scholarly journals The effects of hyaluronate-containing medium on human embryo attachment to endometrial epithelial cells in vitro

2020 ◽  
Vol 2020 (2) ◽  
Author(s):  
Peter T Ruane ◽  
Chelsea J Buck ◽  
Phoebe A Babbington ◽  
Wedad Aboussahoud ◽  
Stéphane C Berneau ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Transfer ◽  
Human Embryo ◽  
Research Network ◽  
Human Embryos ◽  
Ishikawa Cell ◽  
Practitioner Training ◽  
Cell Layers ◽  
Endometrial Epithelial Cells

Abstract STUDY QUESTION Does embryo transfer medium containing hyaluronate (HA) promote the attachment phase of human embryo implantation? SUMMARY ANSWER HA-containing medium does not promote human blastocyst attachment to endometrial epithelial cells in vitro. WHAT IS KNOWN ALREADY Embryo transfer media containing high concentrations of HA are being used to increase implantation and live birth rates in IVF treatment, although the mechanism of action is unknown. STUDY DESIGN, SIZE, DURATION Expression of HA-interacting genes in frozen-thawed oocytes/embryos was assessed by microarray analysis (n = 21). Fresh and frozen human blastocysts (n = 98) were co-cultured with human endometrial epithelial Ishikawa cell layers. Blastocyst attachment and the effects of a widely used HA-containing medium were measured. PARTICIPANTS/MATERIALS, SETTING, METHODS Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Blastocyst-stage embryos were transferred at day 6 to confluent Ishikawa cell layers; some blastocysts were artificially hatched. Blastocyst attachment was monitored from 1 to 48 h, and the effects of blastocyst pre-treatment for 10 min with HA-containing medium were determined. MAIN RESULTS AND THE ROLE OF CHANCE Human embryos expressed the HA receptor genes CD44 and HMMR, hyaluronan synthase genes HAS1–3, and hyaluronidase genes HYAL1–3, at all stages of preimplantation development. Attachment of partially hatched blastocysts to Ishikawa cells at 24 and 48 h was related to trophectoderm grade (P = 0.0004 and 0.007, respectively, n = 34). Blastocysts of varying clinical grades that had been artificially hatched were all attached within 48 h (n = 21). Treatment of artificially hatched blastocysts with HA-containing medium did not significantly affect attachment at early (1–6 h) or late (24 and 48 h) time points, compared with control blastocysts (n = 43). LIMITATIONS, REASONS FOR CAUTION Using an adenocarcinoma-derived cell line to model embryo-endometrium attachment may not fully recapitulate in vivo interactions. The high levels of blastocyst attachment seen with this in vitro model may limit the sensitivity with which the effects of HA can be observed. WIDER IMPLICATIONS OF THE FINDINGS Morphological trophectoderm grade can be correlated with blastocyst attachment in vitro. HA-containing medium may increase pregnancy rates by mechanisms other than promoting blastocyst attachment to endometrium. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by a grant from the Wellbeing of Women, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the Department of Health Scientist Practitioner Training Scheme, and the Ministry of Higher Education, The State of Libya. None of the authors has any conflict of interest to declare.

scholarly journals Coculture of Human Embryos with Autologous Human Endometrial Epithelial Cells in Patients with Implantation Failure1

1999 ◽  
Vol 84 (8) ◽  
pp. 2638-2646
Author(s):  
Carlos Simón ◽  
Amparo Mercader ◽  
Juan Garcia-Velasco ◽  
George Nikas ◽  
Carlos Moreno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Transfer ◽  
Cardiac Activity ◽  
Oocyte Donation ◽  
Pregnancy Rates ◽  
Blastocyst Transfer ◽  
Human Embryos ◽  
Blastocyst Formation ◽  
Ovum Donation ◽  
Endometrial Epithelial Cells

We have developed a coculture system with autologous human endometrial epithelial cells (AEEC) that retained many features of human endometrial epithelium. Implantation failure (IF; >3 previous cycles failed with 3–4 good quality embryos transferred) is a distressing condition in which 2-day embryo transfer repetition is the routine option. The objective of this study was to investigate the basics and to evaluate prospectively the clinical value of embryo coculture on AEEC and blastocyst transfer with their own oocytes [in vitro fertilization (IVF) patients] or with donated oocytes (oocyte donation patients) compared to a routine day 2 embryo transfer for patients with IF. Scanning electron microscopy and mouse embryo assays demonstrate that EEC from fertile and IF patients were morphologically and functionally similar; similar findings were observed in EEC obtained from fresh or frozen endometria. Clinically, 168 IVF cycles were performed in 127 patients with 3.8 ± 0.2 previously failed cycles, and 80 cycles were performed in 57 patients undergoing oocyte donation with 3.0 ± 0.2 previously failed cycles. Twenty IVF patients and 15 ovum donation patients with 3 previously failed cycles in whom a 2-day embryo transfer was performed were used as controls. In 88% of ovum donation cycles, at least 2 blastocysts were available for transfer, with 60.1% blastocyst formation; 2.2 ± 0.1 blastocysts were transferred/cycle, and 36 pregnancies (determined by fetal cardiac activity) were obtained (32.7% implantation and 54.5% pregnancy rates). In 168 IVF cycles, 8.1 ± 0.2 embryos/cycle started coculture, resulting in 49.2% blastocyst formation; 2.3 ± 0.2 blastocysts were transferred/cycle, and 29 clinical pregnancies were obtained (11.8% implantation and 20.2% pregnancy rates). Fifteen cycles were canceled (9%). In oocyte donation patients with IF undergoing 2-day embryo transfer, implantation and pregnancy rates were significantly lower (4.5% and 13.3%; P < 0.01) than with coculture; however, in IVF patients with IF, results with day 2 transfer (10.7% and 35%) were similar to those with coculture. The present study demonstrates that coculture of human embryos with AEEC and blastocyst transfer is safe, ethical, and effective and constitutes a new approach to improve implantation in patients with IF undergoing ovum donation, but not in IVF patients.

scholarly journals Embryonic Regulation of Integrins β3,α 4, and α1 in Human Endometrial Epithelial Cells in Vitro1

1997 ◽  
Vol 82 (8) ◽  
pp. 2607-2616 ◽  
Author(s):  
Carlos Simón ◽  
MarÍa José Gimeno ◽  
Amparo Mercader ◽  
José Enrique O’Connor ◽  
José RemohÍ ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Adhesion Assay ◽  
Fertilization Program ◽  
Human Blastocyst ◽  
Embryonic Regulation ◽  
Endometrial Epithelial Cells

In the present study, we examined the embryonic regulation ofβ 3 integrin in human endometrial epithelial cells (EEC) at the protein level and analyzed putative embryonic factors responsible for this regulation. The model employed is based on a clinical in vitro fertilization program in which single human embryos were cocultured with EEC until blastocyst stage and then transferred back to the uterus. After embryo transfer, EEC wells were divided according to the embryonic status reached: EEC with embryos that achieved the blastocyst stage, EEC with arrested embryos, and EEC without embryos. Immunostaining for β3 was positive in plasma membrane of EEC. Flow cytometry showed a mean percentage ofβ 3-stained cells of 24.1 ± 5.7 in EEC cocultured with embryos that achieved the blastocyst stage (n = 13) vs. 9.5 ± 1.6 (P < 0.05) in those EEC cultured with arrested embryos (n = 12). Immunostaining for α1 and α4 integrins was negative in EEC monolayers studied, regardless of the presence or absence of embryos, and these findings were confirmed by flow cytometry. The possibility that the embryonic IL-1 system and leukemia inhibitory factor were involved in the endometrial β3 up-regulation was investigated by neutralizing experiments demonstrating a significant inhibition of β3-stained cells when EEC monolayers were cultured in the presence of EEC/blastocyst-conditioned media with (n = 4) vs. without (n = 8) antihuman interleukin (IL)-1α + IL-1β (1.65% vs. 14.6%; P < 0.05). Dose-response experiments further demonstrated an up-regulation of β3 positive cells when IL-1α + IL-1β were added to the medium at a concentration of 10 pg/mL compared with control medium without added cytokines (40% vs. 20%, n = 4). The functional relevance of the EEC β3 up-regulation was tested using a mouse blastocyst adhesion assay. More mouse blastocysts attached to EEC previously in contact with human blastocyst (72.7%) compared with those EEC previously in contact with arrested embryos (40%). Our results demonstrate the selective effect of a developing human embryo on EEC expression of β3, which is maximal when a human blastocyst instead of an arrested embryo is considered. Furthermore, the embryonic IL-1 system seems to be involved in the EECβ 3 up-regulation, reinforcing the concept of precise paracrine cross-talk between blastocyst and endometrial epithelium during embryonic implantation.

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 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 Growth factor induced proliferation, migration, and lumen formation of rat endometrial epithelial cells in vitro

2016 ◽  
Vol 62 (3) ◽  
pp. 271-278 ◽  
Author(s):  
Md. Rashedul ISLAM ◽  
Kazuki YAMAGAMI ◽  
Yuka YOSHII ◽  
Nobuhiko YAMAUCHI
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Lumen Formation ◽  
Endometrial Epithelial Cells

scholarly journals Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells

2005 ◽  
Vol 34 (2) ◽  
pp. 517-534 ◽  
Author(s):  
S Hombach-Klonisch ◽  
A Kehlen ◽  
P A Fowler ◽  
B Huppertz ◽  
J F Jugert ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Estrogen Receptor Alpha ◽  
Hormone Receptors ◽  
Steroid Receptors ◽  
Steroid Hormone ◽  
Three Dimensional ◽  
Steroid Hormone Receptors ◽  
Endogenous Estrogen ◽  
Endometrial Epithelial Cells

Information on the regulation of steroid hormone receptors and their distinct functions within the human endometrial epithelium is largely unavailable. We have immortalized human primary endometrial epithelial cells (EECs) isolated from a normal proliferative phase endometrium by stably transfecting the catalytic subunit (hTERT) of the human telomerase complex and cultured these hTERT-EECs now for over 350 population doublings. Active hTERT was detected in hTERT-EECs employing the telomerase repeat amplification assay protocol. hTERT-EECs revealed a polarized, non-invasive epithelial phenotype with apical microvilli and production of a basal lamina when grown on a three-dimensional collagen–fibroblast lattice. Employing atomic force microscopy, living hTERT-EECs were shown to produce extracellular matrix (ECM) components and ECM secretion was modified by estrogen and progesterone (P4). hTERT-EECs expressed inducible and functional endogenous estrogen receptor-alpha (ER-alpha) as demonstrated by estrogen response element reporter assays and induction of P4 receptor (PR). P4 treatment down-regulated PR expression, induced MUC-1 gene activity and resulted in increased ER-beta transcriptional activity. Gene activities of cytokines and their receptors interleukin (IL)-6, leukemia inhibitory factor (LIF), IL-11 and IL-6 receptor (IL6-R), LIF receptor and gp130 relevant to implantation revealed a 17 beta-estradiol (E2)-mediated up-regulation of IL-6 and an E2- and P4-mediated up-regulation of IL6-R in hTERT-EECs. Thus, hTERT-EECs may be regarded as a novel in vitro model to investigate the role of human EECs in steroid hormone-dependent normal physiology and pathologies, including implantation failure, endometriosis and endometrial cancer.

Treatment of Intrauterine Adhesions with Human Amnion Mesenchymal Stromal Cells (hAMSCs) on Promoting Endometrial Regeneration and Repair Via Notch Signaling Pathway Regulation

2020 ◽  
Author(s):  
Jie Yu ◽  
Wenwen Zhang ◽  
Jiayue Huang ◽  
Yating Gou ◽  
Congcong Sun ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Notch Signaling ◽  
Signaling Pathway ◽  
Notch Signaling Pathway ◽  
Intrauterine Adhesions ◽  
Epithelial Markers ◽  
Qrt Pcr ◽  
Endometrial Epithelial Cells

Abstract Background: Human amniotic mesenchymal stem cells(hAMSCs) can repair and improve the damaged endometrium which its aplastic disorder is the main reason for intrauterine adhesions(IUAs).Methods: We conducted in vivo and in vitro experiments. In vivo experiments: 45 female Sprague-Dawley(SD) rats were involved and randomized equally into Sham group, IUA group, Estradiol(E2) group, hAMSCs group, and E2 + hAMSCs group. The effect of hAMSCs and E2 only or combined was evaluated by Hematoxylin-eosin(HE) and Masson staining. The expression of epithelial markers and key proteins of Notch signaling pathway by Immunohistochemistry. In vitro experiments: Firstly, the hAMSCs cells were taken and divided into control group and induced group in which hAMSCs were differentiated into endometrial epithelial cells in induced microenvironment, and extracted their RNA respectively. The expression of epithelial markers and Notch1 messenger RNA (mRNA) was detected by Real-time quantitative polymerase chain reaction(qRT-PCR). and the changes in expression position of Notch intracellular domain(NICD) and expression amount of target gene, hairy enhancer of split 1(Hes1) were detected by Immunofluorescence. Then, Activated and inhibited the Notch signaling pathway while induction, and detected mRNA expression of hAMSCs epithelial markers by quantitative real-time polymerase chainreaction (qRT-PCR) respectively and detected hAMSCs cell cycle by flow cytometric. Results:This study showed that hAMSCs alone or combined with E2 could promote endometrial repair, and Notch signaling pathway a great role in it. And otherwise, the activation or habitation of Notch signaling pathway determines whether hAMSCs could differentiate into endometrial epithelial cells or not.Conclusion: we concluded that activate the Notch signaling pathway promote the differentiation of hAMSCs into endometrial epithelial cells, and further treat IUAs.

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