocytes. Following immunoneutralization of MNSFβprotein in the HCC-94 supernatant, the stimulatory effect of MNSFβon IL-4 secretion from mouse lymphocytes was reduced, while the inhibitory effect on secretion of TNFαwas abrogated. Expression of MNSFβwas detected in both embryonic and endometrial stromal cells, and its immunoneutralization inhibited the hatching and spreading of embryos in anin vitrococulture model. These results indicated that MNSFβmay play critical roles during the peri-implantation process by regulating cytokine secretion of lymphocytes and by mediating the crosstalk between embryonic cells and endometrial stromal cells.

Intracytoplasmic Sperm Injection Allows Normal Pregnancy Rates for Males ≥ 40 With Low Hypo-osmotic Swelling Test Scores Even When Complicated by Very Low Motility Percentage

BACKGROUND: Males with sperm with subnormal hypo-osmotic swelling (HOS) tests (<50%) are generally infertile. Interestingly, the abnormality does not prevent fertilization or the production of morphologically normal embryos. Instead the defect has been clearly demonstrated to inhibit embryo implantation. The defect seems to be related to a toxic factor that causes an impairment to the functional integrity of the sperm membrane which forms the basis of the test to detect this abnormality (impairment of the sperm membrane to allow normal osmosis).

115. DEVELOPMENT OF POTENT AND STABLE PC6 INHIBITORS TO BLOCK EMBRYO IMPLANTATION FOR FEMALE CONTRACEPTION AND PREVENTION OF HIV

Proprotein convertase (PC) 6, a member of the PC family that activate precursor proteins into their active forms, is a critical endometrial factor for embryo implantation. Blocking PC6 production in mice inhibits decidualisation (a critical process of implantation) and inhibits implantation. PCs including PC6 also play a critical role in HIV infection through cleaving HIV envelope precursor protein gp160 into functional gp120 and gp41. PC inhibitors are demonstrated to inhibit HIV transmission via blocking gp160 cleavage. We hypothesised that PC6 is a potential target for the development of female contraception that could also provide protection from HIV infection. One key requirement to prove this concept in an animal model is a potent PC6 inhibitor that is stable in serum. Polyarginine peptide (polyR) is published to be a potent PC6 inhibitor that also inhibits HIV. We have confirmed that polyR inhibits PC6 in vivo and completely blocks decidualisation of human endometrial stromal cells in culture. However, polyR has short serum half-life. The aim of this current study was to generate polyR derivatives that are potent PC6 inhibitors, with increased serum stability. We modified polyR by either PEGylation with different sized PEG (polyethylene glycol) or cyclization, and tested their potency, stability and utility in vivo. Modifications at both terminals of polyR dramatically reduced its PC6 inhibitory potency. PEGylation at the C-terminal, regardless of the PEG size, had no effect. In silico docking experiments showed that N-terminal PEGylation or cyclization affected the binding of polyR to the PC6 active site, but not C-terminal PEGylation. One of the polyR derivatives, C-30kDa-PEG polyR was confirmed to be as potent as the parental peptide but much more stable in serum. Studies are currently in place to inhibit embryo implantation in mice using C-30kDa-PEG polyR and to determine its ability to inhibit HIV.