Embryo-maternal Communication Research Articles

Understanding the morpho-functional features of embryo-maternal communication in canine placenta through in vivo and in vitro studies; implications for canine placenta as a translational model.

Understanding the morpho-functional features of embryo-maternal communication in canine placenta through in vivo and in vitro studies; implications for canine placenta as a translational model.

Extracellular vesicles as mediators of stress response in embryo-maternal communication.

Introduction: The pivotal role of extracellular vesicles (EVs) in facilitating effective communication between the embryo and maternal cells during the preimplantation stage of pregnancy has been extensively explored. Nonetheless, inquiries persist regarding the alterations in EV cargo from endometrial cells under stress conditions and its potential to elicit specific stress responses in trophoblast cells. Thus, the aim of this study was to elucidate the involvement of EV miRNA miRNAs in transmitting stress signals from maternal cells to trophoblasts. Methods: The receptive endometrial epithelium analogue RL95-2 cells were subjected to stress induction with 200µM CoCl2 for 24h before EV isolation. JAr trophoblast spheroids, which serve as embryos, were subjected to treatment with stressed or unstressed EVs derived from RL95-2 cells for 24h. Transcriptomic alterations in the treated JAr spheroids as well as in the untreated group, as a negative control, were investigated by mRNA sequencing. Furthermore, the changes in EV miRNAs were assessed by sequencing EV samples. Results: A comprehensive analysis comparing the miRNA profiles between stressed and unstressed EVs revealed significant changes in 25 miRNAs. Furthermore, transcriptomic analysis of JAr spheroids treated with stressed RL95-2EVs versus unstressed EVs or the untreated group demonstrated 6 and 27 differentially expressed genes, respectively. Pathway enrichment analysis showed that stressed EVs induce alterations in gene expression in trophoblast cells, which is partially mediated by EV microRNAs. Discussion: Our results suggest that EVs can transfer stress signals from endometrial cells to the embryo. These discoveries shed new light on the mechanism underlying implantation failures under stress conditions. Unraveling the role of EVs in transmitting stress signals, can extend our knowledge to pave the way for targeted interventions to manage stress-related implantation failures.

Do extracellular vesicles have specific target cells?; Extracellular vesicle mediated embryo maternal communication.

Extracellular vesicles (EVs) serve as messengers for intercellular communication, yet the precise mechanisms by which recipient cells interpret EV messages remain incompletely understood. In this study, we explored how the origin of EVs, their protein cargo, and the recipient cell type influence the cellular response to EVs within an embryo implantation model. We treated two types of EVs to 6 different recipient cell types and expression of zinc finger protein 81 (ZNF81) gene expression in the recipient cells were quantified using quantitative polymerase chain reaction (qPCR). The proteomic contents of the EV cargos were also analyzed. The results showed that downregulation of the ZNF81 gene was a specific cellular response of receptive endometrial epithelial cells to trophoblast derived EVs. Protein cargo analysis revealed that the proteomic profile of EVs depends on their cell of origin and therefore may affect the recipient cell response to EVs. Furthermore, trophoblastic EVs were found to be specifically enriched with transcription factors such as CTNNB1 (catenin beta-1), HDAC2 (histone deacetylase 2), and NOTCH1 (neurogenic locus notch homolog protein 1), which are known regulators of ZNF81 gene expression. The current study provided compelling evidence supporting the existence of EV specificity, where the characteristics of both the EVs and the recipient cell type collectively contribute to regulating EV target specificity. Additionally, EV protein cargo analysis suggested a potential association between transcription factors and the specific functionality of trophoblastic EVs. This in vitro embryo implantation model and ZNF81 read-out provides a unique platform to study EV specific functionality in natural cell-cell communication.

O-266 Multi-omics landscape of uterine fluid-derived extracellular vesicles across the menstrual cycle

Abstract Study question Do extracellular vesicles (EVs) mirror the cyclic physiological changes in endometrial tissue, reflecting the endometrial maturation along the entire menstrual cycle? Summary answer Multi-omics data highlights the importance of EVs during the mid-secretory phase and EVs cargo reflects the state of the tissue throughout the menstrual cycle. What is known already Extracellular vesicles isolated from uterine fluid (UF) during the mid-secretory phase have been characterized for their pivotal role in embryo-maternal communication, particularly in the context of microRNAs (miRNAs) and mRNA. EVs have been posited as potential biomarkers in a minimally invasive liquid biopsy approach for assessing endometrial receptivity, hopefully replacing the endometrial tissue biopsy-based testing. However, the dynamics of EVs and their cargo throughout the distinct phases of the menstrual cycle remain unknown. This is the first study to elucidate UF-EV content throughout the maturation of the endometrium. Study design, size, duration Whole-exome RNA and small-RNA sequencing was performed on 15 paired samples, comprising UF and endometrial biopsy (EB), obtained from healthy female subjects across the menstrual cycle. This initial dataset was expanded with 35 additional UF samples. Cumulatively, the analysis encompassed samples (EB and UF) across the menstrual cycle’s four distinct phases: proliferative (n = 10), early-secretory (ES) (n = 17), mid-secretory (MS) (n = 20), and late-secretory (LS) (n = 18). Additionally, 20 UF-EV samples were used for flow cytometry surface proteome analysis. Participants/materials, setting, methods Participants were excluded when infertility-associated conditions were found and included when they had at least one child born, with no hormonal medications used for three months before the sampling. EVs were extracted from UF with size-exclusion chromatography (SEC) and subsequently with glycosaminoglycan-based precipitation. The EVs were characterized with nanoparticle tracking analysis, western blot, transmission electron microscopy and flow-cytometry surface proteome analysis (MACSPlex) for 37 surface markers. Single-end RNA and small-RNA sequencing were performed on NextSeq1000. Main results and the role of chance The transcriptome, miRNome, and protein surface markers of UF-EVs were characterized throughout the menstrual cycle. Transcriptome profiling was performed in comparison to the preceding phase. During the ES phase, 5,507 differentially expressed genes (DEGs) were observed, most of them being downregulated and associated with G-coupled-protein signaling, while in the MS phase, 867 DEGs were identified associated with nucleosome assembly and cell differentiation. Interestingly in the LS phase, the histone genes associated with nucleosome assembly were upregulated compared to MS, highlighting that histone dynamics change corresponding to the menstrual cycle. The exploration of gene ontology revealed consistent enrichment of genes related to extracellular vesicles in UF-EV and EBs across the menstrual cycle. The miRNA analysis of UF-EVs and EB showed significant overlap in miRNA expression level especially in the MS phase. Furthermore, we found 16 unique miRNAs in the mid-secretory phase whose targets were associated with embryo morphogenesis and cell adhesion. MACSPlex analysis revealed that CD9 and CD63, canonical EV markers, were significantly abundant during the MS phase, indicative of an enriched secretion of EVs during the preparation of the endometrium for embryo implantation. These insights underscore that the cargo of UF-EVs reflects the dynamic changes in the endometrial tissue. Limitations, reasons for caution The research on UF-EVs faced notable challenges due to the irregular and non-standardized collection methods of UF in gynecological practice. This variability in UF collection introduces potential confounding factors. The study also operated with a limited sample size, underscoring the need for further validation. Wider implications of the findings Our study indicates that the UF-EVs reflect the menstrual cycle-related molecular changes and UF-EV analysis could be potentially used to follow the menstrual cycle-related molecular processes in the uterine environment. Trial registration number not applicable

O-055 Understanding endometrial functionality and embryo-maternal communication via in vitro “OMICs’’ platforms

Abstract The uterus (womb) is perhaps the most fascinating organ in the human body. It undergoes cyclical regeneration of its inner layer, the endometrium, and is notoriously unreceptive to implantation, except for a short period of time known as the window of implantation (WoI), the only time when it becomes receptive to the implantation of an embryo resulting in pregnancy. A common reason for pregnancy failure is implantation failure. Similarly, many in vitro fertilisation (IVF) treatments that do not result in a live birth fail because the embryo does not successfully implant. This is a significant challenge as there is limited understanding of the exact timing and cellular/molecular characteristics of the human endometrium during WoI. Here I will present results on the characterisation of the WoI in humans, contextualised with results from “omics’ platforms. Moreover, we will provide 3D data on the structural development of the human uterus, paving the way to develop innovative culture platforms to investigate embryo-maternal communication.

Effect of 3D and 2D cell culture systems on trophoblast extracellular vesicle physico-chemical characteristics and potency.

The growing understanding of the role of extracellular vesicles (EVs) in embryo-maternal communication has sparked considerable interest in their therapeutic potential within assisted reproductive technology, particularly in enhancing implantation success. However, the major obstacle remains the large-scale production of EVs, and there is still a gap in understanding how different culture systems affect the characteristics of the EVs. In the current study, trophoblast analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension culture (3D) and how the cell growth environment affects the physical, biochemical and cellular signalling properties of EVs produced by them was studied. Interestingly, the 3D system was more active in secreting EVs compared to the 2D system, while no significant differences were observed in terms of morphology, size, and classical EV protein marker expression between EVs derived from the two culture systems. There were substantial differences in the proteomic cargo profile and cellular signalling potency of EVs derived from the two culture systems. Notably, 2D EVs were more potent in inducing a cellular response in endometrial epithelial cells (EECs) compared to 3D EVs. Therefore, it is essential to recognize that the biological activity of EVs depends not only on the cell of origin but also on the cellular microenvironment of the parent cell. In conclusion, caution is warranted when selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs through in vitro studies.

Characterization of oviduct epithelial spheroids for the study of embryo–maternal communication in cattle

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo–maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100–200 μm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 μl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-μL droplet of SOF medium under mineral oil) than in M199/25 (25-μL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1, ESR1, HSPA8, and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo–maternal interactions in cattle.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders

BackgroundWomen with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing.MethodsEndometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student’s t-test or Wilcoxon test.ResultsWe identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia.ConclusionsIdentifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

Extracellular vesicles: emerging paradigms in bovine embryo-maternal communication.

The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.

Extracellular Vesicles: the Next Frontier in Pregnancy Research.

Extracellular vehicles (EVs) have been involved in several aspects of pregnancy, including endometrial receptivity, embryo implantation, and embryo-maternal communication showing them associated with pregnancy disorders, such as preeclampsia, gestational diabetes mellitus, and preterm birth. Further research is warranted to fully comprehend the exact pathophysiological roles of EVs and to develop new therapies targeting EVs thereby improving pregnancy outcomes. Herein, we review the recent knowledge on the multifaceted roles of EVs during pregnancy and address the majority of the molecular interactions between EVs, maternal, and fetal cells with an emphasis on disorders of pregnancy under the influence of EVs. Moreover, we also discuss its applications in clinical trials followed by prospects.

Embryo-maternal communication mediated by extracellular vesicles in the early stages of embryonic development is modified by in vitro conditions

Extracellular vesicles (EVs) have become important in embryo-maternal communication during early development. The aim of this study was to evaluate the effect of an in vitro system on early bidirectional embryo-maternal communication mediated by EVs. For this purpose, two experiments were performed: one to evaluate the effect of embryonic EVs on maternal cells and the second to determine the effect of maternal EVs on early embryonic development. For the first in vitro (IVP) and in vivo (IVV) experiments, bovine blastocysts were selected and individually cultured for 48 h to collect embryonic EVs secreted during days 7–9 of embryonic development. Embryonic EVs were added to the medium of in vitro-cultured bovine endometrial cells to evaluate their effect on the expression pattern of genes associated with endometrial function and response to interferon tau (IFNT). Non-classical interferon-stimulated genes (ISGs) were only induced by in vitro-derived embryos. In the second experiment, EVs released by endometrial cells cultured in vitro (EVC) and collected from uterine fluid (EV-UF) of cows in the early luteal phase were added to the culture medium of bovine embryos produced in vitro during days 5–9 of development. The effect of maternal in vitro or in vivo-derived EVs differs in the quality of bovine embryos produced in vitro during the pre-implantation period. The expression of IFNT in bovine embryos is increased by the effect of EV-UF treatment. Additionally, EV-UF treatment induces a sustained increase in diameter during embryonic development and a tendency towards a greater number of expanded and hatched blastocysts. However, some genes related to embryo quality are induced by EVC treatment.

Zona pellucida removal modifies the expression and release of specific microRNAs in domestic cat blastocysts.

The in vitro culture of domestic cat embryos without the zona pellucida affects their implantation capacity. MicroRNAs (miRNAs) have an important role in embryo-maternal communication and implantation. The objective of this study was to evaluate the expression of specific miRNAs in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were done: (1) domestic cat embryos cultured with the zona pellucida (zona intact control group, ZI); and (2) cultured without the zona pellucida (zona free group, ZF). The cleavage, morula and blastocyst rates were evaluated. The blastocysts and their spent medium were used for miRNA expression analysis using RT-qPCR (miR-21, miR-24, mi25, miR-29, miR-96, miR-98, miR-103, miR-191, miR-196, miR-199, miR-130, miR-155 and miR-302). The pre-mature microRNAs (pre-miRNAs) and miRNAs were evaluated in the blastocysts and only miRNAs were evaluated in the spent medium. No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups (P > 0.05). For miRNAs analysis, miR-103 and miR-191 had the most stable expression and were selected as internal controls. ZF blastocysts had a higher expression of miR-21, miR-25, miR-29 and miR-199 and a lower expression of miR-96 than their ZI counterparts (P < 0.05). Furthermore, higher levels of miR-21, miR-25 and miR-98 were detected in the spent medium of ZF blastocysts (P < 0.05). In conclusion, in vitro culture of domestic cat embryos without the zona pellucida modifies the expression of miR-21, miR-25, miR-29, miR-199 and miR-96 at the blastocyst stage and the release of miR-21, miR-25 and miR-98.

Secretory Proteomic Responses of Endometrial Epithelial Cells to Trophoblast-Derived Extracellular Vesicles.

Synchronized crosstalk between the embryo and endometrium during the periconception period is integral to pregnancy establishment. Increasing evidence suggests that the exchange of extracellular vesicles (EVs) of both embryonic and endometrial origin is a critical component of embryo-maternal communication during peri-implantation. Here, we investigated whether embryonic signals in the form of EVs can modulate the endometrial epithelial cell secretome. Receptive endometrial analog RL95-2 cells were supplemented with trophoblast analog JAr cell-derived EVs, and the secretory protein changes occurring in the RL95-2 cells were analyzed using mass spectrometry. EVs of non-trophoblastic origin (HEK 293 cells) were used as the control EV source to supplement endometrial cells. Trophoblast cell-derived EVs enriched endometrial epithelial cell secretions with proteins that support embryo development, attachment, or implantation, whereas control EVs were unable to induce the same effect. The present study suggests that embryonic signals in the form of EVs may prime receptive endometrial epithelial cells to enrich their secretory proteome with critical proteomic molecules with functional importance for periconception milieu formation.

P-319 Transcriptomic analysis reveals deregulated molecular mechanisms related to miscarriage, preeclampsia, and pregnancy complications during gestational endometrial phase in adenomyosis

Abstract Study question Is there any molecular deregulation in the gestational endometrium of women with adenomyosis responsible for adenomyosis-related pregnancy disorders? Summary answer Women with adenomyosis show deregulated molecular mechanisms in their gestational phase endometrium involved in miscarriage, preeclampsia, and pregnancy complications, causing adenomyosis-related infertility. What is known already Women with adenomyosis are characterized by having defective decidualization, altered endometrial receptivity, impaired embryo-maternal communication, implantation failure and higher risk of preeclampsia and miscarriage. However, molecular mechanisms underlying these infertility-related conditions remain unknown, mainly due to the inaccessibility of obtaining and maintaining these endometrial tissues. We have previously established adenomyosis endometrial organoids and differentiated them into gestational phase, demonstrating the maintenance of disease-specific traits. For this reason, here we use the already established adenomyosis organoid platform to describe the molecular mechanisms deregulated during gestational phase involved in adenomyosis-associated pregnancy disorders, which could be possible infertility therapeutical targets. Study design, size, duration Endometrial organoids from eutopic endometrium of adenomyosis and non-disease (control) women (n = 15/group) were established and differentiated into gestational phase (ADENO-GESTorg and CONTROL-GESTorg) by supplementation with ovarian and pregnancy hormones. An RNA-sequencing was performed. DESeq2 was used to identify differentially expressed genes (DEGs), FDR &amp;lt; 0.05. GO and KEGG analysis were performed to study DEGs implication at biologically functional level. QIAGEN Ingenuity Pathway Analysis (IPA) was used to further identify upregulated and downregulated pathways. Participants/materials, setting, methods Endometrial biopsies were obtained by hysteroscopy from adenomyosis and control patients (n = 15/group) at IVI Valencia Clinics. Patients between 18≤45 years old and BMI≤28 kg/m2 diagnosed with adenomyosis (adenomyosis group) or without adenomyosis nor other uterine pathologies (control group) were included in the study. Main results and the role of chance We identified 1999 DEGs (153 upregulated and 1846 downregulated) in ADENO- compared to CONTROL-GESTorg. Between upregulated genes, CXCL14 restricts trophoblast invasion and outgrowth, CYP24A1 is increased in spontaneous miscarriage and preeclamptic placentas, and PTAFR induces preterm delivery in mice (log2 Fold Change [log2FC] = 1.6, 2.4, 0.8). Among downregulated genes, deregulated PGR expression has been related to severe preeclampsia and recurrent pregnancy loss, whereas defects on ZWINT, ESCO2 and MCM6 expression are associated with high incidence of aneuploidy, leading to miscarriage, infertility, and newborn disorders (log2FC = −2.21, -2.57, -2.53, -2.48). Functional analysis showed deregulated functions as blastocyst growth, in utero embryonic development, developmental maturation and response to oxidative stress, standing out for their possible involvement in pregnancy disorders. IPA predicted a significant inhibition of D-myo-inositol metabolism (z score [z] = −0.7) affecting oocyte and embryo quality, VEGF signaling (z = −1.5) disturbing trophoblast adaptation to hypoxia, ERK/MAPK signaling (z = −4.0) causing embryonic lethality due to placental alterations; and significant activation of PPARα/RXRα (z = 0.5) that gives rise to recurrent miscarriage and preeclampsia, p53 signaling (z = 1.1) related with preeclampsia-complicated pregnancies, and RHOGDI and PTEN signaling (z = 3.6, 3.4) both promoting preeclampsia. Limitations, reasons for caution Although organoid platforms faithfully recapitulate tissue of origin characteristics as well as disease-specific traits, they are still in vitro models that need to be translated into in vivo studies to further corroborate the results obtained. Wider implications of the findings Molecular mechanisms involved in impaired embryo development, pregnancy loss, preeclampsia and placental defects are deregulated in gestational phase endometrium of women with adenomyosis. These deregulated mechanisms could be therapeutic target to develop pharmacological treatments to ameliorate adenomyosis-related infertility. Trial registration number not applicable

Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells.

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

Rhythm of the First Language: Dynamics of Extracellular Vesicle-Based Embryo-Maternal Communication in the Pre-Implantation Microenvironment.

One of the most critical steps in mammalian reproduction is implantation. Embryos with an impaired capacity for embryo-maternal crosstalk are thought to have a reduced potential for implantation. One agent of embryo-maternal communication is extracellular vesicles (EV). EVs are lipid bilayer-bound biological nanoparticles implicated in intercellular communication between many of the known cell types. In the current study, we isolated EVs from trophoblast analogue JAr spheroids and supplemented the EVs with receptive endometrium analogue RL95-2 cells to simulate pre-implantation embryo-maternal dialogue. The transcriptome of the endometrial cells was examined at 30 min, 4 h and 48 h intervals using Oxford Nanopore® technology. At the time points, 30 min, 4 h and 48 h, the endometrial cells showed a significantly altered transcriptome. It seems trophoblast EVs induce a swift and drastic effect on the endometrial transcriptome. The effect peaks at around 4 h of EV supplementation, indicating a generalized effect on cell physiology. Alterations are especially apparent in biological pathways critical to embryonic implantation, such as extracellular matrix-receptor interactions and cytokine-receptor interactions. These observations can be helpful in elucidating the dynamics of embryo-maternal communication in the pre-implantation period.

MicroRNAs as A Biomarker to Predict Embryo Quality Assessment in In Vitro Fertilization.

Embryo selection for in vitro fertilization (IVF) is an effort to increase the success rate of embryo implantation. Factors influencing the success of embryo implantation include embryo quality, endometrial receptivity, embryo characteristics, and maternal interactions. Some molecules have been found to influence these factors, but their regulatory mechanisms are unclear. MicroRNAs (miRNAs) are reported to play an essential role in the embryo implantation process. miRNAs are small non-coding RNAs consisting of only 20 nucleotides that play an essential role in the stability of gene expression regulation. Previous studies have reported that miRNAs have many roles and are released by cells into the extracellular environment for intracellular communication. In addition, miRNAs can provide information related to physiological and pathological conditions. These findings encourage research development in determining the quality of embryos in IVF to increase the implantation success rate. Moreover, miRNAs can provide an overview of embryo-maternal communication and potentially be noninvasive biological markers of embryo quality, which could increase assessment accuracy while reducing mechanical damage to the embryo itself. This review article summarizes the involvement of extracellular miRNAs and the potential applications of miRNAs in IVF.

Extracellular vesicles derived from endometrial epithelial cells deliver exogenous miR-92b-3p to affect the function of embryonic trophoblast cells via targeting TSC1 and DKK3

BackgroundExtracellular vesicles (EVs) could mediate embryo-maternal communication to affect embryo implantation by delivering biology information, including microRNA (miRNA), protein, lipid. Our previous research shows that miR-92b-3p was differentially expressed in EVs of uterine flushing fluids during the embryo implantation period. However, the role of miR-92b-3p from EVs in embryo implantation remains elusive.Materials and methodsEVs were isolated from porcine endometrial epithelial cells (EECs) by ultracentrifugation. MiR-92b-3p mimics and EVs were used to regulate the expression of miR-92b-3p in porcine trophoblast cells (PTr2 cells). Cell proliferation, migration and adhesion analyses were used to observe the phenotype. RT-qPCR, western blot and dual-luciferase reporter assay were used to assess the targets of miR-92b-3p.ResultsIn this study, EVs derived from porcine EECs were identified and could be taken up by PTr2 cells. We found that the EVs derived from EECs transfected with miR-92b-3p mimic (EVs-miR-92b-3p) significantly promoted the proliferation, migration and adhesion of PTr2 cells. We verified that Tuberous sclerosis complex subunit (TSC1) and Dickkopf 3 (DKK3) were the target genes of miR-92b-3p. Moreover, our study showed that miR-92b-3p plays a vital role in PTr2 cells via targeting TSC1 and DKK3. Furthermore, the 3'UTR vectors of TSC1 and DKK3 can rescue the effect of miR-92b-3p on PTr2 cells.ConclusionsTaken together, this study reveals a novel mechanism that EVs derived from porcine EECs treated with miR-92b-3p crosstalk with trophoblasts by targeting TSC1 and DKK3, leading to an enhanced ability for implantation.

Expression dynamics of ISGs and chemokines in maternal whole blood as an indicator of healthy embryonic implantation in buffalo

Successful pregnancy establishment has a major influence on reproductive efficiencies of dairy buffaloes. This success depends on coordinated communications between conceptus and maternally derived cells, and any disturbance in this communication network during this process leads to early embryonic losses. Since, early embryonic losses is one of the prime cause of pregnancy failure in dairy buffaloes, present investigation was aimed to understand the expression dynamics and correlation pattern of Interferon tau stimulated genes and chemokines during peri-implantation period in dairy buffaloes through transcriptional profiling followed by Principal Component Analysis. Our study revealed upregulation of all the candidate genes in maternal whole blood across the parity, emphasizing the involvement of immune responses as signatures for healthy pregnancy. Understanding these embryo-maternal communication signals offers potential to prevent incidences of early embryonic losses and can provide insights on possible targets for therapy too.

Role of extracellular vesicles in intercellular communication during reproduction.

The mammalian reproduction is a process of controlled cellular growth and development regulated by constant communication between the gametes, the subsequent embryo and the maternal system. Extracellular vesicles (EVs) are involved in these communications to a significant degree from the gamete production and maturation to fertilization, embryo development and implantation. They regulate the cellular physiology and the immune reaction to bring about a favourable environment for a successful pregnancy. Deciphering the mechanisms employed in EV-mediated embryo maternal communication could improve our knowledge in mammalian reproduction and increase the efficiency of animal breeding.