- Email: [email protected]
Induction of early decidualization by cadmium, a major contaminant of cigarette smoke
Induction of early decidualization by cadmium, a major contaminant of cigarette smoke To investigate the effect of cadmium (Cd) contamination on endometrial function, human endometrial stromal cells were isolated and cultured with E2 plus P in the presence of Cd, a major contaminant in cigarette smoke, and assayed for PRL concentrations and its messenger RNA expression. Cd significantly increased PRL concentrations in the culture media and significantly up-regulated PRL messenger RNA expression of the endometrial stromal cells, suggesting that Cd stimulates decidualization of the endometrium and may disrupt endometrial environment, causing early decidualization. (Fertil Steril 2009;91:1614–7. 2009 by American Society for Reproductive Medicine.) Key Words: Cadmium, decidualization, prolactin, IGFBP-1
The technique of IVF led to major improvements in the treatment of infertility, although problems of implantation failure remain (1). Decidualization, that is, endometrial stromal differentiation, is indispensable for implantation of the developing blastocyst. Decidualization of human endometrial stromal cells occurs in the late secretory phase of the menstrual cycle and is characterized by morphologic and functional differentiation. Decidual endometrial stromal cells release PRL and insulin-like growth factor binding protein-1 (IGFBP-1), which serve as key markers of decidualization. Cadmium (Cd) is one of the major contaminants of cigarette smoke. Cd is a heavy metal that has no known beneficial role in the human body and is linked to a wide range of harmful effects on mammalian reproduction (2, 3). Timed decidual transformation of endometrial stromal cells is important for successful implantation in human reproduction. In the present study, we performed experiments using an in vitro model of decidualization to investigate the effect of Cd contamination on human endometrium. Endometrial tissues were obtained from 20 patients (ages 41–49 years, 9 in the proliferative phase and 11 in the secretory phase) undergoing hysterectomy for benign gynecologic conditions. It is difficult to obtain an amount of endometrium of younger reproductive-age women, because such women who undergo hysterectomy are rare. There were no differences in the results whether cells were obtained from the proliferative phase or the secretory phase. All patients had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before
Received July 29, 2008; revised December 5, 2008; accepted December 10, 2008; published online February 6, 2009. R.T. has nothing to disclose. H.H. has nothing to disclose. M.M. has nothing to disclose. Y.H. has nothing to disclose. F.N. has nothing to disclose. T.Y. has nothing to disclose. O.T. has nothing to disclose. Y.T. has nothing to disclose. Reprint requests: Mikio Momoeda, M.D., Ph.D., Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (FAX: 03-3816-2017; E-mail: [email protected]).
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surgery. There were no smokers among the patients. The experimental procedures were approved by the Institutional Review Board of the University of Tokyo, and signed informed consent for use of the endometrium was obtained from each woman. Isolation and culture of human endometrial stromal cells and in vitro decidualization were achieved as described previously (4, 5). Human endometrial stromal cells were treated with 2% charcoal-stripped fetal bovine serum in the presence of 10 nmol/L E2 plus 1 mmol/L P in the presence or absence of CdCl2 or 0.1% ethanol vehicle (control). Culture media were collected, and wells were replenished every 3 days. After treatment with E2 and P for 12 days, endometrial stromal cells and culture medium were collected and stored at 80 C until use. Concentrations of PRL in the culture medium were determined with use of enzyme-linked fluorescent assay (Arklay, Kyoto, Japan). Enzyme-linked fluorescent assay is a method of automatic measurement that relies on the same principle as ELISA. Total RNA was extracted from endometrial stromal cells with use of the RNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized from 750 ng of total cellular RNA with use of the First Strand cDNA synthesis kit (Toyobo, Osaka, Japan). The amount of PRL, IGFBP-1, P receptor (PR), and b-actin messenger RNA (mRNA) in endometrial stromal cells was quantified by real-time polymerase chain reaction (PCR) with the Light Cycler rapid thermal cycler system (Roche Diagnostics, Lewes, United Kingdom). Human PR has two distinct isoforms (PR A and PR B). We designed PCR primers for PR within a common sequence area to both subtypes. The PCR products were subcloned into pCR2.1TOPO vector with TOPO TA cloning kit (Invitrogen, Carlsbad, CA), and these vectors were used for the standard of quantitative real-time PCR. We confirmed the identity of the product by DNA sequencing. Statistically significant differences between treatment groups were identified with use of one-way analysis of
Fertility and Sterility Vol. 91, No. 4, Supplement, April 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.12.055
variance. All statistical tests were conducted with use of Statview (SAS Institute, Cary, NC). Achievement of in vitro decidualization was quantified by measuring PRL concentrations in the culture medium of endometrial stromal cells. Prolactin serves as a key marker of decidualization (6). Prolactin was detectable in the culture medium of endometrial stromal cells treated by E2 and P, ensuring that in vitro decidualization was achieved in the present study. We have studied the dose effects of Cd on PRL release from endometrial stromal cells. Prolactin concentrations significantly increased to 2.4-fold at 1 mmol/L CdCl2 compared with E2 and P treatment, although PRL concentrations did not change at 10 nmol/L or 100 nmol/L (Fig. 1A). The amount of PRL mRNA did not change at 10 nmol/L or 100 nmol/L of CdCl2 (Fig. 1B). At 1 mmol/L CdCl2 which is consistent with the doses to increase PRL secretion, PRL mRNA expression was significantly up-regulated to 2.9fold compared with E2- and P-treated cells, which is consistent with the doses to increase PRL secretion. Figure 1C shows the time course of PRL accumulation. With treatment of E2 and P, PRL was not detectable on day 3. The concentration of PRL was 1.3 1.0 ng/mL on day 6 and showed a significant progressive increase on days 9 and 12 (Fig. 1C). Prolactin concentrations were significantly higher in the Cd group on days 6, 9, and 12 compared with the control group (Fig. 1C). Prolactin concentrations in the Cd group on day 9 were equal to PRL concentrations in the control group on day 12. Insulin-like growth factor binding protein-1 is also one of the key markers of decidualization. The expression of IGFBP-1 mRNAwas significantly up-regulated to 3.8-fold and 4.5-fold at 100 nmol/L and 1 mmol/L CdCl2, respectively. Without E2 treatment, PRL mRNA was approximately 79% compared with that in E2- and P-treated cells, showing that E2 is involved in PRL mRNA expression. Interestingly, the effect of Cd was observed without E2 treatment. The stimulatory effect of Cd on decidualization was more than that of 10 nmol/L of E2 or 100 nmol/L of E2 (Fig. 1D). Because decidualization is a P-dependent process, we investigated the amount of PR mRNA in endometrial stromal cells treated with E2 and Cd. Treatment with 10 nmol/L E2 and P significantly up-regulated the expression of PR mRNA to 2.0-fold compared with treatment with P only. However, treatment with Cd did not change the expression of PR mRNA. Because the primers we used did not distinguish between PR A and PR B, it is unknown whether Cd could affect selectively the expression of PR A or PR B. Cd is a heavy metal that has no known beneficial role in the human body (2, 3). Surprisingly Cd significantly elevated PRL levels, indicating that Cd stimulates decidualization in vitro. Treatment with Cd further up-regulated PRL mRNA in the decidual endometrial stromal cells, showing that decidualization is regulated transcriptionally by Cd. In the present study, treatment of Cd also up-regulated IGFBP-1 mRNA in the decidual endometrial stromal cells. Studies in decidualization have revealed that IGFBP-1 gene Fertility and Sterility
expression occurs coincidentally with morphologic changes of fibroblast-like cells to rounded polygonal cells. Insulinlike growth factor binding protein-1 secretion begins on day 10 after the LH peak in vivo (7). It is interesting that this time point is relevant to a marked reduction of endometrial receptivity and a rapidly increasing risk of implantation failure (8). Consequently, Fluhr et al. (5) speculated that this may signal the closing of the so-called implantation window and that IGFBP-1 may have a possible direct role in restricting uterine receptivity. Therefore, Cd may affect the endometrium by up-regulating IGFBP-1 and accelerating the decidualization. There are some reports noting a relationship between Cd and infertility. For example, the rates of sterility in married women in an area of Cd pollution were fivefold significantly higher than those in a control area (9). Among animal data, SC injections of CdCl2 caused a period of sterility in the golden hamster (10). Cd reduced P production of cultured human ovarian granulosa cells (11). There are few direct evidences between human endometrium and Cd in the literature. Yaman et al. (12) compared trace metal concentrations with cancerous and noncancerous human endometrium. There were no significant differences with Cd concentrations (12). There are some studies showing that Cd directly affects endometrium. Johnson et al. (13) revealed that exposure to Cd increased uterine wet weight and induced proliferation of the endometrium in ovariectomized animals. Cd is one of the major contaminants of cigarette smoke. Cd concentration in the serum of smokers is significantly higher than in that of nonsmokers (14). Gala_zyn-Sidorczuk et al. (15) reported that blood and urinary Cd concentrations in smokers were two to four times higher than those in nonsmokers. The reproductive organs of smokers generally are considered to be at an increased risk of exposure to toxic levels of Cd. In the follicular fluid and placentas of smokers, Cd concentrations are higher and P levels are lower than in those of nonsmokers (16, 17). Cd concentration found in follicular fluids of heavy smokers is approximately 70 nmol/L (16). Cd concentration found in the intima of arterial vessel walls of smokers has been reported to be 1.5 mmol/L (15). Cd concentrations obtained in the present study (100 nmol/L and 1 mmol/L) were not this high. Thus, we believe Cd involvement in human reproduction is not negligible. Cigarette smoking long has been known to have an adverse effect on female fertility. Disorders of ovarian function are known to lower the mean age of menopause (18). The pregnancy rate for IVF cycles is worse in heavy smokers compared with nonsmokers (19). Recently, it has been reported that cigarette smoke affects uterine receptiveness. A study of patients having oocyte donation revealed that the pregnancy rate of heavy smokers was significantly lower than that of nonsmokers, suggesting that smoking may affect endometrial function (20). Neal et al. (21) reported lower pregnancy and implantation rates in smokers undergoing IVF cycles in which the number of morphologically good
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FIGURE 1 (A) Effects of CdCl2 on the secretion of PRL from decidualized endometrial stromal cells. Endometrial stromal cells were treated with 10 nmol/L E2 (E) and 1 mmol/L P with or without CdCl2 (10, 100, 1,000 nmol/L). Data are represented as means SEM of three independent experiments. (B) Effects of CdCl2 on PRL mRNA levels in decidualized endometrial stromal cells. Endometrial stromal cells were treated with or without CdCl2 in the presence of E2 and P for 12 days. Prolactin mRNA expression was determined by quantitative real-time reverse transcriptase PCR. Relative expression (PRL mRNA/b-actin mRNA) is shown. Data are represented as means SEM of five independent experiments. (C) Prolactin accumulation by endometrial stromal cells treated with 10 nmol/L E2 and 1 mmol/L P with or without CdCl2 (1 mmol/L). Culture medium was collected every 3 days and assayed for PRL concentrations. Each circle represents the mean of seven independent experiments (mean SEM). Open circle: E2 and P treatment; closed circle: E2, P, and CdCl2 treatment. (D) Effects of CdCl2 on PRL mRNA levels in decidualized endometrial stromal cells. Endometrial stromal cells were treated with or without CdCl2 in the presence or absence of E2 or P for 12 days. Relative expression (PRL mRNA/b-actin mRNA) is shown. Statistically significant differences between treatment groups were identified with use of one-way analysis of variance. All statistical tests were conducted with use of Statview. Data are represented as means SEM of five independent experiments. Significant differences are indicated between matching letters (P< .05).
Tsutsumi. Correspondence. Fertil Steril 2009.
embryos replaced was similar to that in nonsmokers. These data suggest that cigarette smoke disrupts not only ovarian function but also uterine receptiveness. In the present study, Cd treatment resulted in elevated PRL concentrations from the early days of in vitro decidualization. Prolactin concentrations in the Cd group on day 9 were equal to PRL concentrations in the control group on day 12, indicating that Cd induced earlier decidualization and disrupted timed decidualization. These effects may be the cause of the reduction of implantation seen in heavy smokers. The mechanism involved in the stimulatory effect of Cd is yet to be elucidated. It has been reported that Cd mimics estrogenic effects (22). In rats, Cd precipitated early puberty onset, increased uterine weight, and enhanced mammary 1616
Tsutsumi et al.
Correspondence
development (13). Estrogen is one of the inducers of decidualization. However, in the present study, the stimulatory effect of 1 mmol/L Cd was more than those of 10 nmol/L E2 and 100 nmol/L E2, which are the most effective doses of E2 (Fig. 1D). Hence, it is improbable that the estrogenic effect of Cd was involved in the Cd-induced decidualization. The cellular responses to P are mediated predominantly by PR, a member of the superfamily of ligand-inducible transcription factors. Progesterone receptor is also one of the estrogen-responsive genes. Brosens et al. (23) revealed that maintenance of elevated PR levels inhibits initiation of decidualization in human endometrial stromal cells. In the present study, estrogen up-regulated the expression of PR mRNA on day 12. But the addition of Cd did not change Vol. 91, No. 4, Supplement, April 2009
the expression of PR mRNA. Decidualization by Cd may not be induced via PR expression. Furthermore, it also showed that Cd did not mimic estrogenic effect in human in vitro decidualization. In the present study, we demonstrated that Cd stimulated in vitro decidualization of human endometrium. The mechanism involved in the stimulatory effect of Cd is yet to be elucidated. Ryo Tsutsumi, M.D. Hisahiko Hiroi, M.D., Ph.D. Mikio Momoeda, M.D., Ph.D. Yumi Hosokawa, M.D. Fumiko Nakazawa, M.D., Ph.D. Tetstu Yano, M.D., Ph.D. Osamu Tsutsumi, M.D., Ph.D. Yuji Taketani, M.D., Ph.D. Department of Obstetrics and Gynecology, the University of Tokyo, Tokyo, Japan REFERENCES 1. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod 2006;21:3036–43. 2. Thompson J, Bannigan J. Cadmium: toxic effects on the reproductive system and the embryo. Reprod Toxicol 2008;25:304–15. 3. Henson MC, Chedrese PJ. Endocrine disruption by cadmium, a common environmental toxicant with paradoxical effects on reproduction. Exp Biol Med 2004;229:383–92. 4. Harada M, Osuga Y, Takemura Y, Yoshino O, Koga K, Hirota Y, et al. Mechanical stretch upregulates IGFBP-1 secretion from decidualized endometrial stromal cells. Am J Physiol Endocrinol Metab 2006;290:268–72. 5. Fluhr H, Krenzer S, Deperschmidt M, Zwirner M, Wallwiener D, Licht P. Human chorionic gonadotropin inhibits insulin-like growth factor-binding protein-1 and prolactin in decidualized human endometrial stromal cells. Fertil Steril 2006;86:236–8. 6. Garzia E, Borgato S, Cozzi V, Doi P, Bulfamante G, Persani L, et al. Lack of expression of endometrial prolactin in early implantation failure: a pilot study. Hum Reprod 2004;19:1911–6. 7. Licht P, Russu V, Lehmeyer S, Moll J, Siebzehnrubl E, Wildt L. Intrauterine microdialysis reveals cycle-dependent regulation of endometrial insulin-like growth factor binding protein-1 secretion by human chorionic gonadotropin. Fertil Steril 2002;78:252–8.
Fertility and Sterility
8. Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999;340:1796–9. 9. Wu SY, Tian J, Wang MZ, Pan BJ, L€u HD, Wang ZM, et al. The effect of cadmium pollution on reproductive health in females. Zhonghua Liu Xing Bing Xue Za Zhi 2004;25:852–5. 10. Saksena SK, Salmonsen R. Effects of cadmium chloride on ovulation and on induction of sterility in the female golden hamster. Biol Reprod 1983;29:249–56. 11. Paksy K, Rajczy K, Forga´cs Z, La´za´r P, Bernard A, Ga´ti I, et al. Effect of cadmium on morphology and steroidogenesis of cultured human ovarian granulosa cells. J Appl Toxicol 1997;7:321–7. 12. Yaman M, Kaya G, Simsek M. Comparison of trace element concentrations in cancerous and noncancerous human endometrial and ovary tissues. Int J Gynecol Cancer 2007;1:220–8. 13. Johnson MD, Kenney N, Stoica A, Hilakivi-Clarke L, Singh B, Chepko G, et al. Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland. Nat Med 2003;9:1081–4. 14. Bernhard D, Rossmann A, Henderson B, Kind M, Seubert A, Wick G. Increased serum cadmium and strontium levels in young smokers: effects on arterial endothelial cell gene transcription. Arterioscler Thromb Vasc Biol 2006;26:833–8. 15. Gala_zyn-Sidorczuk M, Brzoska MM, Moniuszko-Jakoniuk J. Estimation of Polish cigarettes contamination with cadmium and lead, and exposure to these metals via smoking. Environ Monit Assess 2008;137: 481–93. 16. Zenzes MT, Krishnan S, Krishnan B, Zhang H, Casper RF. Cadmium accumulation in follicular fluid of women in in vitro fertilization–embryo transfer is higher in smokers. Fertil Steril 1995;64:599–603. 17. Piasek M, Blanusa M, Kostial K, Laskey JW. Placental cadmium and progesterone concentrations in cigarette smokers. Reprod Toxicol 2001;15:673–81. 18. Jick H, Porter J, Morrison AS. Relation between smoking and age of natural menopause. Report from the Boston Collaborative Drug Surveillance Program. Lancet 1977;309:1354–5. 19. Lintsen AM, Pasker-de Jong PC, de Boer EJ, Burger CW, Jansen CA, Braat DD, et al. Effects of subfertility cause, smoking and body weight on the success rate of IVF. Hum Reprod 2005;20:1867–75. 20. Soares SR, Simon C, Remohı J, Pellicer A. Cigarette smoking affects uterine receptiveness. Hum Reprod 2007;22:543–7. 21. Neal MS, Hughes EG, Holloway AC, Foster WG. Sidestream smoking is equally as damaging as mainstream smoking on IVF outcomes. Hum Reprod 2005;20:2531–5. 22. Garcia-Morales P, Saceda M, Kenney N, Kim N, Salomon DS, Gottardis MM, et al. Effect of cadmium on estrogen receptor levels and estrogen-induced responses in human breast cancer cells. J Biol Chem 1994;269:16896–901. 23. Brosens JJ, Hayashi N, White JO. Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells. Endocrinology 1999;140:4809–20.
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The technique of IVF led to major improvements in the treatment of infertility, although problems of implantation failure remain (1). Decidualization, that is, endometrial stromal differentiation, is indispensable for implantation of the developing blastocyst. Decidualization of human endometrial stromal cells occurs in the late secretory phase of the menstrual cycle and is characterized by morphologic and functional differentiation. Decidual endometrial stromal cells release PRL and insulin-like growth factor binding protein-1 (IGFBP-1), which serve as key markers of decidualization. Cadmium (Cd) is one of the major contaminants of cigarette smoke. Cd is a heavy metal that has no known beneficial role in the human body and is linked to a wide range of harmful effects on mammalian reproduction (2, 3). Timed decidual transformation of endometrial stromal cells is important for successful implantation in human reproduction. In the present study, we performed experiments using an in vitro model of decidualization to investigate the effect of Cd contamination on human endometrium. Endometrial tissues were obtained from 20 patients (ages 41–49 years, 9 in the proliferative phase and 11 in the secretory phase) undergoing hysterectomy for benign gynecologic conditions. It is difficult to obtain an amount of endometrium of younger reproductive-age women, because such women who undergo hysterectomy are rare. There were no differences in the results whether cells were obtained from the proliferative phase or the secretory phase. All patients had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before
Received July 29, 2008; revised December 5, 2008; accepted December 10, 2008; published online February 6, 2009. R.T. has nothing to disclose. H.H. has nothing to disclose. M.M. has nothing to disclose. Y.H. has nothing to disclose. F.N. has nothing to disclose. T.Y. has nothing to disclose. O.T. has nothing to disclose. Y.T. has nothing to disclose. Reprint requests: Mikio Momoeda, M.D., Ph.D., Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (FAX: 03-3816-2017; E-mail: [email protected]).
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surgery. There were no smokers among the patients. The experimental procedures were approved by the Institutional Review Board of the University of Tokyo, and signed informed consent for use of the endometrium was obtained from each woman. Isolation and culture of human endometrial stromal cells and in vitro decidualization were achieved as described previously (4, 5). Human endometrial stromal cells were treated with 2% charcoal-stripped fetal bovine serum in the presence of 10 nmol/L E2 plus 1 mmol/L P in the presence or absence of CdCl2 or 0.1% ethanol vehicle (control). Culture media were collected, and wells were replenished every 3 days. After treatment with E2 and P for 12 days, endometrial stromal cells and culture medium were collected and stored at 80 C until use. Concentrations of PRL in the culture medium were determined with use of enzyme-linked fluorescent assay (Arklay, Kyoto, Japan). Enzyme-linked fluorescent assay is a method of automatic measurement that relies on the same principle as ELISA. Total RNA was extracted from endometrial stromal cells with use of the RNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized from 750 ng of total cellular RNA with use of the First Strand cDNA synthesis kit (Toyobo, Osaka, Japan). The amount of PRL, IGFBP-1, P receptor (PR), and b-actin messenger RNA (mRNA) in endometrial stromal cells was quantified by real-time polymerase chain reaction (PCR) with the Light Cycler rapid thermal cycler system (Roche Diagnostics, Lewes, United Kingdom). Human PR has two distinct isoforms (PR A and PR B). We designed PCR primers for PR within a common sequence area to both subtypes. The PCR products were subcloned into pCR2.1TOPO vector with TOPO TA cloning kit (Invitrogen, Carlsbad, CA), and these vectors were used for the standard of quantitative real-time PCR. We confirmed the identity of the product by DNA sequencing. Statistically significant differences between treatment groups were identified with use of one-way analysis of
Fertility and Sterility Vol. 91, No. 4, Supplement, April 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.12.055
variance. All statistical tests were conducted with use of Statview (SAS Institute, Cary, NC). Achievement of in vitro decidualization was quantified by measuring PRL concentrations in the culture medium of endometrial stromal cells. Prolactin serves as a key marker of decidualization (6). Prolactin was detectable in the culture medium of endometrial stromal cells treated by E2 and P, ensuring that in vitro decidualization was achieved in the present study. We have studied the dose effects of Cd on PRL release from endometrial stromal cells. Prolactin concentrations significantly increased to 2.4-fold at 1 mmol/L CdCl2 compared with E2 and P treatment, although PRL concentrations did not change at 10 nmol/L or 100 nmol/L (Fig. 1A). The amount of PRL mRNA did not change at 10 nmol/L or 100 nmol/L of CdCl2 (Fig. 1B). At 1 mmol/L CdCl2 which is consistent with the doses to increase PRL secretion, PRL mRNA expression was significantly up-regulated to 2.9fold compared with E2- and P-treated cells, which is consistent with the doses to increase PRL secretion. Figure 1C shows the time course of PRL accumulation. With treatment of E2 and P, PRL was not detectable on day 3. The concentration of PRL was 1.3 1.0 ng/mL on day 6 and showed a significant progressive increase on days 9 and 12 (Fig. 1C). Prolactin concentrations were significantly higher in the Cd group on days 6, 9, and 12 compared with the control group (Fig. 1C). Prolactin concentrations in the Cd group on day 9 were equal to PRL concentrations in the control group on day 12. Insulin-like growth factor binding protein-1 is also one of the key markers of decidualization. The expression of IGFBP-1 mRNAwas significantly up-regulated to 3.8-fold and 4.5-fold at 100 nmol/L and 1 mmol/L CdCl2, respectively. Without E2 treatment, PRL mRNA was approximately 79% compared with that in E2- and P-treated cells, showing that E2 is involved in PRL mRNA expression. Interestingly, the effect of Cd was observed without E2 treatment. The stimulatory effect of Cd on decidualization was more than that of 10 nmol/L of E2 or 100 nmol/L of E2 (Fig. 1D). Because decidualization is a P-dependent process, we investigated the amount of PR mRNA in endometrial stromal cells treated with E2 and Cd. Treatment with 10 nmol/L E2 and P significantly up-regulated the expression of PR mRNA to 2.0-fold compared with treatment with P only. However, treatment with Cd did not change the expression of PR mRNA. Because the primers we used did not distinguish between PR A and PR B, it is unknown whether Cd could affect selectively the expression of PR A or PR B. Cd is a heavy metal that has no known beneficial role in the human body (2, 3). Surprisingly Cd significantly elevated PRL levels, indicating that Cd stimulates decidualization in vitro. Treatment with Cd further up-regulated PRL mRNA in the decidual endometrial stromal cells, showing that decidualization is regulated transcriptionally by Cd. In the present study, treatment of Cd also up-regulated IGFBP-1 mRNA in the decidual endometrial stromal cells. Studies in decidualization have revealed that IGFBP-1 gene Fertility and Sterility
expression occurs coincidentally with morphologic changes of fibroblast-like cells to rounded polygonal cells. Insulinlike growth factor binding protein-1 secretion begins on day 10 after the LH peak in vivo (7). It is interesting that this time point is relevant to a marked reduction of endometrial receptivity and a rapidly increasing risk of implantation failure (8). Consequently, Fluhr et al. (5) speculated that this may signal the closing of the so-called implantation window and that IGFBP-1 may have a possible direct role in restricting uterine receptivity. Therefore, Cd may affect the endometrium by up-regulating IGFBP-1 and accelerating the decidualization. There are some reports noting a relationship between Cd and infertility. For example, the rates of sterility in married women in an area of Cd pollution were fivefold significantly higher than those in a control area (9). Among animal data, SC injections of CdCl2 caused a period of sterility in the golden hamster (10). Cd reduced P production of cultured human ovarian granulosa cells (11). There are few direct evidences between human endometrium and Cd in the literature. Yaman et al. (12) compared trace metal concentrations with cancerous and noncancerous human endometrium. There were no significant differences with Cd concentrations (12). There are some studies showing that Cd directly affects endometrium. Johnson et al. (13) revealed that exposure to Cd increased uterine wet weight and induced proliferation of the endometrium in ovariectomized animals. Cd is one of the major contaminants of cigarette smoke. Cd concentration in the serum of smokers is significantly higher than in that of nonsmokers (14). Gala_zyn-Sidorczuk et al. (15) reported that blood and urinary Cd concentrations in smokers were two to four times higher than those in nonsmokers. The reproductive organs of smokers generally are considered to be at an increased risk of exposure to toxic levels of Cd. In the follicular fluid and placentas of smokers, Cd concentrations are higher and P levels are lower than in those of nonsmokers (16, 17). Cd concentration found in follicular fluids of heavy smokers is approximately 70 nmol/L (16). Cd concentration found in the intima of arterial vessel walls of smokers has been reported to be 1.5 mmol/L (15). Cd concentrations obtained in the present study (100 nmol/L and 1 mmol/L) were not this high. Thus, we believe Cd involvement in human reproduction is not negligible. Cigarette smoking long has been known to have an adverse effect on female fertility. Disorders of ovarian function are known to lower the mean age of menopause (18). The pregnancy rate for IVF cycles is worse in heavy smokers compared with nonsmokers (19). Recently, it has been reported that cigarette smoke affects uterine receptiveness. A study of patients having oocyte donation revealed that the pregnancy rate of heavy smokers was significantly lower than that of nonsmokers, suggesting that smoking may affect endometrial function (20). Neal et al. (21) reported lower pregnancy and implantation rates in smokers undergoing IVF cycles in which the number of morphologically good
1615
FIGURE 1 (A) Effects of CdCl2 on the secretion of PRL from decidualized endometrial stromal cells. Endometrial stromal cells were treated with 10 nmol/L E2 (E) and 1 mmol/L P with or without CdCl2 (10, 100, 1,000 nmol/L). Data are represented as means SEM of three independent experiments. (B) Effects of CdCl2 on PRL mRNA levels in decidualized endometrial stromal cells. Endometrial stromal cells were treated with or without CdCl2 in the presence of E2 and P for 12 days. Prolactin mRNA expression was determined by quantitative real-time reverse transcriptase PCR. Relative expression (PRL mRNA/b-actin mRNA) is shown. Data are represented as means SEM of five independent experiments. (C) Prolactin accumulation by endometrial stromal cells treated with 10 nmol/L E2 and 1 mmol/L P with or without CdCl2 (1 mmol/L). Culture medium was collected every 3 days and assayed for PRL concentrations. Each circle represents the mean of seven independent experiments (mean SEM). Open circle: E2 and P treatment; closed circle: E2, P, and CdCl2 treatment. (D) Effects of CdCl2 on PRL mRNA levels in decidualized endometrial stromal cells. Endometrial stromal cells were treated with or without CdCl2 in the presence or absence of E2 or P for 12 days. Relative expression (PRL mRNA/b-actin mRNA) is shown. Statistically significant differences between treatment groups were identified with use of one-way analysis of variance. All statistical tests were conducted with use of Statview. Data are represented as means SEM of five independent experiments. Significant differences are indicated between matching letters (P< .05).
Tsutsumi. Correspondence. Fertil Steril 2009.
embryos replaced was similar to that in nonsmokers. These data suggest that cigarette smoke disrupts not only ovarian function but also uterine receptiveness. In the present study, Cd treatment resulted in elevated PRL concentrations from the early days of in vitro decidualization. Prolactin concentrations in the Cd group on day 9 were equal to PRL concentrations in the control group on day 12, indicating that Cd induced earlier decidualization and disrupted timed decidualization. These effects may be the cause of the reduction of implantation seen in heavy smokers. The mechanism involved in the stimulatory effect of Cd is yet to be elucidated. It has been reported that Cd mimics estrogenic effects (22). In rats, Cd precipitated early puberty onset, increased uterine weight, and enhanced mammary 1616
Tsutsumi et al.
Correspondence
development (13). Estrogen is one of the inducers of decidualization. However, in the present study, the stimulatory effect of 1 mmol/L Cd was more than those of 10 nmol/L E2 and 100 nmol/L E2, which are the most effective doses of E2 (Fig. 1D). Hence, it is improbable that the estrogenic effect of Cd was involved in the Cd-induced decidualization. The cellular responses to P are mediated predominantly by PR, a member of the superfamily of ligand-inducible transcription factors. Progesterone receptor is also one of the estrogen-responsive genes. Brosens et al. (23) revealed that maintenance of elevated PR levels inhibits initiation of decidualization in human endometrial stromal cells. In the present study, estrogen up-regulated the expression of PR mRNA on day 12. But the addition of Cd did not change Vol. 91, No. 4, Supplement, April 2009
the expression of PR mRNA. Decidualization by Cd may not be induced via PR expression. Furthermore, it also showed that Cd did not mimic estrogenic effect in human in vitro decidualization. In the present study, we demonstrated that Cd stimulated in vitro decidualization of human endometrium. The mechanism involved in the stimulatory effect of Cd is yet to be elucidated. Ryo Tsutsumi, M.D. Hisahiko Hiroi, M.D., Ph.D. Mikio Momoeda, M.D., Ph.D. Yumi Hosokawa, M.D. Fumiko Nakazawa, M.D., Ph.D. Tetstu Yano, M.D., Ph.D. Osamu Tsutsumi, M.D., Ph.D. Yuji Taketani, M.D., Ph.D. Department of Obstetrics and Gynecology, the University of Tokyo, Tokyo, Japan REFERENCES 1. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod 2006;21:3036–43. 2. Thompson J, Bannigan J. Cadmium: toxic effects on the reproductive system and the embryo. Reprod Toxicol 2008;25:304–15. 3. Henson MC, Chedrese PJ. Endocrine disruption by cadmium, a common environmental toxicant with paradoxical effects on reproduction. Exp Biol Med 2004;229:383–92. 4. Harada M, Osuga Y, Takemura Y, Yoshino O, Koga K, Hirota Y, et al. Mechanical stretch upregulates IGFBP-1 secretion from decidualized endometrial stromal cells. Am J Physiol Endocrinol Metab 2006;290:268–72. 5. Fluhr H, Krenzer S, Deperschmidt M, Zwirner M, Wallwiener D, Licht P. Human chorionic gonadotropin inhibits insulin-like growth factor-binding protein-1 and prolactin in decidualized human endometrial stromal cells. Fertil Steril 2006;86:236–8. 6. Garzia E, Borgato S, Cozzi V, Doi P, Bulfamante G, Persani L, et al. Lack of expression of endometrial prolactin in early implantation failure: a pilot study. Hum Reprod 2004;19:1911–6. 7. Licht P, Russu V, Lehmeyer S, Moll J, Siebzehnrubl E, Wildt L. Intrauterine microdialysis reveals cycle-dependent regulation of endometrial insulin-like growth factor binding protein-1 secretion by human chorionic gonadotropin. Fertil Steril 2002;78:252–8.
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