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The G(−2518)A polymorphism of monocyte chemotactic protein-1 (MCP-1) and its serum and peritoneal fluid levels in Korean women with endometriosis
European Journal of Obstetrics & Gynecology and Reproductive Biology 139 (2008) 106–110 www.elsevier.com/locate/ejogrb
The G(2518)A polymorphism of monocyte chemotactic protein-1 (MCP-1) and its serum and peritoneal fluid levels in Korean women with endometriosis Ja Yeon Kim, Hoon Kim, Chang Suk Suh, Seok Hyun Kim, Young Min Choi, Jung Gu Kim * Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yeungun-dong Chongno-Ku, Seoul 110-744, Republic of Korea Received 4 April 2007; received in revised form 27 July 2007; accepted 22 October 2007
Abstract Objectives: To investigate the associations between endometriosis and the G(2518)A polymorphism of monocyte chemotactic protein-1 (MCP-1), and serum and peritoneal fluid MCP-1 levels in Korean women. Study design: The G(2518)A polymorphism of MCP-1 was analyzed by restriction fragment length polymorphism in 105 women with and in 101 women without endometriosis. Serum and peritoneal fluid MCP-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: The genotype frequencies of the MCP-1 G (2518)A polymorphism were GG 36.9%, AG 52.9%, and AA 10.2%. MCP-1 genotype frequencies and serum and peritoneal fluid MCP-1 levels were similar in those with or without endometriosis and were not dependent on disease stage. A significant correlation was found between serum and peritoneal fluid levels of MCP-1. However, no differences were found between MCP-1 genotypes in terms of serum and peritoneal fluid MCP-1 levels. Conclusions: Serum and peritoneal fluid MCP-1 levels and the G (2518)A MCP-1 polymorphism were found not to be associated with endometriosis in Korean women. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Endometriosis; MCP-1; Polymorphism; Serum; Peritoneal fluid
1. Introduction Endometriosis is a common gynecologic condition characterized by the ectopic growth of endometrial tissue outside the uterus. The estimated prevalence of endometriosis is 9–33% in women of reproductive age. Although three main theories – the implantation theory, the metaplastic theory, and the combination theory – have been proposed as etiologies, a large body of evidence suggests that a complex interplay of multiple genetic, environmental, and immunologic factor is involved in the progression of endometriosis [1–4]. * Corresponding author. Tel.: +82 2 2072 3256; fax: +82 2 762 3599. E-mail address: [email protected] (J.G. Kim).
Monocyte chemotactic protein-1 (MCP-1), a specific factor that chemoattracts and activates monocytes and macrophages, is a 76-amino acid protein that is produced by many types of cells including monocytes, lymphocytes, fibroblasts, and smooth muscle cells [5]. Activated monocytes/macrophages secrete many angiogenic and other growth factors, which may promote the growth of endometrial explants and the expressions of numerous proinflammatory molecules [6,7]. Moreover, MCP-1 has been one of the most commonly investigated chemokines during recent years, because in women with endometriosis, the monocyte/macrophage system is considered to play a central role in the maintenance of humoral and cellmediated immunity, and it has been reported that the activity of MCP-1 is elevated in endometriosis patients [8]. Recently,
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2007.10.009
J.Y. Kim et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 139 (2008) 106–110
several investigators reported a relationship between serum [9–11] or peritoneal fluid [11–13] MCP-1 levels and endometriosis, but published results are conflicting. Many studies have been undertaken to identify the candidate genes responsible for endometriosis [3,14,15], but studies in different populations have produced different results, and as yet, the genes responsible for its development have not been identified. In a previous study, we showed that endometriosis was associated with the a2-Heremans Schmidt glycoprotein (AHSG) gene polymorphism [14] and vascular endothelial growth factor (VEGF) gene polymorphism [15] in Korean women. Moreover, recently, two polymorphisms of the MCP-1 distal regulatory region, G(2518)A and A(2076)T, were identified [16], and the polymorphism at position 2518 has been reported to affect the transcriptional activity of the distal regulatory region of MCP-1 and its production by monocytes [16]. Various diseases have been reported to be affected by the MCP-1 G(2518)A polymorphism [17,18]. However, to the best of our knowledge, no investigation has been conducted on the relationship between endometriosis and the MCP-1 G(2518)A polymorphism. The purposes of this study were to investigate the association between endometriosis and the presence of the MCP-1 G(2518)A polymorphism and serum and peritoneal fluid MCP-1 levels in Korean women with and without endometriosis.
2. Materials and methods 2.1. Patients One hundred and five women aged 20–40 years with endometriosis were recruited. Patients underwent laparoscopy for pelvic pain, an ovarian mass, or infertility. The presence of endometriosis was confirmed by laparoscopy and by histological examination of biopsy specimen. No patient had received any medication associated with endometriosis or had any history of pelvic surgery. One hundred and one women aged 20–40 years, who did not have endometriosis based on laparoscopic findings served as controls. All 206 study subjects had regular menstrual cycles. Laparoscopic procedures were performed during the proliferative phase of the menstrual cycle. According to the revised American Fertility Society Classification [19], 62 women had stage I or stage II endometriosis, and 43 had stage III or stage IV endometriosis. This study was approved by the Institutional Review Board of Seoul National University Hospital.
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procedures in accordance with the guidelines of the Declaration of Helsinki. Genomic DNA was extracted from peripheral blood leukocytes using QiaAmp blood kits (Qiagen GmbH, Hilden, Germany). The polymorphic region of the MCP-1 gene was amplified by polymerase chain reaction (PCR) using specific forward (50 -CCGAGATGTTCCCAGCACA30 ) and reverse primers (50 -CTGCTTTGCTTGTGCCT CTT-30 ). Genomic DNA was amplified in one unit of Taq polymerase, 10 mM Tris–HCl buffer (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 20 mM dNTP and 20 pM of each primer. PCR was performed over 35 cycles as follows; a denaturing step at 95 8C for 30 s, annealing at 60 8C for 40 s, and extension at 72 8C for 90 s. The amplified product of 929 base pairs (bp) was digested with restriction endonuclease PvuII and then electrophoresed through an agarose gel containing ethidium bromide. Gels were visualized on a transilluminator under ultraviolet light and photographed. In the absence of the PvuII restriction site (indicating the presence of the (2518)A allele), only a 929 bp fragment was visible in gel, but in the presence of this restriction site (indicating the presence of the (2518)G allele), two fragments were generated, one at 707 bp and the other at 222 bp (Fig. 1). 2.3. Measurements of serum and peritoneal fluid MCP1 Blood samples were immediately centrifuged at 1500 g for 15 min at room temperature, aliquoted, and stored at 70 8C until assay. Peritoneal fluids were obtained from 50 women with endometriosis and from 17 controls, at the time of laparoscopy before any surgical intervention in order to minimize blood contamination. Immediately after collection, peritoneal fluid samples were centrifuged at 1500 g for 15 min at room temperature and supernatants were aliquoted and stored at 70 8C until assayed. MCP-1 was measured using a Quantikine (M) enzyme-linked immunosorbent assay kit (R&D, Minneapolis, USA), according to the manufacturer’s instructions. In brief, after
2.2. Determination of MCP-1 polymorphism Blood samples were collected from an antecubital vein in all subjects of study immediately after laparoscopic
Fig. 1. The MCP-1 gene G(2518)A polymorphism determined by PCRRFLP. AA, AG and GG genotypes are shown. M: DNA ladder, RE(): not treated with restriction enzyme.
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adding serum or peritoneal fluid to wells pre-coated with mouse monoclonal antibody against MCP-1, plates were incubated for 2 h at room temperature, and then washed three times with wash buffer. Polyclonal antibody against MCP-1, conjugated to horseradish peroxidase, was added to the wells and the plates were then incubated for 2 h at room temperature. After further washing, reactions were developed by adding 200 mL of tetramethylbenzidine as chromogen, and incubating for 30 min at room temperature. Reactions were terminated by adding 50 mL of 2N sulfuric acid. Optical densities were determined at 450 nm using a VERSA max Trade Mark enzyme-linked immunosorbent assay reader (Molecular Devices, Sunnyvale, CA). The kit used had a sensitivity of 5 pg/mL, and intra- and inter-assay variations were 5.8% and 4.7%, respectively. 2.4. Statistical analysis Statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL). Genotype distributions among study subjects were tested for compliance with the Hardy–Weinberg equilibrium using the Chi-square test. Allele and genotype frequencies were compared between women with and without endometriosis using the Chi-square test. The Student’s t-test or analysis of variance was used to compare serum and peritoneal fluid MCP-1 levels with respect to the MCP-1 polymorphism. Odds ratios were calculated together with their 95% confidence intervals. The correlation between serum and peritoneal fluid MCP-1 levels was analyzed by simple regression analysis and the Pearson’s correlation coefficient was calculated. p-values of less than 0.05 were regarded significant.
3. Results The genotype frequencies of the MCP-1 polymorphism in the 206 study subjects were GG 36.9%, AG 52.9%, AA 10.2% which were in agreement with the Hardy–Weinberg equilibrium. No differences were noted between women with and without endometriosis in with respect to allele and genotype frequencies (Table 1). Allele and genotype
frequencies in women with early stage endometriosis (stage I and stage II) were not different from those with late stage endometriosis (stage III and stage IV) (Table 2). As shown in Table 3, no significant differences in age or body mass index were observed between women with and without endometriosis. Serum MCP-1 levels in women with endometriosis were not different from those in women without endometriosis. Peritoneal fluid MCP-1 levels were higher than serum MCP-1 levels, and peritoneal fluid MCP-1 levels in women with endometriosis tended to be higher than in controls, but it had no statistical significance. Serum and peritoneal fluid MCP-1 levels were not different in those with early and late stage endometriosis. Serum MCP-1 levels were found to correlate significantly with peritoneal fluid MCP-1 levels (r = 0.347, p < 0.01). However, serum and peritoneal fluid MCP-1 levels were not found to be significantly different according to MCP-1 genotypes.
4. Discussion Endometriosis is considered to be a pathological disorder caused by an interaction of multiple genetic, immunologic, and environmental factors. It has been suggested that several genetic regions are involved in susceptibility to endometriosis, but investigations on these candidate genes have produced conflicting results [3,14,15]. The immuno-inflammatory processes mediated by numerous cytokines and growth factors are now believed to play important roles in the progression of endometriosis. In endometriosis, it has been reported that peritoneal fluid has an increased chemotactic effect on macrophages [20]. As MCP-1 production is believed to recruit monocyte-macrophage lineage cells, it seems plausible that MCP-1 is secreted by metaplastic peritoneal cells in patients with pelvic endometriosis [13]. In the present study, we investigated the association between the MCP-1 G(2518)A polymorphism in the MCP-1 gene and endometriosis in Korean women and compared serum and peritoneal fluid MCP-1 levels in Korean women with and without endometriosis. The MCP-1 gene, a member of the small inducible gene family, is located on chromosome 17, subbands q11.2–q12.
Table 1 Genotype and allele frequencies for MCP-1 G(2518)A polymorphism in women with and without endometriosis Women with endometriosis (n = 105) Genotype GG AG AA GG and AG Allele G A CI: confidence interval.
39 59 7 98
(37.1%) 56.2%) (6.7%) (93.3%)
137 (65.2%) 73 (34.8%)
Women without endometriosis (n = 101) 37 50 14 87
(36.6%) (49.5%) (13.9%) (86.1%)
124 (61.4%) 78 (38.6%)
Odds ratio (95% CI)
p-Values
0.22 0.44 (0.15–1.24)
0.14
1.18 (0.78–1.80)
0.48
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Table 2 Genotype and allele frequencies for MCP-1 G(2518)A polymorphism according to endometriosis stage American fertility society
Odds ratio (95% CI)
Stage I/II (n = 62)
Stage III/IV (n = 43)
Genotype GG AG AA GG and AG
22 36 4 58
17 23 3 40
Allele G A
80 (64.5%) 44 (35.5%)
(35.5%) (58.1%) (6.5%) (93.5%)
(39.5%) (53.5%) (7.0%) (93.0%)
p-Values
0.89
57 (66.3%) 29 (33.7%)
0.92 (0.16–5.54)
0.77
0.93 (0.50–1.72)
0.91
CI: confidence interval.
In the present study, the frequency of the MCP-1 (2518)G allele in Korean women (63.0%) was found to be similar to that in Chinese women (64.0%) [17], which differs from that in Caucasian women (29.0%) [16]. Various immunoinflammatory diseases, including arthritis of systemic lupus erythematosus and systemic sclerosis, have been reported to be associated with the MCP-1 G(2518)A polymorphism [17,18]. However, in the present study, we found no differences in the genotype and allele frequencies of the MCP-1 G(2518)A polymorphism according to the presence or stage of endometriosis. To the best of our knowledge, this study is the first study conducted to establish an association between endometriosis and the MCP-1 gene polymorphism. In the present study, serum MCP-1 levels were not found to be dependent on the presence or stage of endometriosis, which is in complete agreement with the results of Akoum et al. [10], who found no difference between women with endometriosis and controls with respect to plasma MCP-1 levels during the proliferative phase of the menstrual cycle. However, several investigators have reported that serum [9,11] and peritoneal fluid [8,11,12,13] MCP-1 levels were higher in women with endometriosis than in controls. To explain this inconsistency of various studies, several points should be considered. First, the phase of the menstrual cycle at the time of blood or peritoneal fluid collection must be controlled. However, in the majority of previous studies
[9,13,21], serum or peritoneal fluid are collected without regard of the phase of menstrual cycle. Akoum et al. [10] pointed out that the phase of menstrual cycle could affect serum MCP-1 levels by reporting that significant elevation of MCP-1 levels was noted only during the secretory phase of the menstrual cycle. They also reported that MCP-1 secretion by peritoneal macrophages was higher in women with endometriosis than in healthy women controls only during the proliferative phase [8]. In the present study, serum and peritoneal fluid samples, collected only during the proliferative phase of the menstrual cycle, were included to eliminate possible variations. Second, the results of previous studies are inconsistent with regard to the endometriosis stage at which MCP-1 levels were elevated [8,9,10,11,13,21]. In contrast to the findings of Pizzo et al. [11], who found that serum and peritoneal fluid MCP-1 levels were elevated at all stages of endometriosis patients compared to normal controls, other investigators [8,9,10,13,21] have reported that only women with specific stage of endometriosis have elevated MCP-1 levels compared to normal controls. Akoum et al. demonstrated that only women with endometriosis stage I had higher serum MCP-1 levels [10], whereas Gmyrek et al. reported that MCP-1 levels were elevated only in stage III and stage IV endometriosis [9]. Moreover, the clinical endometriosis stage classification systems used and patient inclusion criteria have been inconsistent [8,9,10,11,13,21]. Third, the
Table 3 Demographic data and sera and peritoneal fluid MCP-1 levels in women without and with endometriosis Women without endometriosis (n = 76)
Women with endometriosis Stage I/II (n = 55)
Stage III/IV (n = 39)
Total (n = 94)
Age (years) BMI (kg/m2) Serum MCP-1 (pg/mL)
34.7 0.8 21.6 0.2 348.6 21.4a
34.5 0.8 21.2 0.4 340.4 23.1b
33.5 1.0 21.0 0.5 293.6 9.6 c
34.1 0.6 21.1 0.3 321.0 14.7d
Peritoneal fluid MCP-1 (pg/mL)
(n = 17) 499.7 106.7a
(n = 36) 594.7 68.6b
(n = 14) 643.5 132.5c
(n = 50) 608.3 61.1d
Values are means S.E. a p < 0.05. b p < 0.0001. c p < 0.0001. d p < 0.0001.
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sample sizes of previous studies have been too small, and thus, median values rather than mean values were compared [9,21]. Finally, differences in ethnicity, sample preparation, and methods of measurement may also have contributed to the above-mentioned results differences. It has been reported that IL-1b-treated peripheral blood mononuclear cells from healthy individuals with the MCP-1 (2518)G allele produced more MCP-1 than cells from individuals without the G allele [16]. However, in the present study, no differences were found in serum and peritoneal fluid MCP-1 levels according to the MCP-1 genotypes. These conflicting results could be explained by different methods of sample preparation and measurement, different sample sizes, and race. In conclusion, the present study shows that the MCP-1 G(2518)A polymorphism and serum and peritoneal fluid MCP-1 levels are not associated with endometriosis and that the MCP-1 gene G(2518)A polymorphism does not affect serum and peritoneal fluid MCP-1 levels in Korean women. Therefore, it seems that neither MCP-1 nor its polymorphisms play a substantial role in endometriosis in Korean women. However, our findings should be confirmed in different races and in larger numbers of subjects.
Acknowledgement This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-041-E00224).
References [1] Mathur SP. Autoimmunity in endometriosis: relevance to infertility. Am J Reprod Immunol 2000;44:89–95. [2] Kennedy S, Bennett S, Weeks DE. Affected sib-pair analysis in endometriosis. Hum Reprod Update 2001;7:411–8. [3] Zondervan KT, Cardon LR, Kennedy SH. The genetic basis of endometriosis. Curr Opin Obstet Gynecol 2001;13:309–14. [4] Simpson JL, Bischoff FZ, Kamat A, Buster JE, Carson SA. Genetics of endometriosis. Obstet Gynecol Clin North Am 2003;30:21–40. [5] Yamashiro S, Takeya M, Nishi T, et al. Tumor-derived monocyte chemoattractant protein-1 induces intratumoral infiltration of monocyte derived macrophage subpopulation in transplanted rat tumors. Am J Pathol 1994;145:856–7.
[6] Braun DP, Gebel H, House R, Rana N, Dmowski WP. Spontaneous and induced synthesis of cytokines by peripheral blood monocytes in patients with endometriosis. Fertil Steril 1996;65:1125–9. [7] Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 1996;65:925–30. [8] Akoum A, Turcot-Lemay L, Lemay A, Maheux R, McColl S. Elevated concentration and biologic activity of monocyte chemotactic protein-1 in the peritoneal fluid of patients with endometriosis. Fertil Steril 1996;66:17–23. [9] Gmyrek GB, Sozanski R, Jerzak M, et al. Evaluation of monocyte chemotactic protein-1 levels in peripheral blood of infertile women with endometriosis. Eu J Obstet Gynecol Reprod Biol 2005;122: 199–205. [10] Akoum A, Lemay A, McColl SR, Paradis I, Maheux R. Increased monocyte chemotactic protein-1 level and activity in the peripheral blood of women with endometriosis. Am J Obstet Gynecol 1996;175: 1620–5. [11] Pizzo A, Salmeri FM, Ardita F, Sofo V, Maria T, Marsico S. Behavior of cytokine levels in serum and peritoneal fluid of women with endometriosis. Gynecol Obstet Inv 2002;54:82–7. [12] Arici A, Tazuke SI, Oral E, Olive DL, Attar E. Monocyte chemotactic protein-1 concentration in peritoneal fluid of women with endometriosis and its modulation of expression in mesothelial cells. Fertil Steril 1997;67:1065–72. [13] Yih S, Katabuchi H, Araki M, et al. Expression of monocyte chemoattractant protein-1 in peritoneal endometriotic cells. Virchows Arch 2001;438:70–7. [14] Kim JG, Kim H, Ku SY, Kim SH, Choi YM, Moon SY. Association between human 2-Heremans Schmidt glycoprotein (AHSG) polymorphism and endometriosis in Korean women. Fertil Steril 2004;82:1497–500. [15] Kim SH, Choi YM, Choung SH, Jun JK, Kim JG, Moon SY. Vascular endothelial growth factor gene +405 C/G polymorphism is associated with susceptibility to advanced stage endometriosis. Hum Reprod 2005;20:2904–8. [16] Rovin BH, Lu L, Saxena R. A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophysic Res Commun 1999;259:344–8. [17] Ye DQ, Hu YS, Li XP, et al. The correlation between monocyte chemoattractant protein-1 and the arthritis of systemic lupus erythematosus among Chinese. Arch Dermatol Res 2005;296:366–71. [18] Karrer S, Bosserhoff AK, Weiderer P, et al. The 2518 promoter polymorphism in the MCP-1 gene is associated with systemic sclerosis. J Invest Dermatol 2005;124:92–8. [19] The American Fertility Society Revised classification of endometriosis. Fertil Stertil 1985;43:351–2. [20] Leiva MC, Hasty LA, Pfeifer S, Mastroianni Jr L, Lyttle CR. Increased chemotactic activity of peritoneal fluid in patients with endometriosis. Am J Obstet Gynecol 1993;168:592–8. [21] Akoum A, Lemay A, Brunet C, Hebert J. Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Fertil Steril 1995;63:322–8.
The G(2518)A polymorphism of monocyte chemotactic protein-1 (MCP-1) and its serum and peritoneal fluid levels in Korean women with endometriosis Ja Yeon Kim, Hoon Kim, Chang Suk Suh, Seok Hyun Kim, Young Min Choi, Jung Gu Kim * Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yeungun-dong Chongno-Ku, Seoul 110-744, Republic of Korea Received 4 April 2007; received in revised form 27 July 2007; accepted 22 October 2007
Abstract Objectives: To investigate the associations between endometriosis and the G(2518)A polymorphism of monocyte chemotactic protein-1 (MCP-1), and serum and peritoneal fluid MCP-1 levels in Korean women. Study design: The G(2518)A polymorphism of MCP-1 was analyzed by restriction fragment length polymorphism in 105 women with and in 101 women without endometriosis. Serum and peritoneal fluid MCP-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: The genotype frequencies of the MCP-1 G (2518)A polymorphism were GG 36.9%, AG 52.9%, and AA 10.2%. MCP-1 genotype frequencies and serum and peritoneal fluid MCP-1 levels were similar in those with or without endometriosis and were not dependent on disease stage. A significant correlation was found between serum and peritoneal fluid levels of MCP-1. However, no differences were found between MCP-1 genotypes in terms of serum and peritoneal fluid MCP-1 levels. Conclusions: Serum and peritoneal fluid MCP-1 levels and the G (2518)A MCP-1 polymorphism were found not to be associated with endometriosis in Korean women. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Endometriosis; MCP-1; Polymorphism; Serum; Peritoneal fluid
1. Introduction Endometriosis is a common gynecologic condition characterized by the ectopic growth of endometrial tissue outside the uterus. The estimated prevalence of endometriosis is 9–33% in women of reproductive age. Although three main theories – the implantation theory, the metaplastic theory, and the combination theory – have been proposed as etiologies, a large body of evidence suggests that a complex interplay of multiple genetic, environmental, and immunologic factor is involved in the progression of endometriosis [1–4]. * Corresponding author. Tel.: +82 2 2072 3256; fax: +82 2 762 3599. E-mail address: [email protected] (J.G. Kim).
Monocyte chemotactic protein-1 (MCP-1), a specific factor that chemoattracts and activates monocytes and macrophages, is a 76-amino acid protein that is produced by many types of cells including monocytes, lymphocytes, fibroblasts, and smooth muscle cells [5]. Activated monocytes/macrophages secrete many angiogenic and other growth factors, which may promote the growth of endometrial explants and the expressions of numerous proinflammatory molecules [6,7]. Moreover, MCP-1 has been one of the most commonly investigated chemokines during recent years, because in women with endometriosis, the monocyte/macrophage system is considered to play a central role in the maintenance of humoral and cellmediated immunity, and it has been reported that the activity of MCP-1 is elevated in endometriosis patients [8]. Recently,
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2007.10.009
J.Y. Kim et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 139 (2008) 106–110
several investigators reported a relationship between serum [9–11] or peritoneal fluid [11–13] MCP-1 levels and endometriosis, but published results are conflicting. Many studies have been undertaken to identify the candidate genes responsible for endometriosis [3,14,15], but studies in different populations have produced different results, and as yet, the genes responsible for its development have not been identified. In a previous study, we showed that endometriosis was associated with the a2-Heremans Schmidt glycoprotein (AHSG) gene polymorphism [14] and vascular endothelial growth factor (VEGF) gene polymorphism [15] in Korean women. Moreover, recently, two polymorphisms of the MCP-1 distal regulatory region, G(2518)A and A(2076)T, were identified [16], and the polymorphism at position 2518 has been reported to affect the transcriptional activity of the distal regulatory region of MCP-1 and its production by monocytes [16]. Various diseases have been reported to be affected by the MCP-1 G(2518)A polymorphism [17,18]. However, to the best of our knowledge, no investigation has been conducted on the relationship between endometriosis and the MCP-1 G(2518)A polymorphism. The purposes of this study were to investigate the association between endometriosis and the presence of the MCP-1 G(2518)A polymorphism and serum and peritoneal fluid MCP-1 levels in Korean women with and without endometriosis.
2. Materials and methods 2.1. Patients One hundred and five women aged 20–40 years with endometriosis were recruited. Patients underwent laparoscopy for pelvic pain, an ovarian mass, or infertility. The presence of endometriosis was confirmed by laparoscopy and by histological examination of biopsy specimen. No patient had received any medication associated with endometriosis or had any history of pelvic surgery. One hundred and one women aged 20–40 years, who did not have endometriosis based on laparoscopic findings served as controls. All 206 study subjects had regular menstrual cycles. Laparoscopic procedures were performed during the proliferative phase of the menstrual cycle. According to the revised American Fertility Society Classification [19], 62 women had stage I or stage II endometriosis, and 43 had stage III or stage IV endometriosis. This study was approved by the Institutional Review Board of Seoul National University Hospital.
107
procedures in accordance with the guidelines of the Declaration of Helsinki. Genomic DNA was extracted from peripheral blood leukocytes using QiaAmp blood kits (Qiagen GmbH, Hilden, Germany). The polymorphic region of the MCP-1 gene was amplified by polymerase chain reaction (PCR) using specific forward (50 -CCGAGATGTTCCCAGCACA30 ) and reverse primers (50 -CTGCTTTGCTTGTGCCT CTT-30 ). Genomic DNA was amplified in one unit of Taq polymerase, 10 mM Tris–HCl buffer (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 20 mM dNTP and 20 pM of each primer. PCR was performed over 35 cycles as follows; a denaturing step at 95 8C for 30 s, annealing at 60 8C for 40 s, and extension at 72 8C for 90 s. The amplified product of 929 base pairs (bp) was digested with restriction endonuclease PvuII and then electrophoresed through an agarose gel containing ethidium bromide. Gels were visualized on a transilluminator under ultraviolet light and photographed. In the absence of the PvuII restriction site (indicating the presence of the (2518)A allele), only a 929 bp fragment was visible in gel, but in the presence of this restriction site (indicating the presence of the (2518)G allele), two fragments were generated, one at 707 bp and the other at 222 bp (Fig. 1). 2.3. Measurements of serum and peritoneal fluid MCP1 Blood samples were immediately centrifuged at 1500 g for 15 min at room temperature, aliquoted, and stored at 70 8C until assay. Peritoneal fluids were obtained from 50 women with endometriosis and from 17 controls, at the time of laparoscopy before any surgical intervention in order to minimize blood contamination. Immediately after collection, peritoneal fluid samples were centrifuged at 1500 g for 15 min at room temperature and supernatants were aliquoted and stored at 70 8C until assayed. MCP-1 was measured using a Quantikine (M) enzyme-linked immunosorbent assay kit (R&D, Minneapolis, USA), according to the manufacturer’s instructions. In brief, after
2.2. Determination of MCP-1 polymorphism Blood samples were collected from an antecubital vein in all subjects of study immediately after laparoscopic
Fig. 1. The MCP-1 gene G(2518)A polymorphism determined by PCRRFLP. AA, AG and GG genotypes are shown. M: DNA ladder, RE(): not treated with restriction enzyme.
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adding serum or peritoneal fluid to wells pre-coated with mouse monoclonal antibody against MCP-1, plates were incubated for 2 h at room temperature, and then washed three times with wash buffer. Polyclonal antibody against MCP-1, conjugated to horseradish peroxidase, was added to the wells and the plates were then incubated for 2 h at room temperature. After further washing, reactions were developed by adding 200 mL of tetramethylbenzidine as chromogen, and incubating for 30 min at room temperature. Reactions were terminated by adding 50 mL of 2N sulfuric acid. Optical densities were determined at 450 nm using a VERSA max Trade Mark enzyme-linked immunosorbent assay reader (Molecular Devices, Sunnyvale, CA). The kit used had a sensitivity of 5 pg/mL, and intra- and inter-assay variations were 5.8% and 4.7%, respectively. 2.4. Statistical analysis Statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL). Genotype distributions among study subjects were tested for compliance with the Hardy–Weinberg equilibrium using the Chi-square test. Allele and genotype frequencies were compared between women with and without endometriosis using the Chi-square test. The Student’s t-test or analysis of variance was used to compare serum and peritoneal fluid MCP-1 levels with respect to the MCP-1 polymorphism. Odds ratios were calculated together with their 95% confidence intervals. The correlation between serum and peritoneal fluid MCP-1 levels was analyzed by simple regression analysis and the Pearson’s correlation coefficient was calculated. p-values of less than 0.05 were regarded significant.
3. Results The genotype frequencies of the MCP-1 polymorphism in the 206 study subjects were GG 36.9%, AG 52.9%, AA 10.2% which were in agreement with the Hardy–Weinberg equilibrium. No differences were noted between women with and without endometriosis in with respect to allele and genotype frequencies (Table 1). Allele and genotype
frequencies in women with early stage endometriosis (stage I and stage II) were not different from those with late stage endometriosis (stage III and stage IV) (Table 2). As shown in Table 3, no significant differences in age or body mass index were observed between women with and without endometriosis. Serum MCP-1 levels in women with endometriosis were not different from those in women without endometriosis. Peritoneal fluid MCP-1 levels were higher than serum MCP-1 levels, and peritoneal fluid MCP-1 levels in women with endometriosis tended to be higher than in controls, but it had no statistical significance. Serum and peritoneal fluid MCP-1 levels were not different in those with early and late stage endometriosis. Serum MCP-1 levels were found to correlate significantly with peritoneal fluid MCP-1 levels (r = 0.347, p < 0.01). However, serum and peritoneal fluid MCP-1 levels were not found to be significantly different according to MCP-1 genotypes.
4. Discussion Endometriosis is considered to be a pathological disorder caused by an interaction of multiple genetic, immunologic, and environmental factors. It has been suggested that several genetic regions are involved in susceptibility to endometriosis, but investigations on these candidate genes have produced conflicting results [3,14,15]. The immuno-inflammatory processes mediated by numerous cytokines and growth factors are now believed to play important roles in the progression of endometriosis. In endometriosis, it has been reported that peritoneal fluid has an increased chemotactic effect on macrophages [20]. As MCP-1 production is believed to recruit monocyte-macrophage lineage cells, it seems plausible that MCP-1 is secreted by metaplastic peritoneal cells in patients with pelvic endometriosis [13]. In the present study, we investigated the association between the MCP-1 G(2518)A polymorphism in the MCP-1 gene and endometriosis in Korean women and compared serum and peritoneal fluid MCP-1 levels in Korean women with and without endometriosis. The MCP-1 gene, a member of the small inducible gene family, is located on chromosome 17, subbands q11.2–q12.
Table 1 Genotype and allele frequencies for MCP-1 G(2518)A polymorphism in women with and without endometriosis Women with endometriosis (n = 105) Genotype GG AG AA GG and AG Allele G A CI: confidence interval.
39 59 7 98
(37.1%) 56.2%) (6.7%) (93.3%)
137 (65.2%) 73 (34.8%)
Women without endometriosis (n = 101) 37 50 14 87
(36.6%) (49.5%) (13.9%) (86.1%)
124 (61.4%) 78 (38.6%)
Odds ratio (95% CI)
p-Values
0.22 0.44 (0.15–1.24)
0.14
1.18 (0.78–1.80)
0.48
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Table 2 Genotype and allele frequencies for MCP-1 G(2518)A polymorphism according to endometriosis stage American fertility society
Odds ratio (95% CI)
Stage I/II (n = 62)
Stage III/IV (n = 43)
Genotype GG AG AA GG and AG
22 36 4 58
17 23 3 40
Allele G A
80 (64.5%) 44 (35.5%)
(35.5%) (58.1%) (6.5%) (93.5%)
(39.5%) (53.5%) (7.0%) (93.0%)
p-Values
0.89
57 (66.3%) 29 (33.7%)
0.92 (0.16–5.54)
0.77
0.93 (0.50–1.72)
0.91
CI: confidence interval.
In the present study, the frequency of the MCP-1 (2518)G allele in Korean women (63.0%) was found to be similar to that in Chinese women (64.0%) [17], which differs from that in Caucasian women (29.0%) [16]. Various immunoinflammatory diseases, including arthritis of systemic lupus erythematosus and systemic sclerosis, have been reported to be associated with the MCP-1 G(2518)A polymorphism [17,18]. However, in the present study, we found no differences in the genotype and allele frequencies of the MCP-1 G(2518)A polymorphism according to the presence or stage of endometriosis. To the best of our knowledge, this study is the first study conducted to establish an association between endometriosis and the MCP-1 gene polymorphism. In the present study, serum MCP-1 levels were not found to be dependent on the presence or stage of endometriosis, which is in complete agreement with the results of Akoum et al. [10], who found no difference between women with endometriosis and controls with respect to plasma MCP-1 levels during the proliferative phase of the menstrual cycle. However, several investigators have reported that serum [9,11] and peritoneal fluid [8,11,12,13] MCP-1 levels were higher in women with endometriosis than in controls. To explain this inconsistency of various studies, several points should be considered. First, the phase of the menstrual cycle at the time of blood or peritoneal fluid collection must be controlled. However, in the majority of previous studies
[9,13,21], serum or peritoneal fluid are collected without regard of the phase of menstrual cycle. Akoum et al. [10] pointed out that the phase of menstrual cycle could affect serum MCP-1 levels by reporting that significant elevation of MCP-1 levels was noted only during the secretory phase of the menstrual cycle. They also reported that MCP-1 secretion by peritoneal macrophages was higher in women with endometriosis than in healthy women controls only during the proliferative phase [8]. In the present study, serum and peritoneal fluid samples, collected only during the proliferative phase of the menstrual cycle, were included to eliminate possible variations. Second, the results of previous studies are inconsistent with regard to the endometriosis stage at which MCP-1 levels were elevated [8,9,10,11,13,21]. In contrast to the findings of Pizzo et al. [11], who found that serum and peritoneal fluid MCP-1 levels were elevated at all stages of endometriosis patients compared to normal controls, other investigators [8,9,10,13,21] have reported that only women with specific stage of endometriosis have elevated MCP-1 levels compared to normal controls. Akoum et al. demonstrated that only women with endometriosis stage I had higher serum MCP-1 levels [10], whereas Gmyrek et al. reported that MCP-1 levels were elevated only in stage III and stage IV endometriosis [9]. Moreover, the clinical endometriosis stage classification systems used and patient inclusion criteria have been inconsistent [8,9,10,11,13,21]. Third, the
Table 3 Demographic data and sera and peritoneal fluid MCP-1 levels in women without and with endometriosis Women without endometriosis (n = 76)
Women with endometriosis Stage I/II (n = 55)
Stage III/IV (n = 39)
Total (n = 94)
Age (years) BMI (kg/m2) Serum MCP-1 (pg/mL)
34.7 0.8 21.6 0.2 348.6 21.4a
34.5 0.8 21.2 0.4 340.4 23.1b
33.5 1.0 21.0 0.5 293.6 9.6 c
34.1 0.6 21.1 0.3 321.0 14.7d
Peritoneal fluid MCP-1 (pg/mL)
(n = 17) 499.7 106.7a
(n = 36) 594.7 68.6b
(n = 14) 643.5 132.5c
(n = 50) 608.3 61.1d
Values are means S.E. a p < 0.05. b p < 0.0001. c p < 0.0001. d p < 0.0001.
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sample sizes of previous studies have been too small, and thus, median values rather than mean values were compared [9,21]. Finally, differences in ethnicity, sample preparation, and methods of measurement may also have contributed to the above-mentioned results differences. It has been reported that IL-1b-treated peripheral blood mononuclear cells from healthy individuals with the MCP-1 (2518)G allele produced more MCP-1 than cells from individuals without the G allele [16]. However, in the present study, no differences were found in serum and peritoneal fluid MCP-1 levels according to the MCP-1 genotypes. These conflicting results could be explained by different methods of sample preparation and measurement, different sample sizes, and race. In conclusion, the present study shows that the MCP-1 G(2518)A polymorphism and serum and peritoneal fluid MCP-1 levels are not associated with endometriosis and that the MCP-1 gene G(2518)A polymorphism does not affect serum and peritoneal fluid MCP-1 levels in Korean women. Therefore, it seems that neither MCP-1 nor its polymorphisms play a substantial role in endometriosis in Korean women. However, our findings should be confirmed in different races and in larger numbers of subjects.
Acknowledgement This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-041-E00224).
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