P–379 Human platelet lysate improves trophoblast spheroid attachment to primary endometrial epithelial cells from patients with recurrent implantation failure

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T T T N Nguyen ◽  
Y S S Kwok ◽  
S Russell ◽  
C Librach
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Fluorescent Microscopy ◽  
Endometrial Receptivity ◽  
Platelet Lysate ◽  
Implantation Failure ◽  
Trophoblast Cells ◽  
Recurrent Implantation Failure ◽  
Endometrial Epithelial Cells

Abstract Study question Could non-autologous platelet lysate (PL) increase attachment of HTR–8 spheroids in vitro to primary endometrial epithelial cells (EECs) from patients with recurrent implantation failure (RIF)? Summary answer Increased quantity of HTR–8 spheroids attached to primary EECs, isolated from patients with RIF, suggests in vitro treatment with non-autologous PL could improve endometrial receptivity. What is known already Inadequate endometrial receptivity and thickness are major causes for RIF. Recent studies suggest that platelet-rich plasma (PRP) may improve pregnancy outcomes for RIF and/or thin endometrium (TE) patients. Our previous results show that a commercially sourced and non-autologous human PRP/PL (HPL) promotes EC proliferation in vitro, suggesting that HPL may help to standardize future clinical treatments. In addition to EC proliferation, HPL treatment may improve embryo attachment to primary EECs isolated from patients with a history of RIF. In vitro attachment assays with trophoblast spheroids (embryo model) could help elucidate the effect of HPL on endometrial receptivity in RIF patients. Study design, size, duration Endometrial tissue was collected from nine RIF patients at the CReATe Fertility Centre, Toronto, Canada (Veritas REB#16580): five with (RIF+TE) and four without a TE (RIF only). Primary EECs were enzymatically isolated and treated with serum-free culture media (SFM) or 1% HPL in SFM for 48 hours before performing the attachment assay. Trophoblast cells (HTR–8/SVneo) were grown in suspension on a rocker to form 70–100 uM spheroids over 24 hours before use in the assay. Participants/materials, setting, methods Spheroids were fluorescently labelled with calcein-AM for 30 minutes and size-selected to capture spheroids similar in size to a human blastocyst. Spheroids were seeded on top of EEC monolayers and calcein fluorescence was immediately measured by a spectrophotometer. Following the 1-hour incubation, unattached spheroids were aspirated, and fluorescence was measured again. Spheroids were also individually quantified by fluorescent microscopy and ImageJ™ software. The percentage of spheroid attachment was calculated for calcein fluorescence and ImageJ™ quantification. Main results and the role of chance The HTR–8/SVneo cell line, derived from human first-trimester extravillous trophoblast cells (EVT), has been shown to be a suitable cell line to assess adhesion and invasion in vitro. Trophoblast spheroids generated from this cell line visually resembled a blastocyst and maintained expression of the EVT and implantation biomarkers: GATA3, ITGA5, and LIF. Primary EECs, treated for 48 hours with SFM supplemented with 1% commercially sourced and non-autologous HPL, overall exhibited increased attachment to HTR–8 spheroids. The percentage of spheroid attachment, as measured by fluorescence alone, significantly increase from 47.98% to 64.27% (P < 0.01) of seeded spheroids in RIF+TE EEC cultures, and from 48.12% to 85.77% (P < 0.001) of seeded spheroids in RIF only EEC cultures. Quantification by fluorescent microscopy and ImageJ™ software for individual calcein-stained spheroids, revealed a significant increase in spheroid attachment, from 57.52% to 86.5% (P < 0.01) in RIF+TE EEC cultures, and from 42.58% to 68.90% (P < 0.01) in RIF only EEC cultures. Limitations, reasons for caution Although there was a positive correlation between calcein fluorescence and spheroid quantity, quantification by fluorescence alone may be unreliable due to the variable numbers of cells in each spheroid. Our data suggest a more precise increase in attachment is detected when quantified by fluorescent microscopy and ImageJ™ software. Wider implications of the findings: We report a method for functional assessment of endometrial receptivity in vitro. HPL appears to promote implantation in RIF patients in a model of embryo attachment. We predict that the observed increase in attachment is due to increased endometrial receptivity gene expression, which will be our next investigative avenue. Trial registration number N/A

scholarly journals Increased expression of HMGB1 in the implantation phase endometrium is related to recurrent implantation failure

2021 ◽  
Author(s):  
Mi Han ◽  
Yi Cao ◽  
Wenjie Zhou ◽  
Mingjuan Zhou ◽  
Xiaowei Zhou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Underlying Mechanism ◽  
Endometrial Receptivity ◽  
In Vitro Assays ◽  
Control Group ◽  
Implantation Failure ◽  
Recurrent Implantation Failure ◽  
Tubal Infertility ◽  
Endometrial Stromal Cells

Abstract Impaired endometrial receptivity is the main cause of recurrent implantation failure (RIF), however, its underlying mechanism is unclear. In this study, we found that HMGB1 expression was significantly decreased in the implantation phase endometrium in the control group (patients with tubal infertility who successfully achieved conception after the first embryo transfer) (P = 0.006). However, the expression levels of HMGB1 mRNA and protein were significantly upregulated during the implantation phase in endometrial tissues obtained from patients with RIF compared to those in the control group (P = 0.001), consistent with the results of genome-wide expression profiling. Moreover, in vitro assays showed that increased expression of HMGB1 in human endometrial epithelial cells cause marked deficiency in supporting blastocysts and human embryonic JAR cell adhesion, mimicking the process of embryo adhesion. However, overexpression of HMGB1 had no effect on cell proliferation and in-vitro decidualization in a human endometrial stromal cell line (T-HESCs) and in primary human endometrial stromal cells (HESCs). These findings indicate that increased HMGB1 levels suppressed the adhesion capability of epithelial cells, contributing to impaired endometrial receptivity in patients with recurrent implantation failure. This characteristic can be used as a target for detecting and treating recurrent implantation failure in clinical practice.

scholarly journals GnRH antagonist alters the migration of endometrial epithelial cells by reducing CKB

Reproduction ◽  
2020 ◽  
Vol 159 (6) ◽  
pp. 733-743 ◽  
Author(s):  
Qian Chen ◽  
Yong Fan ◽  
Xiaowei Zhou ◽  
Zheng Yan ◽  
Yanping Kuang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Implantation Rate ◽  
Endometrial Receptivity ◽  
Ishikawa Cells ◽  
Atp Generation ◽  
And Migration ◽  
Insight Into ◽  
Endometrial Epithelial Cells

Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

scholarly journals Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

Reproduction ◽  
2019 ◽  
pp. 53-64 ◽  
Author(s):  
Yumiko Miyazaki ◽  
Akihito Horie ◽  
Hirohiko Tani ◽  
Masashi Ueda ◽  
Asuka Okunomiya ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chondroitin Sulfate ◽  
Cell Line ◽  
Human Embryo ◽  
Embryo Implantation ◽  
Cancer Cell Line ◽  
Promoting Effect ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

scholarly journals Expression and significance of miR-30d-5p and SOCS1 in patients with recurrent implantation failure during implantation window

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuhao Zhao ◽  
Dongmei He ◽  
Hong Zeng ◽  
Jiefeng Luo ◽  
Shuang Yang ◽  
...  
Keyword(s):  
Endometrial Biopsy ◽  
Endometrial Receptivity ◽  
Leukaemia Inhibitory Factor ◽  
In Vitro Fertilisation ◽  
Control Group ◽  
Implantation Failure ◽  
Recurrent Implantation Failure ◽  
Tissue Samples ◽  
Implantation Window

Abstract Background Poor endometrial receptivity is a major factor that leads to recurrent implantation failure. However, the traditional method cannot accurately evaluate endometrial receptivity. Various studies have indicated that microRNAs (miRNAs) are involved in multiple processes of embryo implantation, but the role of miRNAs in endometrial receptivity in patients with recurrent implantation failure (RIF) remains elusive. In the present study, we investigated the presence of pinopodes and the roles of miR-30d-5p, suppressor of cytokine signalling 1 (SOCS1) and the leukaemia inhibitory factor (LIF) pathway in women with a history of RIF during the implantation window. Methods Endometrial tissue samples were collected between January 2018 to June 2019 from two groups of women who underwent in vitro fertilisation and embryo transfer (IVF-ET) or frozen ET. The RIF group included 20 women who underwent ≥ 3 ETs, including a total of ≥ 4 good-quality embryos, without pregnancy, whereas the control group included 10 women who had given birth at least once in the past year. An endometrial biopsy was performed during the implantation window (LH + 7). The development of pinopodes in the endometrial biopsy samples from all groups was evaluated using scanning electron microscopy (SEM). Quantitative reverse transcription-polymerase chain reaction and western blotting were used to investigate the expression levels of miR-30d-5p, SOCS1, and the LIF pathway. Results The presence of developed pinopodes decreased in patients with RIF on LH + 7. The expression level of miR-30d-5p decreased in the endometria during the implantation window of patients with RIF, whereas the mRNA and protein levels of SOCS1 were significantly higher in the RIF group than in the control group. Furthermore, a negative correlation was observed between the expression of miR-30d-5p and SOCS1 (r2 = 0.8362). In addition, a significant decrease in LIF and p-STAT3 expression was observed during the implantation window in patients with RIF. Conclusions MiR-30d-5p and SOCS1 may be potential biomarkers for endometrial receptivity. Changes in pinopode development and abnormal expression of miR-30d-5p, SOCS1 and LIF pathway in the endometrium could be the reasons for implantation failure.

scholarly journals FBXO18/Circrna211/Mir-431/CSF1 Axis Regulates the Establishment of Endometrial Receptivity Though Activating MAPK and CSF1R/PI3K/AKT/Mtor Pathways in Dairy Goats

2021 ◽  
Author(s):  
Lichun Yang ◽  
Xiaorui Liu ◽  
Lei Zhang ◽  
Danni Li ◽  
Guili Li ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Regulatory Network ◽  
Target Genes ◽  
Endometrial Receptivity ◽  
Physiological Status ◽  
Dairy Goats ◽  
In Vivo Models ◽  
Endometrial Epithelial Cells

Abstract Background: Endometrial epithelial cells proliferation and secretion of various cytokines have a strong impact on the formation of receptive endometrium, which is known as a physiological status that allows an activated embryo to attach to the endometrium for a limited time. Circular RNAs and miRNAs can be involved in the dynamic physiological changes of endometrium by regulating relevant functional target genes in the uterus. Our work presented here with the ultimate purpose of revealing the latent molecular mechanism of FBXO18/circRNA211/miR-431/CSF1 axis in the establishment of endometrial receptivity of dairy goats.Results: In vitro, we found a regulatory network of FBXO18/circRNA211/miR-431/CSF1 in goat endometrial epithelial cells that circRNA211 severed as a sponge for miR-431, resulting in weakening the inhibition of miR-431 on target genes CSF1 and FBXO18. FBXO18/circRNA211/miR-431/CSF1 axis promoted the proliferation through regulating the key proteins of Ras, Raf, MEK, ERK in MAPK pathway via CCK-8, EdU, flow cytometry and Western blot assays. Furthermore, FBXO18/circRNA211/miR-431/CSF1 axis activated the phosphorylation of key proteins PI3K, AKT and mTOR in PI3K-mTOR pathway by CSF1R, thereby promoting the establishment of endometrial receptivity. In vivo models, mice injected with miR-431 agomir showed that the endometrial thickness and the number of pinopodes were significantly decreased by HE staining and scanning electron microscope. Immunohistochemistry results showed that VEGF and OPN proteins were down-regulated and MUC1 protein was up-regulated under the treatment of miR-431 agomir. Further study demonstrated that miR-431 inhibited embryo implantation by impeding the establishment of endometrial receptivity.Conclusion: Ultimately, our study revealed a regulatory mechanism of FBXO18/circRNA211/miR-431/CSF1 axis in goat endometrial epithelial cells. This circRNA/miRNA/mRNA regulatory network presented here in vitro and in vivo models may provide a novel insight into the potentially regulating endometrium biological functions and promoting the formation of endometrium receptivity.

scholarly journals Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ryo Yokomizo ◽  
Yukiko Fujiki ◽  
Harue Kishigami ◽  
Hiroshi Kishi ◽  
Tohru Kiyono ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Stromal Cells ◽  
Therapeutic Option ◽  
Three Dimensional ◽  
Fertility Treatment ◽  
Feeder Cells ◽  
Endometrial Stromal Cells ◽  
Thin Endometrium ◽  
Endometrial Epithelial Cells

Abstract Background Thin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro. Methods We prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells. Results Co-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model. Conclusions We herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.

scholarly journals Association between HOX Transcript Antisense RNA Single-Nucleotide Variants and Recurrent Implantation Failure

2021 ◽  
Vol 22 (6) ◽  
pp. 3021
Author(s):  
Jeong Yong Lee ◽  
Eun Hee Ahn ◽  
Hyeon Woo Park ◽  
Ji Hyang Kim ◽  
Young Ran Kim ◽  
...  
Keyword(s):  
Antisense Rna ◽  
Coagulation Factors ◽  
Healthy Controls ◽  
Implantation Failure ◽  
Single Nucleotide Variants ◽  
Recurrent Implantation Failure ◽  
Single Nucleotide ◽  
Vitro Fertilization ◽  
And Control

Recurrent implantation failure (RIF) refers to the occurrence of more than two failed in vitro fertilization–embryo transfers (IVF-ETs) in the same individual. RIF can occur for many reasons, including embryo characteristics, immunological factors, and coagulation factors. Genetics can also contribute to RIF, with some single-nucleotide variants (SNVs) reported to be associated with RIF occurrence. We examined SNVs in a long non-coding RNA, homeobox (HOX) transcript antisense RNA (HOTAIR), which is known to affect cancer development. HOTAIR regulates epigenetic outcomes through histone modifications and chromatin remodeling. We recruited 155 female RIF patients and 330 healthy controls, and genotyped HOTAIR SNVs, including rs4759314, rs920778, rs7958904, and rs1899663, in all participants. Differences in these SNVs were compared between the patient and control groups. We identified significant differences in the occurrence of heterozygous genotypes and the dominant expression model for the rs1899663 and rs7958904 SNVs between RIF patients and control subjects. These HOTAIR variants were associated with serum hemoglobin (Hgb), luteinizing hormone (LH), total cholesterol (T. chol), and blood urea nitrogen (BUN) levels, as assessed by analysis of variance (ANOVA). We analyzed the four HOTAIR SNVs and found significant differences in haplotype patterns between RIF patients and healthy controls. The results of this study showed that HOTAIR is not only associated with the development of cancer but also with pregnancy-associated diseases. This study represents the first report showing that HOTAIR is correlated with RIF.

scholarly journals Modulation of IGF2 Expression in the Murine Thymus and Thymic Epithelial Cells Following Coxsackievirus-B4 Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 402
Author(s):  
Hélène Michaux ◽  
Aymen Halouani ◽  
Charlotte Trussart ◽  
Chantal Renard ◽  
Hela Jaïdane ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Thymic Epithelial Cells ◽  
Altered Expression ◽  
Coxsackievirus B4 ◽  
Gene Coding ◽  
Self Tolerance ◽  
Stat3 Phosphorylation ◽  
The Stability

Coxsackievirus B4 (CV-B4) can infect human and murine thymic epithelial cells (TECs). In a murine TEC cell line, CV-B4 can downregulate the transcription of the insulin-like growth factor 2 (Igf2) gene coding for the self-peptide of the insulin family. In this study, we show that CV-B4 infections of a murine TEC cell line decreased Igf2 P3 promoter activity by targeting a region near the transcription start site; however, the stability of Igf2 transcripts remained unchanged, indicating a regulation of Igf2 transcription. Furthermore, CV-B4 infections decreased STAT3 phosphorylation in vitro. We also showed that mice infected with CV-B4 had an altered expression of Igf2 isoforms as detected in TECs, followed by a decrease in the pro-IGF2 precursor in the thymus. Our study sheds new light on the intrathymic regulation of Igf2 transcription during CV-B4 infections and supports the hypothesis that a viral infection can disrupt central self-tolerance to insulin by decreasing Igf2 transcription in the thymic epithelium.

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