A new osteogenic protein isolated from Dioscorea opposita Thunb accelerates bone defect healing through the mTOR signaling axis.

Delayed bone defect repairs lead to severe health and socioeconomic impacts on patients. Hence, there are increasing demands for medical interventions to promote bone defect healing. Recombinant proteins such as BMP-2 have been recognized as one of the powerful osteogenic substances that promote mesenchymal stem cells (MSCs) to osteoblast differentiation and are widely applied clinically for bone defect repairs. However, recent reports show that BMP-2 treatment has been associated with clinical adverse side effects such as ectopic bone formation, osteolysis and stimulation of inflammation. Here, we have identified one new osteogenic protein, named 'HKUOT-S2' protein, from Dioscorea opposita Thunb. Using the bone defect model, we have shown that the HKUOT-S2 protein can accelerate bone defect repair by activating the mTOR signaling axis of MSCs-derived osteoblasts and increasing osteoblastic biomineralization. The HKUOT-S2 protein can also modulate the transcriptomic changes of macrophages, stem cells, and osteoblasts, thereby enhancing the crosstalk between the polarized macrophages and MSCs-osteoblast differentiation to facilitate osteogenesis. Furthermore, this protein had no toxic effects in vivo. We have also identified HKUOT-S2 peptide sequence TKSSLPGQTK as a functional osteogenic unit that can promote osteoblast differentiation in vitro. The HKUOT-S2 protein with robust osteogenic activity could be a potential alternative osteoanabolic agent for promoting osteogenesis and bone defect repairs. We believe that the HKUOT-S2 protein may potentially be applied clinically as a new class of osteogenic agent for bone defect healing.

Attachment of a trophoblastic spheroid onto endometrial epithelial cells predicts cumulative live birth in women aged 35 and older.

OBJECTIVE: To evaluate the attachment rate of a human embryonic stem cell-derived trophoblastic spheroid onto endometrial epithelial cells in predicting the cumulative live birth rate of an in vitro fertilization (IVF) cycle. DESIGN: A prospective observational study. SETTING: University hospital and research laboratory. PATIENT(S): A total of 240 infertile women from 2017-2021. INTERVENTION(S): Infertile women with regular cycles attending for IVF were recruited. An endometrial aspirate was collected from a natural cycle 1 month before IVF to determine the BAP-EB attachment rate. MAIN OUTCOME MEASURE(S): Cumulative live birth rates from a stimulated cycle and its derived frozen embryo transfer cycles within 6 months of ovarian stimulation were obtained. RESULT(S): The BAP-EB attachment rate in women who attained a cumulative live birth was similar to that in those who did not. When women were stratified by age into <35 years and ≥35 years, the BAP-EB attachment rate was significantly higher only in women aged ≥35 years having a live birth when compared with those in the same age group without a live birth. Receiver operating characteristic curve analysis of BAP-EB attachment rate in predicting cumulative live birth showed the areas under the curve of 0.559 (95% confidence interval [CI], 0.479-0.639), 0.448 (95% CI, 0.310-0.585), and 0.613 (95% CI, 0.517-0.710) for all ages, an age of <35 years, and an age of ≥35 years, respectively. CONCLUSION(S): The BAP-EB attachment rate offers only a very modest prediction of the cumulative live birth rate in women aged ≥35 years undergoing IVF. CLINICAL TRIAL REGISTRATION NUMBER: NCT02713854 (https://clinicaltrials.gov/ct2/show/NCT02713854; Date of registration, March 21, 2016; date of enrollment of the first subject, August 1, 2017).

Integrated MicroRNA and Secretome Analysis of Human Endometrial Organoids Reveal the miR-3194-5p/Aquaporin/S100A9 Module in Regulating Trophoblast Functions.

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.

Endometrial stromal cells from women with repeated implantation failure display impaired invasion towards trophoblastic spheroids.

In brief: Implantation failure can occur even after the transfer of good-quality embryos. This study showed that the migration of human endometrial stromal cells towards embryonic trophoblasts is higher in women with live births in the first in vitro fertilization cycle than those with repeated implantation failure, suggesting that the chemotactic response of stroma cells is associated with successful pregnancy. Abstract: The success rate of in vitro fertilization (IVF) remains limited in some women despite transfers of good-quality embryos in repeated attempts. There is no reliable tool for assessing endometrial receptivity. This study aimed to assess the interaction between decidualized human primary endometrial stromal cells (1°-EnSC) and human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) and to compare the invasion ability of decidualized 1°-EnSC towards BAP-EB between women attaining live birth in the first IVF cycle and those with repeated implantation failure (RIF). The invasion of the decidualized human endometrial cell line (T-HESC) and 1°-EnSC towards BAP-EB was studied. Real-time quantitative PCR and immunocytochemistry were employed to determine the expression of decidualization markers at mRNA and protein levels, respectively. Trophoblast-like BAP-EB-96h, instead of early trophectoderm (TE)-like BAP-EB-48h, facilitated the invasion ability of decidualized T-HESC and decidualized 1°-EnSC. Human chorionic gonadotropin at supra-physiological levels promoted the invasiveness of decidualized 1°-EnSC. The extent of BAP-EB-96h-induced invasion was significantly stronger in decidualized 1°-EnSC from women who had a live birth in the first IVF cycle when compared to those with RIF. While no difference was found in the expression of decidualization markers, PRL and IGFBP1 among two groups of women, significantly lower HLA-B was detected in the non-decidualized and decidualized 1°-EnSC from women with RIF. Collectively, the findings suggested that the invasion of decidualized 1°-EnSC towards trophoblast-like BAP-EB-96h was higher in women who had a live birth in the first IVF cycle than those with RIF.

Aberrant Transferrin and Ferritin Upregulation Elicits Iron Accumulation and Oxidative Inflammaging Causing Ferroptosis and Undermines Estradiol Biosynthesis in Aging Rat Ovaries by Upregulating NF-Κb-Activated Inducible Nitric Oxide Synthase: First Demonstration of an Intricate Mechanism.

We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases.

Expression of microRNA let-7 in cleavage embryos modulates cell fate determination and formation of mouse blastocysts†.

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ~75% of the resulting blastocysts possessed GFP+ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3'UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

ACE inhibitors on ACE1, ACE2, and TMPRSS2 expression and spheroid attachment on human endometrial Ishikawa cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells via receptor angiotensin-converting enzyme 2 (ACE2) and co-receptor transmembrane serine protease 2 (TMPRSS2). However, patients with SARS-CoV-2 infection receiving ACE1 inhibitors had higher ACE2 expression and were prone to poorer prognostic outcomes. Until now, information on the expression of ACE1, ACE2, and TMPRSS2 in human endometrial tissues, and the effects of ACE inhibitors on embryo implantation are limited. We found human endometria expressed ACE1, ACE2, and TMPRSS2 transcripts and proteins. Lower ACE1, but higher ACE2 transcripts were found at the secretory than in the proliferative endometria. ACE1 proteins were weakly expressed in endometrial epithelial and stromal cells, whereas ACE2 and TMPRSS2 proteins were highly expressed in luminal and glandular epithelial cells. However, ACE1 and TMPRSS4 were highly expressed in receptive human endometrial epithelial (Ishikawa and RL95-2) cells, but not in non-receptive AN3CA and HEC1-B cells. Treatment of human endometrial epithelial cells with ACE1 (Captopril, Enalaprilat, and Zofenopril) or ACE2 (DX600) inhibitors did not significantly alter the expression of ACE1, ACE2 and TMPRSS2 transcripts and spheroid (blastocyst surrogate) attachment onto Ishikawa cells in vitro. Taken together, our data suggest that higher ACE2 expression was found in mid-secretory endometrium and the use of ACE inhibitors did not alter endometrial receptivity for embryo implantation.

High-Throughput In Vitro Screening Identified Nemadipine as a Novel Suppressor of Embryo Implantation.

Current contraceptive methods interfere with folliculogenesis, fertilization, and embryo implantation by physical or hormonal approaches. Although hormonal contraceptive pills are effective in regulating egg formation, they are less effective in preventing embryo implantation. To explore the use of non-hormonal compounds that suppress embryo implantation, we established a high-throughput spheroid-endometrial epithelial cell co-culture assay to screen the Library of Pharmacologically Active Compounds (LOPAC) for compounds that affect trophoblastic spheroid (blastocyst surrogate) attachment onto endometrial epithelial Ishikawa cells. We identified 174 out of 1280 LOPAC that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 compounds, we found the one with the lowest cytotoxicity in Ishikawa cells, P11B5, which was later identified as Nemadipine-A. Nemadipine-A at 10 µM also suppressed BeWo spheroid attachment onto endometrial epithelial RL95-2 cells and primary human endometrial epithelial cells (hEECs) isolated from LH +7/8-day endometrial biopsies. Mice at 1.5 days post coitum (dpc) treated with a transcervical injection of 100 µg/kg Nemadipine-A or 500 µg/kg PRI-724 (control, Wnt-inhibitor), but not 10 µg/kg Nemadipine-A, suppressed embryo implantation compared with controls. The transcript expressions of endometrial receptivity markers, integrin αV (ITGAV) and mucin 1 (MUC1), but not ß-catenin (CTNNB1), were significantly decreased at 2.5 dpc in the uterus of treated mice compared with controls. The reduction of embryo implantation by Nemadipine-A was likely mediated through suppressing endometrial receptivity molecules ITGAV and MUC1. Nemadipine-A is a potential novel non-hormonal compound for contraception.

Overexpression of Lin28a induces a primary ovarian insufficiency phenotype via facilitation of primordial follicle activation in mice.

Lin28a is an RNA binding protein and increasing evidence has indicated its role in regulating female fertility. Lin28a has been reported to be involved in ovarian follicle activation. However, its role and mechanisms in regulating primordial follicle activation have not yet been explored. To test whether overexpression of Lin28a activates ovarian primordial follicles, studies were conducted in wild type (WT) and Lin28a Tg mice. Female Lin28a Tg mice at 4-month old exhibited significantly smaller litter size and fewer ovulated oocytes when compared with the WT mice. By 6-month of age, these parameters in Lin28a Tg mice were less than 20% of the WT mice. At postnatal day (PD) 14, the number of primordial follicles was significantly decreased but the number of primary follicles was significantly increased in the transgenic mice. The number of primordial follicles, secondary and antral follicles in these mice were drastically reduced at PD21. In the ovary of Lin28a Tg mice, there were activation of Wnt/ß-catenin signaling and its downstream mTOR pathway. Interestingly, overexpression of Lin28a, which can also act as transcriptional activator, activated Wnt signaling through enhancing the transcription of Wnt co-receptor LRP5. In conclusion, overexpression of Lin28a induced a primary ovarian insufficiency phenotype in long term via facilitating Wnt/ß-catenin signaling leading to activation of primordial follicles.

Single-Cell RNA-Sequencing Reveals Interactions between Endometrial Stromal Cells, Epithelial Cells, and Lymphocytes during Mouse Embryo Implantation.

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.

Bisphenol A Analogues Suppress Spheroid Attachment on Human Endometrial Epithelial Cells through Modulation of Steroid Hormone Receptors Signaling Pathway.

Bisphenol A (BPA) is a well-known endocrine disruptor, widely used in various consumer products and ubiquitously found in air, water, food, dust, and sewage leachates. Recently, several countries have restricted the use of BPA and replaced them with bisphenol S (BPS) and bisphenol F (BPF), which have a similar chemical structure to BPA. Compared to BPA, both BPS and BPF have weaker estrogenic effects, but their effects on human reproductive function including endometrial receptivity and embryo implantation still remain largely unknown. We used an in vitro spheroid (blastocyst surrogate) co-culture assay to investigate the effects of BPA, BPS, and BPF on spheroid attachment on human endometrial epithelial cells, and further delineated their role on steroid hormone receptor expression. We also used transcriptomics to investigate the effects of BPA, BPS, and BPF on the transcriptome of human endometrial cells. We found that bisphenol treatment in human endometrial Ishikawa cells altered estrogen receptor alpha (ERα) signaling and upregulated progesterone receptors (PR). Bisphenols suppressed spheroid attachment onto Ishikawa cells, which was reversed by the downregulation of PR through PR siRNA. Overall, we found that bisphenol compounds can affect human endometrial epithelial cell receptivity through the modulation of steroid hormone receptor function leading to impaired embryo implantation.

Impairment of caprine oocyte maturation in vitro and alteration of granulosa cells functions by widely used fungicide mancozeb.

Mancozeb is classified as an endocrine disruptor; thus the present study was carried out to investigate the impact of mancozeb on mammalian ovarian functions using in vitro caprine oocyte maturation and granulosa cell culture models. Caprine cumulus oocyte complexes (COCs) and granulosa cells were cultured under standard culture conditions and treated with mancozeb concentrations of 0.3, 3, and 30 µg/ml along with a control for 24 h and assessed. Granulosa cell viability and progesterone concentration in spent culture media after treatments were also assessed. Mancozeb significantly decreased (P < 0.05) the oocytes cumulus expansion and the maturation of caprine oocytes. Marked changes in granulose cell morphology were observed with 30 µg/ml mancozeb and significantly reduced (P < 0.05) cell viability. Interestingly, the same concentrations significantly increased (P < 0.05) the progesterone secretion by the cells. Significant reduction of granulosa cells viability and reduction of cumulus expansion and suppression of metaphase plate formation in oocyte can impair the fertilization ability and developmental potential of the oocytes. High progesterone concentration due to mancozeb treatment may suppress LH surge and suppress ovulation. In conclusion, mancozeb suppresses granulosa cells viability, reduces cumulus expansion, and suppresses metaphase plate formation but induces progesterone secretion from granulosa cells that may inhibit LH surge for ovulation process.

Expression of membrane protein disulphide isomerase A1 (PDIA1) disrupt a reducing microenvironment in endometrial epithelium for embryo implantation.

Various proteins in the endometrial epithelium are differentially expressed in the receptive phase and play a pivotal role in embryo implantation. The Protein Disulphide Isomerase (PDI) family contains 21 members that function as chaperone proteins through their redox activities. Although total PDIA1 protein expression was high in four common receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) endometrial epithelial cell lines, significantly higher membrane PDIA1 expression was found in non-receptive AN3CA cells. In Ishikawa cells, oestrogen up-regulated while progesterone down-regulated membrane PDIA1 expression. Moreover, mid-luteal phase hormone treatment down-regulated membrane PDIA1 expression. Furthermore, oestrogen at 10 nM reduced spheroid attachment on Ishikawa cells. Interestingly, inhibition of PDIA1 function by bacitracin or 16F16 increased the spheroid attachment rate onto non-receptive AN3CA cells. Over-expression of PDIA1 in receptive Ishikawa cells reduced the spheroid attachment rate and significantly down-regulated integrin ß3 levels, but not integrin αV and E-cadherin. Addition of reducing agent TCEP induced a sulphydryl-rich microenvironment and increased spheroid attachment onto AN3CA cells and human primary endometrial epithelial cells collected at LH+7/8 days. The luminal epithelial cells from human endometrial biopsies had higher PDIA1 protein expression in the proliferative phase than in the secretory phase. Our findings suggest oestrogen and progesterone regulate PDIA1 expression, resulting in the differential expressions of membrane PDIA1 protein to modulate endometrial receptivity. This suggests that membrane PDIA1 expression prior to embryo transfer could be used to predict endometrial receptivity and embryo implantation in women undergoing assisted reproduction treatment.

Differential expression of protein disulfide isomerase (PDI) in regulating endometrial receptivity in humans.

Estrogen and progesterone regulate the expression of endometrial proteins that determine endometrial receptivity for embryo implantation. The protein disulfide isomerase (PDI) family of proteins play a diverse role in regulating protein modification and redox function. Although the role of PDIs in cancer progression has been widely studied, their role in endometrial receptivity is largely unknown. We have focused on the expressions of PDIA1, PDIA2, PDIA3, PDIA4, PDIA5, and PDIA6 isoforms in endometrial epithelium under the influence of estrogen and progesterone and investigated their functional role in regulating endometrial receptivity. We found PDIA1-6 transcripts were expressed in endometrial epithelial Ishikawa, RL95-2, AN3CA, and HEC1-B cell lines. The expression of PDIA1 was low and PDIA5 was high in HEC1-B cells, whereas PDIA2 was high in both AN3CA and HEC1-B cells. In Ishikawa cells, estrogen (10 and 100 nM) upregulated PDIA1 and PDIA6, whereas estrogen (100 nM) downregulated PDIA4 and PDIA5; and progesterone (0.1 and 1 µM) downregulated transcript expressions of PDIA1-6. In human endometrial samples, significantly lowered transcript expressions of PDIA2 and PDIA5 were observed in the secretory phase compared with the proliferative phase, whereas no change was observed in the other studied transcripts throughout the cycle. Inhibition of PDI by PDI antibody (5 and 10 µg/mL) and PDI inhibitor bacitracin (1 and 5 mM) significantly increased the attachment of Jeg-3 spheroids onto AN3CA cells. Taken together, our study suggests a role of PDI in regulating endometrial receptivity and the possibility of using PDI inhibitors to enhance endometrial receptivity.

The fungicide Mancozeb reduces spheroid attachment onto endometrial epithelial cells through downregulation of estrogen receptor ß and integrin ß3 in Ishikawa cells.

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 µg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 µg/mL and ETU at 300 µg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 µg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor ß and integrin ß3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor ß and integrin ß3 expression of endometrial epithelial cells.

Human embryonic stem cells as an in vitro model for studying developmental origins of type 2 diabetes.

The developmental origins of health and diseases (DOHaD) is a concept stating that adverse intrauterine environments contribute to the health risks of offspring. Since the theory emerged more than 30 years ago, many epidemiological and animal studies have confirmed that in utero exposure to environmental insults, including hyperglycemia and chemicals, increased the risk of developing noncommunicable diseases (NCDs). These NCDs include metabolic syndrome, type 2 diabetes, and complications such as diabetic cardiomyopathy. Studying the effects of different environmental insults on early embryo development would aid in understanding the underlying mechanisms by which these insults promote NCD development. Embryonic stem cells (ESCs) have also been utilized by researchers to study the DOHaD. ESCs have pluripotent characteristics and can be differentiated into almost every cell lineage; therefore, they are excellent in vitro models for studying early developmental events. More importantly, human ESCs (hESCs) are the best alternative to human embryos for research because of ethical concerns. In this review, we will discuss different maternal conditions associated with DOHaD, focusing on the complications of maternal diabetes. Next, we will review the differentiation protocols developed to generate different cell lineages from hESCs. Additionally, we will review how hESCs are utilized as a model for research into the DOHaD. The effects of environmental insults on hESC differentiation and the possible involvement of epigenetic regulation will be discussed.

Decidual glycodelin-A polarizes human monocytes into a decidual macrophage-like phenotype through Siglec-7.

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.

Human embryonic stem cell-derived blastocyst-like spheroids resemble human trophectoderm during early implantation process.

OBJECTIVE: To study the use of human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) as human blastocyst surrogates for studying early implantation and trophoblast development. DESIGN: Laboratory study. SETTING: University research laboratory. PATIENT(S): Infertile in vitro fertilization patients donating endometrial aspirates and human embryonic stem cells (hESCs: VAL3 and H9/WA09). INTERVENTION(S): In BAP-EB derived from hESC, transcriptomes analyzed by next-generation RNA sequencing, effects of Hippo signaling pathway studied by a YAP inhibitor, comparison of attachment of BAP-EB onto primary endometrial epithelial cells (EEC) collected at prereceptive and receptive phases, and antibody blocking assay used to study the molecule(s) involved in BAP-EB attachment. MAIN OUTCOME MEASURE(S): Gene expression profiles and endometrial cell attachment rates. RESULT(S): The BAP-EB differentiation protocol for VAL3 could be used to induce trophoblast differentiation in another hESC line, H9. Transcriptomic analysis showed that the epiblast signature gene expression was reduced while that of the trophoblast was induced during BAP-EB differentiation. Specifically, trophectoderm signature genes were induced in BAP-EB at 48 hours and 72 hours after induction of differentiation. The Hippo signaling pathway was one of the pathways induced during BAP-EB differentiation, and YAP1 inhibitor statistically significantly reduced attachment, outgrowth, and trophoblast gene expressions of BAP-EB. A statistically significantly higher number of BAP-EB derived from both VAL3 and H9 attached onto receptive EEC than prereceptive EEC. The antibody blocking assay demonstrated that endometrial E-cadherin might be critical in early implantation. CONCLUSION(S): The data suggest that BAP-EB possesses a trophectoderm-like signature, which supports the use of BAP-EB as a blastocyst surrogate for the study of trophoblast development and endometrial receptivity.

In vitro and in vivo impairment of embryo implantation by commonly used fungicide Mancozeb.

The fungicide Mancozeb is an endocrine-disrupting chemical and the mode of action of Mancozeb on embryo implantation is largely unknown. Mancozeb (1 and 3 µg/ml) significantly reduced Jeg-3 trophoblastic spheroids attachment to endometrial epithelial Ishikawa cells. Mancozeb treatment from gestation day (GD) 1 to GD8 or from GD4 to GD8 significantly lowered the number of implantation sites with higher incidence of morphological abnormalities in the reproductive tissues. However, these were not seen in the treatment from GD1 to GD4. Mancozeb at 30 mg/kg BW/d did not alter the expression of p53, COX-2, or PGFS transcripts in the uterus, but down-regulated the PGES transcript and protein. Mancozeb treatment in human endometrial stromal cells did not alter the decidualization response, but the morphological transformation was impaired. Taken together, exposure to Mancozeb affected embryo implantation probably through the modulation of decidualization and to delineate the exact mode of action needs further investigations.

Chemical constituents from the thorns of Gleditsia sinensis and their cytotoxic activities.

A new aromatic glycoside (1) and a new natural product, neolignan (2), along with twenty-three known compounds (3-25), were isolated from the thorns of Gleditsia sinensis. According to the spectroscopic analyses (IR, UV, HRESIMS, NMR and ECD), the structures of isolates were elucidated. Herein, compounds 4, 6-8, 10-13, 15, 16, 18, 20, 23 were isolated from the plant of G. sinensis for the first time. Moreover, compounds 4, 6, 15 and 24 showed cytotoxic effects on human ovarian cancer (SKOV-3) cells with IC50 values of 24.83 ± 4.90, 48.86 ± 9.11, 80.13 ± 5.62, 15.38 ± 2.21 µM, respectively. [Formula: see text].