Reduced expression and concomitant promoter hypermethylation of HOXA10 in endometrium from women wearing intrauterine devices

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1 Reduced expression and concomitant promoter hypermethylation of HOXA10 in endometrium from women wearing intrauterine devices Yuan Lu, M.D., Ph.D., a Jichan Nie, M.D., a Xishi Liu, M.D., Ph.D., a and Sun-Wei Guo, Ph.D. b a Department of Gynecology, Shanghai OB/GYN Hospital, and Department of Gynecology and Obstetrics, Shanghai Medical School, Fudan University; and b Renji Hospital, and the Institute of Obstetric and Gynecologic Research, Shanghai Jiao Tong University School of Medicine, Shanghai, People s Republic of China Objective: To determine whether prolonged intrauterine device (IUD) usage is associated with decreased HOXA10 expression and increased methylation in the endometrium. Design: Observational study. Patient(s): Women wearing IUDs and not wearing IUDs. Intervention(s): Immunohistochemistry of HOXA10 and methylation-specific PCR. Main Outcome Measure(s): Endometrial HOXA10 expression levels and methylation frequency. Result(s): The IUD usage was associated with decreased endometrial HOXA10 expression, concordant with higher frequency of HOXA10 promoter hypermethylation. The HOXA10 hypermethylation was associated with the duration of IUD usage, irrespective age, gravidity, parity, and IUD type. Conclusion(s): Prolonged IUD use may induce endometrial HOXA10 hypermethylation and reduced expression. These results suggest a possible novel mode of action for IUD, a possibility to rectify the aberrant methylation through pharmacologic means, and a possible noninvasive way for detection of aberrant methylation at HOXA10. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Contraception, endometrium, expression, HOXA10, IUD, methylation The intrauterine devices (IUDs) are the world s most widely used method of reversible birth control for women. In mainland China, IUDs are used by 45% of married women (1). Much of their popularity come from their low failure rate, long duration of action, low cost, and, above all, efficacy (2). Thus, IUDs have been suggested as an alternative to female sterilization, especially in younger women who are more likely to experience regret after sterilization (3). Despite their worldwide popularity and proven efficacy, their exact mechanism to prevent pregnancy has not been fully elucidated (4). It is thought that the contraceptive effects of IUDs may be due to a sterile inflammatory reaction in the uterine cavity that interferes with sperm function, therefore that fertilization is less likely to occur (5 7). The IUDs may also interfere with implantation, but the extent to which this contributes to their contraceptive action is unknown. Although considered a reversible contraceptive method, the pregnancy rates (PR) after IUD removal appear to be lower than that in a control population (8 10), especially for prolonged IUD usage (11). One recent genomewide gene expression profiling study Received July 10, 2009; revised September 7, 2009; accepted September 10, 2009; published online November 1, Y.L. has nothing to disclose. J.N. has nothing to disclose. X.L. has nothing to disclose. S.-W.G. has nothing to disclose. Yuan Lu and Jichan Nie contributed equally to this article. Supported by grants C / (Y.L.) and (S.-W.G.) from the National Science Foundation of China, and the Pujian Project and grant from the Shanghai Science and Technology Commission (S.-W.G.). Reprint requests: Sun-Wei Guo, Ph.D., Institute of Obstetric and Gynecologic Research, Shanghai Jiao Tong University School of Medicine, and Renji Hospital, 145 Shandong Zhong Road, Shanghai , People s Republic of China (FAX: ; hoxa10@gmail.com). reported that 1 year after 2-month usage of IUD and subsequent removal only about 80% of the genes recovered their normal expression profile, but other genes remained dysregulated (12). However, little is known about any transcriptional or epigenetic changes in endometrium after prolonged use of IUDs. Recently, Tetrault et al. (13) reported that the use of copper IUD was associated with decreased endometrial HOXA10 expression. They also reported that age and the menstrual phase, but not the duration of IUD usage, were associated with HOXA10 expression. The HOX genes are highly conserved transcriptional regulators that play an essential role in determining tissue identity during embryonic development and are involved in endometrial development and endometrial receptivity (14, 15). In particular, HOXA10 is expressed in endometrium and has been shown to be essential for embryo implantation in mice and humans (15). In the endometrium from women with endometriosis HOXA10 expression is reduced, which may be responsible for endometriosis-associated subfertility (16, 17). We have previously shown that, concordant with reduced endometrial HOXA10 expression in women with endometriosis, the HOXA10 promoter region is hypermethylated (18). Endometriosis-induced HOXA10 hypermethylation in endometrium has since been confirmed in animal models of endometriosis (19, 20). Because DNA methylation is associated with gene silencing (21), naturally the question arises as whether IUD usage can induce HOXA10 hypermethylation in endometrium, which could be a proximate cause for decreased endometrial HOXA10 expression. In addition, as only the copper IUD was examined, it is unclear whether the use of noncopper IUDs could also be associated with decreased HOXA10 expression. Finally, as the average duration of IUD usage reported by Tetrault et al. (13) was 17 months, it is unclear whether the similar pattern still holds for longer duration of IUD usage, and if so, whether this is associated with HOXA10 methylation /$36.00 Fertility and Sterility â Vol. 94, No. 5, October doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 In this study, we examined endometrial HOXA10 expression levels by immunohistochemistry and HOXA10 methylation status in IUD users and nonusers. We sought to investigate as whether prolonged IUD usage is associated with decreased HOXA10 expression and increased methylation in the endometrium. We also sought to correlate HOXA10 expression levels and HOXA10 methylation status, and, if so, whether longer duration of IUD usage is associated with higher frequency of methylation. MATERIALS AND METHODS Patients and Tissue Samples Thirty-seven women wearing IUDs for varying durations were recruited to this study. They were seen at Shanghai OB/GYN Hospital, Fudan University Shanghai Medical College, from Among them, 27 (73.0%) wore older, O-shaped, IUDs made of inert metals, and the other 10 (27.0%) wore newer IUDs made of copper, with 4, 3, 2, and 1 being V-, r-, I-, and T-shaped IUDs, respectively. These women came to the hospital for several reasons. Seven (18.9%) wanted their IUDs removed due to spotting/bleeding, another 7 (18.9%) wanted to have children, and the other 23 (62.2%) wanted removal because they had worn IUDs for more than 10 years. The control population was 27 roughly age- and menstrual cycle-matched women who had no IUD use for more than 10 years. These women complained of mild menorrhagia, but their hemoglobin levels were all R110 g/l, indicating no signs of anemia. All women in the study and control groups were premenopausal and had no hormone therapy for R6 months before tissue collection. They all underwent ultrasonographic and gynecological examinations to rule out other conditions such as uterine myoma, adenomyosis, endometriosis, uterine hypertrophy, endometrial lesions, and gynecological malignancy. The menstrual phase at the time of tissue harvesting was dated using Noyes s criteria and was grouped either in the proliferative or secretory phase. In the group of women wearing IUDs, 30 were in the proliferative phase and 7 were in the secretory phase. In the control group all women had regular, apparently normal menstrual cycles (27 35 days) with 17 being in the proliferative phase and 10 in the secretory phase, giving no indication of any hormonal disturbances. For each patient recruited to the study, information was collected from medical charts and interviewing patients on age at curettage, type of IUDs (for study group), duration of IUD usage (if any), gravidity, and parity. All endometrial tissue samples, harvested by curettage, were examined histologically. These were free of hyperplasia, atypical hyperplasia, and endometrial cancer. Half of the tissue samples were paraffin-embedded in tissue blocks, and the remaining half was used for methylation analysis. This study was approved by the institutional ethics review board of Shanghai OB/GYN Hospital. Immunohistochemistry Consecutive 4-mm sections were obtained from each paraffin-embedded tissue block, with the first resultant slide stained with hematoxylin and eosin (H & E) for endometrial dating and for pathologic diagnosis, if any, and the subsequent slides stained for HOXA10. Routine deparaffinization and rehydration procedures were performed. The goat polyclonal antibodies against HOXA10 (sc-17159; Santa Cruz Biotechnology, Santa Cruz, CA), diluted to 1:50, were used as a primary antibody. For antigen retrieval, the slides were heated at 98 C in an ethylenediaminetetraacetic acid (EDTA) buffer (ph 9.0) for a total of 30 minutes and cooled at room temperature. Sections were then incubated overnight with the primary antibody at 4 C. After slides were rinsed, the biotinylated secondary antibody, Supervision Universal (antisheep) Detection Reagent of horse radish peroxidase (HRP) (# ; KPL, Gaithersburg, MD), was incubated at room temperature for 30 minutes. The bound antibody complexes were stained for 3 5 minutes or until appropriate for microscopic examination with diaminobenzidine, counterstained with hematoxylin, and mounted. Immunoreactivity staining was characterized quantitatively by digital image analysis using Image Pro-Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD) as reported previously (22, 23) without prior knowledge of any of the clinicopathological information. Immunostaining scores were evaluated independently by two persons (Y.L. and J.C.N.). Discrepancies, if they occurred, were resolved by consensus. DNA Extraction and Methylation-Specific PCR Fresh endometrial tissue samples were digested by proteinase K overnight at 50 C and genomic DNA was isolated using the Genomic DNA isolation kit (Shenerg Biocolor, Shanghai, China) following the manufacturer s instructions. Two fragments, F1 and F2, located, respectively, in the promoter region and the first intronic region of HOXA10, previously reported to be methylated in endometrium of women with endometriosis (18), were examined by methylation-specific polymerase chain reaction (PCR) for methylation. Sodium bisulfite conversion of genomic DNA was performed as instructed by the EZ DNA Methylation Gold Kit (Zymo Research, San Francisco, CA). After bisulfite conversion, methylation-specific PCR for HOXA10 was performed using the primers described previously (18). CpGenome-Universal Methylated DNA (Chemicon, Schwalbach, Germany) was used as the methylated control and CpGenome-Universal unmethylated DNA (Chemicon) was used as the unmethylated control. A null control with all reagents except DNA templates was also used. The PCR conditions were as follows: 95 C for 3 minutes, 40 cycles (95 C for 30 seconds, 60 C for 30 seconds, and 72 C for 30 seconds), and 72 C for 10 minutes. When the CpG sites in the region, analyzed by methylation-specific PCR are methylated, the M (methylated) band would show up. On the other hand, the U (unmethylated) band would be present when the sites are unmethylated. Occasionally, both bands could be present if the sites are partially methylated. Statistical Analysis The comparison of distributions of continuous variables between two or among three or more groups was made using the Wilcoxon test and Kruskal-Wallis test, respectively. Pearson s or Spearman s rank correlation coefficient was used when appropriate. To evaluate which factors are associated with the HOXA10 expression level, a multiple linear regression analysis was used in conjunction with a stepwise regression, with the HOXA10 expression level square-transformed to improve normality. To evaluate the effect of the duration of IUD usage and other factors on the probability of methylation, a multiple logistic regression model was used. Because the number of women using copper IUDs was small, we lumped all women with copper IUDs into one group (copper IUDs), irrespective of shape. Hence the type of IUDs was grouped into two: IUDs made of inert metals (noncopper) and of copper. The values P<.05 were considered statistically significant. All computations were made with R (24) ( RESULTS The IUD users and nonusers recruited to this study were comparable with age and menstrual phase. However, they differed significantly in gravidity and parity, with the IUD users having higher parity and gravidity (Table 1). HOXA10 Expression in IUD and Non-IUD Groups Consistent with published reports (13, 25), endometrial HOXA10 expression was localized in the nucleus of glandular epithelial and stromal cells, irrespective of the menstrual phase (Fig. 1). In addition, HOXA10 expression was significantly higher in the secretory phase than that in the proliferative phase in the endometrium from non-iud users (P¼.009), but no such difference was found in IUD users (P¼.65). There was no difference in HOXA10 expression between women wearing different types of IUDs (P¼.62). The Effect of Age, Menstrual Phase, and Duration of IUD Usage on Endometrial HOXA10 Expression As reported by Tetrault et al. (13), the HOXA10 expression was significantly lower in the endometrium from IUD users than that from 1584 Lu et al. HOXA10 hypermethylation in women wearing IUDs Vol. 94, No. 5, October 2010

3 TABLE 1 Comparison of characteristics between IUD users and nonusers. Variable Women not wearing IUD (n [ 27) Women wearing IUD (n [ 37) P value in assessing the difference Age, y Mean (SD) (9.4) (6.5) Range Menstrual phase Proliferative 17 (63.0%) 30 (81.1%).15 Secretory 10 (37.0%) 7 (18.9%) Duration of IUD usage, y Not applicable Not applicable Mean (SD) 13.6 (6.8) Range Median 15 %10 12 >10 25 IUD type Not applicable Not applicable O type (inert metals) 27 (73.0%) Copper 10 (27.0%) Gravidity 0 5 (18.5%) 0 (0%) 1 4 (14.8%) 7 (18.9%) R2 18 (66.7%) 30 (81.1%).026 Parity 0 6 (22.2%) 0 (0%) 1 13 (48.1%) 36 (97.3%) R2 8 (29.6%) 1 (2.7%) Methylation status in fragment 1 region Unmethylated or lightly methylated 19 (70.4%) 9 (24.3%) Methylated 8 (29.6%) 28 (75.7%).005 Methylation status in fragment 2 region Unmethylated or lightly methylated 18 (66.7%) 11 (29.7%) Methylated 9 (33.3%) 26 (70.3%).005 Methylation status in both regions Unmethylated or lightly methylated 19 (70.4%) 12 (32.4%) Methylated 8 (29.6%) 25 (67.6%).005 nonusers (P¼.0008). We also found that the lower HOXA10 expression level was associated with longer duration of IUD usage (P¼.003; Fig. 2). Although the difference in HOXA10 expression, as well as in gravidity and parity, was significant between IUD users and nonusers (Table 1), we did not find any correlation between HOXA10 expression level and gravidity (r ¼ 0.09, P¼.50) and parity (r ¼ 0.16, P¼.21). To identify factors potentially influencing the HOXA10 expression, we carried out a multiple linear regression analysis of square-transformed HOXA10 expression level incorporating age, menstrual phase, duration of IUD usage, type of IUD (copper vs. noncopper), IUD usage (yes or no), gravidity, and parity as covariates. We found that IUD usage was the only covariate significantly associated with the HOXA10 expression level (P¼.025). Therefore, IUD usage was the only factor that was associated with the HOXA10 expression level. The Effect of HOXA10 Promoter Methylation Status on HOXA10 Expression We carried out methylation-specific PCR analyses and found that in fragment 1 of the HOXA10 promoter, about 70% of IUD users had methylation, whereas only 24% of non-iud users did, and the difference was statistically significant (P¼.005; Table 1). The pattern was similar in fragment 2 or when the methylation status was defined to be methylated in both fragments (Table 1, Fig. 3). There was a statistically significant difference in HOXA10 expression levels between women with and without methylated fragment 1 (P¼ ) or fragment 2 (P¼ ), or both (P¼ ). In fact, a multiple linear regression analysis incorporating age, menstrual phase, duration of IUD usage, type of IUD, gravidity, parity, and methylation status as covariates revealed that the methylation status at either fragment 1 or 2, or both, was the only covariate that was significantly associated with the HOXA10 expression level (P¼ , , and , respectively). This suggests that methylation status is the only variable that determines the HOXA10 expression level, eclipsing gravidity, parity, duration of IUD usage, and age. Methylation in the HOXA10 Promoter and Duration of IUD Usage We found that longer duration of IUD usage was associated with a higher frequency of methylation in the HOXA10 promoter region. Specifically, the methylation frequency in fragment 1 was 29.6%, 58.3%, and 84%, respectively, in the control group, the %10-year IUD usage, and the >10-year IUD usage groups (P¼.0003). The corresponding numbers in fragment 2 or both were 33.3%, 58.3%, Fertility and Sterility â 1585

4 FIGURE 1 Immunohistochemical staining of HOXA10 in the endometrium. (A) Negative control using Tris-buffered saline (TBS) instead of the primary antibody in an endometrium tissue sample without intrauterine device (IUD) usage. (B) HOXA10 staining in the endometrium of a control patient without IUD usage. (C) HOXA10 staining in the endometrium of a woman wearing IUD. All magnifications, 400. and 76.0% (P¼.008), and 29.6%, 50.0%, and 76.0% (P¼.003), respectively. These results suggest that the duration of IUD usage was correlated with the frequency of HOXA10 methylation. We found that women who had methylation in fragment 1, 2, or both, in the promoter region of HOXA10 gene actually had longer duration of IUD usage (P¼.0002,.0014, and.0006, respectively). This suggests that the HOXA10 promoter hypermethylation may be induced by prolonged IUD usage. We carried out a multiple logistic regression to determine which factors affect the methylation status, incorporating age, duration of IUD usage, gravidity, parity, and type of IUDs. In all cases we found FIGURE 2 Boxplot of endometrial HOXA10 expression levels among IUD users with different duration of usage and nonusers. that duration of IUD usage is the only factor that is associated with the methylation status, with P¼.0007 for fragment 1, P¼.002 for fragment 2, and P¼.001 for both. The odds ratio for having a methylated state for every 10 years of IUD usage ranged from 2.98 for fragment 2 to 3.75 for fragment 1. This suggests that the duration of IUD usage completely eclipses other factors in inducing methylation in the HOXA10 promoter, and that for every 10 years of IUD usage, the risk of having HOXA10 methylation is increased by about threefold. DISCUSSION At present only a few factors have been identified to cause aberrant methylation (26), which include inflammation, viral infection, and prolonged transcriptional suppression (27 32). To our best FIGURE 3 Representative methylation-specific polymerase chain reaction (PCR) (MSP) results in the F1 and F2 fragments of HOXA10 using bisulfite-treated endometrial tissue sample from a non-iud user and an IUD user. U ¼ reactions using the HOXA10 primer set specific for the unmethylated CpG sites giving rise to a 260-bp band; M ¼ reactions using the primer set specific for the methylated CpG sites giving rise to a 260-bp band. Marker is a molecular marker Lu et al. HOXA10 hypermethylation in women wearing IUDs Vol. 94, No. 5, October 2010

5 knowledge this is the first report that wearing IUDs for an extended period is associated with, and likely responsible for, higher frequency of DNA methylation in the endometrium. The IUD usage reportedly results in a significant influx of inflammatory cells in the uterine cavity and the tubal wall (5, 6, 33). Stimulation with one proinflammatory cytokine, interleukin (IL)-1b, which is known to be involved in the inflammatory immune response (34), is reported to result in nearly 90% decrease in HOXA10 expression in decidual cells (35). It is thus speculated that the IUD s local foreign body effects may stimulate the production of proinflammatory cytokines, such as IL-1b, leading to decreased endometrial HOXA10 expression (13). Along this line, prolonged IUD usage could result in persistent suppression of HOXA10 expression, which in turn induces HOXA10 promoter hypermethylation, ultimately leading to silencing of its expression. Our data support this scenario. Although aging is a known factor that is associated with methylation of certain genes (27, 28), we found that it is the duration of IUD usage, not age, that is significantly associated with, and possibly responsible for, HOXA10 methylation. The finding that menstrual phase-dependent HOXA10 expression is seen only in non-iud users, not in IUD users, is likely due to the difference in frequency of methylation, or, more precisely, in frequency of silenced HOXA10 gene. When most women with IUDs have HOXA10 methylated and thus silenced, we would not see a cycle-dependent pattern of expression. In contrast, when most women still have a normally functional HOXA10 gene, the cycle-dependent expression pattern would be the norm. Our results suggest that promoter hypermethylation may be a more proximate cause for decreased HOXA10 expression associated with IUD usage. We also provided evidence that the duration of IUD usage is the single most important factor associated with HOXA10 methylation, more important than other factors such as age and IUD types. Our results suggest that one possible mode of action for contraception is through inhibition of HOXA10 expression, first transcriptionally but later on epigenetically, thereby interfering with or hindering the implantation process. This seems to be consistent with reports from several long-term studies that when pregnancy occurs in IUD users, the embryo is more likely to be ectopic than in women using no contraception at all or in those who become pregnant while taking oral contraceptives (OC) (2, 36). Although our results suggest that prolonged IUD usage may result in HOXA10 promoter hypermethylation in the endometrium and thus decrease the chance for pregnancy even after removal of IUDs, our results also suggest a possible way to rectify this aberration, that is, by demethylation agents, or possibly histone deacetylase inhibitors (37), or both, as seen in the reactivation of E-cadherin in endometriotic cells (38). This could be achieved, for example, by local administration of these compounds without affecting other untoward organs or tissues. At the very least, our results suggest that one possible noninvasive way to evaluate whether HOXA10 gene is methylated or not is through the analysis of menstrual blood, which is abundant, easy, and inexpensive to retrieve. In summary we have found that IUD usage, irrespective of age and IUD types, is associated with a higher frequency of HOXA10 promoter hypermethylation, concordant with decreased HOXA10 expression. 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