Cell adhesion in endometrial epithelium is regulated to maintain the continuity and protectiveness of the luminal covering cell layer while permitting interstitial implantation of the embryo during a restricted period of about 4 days. Many apparently normal embryos fail to implant, and epithelial-embryo adhesion remains a poorly understood phenomenon. After menstruation, epithelial regeneration occurs by epiboly from the basal residues of glands, an activity that requires migration on extracellular matrix as well as cell-cell cohesion. Here we review current knowledge of adhesion molecules in the epithelium.
Successful embryo implantation depends on the quality of the embryo, as well as on the receptivity of the endometrium. The aim of this study was to investigate the endometrial gene expression profile in women with unexplained infertility in comparison with fertile controls at the time of embryo implantation in order to find potential predictive markers of uterine receptivity and to identify the molecular mechanisms of infertility. High-density oligonucleotide gene arrays, comprising 44 000 gene targets, were used to define the endometrial gene expression profile in infertile (n = 4) and fertile (n = 5) women during the mid-secretory phase (day LH +7). Microarray results were validated using real-time PCR. Analyses of expression data were carried out using non-parametric methods. Hierarchical clustering and principal component analysis showed a clear distinction in endometrial gene expression between infertile and fertile women. In total we identified 145 significantly (>3-fold change) up-regulated and 115 down-regulated genes in infertile women versus controls. Via Database for Annotation, Visualization and Integrated Discovery functional analysis we detected a substantial number of dysregulated genes in the endometria of infertile women, involved in cellular localization (21.1%) and transport (18.8%) and transporter activity (13.1%) and with major localization in extracellular regions (19.2%). Ingenuity Pathways Analysis of the gene list showed dysregulation of gene pathways involved in leukocyte extravasation signalling, lipid metabolism and detoxification in the endometria of infertile women. In conclusion, endometrial gene expression in women with unexplained infertility at the time of embryo implantation is markedly different from that in fertile women. These results provide new information on genes and pathways that may have functional significance as regards to endometrial receptivity and subsequent embryo implantation.
Mammalian target of rapamycin (MTOR) is a protein kinase that plays a central role in cell growth and proliferation. It is a part of the signaling network transmitting growth factor signaling to translational control. Previous studies have shown that MTOR is involved in embryo implantation, but its expression in the uterus and its role in implantation are unclear. Here, we have investigated the expression and role of MTOR in mouse uterus during early pregnancy. RT-FQ PCR showed that the mRNA levels of Mtor in endometria of pregnant mice were higher than those of nonpregnant mice. The mRNA levels in the pregnant mice gradually increased from D3 of pregnancy, reached maximum on D5, and then declined afterward. In situ hybridization and immunohistochemical analysis showed that the mRNA and protein of MTOR were mainly located in stromal cells. The levels of the expressed MTOR protein correlate with those of mRNA. The number of implantation sites was greatly decreased by the intrauterine injection with rapamycin in the early morning of D4 of the pregnancy. These findings suggest that MTOR may play an important role in embryo implantation in mice.
There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression ( P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.
Pregnancy is dependent upon the endometrium acquiring a receptive phenotype that facilitates apposition, adhesion and invasion of a developmentally competent embryo. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry of mid-secretory endometrial biopsies revealed a 28 kDa protein peak that discriminated highly between samples obtained from women with recurrent implantation failure and fertile controls. Subsequent tandem mass spectroscopy unambiguously identified this peak as apolipoprotein A-I (apoA-I), a potent anti-inflammatory molecule. Total endometrial apoA-I levels were, however, comparable between the study and control group. Moreover, endometrial apoA-I mRNA expression was not cycle-dependent although there was partial loss of apoA-I immunoreactivity in luminal and glandular epithelium in mid-secretory compared with proliferative endometrial samples. Because of its putative anti-implantation properties, we examined whether endometrial apoA-I expression is regulated by embryonic signals. Human chorionic gonadotrophin (hCG) strongly inhibited apoA-I expression in differentiating explant cultures but not when established from eutopic endometrium from patients with endometriosis. Pelvic endometriosis was associated with elevated apoA-I mRNA levels, increased secretion by differentiating eutopic endometrial explant cultures and lack of hCG-dependent down-regulation. To corroborate these observations, we examined endometrial apoA-I expression and its regulation by hCG in a non-human primate model of endometriosis. As in humans, hCG strongly inhibited endometrial apoA-I mRNA expression in disease-free baboons, but this response was entirely lost upon induction of pelvic endometriosis. Together, these observations indicate that perturbations in endometrial apoA-I expression, modification or regulation by paracrine embryonic signals play a major role in implantation failure and infertility.
Objective To assess the benefit of selecting blastocysts for cryotransfer based upon prior comparative genomic hybridization (CGH) karyotyping of blastomeres derived from their cleaved embryos of origin. Implantation and birth rates per transfer of previtrified CGH-tested blastocysts were compared with those following the transfer of nonCGH-tested fresh and warmed embryos. Design In vitro studies. Setting Private infertility clinic. Patient(s) Women undergoing infertility treatment. Intervention(s) Three groups of women with similar clinical and demographic characteristics were compared. Group A underwent transfer of warmed blastocysts derived from CGH-normal day 3 embryos. Group B underwent embryo transfer of warmed blastocysts derived from nonkaryotyped vitrified embryos. Group C underwent fresh transfers with non-CGH-tested blastocysts. Main Outcome Measure(s) Implantation and birth rates per embryo after the cryotransfer of CGH-tested blastocysts. Result(s) The birth rate per transfered blastocyst in group A was 48%, versus 15% for group B and 19% for group C. The birth rate per embryo transfer was 60% for group A, and 33% for group B and 36% for group C. The miscarriage rate was 4% in group A, 8% in group B, and 12% in group C. Conclusion(s) The transfer of previously vitrified blastocysts derived from CGH-normal embryos significantly improves implantation and birth rates per embryo transfered and reduces the miscarriage rate. Vitrification does not compromise this enhancement.
Objective The aim of this study was to identify a novel implantation-related molecule and to examine EMO2 expression in the mouse uterus during the peri-implantation period. Design Experimental study. Setting Research laboratory. Animal(s) Adult ICR mice aged 6–8 weeks. Intervention(s) Adult female mice were mated with fertile males to achieve pregnancy. Implantation was delayed by ovariectomizing pregnant mice on day 4 and administering P during days 5–7; implantation was then initiated by administering E2 . Pseudopregnant mice were obtained by mating females with vasectomized males. Main Outcome Measure(s) The tissue distribution of EMO2 mRNA was detected by reverse transcriptase–polymerase chain reaction, and the uterine expression pattern of the EMO2 protein was determined by immunohistochemistry. Result(s) The full cDNA sequence of EMO2 was registered in GenBank (AY372183). EMO2 mRNA expression was observed in all mouse tissues tested. The expression of the EMO2 protein was predominately localized in decidual cells at the implantation site during days 5–6 of pregnancy, and its expression was induced by the active blastocyst and artificially induced decidualization. Conclusion(s) Our data indicate that EMO2 may play a key role in the mouse embryo implantation process.
Abstract Objectives LAT-1 (L-type amino acid transporter 1) is a system L, Na+ -independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and is related to tumor invasion. Primary rat hepatocytes in culture increase LAT-1 mRNA in response to amino acid depletion. Transformed hepatic cell lines demonstrate constitutive expression of LAT-1. These observations suggest that LAT-1 expression confers a growth and survival advantage under limited amino acid availability. LAT-1 is highly expressed in the placenta. It has been shown previously that amino acids are fundamental regulators of cell function and energy metabolism in pre-implantation embryos. Our objectives were to analyze qualitatively and quantitatively LAT-1 expression in pre-implantation stages of mouse embryo development and to identify cell types expressing LAT-1 in post-implantation stages. Methods LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry. Results Our results show increasing mRNA levels of LAT-1 as the embryo develops from zygote to blastocyst with highest levels at hatching blastocyst. Expression studies of LAT-1 on microdissected samples from developing mouse placenta show highest levels of LAT-1 mRNA in trophoblast giant cells (TGCs) at the time of implantation (E7.5), followed by maternal decidua, ectoplacental cone and epiblast. At later stages of development (E9.5 and E11.5) no differential expression of LAT-1 was observed. In situ hybridization and immunohistochemistry also showed differential expression of LAT-1 mRNA and protein, respectively, with darkest staining in TGCs at E7.5. By E9.5 and E11.5 mRNA expression was no longer preferentially localized to TGCs, hybridization was equal across the different cell types and regions. LAT-1 protein expression, however, still showed highest intensity of staining in TGCs at E9.5 and E11.5. Conclusions Since trophoblast giant cells are invasive cells that displace and phagocytose the uterine epithelial cells, these data suggest that LAT-1 may play a role in the invasive phenotype. The mechanism of LAT-1 regulation during placentation, therefore, might provide valuable clues to its role in tumor progression and invasion.
Cytosolic phospholipase A2 (cPLA2, PLA2G4A) catalyzes the release of arachidonic acid for prostaglandin synthesis by cyclooxygenase 1 (PTGS1) and cyclooxygenase 2 (PTGS2). Mice with Pla2g4a deficiency have parturition delay and other reproductive deficits, including deferred onset of implantation, crowding of implantation sites, and small litters. In this study, we examined the contribution of PLA2G4A to parturition in mice. Pla2g4a mRNA and protein expression were discretely localized in the term and preterm uterine luminal epithelium and colocalized with Ptgs1 , but not Ptgs2 , expression. The levels of PGE2, PGF2alpha, 6-keto-PGF1alpha, and TxB2 were significantly decreased in Pla2g4a -null uterine tissues, similar to Ptgs1 -null uteri, consistent with predominance of PLA2G4A-PTGS1-mediated prostaglandin synthesis in preparation for murine parturition. Litter size was strongly associated with the timing of parturition in Pla2g4a -null mice but could not fully account for the parturition delay. Pla2g4a -null females that received PGE2 + carbaprostacyclin at the time of implantation delivered earlier (20.5 Â± 0.2 days vs. 21.6 Â± 0.2 days, P < 0.01), although litter size was not improved (4.6 vs. 4.4 pups per litter, P = 0.6). After correction for small litter size, multivariate analysis indicated that Pla2g4a -null mice given prostaglandin treatment to improve implantation timing had gestational length that was similar to wild-type and Pla2g4a heterozygous mice. These results indicate that, despite specific Pla2g4a expression and function in term gestation uteri, the delayed parturition phenotype in Pla2g4a -null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy.