Endometrial Coculture Research Articles

Human endometrial co-culture system reveals novel cell-specific effects of nonoxynol-9 on endometrial transcriptome and chemokine secretion

Detrimental effects of the spermicide Nonoxynol-9 (N9) on the lower female reproductive tract are well documented. However, whether N9 alters the endometrial transcriptome and chemokine secretion is unknown. Herein, we studied the effects of N9 on human endometrial epithelial cells (eEC) and stromal fibroblasts (eSF).

Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

BackgroundsPrevious studies suggested that endocrine disruptors (ED) are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking.MethodsBisphenol A (BPA) and Aroclor 1254 (polychlorinated biphenyls) were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations.ResultsAt 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro.ConclusionsIn conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors.

Increased expression of macrophage colony–stimulating factor and its receptor in patients with endometriosis

To investigate the expression and regulation of colony-stimulating factor 1 (CSF-1) and its receptor, C-FMS, in endometriosis. In vivo and vitro study. University-based academic medical center. Reproductive-age women undergoing surgery for benign conditions. Peritoneal and endometrial tissue samples were obtained. CSF-1 and C-FMS expression. Significantly higher CSF-1 levels were found in peritoneal fluid of patients with endometriosis compared with control subjects. Ectopic endometriotic tissue had 3.5-fold and 1.7-fold increases in CSF-1 and C-FMS expression, respectively, compared with eutopic tissue. Coculture of endometrial cells from either established cell lines or patient samples with peritoneal mesothelial cells (PMCs) led to increased expression of CSF-1 and C-FMS. A higher but nonsignificant increase in levels of C-FMS and CSF-1 was found in cocultures of endometrial epithelial cells from patients with endometriosis compared with those without endometriosis. Increased CSF-1 levels may contribute to endometriosis lesion formation and progression. Elevation in CSF-1 after coculture of endometrial cells with PMCs suggests that endometrial tissue may be a source of peritoneal CSF-1. Increased C-FMS expression in endometrial cells from women with endometriosis cocultured with PMCs suggests that endometrial tissue involved in lesion formation is highly responsive to CSF-1 signaling.

The use of autologous endometrial co-culture (AECC) improves outcome in young poor responders with multiple failed IVF cycles

Young women with multiple failed in vitro fertilization (IVF) attempts are often given a poor prognosis for success. The aim of this study is to analyze the effectiveness of AECC in improving pregnancy rates in young patients (<35) with multiple failed IVF cycles (>2) and proven poor response (<5 oocytes retrieved). Retrospective case control 120 patients were included in the study. 23 patients utilized AECC (cases) and 97 patients utilized traditional culture media (controls). Controls were matched for age, cycle response and ovarian reserve testing. All patients had < 5 oocytes retrieved in previous cycles. Patients undergoing AECC underwent luteal biopsy (5-12 days after LH surge) prior to the treatment cycle, with glandular (G) and stromal (S) endometrial cells isolated by enzymatic digestion and separated based on differential sedimentation rate. These cells were cryopreserved, then plated as a 50%/50% combination of G and S cells prior to embryo exposure. Patients utilizing AECC had all of their embryos grown on AECC until embryo transfer (ET). All patients underwent a day 3 ET. Paired t tests and Chi-square test were used in analysis. P<0.05 was considered statistically significant. Results are shown in table 1. Patients were similar in age and response. In the AECC group, more embryos developed and progressed to transfer. The AECC group also showed improved clinical pregnancy rates.Tabled 1AECC n = 23No AECC n = 97P valueAge33.1 ± 1.833.4 ± 2.5NSPrevious failed cycles4.3 ± 2.53.1 ± 1.30.042Oocytes4.1 ± 0.93.8 ± 1.0NSMature oocytes3.4 ± 1.13.4 ± 1.2NSFertilized 2PN2.7 ± 0.952.5 ± 1.2NSEmbryos progressed to ET*91%80%0.02Implantation rate26.1%14.2%NSClinical pregnancy43.5%21.7%0.03Table 1. *ET (Embryo transfer) Open table in a new tab Table 1. *ET (Embryo transfer) Utilization of AECC in young IVF patients with prior poor response and multiple failed IVF cycles appears to improve pregnancy outcome. A prospective randomized controlled trial is currently ongoing to evaluate this finding.

Co-Culture Techniques in Assisted Reproduction: History, Advances and the Future

The co-culture technique was first implemented in animal models in the mid-sixties in an attempt to improve the suboptimal conditions provided by culture media. Since then, autologous endometrial co-culture was introduced in human IVF, and different outcomes were reported regarding blastulation, fragmentation and implantation rates. Various growth factors and cytokines were proposed to be involved in the mechanism by which co-culture systems improved embryo development. However, randomized controlled trials are still warranted to provide evidence on the efficacy of endometrial co-cultures in the era of modern sequential- and monoculture media.

Genistein effects on stromal cells determines epithelial proliferation in endometrial co-cultures

Background Estrogen is the leading etiologic factor for endometrial cancer. Estrogen-induced proliferation of endometrial epithelial cells normally requires paracrine growth factors produced by stromal cells. Epidemiologic evidence indicates that dietary soy prevents endometrial cancer, and implicates the phytoestrogen genistein in this effect. However, results from previous studies are conflicting regarding the effects of genistein on hormone responsive cancers. Methods The effects of estrogen and genistein on proliferation of Ishikawa (IK) endometrial adenocarcinoma cells were examined in co-cultures of IK cells with endometrial stromal cells, recapitulating the heterotypic cell-to-cell interactions observed in vivo. The roles of estrogen receptor (ER)α and ERβ were evaluated using ERα and ERβ specific agonists. ER activation and cell proliferation in the IK epithelial cells were determined by alkaline phosphatase assay and Coulter counter enumeration, respectively. Results Both estrogen and genistein increased estrogen receptor-induced gene activity in IK cells over a range of concentrations. Estrogen alone but not genistein increased IK proliferation in co-cultures. When primed by estrogen treatment, increasing concentrations of genistein produced a biphasic effect on IK proliferation: nM concentrations inhibited estrogen-induced proliferation while μM concentrations increased proliferation. Studies with an ERβ-specific agonist produced similar results. Genistein did not influence the effects of estrogen on IK proliferation in monoculture. Conclusions Our study indicates that nutritionally relevant concentrations (nM) of genistein inhibit the proliferative effects of estrogen on endometrial adenocarcinoma cells presumably through activation of stromal cell ERβ. We believe that sub-micromolar concentrations of genistein may represent a novel adjuvant for endometrial cancer treatment and prevention.

Genistein effects on stromal cells determines epithelial proliferation in endometrial co-cultures

Genistein effects on stromal cells determines epithelial proliferation in endometrial co-cultures

On-Chip Single Embryo Coculture With Microporous-Membrane-Supported Endometrial Cells

In vitro culture (IVC) of the mammalian embryo is an essential technique in reproductive technology and other related life science disciplines. Although embryos are usually cultured in groups, a single embryo culture has been highly desired for IVC to investigate developmental processes. In this study, we proposed and developed the first single embryo coculture device, which allows making an array of a single embryo coculture with endometrial cells by controlling the culture environment in a microfluidic device. To realize this concept, we investigated three key issues: selection of a culture medium for the embryo coculture with endometrial cells using a mouse embryo and endometrial cells, evaluation of an on-microporous-membrane coculture of endometrial cells and an embryo to control the polarization of endometrial cells on the membrane, and evaluation of the coculture of endometrial cells and the embryo in the microfluidic device. We successfully obtained an array of a single coculture of embryo with endometrial cells in a microfluidic device. This concept will open and enhance the management of an individual embryo for assisted reproductive technology, livestock breeding, and fundamental stage research by further development.

Excessive ovarian stimulation up-regulates the Wnt-signaling molecule DKK1 in human endometrium and may affect implantation: an in vitro co-culture study

High serum estradiol (E2) levels following ovarian stimulation lead to reduced implantation and pregnancy rates, yet the underlying mechanisms remain unknown. We investigated if aberrant expression of genes in the Wnt-signaling pathway may be involved. Microarray and real-time PCR analysis were performed to analyze gene expression profiles of endometrial samples taken at day hCG + 7 in stimulated cycles, and days LH + 7 and LH + 10 in natural cycles. Expression of several Wnt-signaling transcripts, including Dickkopf homolog 1 (DKK1), DKK2 and secreted frizzled-related protein 4 (sFRP4), was analyzed throughout the menstrual cycle. JAr spheroid/Ishikawa endometrial cell co-culture experiments were established to study effects of DKK1 on spheroid attachment in vitro. We identified 351 differentially expressed genes. Endometrial samples taken at hCG + 7 had similar expression profiles to those at LH + 10. DKK1 transcripts were up-regulated and DKK2 and sFRP4 were down-regulated in the stimulated compared with LH + 7 group (all P < 0.05). DKK1 transcripts were low in proliferative phase (PS) and increased in late-secretory phase (LS, P < 0.05), although DKK2 peaked in mid-secretory phase (P < 0.05). sFRP4 transcripts were high in PS. Treatment of spheroid with recombinant human DKK-1 protein dose-dependently suppressed (P < 0.05 versus control) spheroids attachment onto endometrial cells (associated with decreased beta-catenin protein): this suppression was nullified by anti-DKK1 antibody. Gene expression patterns in stimulated cycles resembled those of LS in natural cycles, when the implantation window is about to close, suggesting high serum E2 and/or progesterone concentrations may advance endometrial development, altering the implantation window and possibly decreasing pregnancy rate. Aberrant expression of DKK1 might impair embryo attachment and implantation in vivo.

Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure

To confirm the beneficial effect of endometrial coculture in patients with repeated failures with assisted reproductive techniques (ART). Patients with previous failures were offered a repetition of ART in conjunction with autologous endometrial coculture. Private fertility center. Sixty-eight couples who had attempted 92 cycles of IVF or intracytoplasmic sperm injection without obtaining an evolutive pregnancy. Patients repeated one cycle of ART with concomitant endometrial coculture of their embryos. Comparative pregnancy and delivery rates in conventional ART cycles vs. cycles with autologous endometrial coculture. In the previous 92 cycles (146 ETs, fresh plus frozen) only 8 pregnancies were initiated, and all ended in spontaneous abortion. Upon repeating 68 cycles (76 ETs) using coculture, 39 pregnancies were obtained, of which 19 resulted in live births, 10 are ongoing evolutive pregnancies, and 10 ended in spontaneous abortions. These results confirm the usefulness of autologous endometrial coculture for the treatment of patients with repeated implantation failure.

The use of autologous endometrial co-culture in patients attempting assisted reproduction (AR) techniques for the first time

The use of autologous endometrial co-culture in patients attempting assisted reproduction (AR) techniques for the first time

Live births in poor prognosis IVF patients using a novel non-contact human endometrial co-culture system

Patients with repeated implantation failures or poor embryo quality may benefit from embryo culture using the co-culture technique; growth factors secreted by co-culture cells may act as survival factors. Autologous endometrial co-culture has been suggested as a safe alternative to animal cells for co-culture of human embryos. However, the technique is fairly labour intensive and its effectiveness can vary from patient-to-patient. This study presents clinical outcome data on a novel non-contact co-culture system using a human endometrial cell line rather than autologous tissue. Embryos from 316 poor prognosis patients with repeated IVF failures, previous cycles with poor embryo quality or advanced maternal age were cultured in Transwell chambers with a monolayer of endometrial cells. The clinical pregnancy rate in patients less than 39 years of age was 53% and for patients aged between 39 and 42 years it was 33%. To date, 76 patients have delivered 111 healthy infants with no congenital anomalies and 18 pregnancies are ongoing. This is the first report on the potential benefits of a non-contact co-culture system in the IVF laboratory. This study shows that an established human endometrial cell line can be used to obtain the benefits of co-culture without the potential disadvantages associated with using autologous endometrial tissue.

Effects of Tibolone Metabolites on Human Endometrial Cell Lines in Co-culture

In human endometrium, cell proliferation is regulated by ovarian steroids through heterotypic interactions between stromal and epithelial cells populating this tissue. The authors test the proliferative effects of tibolone and its metabolites using endometrial co-cultures that mimic the normal proliferative response to hormones. They found that both the Delta(4)-tibolone metabolite and the pure progestin ORG2058 counteract estradiol-driven epithelial cell proliferation. Surprisingly, the estrogen receptor binding 3-hydroxyl-metabolites of tibolone also counteracted estradiol-driven proliferation. Inhibition of proliferation by 3beta-OH-tibolone was abrogated by low doses of the progesterone receptor antagonist mifepristone. This suggests that 3beta-OH-tibolone is converted to a progestagenic metabolite. The authors found that the stromal cells used in the co-cultures express high levels of the ketosteroid dehydrogenase AKR1C2, which is able to oxidize 3beta-OH-tibolone back to tibolone. Thus, the unexpected progestagenic effect of 3beta-OH-tibolone in these co-cultures may be due to metabolic activity present in the stromal cells of the co-cultures.

Do cytokine levels in the supernatant from conditioned media (CM) of embryos grown in autologous endometrial coculture (AECC) correlate with pregnancy outcomes after in vitro fertilization (IVF)?

AECC has been shown to improve outcomes in patients with multiple failures after IVF. AECC works to enhance in vitro conditions in several ways including the elaboration of cytokines by endometrial cells. IL-2, IL-12p70, IL-4, IL-5 are known to be produced by the endometrium. The aim of this study was to determine the association of these cytokines with IVF outcomes. In 92 IVF patients, supernatant from AECC was tested for the presence of IL-2, IL-12p70, IL-4 and IL-5. From a luteal endometrial biopsy performed prior to IVF, glandular and stromal cells were isolated, separated, then plated in a 50/50 combination. After one week in which the culture medium was changed every 2–3 days, the cells were cryopreserved to be thawed 3 days prior to embryo placement. If more than 6 oocytes fertilized normally, embryos were randomly grown on AECC cells (75%) and conventional media (25%), otherwise, all embryos were grown on AECC. Immediately after embryo removal before transfer, the CM was collected and cryopreserved. Only CM after embryo exposure was tested as previous research has shown that the presence of embryos does not alter cytokine levels from CM with AECC. Cytokine levels were measured utilizing commercially available enzyme linked immunoassays. The average age of the patients was 37.2 ± 4.4 years. Mean numbers of MIIs, 2PNs, and embryos transferred were 12.0 ± 6.6, 7.3 ± 4.8, and 3.3 ± 1.4 respectively. The clinical pregnancy rate was 44.6% and implantation rate was 22.9%. Embryos grown on AECC demonstrated a significant improvement in the mean number of blastomeres and mean fragmentation rate when compared to embryos grown in conventional media (5.8 ± 1.2 vs. 5.0 ± 1.9; 10.5% ± 7.1 vs. 17.6% ± 13.8; P<0.01). The presence of IL-2, IL-12p70, IL-4 and IL-5 was detected in 84.8%, 94.6%, 98.9%, and 95.7% of samples, respectively. Although cytokine levels in the CM did not correlate with either mean number of blastomeres or mean fragmentation rate, IL-12p70 levels were decreased in CM from embryos of good quality in comparison to levels in embryos of poor quality (1.0 ± 1.0 vs. 2.0 ± 3.1; P<0.05). No association was found between cytokine levels and pregnancy outcomes. Significant improvement in blastomere number and fragmentation rate was achieved with AECC. All of the evaluated cytokines were expressed by cells in the AECC. Cytokine levels did not correlate with pregnancy outcomes. However, IL-12p70 levels were decreased in CM from good quality embryos.

A case control study evaluating autologous endometrial coculture (AECC) as an effective tool for young patients (age

Young women with multiple failed IVF cycles and poor ovarian reserve are generally given a grim prognosis for IVF success. CC of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures after IVF-ET. Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. Earlier studies have demonstrated improved embryo quality in patients utilizing AECC. In this study, we analyzed the effectiveness of AECC in improving pregnancy rates in 207 cycles of IVF-ET in women with multiple failed IVF cycles and poor ovarian reserve.

Autologous endometrial co-culture improves results in a population of patients with reduced rate of success in ART

Autologous endometrial co-culture improves results in a population of patients with reduced rate of success in ART

P-970: Autologous endometrial co-culture (AECC) in patients with IVF failure: Negative correlations of outcome with interleukin-2

Co-culture of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures. Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. The endometrial cells elaborate multiple cytokines including IL-2, IL-3 and IL-5. The specific aim of this study was to determine if IL-2, IL-3 and IL-5 levels as measured in the supernatants of conditioned media (CM) of embryos co-cultured in AECC is predictive of IVF outcome.

P-268: Can recurrent implantation failure cases be the target population for autologous endometrial coculture? Preliminary results from 389 cycles

Although in vitro fertilization techniques are now well established and being used worldwide, relatively lower success rates can partially be attributable to gamete and embryo quality as well as sub-optimal embryo culture conditions. Autologous endometrial coculture (AEC) is a recently proposed culture technique that can promote the development of embryos using patients own endometrial cells. In this study , a possible benefit of AEC in cases with at least two previous unsuccessful ART attempts (Recurrent Implantation Failure; RIF) were analyzed by a) comparing the embryo development in past and present cycles (conventional culture medium vs.

P-244: Co-culture of human embryos with a novel endometrial cell line : Clinical outcome data

Despite improvements in IVF culture media formulations, patients with repeated implantation failures or poor embryo quality may still potentially benefit from the coculture technique. Growth factors secreted by coculture cells may act as survival factors and aid in the development of struggling embryos. Autologous endometrial co-culture has been been suggested as a safe alternative to animal cells for co-culture of human embryos. The technique is fairly labor intensive, requiring endometrial biopsy and preparation of a patient’s cells before their scheduled retrieval.

O-10: Prospective randomized trial of autologous endometrial coculture versus traditional IVF: An interim analysis

One factor that may contribute to the poor success rates in IVF may be the suboptimal culture conditions in which fertilization and early embryonic growth occur. A significant amount of research has focused on tailoring the in vitro culture media to the requirements of the human embryo. Unfortunely, these media are devoid of many factors expressed in vivo (ie growth factors). Therefore, in an effort to more closely mimic the in vivo preimplantation environment the growth of embryos on a layer of cells has been developed (coculture).