Cultures Of Endometrial Epithelial Cells Research Articles

Porcine reproductive and respiratory syndrome virus induces tight junction barrier dysfunction and cell death in porcine glandular endometrial epithelial cells

Reproductive failure caused by porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by embryonic death and weak-born piglets and is associated with placental cell apoptosis and impairment of endometrial integrity. Here, we aimed to determine whether endometrial epithelial barrier function and viability were altered following PRRSV type 1 or type 2 infection. PRRSV inoculation was examined at the apical or basolateral side of porcine glandular endometrial epithelial cell cultures isolated from 4- to 6-month-old PRRSV-free herd gilts (n = 7 pigs). On the apical side, four days postinfection (4 dpi) with type 2 PRRSV, transepithelial electrical resistance decreased by 31% ± 5%, and paracellular permeability to fluorescein isothiocyanate-dextran (4 kDa) increased by 10-fold as compared with the mock and type 1 infection. Real-time polymerase chain reaction results revealed that both PRRSV types upregulated the mRNA expression of the barrier builder tight junction protein (TJ) Cldn5, but downregulated pore-forming TJ Cldn7. Additionally, the expression of other TJ genes, i.e., Cldn3 and Cldn8, was differentially increased by PRRSV type 1 and that of zonula occludens-1 was increased by PRRSV type 2. MTT assays indicated an increase in porcine glandular endometrial epithelial cell culture at 2–6 dpi following type 2 infection. Analysis of apoptosis using Annexin/propidium iodide staining combined with flow cytometry showed that the percentage of viable cells decreased, accompanied by a significantly higher dead cell population following PRRSV type 2 infection at 2–4 dpi. PRRSV type 1 infection also induced dead cells (>4%) at 2 dpi; however, the cell population recovered at 4 dpi. In conclusion, PRRSV type 2 infection caused more severe TJ barrier dysfunction and reduced cell viability compared with PRRSV type 1 infection in the porcine endometrium. Impairment in the membrane integrity of the maternal glandular endometrium may be the underlying mechanism of PRRSV-induced reproductive failure in pregnant sows.

Culture of endometrial epithelial cells collected by a cytological brush in vivo

In cattle, mechanistic studies of endometrial function rely on cell lines or primary culture of cells harvested postmortem. Understanding the endometrial physiology in dairy cows is essential, because approximately 50% of pregnancies are lost in the first 3 wk of gestation for unknown reasons. The objective was to validate an in vivo, minimally invasive, and estrous cycle stage–specific method to obtain endometrial luminal epithelial cells for culture. The uterine body of 26 cows was sampled using a cytology brush (cytobrush) 4 d after estrus. The viability of cells was measured by flow cytometry (80% live cells) and epithelial identity was determined by anti-vimentin and anti-cytokeratin immunofluorescence and quantitative PCR for KRT18 and VIM. A pool of cells from 15 animals was passaged 4 times in culture until confluent and then treated with 0, 0.1, 1, or 10 ng/mL of recombinant bovine interferon-tau (rbIFN-τ). The relative expression of transcripts related to IFN-τ signaling (IFNAR1), early (IRF2) and late (ISG15, OAS1) response to IFN-τ stimulus, and other IFN-τ–stimulated genes (CCL8, CXCL10, and FABP3) was measured by quantitative PCR. The relative expression of KRT18 transcripts was similar across passages; the relative expression of VIM increased at passage 2, and IFNAR1 transcripts decreased in cultured compared with that in fresh cells. The relative expression of ISG15, OAS1, CCL8, and FABP3 increased in response to rbIFN-τ. In conclusion, culture of endometrial luminal cells collected by cytobrush was feasible, generating a monolayer enriched in epithelial cells, and therefore constitutes a novel model by which to study endometrial luminal epithelial cell function, including responses to IFN-τ.

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

BackgroundThin 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.MethodsWe 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.ResultsCo-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.ConclusionsWe 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.

Isolation, Culture, and Characterization of Primary Bovine Endometrial, Epithelial, and Stromal Cells for 3D In Vitro Tissue Models.

Efficient isolation, characterization, and culture of endometrial epithelial cells and stromal fibroblasts from calf uteri collected at the slaughterhouse is key to develop useful 3D culture tissue models to investigate uterine physiology and pathology without the need of performing invasive procedures to recover tissue samples.Here we provide a detail methodology that gives consistently pure and viable populations of distinct primary bovine endometrial cells.

Exosomes and soluble secretome from hormone-treated endometrial epithelial cells direct embryo implantation.

A successful pregnancy requires a synchronous dialogue between endometrium and embryo within the endometrial milieu. The aim of this study was to assess the role in the implantation of mediators in the endometrial milieu. Total secretome (TS), soluble secretome (SS) and small extracellular vesicles (containing exosomes) were generated from hormonally primed human endometrial epithelial cell culture medium. Human trophectoderm stem cell-derived spheroids were cultured with TS, SS or exosomes (30 µg/ml) on hormonally primed epithelial cells, with exosomes significantly increasing cell adhesion and outgrowth. Furthermore, F1 mouse 2-cell embryos were cultured in groups for 48 h followed by culture with each secretome fraction (30 µg/ml) for 48 h. Blastocyst cell number and hatching were quantified. In addition, blastocysts were further cultured on a fibronectin matrix for 72 h or transferred to recipient mice (with corresponding secretomes) with embryo implantation assessed after 6 days. Exosomes significantly increased total cell number in mouse embryos and complete hatching from zona pellucida, with both exosomes and SS significantly enhancing mouse embryo outgrowth. Importantly, exosomes increased the embryo implantation rate in comparison to other secretome fractions (normalized based on treatment amount) from the endometrial epithelia. These data indicate that endometrial epithelial exosomes support embryo growth, development and implantation while the SS has selective involvement specifically on mouse embryo outgrowth. This finding provides new insights into the molecular differences of endometrial secretome components in implantation and early embryo development and may implicate endometrial exosomes in the pathophysiology of implantation failure in infertility.

Melatonin activity and receptor expression in endometrial tissue and endometriosis.

Are melatonin receptors (melatonin receptor 1A (MR1A) and melatonin receptor 1B (MR1B)) expressed in human endometrium and endometriotic tissue, and does melatonin affect endometrial cell proliferation? Melatonin receptors are expressed in human eutopic endometrium, endometriomas and peritoneal lesions, although to different extents, and melatonin treatment attenuated estradiol-induced endometrial epithelial cell proliferation in culture. Melatonin decreased endometriotic lesion volume in a rat model of endometriosis. Melatonin treatment reduced pain scores in and analgesic use by women with endometriosis. Basic science study using human endometrial tissue and an endometrial epithelial cell line. Measurement of melatonin receptor expression (mRNA and protein) in women with surgically confirmed endometriosis (endometrioma (n = 20) or peritoneal lesion (n = 11) alone) and women without surgical evidence of endometriosis (control, n = 15). Collection of endometrial and endometriotic tissue samples, gynecologic history and demographic information. Quantification of estradiol (1.0nM) and melatonin (0.1nM-1.0μM) ± estradiol-induced endometrial epithelial cell proliferation in cultures of endometrial epithelial cells (CRL-1671) following 24 and 48hours of culture. MR1A and MR1B were localized by immunohistochemistry in glandular epithelial cells of endometrial biopsies from women with and without endometriosis. Both receptors were expressed in eutopic and ectopic endometrial tissue. mRNA expression of MR1A and MR1B was significantly greater in peritoneal lesions than in either endometriomas or eutopic endometrium. However, protein expression of MR1A was decreased in peritoneal lesions compared to control eutopic endometrium, whereas MR1B expression did not differ between the groups. Melatonin (0.1nM-1.0μM) treatment inhibited estradiol (1.0nM)-induced endometrial epithelial cell proliferation at 48hours but not 24hours of culture. Beneficial effects of melatonin seen in culture have yet to be comprehensively evaluated in women with endometriosis. Our data suggest that melatonin may be useful as an adjunct to current endometriosis treatments. This study was supported by the Canadian Institutes of Health Research (grant MOP142230 to W.G.F.). A.A.M. is supported by a resident research grant through the Physicians Services Incorporated Foundation. The authors have no conflicts of interest.

Hypomethylation of the GSTM1 promoter is associated with ovarian endometriosis.

Is the methylation status of the glutathione S-transferase M1 (GSTM1) promoter region altered in patients with ovarian endometriosis, and does this affect the expression of GSTM1 in their endometrial tissues? The promoter region of GSTM1 was significantly hypomethylated in the ectopic and eutopic endometrium of patients with ovarian endometriosis and this was associated with higher expression of GSTM1 mRNA. GSTM1, a member of the glutathione S-transferase family, is primarily known as a detoxification enzyme, but it has also been shown to negatively regulate apoptosis-related signalling cascades through protein-protein interactions with apoptosis signal-regulating kinase-1. This is a case-control study between September 2013 and December 2016, involving 65 patients with ovarian endometriosis and 53 women without endometriosis. We analysed the methylation status and expression levels of GSTM1 in the ectopic and eutopic endometrium of patients with ovarian endometriosis and the endometrium of women without endometriosis. In addition, we collected endometrial samples from 12 women without endometriosis for endometrial epithelial cell cultures. Methylation levels of the GSTM1 promoter region in the ectopic and eutopic endometrial tissues of patients with ovarian endometriosis and the endometrial tissues of women without endometriosis were analysed by pyrosequencing. The expression of GSTM1 mRNA and protein in endometrial tissues was investigated by RT-qPCR and immunohistochemistry, respectively. Primary cell culture, gene transfection, Cell Counting Kit-8 assay and flow cytometry were used to analyse the effect of GSTM1 on viability and apoptosis in endometrial epithelial cells. Compared with that in the endometrium of women without endometriosis, the GSTM1 promoter region was significantly hypomethylated in the ectopic and eutopic endometrium of patients with ovarian endometriosis. Additionally, GSTM1 mRNA and protein levels were significantly higher in the ectopic and eutopic endometrium than in the control endometrium. Moreover, the methylation levels of the GSTM1 promoter region were significantly negatively correlated with the mRNA expression of GSTM1. Furthermore, in vitro results suggested that the over-expression of GSTM1 could significantly increase viability and inhibit apoptosis in endometrial epithelial cells following hormone treatment and withdrawal. Due to restrictions in the isolation and culture of pure populations of endometrial epithelial cells, as well as limitations in the number of passages possible in primary cells, we could not explore the underlying molecular mechanism by which GSTM1 modulates apoptosis in endometrial cells. This study provides new evidence to support the notion that endometriosis may be an epigenetic disease. This work was supported by grants from the Natural Science Foundation of Hebei Province (Grant number: H2018206200) and the Department of Education of Hebei Province (Grant number: CXZZBS2017114). The authors have no conflicts of interest to declare.

Sexual Dimorphism of miRNAs Secreted by Bovine In vitro-produced Embryos.

Sexual dimorphism of bovine blastocysts has previously been observed through differences in development, cell death, metabolism, telomere length, DNA methylation, and transcriptomics. However, dimorphism in the secretion of miRNAs to culture media has not yet been evaluated. The objectives of this study were to determine if sex-specific blastocyst miRNA secretion occurs and to further investigate the role these miRNAs may have in the interaction between a blastocyst and the maternal environment. In vitro embryo culture was performed and media from male and female blastocysts was collected into sex-specific pools. Profiling of 68 miRNAs revealed a total of eight miRNAs that were differentially expressed between female and male-conditioned media. Validation by qPCR confirmed higher expression of miR-22 (P < 0.05), miR-122 (P < 0.05), and miR-320a (P < 0.05) in female media for three additional biological replicates. To examine the potential roles of secreted miRNAs to the media in communication with the maternal environment, miR-22, miR-122, and miR-320a were each supplemented to four replicates of primary bovine endometrial epithelial cell culture. Uptake of miR-122 (P < 0.05) and miR-320a (P < 0.05) was detected, and a trend of uptake was detected for miR-22 (P > 0.05). Further, expression of the progesterone receptor transcript, a predicted target of all three miRNAs, was found to be upregulated in the cells following supplementation of miR-122 (P < 0.05) and miR-320a (P < 0.05), and a trend upregulation of the transcript was observed following miR-22 (P > 0.05) supplementation. This work demonstrates that male and female conceptuses are able to differentially secrete miRNAs at the blastocyst stage and that these miRNAs have the ability to induce a transcriptomic response when applied to maternal cells. This knowledge builds on the known dimorphic differences in conceptuses at the blastocyst stage and demonstrates a role for blastocyst-secreted miRNAs in cell–cell communication.

The Flavonol Quercetin Improves Endometrial Barrier Function through the Expression of Tight Junction Proteins

Quercetin, the most abundant dietary flavonoid, shows the promotive and protective effects on intestinal tight junction (TJ) barrier function; however, little has been known about its effects in endometrium. The present study aims to investigate the potential effect of quercetin on endometrial TJ barrier function using the immortalized porcine glandular endometrial epithelial cell culture. The TJ barrier function was determined by transepithelial electrical resistance (TER) and fluorescein isothiocyanate‐dextran flux (FD‐4 and FD‐40) across the monolayer. The mRNA expression of TJ proteins, claudin‐2, ‐4, ‐5 and cytoskeletal zona occludens‐1 (ZO‐1) was evaluated by real‐time reverse transcription‐polymerase chain reaction. Quercetin (1, 10 and 100 μM) showed a concentration‐dependent increase in TER with markedly observed in the first 1–2 h. However, treatment with quercetin for 48 h had no significant change in the TER but a markedly decrease in FD4 and FD40 fluxes. Treatment with quercetin (10 μM) for 48 h also increased claudin‐2, ‐4 ‐5 and ZO‐1 mRNA whereas higher concentration only increased the ZO‐1 expression. Thus the increased claudin‐4, ‐5 and ZO‐1 appeared to promote the TJ assembly leading to decreased paracellular permeability. The present results indicate the promotive effects of quercetin on endometrial barrier function and possibly innate immune defense protecting against invading pathogens.Support or Funding InformationThis work was supported by Srinakharinwirot University Research Grant.

The involvement of osteopontin and matrix metalloproteinase- 9 in the migration of endometrial epithelial cells in patients with endometriosis.

BackgroundEndometriosis, which shares certain characteristics with cancers, may cause abnormal expression of proteins involved in cell migration. Endometrial epithelial cells (EECs) are believed to play an important role in endometriotic migration. The aim of this study was to investigate the relationship between the expression of osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9) in endometriotic migration.MethodsWe performed primary culture of EECs and investigated the expression of OPN and MMP-9 in EECs regulated by 17beta-estradiol (E2). OPN-specific siRNA interference was used to down-regulate OPN and to explore the corresponding change in MMP-9 expression. Real-time RT-PCR, western blot analysis and flow cytometry were used to determine the expression levels of OPN and MMP-9. Gelatin zymography was performed to observe the enzymatic activity of MMP-9 in conditioned media. Transwell and wound scratch assays were performed to investigate the migration ability of EECs.ResultsThe expression levels of OPN and MMP-9 in normal EECs (NEECs) were inferior to those in EECs from patients with endometriosis (EEECs). The expression levels of OPN and MMP-9 from stage III/IV EEECs and secretory-phase EECs were higher than those of stage I/II EEECs or proliferative-phase EECs. The expression levels of OPN and MMP-9 in EEECs were increased by E2 treatment and remarkably decreased by siRNA interference. Active MMP-9 expression increased with E2 treatment and decreased with siRNA treatment in EEECs compared with the same treatments in NEECs. The migratory abilities of EEECs were enhanced after cells were treated with E2; in contrast, these abilities were reduced by siRNA interference. In NEECs, active MMP-9 and cellular migration abilities were only minimally influenced by E2 and siRNA treatment.ConclusionsThe present study suggests that the up-regulation of MMP-9 via activation of OPN induced by estrogen may correlate with the migration of endometrial epithelial cells in patients with endometriosis.

Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts.

Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency.

Effects of conjugated linoleic acids on prostaglandin secretion by bovine endometrial epithelial cells in vitro

To determine the effects of 2 conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12) on synthesis of prostaglandin (PG) E(2) and F(2α) and expression of prostaglandin H synthase-2 (PGHS-2) of adult and fetal bovine endometrial epithelial cells in vitro. Primary cultures of endometrial epithelial cells obtained from 4 adult cows and 4 fetal bovine carcasses. Cells were exposed to 0, 50, 100, or 200μM cis-9, trans-11 or trans-10, cis-12 CLA isomers for 24 hours. Culture media collected before and after 6 hours of stimulation of cells with phorbol 12-myristate 13-acetate were assayed to detect PGE(2) and PGF(2α) via ELISA. After stimulation, cells were collected for western blot analysis to quantify PGHS-2. Concentrations of PGF(2α) and PGE(2) were significantly lower in culture media of adult and fetal endometrial epithelial cells exposed to any concentration of either CLA than they were in media of cells not exposed to CLAs. The trans-10, cis-12 CLA isomer seemed to decrease PG production more markedly than did the cis-9, trans-11 CLA isomer. Most concentrations of both CLAs significantly reduced culture media PGE(2):PGF(2α) concentration ratios of cells. Exposure of cells to CLAs did not affect expression of PGHS-2 protein. Results of this study indicated CLAs significantly decreased PGF(2α) and PGE(2) concentrations and PGE(2):PGF(2α) concentration ratios for cultures of adult and fetal endometrial epithelial cells with no apparent effect on PGHS-2 expression. Similar effects in cows could have effects on maternal recognition of pregnancy and immune function.

Regulatory mechanism underlying cyclic changes in mouse uterine bicarbonate secretion: role of estrogen

Our previous study has demonstrated that bicarbonate in the uterine fluid plays an indispensable role in sperm capacitation. However, the cellular mechanisms underlying the formation of bicarbonate-rich uterine fluid and the regulatory mechanism remained largely unknown. In this study, the expression profiles of bicarbonate transport/production proteins, the cystic fibrosis transmembrane conductance regulator (CFTR), SLC26A6, carbonic anhydrase 2 (CAR2, CA2) and CAR12 (CA12), throughout the estrous cycle, were examined in the mouse uterus by western blot. The results showed that the maximum expression levels of the proteins examined were observed at estrus. Luminal surface pH measurements showed that the resting uterine surface pH at estrus was significantly higher than that at diestrus, which could be reduced significantly by CFTR blocker, diphenylamine-2,2'-dicarboxylic acid, SLC26A6 inhibitor, 4',4'-diisothiocyanostilbene-2',2'-disulfonic acid, and CA inhibitor, acetazolamide. In ovariectomized mice and primary culture of endometrial epithelial cells, estrogen could upregulate CFTR, SLC26A6, CAR2, and CAR12 expression with a corresponding increase in the bicarbonate-dependent short-circuit current (I(sc)) and endometrial surface pH. The present results have demonstrated dynamic changes in uterine bicarbonate secretion and expression of the proteins involved in bicarbonate secretion during the estrous cycle and suggested a novel role of estrogen in regulating uterine bicarbonate transport, which may be important for successful reproduction.

Effect of vascular endothelial growth factor and interleukin-1β on apoptosis in endometrial cell cultures from patients with endometriosis and controls

The aim of this study was to evaluate the effect of vascular endothelial growth factor (VEGF) and interleukin-1beta (IL-1beta) on apoptosis induced by leuprolide acetate (LA) in endometrial epithelial cell cultures from patients with endometriosis. Primary endometrial epithelial cell cultures were obtained from uterine endometrial biopsies of patients with endometriosis and control women. Endometrial epithelial cells were incubated with LA; a combination of LA and VEGF; a combination of LA and IL-1beta; or in basal conditions. LA was added 3h prior to addition of VEGF and IL-1beta. After stimulation, the percentage of apoptotic cells was evaluated by the acridine orange-ethidium bromide technique and Bax expression was assessed by western blot. Treatment with LA enhanced the percentage of apoptotic cells in endometrial epithelial cells from subjects with endometriosis and control subjects. Addition of either VEGF or IL-1beta after exposure to LA restored the percentage of apoptotic cells to basal levels. Moreover, treatment with LA increased Bax expression in endometrial epithelial cells from patients with endometriosis. This effect was reverted by the addition of either VEGF or IL-1beta. Our results show that VEGF and IL-1beta reduce apoptosis and decrease Bax expression in endometrial epithelial cells from patients with endometriosis. This study suggests that VEGF and IL-1beta may protect endometriotic cells from undergoing apoptosis in addition to exerting their pro-angiogenic role.

Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system

To compare embryologic and clinical outcomes in terms of preimplantation development, implantation, pregnancy rates, and secretome profile of implanted blastocysts from the preimplantation genetic diagnosis program grown in sequential versus endometrial epithelial cell (EEC) coculture system. Retrospective clinical study and prospective experimental study. In vitro fertilization clinical unit and university research laboratory. Blastomere biopsy, embryo culture, blastocyst transfer, and protein analysis of the media conditioned from implanted embryos obtained from coculture and sequential systems. Clinical study: blastocyst, implantation, and gestation rates in own and donated oocytes. Experimental study: differential protein analysis of implanted embryos grown in coculture system versus sequential system. Of the 12,377 embryos analyzed, the blastocyst rates were 56.0% versus 45.9% in the coculture versus the sequential system, respectively, with own oocytes. With ovum donation, the rates were 70.5% versus 56.4%, respectively. Reproductive outcomes in terms of pregnancy rates (39.1% vs. 27.5%) and implantation rates (33.3% vs. 20.9%,) were statistically higher in EEC coculture versus sequential media. Furthermore, the protein profile of the EEC coculture versus the sequential system was obtained. Interleukin-6 (IL-6) was the most secreted protein by the EEC culture. Further ELISA experiments showed that the IL-6 present in the sequential medium diminished in implanted blastocysts. The coculture system favors blastocyst development and implantation rates, given the contribution of the factors secreted by endometrial epithelial cells, such as IL-6.

Seminal plasma stimulates cytokine production in endometrial epithelial cell cultures independently of the presence of leucocytes

We examined the effects of normal and leucocyte-positive semen on cytokine expression in endometrial epithelial cell cultures. Cytokines in pooled seminal plasma (SP) samples of men with normal semen parameters without (n = 9) and with leucocytospermia (LCS) (n = 9) were determined. Cultures of epithelial endometrial cells of women (n = 7) in the secretory phase were incubated with 10% SP for 24 h. Cytokines in culture supernatants and mRNA concentrations in the cultured cells were determined. Mean concentrations of interleukin (IL)-1beta and transforming growth factor (TGF)-beta1 were significantly higher (P < 0.05) in culture media with SP of men with normal semen parameters and LCS compared with control. Accordingly, a significant increase (P < 0.05) in mRNA concentrations (amol ml(-1)) of IL-1beta and TGF-beta1 could be detected. The mean TGF-beta1 and IL-1beta concentrations in the culture supernatant with 10% SP from patients with normal semen parameters were 24-fold (P < 0.05) and 2-fold higher respectively when compared with control. The mean protein concentration of TGF-beta1 measured in the supernatant with SP of men with LCS was not significantly reduced as compared with the supernatant with normal SP. In conclusion, our experiments support the concept of an endometrial sensitation effect by semen.

Application of the ciclooxygenase-2 inhibitor, celecoxib, for the treatment of endometriosis: an in-vitro study

Cyclooxygenase-2 (COX-2) specific inhibitors block cell growth and induce apoptosis and cell arrest in various tumour cell lines. Also, it has been shown that COX-2 inhibitors, such as celecoxib, are potent inhibitors of angiogenesis, both in-vitro and in-vivo. We have previously shown that celecoxib inhibits proliferation and induces apoptosis in cultured endometrial epithelial cells (EEC) from patients with endometriosis (EDT) and controls. In the present study we evaluated the effect of celecoxib on in-vitro vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) secretion, as well as on COX-2 expression in EEC from EDT patients. Experimental in vitro study. Endometrial biopsies were taken during the proliferative phase in 13 untreated patients with EDT diagnosed by laparoscopy. Purification and culture of EEC were done according to the technique described by Meresman et al. (Fertil Steril, 2003). Cultures were treated with celecoxib in doses between 25 and 100 mM. COX-2 expression was assesed by western blot and immunocytochemistry. VEGF and PGE2 secretion were evaluated by ELISA. Statistical analysis was done by Kruskal-Wallis nonparametric ANOVA test, and Dunn´s multiple comparisons test. COX-2 expression was shown by immunocytochemistry in all celecoxib treated EEC cultures and in basal conditions. Western blot analysis for COX-2 showed no statistical change in protein expression in 25–75 mM celecoxib treated EEC. However, at 100 mM there was a significant increase in COX-2 expression (P<0.05). To determine whether COX-2 activity was affected by celecoxib treatment, PGE2 production was measured in conditioned medium from EEC after celecoxib treatment (25, 50 and 100 mM). All doses of celecoxib significantly inhibited COX-2 activity (P<0.001 vs. basal). We also evaluated the levels of VEGF in EEC treated with celecoxib or vehicle (basal). Celecoxib treatment significantly reduced VEGF levels in EEC from patients with EDT (25 mM, P<0.001; 50 mM, P<0.05 and 100 mM, P<0.001 vs. basal). Our data suggest that removal of negative feedback by celecoxib treatment results in COX-2 induction in EEC from patients with EDT. We also showed that treatment with celecoxib reduced VEGF levels as well as PGE2 production. These findings support the investigation of COX-2 inhibitors as a treatment option in EDT.

Sequential co-culture of preimplantation embryos with oviductal epithelial cells first is more effective than that with endometrial epithelial cells first in human

To address the physiological relevance of the process that fertilized embryos gradually move from the oviduct to the uterine cavity during their development into the blastocyst. We studied preimplantation embryo development in vitro using two sequential co-culture conditions. One hundred and two surplus frozen embryos (G2 or G3 based on the Veeck's classification) in the IVF program were used for the co-culture. Such embryos at 4 cell stage were randomly allocated into two groups as follows: the oviductal (2 days)-endometrial (2 days) sequential co-culture group (OEG) and the endometrial (2 days)-oviductal (2 days) sequential co-culture group (EOG). SV40TAg-transfected oviductal or endometrial epithelial cell lines were used for co-culture. Cells were cultured in RPMI1640 supplemented with 10% FCS. The medium was replaced 24 h before co-culture was started. Only cells at 80% confluence in 6-well plates (Falcon) were used for embryo co-culture. Conditioned medium for 48 h cell culture was assayed for bFGF, HGF (hepatocyte growth factor), TGF-β1, IGF-I, IL-1β, IL-4, IL-6 and IL-8 with EIA (for bFGF, TGF-β1, IL-1β, IL-6) or IRMA (for IGF-I) or ELISA (for IL-4, IL-8 and HGF). Differences in embryonic development between OEG and EOG were compared with Fisher's exact test. Differences in concentrations between culture conditions were compared with two-tailed t-test. Of 46 and 43 frozen-thawed embryos with good quality (3 or 4 blastomeres were viable after thawing) in OEG and EOG, 31 (67.4%) and 26 (60.5%) embryos reached the morula stage (N.S.), 23 (50.0%) and 12 (27.9%) embryos reached the early blastocyst stage (P<0.05), and 8 (17.4%) and 6 (14.0%) reached the expanded blastocyst stage (N.S.), respectively. IL-1β and HGF concentrations were significantly higher in the conditioned medium of the oviductal epithelial cell culture. IL-6 and IL-8 concentrations were significantly higher in the conditioned medium of the endometrial epithelial cell culture. IGF-I and IL-4 were not detected in each assay. Although both oviduct and uterus are developed from the Müllerian tubercle, each tissue might play differentiated roles in the preimplantation embryo growth. We could not show clear difference in the development into expanded blastocyst stage possibly due to the limitation of our co-culture conditions that did not include hormonal regulation or stromal cells.

Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls

Our purpose was to evaluate the effect of the GnRH agonist (GnRHa), leuprolide acetate (LA), and the GnRH antagonist (GnRHant), Antide, on apoptosis and expression of apoptosis-related proteins in endometrial epithelial cell (EEC) cultures from patients with endometriosis and controls (infertile women without endometriosis). Biopsy specimens of eutopic endometrium were obtained from 22 patients with endometriosis and from 14 women that served as controls. Apoptosis was examined in EEC after incubation with LA and Antide. Bax, Bcl-2, Fas and FasL expression was evaluated after exposure to LA, Antide or a combination of both. The percentage of apoptotic cells (%ApC) was assessed by the acridine orange-ethidium bromide technique, and protein expression was evaluated by western blot and immunocytochemistry. LA 100 and 1000 ng/ml increased the %ApC in EEC from patients with endometriosis (both P < 0.05) and controls (p < 0.05 and P < 0.01, respectively). Antide 10(-5) M increased the %ApC in EEC from patients with endometriosis and controls (P < 0.01). In EEC from women with endometriosis, Bax expression increased after treatment with LA, Antide and LA + Antide (P < 0.05, P < 0.001 and P < 0.001), whereas Bcl-2 expression decreased after exposure to LA and Antide (P < 0.001 and P < 0.01). FasL expression increased after LA, Antide and LA + Antide treatments (P < 0.01, P < 0.001 and P < 0.01). No significant changes were observed on Fas expression. GnRH analogues enhanced apoptosis in EEC, and this was accompanied by an increase in expression of the pro-apoptotic proteins Bax and FasL and a decrease in expression of the anti-apoptotic protein Bcl-2.

P-346: Ciclooxygenase-2 (COX-2) inhibitor induces apoptosis and inhibits cell proliferation in endometrial epithelial cells from patients with endometriosis (EDT)

There is room for improvement in the medical therapy of EDT. COX-2 inhibitors are lately emerging as a new generation of therapeutic drugs in cancer treatment in combination with chemotherapy. Celecoxib, a potent selective COX-2 inhibitor, strongly suppresses cell proliferation and induces cell death in different tumor cell lines and in oncogenic animal models (Bundred N et al, Br J Cancer,93 Suppl 1:S10, 2005). In addition, Efstathiou et al. have shown that Celecoxib significantly suppresses endometriosis in a murine model (Fertil Steril 83:171, 2005). In this context, the purpose of this study was to evaluate the effect of Celecoxib on cell proliferation, apoptosis and COX-2 protein expression in endometrial epithelial cell (EEC) cultures from patients and controls. Apoptosis, cell proliferation and COX-2 expression were evaluated in Celecoxib treated EEC from EDT patients and controls. Endometrial biopsies were taken during the proliferative phase in 14 untreated patients with EDT diagnosed by laparoscopy and in 8 Controls (without EDT). Purification and culture of EEC were done according to the technique described by Meresman et al. (Fertil Steril 80, suppl 2:702, 2003). Cultures were treated with Celecoxib in different doses between 10 and 100μM. Percentages of apoptotic cells were measured using the acridine orange - ethidium bromide technique and cell proliferation was evaluated by 3H-Thymidine incorporation. Preliminary studies were performed to assess COX-2 expression by western blot in proteins extracted from 25, 40, 50, 75 and 100 μM Celecoxib treated EEC cultures from patients with endometriosis. Comparisons among groups were performed by Kruskal-Wallis nonparametric ANOVA test, and Dunn′s multiple comparisons test. Celecoxib 50, 75, and 100 μM induced apoptosis and inhibited cell proliferation in EEC from patients with EDT and controls. Lower concentrations of Celecoxib did not induce any significant changes in cell proliferation rates and apoptosis levels. We found no differences in any of parameters studied between cell cultures from patients and controls (see table 1, Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs. basal). In addition, COX-2 expression was similar in all Celecoxib treated EEC cultures.Tabled 1 Our data showed that Celecoxib produced a significant and positive effect on apoptosis and cell proliferation on EEC. This growth inhibitory effect appears to be independent of the COX-2 protein expression. These findings support the further investigation of COX-2 inhibitors as a treatment option in EDT.