Molecular Human Reproduction, Vol. 9, No. 12, pp. 785-791, 2003
© European Society of Human Reproduction and Embryology 2003; all rights reserved
Seminal plasma induces mRNA expression of IL-1ß, IL-6 and LIF in endometrial epithelial cells in vitro
1Department of Obstetrics and Gynaecology, Klinikum der Universität München-Großhadern, Marchioninistraße 15, 81377 München, and 2Department of Gynecologcical Endocrinology and Reproductive Medicine, Universitaetsklinikum Heidelberg, Voßstraße 9, 69115 Heidelberg, Germany 4Both authors contributed equally to this work
3 To whom correspondence should be addressed. e-mail: michael.von.wolff{at}med.uni-heidelberg.de
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Abstract |
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The influence of seminal plasma on the mRNA expression of cytokines in human endometrial epithelial and stromal cells and the cytokine production of spermatozoa were investigated in vitro. Seminal plasma and spermatozoa were collected from healthy volunteers and were screened by enzyme-linked immunosorbent assay for cytokines. Epithelial and stromal cells from fertile women were cultured on matrigel or polystyrol and incubated with pooled seminal plasma or with transforming growth factor ß1 (TGF-ß1), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), which were found to be significantly concentrated in seminal plasma. Endometrial cytokine expression was analysed by RNase protection assay and supported by RT–PCR. Supernatants of highly purified spermatozoa did not contain detectable levels of IL-1ß, IL-6 and VEGF. Screening of seminal plasma revealed concentrations >10-fold above the serum level for TGF-ß1, IL-8 and VEGF. Incubation of epithelial cells with 0.1, 1 and 10% seminal plasma resulted in concentration-dependant stimulation of IL-1ß, IL-6 and LIF mRNA expression. Maximum stimulation was found in epithelial cells from tissue samples taken in the mid secretory phase. Epithelial mRNA expression of IL-1ß, IL-6 and LIF increased by stimulation with TGF-ß1 and IL-8, but not with VEGF. In conclusion, seminal plasma stimulates expression of pro-inflammatory cytokines in endometrial epithelial cells in vitro. This effect might at least in part be exerted by TGF-ß1 and IL-8, abundantly present in seminal plasma. The in-vivo physiological relevance of these in-vitro studies remains to be determined.
Key words: endometrium/interleukin 1/interleukin 6/leukaemia inhibitory factor/seminal plasma
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Introduction |
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The implantation of the blastocyst is dependant on adequately proliferated, transformed and functionally unimpaired endometrium and is facilitated through a well balanced network of mediators in epithelial, stromal and immune cells. Several of these mediators such as the pro-inflammatory cytokines interleukin 6 (IL-6) (Tabibzadeh et al., 1995; von Wolff et al., 2002a), IL-1ß (Simón et al., 1993; von Wolff et al., 2000) and leukaemia inhibitory factor (LIF) (Charnock-Jones et al., 1994; von Wolff et al., 2000) are up-regulated during the pre-implantation period. Their expression is suppressed in infertile patients and patients with recurrent early abortions (Laird et al., 1997; Lim et al., 2000; von Wolff et al., 2000), indicating a role in implantation and early pregnancy. IL-6, IL-1ß and LIF belong to the complex and still poorly understood network of endometrial cytokines, which are regulated by several paracrine factors such as IL-1ß and tumour necrosis factor
(TNF) (Tabibzadeh et al., 1989; von Wolff et al., 2002b). Even though biologically active cytokines are found in abundance in seminal plasma and have been shown to be secreted by spermatozoa, the function of seminal plasma and spermatozoa in the regulation of endometrial function and in the process of implantation has never been studied in humans. Human seminal plasma contains high concentrations of cytokines and prostaglandins such as IL-8, vascular endothelial growth factor (VEGF), G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor ß (TGF-ß) and prostaglandin E2 (Srivastava et al., 1996; Huleihel et al., 1999; Gutsche et al., 2002). They are found at increased or reduced concentrations in sperm of infertile men (Dousset et al. 1997; Huleihel et al., 1996, 1999; Eggert-Kruse et al., 2001), indicating that they play a role in the process of reproduction. Human spermatozoa have been shown to produce IL-1 and IL-6 (Huleihel et al., 2000a,b), which might stimulate endometrial cells while ascending the uterine cavity. Most cytokines identified in seminal plasma and spermatozoa have also been detected in human endometrium and have been suggested to play a role in the regulation of endometrial function (Horowitz et al., 1993; Simón et al., 1993; Tabibzadeh et al., 1995; Torry et al., 1996; Vandermolen and Gu, 1996; Arici et al., 1998; Zhao and Chegini, 1999).
In-vitro experiments in mice support the hypothesis that seminal plasma plays a role in the regulation of implantation by regulating endometrial epithelial cells: seminal plasma of mice contains high concentrations of TGF-ß, which stimulates the release of pro-inflammatory cytokines, including GM-CSF (Tremellen et al., 1998), in vivo and in vitro. Regulation of GM-CSF by seminal plasma appears to play an important role in the development of the embryo. Fertility is compromised in genetically GM-CSF-deficient mice (Robertson et al., 1999) and GM-CSF promotes blastomere viability in murine preimplantation embryos (Robertson et al., 2001; Robertson and Sharkey, 2001).
A growing body of evidence indicates that human seminal plasma and spermatozoa also play a role in the regulation of implantation: seminal plasma has been shown in vitro to suppress T- and B-cell proliferation, neutrophil and macrophage phagocytic activity and killer cell activity (Thaler, 1989), and by interfering with IgG-Fc-mediated effector functions, to control in vitro potentially harmful antipaternal immune responses (Thaler, 1992). Clinical evidence is given by Bellinge et al. (1986), who deposited semen in the vagina of patients undergoing IVF treatment at the time of oocyte fertilization and found an implantation rate of 53%, compared with 23% in the control group. Coulam and Stern (1995) performed a placebo-controlled clinical trial, depositing vaginal capsules containing seminal plasma or placebo and described implantation rates of 80% in the group of patients treated with seminal plasma, compared with 67% in the placebo group. Tremellen et al. (2000) randomized patients either to abstain or to engage in vaginal intercourse and found similar pregnancy rates, but a significantly higher proportion of viable embryos at 6–8 weeks of pregnancy in the group engaged in vaginal intercourse.
However, even though experiments in mice indicate that seminal plasma has the potential to modify endometrial function, it is still under debate whether such effects also take place in humans. Unlike in mice, the human cervix produces cervical mucous which is believed to prevent high concentrations of spermatozoa and seminal plasma to be flushed into the uterine cavity during intercourse. On the other hand, experiments by Leyendecker et al. (1996) and Kunz et al. (1997) have demonstrated that technetium-labelled albumin macrospheres, placed at the external os of the cervix, reached the tubes within 1 min due to subendometrial and myometrial peristaltic waves. These experiments support the hypothesis that not only spermatozoa and seminal plasma are transported passively through the cervix and reach the uterine cavity where they may interact with endometrial components.
Assuming that seminal plasma ascends through the cervix into the uterine cavity, we suggest that seminal plasma plays a role in the regulation of endometrial function by directly stimulating endometrial epithelial cells. Interestingly, in patients, undergoing IVF or ICSI, seminal plasma and spermatozoa are not getting into contact with the uterine cavity. The blastocyst is retransferred without any seminal plasma and spermatozoa and patients usually abstain from sexual intercourse before and after oocyte retrieval. The lack of stimulation of endometrial cells by spermatozoa and seminal plasma might contribute to the limited implantation rates in IVF and ICSI.
As these studies suggest that components of semen play a role in the regulation of endometrial function and implantation, we intended to analyse in vitro the potential of seminal plasma and spermatozoa to directly stimulate endometrial cytokines.
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Materials and methods |
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Collection of material
Endometrial tissues from regularly cycling women were collected at different phases of the menstrual cycle after hysterectomy. None of the patients was hormonally stimulated. Ethical approval according to the principals set out in the Declaration of Helsinki was obtained. For separation of endometrial and stromal cells, tissue was stored in medium until further processing. Serum was collected from each patient at the time of hysterectomy and stored at –20°C. All samples were dated according to the last menstrual period, the histological criteria of Noyes et al. (1950), and the serum levels of estradiol and progesterone. The menstrual cycle was divided into three phases: the proliferative phase (days 1–14), the early and mid secretory phase (days 15–24) and the late secretory phase (days 25–28).
Ejaculates from six healthy volunteers with normal spermiogram according to the WHO criteria were collected and liquified. Sperm were purified with the swim-up technique described by Richter et al. (1999). To exclude persistent cytokine contamination by seminal plasma, a fraction of sperm was double purified by density gradient centrifugation and the swim-up technique. Seminal plasma was extracted by centrifugation of ejaculates at 700 g for 10 min. A second round of centrifugation was performed at 10 000 g for 10 min. The supernatant, seminal plasma, was collected, pooled and stored at –20°C.
Cell culture
Isolation and culture of endometrial tissue was performed according to Classen-Linke et al. (1997) and von Wolff et al. (2002a,b). In brief, endometrial tissue was taken from premenopausal patients undergoing hysterectomy for benign reasons. Separation of stromal and epithelial cells was performed by filtration of minced endometrium through a 180 µm nylon membrane (Millipore, Eschborn, Germany) followed by a 40 µm nylon sieve (Becton Dickinson, NJ, USA).
Epithelial glands were trapped on the second sieve and seeded at a density of 6000 endometrial glands/cm2 on 12 mm diameter filters (pore size 0.4 µm) in Millicell CM (Millipore) inserts, previously coated with 0.1 ml of 1:4 diluted matrigel without phenol red (Becton Dickinson). Inlets were placed in 24-well tissue culture plates (Falcon, Oxnard, USA) and incubated in DMEM/F-12 Ham medium without phenol red (Sigma, Deisenhofen, Germany), containing antibiotics, antimycotics and 10% fetal calf serum (FCS; Gibco, Eggenstein, Germany), at 37°C and 5% CO2 until 50–90% confluence was reached.
Stromal cells that passed through the 40 µm sieve were thoroughly washed and purified by extraction of immune and endothelial cells using Epithelial enrich-, CD14-, CD56-, CD45- and CD31-positive magnetic Dynabeads (Dynal, Oslo, Norway). To further reduce contamination of cell cultures by non-stromal cells, cells were passaged once: cells were cultured for 2 days, released by incubation with 10% trypsin (Gibco) and seeded in 24-well tissue culture plates (Falcon) with a density of 100 000 cells/well. All cells were cultured for 4–5 days in the presence of 17ß-estradiol (10–8 mol/l = 2.7 ng/ml) and progesterone (10–6 mol/l = 310 ng/ml) until subconfluency was reached.
To analyse the effect of seminal plasma on endometrial cytokine expression, we first incubated epithelial and stromal cells with 10% seminal plasma for 1, 3, 6 and 24 h and found maximum mRNA stimulation after 3 h of incubation (n = 5). Epithelial and stromal cells were then incubated with 0.1, 1 and 10% of seminal plasma for 3 h to analyse the concentration-dependant stimulation of endometrial cytokine expression (n = 8). Incubation with DMEM/F-12 Ham medium without seminal plasma (0%) or stimulation with heat-denatured seminal plasma were used as negative controls.
The effect of those cytokines found in seminal plasma at high concentrations was analysed by incubating epithelial cells with recombinant TGF-ß1, IL-8 and VEGF at concentrations already described by others (Srivastava et al., 1996; Loras et al., 1999) and determined in our pool of seminal plasma (TGF-ß1, 1500 pg/ml; IL-8, 2000 pg/ml; VEGF, 770 000 pg/ml; Sigma) (n = 4). To further control the effect of recombinant cytokines on epithelial cytokine expression, epithelial mRNA expression of IL-1ß, IL6 and LIF was analysed after incubation with recombinant TGF-ß1 and after incubation with TGF-ß1 (1500 pg/ml) mixed with blocking anti-TGF-ß1 antibody (5 µg/ml; R&D Systems).
To analyse cytokine secretion by spermatozoa, 3x106 spermatozoa were incubated in 1 ml of IVF medium (Stefan Gück GmbH, Berlin, Germany) at 37°C and 5% CO2 for 24 h (n = 3). The supernatants were stored at –20°C until enzyme-linked immunosorbent assay (ELISA) was performed.
Isolation of RNA and RNase protection assay
Spermatozoa were purified and endometrial cells were washed (epithelial cells) or scraped (stromal cells) out of the culture wells, transferred into sterile cups with Trizol (Life Technologies, Karlsruhe, Germany) and stored at –70°C before RNA isolation was performed. Total RNA was isolated by Trizol, based on phenol/chloroform extraction. The content of RNA was quantified by UV spectophotometry and checked for degradation by agarose gel electrophoresis.
Multiprobe RNase protection assay (RPA) was performed as described elsewhere (von Wolff et al., 2000), using the Riboquant Kit (Pharmingen, San Diego, CA, USA), which allowed comparison and quantification of several mRNA species in single RNA samples. Templates were purchased from Pharmingen where they were generated by subcloning cDNA strands into plasmids containing bacteriophage promoters. By incorporating probes for the housekeeping gene, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), and the ribosomal RNA, L32, the levels of individual mRNA species could be compared between samples. The probes were internally labelled by in-vitro transcription with [-32P]UTP (3000 Ci/mmol; 10 mCi/ml; Amersham Life Science Inc., IL, USA). In-vitro transcription was performed by incubating probes with T7 RNA polymerase, nucleotides and dithiothreitol for 1 h at 37°C. After terminating the reaction by addition of DNase for 30 min, probes were purified by phenol/chloroform extraction. The efficiency of in-vitro transcription was quantified in a scintillation counter. A probe concentration of 6x105 c.p.m. was used for each RPA. For the RPA, 1 µg of total RNA was dried in a vacuum evaporator centrifuge and diluted in hybridization buffer. RNA samples were mixed with the probe set and hybridized for 14–16 h at 56°C. To reduce assay variability, 40 samples were analysed at once. Non-hybridized RNA and free probes were digested by incubation with RNase A and T1 for 45 min at 30°C. Enzyme activity was stopped by addition of proteinase K. The non-digested, RNase-protected mRNAs were purified by phenol/chloroform extraction and resolved on a denaturating 6% acrylamide gel for 2 h at 50 W. Unprotected probes were loaded as size markers. Gels were dried for 1 h at 80°C and exposed to a film (Kodak X-AR; Kodak, Rochester, NY, USA) with an intensifying screen for 6 h to 7 days. The identity of the RNase-protected bands was established by comparing their size with the size of the bands of the positive control samples (Pharmingen) and by comparing the migration distance of the bands with those of the probes. The migration distances of the probes were 27–29 nucleotides shorter due to flanking sequences in the probes that did not hybridize to the target RNAs. Semi-quantification of the level of mRNA of various cytokines was achieved by normalizing the optical densities of the specific bands to the optical densities of the housekeeping genes, GAPDH and L32. The optical density of the specific protected bands was expressed as relative values. A 2-fold increase of the relative optical density values corresponded, on average, to a 1.7-fold increase of the specific mRNA, as determined in several dilution series.
RT–PCR
RT–PCR was performed as described elsewhere (von Wolff et al., 2002a,b). In brief, first strand cDNA was synthesized using First Strand Synthesis Kits (Roche Diagnostics), followed by PCR (PCR Core Kit; Roche Diagnostics). The housekeeping gene cytochrome oxidase subunit I (CO-I) was co-amplified in a companion tube. Primers were synthesized by MWG Biotech (Eberberg, Germany) or were purchased from Clontech (Palo Alto, CA, USA) (IL-6). The identity of the PCR products was confirmed by sequencing. The CO-I primers yielded a 268 bp product: sense (5'-CGTCACAGCCCATGCATTTG-3') and antisense (5'-GGTTAGGTCTACGGAGGCTC-3'); the TNF primers yielded a 254 bp product: sense (5'-CGAGTGACAAGCCTGTAGCC-3') and antisense (5'-GTTGACCTTGGTCTGGTAGG-3'); the IL-1ß primers yielded a 263 bp product: sense (5'-GGATATGGAGCAACAAGTGG-3') and antisense (5'-AGTTACCAGTTGGGGAACTG-3'); the IL-6 primers yielded a 628 bp product: sense (5'-ATGAACTCCTTCTCCACAAGCGC-3') and antisense (5'-GAAGAGCCCTCAGGCTGGACTG-3'); the LIF primers yielded a 477 bp product: sense (5'-CAGCTCAATGGCAGTGCCAA-3') and antisense (5'-GTTCACAGCACACTTCAAGAC-3'). The numbers of cycles were within the linear logarithmic phase of the amplification curve. Thirty five cycles were used for TNF and LIF, 34 cycles for IL-1ß and IL-6, 28 cycles for CO-I, and amplified as follows: 1 min at 92°C, 1 min at 58°C (TNF, IL-1ß, IL-6, LIF) or 51°C (CO-I) and 1 min at 72°C. The PCR products were separated electrophoretically in a 1% agarose gel.
ELISA
Concentrations of IL-1ß, IL-1RA, IL-2, IL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-8, IL-10, TNF, free TGF-ß1, TGF-ß2, G-CSF, GM-CSF and VEGF in seminal plasma and IL-1ß, IL-6 and VEGF in culture supernatants of spermatozoa were determined using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA). ELISAs were performed according to the instructions of the manufacturer.
Statistical analysis
The Friedman test or Mann–Whitney test for non-related subjects (Figure 4) were used for analysis. Significance was established at the P < 0.05 (*), P < 0.01 (**) or P < 0.001 (**) level.
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Cytokine secretion and mRNA expression by spermatozoa
Spermatozoa were first purified by the swim-up technique and incubated for 24 h. Culture supernatants contained 25 pg/ml VEGF (18 pg/100 000 spermatozoa). Concentrations of IL-1ß and IL-6 were below the limit of detection (IL-1ß, 2 pg/ml; IL-6, 6 pg/ml). To exclude persistent contamination of spermatozoa by seminal plasma, we double purified spermatozoa by density gradient centrifugation followed by the swim-up technique before incubating for 24 h. Concentrations of VEGF in supernatants were below the limit of detection (VEGF, 16 pg/ml).
Cytokine mRNA expression by spermatozoa was excluded by 40 cycles of RT–PCR. RT–PCR of the housekeeping gene ß-actin showed a strong signal, whereas mRNA expression of IL-1ß and IL-6 was not detectable.
Cytokine concentration in seminal plasma
Concentration of cytokines in a pooled seminal plasma sample of healthy donors was analysed by ELISA. Concentrations of free TGF-ß1, IL-8 and VEGF were 
10-fold above average serum levels: free (not latent) TGF-ß1, 383 pg/ml (serum, 30–50 pg/ml); IL-8, 1456 pg/ml (serum, 0–32 pg/ml); VEGF, 772 000 pg/ml (serum, 62–707 pg/ml). Concentrations of TGF-ß2, IL-1ß, IL-6, G-CSF and GM-CSF were moderately higher than serum levels: TGF-ß2, 148 pg/ml (serum, 0–20 pg/ml); IL-1ß, 19 pg/ml (serum, 0–2 pg/ml); IL-6, 118 pg/ml (serum, 0–51 pg/ml); G-CSF, 250 pg/ml (serum, 9–51 pg/ml); GM-CSF, 5.6 pg/ml (serum, 0–2.2 pg/ml). Concentrations of IL-2, IL-2 receptor, IL-3, IL-4, IL-6 receptor, IL-10 and TNF were within the range of serum levels.
Effect of seminal plasma on endometrial cytokine expression
Epithelial and stromal cells were stimulated with different concentrations of seminal plasma. mRNA expression of TNF, IL-1ß, IL-6, LIF, VEGF, M-CSF, G-CSF and GM-CSF and GAPDH and L32 in epithelial and stromal cells was analysed by multiprobe RPA (n = 8). mRNA expression of IL-1ß, IL-6 and LIF significantly increased in early to mid secretory phase epithelial cells in a concentration-dependent manner. Cytokine mRNA expression increased 2-fold for IL-1ß, 2.5-fold for IL-6 and 2.2-fold for LIF by stimulation with 10% seminal plasma for 3 h (Figures 1 and 2). Stimulation of mRNA expression was confirmed by RT–PCR (Figure 3). mRNA expression of VEGF, M-CSF, G-CSF and GM-CSF and of the housekeeping genes, GAPDH and L32, was not stimulated by seminal plasma in epithelial cells. Heat denaturation of seminal plasma completely abolished the effect of seminal plasma on epithelial cells (Figure 1). In contrast to epithelial cells, cytokine mRNA expression in stromal cells was not stimulated by seminal plasma.
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To analyse the stimulatory effect of seminal plasma on proliferative phase and secretory phase epithelial cells, cells from different phases of the menstrual cycle were incubated with seminal plasma (n = 33; Figure 4). Epithelial cells from the proliferative and late secretory phases showed only weak stimulation of cytokine mRNA expression, whereas stimulation of cytokine mRNA expression in cells from the early and mid secretory phase was significantly higher (P < 0.01). Cytokine mRNA expression increased in cells from the secretory phase, 2.2- (IL-1ß), 2.5- (IL-6) and 2.2-fold (LIF) in comparison with unstimulated controls.
Effect of seminal plasma constituents on cytokine expression in endometrial epithelial cells
To analyse the stimulatory effect of those cytokines found in seminal plasma at high concentrations, we incubated epithelial cells with recombinant TGF-ß1, IL8 and VEGF at concentrations comparable with those found in the pool of seminal plasma used in the previous experiments (n = 4; Figure 5). The stimulatory effect of these cytokines on mRNA expression of IL-1ß, IL-6 and LIF was slightly reduced, as compared with 10% seminal plasma (control) (Figure 5): concentrations of IL-1ß mRNA were 
20% lower if incubated with TGF-ß1 or IL-8, as compared with the control. Concentrations of IL-1ß mRNA (if incubated with VEGF) and IL-6 and LIF mRNA (if incubated with TGF-ß1, IL-8 and VEGF) were 55% lower as compared with the control.
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As the maximum stimulatory effect was found for TGF-ß1, TGF-ß1 was chosen to analyse the stimulatory effect of a combination of seminal plasma and inhibiting anti-TGF-ß antibody. Combining seminal plasma with inhibiting anti-TGF-ß1 antibody reduced the stimulatory effect of seminal plasma on mRNA expression of IL-1ß by 56%, IL-6 by 37% and LIF by 59% (data not shown).
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Discussion |
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The results of this study provide for the first time experimental evidence that seminal plasma regulates cytokine expression in human endometrial epithelial cells in vitro. These results support the hypothesis that seminal plasma contributes to the regulation of endometrial function, assuming that human seminal plasma ascends through the cervix into the uterine cavity during sexual intercourse.
Our experiments further suggest that cytokines produced by spermatozoa do not contribute to the regulation of human endometrium. We excluded IL-1ß and IL-6 mRNA expression in spermatozoa by RPA and by RT–PCR. Furthermore, spermatozoa did not produce or secrete measurable concentrations of IL-1ß, IL-6 and VEGF. As expected from these results, the supernatants of spermatozoa did not stimulate mRNA expression of IL-1ß, IL-6 and LIF in endometrial cells. Even though these results provide evidence that spermatozoa do not contribute to the regulation of human endometrium, it cannot be excluded that spermatozoa influence the regulation of human endometrium either by soluble factors, not analysed in our experiments, or by close endometrial contact in vivo. However, as only 1 in 1000 sperm reach the uterine cavity (Settlage et al., 1973), the impact of spermatozoa on endometrial function can be assumed to be limited.
Bearing in mind that seminal plasma reaches the uterine cavity, we performed several experiments to analyse the effect of seminal plasma on endometrial cells. Incubation of stromal and epithelial cells with seminal plasma at different concentrations revealed that cytokine mRNA expression in stromal cells was not stimulated by seminal plasma, whereas epithelial mRNA expression of IL-1ß, IL-6 and LIF was increased by seminal plasma. Incubation of epithelial cells with cytokines such as TGF-ß, IL-8 and VEGF, which were found in seminal plasma at high concentrations, resulted in reduced stimulation of cytokine mRNA expression in comparison with seminal plasma. These experiments indicate that cytokine expression in human endometrial epithelial cells in vitro is not regulated exclusively by single cytokines. We propose that regulation of epithelial cytokine expression in vitro is under the control of a broad spectrum of several mediators in seminal plasma.
Regulation of epithelial cytokine production by seminal plasma has previously been studied in mice and rabbits. Tremellen et al. (1998) described stimulation of epithelial GM-CSF by seminal plasma and by recombinant TGF-ß: GM-CSF injected into the uterine lumen was found to elicit a dose-dependant accumulation of macrophages and granulocytes in endometrium (Robertson et al., 2000). An inflammatory reaction by semen was also observed in equine endometrium (Troedsson et al., 2001). In support of these studies indicating that seminal plasma evokes an inflammatory reaction, we found stimulation of the pro-inflammatory cytokines IL-1ß, IL-6 and LIF in epithelial cells by human seminal plasma. However, the pattern of cytokines stimulated by human seminal plasma was different from that described by Tremellen et al. (1998). They described in mice, stimulation of GM-CSF in endometrial epithelial cells, whereas GM-CSF was not stimulated in our experiments. These differences might, however, be due to different species and due to technical differences.
Studies from murine models have provided evidence that pregnancy rejection is mediated by T-helper-1 (TH-1) cytokines, whereas a T-helper-2 (TH-2) cytokine response confers protection (Wegmann et al., 1993). Further evidence was given by Lim et al. (2000) and von Wolff et al. (2000), who demonstrated that women with recurrent miscarriage exhibit primarily TH-1 cytokines, whereas healthy women exhibit decreased TH-1 and increased TH-2 cytokines. These studies support our findings, revealing that seminal plasma stimulates mRNA expression of the TH-2 cytokines IL-6 and LIF while not affecting the TH-1 cytokine TNF. Previous experiments had demonstrated the capacity of human seminal plasma to skew T-cell cytokine synthesis in a type 2 manner (Kelly et al., 1997). Furthermore, prostaglandin E, which is particularly abundant in human semen, acts to programme a type 2-inducing phenotype in dendritic cells (Kapsenberg et al., 1999) and in T-cell lines (Betz and Fox, 1991).
Our experiments revealed endometrial stimulation of IL-1ß, IL-6 and LIF, which have been shown in several studies to play a role in the regulation of endometrial function and implantation. Endometrial IL-6 is expressed at increasing concentrations in endometrial epithelial cells (Tabibzadeh et al., 1995; von Wolff et al., 2002a,b) and was found at reduced concentrations in the mid secretory phase in patients with recurrent miscarriages (Lim et al., 2000; von Wolff et al., 2000). IL-1 seems to play an important role in murine implantation (Simón et al., 1998) and shows an endometrial expression pattern similar to IL-6 (Simón et al., 1993; von Wolff et al., 2000). IL-1ß stimulates IL-6 expression (Laird et al., 1994; von Wolff et al., 2002a,b) and is suppressed in the mid secretory phase of patients with recurrent early abortions (von Wolff et al., 2000). Endometrial LIF is expressed at maximum concentrations during the secretory phase of the cycle (Charnock-Jones et al., 1994; Vogiagis et al., 1996) and LIF concentrations were decreased in women with unexplained infertility (Laird et al., 1997). Moreover, LIF was demonstrated in mice to be essential for implantation of the blastocyst (Stewart et al., 1992).
Even though our experiments suggest seminal plasma to play a role in ‘priming’ of endometrium before implantation, this suggestion is based on the assumption that significant amounts of seminal plasma reach the uterine cavity. However, as already stated above, this matter is still under debate. An ‘insuck’ of seminal plasma by the uterus was suggested by Hartman (1957), and experimentally supported by Egli and Newton (1961). However, Walton (1930) and Asch et al. (1977), excluded ascension of seminal plasma into the uterine cavity in rabbits. These studies are in contradiction to those by Leyendecker at al. (1996) and Kunz et al. (1997), who demonstrated that technetium-labelled albumin macrospheres, placed at the external os of the cervix, reached the tubes within 1 min due to subendometrial and myometrial peristaltic waves. The contradicting findings might be due to very little amounts of seminal plasma in the uterine cavity which were undetectable in some of the studies. We demonstrated stimulation of endometrial epithelial cells by seminal plasma at concentrations of 1 and 10%, whereas concentrations of 0.1% did not sufficiently stimulate enodometrial cytokine expression. This raises the question as to whether the effect of seminal plasma is not only based on direct endometrial stimulation by intrauterine seminal plasma but also on indirect stimulation through other pathways. Robertson et al. (2002), suggested that seminal plasma might elicit the production of pro-inflammatory cytokines by endometrial epithelial cells in the outer endocervical canal or cervical ectropion, thereby leading to the formation of a post-coital cervical inflammatory response. The cervical inflammatory cells may then alter the local uterine immune response, which in turn indirectly affects blastocyst function.
In summary, seminal plasma stimulates expression of pro-inflammatory cytokines in endometrial epithelial cells in vitro. To analyse the clinical relevance of seminal plasma in assisting successful implantation, further studies, such as vaginal and cervical application of seminal plasma in patients undergoing in-vitro fertilization, are needed.
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Acknowledgements |
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The authors thank Dr F.Graf von Buquoy from the Department of Obstetrics and Gynaecology of the Krankenhaus Dritter Orden in Munich and Professor Dr W.Jonatha from the Krankenhaus Harlaching in Munich who kindly provided endometrial tissue samples. We would like to thank Dr P.Lohse, Institute of Clinical Chemistry, Klinikum der Universität München-Großhadern, for his support with RPAs, Dr D.Daniel from the Department of Immunology, Klinikum Heidelberg, for the analysis of cytokines in seminal plasma and Mrs M.Fileki, Munich, Mrs J.Jauckus and Mrs A.Schadwinkel, Heidelberg, for their help with our cell culture experiments, RPAs and RT–PCRs.
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References |
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Submitted on August 15, 2002; resubmitted on July 22, 2003. accepted on July 28, 2003
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