I-21: The Expression of TLRs in Follicular Cells of Poor Ovarian Responder

Background: One of the most annoying problems in IVF is poor ovarian response. It is anticipated that 5-18% of all IVF cycle are affected by poor response to ovarian hyperstimulation. Poor response to goandotropin may lead to decline in the pool of embryos for transfer or cryoperservation, and decrease pregnancy rates. Different mechanisms explain poor ovarian response for example decreased number of FSH receptors in granulosa cells, anti-FSH IgA and IgG potentially exerting a local FSH antagonizing effect in maturing follicles, the presence of a specific FSH receptor-binding inhibitor in the follicular fluid, the higher FSH threshold to stimulation follicle development. As mentioned above, likely immune system is involved in the mechanism of poor responder and affects the steroidogenesis. First line of immune system is pattern recognition receptors (PRRs) as a compartment of innate immunity. The most important group of PRRs which also identified in female reproductive tract is Toll like receptors (TLRs) family. So far, TLR1-10 are characterized in human. TLR1, 2, 4, 5, 6 and TLR10 are expressed on the cell membrane and they recognize lipid and protein ligands. TLR3, 7, 8 and TLR9 can detect nucleic acid from pathogens are found in endosomal compartment. In addition these receptors recognize a broad range of endogenous ligands including heat shock proteins, hyaloronan, host RNA, and reactive oxygen species (ROS). So TLRs play abundant immunologic and physiologic roles in reproductive system. Therefore, the aim of this study is to investigate TLR1.2, 3, 5, 7, 8 genes expression in follicular cells obtained from ovarian poor response women in compare to normal women. Materials and Methods: All procedures were approved by the Royan Ethics committee and informed consent was obtained prior to the collection of samples. Forty patients (20 infertile ovarian poor responder patients and 20 normal women with male factor infertility) underwent controlled ovarian stimulation with monitoring E2 levels and pelvic ultrasounds. Gonadotropin doses were then adjusted accordingly and monitoring was continued until patients received 10,000 IU hCG intramuscular. Oocyte retrieval was performed approximately 36 h after hCG. The follicular fluid was obtained from the largest follicle (>18 mm) visualized on ultrasound before using any flushing medium. This follicle was aspirated with a 17-gauge Cook needle attached to 100 mm Hg pump-operated aspirator. It was the first puncture of the oocyte retrieval. The follicular fluid was transferred to a sterile Petri dish, and the oocytes were then removed. The follicular fluid was placed into a 15- mL conical tube and centrifuged at 300g for 5 min. The supernatant was removed for further proteomic study. Total RNA was extracted separately from cellular pallet in each group using TRI reagent and treated with DNaseI. First strand cDNA synthesis was performed using oligo dT primers and Superscript II reverse transcriptase system. RT-PCR and Quantitative PCR was performed using the prepared cDNA and primer for TLR1-10. Relative TLR expression quantities were compared between two groups. The threshold cycle values were normalized against the threshold value of human β-actin. Differences in normalized expression values between samples were tested for significance using ANOVA statistical test. The results were expressed as mean ± SEM. The level of statistical significance was set at p < 0.05. Results: TLR1-10 genes were expressed in follicular cell of both, case and control groups. The mean relative expression of TLR1, 2, 4, 6 genes were significantly higher in poor ovarian responder, TLR5 and 8 expressions were higher in poor ovarian responder but not significant. TLR 3 , 7, 9 and TLR10 was lower in patients with poor ovarian responder in compare to normal women. Conclusion: Our findings suggested that TLRs are involved in pathophysiology of ovarian poor response. It’s been proposed that inflammatory markers such as IL6, IL8 and TNF-α are vary in poor responders. Since these markers are produced by TLRs signaling, therefore it’s possible that these changes are a result of TLRs activation in poor responders. Keywords: Follicular Cells, Innate Immunity, Ovarian Poor Response, TLR