{"paper_id":"0da32865-de47-463a-9cc9-bd19e255bbb6","body_text":"Infection within the upper regions of the female\nreproductive tract (FRT), particularly the fallopian\ntubes, can have serious consequences such as\nchronic pelvic inflammation, infertility and pregnancy\ncomplications ( 1 ,  2 ). For example, sexually\ntransmitted diseases (STDs) that infect the upper\nregions of the FRT are a major worldwide health\nproblem ( 3 ,  4 ). Approximately 8% of females\nannually develop pelvic inflammatory disease\n(PID) and after re-infection, this risk increases by\n40-70% ( 5 ) . It is thus of paramount importance that infectious agents are quickly recognised and\nremoved from the upper parts of the FRT. Characterization\nof the defense systems present within\nthe FRT will assist the development of effective\ntherapies or vaccination strategies against STDs.\nThe innate immune system is the first line of defense\nagainst infection. This system is able to identify\nwhat is foreign or non-self and produces adequate\nresponses that lead to the pathogens being\nsuppressed. Toll like receptors ( TLRs ) are a family\nof pattern recognition receptors that recognise\npathogen-associated molecular patterns (PAMP)\nand constitute a major part of the innate immune\nsystem ( 6 ,  7 ). Until now, eleven members of this\nreceptor family have been discovered in humans.\nOf these,  TLR1–9  are conserved between human\nand mouse. Using various methods including ectopic\nexpression of mammalian cDNA in cell\nlines, some of the activating ligands of  TLRs  have\nbeen discovered. Each individual  TLR  is known to\ndetect molecules (ligands) from varying classes of\nmicrobial agents ( 8 ,  9 ). Some  TLRs  like  TLR2  and\nits associated receptors  TLR1  and  TLR6  mainly\nreact against Gram-positive bacteria by detecting\nmolecules from mycobacteria and Gram-positive\nbacteria ( 10 - 12 ). Some PAMP, like lipoteichoic\nacid (LTA), can be detected only by  TLR2  ( 11 ).\nIn contrast,  TLR1  associates with  TLR2  to recognize\ntriacylated lipoproteins ( 13 ), whereas\n TLR2  together with  TLR6  detects diacylated\nlipoproteins and peptidoglycans ( 14 - 16 ).  TLR4 \nrecognises lipopolysaccharides (LPS) which\nare present in Gram-negative bacteria ( 17 - 19 ).\n TLR5  recognises bacterial flagellin ( 20 ). Other\n TLRs  mainly react against viruses. For example,\n TLR3  recognises RNA from double stranded\nRNA viruses ( 21 ,  22 ). Also,  TLR7  and 8 recognise\nRNA from single stranded RNA viruses and\nantiviral compounds such as imidazoquinolines\n( 16 ,  19 ,  23 ), whilst  TLR9  recognises unmethylated\nCpG DNA found richly in prokaryotic genomes\nand DNA viruses ( 19 ,  24 ). Like  TLR1 \nand  TLR6 ,  TLR10  is another  TLR2 -associated\nreceptor and also highly homologous to  TLR2 ,\nhowever, the function of this  TLR  is not completely\nunderstood ( 25 - 27 ). In addition, it was\nrevealed that the  TLR  pathway has physiological\nrelevance to human fertility  TLR  by playing\nroles in ovulation, sperm capacitation, fertilization\nand pregnancy ( 28 - 31 ). They were also\nshown to play a role in the pathophysiology of\nrelevant disorders including endometriosis and\npoor ovarian response ( 32 ,  33 ).\nSeveral studies have investigated the presence\nand the role of  TLRs  in the male and FRTs\n( 34 - 43 ). Furthermore, the expression of  TLRs \nin endometrial cell lines have been shown by\nAbussahoud et al. ( 44 ). It is evident that sex\nhormones modulate cells and their immune response\npotential, but varies throughout the FRT\n( 45 ). Reports by us and others have demonstrated\nthe existence of  TLRs  in FRT and the\ncycle-dependent expression of  TLRs  in the endometrium\n( 34 ,  46 ). The effect of estradiol has\nalso been showed in endometrial cells in several\nstudies ( 47 - 49 ).\nThe cycle-dependent expression of  TLRs  in FRT\nimplicates a role for sex hormones in regulation\nof  TLR  function in FRT. Here we used a fallopian\ntube epithelial cell line (OE-E6/E7) ( 50 ) to investigate\nthe role of two sex hormones (estradiol and\nprogesterone) in modulating  TLR  expression in\nthe fallopian tube. This cell line has characteristics\nof human fallopian tube epithelial cells, including\nmorphology, receptor expression and hormonal\nresponses ( 50 ). Thereafter, the effect of the sex\nhormones and their combination as well as their\nantagonists on expression of  TLR1-10  in fallopian\ntube cells was investigated. This was done by testing\n TLR  whether  TLR  expression is altered in the\npresence of sex hormones.\n\nThis investigation was an experimental study\napproved by the Royan Institute Ethics Committee.\nInformed written consent was obtained\nprior to the collection of tissue samples. Human\nfallopian tube tissues were collected from\n9 patients undergoing total abdominal hysterectomy\nfor benign gynaecological conditions.\nThe mean age of the women taking part in the\nstudy was 42 (range of 33-56) years with all in\nthe secretory phase of their menstrual cycle. For\ngenomic studies, fallopian tube tissue samples\nwere immediately placed in RNAlater (Ambion,\nUK) and stored for 24 hours at 4˚C followed\nby immersion and storage in liquid nitrogen until\nthe time of processing.\nAntibodies and peptides used in the experiments\nwere obtained from Santa Cruz Biotechnology\nInc. (USA). These were goat polyclonal\nantibodies specific for N-terminal domains of\n TLR1, TLR2, TLR3, TLR5  and  TLR6 , and a goat\npolyclonal antibody specific for the C-terminal\ndomain of  TLR4 . Blocking peptides specific for\nthe respective antibodies were used to detect\nnon-specific staining.\nFor immunostaining, OE-E6/E7 cells were\ncultured in four well chamber slides. They were\ncultured at 37˚C in Dulbecco’s Modified Eagle\nMediumF12 (DMEM-F12) culture medium (Invitrogen,\nUK) supplemented with 1% penicillin\nand streptomycin (Sigma-Aldrich, UK), 10% fetal\ncalf serum (FCS, Invitrogen, USA) and L-glutamine\n(Invitrogen, USA) in 5% CO 2  atmosphere.\nAt confluency, the slides were washed five times\nwith Ca 2+  and Mg 2+  free phosphate buffered saline\n(PBS, Gibco, USA), fixed with 5% formalin and\nstored at 4˚C until use.\nFormalin-fixed slides were washed in PBS and\nthen stained using a Vectastain Elite ABC peroxidase\nkit (Vector Laboratories Ltd, UK). In addition,\nto avoid non-specific binding, an avidin/\nbiotin blocking kit (Vector) was used. Briefly,\nslides were blocked for 1 hour at room temperature\nin PBS solution containing 0.2% v/v horse\nserum and 25% v/v avidin supplied in the blocking\nkit. The block was subsequently removed\nand slides were incubated for 2 hours at room\ntemperature with primary antibody at an appropriate\ndilution using antibody diluent media (Dakocytomation\nLtd, UK) and 250 ml biotin per ml\nof diluted antibody. Binding was then visualized\nby incubating the slide with peroxidase substrate\n3-amino-9-ethylcarbazole (AEC) (Vector) for 10\nminutes, washed in distilled water for 3 minutes\nand counterstained in 10% haematoxylin for 10\nminutes. Slides were finally washed in tap water\nfor 2 minutes and mounted with Aquamount\n(VWR International, UK).\nOptimal staining was achieved by incubating\nslides with different concentrations of  TLR  antibodies\n( TLR1, TLR2, TLR3, TLR4, TLR5, TLR6 \nwith 4,  4 ,  10 ,  10 , 4 and 10 μg/ml respectively).\nNegative control sections were obtained by blocking\nthe primary antibody with the corresponding\nspecific peptide. Immunostained sections were\nexamined using an Olympus BH2 microscope\n(Olympus, UK).\nTLR  expression in the OE-E6/E7 cell line was\ninvestigated and compared to fallopian tube tissue\nsamples. OE-E6/E7 cells were cultured at 37˚C in\nDMEM (F12) supplemented with 1% penicillin\nand streptomycin, 10% FCS and L-glutamine in\n5% CO 2  atmosphere. At confluency, cells were\nwashed with Ca 2+  and Mg 2+  free PBS and then harvested\nusing trypsin-Ethylenediaminetetraacetic\nacid (EDTA, Invitrogen, USA) pelleted by centrifugation\nat 300 g for 5 minutes. One ml of TRIreagent\n(Sigma, UK) was added onto the pellet\n(5×106 cells). Thereafter, total RNA from pelleted\ncells was extracted following the standard protocol\nsupplied by the manufacturer.\nOn the day of the experiment, fallopian tube tissues\nwere removed from RNAlater and homogenised\nin 3 ml of TRI reagent using an Ultra-Turrax\nhomogenizer for 2 minutes following the standard\nprotocol supplied by the manufacturer.\nTotal RNA obtained from OE-E6/E7 cells and\nfallopian tube tissue samples (by using chloroform\nand isopropanol) were then treated with DNase I\n(DNA-free TM , Ambion, USA) to remove genomic\nDNA contamination from the samples. First strand\ncDNA was synthesized by reverse transcription\nusing oligodT primers (Metabion, Germany) and\nSuperScript II (200 U/μl, Invitrogen, USA). RT\ncontrols were prepared without the enzyme (nonreverse-\ntranscribed controls).\nPolymerase chain reaction (PCR) was performed\nusing prepared cDNA, Platinum Blue\nPCR Super Mix (Invitrogen, USA) and primers\nfrom Metabion ( Table 1 ). The amplification\nwas run for 40 cycles under the following conditions:\ninitial heat at 95˚C for 30 secends, 59˚C\nto 65˚C for 30 seconds and final annealing at\n72˚C for 30 seconds. All experiments included\nreverse transciptase (RT) controls as well as\nnegative controls (no cDNA). PCR products\nwere visualised on a 1.2 % agarose gel. All amplified\nPCR products were sequenced to confirm\nthe identity of the amplified product.\nSequence of primers used in this study\nTLR ; Toll like receptor.\nTo investigate the effect of estradiol and progesterone\non  TLR  expression in the OE-E6/E7\ncell line, OE-E6/E7 cells were cultured again in\ntriplicates at 37˚C in DMEM (F12) culture medium\nand water soluble estradiol and progesterone\n(Sigma-Aldrich, UK) to reach the final concentrations\nof 0.1, 1, 10, 100 nM and 1, 10, 100, 1000\nnM for estradiol and progesterone respectively. In\naddition, the following combinations of these two\n(final concentrations) were used in four groups of:\ncontrol (C, without any additional treatment of sex\nhormones), menstruation (M, 1 nM progesterone\nand 0.1 nM estradiol), pre-ovulation (P, 6.5 nM\nprogesterone and 1.5 nM estradiol) and window of\nimplantation (W, 35 nM progesterone and 1 nM\nestradiol) in 5% CO 2  atmosphere in 75 ml flasks\nfor 24 hours in the absence of phenol red and serum.\nIn the next step, the effect of sex hormone antagonists\non  TLR  expression was evaluated. To do\nthis, OE-E6/E7 cells were divided in two groups.\nOne group was pre-treated for 2 hours with 1 μM\nICI 182, 780 (fulvestrant, an estradiol antagonist,\nSigma-Aldrich, UK) and the other pre-treated for 2\nhours with 0.1 μM RU486 (mifepristone, a progesterone\nantagonist, Tocris, USA). After pre-treatment,\nboth groups were treated again separately\nwith a combination of estradiol and progesterone\nbased on the four treatment groups (C, M, P and\nW) in 5% CO 2  atmosphere in 75 ml flasks for 24\nhours in the absence of phenol red and serum.\nCells were next treated for RNA isolation and\ncDNA synthesis following the same protocol mentioned\nabove. qRT-PCR was performed using the\ncDNA prepared from the estradiol and progesterone\ntreatment experiments, same primers as in table\n1 and SYBR Green Jump Start (Sigma, UK)\nmaster mix (containing 10 μl SYBR Green, 7 μl\nWater, 1 μl of each primer and 1 μl cDNA). The\nPCR amplification was performed under the following\nconditions: 50 cycles of 95˚C for 30 seconds,\n59˚C to 63˚C for 30 seconds and 72˚C for\n30 seconds. All experiments included RT controls\nand negative controls (no cDNA). qRT-PCR was\nperformed on a Mx3005P QPCR machine (Stratagene,\nGermany) and results were analyzed using\nMxPro QPCR software version 4.01. In preliminary\nexperiments, the efficiency of the primer sets\nof each Q-PCR reaction was established. Variation\nin  Beta-actin  and  GAPDH  expression as two\nhousekeeping genes was also tested.\nThe qRT-PCR data were analyzed using the\ncomparative CT method ( 51 ). The fold change was\ncalculated as FC=2 -ΔΔCT .\nThe results were expressed as mean ± SEM. Significance\ntesting was performed by one-way ANOVA\nwith Tukey’s multiple comparison test. P<0.05\nwas considered significant.\n\nFigure 1  shows the results of RT-PCR of  TLR1-\n10  genes in the human fallopian tube tissue.  Figure\n2  shows the results of RT-PCR of  TLR1-6  genes in\nOE-E6/E7 cells. Size of all amplified PCR products\nwere as predicted and sequencing verified\ncorrect amplification of each gene. No product was\namplified in negative control samples, indicating\nabsence of genomic DNA contamination.\nFormalin-fixed slides were used to study the distribution\nof  TLR1-6  in OE-E6/E7 cells. Positive immunostaining\nfor all six  TLRs  was observed with  TLR1 ,\n2, 4 and  TLR6  displaying moderate staining in this\nepithelial cell line. However, strong staining for  TLR3 \nand  TLR5  was observed. The immunocytochemical\nlocalisation of  TLR1-6  is shown in  figure 3 .\nA stable expression of  Human ß-actin  and  GAPDH \ngenes with variable hormonal treatments was observed\n( Fig .4 ).The quantitative expression profiles of\n TLR1-6  genes in OE-E6/E7 cells treated with varying\nconcentrations of estradiol and progesterone are\nshown in figures  5  and  6  respectively. The relative expression\nof  TLR1-6  genes did not significantly differ\nin response to different concentrations of either estradiol\nor progesterone. The mean relative expression of\n TLR1-6  differed considerably between P, M, W and\nC groups ( Fig .7 ). The Pattern of  TLR  expression in\nP, M and W were significantly different among P, M\nand W groups. Also, by using 1 μM of fulvestrant\n(estradiol antagonist) or 0.1 μM mifepristone (progesterone\nantagonist) 2 hours prior to the combined\nestradiol and progesterone treatment, the mean relative\nexpression of  TLR1-6  did not differ markedly in\nC, P, M and W groups (Figes .8 ,  9 ).\nThe expression of  TLR1-10  genes in the human fallopian tube tissue. Each pair of primers produced a specific product with the specific\npredicted size observed in the test (T) samples. C; Control samples (samples without using cDNA) and  TLR ;  Toll like receptor .\nThe expression of  TLR1-6  genes in the human fallopian tube cell line (OE-E6/E7). Each pair of primers produced a specific product with\nthe specific predicted size observed in the test (T) samples. C; Control samples (samples without using cDNA) and  TLR ;  Toll like receptor .\nImmunohistochemical staining of  TLR1-6  in the OE-E6/E7 cell line. Positive staining is red, negative staining is blue. Small inserts\nshow blocking of anti  TLR1-6  antibodies with respective specific peptides.  TLR ;  Toll like receptor .\nThe stable expression of GAPDH and β-actin in OE-E6/E7 under variable hormonal treatments. E; Estradiol, P; Progestrone, C; Control,\nM; Menstruation and W; Window of implantation.\nExpression of  TLR1-6  in OE-E6/E7cultured with estradiol. Mean ± SEM of normalized expression values (with β-actin) for  TLR1-6 \ngenes in OE-E6/E7 cultured with different concentrations of estradiol (control: 0 nM, E 0.1: 0.1 nM, E1: 1 nM, E10: 10 nM and E100: 100\nnM or 1 μM). No significant results were obtained.  TLR ;  Toll like receptor .\nExpression of  TLR1-6  in OE-E6/E7cultured with progesterone. Mean ± SEM of normalized expression values (with β-actin) for  TLR1-\n6  genes in OE-E6/E7 cultured with different concentrations of progesterone (control: 0 nM, P1: 1 nM, P10: 10 nM, P100: 100 nM and\nP1000: 1000 nM or 1 μM). No significant results were obtained.  TLR ;  Toll like receptor .\nExpression of  TLR1-6  in OE-E6/E7 cultured with a combination of estradiol and progesterone concentrations. Mean ± SEM\nof normalized expression values (with β-actin) for  TLR1-6  genes in OE-E6/E7 cultured with a combination of estradiol and progesterone.\nControl (C, without any additional treatment of sex hormone), menstruation (M, 1 nM progesterone and 0.1 nM estradiol), pre-ovulation\n(P, 6.5 nM progesterone and1.5 nM estradiol) and window of implantation (W, 35 nMprogesterone and 1 nM estradiol) (control: 0 nM, P1:\n1 nM, P10: 10 nM, P100: 100 nM and P1000: 1000 nM or 1 μM). Star denotes statistically significant differences.  TLR ;  Toll like receptor .\nExpression of  TLR1-6  in OE-E6/E7 precultured with fulvestrant, then cultured with a combination of estradiol and progesterone\nconcentrations. Mean ± SEM of normalized expression values (with β-actin) for  TLR1-6  genes in OE-E6/E7 pre-cultured with fulvestrant\nfor 2 hours, then cultured with a combination of estradiol and progesterone in 4 groups (C, M, P, W). No significant results were obtained.\n TLR ;  Toll like receptor .\nExpression of  TLR1-6  in OE-E6/E7 pre-cultured with mifepristone and cultured with a combination of estradiol and progesterone in\n4 groups (C, M, P, W). Mean ± SEM of normalized expression values (with β-actin) for  TLR1-6  genes. No significant results were obtained.\n TLR ;  Toll like receptor .\n\nEpithelial cells are the first layer of defense against pathogens ascending FRT. Considering their role in protecting the tract from the external environment,  TLRs  are expected to be present in this tissue. Several studies have investigated the presence and the role of  TLRs  in FRT especially within the fallopian tube ( 34 , 42 , 52 ). Previously, Pioli et al. ( 39 ), showed the presence of  TLR1-6  in the FRT as well as the accessory molecule CD14 and the molecular adapter MyD88 both of which are needed for  TLR  signalling. They also observed expression of  TLR1-6  at the transcript level in fallopian tubes. In agreement with this report, we previously showed the localization of  TLR1-6  in the fallopian tube using immunohistochemistry ( 36 ). Both observations are in agreement with our findings in this study that demonstrate the presence of  TLR1-6  transcripts in human fallopian tube tissues and cell line. The expression of  TLR4  protein and transcript has been detected in human endometrial epithelial cells, stromal cells ( 38 ) and fallopian tube stromal fibroblasts ( 53 ). However,  TLR4  expression was not detected in fallopian tube epithelial cells by Itoh et al. ( 53 ). In another study Hart et al. ( 42 ), reported the expression of  TLR7–9  in the fallopian tube, uterine, endometrium, cervix and ectocervix, while  TLR10  expression was restricted to the fallopian tube.\nIn this study, although expression of  TLR1-10  was detected in human fallopian tube tissues, expression of only  TLR1-6  was detected in OE-E6/ E7 cells. This may be due to difference in features of the fallopian tube tissue and this specific cell line. Fallopian tube tissue contains an epithelial layer, stroma and capillaries supplying blood. Within the epithelial layer of the fallopian tube tissue, some of the cells are ciliated and some are known as secretory cells. The OE-E6/E7 cell line is only an isolated fallopian tube epithelial layer cell line. Therefore, the expression of  TLR  genes and their level of expression in this cell line is likely to be different compared with the original tissue consisting of different types of cells. The other potential explanation for the absence of expression of some  TLRs  in OE-E6/E7 cells may be due to differences between immortalised cell lines and their original parental primary cells. It is known that cell lines may undergo changes due to the process of immortalization. However, the characteristics of this immortalized cell line have been compared to the parental human fallopian tube epithelium in several investigations. For example, human oviduct-specific glycoprotein, estradiol receptors and cytokeratin molecules have been found to be produced in both primary fallopian tube cells and OE-E6/E7 cells ( 50 , 54 , 55 ).\nThe FRT environment is under the control of sex hormones during the menstrual cycle. Sex hormones not only regulate anatomical and histological characteristics of this tract ( 56 , 57 ), they are also involved in the influx and localization of immune cells in this tract ( 58 , 60 ). For example, uterine natural killer (uNK) cells are found in the human uterus in large numbers and are spread throughout the endometrium with increasing numbers as the menstrual cycle progresses ( 61 , 62 ). In addition, the adaptive immune system is also influenced by the changing levels of sex hormones during the menstrual cycle. Antigen presentation has been shown to be suppressed in response to increasing concentrations of estradiol ( 63 , 64 ). Sex hormones including estradiol and progesterone tightly control the distribution of macrophages within the endometrium ( 65 , 68 ). Another study demonstrated that estradiol, which is secreted by the ovary during the menstrual cycle, modulates epithelial cells and other immune cells in FRT directly and indirectly to regulate a wide range of immune functions specific to each site in FRT ( 69 ). Nasu and Narahara ( 49 ) also showed that antigen-presenting cells in the uterus and vagina are responsive to estradiol where antigen presentation as well as co-stimulatory molecule expression is inhibited by estradiol. Furthermore, they suggested that antigen-presenting cells in the uterus and vagina respond to selected  TLR  agonists with altered antigen presentation.\nIt is therefore likely that the action of  TLRs  is also modified by changing concentrations of estradiol and progesterone. Although all  TLR  molecules are expressed throughout the cycle, the majority of these genes are expressed at their lowest level during menstrual and proliferative stages of the cycle ( 34 ). At follicular stage of the cycle, while progesterone levels are at their lowest, estradiol levels are at their highest. This may indicate an inhibitory effect of estradiol and/or enhancing influence of progesterone on the expression of  TLR  molecules in FRT, especially in the endometrium. It is plausible that this alteration in  TLR  gene expression may even influence the function of  TLRs  in mediating innate immune responses in FRT. To test these hypotheses, we examined the effect of sex hormones on  TLR  expression in the OE-E6/ E7 cell line. We used various concentrations of estradiol and progesterone to determine the effects of these sex hormones on  TLR  expression. No significant alteration in the relative expression of  TLR1-6  transcripts was observed in the fallopian tube cell line in response to different concentrations of estradiol and progesterone. These results agreed with the findings of Lesmeister et al. ( 47 ) who also showed that  in vitro  treatment of endometrial epithelial cell line (RL95-2) with 17betaestradiol did not have an effect on  TLR3  transcript or protein expression. Also, in a recent study on human uterine epithelial cells and the ECC-1 uterine epithelial cell line, Nasu and Narahara ( 49 ) demonstrated that estradiol either alone or prior to treatment with poly (I:C), had no effect on the expression of interferon β (IFNβ) or interferonstimulated genes (ISG). However, another study showed that production and secretion of protective antimicrobials including human β defensin-2 (HBD2) and secretory leukocyte protease inhibitor (SLPI) are directly upregulated by estradiol. On the contrary, estradiol inhibited LPS and poly (I:C)induced secretion of macrophage inhibitory factor (MIF), interleukin 6 (IL-6) and IL-8 in primary uterine epithelial cells ( 70 ).\nIn the next step of our study, combined effect of estradiol and progesterone was evaluated. Expression of  TLR1-6  was higher when the level of sex hormones in culture media was comparable to their concentration in serum during the window of implantation (group W) in the menstrual cycle. These results agreed with our previous findings where higher expression of  TLRs  was observed during the secretory phase in human endometrium ( 34 , 71 ). These findings demonstrated a stimulatory effect of co-presence of both sex hormones (M, P and W) on  TLR1-6  expression.\nA recent study reported the fluctuation of  TLR  responsiveness in peripheral blood throughout the menstrual cycle ( 72 ). In agreement with our data, during the follicular phase, lower levels of IL-6 and tumor necrosis factor (TNF)-α following stimulation with the  TLR2  agonist, lower levels of IL-1β, IL-6 and TNF-α following stimulation with the  TLR4  agonist LPS, and lower levels of IL-1β and TNF-α following stimulation of whole blood with the  TLR5  agonist flagellin was observed when compared with the early luteal phase ( 72 ). Jorgenson et al. ( 46 ) also illustrated that  TLR3  transcript level in primary endometrial epithelial cells is menstural cycle-dependent. They showed  TLR3  was expressed throughout the menstrual cycle but at its highest during the secretory phase of the cycle. In another study, Yao et al. ( 73 ) reported that except  TLR11 , the expression of  TLR1-10  is cycle-dependent in mouse. Whether the above mentioned findings are due to estradiol, progesterone or their combined effect on FRT epithelium remains to be deciphered.\nWhen fulvestrant (estradiol antagonist) or mifepristone (progesterone antagonist) was used, the combined effect of estradiol and progesterone on  TLR  expression was inhibited. These data thus confirm our results that on the expression of  TLR16  is not affected by progesterone and estradiol individually  TLR1-6  but  TLR1-6  under the synergistic/additive effect of the combination of estradiol and progesterone.\nIn performing this investigation, we took several precautionary measures. The effect of hormones in cell culture experiments was tested in the absence of phenol red and serum. Phenol red has estradiolic properties and serum may contain small molecules with estradiolic effects and if present, may hamper the results of experiments.\nIn  in vitro  culture systems and particularly in the presence of blood or serum samples, progesterone degrades quickly ( 74 ). To avoid early degradation of hormones during our cell culture experiments, we used water soluble estradiol and progesterone (Cyclodextrin-encapsulated 17β-estradiol and progesterone) which are stable and tested for cell culture applications. These compounds have been used in several similar investigations ( 75 , 79 ). Furthermore, our results clearly demonstrated that these compounds do not alter  TLR  gene expression in cultured cells.\nIt seems that the pattern of  TLR  expression under different concentrations of estradiol and progesterone (mimicking those of the menstrual cycle) is similar to the pattern of  TLR  expression in endometrial tissue during the menstrual cycle ( 34 ). On the other hand, higher expression of  TLRs  in the proliferative phase compared with menstruation as well as their higher expression in the secretory phase than other phases show that safety in the human fallopian tube at the time of ovulation or early embryo development is a key factor.\nFuture studies should be directed towards understanding the role of signalling pathways that enable estradiol and progesterone to modulate the expression and function of  TLRs  in FRT.\n\nThis study firmly points to the involvement of sex hormones in modulation of  TLR  gene expression in human fallopian tube cells. Further experiments should be undertaken to reveal the regulatory mechanism(s) and signalling pathway(s) responsible for the effect of sex hormones in modulating innate immunity in human FRT.","source_license":"CC-BY-4.0","license_restricted":false}