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Pregnancy involves a wide range of adaptations in the maternal body. Maternal immune tolerance toward the foreign fetus is critical for a successful pregnancy. Decidual macrophages are the primary antigen-presenting and phagocytic cells responsible for antigen presentation and apoptotic cell removal. Their phenotype changes dynamically during pregnancy. Placenta-derived exosomes are small vesicles carrying active biological molecules such as microRNAs, proteins, and lipids. The placenta-derived exosomes have been implicated in endothelial cell activation, smooth muscle cell migration, and T-cell apoptosis, but it is unknown whether placenta-derived exosomes would affect the development and functions of decidual macrophages. In this study, we reported that placenta-derived exosomes stimulated macrophage polarization into alternatively activated (M2) macrophages. Mechanistically, miRNA-30d-5p from the placenta-derived exosomes induced macrophage polarization to the M2 phenotype by targeting histone deacetylase 9. Furthermore, the conditioned medium of placenta-derived exosome–treated macrophages promoted trophoblast migration and invasion. By contrast, the conditioned medium impaired the ability of endothelial cell tube formation and migration. Placenta-derived exosome–treated macrophages had no impact on T-cell proliferation. Together, we demonstrated that placenta-derived exosomes polarize macrophages to acquire a decidua-like macrophage phenotype to modulate trophoblast and endothelial cell functions.

Implantation is a limiting factor for treatment success in assisted reproduction. Both embryonic and endometrial factors contribute to implantation. Embryonic factors have often been ignored in previous studies about the role of endometrium in implantation. In this study, we sought to identify the endometrial genes associated with negative pregnancy outcomes following the transfer of a single euploid blastocyst.Computational analyses of the transcriptomes of mid-secretory endometria from nine pregnant and seven non-pregnant patients in a cycle preceding the transfer of a single euploid blastocyst in a vitrified-warmed cycle were performed.Principal component analysis of two reported endometrial receptivity gene sets showed close clustering of the pregnant and non-pregnant samples. Differential gene expression analysis and co-expression module analysis identified 131 genes associated with the pregnancy status. The endometrial signatures identified highlight the importance of immune and metabolic regulation in pregnancy outcome. Network analysis identified 20 hub genes that could predict pregnancy outcomes with 88.9% sensitivity and 85.7% specificity. Single-cell gene expression analysis highlighted the regulation of endometrial natural killer cells, T cells, and macrophages during embryo implantation. Immune cell abundance analysis supported the dysregulation of cytotoxic immune cells in the endometria of non-pregnant women.We reported the first endometrial gene signature associated with pregnancy after elimination of embryo aneuploidy and highlighted the importance of the endometrial immune microenvironment and metabolic status in pregnancy outcomes. This article is protected by copyright. All rights reserved.

The placenta is important for fetal development in mammals, and spatial transcriptomic profiling of placenta helps to resolve its structure and function. In this study, we described the landscape of spatial transcriptome of human placental villi obtained from two pregnant women at the first trimester using the modified Stereo-seq method applied for paraformaldehyde (PFA) fixation samples. The PFA fixation of human placenta villi was better than fresh villi embedded in optimum cutting temperature (OCT) compound, since it greatly improved tissue morphology and the specificity of RNA signals. The main cell types in chorionic villi such as syncytiotrophoblasts (SCT), villous cytotrophoblasts (VCT), fibroblasts (FB), and extravillous trophoblasts (EVT) were identified with the spatial transcriptome data, whereas the minor cell types of Hofbauer cells (HB) and endothelial cells (Endo) were spatially located by deconvolution of scRNA-seq data. We demonstrated that the Stereo-seq data of human villi could be used for sophisticated analyses such as spatial cell-communication and regulatory activity. We found that the SCT and VCT exhibited the most ligand-receptor pairs that could increase differentiation of the SCT, and that the spatial localization of specific regulons in different cell types was associated with the pathways related to hormones transport and secretion, regulation of mitotic cell cycle, and nutrient transport pathway in SCT. In EVT, regulatory pathways such as the epithelial to mesenchyme transition, epithelial development and differentiation, and extracellular matrix organization were identified. Finally, viral receptors and drug transporters were identified in villi according to the pathway analysis, which could help to explain the vertical transmission of several infectious diseases and drug metabolism efficacy. Our study provides a valuable resource for further investigation of the placenta development, physiology and pathology in a spatial context.

Purpose: This retrospective cohort study aimed to investigate the value of preimplantation genetic testing for aneuploidy (PGT-A) as a screening test for patients suffered from unexplained recurrent implantation failure (RIF). Methods: After screening patients in one reproductive medicine center, twenty-nine, eighty-seven and thirty-eight women (Results: The live birth rate per transfer was significantly higher in the RIF+PGT-A group than that in the RIF+NO PGT-A group (47.4% vs 31.6%) after adjusting the confounder (the maternal age). After 3 cycles of FET, RIF+PGT-A group had a significantly higher conservative cumulative live birth rate (CLBR) compared to the RIF+PGT-A group (69.0% vs 42.5%, odds ratio [OR] 3.777, p=0.005), but had similar CLBR compared to the NO RIF+PGT-A group (69.0% vs 81.6%). The optimal CLBR in the RIF+PGT-A patients was similar to those in the other 2 groups. The miscarriage rate per clinical pregnancy was not different between the RIF+PGT-A and RIF+NO PGT-A, RIF+PGT-A and NO RIF+PGT-A groups. Conclusion: Although PGT-A could land on the non-beneficial side after three blastocyst FETs, it did be superior in reducing the number of transfer cycles required to achieve a similar live birth rate. Further studies to identify the RIF patients who would benefit most from PGT-A are necessary.

During implantation, a symphony of interaction between the trophoblast originated from the trophectoderm of the implanting blastocyst and the endometrium leads to a successful pregnancy. Defective interaction between the trophoblast and endometrium often results in implantation failure, pregnancy loss, and a number of pregnancy complications. Owing to ethical concerns of using in vivo approaches to study human embryo implantation, various in vitro culture models of endometrium were established in the past decade ranging from two-dimensional cell-based to three-dimensional extracellular matrix (ECM)/tissue-based culture systems. Advanced organoid systems have also been established for recapitulation of different cellular components of the maternal–fetal interface, including the endometrial glandular organoids, trophoblast organoids and blastoids. However, there is no single ideal model to study the whole implantation process leaving more research to be done pursuing the establishment of a comprehensive in vitro model that can recapitulate the biology of trophoblast-endometrium interaction during early pregnancy. This would allow us to have better understanding of the physiological and pathological process of trophoblast-endometrium interaction during implantation.

In-vitro fertilization is an effective treatment for various causes of infertility. However, management of women with poor ovarian response or premature ovarian insufficiency remains challenging because these women have underdeveloped small ovarian follicles that do not respond to hormone treatment. In-vitro activation of small follicles has been developed but its efficiency has much room for improvement. In the current study, we provide several lines of evidence showing that curcumin, an FDA-approved traditional medicine, can specifically promote the development of mouse ovarian follicles from the primary to secondary stage, which greatly potentiates these small follicles for subsequent in-vivo development into antral follicles that can be ovulated. Mechanistically, we show that curcumin promotes the proliferation and differentiation of granulosa cells and the growth of oocytes by activating the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Most importantly, we show that in-vitro treatment of human ovarian tissues with curcumin can promote the in-vivo survival and development of small human ovarian follicles, showing that curcumin can be used as a potential drug to increase the success rate of in-vitro activation of small human follicles. We thus identify curcumin as a novel potential drug for promoting the development of small human ovarian follicles for infertility treatment.

One objective of human genetics is to unveil the variants that contribute to human diseases. With the rapid development and wide use of next-generation sequencing (NGS), massive genomic sequence data have been created, making personal genetic information available. Conventional experimental evidence is critical in establishing the relationship between sequence variants and phenotype but with low efficiency. Due to the lack of comprehensive databases and resources which present clinical and experimental evidence on genotype-phenotype relationship, as well as accumulating variants found from NGS, different computational tools that can predict the impact of the variants on phenotype have been greatly developed to bridge the gap. In this review, we present a brief introduction and discussion about the computational approaches for variant impact prediction. Following an innovative manner, we mainly focus on approaches for non-synonymous variants (nsSNVs) impact prediction and categorize them into six classes. Their underlying rationale and constraints, together with the concerns and remedies raised from comparative studies are discussed. We also present how the predictive approaches employed in different research. Although diverse constraints exist, the computational predictive approaches are indispensable in exploring genotype-phenotype relationship.

Chemerin is an adipokine that regulates metabolism in pregnancy. An elevation of serum chemerin level is associated with pregnancy complications. Consistently, we demonstrated that the chemerin expression was increased in placenta of preeclamptic patients at deliveries. The G protein-coupled receptor chemokine-like receptor 1 (CMKLR1) mediates the actions of chemerin. The functions of the chemerin-CMKLR1 axis in maintaining pregnancy are still unknown. In this study, we demonstrated that CMKLR1 was expressed in the decidual natural killer (dNK) cells and chorionic villi of human. Chemerin suppressed the proliferation of the dNK cells in vitro. Specific antagonist of CMKLR1, α-Neta abolished the suppressive effect of spent medium from chemerin-treated dNK cells culture on extravillous trophoblast invasion. Activation of the chemerin-CMKLR1 axis promoted fusion and differentiation of human cytotrophoblast to syncytiotrophoblast in vitro. We generated Cmklr1 knockout mice and showed that the Cmklr1 deficiency negatively affected pregnancy outcome in terms of number of implantation sites, litter size and fetal weight at birth. Histologically, the Cmklr1 deficiency impaired formation of the syncytiotrophoblast layer II, induced enlargement of the maternal lacunae in the labyrinth, increased the diameter of the spiral arteries and increased trophoblast invasion in the decidua. The Cmklr1 deficient placenta also displayed an increased number of dNK cells and serum IL-15 level. In summary, the chemerin-CMKLR1 axis regulated placental development and spiral artery remodeling in early pregnancy.

Objective Vitamin D receptors are present in the female reproductive tract. Studies on the association between serum vitamin D level and pregnancy rate of in vitro fertilization (IVF) showed inconsistent results and focused on a single fresh or frozen embryo transfer cycle. The objective of our study was to evaluate if serum vitamin D level before ovarian stimulation was associated with the cumulative live birth rate (CLBR) of the first IVF cycle. Design Retrospective cohort study. Methods Women who underwent the first IVF cycle from 2012 to 2016 at a university-affiliated reproductive medicine center were included. Archived serum samples taken before ovarian stimulation were analyzed for 25(OH)D levels using liquid chromatography-mass spectrometry. Results In total, 1113 had pregnancy outcome from the completed IVF cycle. The median age (25th–75th percentile) of the women was 36 (34–38) years and serum 25(OH)D level was 53.4 (41.9–66.6) nmol/L. The prevalence of vitamin D deficiency (less than 50 nmol/L) was 42.2%. The CLBR in the vitamin D-deficient group was significantly lower compared to the non-deficient group (43.9%, 208/474 vs 50.9%, 325/639, P = 0.021, unadjusted), and after controlling for women’s age, BMI, antral follicle count, type and duration of infertility. There were no differences in the clinical/ongoing pregnancy rate, live birth rate and miscarriage rate in the fresh cycle between the vitamin D deficient and non-deficient groups. Conclusions Vitamin D deficiency was prevalent in infertile women in subtropical Hong Kong. The CLBR of the first IVF cycle in the vitamin D-deficient group was significantly lower compared to the non-deficient group.

Mitochondria are highly dynamic organelles and their activity is known to be regulated by changes in morphology via fusion and fission events. However, the role of mitochondrial dynamics on cellular differentiation remains largely unknown. Here, we explored the molecular mechanism of mitochondrial fusion during spermatogenesis by generating an Mfn2 (mitofusin 2) conditional knock-out (cKO) mouse model. We found that depletion of MFN2 in male germ cells led to disrupted spermatogenesis and meiosis during which the majority of Mfn2 cKO spermatocytes did not develop to the pachytene stage. We showed that in these Mfn2 cKO spermatocytes, oxidative phosphorylation in the mitochondria was affected. In addition, RNA-Seq analysis showed that there was a significantly altered transcriptome profile in the Mfn2 deficient pachytene (or pachytene-like) spermatocytes, with a total of 262 genes up-regulated and 728 genes down-regulated, compared with wild-type (control) mice. Pathway enrichment analysis indicated that the peroxisome proliferator-activated receptor (PPAR) pathway was altered, and subsequent more detailed analysis showed that the expression of PPAR α and PPAR γ was up-regulated and down-regulated, respectively, in the MFN2 deficient pachytene (or pachytene-like) spermatocytes. We also demonstrated that there were more lipid droplets in the Mfn2 cKO cells than in the control cells. In conclusion, our study demonstrates a novel finding that MFN2 deficiency negatively affects mitochondrial functions and alters PPAR pathway together with lipid metabolism during spermatogenesis and meiosis.

The decidualization of endometrial stromal cells (ESCs) is an essential process facilitating embryo implantation. However, the roles of non-decidualized and decidualized ESCs in regulating the microenvironment of a receptive endometrium remain unclear. We investigated single-cell transcriptomic changes in the uterus of a CD-1 mouse model at the post-implantation stage. The implantation and inter-implantation sites of the uteruses of pregnant mice at 4.5 and 5.5 days post-coitum were dissected for single-cell RNA sequencing. We identified eight cell types: epithelial cells, stromal cells, endothelial cells, mesothelial cells, lymphocytes, myocytes, myeloids, and pericytes. The ESC transcriptome suggests that the four ESC subtypes are involved in the extracellular remodeling during implantation. The trajectory plot of ESC subtypes indicates embryo implantation that involves a differentiation pathway from undifferentiated ESCs (ESC 1) to decidualized ESCs (DEC ESCs), with distinct signaling pathways between the ESC subtypes. Furthermore, the ligand-receptor analysis suggests that ESCs communicate with epithelial cells and immune cells through nectin and ICAM signaling. Collectively, both decidualized and non-decidualized ESCs may regulate the endometrial microenvironment for optimal endometrial receptivity and immune tolerance. This study provides insights on the molecular and cellular characteristics of mouse ESCs in modulating the epithelial and lymphocyte functions during early embryo implantation.

Sperm selection in the female reproductive tract (FRT) is sophisticated. Only about 1,000 sperm out of millions in an ejaculate reach the fallopian tube and thus have a chance of fertilizing an oocyte. In assisted reproduction techniques, sperm are usually selected using their density or motility, characteristics that do not reflect their fertilization competence and, therefore, might result in failure to fertilize the oocyte. Although sperm processing in in vitro fertilization (IVF) and intrauterine insemination (IUI) bypasses many of the selection processes in the FRT, selection by the cumulus mass and the zona pellucida remain intact. By contrast, the direct injection of a sperm into an oocyte in intracytoplasmic sperm injection (ICSI) bypasses all natural selection barriers and, therefore, increases the risk of transferring paternal defects such as fragmented DNA and genomic abnormalities in sperm to the resulting child. Research into surrogate markers of fertilization potential and into simulating the natural sperm selection processes has progressed. However, methods of sperm isolation - such as hyaluronic acid-based selection and microfluidic isolation based on sperm tactic responses - use only one or two parameters and are not comparable with the multistep sperm selection processes naturally occurring within the FRT. Fertilization-competent sperm require a panel of molecules, including zona pellucida-binding proteins and ion channel proteins, that enable them to progress through the FRT to achieve fertilization. The optimal artificial sperm selection method will, therefore, probably need to use a multiparameter tool that incorporates the molecular signature of sperm with high fertilization potential, and their responses to external cues, within a microfluidic system that can replicate the physiological processes of the FRT in vitro.

Capacitated spermatozoa initiate fertilization by binding to the zona pellucida (ZP). Defective spermatozoa-ZP binding causes infertility. The sialyl-Lewis(x) (SLeX) sequence is the most abundant terminal sequence on the glycans of human ZP glycoproteins involving in spermatozoa-ZP binding. This study aimed to identify and characterize the SLeX-binding proteins on human spermatozoa. By using affinity chromatography followed by mass spectrometric analysis, chromosome 1 open reading frame 56 (C1orf56) was identified to be a SLeX-binding protein of capacitated spermatozoa. The acrosomal region of spermatozoa possessed C1orf56 immunoreactive signals with intensities that increased after capacitation indicating translocation of C1orf56 to the cell surface during capacitation. Treatment with antibody against C1orf56 inhibited spermatozoa-ZP binding and ZP-induced acrosome reaction. Purified C1orf56 from capacitated spermatozoa bound to human ZP. A pilot clinical study was conducted and found no association between the percentage of capacitated spermatozoa with C1orf56 expression and in vitro fertilization (IVF) rate in assisted reproduction treatment. However, the percentage of C1orf56 positive spermatozoa in the acrosome-reacted population was significantly (P < 0.05) lower in cycles with a fertilization rate < 60% when compared to those with a higher fertilization rate, suggesting that C1orf56 may have functions after ZP-binding and acrosome reaction. A larger clinical trial is needed to determine the possible use of sperm C1orf56 content for the prediction of fertilization potential of sperm samples.

Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140bCD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear.In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140bTranscriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells.In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b

Decidual macrophages constitute 20-30% of the total leukocytes in the uterus of pregnant women, regulating the maternal immune tolerance and placenta development. Abnormal number or activities of decidual macrophages (dMs) are associated with fetal loss and pregnancy complications, such as preeclampsia. Monocytes differentiate into dMs in a decidua-specific microenvironment. Despite their important roles in pregnancy, the exact factors that regulate the differentiation into dMs remain unclear. Glycodelin-A (PAEP, hereafter referred to as GdA) is a glycoprotein that is abundantly present in the decidua, and plays an important role in fetomaternal defense and placental development. It modulates the differentiation and activity of several immune cell types residing in the decidua. In this study, we demonstrated that GdA induces the differentiation of human monocytes into dM-like phenotypes in terms of transcriptome, cell surface marker expression, secretome, and regulation of trophoblast and endothelial cell functions. We found that Sialic acid-binding Ig-like lectin 7 (Siglec-7) mediates the binding and biological actions of GdA in a sialic acid-dependent manner. We, therefore, suggest that GdA, induces the polarization of monocytes into dMs to regulate fetomaternal tolerance and placental development.

Cilium and flagellum beating are important in reproduction and defects in their motion are associated with ectopic pregnancy and infertility. Adrenomedullin (ADM) is a polypeptide present in the reproductive system. This report demonstrates a novel action of ADM in enhancing the flagellar/ciliary beating of human spermatozoa and rat oviductal ciliated cells. At the concentration found in the seminal plasma, it increases the progressive motility of spermatozoa. ADM binds to its classical receptor, calcitonin receptor-like receptor/receptor activity-modifying protein complex on spermatozoa. ADM treatment increases the protein kinase A activities, the cyclic adenosine monophosphate, and nitric oxide levels of spermatozoa and oviductal cells. Pharmacological activators and inhibitors confirmed that the ADM-induced flagella/ciliary beating was protein kinase A dependent. Whereas nitric oxide donors had no effect on sperm motility, they potentiated the motility-inducing action of protein kinase A activators, demonstrating for the first time the synergistic action of nitric oxide and protein kinase A signaling in flagellar/ciliary beating. The ADM-induced motility enhancement effect in spermatozoa also depended on the up-regulation of intracellular calcium, a known key regulator of sperm motility and ciliary beating. In conclusion, ADM is a common activator of flagellar/ciliary beating. The study provides a physiological basis on possible use of ADM as a fertility regulation drug.

Successful embryo implantation requires a synchronized dialogue between a competent blastocyst and the receptive endometrium, which occurs in a limited time period known as the "window of implantation." Recent studies suggested that down-regulation of olfactomedin 1 (OLFM1) in the endometrium and fallopian tube is associated with receptive endometrium and tubal ectopic pregnancy in humans. Interestingly, the human chorionic gonadotropin (hCG) induces miR-212 expression, which modulates OLFM1 and C-terminal binding protein 1 (CTBP1) expressions in mouse granulosa cells. Therefore, we hypothesized that embryo-derived hCG would increase miR-212 expression and down-regulate OLFM1 and CTBP1 expressions to favor embryo attachment onto the female reproductive tract. We found that hCG stimulated the expression of miR-212 and down-regulated OLFM1 but not CTBP1 mRNA in both human endometrial (Ishikawa) and fallopian (OE-E6/E7) epithelial cells. However, hCG suppressed the expression of OLFM1 and CTBP1 proteins in both cell lines. The 3'UTR of both OLFM1 and CTBP1 contained binding sites for miR-212. The miR-212 precursor suppressed luciferase expression, whereas the miR-212 inhibitor stimulated luciferase expression of the wild-type (WT)-OLFM1 and WT-CTBP1 reporter constructs. Furthermore, hCG (25 IU/ml) treatments stimulated trophoblastic (Jeg-3) spheroid (blastocyst surrogate) attachment onto Ishikawa and OE-E6/E7 cells. Transfection of miR-212 precursor increased Jeg-3 spheroid attachment onto Ishikawa cells and decreased OLFM1 and CTBP1 protein expressions, whereas the opposite occurred with miR-212 inhibitor. Taken together, hCG stimulated miR-212, which in turn down-regulated OLFM1 and CTBP1 expression in fallopian and endometrial epithelial cells to favor spheroid attachment.

Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is an important process during placentation. Dysregulation of the process is associated with a wide range of pregnancy complications. Adrenomedullin (ADM) is a polypeptide expressed most abundantly in first-trimester placentas. We hypothesized that ADM modulated the invasion of human EVCT. Our results showed that ADM enhanced invasion and migration but not proliferation in two EVCT cell lines, JEG-3 and TEV-1. Similar observation can also be obtained in primary EVCTs. JEG-3 and TEV-1 cells expressed ADM receptor components as demonstrated by immunostaining, Western blotting, and RT-PCR. The ADM antagonist ADM(22-52) (ADM C-terminal 22-52 amino acid fragment) suppressed ADM-induced invasion and migration, confirming that ADM exerted its biological effects through its classical receptors. The stimulatory effect of ADM on EVCT invasiveness was associated with induction (P0.05) of urokinase plasminogen activator (uPA) and nitric oxide synthase (NOS) expression and activity. Silencing of uPA by siRNA transfection abolished the stimulatory effect of ADM, suggesting that uPA is the key mediator for ADM-induced invasion. The involvement of NO in enhancing the invasion and biosynthesis of uPA in EVCT cell lines was confirmed by using pharmacological inhibitors of NOS and NO donors. ADM-mediated NO production also increased protein S-nitrosylation of JEG-3 cells. S-nitrosylation activated uPA in vitro and induced a higher proteinase activity. These findings provide indications that ADM and its downstream NO signaling may play an important role in modulating human EVCT functions.

The female reproductive system is a complex system. Epithelia of the female reproductive system including the ovaries, the oviduct, and the uterus are important sites for follicular development, ovulation, fertilization, implantation, and embryo development. They are also able to synthesize and secrete various hormones, growth factors, and cytokines, which are essential to women's health, sexuality, and reproduction. Conversely, their dysfunction has been implicated in disorders such as infertility, endometriosis, and many other gynecological diseases, as well as cancer. In this chapter, we describe detailed procedures for establishing and maintaining primary cultures of human ovarian surface epithelium, oviductal epithelium, and endometrium. We also provide protocols for cell immortalization, clonal isolation, and in coculture with stromal cells. These cultures can be useful models for investigating the molecular and cellular functions of these epithelia in both normal and pathological states.

OBJECTIVE Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities. RESEARCH DESIGN AND METHODS GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured. RESULTS GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6–sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA. CONCLUSIONS Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.

To review the outcomes of frozen-thawed embryo transfer cycles. DESIGN. Retrospective review.Tertiary assisted reproduction centre, Hong Kong.Subfertile patients undergoing frozen-thawed embryo transfer between July 2005 and December 2007.Clinical and ongoing pregnancy rates.A total of 983 frozen-thawed embryo transfer cycles performed during the study period were reviewed. The clinical pregnancy and ongoing pregnancy rates were 35% and 30%, respectively. Factors associated with successful outcome included younger maternal age (=35 years) and 4 or more blastomeres at replacement, but not the method of insemination, the cause of subfertility, or the type of frozen-thawed embryo transfer cycle. The overall multiple pregnancy rate was 18%. For cycles with a single embryo replaced, embryos having 4-cell or higher stages at replacement gave an ongoing pregnancy rate of 25%, whereas those with less than 4 cells had a significantly lower ongoing pregnancy rate of 5% only. Blastomere lysis after thawing significantly reduced the clinical pregnancy and ongoing pregnancy rates of cycles with one embryo replaced.Clinical pregnancy and ongoing pregnancy rates of frozen-thawed embryo transfer cycles were 35% and 30%, respectively. Higher pregnancy rates were associated with younger maternal age (=35 years), blastomere numbers of 4 or more, and no blastomere lysis after thawing.

Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.

Somatic cell-embryo coculture enhances embryo development in vitro by producing embryotrophic factor(s) and/or removing harmful substances from the culture environment. Yet, the underlying molecular mechanisms on how somatic cells remove the toxicants from the culture medium remain largely unknown. By using suppression subtractive hybridization, we identified a number of mouse oviductal genes that were up-regulated when developing preimplantation embryos were present in the oviduct. Epoxide hydrolase 1, microsomal (Ephx1 previously known as mEH) was one of these genes. EPHX1 detoxifies genotoxic compounds and participates in the removal of reactive oxygen species (ROS). The transcript of Ephx1 increases in the oviductal epithelium at the estrus stage and in Day 3 of pregnancy as well as in the uterus of ovariectomized mice injected with estrogen or progesterone. Human oviductal epithelial cells OE-E6/E7 express EPHX1 and improve mouse embryo development in vitro. Addition of an EPHX1 inhibitor, cyclohexene oxide (CHO) or 1,1,1-trichloropropene 2,3-oxide (TCPO), to the culture medium increased intracellular and extracellular ROS levels of OE-E6/E7 cells and suppressed the beneficial effect of the cells on embryo development; CHO and TCPO at these concentrations had no adverse effect on OE-E6/E7 growth and embryo development in vitro. Taken together, EPHX1 in oviductal cells may enhance the development of cocultured embryos by protecting them from oxidative stress. Our result supports the notion that somatic cell coculture may enhance embryo development via removal of deleterious substances in the culture medium.

To report the type and frequency of chromosomal anomalies and Y-microdeletions among Hong Kong Chinese subfertile men with sperm concentrations lower than 5 million/mL. DESIGN. Retrospective study.A reproductive centre in Hong Kong.A total of 295 Chinese subfertile men who underwent both karyotyping and Y-microdeletion studies from 2000 to 2007 were categorised as having non-obstructive azoospermia (n=71), very severe oligospermia (sperm concentration0 andor=2 million/mL, n=158), and severe oligospermia (sperm concentration2 and5 million/mL, n=66).Karyotyping and Y-microdeletion studies.The prevalence of chromosomal anomalies and Y-microdeletions in the study population were 8.5% (25/295; 95% confidence interval, 5.6-12.3%) and 6.4% (19/295; 3.9-9.9%), respectively. The total prevalence of chromosomal anomalies and Y-microdeletions was 13.2% (39/295; 95% confidence interval, 9.6-17.6%) as five cases of non-obstructive azoospermia showed both Y structural alterations and AZFbc deletion. The corresponding figures for chromosomal anomalies in the groups with non-obstructive azoospermia, very severe oligospermia, and severe oligospermia were 21.1% (15/71; 95% confidence interval, 12.3-32.4%), 5.7% (9/158; 2.6-10.5%), and 1.5% (1/66; 0.0-8.2%). While for Y-microdeletions they were 8.5% (6/71; 3.2-17.5%), 8.2% (13/158; 4.5-13.7%) and 0% (0/66; 0.0-4.4%), respectively. The respective overall prevalence rates for chromosomal anomalies and Y-microdeletions in these groups were: 22.5% (16/71; 13.5-34.0%), 13.9% (22/158; 8.9-20.3%), and 1.5% (1/66; 0.0-8.2%).Our findings strongly support the recommendation for both karyotyping and Y-microdeletion analyses in subfertile men with sperm concentrations of 2 million/mL or lower before they undergo assisted reproduction treatment.

Heterotrimeric G proteins play a key role in membrane-mediated cell-signalling and hormonal regulation. Our earlier studies gave evidence of G protein subunit Galpha(i2) being under hormonal regulation in human in vivo. In this study, we used immortalized human oviduct epithelial cell line OE-E6/E7 as a model to study the hormonal regulation of Galpha(i2). We aimed at clarifying whether estradiol or progesterone could individually regulate the expression of Galpha(i2) and its potential signalling partners. Furthermore, we aimed to investigate which sex hormone receptors could potentially mediate the gene regulation in OE-E6/E7 cell line. OE-E6/E7 cells were cultured for 5 days with different concentrations of estradiol or progesterone. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using cDNA of the hormone-treated cells to reveal any changes in gene expression. The presence of potential receptor targets in these cells was studied using PCR. Our data clearly showed that low concentrations of estradiol up-regulated the expression of Galpha(i2) gene and down-regulated the expression of membrane progesterone receptor mPRalpha gene in OE-E6/E7 cell line. Progesterone had no significant effect on Galpha(i2) gene expression, but it caused up-regulation of mPRalpha gene expression. In conclusion, it appears that sex hormones regulate the expression of Galpha(i2) and mPRalpha genes in a reverse manner in OE-E6/E7 cells. Our results suggest that estrogen receptor ERbeta mediates the regulatory effects of estradiol in these cells.

Glycodelin is a glycoprotein with three isoforms, namely glycodelin-S, glycodelin-A and glycodelin-F. They have similar protein core but different glycan side chains. Spermatozoa are exposed to these isoforms during their passage to the fertilization site. They first encounter glycodelin-S in the seminal plasma. Data suggest that glycodelin-S suppresses albumin-induced cholesterol loss and maintains the spermatozoa in an uncapacitated state before they enter into the cervical canal where glycodelin-S is removed. This allows albumin in the uterine cavity to initiate capacitation. In the fallopian tube, the spermatozoa are exposed to glycodelin-A and -F produced by the fallopian tube. Glycodelin-A is an endogenous glycoprotein that inhibits the binding of spermatozoa to the zona pellucida. Glycodelin-A may protect the spermatozoa from maternal immune attack by its immunosuppressive activity. Glycodelin-F is the main glycodelin isoform in the follicular fluid. Similar to glycodelin-A, it inhibits spermatozoa-zona pellucida binding. The biological significance of the anti-fertilization activity of glycodelin-A and -F remains to be established. Glycodelin-F also suppresses progesterone-induced acrosome reaction. This is important to prevent premature acrosome reaction when the spermatozoa are swimming through the cumulus mass towards the oocyte and become exposed to progesterone produced by the cumulus cells. In summary, different isoforms of glycodelin act in succession to modulate different aspects of sperm function, and thereby, contribute to the success of fertilization.

To report our experience in preimplantation genetic diagnosis (PGD) for the exclusion of homozygous alpha degrees thalassemia.PGD was performed on nine couples with alpha degrees thalassemia genotype undergoing assisted reproduction. Oocytes were aspirated after ovarian stimulation and fertilized by intracytoplasmic sperm injection. One or two blastomeres were biopsied from the six- to eight-cell embryo. Single cell multiplex PCR of the normal and alpha degrees thalassemia alleles was performed for first round, followed by semi-nested PCR of the respective alleles using 5'-end labelled fluorescent primers. Only those embryos with a blastomere diagnosed as having at least one normal allele were selected for transfer.One hundred and twenty-six blastomeres from 82 embryos were analyzed. The rates of allele dropout was 10.2% and PCR failure 12.7%. Fifty-eight embryos (70.7%) had at least one normal allele, of which 31 were transferred to 13 prepared cycles and one triplet pregnancy achieved. The triplets showed no ultrasound features of homozygous alpha degrees thalassemia at 18 weeks and were delivered in healthy condition by caesarean section at 34 weeks. Their genotypes were confirmed by cord blood analysis.PGD for alpha degrees thalassemia is possible by single cell PCR. The transfer and successful implantation of unaffected embryos ensure birth of disease-free babies.

Spermatogenesis is a complicated process of germ cell differentiation, involving programmatic expression of diverse cell type- and developmental stage-specific genes. To date, the vitamin-A-deficiency (VAD) rats and postnatal rats are two models commonly used to study spermatogenesis. In the present study, we studied the expression of 1185 known genes in the vitamin-A-deficient and retinol-reinitiated spermatogenesis of rat testis using Clontech Atlas rat cDNA expression arrays. The mRNA expression patterns of post-vitamin-A (PVA) testis on Days 15 and 35 were compared with those of the spermatogenic arrested rat testis on Day 0. About 9% (110/1185) of the genes studied were highly expressed. When compared with VAD rat testis on Day 0, 20 and 31 genes were differentially expressed by a factor of twofold or greater on Days 15 and 35, respectively. Four genes (cytochrome P450 17, sulfated glycoprotein 2, protein kinase inhibitor, and cathepsin L) that play important roles in spermatogenesis were selected and their gene expression patterns were confirmed by semiquantitative reverse transcription-polymerase chain reaction. Comparison of the expression patterns of these genes between PVA-VAD and postnatal rat testis in developmentally matched stages revealed substantial differences during the early stages of spermatogenesis. This discrepancy could be caused by either the presence of arrested but mature somatic cells in the PVA-VAD testis that may contribute to a unique gene expression pattern in this model or the direct effect of retinol on spermatogenesis. Therefore, caution is needed in interpreting the gene expression data using the PVA-VAD and postnatal rat models in studying spermatogenesis in rat testes.

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.