N-glycosylation of uterine endometrium determines its receptivity

Glycosylation alters the molecular and functional features of glycoproteins, which is closely related with many physiological processes and diseases. During “window of implantation”, uterine endometrium transforms into a receptive status to accept the embryo, thereby establishing successful embryo implantation. In this article, we aimed at investigating the role of N-glycosylation, a major modification type of glycoproteins, in the process of endometrial receptivity establishment. Results found that human uterine endometrial tissues at mid-secretory phase exhibited Lectin PHA-E+L (recognizes the branched N-glycans) positive N-glycans as measured by the Lectin fluorescent staining analysis. By utilizing in vitro implantation model, we found that de-N-glycosylation of human endometrial Ishikawa and RL95-2 cells by tunicamycin (inhibitor of N-glycosylation) and peptide-N-glycosidase F (PNGase F) impaired their receptive ability to human trophoblastic JAR cells. Meanwhile, N-glycosylation of integrin αvβ3 and leukemia inhibitory factor receptor (LIFR) are found to play key roles in regulating the ECM-dependent FAK/Paxillin and LIF-induced STAT3 signaling pathways, respectively, thus affecting the receptive potentials of endometrial cells. Furthermore, in vivo experiments and primary mouse endometrial cells-embryos coculture model further verified that N-glycosylation of mouse endometrial cells contributed to the successful implantation. Our results provide new evidence to show that N-glycosylation of uterine endometrium is essential for maintaining the receptive functions, which gives a better understanding of the glycobiology of implantation.     

Decreased ANGPTL4 impairs endometrial angiogenesis during peri‐implantation period in patients with recurrent implantation failure

Insufficient endometrial angiogenesis during peri‐implantation impairs endometrial receptivity (ER), which contributes to recurrent implantation failure (RIF) during in vitro fertilization and embryo transfer (IVF‐ET). Angiopoietin‐like protein 4 (ANGPTL4) acts as a multifunctional secretory protein and is involved in the regulation of lipid metabolism and angiogenesis in various tissues including the endometrium. Herein, we found decreased ANGPTL4 expression in endometrial tissue and serum during peri‐implantation period in 18 RIF‐affected women with elevated uterine arterial impedance (UAI) compared with the pregnancy controls. ANGPTL4 and peroxisome proliferator‐activated receptor gamma (PPARγ) expression were up‐regulated upon decidualization on human endometrial stromal cells (HESCs). Rosiglitazone promoted the expression of ANGPTL4 in HESCs and human umbilical vein endothelial cells (HUVECs) via PPARγ. ANGPTL4 promoted the proliferation, migration and angiogenesis of HUVECs in vitro. Our results suggest that decreased abundance of ANGPTL4 in endometrial tissues impairs the endometrial receptivity via restraining endometrial angiogenesis during decidualization; while rosiglitazone‐induced ANGPTL4 up‐regulation in hESCs and HUVECs through PPARγ. Therefore, ANGPTL4 could be a potential therapeutic approach for some RIF‐affected women with elevated UAI.     

Clock gene Bmal1 in mice embryo is dispensable for early embryo development but critical for live birth

In adult animals, the significance of circadian clocks in the regulation of physiology is well established. However, the physiological roles of embryonic clock genes on early embryo development, implantation and perinatal survival are still unclear. In the present study, using genotyping, embryo culture and transfer, the early embryo development, implantation, and perinatal survival of Bmal1+/+, Bmal1+/? and Bmal1?/? embryo were studied. At cleavage stage, the genotype ratio of Bmal1+/+, Bmal1+/? and Bmal1?/? embryo was 1:1.97:0.95, respectively (p > 0.05). Morula or early blastocyst developmental ratio was 83.8 ± 14.3, 87.1 ± 9.2 and 88.7 ± 14.5%, respectively (p > 0.05). After transferring of the three types of embryos to pseudopregnant wild-type mice, the implantation sites 4 days later was 7.7 ± 0.9, 7.2 ± 1.2 and 7.5 ± 0.5 (n = 4, F = 0.265, p = 0.773). Mean litter size of the mice after transferring with the three types of embryos was 5.5, 6.0, and 3.0 (n = 3, F = 30.3, p = 0.001). The development of Bmal1 null embryos was not impaired in preimplantation and early implantation stages, but the litter size had a trend to decrease.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Embryotrophic effects of extracellular vesicles derived from outgrowth embryos in pre‐ and peri‐implantation embryonic development in mice

Recently, many studies have investigated the role of extracellular vesicles (EVs) on reproductive events, including embryo development and death, oviduct–embryo crosstalk, in vitro fertilization and others. The aim of this study was to demonstrate whether outgrowth embryo–derived EVs function as bioactive molecules and regulate mouse embryonic developmental competence in vitro and implantation potential in utero. The EVs from mouse outgrowth embryos on 7.5 days postcoitum were detected and selectively isolated to evaluate the embryotrophic functions on preimplantation embryos. Developmental outcomes such as the percentage of blastocyst formation, hatching, and trophoblastic outgrowth were assessed. Furthermore, the total cell number and apoptotic index of blastocysts, which were incubated with EVs during the culture period, were evaluated by fluorescence microscopy. Implantation potential in utero was investigated following embryo transfer. The EVs from outgrowth embryo–conditioned media have rounded membrane structures that range in diameter from 20 to 225 nm. Incubation with EVs improved preimplantation embryonic development by increasing cell proliferation and decreasing apoptosis in blastocysts. Moreover, the implantation rates following embryo transfer were significantly higher in EV–supplemented embryos compared with the control. Collectively, EVs from outgrowth embryo could enhance the embryonic developmental competence and even implantation potential in mice.     

Cynomolgus monkey embryo model captures gastrulation and early pregnancy

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Maternal age effect on early human embryonic development and blastocyst formation

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([expanded blastocysts/blastocysts]*100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (<30 years, 82%; ≥40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. © 1996 Wiley-Liss, Inc.     

Potential innate immunity-related markers of endometrial receptivity and recurrent implantation failure (RIF)

The successful implantation of the embryo into a receptive endometrium is essential for the establishment of a viable pregnancy while recurrent implantation failure (RIF) is a real challenge in assisted reproduction. The maternal innate immune system, specifically the Toll-like receptors (TLRs), are involved in maintaining immunity in the female reproductive tract (FRT) required for fertility. In this study, we aimed to investigate the importance of innate immunity-related gene expression in the regulation of human fertility and as a prediction of potential outcome of in vitro fertilization - embryo transfer (IVF-ET), thus, we assessed the gene expression levels of TLR signalling molecules using quantitative real-time PCR between endometrial biopsies of healthy fertile women, and the patients experiencing RIF. Interestingly, our results showed that, TRIB2 and TLR9 genes were differentially expressed between the endometrial biopsies of healthy women and those with RIF. However, comparing expression levels of same genes between pre-receptive and receptive healthy endometrial biopsies showed different genes (ICAM1, NFKBIA, VCAM1, LIF, VEGFB, TLR5) had significantly altered expression, suggesting their involvement in endometrial receptivity. Thus, further investigations will enable us to better understand the role of these genes in the biology of FRT and as a possible target for the improvement of infertility treatments and/or development of non-hormonal contraception.     

Calcitonin administration improves endometrial receptivity via regulation of LIF,Muc-1 and microRNA Let-7a in mice

Calcitonin (CT) is one of the factors affecting the embryo implantation, but its effects on the implantation window have not been fully investigated. The current study investigated the effects of CT on the endometrium receptivity by morphological study and evaluation of leukemia inhibitory factor (LIF), mucin 1 (Muc-1), and microRNA (miRNA) Let-7a in the ovarian stimulation and the normal ovarian cycle. Then the mechanism of the CT effects through the mammalian target of rapamycin (mTOR) signaling pathway was studied by using PP242. A total of 64 BALB/c mice were divided into the normal ovarian cycle and ovarian stimulation groups. Each group consisted of four subgroups: control, calcitonin, PP242, and calcitonin+PP242. CT and PP242 were injected on the fourth of pregnancy into the mice and 24 hr later all the mice were killed. The uterine tissue samples were used for morphological analysis, and endometrial cells were mechanically isolated for evaluation of gene and protein expression. The results showed that ovarian stimulation induced mTOR phosphorylation as well as increased expression of the Let-7a miRNA. In addition, CT injection increased the expression of LIF and miRNA Let-7a in ovarian stimulation similar to that in normal ovarian cycles. However, injection of PP242 reduced expression of miRNA Let-7a and increased Muc-1 expression in ovarian stimulation group. In conclusion, the administration of CT improved endometrial receptivity in mice. This phenomenon occurred by upregulation of LIF, miRNA Let-7a and downregulation of Muc-1 via mTOR signaling pathway.     

Impact of endometriosis on embryo quality and endometrial receptivity in women undergoing assisted reproductive technology

ART is an important treatment method for infertile patients with endometriosis. However, the effects of endometriosis on embryo quality and endometrial receptivity remain unclear. Thus, we aimed to simultaneously investigate the impact of endometriosis and its stage on embryo quality and endometrial receptivity in women undergoing ART. We retrospectively analyzed the data from patients with and without endometriosis who underwent oocyte retrieval and/or high-quality embryos transfer between July 2015 and December 2020, including 1312 IVF cycles and 608 IVF or frozen-thawed embryo transfer (FET) cycles, respectively. The endometriosis group had a lower percentage of good cleavage-stage embryos and fertilization rates than those in the control group (p = 0.038 and 0.008, respectively). The number of retrieved oocytes, MII oocytes, cleavage, blastocysts, and blastulation rates was comparable between two groups. We found no significant difference in clinical pregnancy, implantation, live birth, miscarriage, or multiple pregnancy rates between the two groups among patients who transferred high-quality embryos. Stratification analysis showed that patients with stage III-IV endometriosis had fewer retrieved oocytes than those with stage I-II endometriosis (p = 0.012) and marginally fewer retrieved oocytes than the control group (p = 0.051). The stage I-II group had the lowest percentage of good cleavage-stage embryos, which was significantly lower than that of the control group (p = 0.043). In FET cycles, patients with stage III-IV endometriosis had a higher miscarriage rate than those in the control group (p = 0.023). Our results suggest that endometriosis does not alter endometrial receptivity but affects embryo quality, oocyte fertilization ability, and ovarian response.     

Fate of hamster oviductin in the oviduct and uterus during early gestation

Oviductins are a family of glycoproteins which are synthesized and secreted by oviductal secretory cells and which, upon their secretion in the lumen of the oviduct, become associated with postovulatory oocytes and developing embryos. Recently, we showed that hamster oviductin is maximally secreted in the oviduct at the time of ovulation and is later associated with a certain population of uterine epithelial cells, where it is subsequently endocytosed and degraded. In light of these results, this study was conducted to follow the fate of hamster oviductin in the oviduct and uterus during early gestation. Using a monoclonal antibody against hamster oviductin, immunofluorescence and immunogold labeling revealed that during early gestation, immunoreactivity to oviductin in the uterus gradually diminished to an almost total disappearance at time of implantation. However, the strong labeling intensity remained unchanged in the oviduct. Biochemical analyses demonstrated that a degradation of oviductin occurs in the uterus, and a loss of immunoreactivity was also observed as gestation progressed, so that by the time of implantation, immunoreactivity to oviductin was barely detectable. The decrease of oviductin along the uterine epithelium at the time of blastocyst attachment and its final disappearance at implantation suggest that this glycoprotein could be a potential modulator of uterine receptivity. Mol. Reprod. Dev. 46:306–317, 1997. © 1997 Wiley-Liss, Inc.     

Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate

Oxidative damage due to the production of reactive oxygen species (ROS) is one of a number of culture-induced stresses which may compromise preimplantation embryo development in vitro. Ethylenediaminetetraacetic acid (EDTA), reduced oxygen tension, superoxide dismutase (SOD) and catalase (CAT) offer protection against oxidative stress, but few attempts have been made to determine which of these agents, or which combination, is the most effective. In particular, no systematic investigation of their actions and interactions has been made using a multifactorial experimental design. Murine zygotes were cultured in the presence or absence of 10 miccroM EDTA, SOD (100-7,000 U/ml) and CAT (50-100 U/ml) at atmospheric (20%) and reduced (5%) oxygen tensions. Blastocyst formation and hatching rates (at various time points), and cell numbers were recorded, whilst parallel groups of embryos had their consumption of pyruvate, a hydrogen peroxide scavenger, measured. All parameters interacted significantly and affected blastocyst formation, hatching rate and cell numbers but the effect of EDTA was the most pronounced. There were beneficial effects of 5% O2, CAT and SOD, while 20% O2 had a deleterious effect on development. EDTA improved blastocyst formation and hatching rates but paradoxically led to a reduction in cell number. 5% O2 was the next most significant parameter to enhance embryo development and also increased cell numbers. No differences in pyruvate uptake were apparent between the various treatment groups. The results suggest that embryo culture in EDTA-free medium under 5% O2 provides the most practical and physiological conditions for in vitro murine embryo culture.     

哺乳动物早期胚胎端粒和端粒酶重编程

端粒位于真核染色体末端,是稳定染色体末端的重要元件。端粒酶(TER)是一种特殊的细胞核糖核蛋白(RNP)反转录酶(RT),其核心酶包括蛋白亚基和RNA元件。在DNA复制过程中的端粒丢失可以被有活性的端粒酶修复回来。哺乳动物端粒酶在发育中受调控,端粒的重编程可能是由于早期胚胎不同时期的端粒酶活性而造成的。因此,研究端粒和端粒酶重编程在早期胚胎发育中是非常重要的。该文综述了端粒和端粒酶的结构和功能,及其与哺乳动物早期胚胎发育的关系,并在此基础上展望了端粒和端粒酶在克隆动物胚胎发育的基础研究。     

小鼠植入前胚胎致密化与囊胚形成的分子调控

哺乳动物胚胎植入前的发育中致密化和囊胚形成分别标志着第一次、第二次细胞分化（即细胞命运决定）的起始,是胚胎正常发育的必要条件。因此对影响致密化和囊胚形成的蛋白及调节因子的研究尤为重要。本文探讨了与致密化相关的细胞黏附蛋白、连接蛋白、细胞骨架等分子和囊胚形成相关的紧密连接蛋白、钠钾三磷酸腺苷激酶等分子的一系列调控,以及致密化和囊胚形成在细胞命运决定中的重要作用。     

组蛋白甲基化修饰酶与早期胚胎发育

早期胚胎发育是胚胎发育中细胞分裂与分化最为活跃的时期,也是合子型基因大规模转录的时期,而此时组蛋白的甲基化修饰也显示出动态学的变化。这一时期,在细胞内外信号的共同调控下,经历着一系列基因的激活与抑制,许多调控机制参与其中的调控。而近年来的研究表示,表观遗传学调控显示越来越重要的作用。组蛋白甲基化修饰是表观遗传学重要调控机制之一,在胚胎的早期发育过程中扮演着重要的角色。就近年来组蛋白甲基化修饰酶在早期胚胎发育过程中的作用与功能做一简要综述。     

Quantitative analysis of retromer complex-related genes during embryo development in the mouse

The retromer complex is a heteropentameric protein unit associated with retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Functional silencing study of the Vps26a gene indicated the important role of the retromer complex during early developmental stages in the mouse. However, individual expression patterns and quantitative analysis of individual members of the retromer complex during the early developmental stages has not been investigated. In this study, we conducted quantitative expression analysis of six retromer complex genes (Vps26a, Vps26b, Vps29, Vps35, Snx1, and Snx2) and one related receptor gene (Ci-mpr) during the eleven embryonic stages with normal MEF (mouse embryonic fibroblast) and Vps26a(-/-) MEF cells. Remarkably, except for Vps26a (maternal expression pattern), all tested genes showed maternal-zygotic expression patterns. And five genes (Vps26b, Vps29, Vps35, Snx2, and Ci-mpr) showed a pattern of decreased expression in Vps26a(-/-) MEF cells by comparative analysis between normal MEF and Vps26a(-/-) MEF cells. However, the Snx1 gene showed a pattern of increased expression in Vps26a(-/-) MEF cells. From our results, we could assume that retromer complex-related genes have important roles during oocyte development. However, in the preimplantation stage, they did not have significant roles.