Endometrial extracellular vesicles of recurrent implantation failure patients inhibit the proliferation, migration, and invasion of HTR8/SVneo cells.

PURPOSE: Endometrial extracellular vesicles are essential in regulating trophoblasts' function. This study aims to investigate whether endometrial extracellular vesicles (EVs) from recurrent implantation failure (RIF) patients inhibit the proliferation, invasion, and migration of HTR8/SVneo cells. METHODS: Eighteen RIF patients and thirteen fertile women were recruited for endometria collection. Endometrial cells isolated from the endometria were cultured and modulated by hormones, and the conditioned medium was used for EV isolation. EVs secreted by the endometrial cells of RIF patients (RIF-EVs) or fertile women (FER-EVs) were determined by Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence-labeled EVs were used to visualize internalization by HTR8/SVneo cells. RIF-EVs and FER-EVs were co-cultured with HTR8/SVneo cells. Cell Counting Kit-8, transwell invasion, and wound closure assays were performed to determine cellular proliferation, invasion, and migration, respectively, in different treatments. RESULTS: RIF-EVs and FER-EVs were bilayer membrane vesicles, ranging from 100 to 150 nm in size, that expressed the classic EV markers Alix and CD9. RIF-EVs and FER-EVs were internalized by HTR8/SVneo cells within 2 h. The proliferation rate in the FER-EV group was significantly higher than that in the RIF-EV group at 20 µg/mL. Moreover, the invasion and migration capacity of trophoblast cells were decreased in the RIF-EV group relative to the FER-EV group at 20 µg/mL. CONCLUSION: Endometrial EVs from RIF patients inhibited the functions of trophoblasts by decreasing their proliferation, migration, and invasive capacity. Such dysregulations induced by RIF-EVs may provide novel insights for better understanding the pathogenesis of implantation failure.

Prevalent genotypes of methylenetetrahydrofolate reductase (MTHFR) in recurrent miscarriage and recurrent implantation failure.

OBJECTIVE: To evaluate the association of two common methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with recurrent miscarriage (RM) and repeated implantation failure (RIF) METHODS: The study comprised of 521 patients, with a history of RM (n = 370) or RIF (n = 151). One hundred forty-four women with fallopian tube blockages who had successfully conceived after the first in vitro fertilization embryo transfer treatment served as the control group. The MTHFR alleles, genotypes, and haplotypes were assessed in different groups. RESULTS: There was no difference in allele frequency and distribution of MTHFR polymorphisms between case and control patients. The 1298AA genotype was represented in a higher frequency, and 1298AC genotype was significantly lower in subfertile group when compared to the control group. A significant relationship was found between the 1298AC genotype and the RIF subgroup. The haplotype 677CC/1298AA was overrepresented in the RM subgroup (> 2 times) and haplotype 677CC/1298AC was underrepresented in the RIF subgroup (P < 0.05). Nevertheless, these two haplotypes were not connected to fertilization and embryo cleavage rates. CONCLUSION: Our findings indicate that the MTHFR gene polymorphism might play a role in the etiology of patients with RM or RIF. No adverse effects of different MTHFR haplotypes on embryo development were detected. Further studies on the biological role are needed to better understand the susceptibility to pregnancy complications.

MicroRNA-200a locally attenuates progesterone signaling in the cervix, preventing embryo implantation.

Although cervical pregnancy and placenta previa, in which the embryo and placenta embed in or adjacent to the cervix, are life-threatening complications that result in massive bleeding and poor pregnancy outcomes in women, the incidence of these aberrant conditions is uncommon. We hypothesized that a local molecular mechanism is normally in place to prevent embryo implantation in the cervix. The ovarian hormones progesterone (P(4)) and estrogen differentially direct differentiation and proliferation of endometrial cells, which confers the receptive state for implantation: P(4) dominance causes differentiation of the luminal epithelium but increases stromal cell proliferation in preparation of the uterus for implantation. In search for the cause of cervical nonresponsiveness to implantation, we found that the statuses of cell proliferation and differentiation between the uterus and cervix during early pregnancy are remarkably disparate under identical endocrine milieu in both mice and humans. We also found that cervical levels of progesterone receptor (PR) protein are low compared with uterine levels during this period, and the low PR protein levels are attributed to elevated levels of microRNA(miR)-200a in the cervix. These changes were associated with up-regulation of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (200α-HSD) and down-regulation of its transcriptional repressor signal transducer and activator of transcription 5 in the cervix. The results provide evidence that elevated levels of miR-200a lead to down-regulation of P(4)-PR signaling and up-regulation of (200α-HSD) in the cervix, rendering it nonresponsive to implantation. These findings may point toward not only the physiological but also the pathological basis of the cervical milieu in embryo implantation.

Blastocyst implantation failure relates to impaired translational machinery gene expression.

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF.

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy.

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.

Differential expression of transcriptional repressor snail gene at implantation site in mouse uterus.

The snail superfamily of zinc-finger transcription factors is involved in pronounced cell movements during both embryonic development and tumor progression. This study was to examine snail expression in mouse uterus during early pregnancy and its regulation under pseudopregnancy, delayed implantation, steroid hormone treatment, and artificial decidualization by in situ hybridization and immunohistochemistry. There was a low level of snail mRNA signal and immunostaining in mouse uteri on day 1-4 of pregnancy. When embryo implanted on day 5, both snail mRNA signal and immunostaining were strongly detected in the subluminal stroma immediately surrounding the implanting blastocyst, but not detected in the inter-implantation sites. Under delayed implantation, there was no detectable snail expression. After delayed implantation was terminated by estrogen treatment and embryo implanted, there was a strong level of snail mRNA and immunostaining in the subluminal stroma surrounding the implanting blastocyst, which was similar to that on day 5 of pregnancy. Furthermore, there was no detectable snail expression in mouse uterus on day 5 of pseudopregnancy. From day 6-8 of pregnancy, both snail mRNA signal and immunostaining were detected in the decidua. Our data suggest that snail may play an important role during mouse embryo implantation.

Alternate metabolism during the dauer stage of the nematode Caenorhabditis elegans.

When environmental conditions are unsuitable to support nematode reproduction, Caenorhabditis elegans arrests development before the onset of sexual maturity and specialised 'dauer' larvae, adapted for dispersal, and extended diapause are formed. Dauer larvae do not feed and their metabolism is dependent on internal food reserves. Adult worms which express defects in the insulin/insulin-like growth factor receptor DAF-2 also display enhanced longevity. Whole genome mRNA expression profiling has demonstrated that C. elegans dauer larvae and daf-2 adults have similar transcription profiles for a cohort of longevity genes. Important components of this enhanced longevity system are the alpha-crystallin family of small heat shock proteins, anti-ROS defence systems, increased activity of cellular detoxification processes and possibly also increased chromatin stability and decreased protein turnover. Anaerobic fermentation pathways are upregulated in dauer larvae, while long-lived daf-2 adults appear to have normal oxidative metabolism. Anabolic pathways are down regulated in dauer larvae (and possibly in daf-2 adults as well), and energy consumption appears to be diverted to enhanced cellular maintenance and detoxification processes in both systems.

Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation.

Heparin-binding EGF-like growth factor (HB-EGF) is a newly discovered member of the EGF family of growth factors. HB-EGF can bind to two loci on cell surfaces, heparan sulphate proteoglycans and EGF-receptor (EGF-R), and either one or both of these interactions could play a role in cell-cell interactions. In the rodent, increased endometrial vascular permeability at the site of blastocyst apposition is considered to be an earliest discernible prerequisite event in the process of implantation and this event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium. This investigation demonstrates that the HB-EGF gene is expressed in the mouse uterine luminal epithelium surrounding the blastocyst 6-7 hours before the attachment reaction that occurs at 2200-2300 hours on day 4 of pregnancy. It was further demonstrated that this gene is not expressed in the luminal epithelium at the site of the blastocyst apposition during the progesterone-maintained delayed implantation, but is readily induced in the luminal epithelium surrounding an activated blastocyst after termination of the delay by an estrogen injection. In vitro studies showed that HB-EGF induced blastocyst EGF-R autophosphorylation, and promoted blastocyst growth, zona-hatching and trophoblast outgrowth. These results suggest possible interactions between the uterine HB-EGF and blastocyst EGF-R very early in the process of implantation, earlier than any other embryo-uterine interactions defined to date at the molecular level.