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Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

To determine the importance of testis-specific, Y-encoded-like 1 (TSPYL1) in survival and male factor fertility in mice.

Ectopic pregnancy is the major cause of maternal morbidity and mortality in the first trimester of pregnancy. Tubal ectopic pregnancy (TEP) accounts for nearly 98% of all ectopic pregnancies. TEP is usually associated with salpingitis but the underlying mechanism in salpingitis leading to TEP remains unclear. Adrenomedullin (ADM) is a peptide hormone abundantly expressed in the fallopian tube with potent anti-inflammatory activities. Its expression peaks at the early luteal phase when the developing embryo is being transported through the fallopian tube. In the present study, we demonstrated reduced expression of ADM in fallopian tubes of patients with salpingitis and TEP. Using macrophages isolated from the fallopian tubes of these women, our data revealed that the salpingistis-associated ADM reduction contributed to aggravated pro-inflammatory responses of the tubal macrophages resulting in production of pro-inflammatory and pro-implantation cytokines IL-6 and IL-8. These cytokines activated the expression of implantation-associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. In conclusion, this study provided evidence for the role of ADM in the pathogenesis of TEP through regulating the functions of tubal macrophages.

Ectopic pregnancy is the major cause of maternal morbidity and mortality in the first trimester of pregnancy. Tubal ectopic pregnancy (TEP) accounts for nearly 98% of all ectopic pregnancies. TEP is usually associated with salpingitis but the underlying mechanism in salpingitis leading to TEP remains unclear. Adrenomedullin (ADM) is a peptide hormone abundantly expressed in the fallopian tube with potent anti-inflammatory activities. Its expression peaks at the early luteal phase when the developing embryo is being transported through the fallopian tube. In the present study, we demonstrated reduced expression of ADM in fallopian tubes of patients with salpingitis and TEP. Using macrophages isolated from the fallopian tubes of these women, our data revealed that the salpingistis-associated ADM reduction contributed to aggravated pro-inflammatory responses of the tubal macrophages resulting in production of pro-inflammatory and pro-implantation cytokines IL-6 and IL-8. These cytokines activated the expression of implantation-associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. In conclusion, this study provided evidence for the role of ADM in the pathogenesis of TEP through regulating the functions of tubal macrophages.

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

Human endometrium undergoes cycles of proliferation and differentiation throughout the reproductive years of women. The endometrial stem/progenitor cells contribute to this regenerative process. They lie in the basalis layer of the endometrium next to the myometrium. We hypothesized that human myometrial cells provide niche signals regulating the activities of endometrial mesenchymal stem‐like cells (eMSCs). In vitro coculture of myometrial cells enhanced the colony‐forming and self‐renewal ability of eMSCs. The cocultured eMSCs retained their multipotent characteristic and exhibited a greater total cell output when compared with medium alone culture. The expression of active β‐catenin in eMSCs increased significantly after coculture with myometrial cells, suggesting activation of WNT/β‐catenin signaling. Secretory factors in spent medium from myometrial cell culture produced the same stimulatory effects on eMSCs. The involvement of WNT/β‐catenin signaling in self‐renewal of eMSCs was confirmed with the use of WNT activator (Wnt3A conditioned medium) and WNT inhibitors (XAV939 and inhibitor of Wnt Production‐2 [IWP‐2]). The myometrial cells expressed more WNT5A than other WNT ligands. Recombinant WNT5A stimulated whereas anti‐WNT5A antibody suppressed the colony formation, self‐renewal, and T‐cell factor/lymphoid enhancer‐binding factor (TCF/LEF) transcriptional activities of eMSCs. Moreover, eMSCs expressed FZD4 and LRP5. WNT5A is known to activate the canonical WNT signaling in the presence of these receptor components. WNT antagonist, DKK1, binds to LRP5/6. Consistently, DKK1 treatment nullified the stimulatory effect of myometrial cell coculture. In conclusion, our findings show that the myometrial cells are niche components of eMSCs, modulating the self‐renewal activity of eMSCs by WNT5A‐dependent activation of WNT/β‐catenin signaling. Stem Cells2019;37:1455–1466 Myometrial cells are niche cells of endometrial mesenchymal stem cells. The myometrial cells secrete WNT5A that interacts with WNT receptors FZD4 and LRP5, resulting in activation of the WNT/β‐catenin signaling, enhancement of the self‐renewal and proliferation activities of the endometrial mesenchymal stem cells. Thus, WNT5A plays a pivotal role in expansion and repopulation of stem/progenitor cells in human endometrium.

Shallow extravillous trophoblast (EVT) invasion is central to the pathophysiology of many pregnancy complications. Invasion is mediated partially by matrix metalloproteinases (MMPs). MMP-2 is highly expressed in early pregnancy. MMP activity can be regulated by proinflammatory cytokines, which also induce endoplasmic reticulum (ER) stress in other cells. We investigated whether proinflammatory cytokines regulate MMP-2 activity through ER stress response pathways in trophoblast before exploring potential regulatory mechanisms. There was increased immunoreactivity of heat shock 70-kDa protein 5, also known as 78-kDa glucose regulated protein, in cells of the placental bed, including EVTs, in cases of early-onset preeclampsia compared with normotensive controls. Treating EVT-like JEG-3 and HTR8/SVneo cells with ER stress inducers (tunicamycin and thapsigargin) suppressed MMP2mRNA and protein expression, secretion, and activity and reduced their invasiveness. A cocktail of proinflammatory cytokines (IL-1β, tumor necrosis factor-α, and interferon-γ) suppressed MMP-2 activity in JEG-3 cells and was accompanied by activation of the PKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2A (EIF2A) arm of the ER stress pathway. Knockdown of ATF4, a downstream transcriptional factor of the PERK-EIF2A pathway, by small interference RNA, restored MMP2expression but not cellular proteins. However, suppression of EIF2A phosphorylation with a PERK inhibitor, GSK2606414, under ER stress, restored MMP-2 protein. ER stress regulates MMP-2 expression at both the transcriptional and translational levels. This study provides the first mechanistic linkage by which proinflammatory cytokines may modulate trophoblast invasion through ER stress pathways.

Successful placentation requires delicate communication between the endometrium and trophoblasts. The invasion and integration of trophoblasts into the endometrium during early pregnancy are crucial to placentation. Dysregulation of these functions is associated with various pregnancy complications, such as miscarriage and preeclampsia. The endometrial microenvironment has an important influence on trophoblast cell functions. The precise effect of the endometrial gland secretome on trophoblast functions remains uncertain. We hypothesized that the hormonal environment regulates the miRNA profile and secretome of the human endometrial gland, which subsequently modulates trophoblast functions during early pregnancy. Human endometrial tissues were obtained from endometrial biopsies with written consent. Endometrial organoids were established in matrix gel under defined culture conditions. They were treated with hormones mimicking the environment of the proliferative phase (Estrogen, E2), secretory phase (E2+Progesterone, P4), and early pregnancy (E2+P4+Human Chorionic Gonadotropin, hCG). miRNA-seq was performed on the treated organoids. Organoid secretions were also collected for mass spectrometric analysis. The viability and invasion/migration of the trophoblasts after treatment with the organoid secretome were determined by cytotoxicity assay and transwell assay, respectively. Endometrial organoids with the ability to respond to sex steroid hormones were successfully developed from human endometrial glands. By establishing the first secretome profiles and miRNA atlas of these endometrial organoids to the hormonal changes followed by trophoblast functional assays, we demonstrated that sex steroid hormones modulate aquaporin (AQP)1/9 and S100A9 secretions through miR-3194 activation in endometrial epithelial cells, which in turn enhanced trophoblast migration and invasion during early pregnancy. By using a human endometrial organoid model, we demonstrated for the first time that the hormonal regulation of the endometrial gland secretome is crucial to regulating the functions of human trophoblasts during early pregnancy. The study provides the basis for understanding the regulation of early placental development in humans.

Direct in vivoinvestigation of human placenta trophoblast’s susceptibility to SARS-CoV-2 is challenging. Here we report that human trophoblast stem cells (hTSCs) and their derivatives are susceptible to SARS-CoV-2 infection, which reveals heterogeneity in hTSC cultures. Early syncytiotrophoblasts (eSTBs) generated from hTSCs have enriched transcriptomic features of peri-implantation trophoblasts, express high levels of angiotensin-converting enzyme 2 (ACE2), and are productively infected by SARS-CoV-2 and its Delta and Omicron variants to produce virions. Antiviral drugs suppress SARS-CoV-2 replication in eSTBs and antagonize the virus-induced blockage of STB maturation. Although less susceptible to SARS-CoV-2 infection, trophoblast organoids originating from hTSCs show detectable viral replication reminiscent of the uncommon placental infection. These findings implicate possible risk of COVID-19 infection in peri-implantation embryos, which may go unnoticed. Stem cell-derived human trophoblasts such as eSTBs can potentially provide unlimited amounts of normal and genome-edited cells and facilitate coronavirus research and antiviral discovery.

Intestinal adaptation from parenteral to enteral nutrition is crucial for survival and growth of newborns. Rapid feeding-induced gut maturation occurs immediately after birth in both preterm and term neonates, but it remains unclear whether the responses depend on factors related to birth transition (e.g. bacterial colonization, endocrine, and metabolic changes). We hypothesized that enteral feeding matures the immature intestine, even in fetuses before birth. Hence, control pig fetuses were compared with fetuses fed with milk formula for 24 h in utero. Gel-based proteomics showed that feeding-induced changes in 38 proteins, along with marked increases in intestinal mass and changes in activities of brush border enzymes. Physiological functions of the identified proteins were related to enterocyte apoptosis (e.g. caspase 1) and nutrient metabolism (e.g. citric acid cycle proteins). Many of the differentiated proteins were similar to those identified previously in preterm pigs fed with the same formula after birth, except that effects on proteins related to inflammatory lesions (e.g. heat shock proteins) were absent. Our results show that enteral feeding, independently of the birth transition, induces marked gut maturation and proteome change in the immature intestine. Hence, immediate postnatal feeding-induced gut changes are largely independent of factors related to the birth transition.

Intestinal adaptation from parenteral to enteral nutrition is crucial for survival and growth of newborns. Rapid feeding-induced gut maturation occurs immediately after birth in both preterm and term neonates, but it remains unclear whether the responses depend on factors related to birth transition (e.g.bacterial colonization, endocrine, and metabolic changes). We hypothesized that enteral feeding matures the immature intestine, even in fetuses before birth. Hence, control pig fetuses were compared with fetuses fed with milk formula for 24 h in utero. Gel-based proteomics showed that feeding-induced changes in 38 proteins, along with marked increases in intestinal mass and changes in activities of brush border enzymes. Physiological functions of the identified proteins were related to enterocyte apoptosis (e.g.caspase 1) and nutrient metabolism (e.g.citric acid cycle proteins). Many of the differentiated proteins were similar to those identified previously in preterm pigs fed with the same formula after birth, except that effects on proteins related to inflammatory lesions (e.g.heat shock proteins) were absent. Our results show that enteral feeding, independently of the birth transition, induces marked gut maturation and proteome change in the immature intestine. Hence, immediate postnatal feeding-induced gut changes are largely independent of factors related to the birth transition.

Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance of various degrees that is first detected during pregnancy and disappeared after pregnancy. It is the most common metabolic disorder of pregnancy, occurring in up to 13% and 7% of the pregnancies in the local and United States population, respectively. Women who developed GDM had experienced abnormally high fetal mortality and other risk such as spontaneous abortion and placental alternation. The abnormal carbohydrate metabolism may contribute to the pathology of diabetic complications. Glycodelin-A (GdA) is a glycoprotein belonging to the lipocalin family with contraceptive, immunosuppressive and morphogenic properties. GdA is well accepted to be involved in feto-maternal defense as it is abundant in the decidua and modulate the activities of T cell, B cell and NK cell. Since the glycosylation of GdA is essential for its immunosuppressive activities, we hypothesis that gestational diabetic pregnancy may alters the glycosylation of GdA, and thereby leading to defective immunosuppression. In this study, GdA in amniotic fluid from women having normal glucose tolerance (N.GdA) or GDM (D.GdA) were purified with our established protocol. Our results showed that N.GdA and D.GdA shared the same protein core but with different glycosylation as demonstrated by their differential lectin binding patterns. In addition, D.GdA has lower ability in suppressing the proliferation of Jurkat cells and peripheral blood mononuclear cells (PBMCs) as compared to N.GdA. This reduced inhibitory effect on lymphocytes by D.GdA was associated with impaired ability to induce cell death and to inhibit IL-2 secretion. Taken together, our data suggest that the immunosuppressive activities of D.GdA on lymphocytes were impaired as compared to N.GdA due to change in glycosylation. Further investigation of the type and quantity of a particular GdA-derived glycan which affecting its biological activity will enhance our understanding of the cause of pregnancy loss in diabetic mother, and provide the basis for the development of new treatment strategies.

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 ADM22–52(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 (P< 0.05) 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.

Macrophages contribute to 20-30% of the overall population of leukocytes at the maternal-fetal interface. They are in close proximity with the trophoblast which take part in remodeling of placental tissue, clearing apoptotic cells and controlling trophoblast functions. Glycodelin isoforms comprise a group of secretory glycoproteins with four well defined glycoforms, each with a distinct glycosylation pattern. Glycodelin-A (GdA) is a uterine isoform with glycosylation-dependent immunomodulatory activities that contributes to feto-maternal defense. It is abundantly synthesized in the decidua during the first trimester of pregnancy. In the present study, we hypothesized that GdA may exert immunoregulatory activities on monocyte/macrophage and promotes their development into unique phenotypes for maintaining pregnancy. Monocytic cell line THP-1 and monocytes extracted from human female peripheral blood were used as study model. Macrophages were obtained from peripheral blood or by in vitro differentiation of the monocytes using granulocyte-macrophage colony-stimulating factor. Our results showed that GdA did not affect the proliferation and viability of monocytes and macrophages. GdA was demonstrated to up-regulate the interleukin (IL)-6 secretions in macrophage and monocyte as determined by ELISA and flow cytometry. Suppression of extracellular signal-regulated kinases (ERK) signaling abolished the stimulatory effect of GdA, indicating that GdA increased IL-6 production in monocytes/macrophages via the ERK activation. Interestingly, it has been reported that IL-6 is essential for the process of implantation, trophoblast invasion and embryo development. The inclusion of GdA also increased the indoleamine 2,3-dioxyenase (IDO) expression of macrophage, which subsequently inhibited the lymphocyte proliferation in a co-culture study. IDO is a marker of decidual macrophage with immunosuppressive activity through depriving the access to tryptophan by immune cells. Taken together, the present data propose a novel role of GdA in modulating the cytokine profile and development of monocytes/macrophages in order to facilitate human pregnancy. This work was supported in part by grants from the Small Project Funding, The University of Hong Kong, Hong Kong (ref: 201007176033).(poster)

The development of the placenta involves a series of complex, coordinated interactions between the fetal trophoblast cells and the maternal decidual cells. The invasion of the trophoblasts into the endometrium is crucial to placentation. The key cell in human placentation is the cytotrophoblast. After nidation, the cytotrophoblast differentiates into the villous cytotrophoblasts and the extravillous cytotrophoblasts (EVCTs). The invasive EVCTs appear as migratory cell columns invading through the endometrium. One of the invasive phenotypes of EVCTs is increased production of a set of extracellular matrix-degrading enzymes. Matrix metalloproteinases (MMP)-2 and MMP-9 are the most studied matrix-degrading enzymes in placental invasion and their proteinase activities are balanced by the tissue specific inhibitors of the MMPs (TIMP)-1 and TIMP-2. Glycodelin-A (GdA), a glycoprotein synthesized by the endometrial glands but not the trophoblast, is upregulated during first trimester which coincides with the time of trophoblast invasion. We hypothesize that glycodelin-A is a paracrine regulator generated by the deciduas that fine-tune the invasion process of human trophoblast. Trans-well invasion assay was used to examine the effect of GdA on the invasiveness of the immortalized first trimester extravillous cytotrophoblast (TEV-1) cell line. MMP2/MMP9 and TIMP1/TIMP2 secretions and activities were determined by ELISA and gelatin zymographic analysis respectively. Our results show that GdA significantly reduced the invasiveness and MMP2 secretion while increased the activities of TIMP-1 and TIMP-2 of TEV-1. No significant effect on MMP9 secretion was observed. Taken together, GdA may produce an inhibitory effect on trophoblast invasion by regulating the MMP pathways. Further works are required to characterize the biological role of GdA in trophoblast invasion.