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Mol. Hum. Reprod. Advance Access originally published online on December 18, 2006
Molecular Human Reproduction 2007 13(2):135-140; doi:10.1093/molehr/gal106
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Interleukin-10 gene promoter polymorphisms and their protein production in peritoneal fluid in patients with endometriosis

X. Zhang, P. Hei, L. Deng and J. Lin1

Department of Gynecology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou Zhejiang, P. R. China

1 To whom correspondence should be addressed at: Department of Gynecology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou Zhejiang, 310006, P. R. China. E-mail: xinmei6{at}yahoo.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although associations of interleukin-10 (IL-10) gene promoter polymorphisms and their protein production with endometriosis risk have been reported, the correlations remain controversial. The objective of this study was to determine IL-10 gene promoter polymorphisms at -1082, -819 and -592 sites and their protein production in peritoneal fluid (PF) in patients with and without endometriosis. IL-10 gene promoter polymorphisms at -1082 site were detected by amplification refractory mutation system (ARMS)–PCR and that at -819 and -592 sites was genotyped by restriction fragment length polymorphism (RFLP)-PCR. Protein levels of IL-10 in PF were measured by enzyme-linked immunosorbent assay (ELISA). There were no significant differences in the genotype and allele frequencies of IL-10 gene promoter polymorphisms at position -1082 between the endometriosis and the control groups. However, the frequency of -819 or -592 C alleles was significantly increased in patients with endometriosis compared with controls. The protein levels of IL-10 in PF were statistically higher in the endometriosis group than in the control group. Moreover, the polymorphisms at -1082, -819 and -592 sites were associated with protein levels of IL-10 in PF in the endometriosis group while in the control group only the polymorphisms at position -1082 correlated with protein levels. Increased frequency of -819 or -592 C allele and increased protein production of IL-10 in PF in patients with endometriosis compared with controls and correlations of polymorphisms at -819 and -592 sites with protein levels of IL-10 in PF in patients with endometriosis may suggest that polymorphisms at -819 and -592 sites and their protein production are associated with endometriosis risk.

Key words: endometriosis/interleukin-10/gene polymorphism/peritoneal fluid/protein production


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Endometriosis is a common gynaecologic disease in women of reproductive age, which may result in dysmenorrhoea, dyspareunia, pelvic pain or infertility. The exact aetiology and pathogenesis of endometriosis remains unclear; however, both environmental and genetic factors have been implicated in this disorder (Lin et al., 2003aGo). Recently, several lines of genetic-association studies have revealed associations between the development of endometriosis and certain genetic polymorphisms (Lin et al., 2003bGo; Hsieh et al., 2005Go; Luisi et al., 2006Go), although the genes that play a role in susceptibility to the development and progression of endometriosis are unknown. However, cytokine-mediated immune and inflammatory responses have been considered to play an important role in the pathogenesis of endometriosis (Iwabe et al., 2002Go; Wu and Ho, 2003Go). The role of cytokines has been supported by studies on protein expression of various cytokines. For example, some cytokines such as interleukin (IL)-1 (Ho et al., 1997aGo), IL-6 (Zhang et al., 2005Go), IL-8 (Calhaz-Jorge et al., 2003Go), leptin (Matarese et al., 2000Go), vascular endothelial growth factor (VEGF) (Mahnke et al., 2000Go) and tumor necrosis factor (TNF)-{alpha} (Zhang et al., 2005Go) are increased, and other cytokines such as interferon-{gamma} (IFN-{gamma}) (Ho et al., 1996Go), IL-18 (Zhang et al., 2004Go) and IL-13 (McLaren et al., 1997Go) are decreased in peritoneal fluid (PF) of women with endometriosis. It is apparent that abnormal production of protein by cytokines may play a crucial role in the pathogenesis of this disease.

IL-10 is an important immunoregulatory cytokine mainly produced by macrophages, monocytes, T cells, B cells, dendritic cells, mast cells and eosinophils. As a T-cell helper type 2 (Th2) cell-derived cytokine, IL-10 has been shown to inhibit the secretion of Th1 cell-derived cytokines, limit the inflammatory responses and regulate the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells and mast cells (Gallagher et al., 2003Go; Li and He, 2004Go; Nunez et al., 2006Go). The gene encoding IL-10 has been identified on chromosome 1q31–32 (Opdal, 2004Go). Several polymorphic sites within the IL-10 gene promoter region including three biallelic polymorphisms at positions -1082, -819 and -592 from the transcription start site and two microsatellite polymorphisms have been described (Eskdale et al., 1997Go; Turner et al., 1997Go). Twin studies and family studies have shown that >70% of the observed variability of IL-10 secretion is explained by genetic factors (Westendorp et al., 1997Go). Previous in vitro studies using peripheral blood mononuclear cells (PBMCs) have suggested that -1082 G/G, -819 C/C, -592 C/C and GCC/GCC genotypes are associated with higher IL-10 production than other genotypes (Turner et al., 1997Go). Recently, Kitawaki et al. (2002)Go have demonstrated a positive correlation between -592 C allele in the IL-10 gene promoter region and autoantibodies to carbonic anhydrase II in patients with endometriosis suggestion a possible link of -92 C allele with susceptibility to endometriosis. Interestingly, some authors have reported that protein levels of IL-10 in PF are significantly increased in patients with endometriosis compared with controls (Punnonen et al., 1996Go; Ho et al., 1997bGo), although this has not been found in all studies (Rana et al., 1996Go; McLaren et al., 1997Go). In this study, we aimed to investigate whether IL-10 gene promoter polymorphisms are associated with IL-10 protein production in PF and endometriosis risk and thus may have a role in the pathogenesis of endometriosis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Study subjects
Two hundred and three consecutive patients undergoing laparoscopy or laparotomy from January 2004 through December 2004 at the Women’s Hospital were recruited for this study. Gynaecologic indications for laparoscopy or laparotomy included evaluation of infertility, abdominal pain, pelvic mass, elective tubal sterilization or anastomosis. The control group consisted of 84 patients who were healthy, fertile women with normal pelvic anatomy undergoing tubal sterilization or anastomosis. The case group consisted of 119 patients diagnosed with different stages of endometriosis by surgical findings and histological examinations of resected lesions. All the patients had normal regular menstrual cycles and showed no evidence of acute inflammation. The day of the menstrual cycle was established from the patient’s menstrual history and verified by histologic examination of the endometrium in the study and control groups. None of the patients had used hormonal drugs for at least 3 months before surgery. Clinical data, menstrual history and obstetric history were collected for all study participants. The operative findings regarding the presence, location, volume and degree of endometriosis and the condition of the pelvis, tubes, ovaries, pouch of Douglas and bowels were recorded. The severity of endometriosis found was evaluated and graded according to the Revised American Society for Reproductive Medicine criteria (ASRM) (American Society for Reproductive Medicine, 1997Go). Forty-nine patients had minimal or mild (Stage I + II) and 70 had moderate or severe (Stage III + IV) stages of the disease. Moreover, all study subjects were Han Chinese women originating from Zhejiang Province. The average age of the 119 women with endometriosis who had a laparoscopy (n = 95) or laparotomy (n = 24) was 36.5 ± 5 years and that of the 84 women without endometriosis who had a laparoscopy (n = 51) or laparotomy (n = 33) was 33.8±3 years. Informed consent was obtained from each participant before surgery. The study was conducted under the institutional review board of the Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China.

Determination of IL-10 protein levels in PF
During surgery, PF was firstly aspirated from the posterior cul-de-sac avoiding blood contamination and before any irrigation. PF was collected in a sterile syringe and centrifuged at 600 g for 10 min at 4°C. The supernatant was collected, aliquoted and stored at –70°C until use. Protein levels of IL-10 in PF were measured by enzyme-linked immunosorbent assay (ELISA) using a human IL-10 ELISA kit (Bender MedSystems Diagnostics GmbH, Vienna, Austria) designed specifically to measure IL-10 in body fluids according to the manufacturer’s instructions. The sensitivity of the assay was <1.5 pg/ml, and the intra- and inter-assay coefficients of variation were 3.2% and 5.6, respectively. To avoid subjective bias, an experienced technician blind to the status of the subjects at laboratory conducted the detection of IL-10 protein levels in PF. For each sample, duplicate samples of PF were assayed.

DNA preparation and IL-10 genotyping
Genomic DNA was extracted and purified from whole blood lymphocytes using a blood DNA Kit (Omega Bio-Tek Co., USA) according to the manufacturer’s instructions. IL-10 gene promoter polymorphism at -1082 site (IL-10-1082) was detected by amplification refractory mutation system (ARMS)–PCR and that at -819 and -592 sites was genotyped by restriction fragment length polymorphism (RFLP)–PCR. For genotyping IL-10-1082, the PCR primer sequences were as follows: (i) primer A (sense): 5'-ACTACTAAGGCTTCTTTGGGAA-3'; (ii) primer G (sense): 5'-CTACTAAGGCTTCTTTGGGAG-3' and (iii) generic primer (antisense): 5'-CAGTGCCAACTGAGAATTTGG-3'. The PCR was performed in a 25-µl reaction mixture containing x10 PCR buffer (500 KCl, 100 Mm Tris–HCl, 1.0% Triton X-100; Promega) 2.5 µl, 25 mM MgCl2 1.5 µl, 10 mM dNTPs 0.5 µl, each primer (10 pmol/ml) 1.0 µl, 200 ng of template DNA and Tag polymerase (2U/µl) 0.5 µl. The PCR cycle conditions were 94°C for 5 min, followed by 35 cycles at 94°C for 45 s, 58°C for 45 s and 72°C for 45 s, with a final extension step at 72°C for 10 min. The amplified ARMS–PCR products (PCR product size = 258 bp) were identified by gel electrophoresis on 2% agarose gels stained with ethidium bromide. For genotyping IL-10-819 or -592, DNA fragments were first amplified by use of primer pairs 5'-TCATTCTATGTGCTGGAGATGG-3' and 5'-TGGGGGAAGTGGGTAAGAGT-3' for IL-10-819 and 5'-CCTAGGTCACAGTGACGTGG-3' and 5'-CCTAGGTCACAGTGACGTGG-3' for IL-10-592. The PCR was performed in 25 µl reaction mixture containing x10 PCR buffer (500 KCl, 100 Mm Tris–HCl, 1.0% Triton X-100; Promega) 2.5 µl, 25 mM MgCl2 1.5 µl, 10 mM dNTPs 1.0 µl, each primer (10 pmol/ml) 1.0 µl, 200 ng of template DNA and Tag polymerase (2U/µl) 1.0 µl. The PCR cycle conditions were 94°C for 5 min, followed by 35 cycles at 94°C for 45 s, 60°C for 45 s (IL-10-819) or 30 s (IL-10-592) and 72°C for 45 s, with a final extension step at 72°C for 10 min. The amplified PCR products for IL-10-819 and IL-10-592 were 209 and 412 bp, respectively. After PCR application, a 10 µl aliquot of PCR product for IL-10-819 in 20 µl reaction containing 0.4 µl Mae III (Promega) was digested for 16 h at 55°C and for IL-10-592 containing 0.5 µl Rsa I (Promega) at 37°C for 16 h. The products were resolved and separated for 1 h at 80V (IL-10-819) or 100V (IL-10-592) on a 3% (IL-10-819) or 2% (IL-10-592) agarose gel stained with ethidium bromide. For IL-10-819, the resolved band at 209 bp was for TT genotype, at 125 and 84 bp for CC and at 209, 125 and 84 bp for TC. For IL-10-592, the resolved band at 419 bp was for CC genotype, at 236 and 176 bp for AA and at 412, 236 and 176 bp for AC.

Statistical analysis
For statistical analysis, all data were non-parametric and described as medians (ranges). The medians of IL-10 protein levels in PF were compared using Mann–Whitney U test. Endometriosis and control populations were tested for conformity to Hardy–Weinberg equilibrium using the {chi}2 test between observed and expected frequencies. The differences in the distribution of phenotypes and allele frequencies were analyzed using the {chi}2 test or Fisher’s exact test as appropriate. All odds ratios (OR) were calculated as estimates of the relative risk, and confidence intervals (CI) were calculated at the 95% level (95% CI). Associations between the protein levels and the polymorphisms were examined using Mann–Whitney U test or Kruskal–Wallis H test as appropriate. All statistical analyses were performed using Statistics Package for window SPSS version 12.0. A P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Blood samples were obtained from all patients undergoing laparoscopy or laparotomy. However, 27 of 84 control group PF specimens and 24 of 119 endometriosis group PF specimens were contaminated with blood or inadequate for analysis and were therefore excluded. This left a control group with 84 blood samples and 57 PF specimens and an endometriosis group with 119 blood samples (49 blood samples for the minimal/mild group and 70 for the moderate/severe group) and 95 PF specimens (40 PF specimens for the minimal/mild group and 55 for the moderate/severe group). First, Hardy–Weinberg equilibrium test was used to test for the genotype distribution of polymorphisms at positions -1082, -819 and -592 of IL-10 gene promoter region between observed and expected frequencies in the endometriosis and control groups. The {chi}2 test showed that the endometriosis group and the control group were in Hardy–Weinberg equilibrium, indicating that population selected from the endometriosis and control groups had successful matching.

The genotype and allele frequencies of IL-10-1082 were 89.3% for A/A genotype, 10.7% for A/G genotype, 0% for G/G genotype, 94.6% for A allele and 5.4% for G allele in the control group and 86.6% for A/A genotype, 12.6% for A/G genotype, 0.8% for G/G genotype, 92.9% for A allele and 7.1% for G allele in the endometriosis group (Table I). There were no significant differences in the genotype and allele frequencies of IL-10-1082 between the endometriosis and control groups (P > 0.05, Table II). The distribution of IL-10-819 genotype and allele frequencies was the same as IL-10-592, indicating that -819 T allele was closely linked with -592 A allele. The genotype and allele frequencies of IL-10-819 (or -592) were 45.2% for T/T (or A/A) genotype, 41.7% for T/C (or A/C) genotype, 13.1% for C/C (or C/C) genotype, 66.1% for T (or A) allele and 33.9% for C (or C) allele in the control group, and 29.4% for T/T (or A/A) genotype, 52.1% for T/C (or A/C) genotype, 18.5% for C/C (or C/C) genotype, 55.5% for T (or A) allele and 44.5% for C (or C) in the endometriosis group (Table I). The C allele frequency of IL-10-819 or -592 in the endometriosis group was significantly higher than that in the control group (OR = 1.564, 95% CI 1.039–2.354, P = 0.032), although no differences of the genotype frequencies of IL-10-819 or -592 between the two groups were found (P > 0.05, Table II).


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  		Table I. Frequencies of interleukin-10 (IL-10) alleles at position -1082 and frequency distribution of genotypes, including polymorphisms at positions -819 and -592 in patients with and without endometriosis

 

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  		Table II. The results of Pearson Chi-Square Test analysis on frequency distribution of genotypes and alleles at positions -1082, -819 and -592 of interleukin-10 (IL-10) gene polymorphisms in patients with and without endometriosis

 

The GCC/GCC genotype (high IL-10 production) was only found in one endometriosis patient but not found in the control group. Yet, the ACC/ACC, ACC/ATA, ATA/ATA genotypes (low IL-10 production) had a higher frequency both in the endometriosis group (86.5%) and in the control group (89.2%), whereas the GCC/ACC, GCC/ATA genotypes (intermediate IL-10 production) had a lower frequency both in the endometriosis group (12.6%) and in the control group (10.7%). The frequencies of haplotype GCC (high), ACC (intermediate) and ATA (low) were 5.4, 28.5 and 66.1% in the control group and 7.1, 37.4 and 55.5% in the endometriosis group. There were no statistically significant differences in the frequencies of haplotype and haplotype inheritance between the endometriosis and the control groups (P > 0.05, Tables I and II).

When staging the endometriosis, we divided the endometriosis group into two subgroups: the moderate/severe group and the minimal/mild group. The moderate/severe group exhibited a higher C/C genotype frequency of IL-10-819 or -592 than the minimal/mild group (OR = 2.139, 95% CI 1.347–3.427, P = 0.002) and the control group (OR = 2.096, 95% CI 1.338–3.068, P = 0.002). Moreover, we observed a higher C allele frequency of IL-10-819 or -592 in the moderate/severe group than in the minimal/mild group (OR = 2.494, 95% CI 1.453–4.279, P = 0.001) and in the control group (OR = 2.247, 95% CI 1.417–3.562, P = 0.001). In addition, we observed a statistically higher ACC haplotype frequency in the moderate/severe group than in the minimal/mild group (OR = 1.976, 95% CI 1.213–2.979, P = 0.006) and in the control group (OR = 1.231, 95% CI 1.012–2.125, P = 0.017).

The median (range) levels of IL-10 protein in PF were 1.59 pg/ml (0–24.49) in the control group, and 5.41 pg/ml (0–705.44) in the endometriosis group. There was statistically significant difference in the protein levels of IL-10 in PF between the two groups (Z = –3.744, P = 0.000). When categorized by the stage of endometriosis, the median (range) levels of IL-10 protein in PF were 2.38 pg/ml (0–692.32) in the minimal/mild group and 7.55 pg/ml (0.13–705.44) in the moderate/severe group, respectively. There was a statistically significant higher protein level of IL-10 in PF in the moderate/severe group than in the minimal/mild group (Z = –3.02, P = 0.003) and in the control group (Z = –4.608, P < 0.001; Figure 1) .


Figure 1
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  		Figure 1. Protein levels of IL-10 in peritoneal fluid (PF) in patients with and without endometriosis. Levels were described as median with ranges 25 and 75% percentiles.

 

The G/G, A/G and A/A genotypes of IL-10-1082 were associated with high, intermediate and low protein levels of IL-10 in PF, respectively, and -1082 G and C alleles with high and low levels in all groups (Tables III and IV). In turn, the -819 C/C, T/C and T/T (or -592 C/C, A/C and A/A) genotypes were correlated with high, intermediate and low protein levels of IL-10 in PF, respectively, and -819 C (or -592 C) and T (or -592 A) alleles with high and low protein levels of IL-10 in PF in all groups except for the control group and the minimal/mild group. Moreover, the GCC, ACC and ATA haplotypes were associated with high, intermediate and low protein levels of IL-10 in PF, respectively, and the GCC/ACC, GCC/ATA genotypes and the ACC/ACC, ACC/ATA, ATA/ATA genotypes with high and low levels in all groups.


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  		Table III. Genotypes and alleles at positions -1082, -819 and -592 of interleukin-10 (IL-10) gene polymorphisms associated with their protein levels in peritoneal fluid (PF) in patients with and without endometriosis

 

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  		Table IV. The results of Mann–Whitney U Test (or Kruskal–Wallis H test) analysis on peritoneal fluid (PF) protein levels associated with frequency distribution of IL-10-1082, -819 and -592 genotypes and alleles in patients with and without endometriosis

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
This study showed that the distribution of genotype and allele frequencies of IL-10-1082 was not statistically different between the endometriosis patients and the controls. However, the frequency of -819 or -592 C allele was significantly increased in the patients with endometriosis compared with the controls, although no difference in the genotype frequencies of IL-10-819 or -592 was observed. When stratified by the stage of endometriosis, the C/C genotype and C allele frequencies of IL-10-819 or -592 and ACC haplotype were all statistically higher in the patients with moderate/severe endometriosis than in the patients with minimal/mild endometriosis and in the controls. It is suggested that -819 or -592 C/C genotype and C allele and ACC (-1082 A/-819 C/-592 C) haplotype may have associations with susceptibility to endometriosis.

It has been demonstrated that ethnic differences between populations may affect the distribution of genotypes and haplotypes of IL-10, and thus account for the different associations of IL-10 polymorphisms with disease risk (Albuquerque et al., 2004Go). In the present study, we observed linkage disequilibrium between alleles. Alleles -819 T and -592 A were seen together, which was in agreement with the previous reported results (Albuquerque et al., 2004Go; Ma et al., 2005Go; Kusumoto et al., 2006Go; Scarpelli et al., 2006Go). The polymorphisms within the IL-10 gene promoter region have been shown to affect the protein production of IL-10 (Turner et al., 1997Go); however, the polymorphisms in the IL-10 gene promoter region affecting IL-10 production is dependent on the cell type and stimulation used (Tone et al., 2000Go). In our study, we found that the G/G, A/G and A/A genotypes of IL-10-1082 were associated with high, intermediate and low protein levels of IL-10 in PF, respectively, and -1082 G and C alleles with high and low levels in the endometriosis and control groups. This result agrees with the findings of Turner et al. (1997)Go that the polymorphisms within the IL-10 gene promoter region are associated with IL-10 protein production in PBMCs. However, we did not observe any associations between IL-10-819 or -592 polymorphisms and their protein production in PF in the controls. The correlations between the polymorphisms of IL-10-819 and -592 with IL-10 protein production in PF only found in the endometriosis group in our study may suggest a role of IL-10-819 or -592 polymorphisms in IL-10 protein production in PF in this disorder.

It has been reported that genetic factors can account for between 50 and 75% of the observed variability of IL-10 production (Opdal, 2004Go). Elevated macrophage activities could also increase IL-10 protein production by macrophages in PF in the patients with endometriosis (Rana et al., 1996Go; Ho et al., 1997bGo; McLaren et al., 1997Go; Wu et al., 1999Go; Wu and Ho, 2003Go). Therefore, increased IL-10 protein levels in PF in the patients with endometriosis as compared with the controls (Punnonen et al., 1996Go; Ho et al., 1997bGo; Tabibzadeh et al., 2003Go) and that associated with the disease stage in our study may imply linkage with the C allele carriers of IL-10-819 or -592 and the increased macrophage activities in PF of this disorder. As a major homeostatic regulator of inflammation, immune responses and prevention of autoimmunity, IL-10 has been shown to regulate T-cell differentiation and proliferation by inhibiting IL-2 and IFN-{gamma} release from activated T-cell clones (Ebert, 2000Go). It has also been found to play the role of Th2 cell-derived cytokines and inhibit the secretions of Th1 cell-derived cytokines (Li and He, 2004Go; Nunez et al., 2006Go) and reduce peritoneal T-cell activation (Ho et al., 1997bGo) and major histocompatibility complex (MHC-II) expression pattern. Therefore, increased IL-10 protein production in PF in the patients with endometriosis may suggest an important role for IL-10 in the pathogenesis of this disease by down-regulating peritoneal cell immune response and secretions of Th1 cell-derived cytokines (Lee et al., 2005Go; Ulukus and Arici, 2005Go) and interfering with peritoneal inflammatory response (Tabibzadeh et al., 2003Go; Agic et al., 2006Go).

In conclusion, this is the first study, to our knowledge, to assess the associations of IL-10 gene promoter polymorphisms and their protein production in PF with endometriosis risk. The preliminary data suggest that IL-10-819 or -592 C/C genotype and C allele and ACC haplotype may be correlated with susceptibility to endometriosis. It also suggests that increased IL-10 protein production in PF associated with the polymorphisms of IL-10-819 or -592 may imply a role for IL-10 in the pathogenesis of endometriosis. However, the small sample size in our study necessitates that this finding be validated in a larger study. More data are also needed to determine whether this finding applies to the general endometriosis population.


   Acknowledgements
 
This work was supported by Zhejiang nature science fund (2003-347), and we are grateful to Dr Susan Evans at the endometriosis care center of Australia for helping us with our English.


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on June 10, 2006; accepted on July 11, 2006.


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