Sarah Elliott

Expression and function of Toll-like receptors in human endometrial epithelial cell lines

In mammals, Toll-like receptors (TLRs) are the principal family of innate immune pattern recognition receptors (PRRs). The main function for TLRs is the detection of molecular patterns associated with invading pathogens. We investigated TLR expression and function in three established human endometrial epithelial cell lines, including hTERT-EEC, HEC-1B and Ishikawa cells, and clarified the application of these endometrial cell lines as in vitro models for studying TLR expression and function in the female reproductive tract. TLR gene expression was examined by RT-PCR and protein localization by immunohistochemistry. Our results showed that TLR expression in these cell lines is comparable to published literature on TLR expression in primary human endometrial tissue. TLR function was investigated by the detection of IL-6 and IL-8 production by ELISA in response to TLR2, TLR3, TLR5, TLR7 and TLR9 ligands. We found that hTERT-EEC cells were responsive to TLR5 ligand and HEC-1B cells respond to TLR3 and TLR5 ligands. In contrast, Ishikawa cells respond only to PMA/I which was used as a positive control for IL-8 production. Finally, we investigated the influence of flagellin as a TLR5 stimulant on TLR5 expression in these cell lines by QPCR. Our results showed that the endometrial cell lines showed a tendency for increased TLR5 expression in response to flagellin stimulation and in hTERT-EEC cells this tendency was statistically significant. These results suggest that hTERT-EEC, HEC-1B and Ishikawa cell lines can be used as in vitro models to investigate innate immune responses of endometrial cells in the female reproductive tract.

Altered patterns of differentiation in karyotypically abnormal human embryonic stem cells.

Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.

Activation of Toll-like receptor 5 decreases the attachment of human trophoblast cells to endometrial cells in vitro

BACKGROUND Embryo implantation in the uterus involves the trophoblast cells apposing and adhering to, then invading across the epithelium lining of the endometrium. However, ethical concerns regarding experimentation with primary human tissue during this period of life necessitates creation of in vitro models for understanding the basic mechanisms involved. Toll-like receptors (TLRs) play a crucial role in defence against pathogens invading the female reproductive tract. The objective of this study is to establish and optimize an in vitro model for studying human endometrial embryonic interactions and to understand the effect of TLR5 stimulation on the attachment of trophoblast cells to endometrial cells. METHODS By using a human telomerase immortalized endometrial epithelial cell line (hTERT-EECs) and choriocarcinoma human trophoblast cells (JAr cells), an in vitro assay of human implantation was established. In order to investigate the impact of TLR5 stimulation on attachment in this assay, bacterial flagellin was applied to the endometrial and trophoblast cells. In order to block TLR5 in the endometrial and trophoblast cells, TLR5 function-blocking antibody was applied to the cells prior to flagellin treatment. RESULTS The results demonstrated that JAr spheroids attached to hTERT-EECs in a time and concentration-dependent manner. Our results also demonstrated that treatment of endometrial cells with flagellin, suppressed the attachment of JAr spheres to the endometrial cells. Application of TLR5 function-blocking antibody significantly restored the attachment of JAr spheres to the endometrium. CONCLUSIONS These data suggest a novel mechanism by which the presence of intrauterine infection through TLR5 activation may result in implantation failure. These data may provide a new opportunity in the management of infertility cases.

Serum relaxin levels are reduced in pregnant women with a history of recurrent miscarriage, and correlate with maternal uterine artery Doppler indices in first trimester

OBJECTIVES Defective implantation is a mechanism for recurrent pregnancy loss (RPL). We sought to determine whether the serum expression of human relaxin-2 (RLX) is impaired in women with a history of RPL. STUDY DESIGN Employing a prospective case-controlled design we studied 20 pregnant women with a history of RPL and 20 age-matched women with no history of RPL (NRPL). We measured serum relaxin-2 levels by ELISA at 6-8, 10-12, 20, and 34 weeks gestation and in cord blood, and maternal uterine artery Doppler resistance index (RI) at >or=10 weeks gestation. RESULTS Relaxin rose to a peak at 12 weeks, and gradually declined towards term. At all gestations, women with a history of RPL had lower RLX levels than women without. At 10-12 weeks gestation, uterine artery RI correlated with serum RLX for both RPL and NRPL. In the NRPL group at 10-12 weeks the presence of a notched waveform was associated with higher RLX levels than the absence of a notch (mean 2.1 ng/ml vs. 1.3 ng/ml, P<0.05) and also at 20 weeks (2.1 ng/ml vs. 0.95 ng/ml, P<0.05) but no such difference was seen in the RPL group. Umbilical venous RLX was 4-fold higher in the RPL group than the NRPL group. CONCLUSION Women with a history of RPL demonstrate attenuated levels of serum RLX across all pregnancy trimesters. How dysregulated RLX metabolism may contribute to adverse pregnancy outcome in RPL requires further investigation.

Circulating levels of matrix proteases and their inhibitors in pregnant women with and without a history of recurrent pregnancy loss

BackgroundWe have recently shown that serum relaxin-2 levels are attenuated in women with a history of recurrent pregnancy loss (RPL). We sought to determine whether a history of RPL is also associated with changes in serum matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMP) -1 and -2.MethodsWe obtained serum from 20 pregnant women with a history of RPL and 20 age-matched pregnant women with no history of RPL (NRPL) at 6-8, 10-12, 20, and 34 weeks gestation, and from cord blood. We quantified total serum concentrations of MMP-1, MMP-3, MMP-9 and TIMP-1 and TIMP-2 by ELISA. We determined whether these serum marker levels were associated with a history of RPL and delivery before 37 weeks gestation.ResultsThere was no difference in the rates of miscarriage, preterm birth or prelabour rupture of fetal membranes between RPL and NRPL. However babies born to RPL were lighter than those born to NRPL. Serum MMP-1, 9, and TIMP-1 did not differ between RPL and NRPL but MMP-3 was higher in RPL vs. NRPL at 6-8 weeks (P < 0.05). Serum TIMP-2 levels were higher in RPL women at all gestations (P < 0.01). The ratio of RLX-2 (reported previously) to TIMP-2 at 10-12 weeks gestation was more strongly associated with a history of RPL than either peptide separately - area under the ROC curves for RLX-2 0.79 (95% CI 0.57 to 0.92), TIMP-2 0.83 (95% CI 0.63 to 0.95), and for RLX-2:TIMP-2 ratio 0.92 (95% CI 0.74 to 0.99).ConclusionsWomen with a history of RPL demonstrate increased serum TIMP-2 and reduced RLX-2 during a subsequent viable pregnancy. Determination of both markers in early pregnancy enhances the discrimination of women with a history of RPL. These observations suggest roles for these two peptides in early implantation and placental development. Whether these may prove to be reliable early predictive markers for subsequent pregnancy loss in the index pregnancy is unknown and will require further studies.

Activation of Toll-like receptor 3 reduces actin polymerization and adhesion molecule expression in endometrial cells, a potential mechanism for viral-induced implantation failure

STUDY QUESTION Does activation of endometrial Toll-like receptor 3 (TLR 3) affect cell receptivity to trophoblast adhesion? SUMMARY ANSWER TLR 3 activation in vitro reduces the attachment of trophoblast cells to endometrial cells by altering the cell cytoskeleton and reducing the expression of adhesion molecules in human endometrial cells. WHAT IS KNOWN ALREADY It is well documented that the presence of an infection at the time of implantation can lead to implantation failure. The female reproductive tract recognizes invading micro-organisms through the innate pathogen recognition receptors such as the TLRs. STUDY DESIGN, SIZE, DURATION Poly I:C was used as a TLR 3-specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. The experiments were performed in three replicates on three separate days. PARTICIPANTS/MATERIALS, SETTING, METHODS An in vitro assay was developed using RL95-2 (a human endometrial cell line) and JAr (a human trophoblast cell line) cells. Initially, the percentage of attached JAr spheroids to RL95-2 was measured in response to TLR 3 activation. Next, actin polymerization in RL95-2 cells was assessed in response to TLR 2/6, 3 and 5 activation. Phalloidin was used to assess the mean fluorescence intensity of F-actin by flow cytometry or confocal microscopy. Secondly, the influence of TLR 2/6, 3 and 5 activation on the expression of cluster of differentiation 98 (CD98) and β3 integrin was determined. To further understand through which pathways the TLR 3-induced alterations occur, inhibitors were applied for Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), myeloid differentiation primary response 88 (MYD88), mitogen-activated protein kinases (MAPK) and nuclear factor pathways. MAIN RESULTS AND THE ROLE OF CHANCE We observed that stimulation of TLR 3 in endometrial cells with different concentrations of Poly I:C led to a reduction in the percentage of trophoblasts attached to the endometrial cells in a dose-dependent manner (P < 0.05). This decrease was consistent in the Poly I:C treated group regardless of the co-incubation time (P < 0.05). In addition, our results demonstrated that actin polymerization and CD98 expression significantly decreased only in response to TLR 3 activation (P < 0.05). Activation of endometrial cells with TLR 2/6, 3 and 5 significantly reduced β3 integrin expression (P < 0.05). These alterations were shown to work via MYD88-MAPK pathways (P < 0.05). LIMITATIONS, REASONS FOR CAUTION This study has been performed in vitro. Future in vivo studies will be required in order to confirm our data. WIDER IMPLICATIONS OF THE FINDINGS This is a novel discovery which extends our current knowledge concerning diagnosis and treatment of viral-induced infertility cases. STUDY FUNDING/COMPETING INTERESTS This research was supported by the COST Action FA1201 (GEMINI) by granting a Short Term Scientific Mission and the Instituto de Salud Carlos III by granting Grant PI11/01645. The authors have no conflict of interest to declare.

Local Activation of Uterine Toll-Like Receptor 2 and 2/6 Decreases Embryo Implantation and Affects Uterine Receptivity in Mice

ABSTRACT Embryo implantation is a complex interaction between maternal endometrium and embryonic structures. Failure to implant is highly recurrent and impossible to diagnose. Inflammation and infections in the female reproductive tract are common causes of infertility, embryo loss, and preterm labor. The current work describes how the activation of endometrial Toll-like receptor (TLR) 2 and 2/6 reduces embryo implantation chances. We developed a morphometric index to evaluate the effects of the TLR 2/6 activation along the uterine horn (UH). TLR 2/6 ligation reduced the endometrial myometrial and glandular indexes and increased the luminal index. Furthermore, TLR 2/6 activation increased the proinflammatory cytokines such as interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 in UH lavages in the preimplantation day and IL-1 receptor antagonist in the implantation day. The engagement of TLR 2/6 with its ligand in the UH during embryo transfer severely affected the rate of embryonic implantation (45.00% ± 6.49% vs. 16.69% ± 5.01%, P < 0.05, control vs. test, respectively). Furthermore, this interference with the embryo implantation process was verified using an in vitro model of human embryo implantation where trophoblast spheroids failed to adhere to a monolayer of TLR 2- and TLR 2/6-activated endometrial cells. The inhibition of TLR receptors 2 and 6 in the presence of their specific ligands restored the ability of the spheroids to bind to the endometrial cells. In conclusion, the activation of the innate immune system in the uterus at the time of implantation interfered with the endometrial receptivity and reduced the chances of implantation success.

Effects of spermatozoa-oviductal cell coincubation time and oviductal cell age on spermatozoa-oviduct interactions.

The oviduct plays a crucial role in sperm storage, maintenance of sperm viability and sperm transport to the site of fertilisation. The aim of the present study was to investigate the effects of oviductal cell culture passage number, oviductal cell age and spermatozoa-oviduct coincubation times on gene expression in oviductal cells. Immortalised oviductal epithelial cells (OPEC) obtained from two different cell passages (36 and 57) were subcultured three times with and without spermatozoa for 24 h (control group). In a second study, OPEC were cocultured with spermatozoa for different time intervals (0, 4, 12 and 24 h). Expression of adrenomedullin (ADM), heat shock 70 kDa protein 8 (HSPA8) and prostaglandin E synthase (PGES) in OPEC was measured by quantitative polymerase chain reaction. The expression of ADM and HSPA8 was decreased significantly in OPEC cells from Passage 57, particularly in the later subculture group. These effects on HSPA8, but not ADM, expression in OPEC were further altered after coculture with spermatozoa for 24 h. We also demonstrated that spermatozoa-oviduct coculture for 12 and 24 h resulted in significantly higher expression of ADM, HSPA8 and PGES in OPEC. Overall, the data suggest that the OPEC lose some of their properties as a result of oviductal cell aging and that there are spermatozoa-oviduct interactions leading to increased oviductal cell gene expression.

Characterisation of an in vitro system to study maternal communication with spermatozoa

In vivo, gamete maturation, fertilisation and early embryonic development take place inside the oviduct. Several studies have indicated that local responses towards gametes and embryos are generated by the maternal reproductive tract. However, no defined in vitro model currently exists to allow detailed and systematic investigation of maternal communications with gametes and embryos. Therefore, we characterised an in vitro model based on the interaction of boar spermatozoa with an immortalised porcine oviduct epithelial cell line to evaluate different factors that may affect this model. The factors tested were sperm viability, source of spermatozoa, cell passage effect and the effect of reproductive and non-reproductive epithelial cells in the interaction with spermatozoa. After 24 h of co-incubation, RNA was extracted and used to synthesise cDNA for quantitative real-time PCR. Alteration in the expression of genes such as adrenomedullin, heat-shock 70-kDa protein 8 and prostaglandin E synthase was considered as the end point of this assay. The results showed that sperm viability and cell passage number had an effect on oviductal gene expression in response to spermatozoa. Oviductal cells showed significant alterations in gene expression when compared with non-reproductive epithelial cells. The simple in vitro system described here has potential application for further studies in our understanding of mechanisms involved in maternal interactions with spermatozoa.

Understanding the dynamics of Toll-like Receptor 5 response to flagellin and its regulation by estradiol

Toll-like receptors (TLRs) are major players of the innate immune system. Once activated, they trigger a signalling cascade that leads to NF-κB translocation from the cytoplasm to the nucleus. Single cell analysis shows that NF-κB signalling dynamics are a critical determinant of transcriptional regulation. Moreover, the outcome of innate immune response is also affected by the cross-talk between TLRs and estrogen signalling. Here, we characterized the dynamics of TLR5 signalling, responsible for the recognition of flagellated bacteria, and those changes induced by estradiol in its signalling at the single cell level. TLR5 activation in MCF7 cells induced a single and sustained NF-κB translocation into the nucleus that resulted in high NF-κB transcription activity. The overall magnitude of NF-κB transcription activity was not influenced by the duration of the stimulus. No significant changes are observed in the dynamics of NF-κB translocation to the nucleus when MCF7 cells are incubated with estradiol. However, estradiol significantly decreased NF-κB transcriptional activity while increasing TLR5-mediated AP-1 transcription. The effect of estradiol on transcriptional activity was dependent on the estrogen receptor activated. This fine tuning seems to occur mainly in the nucleus at the transcription level rather than affecting the translocation of the NF-κB transcription factor.