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Endocrine disruptors and human reproductive failure: the in vitro effect of phthalates on human luteal cells
Endocrine disruptors and human reproductive failure: the in vitro effect of phthalates on human luteal cells Federica Romani, M.D.,a Anna Tropea, M.D., Ph.D.,a Elisa Scarinci, M.D.,a Alex Federico, M.D.,a Cinzia Dello Russo, M.D., Ph.D.,b Lucia Lisi, Ph.D.,b Stefania Catino, M.L.T.,b Antonio Lanzone, M.D.,a and Rosanna Apa, M.D., Ph.D.a a Cattolica del Sacro Cuore; and Istituto di Ginecologia ed Ostetricia, Universita Cattolica del Sacro Cuore, Rome, Italy
b
Istituto di Farmacologia, Universita
Objective: To evaluate the influence of phthalates on human luteal cell function. Design: Laboratory study. Setting: University hospital. Patient(s): Twenty-three normally menstruating patients in the midluteal phase. Intervention(s): Human luteal cells isolated from corpora lutea for primary cultures. Main Outcome Measure(s): Progesterone (P4) and prostaglandin release assayed by enzyme immunoassay, vascular endothelial growth factor (VEGF) secretion by enzyme-linked immunosorbent assay (ELISA), and VEGF mRNA expression by real-time polymerase chain reaction. Result(s): We investigated the effect of di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) on basal and hCG-induced progesterone (P4) release, as well as DEHP effect on the balance between prostaglandin (PG) E2, vascular endothelial growth factor (VEGF)-luteotrophic factors, and the luteolitic PGF2a in isolated human steroidogenc cells. Phthalates influence on VEGF expression has been also evaluated. DEHP, DBP, and BBP were able to reduce both basal and hCG-stimulated P4 as well as PGE2 release. PGF2a release was reduced after DEHP incubation. VEGF protein release was decreased by the incubation with the tested phthalates. VEGF mRNA expression was not affected by DEHP, DBP, and BBP. As expected, both hCG and cobalt chloride were able to induce P4 release and VEGF release and mRNA expression in human luteal cells respectively. Conclusion(s): The results show the ability of phthalates to affect luteal steroidogenesis as well as the balance between luteotrophic and luteolytic factors suggesting an interference of phthalates in human luteal function. These data may contribute to clarify the classically known impaired reproductive health observed after Use your smartphone phthalates exposure. (Fertil SterilÒ 2014;102:831–7. Ó2014 by American Society for Reproducto scan this QR code tive Medicine.) and connect to the Key Words: Corpus luteum, endocrine disruptors, luteal function Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/romanif-endocrine-disruptors-phthalates-luteal-cells/
P
hthalates are synthetic chemicals that belong to the endocrine disruptor family (1), and their high production volume has resulted in
ubiquitous human exposure (2). Globally, more than 18 billion pounds of phthalates are used each year, primarily as plasticizers in flexible poly-
Received March 6, 2014; revised May 13, 2014; accepted May 27, 2014; published online July 10, 2014. F.R. has nothing to disclose. A.T. has nothing to disclose. E.S. has nothing to disclose. A.F. has nothing to disclose. C.D.R. has nothing to disclose. L.L. has nothing to disclose. S.C. has nothing to disclose. A.L. has nothing to disclose. R.A. has nothing to disclose. Reprint requests: Federica Romani, M.D., Cattedra di Fisiopatologia della Riproduzione Umana, Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy (E-mail: romani. Universita [email protected]). Fertility and Sterility® Vol. 102, No. 3, September 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.05.041 VOL. 102 NO. 3 / SEPTEMBER 2014
discussion forum for this article now.*
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vinyl chloride (PVC) products (2, 3); used to impart flexibility to plastics, they can be found in most PVC products including vinyl upholstery, tablecloths, shower curtains, several sprays including pesticides, solvents, and soft-squeeze children's toys. The use of these compounds also is approved in several medical devices such as tubing, blood bags, and vinyl gloves, and they also can be found as additives in cosmetics products such as lotion, perfume, and nail polish. 831
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY The high production volume and common use of phthalates has resulted in humans commonly being exposed to phthalates on a daily basis through ingestion, inhalation, and dermal contact (4–6). Because these plasticizers cannot form strong molecular bonds with polymers, they are rapidly leached into the environment and have become a widespread environmental contaminate (2, 7–10). Dozens of phthalates have been developed over the years, and an association between phthalate exposure and adverse reproductive health outcomes has been suggested (11). Interestingly, phthalates have been demonstrated to reduce fertility in both humans and animal models (12), and occupational exposure to phthalates has been associated with decreased pregnancy rates, increased miscarriages, and other gestational complications (13, 14). Moreover, it has been demonstrated that these plasticizers negatively modulate oocyte growth, ovulation, and embryonic development, leading to impaired ovarian function (12, 15). Interference with ovarian steroid synthesis and metabolism could partially explain these effects. Indeed, phthalates exert antiandrogenic or estrogenic effects in vitro, impairing 17bestradiol affinity to its receptors (2, 16–18) and modulating steroid biosynthesis and metabolism by acting as a peroxisome proliferator-activated receptor (PPAR) agonist as well (11). Of note, among the phthalates, di-(2-ethylhexyl) phthalate (DEHP) is well known to be the most abundant and potent reproductive toxicant chemical (Center for the Evaluation of Risks to Human Reproduction-CERHR 2000). Nevertheless, women of reproductive age have a specific exposure risk profile to di-n-butyl phthalate (DBP), which is commonly used in many beauty products (2, 19). Finally, exposure to n-butyl benzyl phthalate (BBP) is well known to be associated with reproductive alterations in males as well as with gynecologic diseases such as endometriosis (20, 21). To expand the available data on impaired reproductivehormone balance resulting from phthalates exposure, our in vitro study investigated the potential effects of DEHP, DBP, and BBP on human luteal cell function. The corpus luteum is a unique transient reproductive gland essential for the establishment and maintenance of early pregnancy. In particular, corpus luteum function is a hormone-dependent phenomenon. Both alpha and beta estrogen receptors as well as PPAR are expressed in the corpus luteum. Through these receptors, xenoestrogens such as phthalates can exert their influence on luteal function. We investigated whether these plasticizers can affect both basal and gonadotropinstimulated luteal progesterone (P4) production in highly purified human luteal cells. Moreover, to evaluate the possible alteration of the balance between intraovarian luteotrophic and luteolytic regulators, we analyzed the effect of DEHP, DBP, and BBP on the luteal release of vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2)—both local luteotrophic factors (19, 20)—and PGF2a, a classic luteolytic modulator (22, 23). In addition, we examined the effect of DEHP, DBP, and BBP in modulating VEGF luteal mRNA expression.
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MATERIALS AND METHODS Cell Cultures Corpora lutea were obtained from 23 normally menstruating women (25 to 38 years old) in the midluteal phase (days 5 to 6 from ovulation) at the time of surgery for nonendocrine gynecologic diseases. The protocol was approved by the institutional review board of Universita Cattolica del Sacro Cuore in Rome, and all patients provided written informed consent. The corpora lutea were dated on the basis of the presumptive day of ovulation (day 0), as determined by urinary luteinizing hormone (LH) peak, ultrasound detection of corpus luteum or disappearance of the dominant follicle, and a rise in the plasma P4 concentration. The human luteal cells cultures were performed as previously reported elsewhere (24). Briefly, luteal tissue was enzymatically dissociated in nutrient mixture F-12 (Flow Laboratories)/Hepes (Sigma-Aldrich) medium containing type IV collagenase (200 U/mL) (Sigma-Aldrich). The identify of both large and small human luteal cells was confirmed by their positive staining for lipids with oil red O14 (25). Isolated human luteal cells (250,000 cells/mL) were plated on 48-well dishes for enzyme-linked immunosorbent assay (ELISA) or enzyme immuno assay (EIA) or for real-time polymerase chain reaction (PCR) and cultured for 24 hours in 5% CO2 and 95% air at 37 C. At the end of isolation and 24 hours after all the treatments, human luteal cells were counted in a hemocytometer, and their viability was determined by trypan blue exclusion test. Neither treatment nor solvent for dissolving tested substances affected either the cell count or cell viability. Human luteal cells were incubated for 24 hours with serum-free medium alone (control), human chorionic gonadotropin (hCG, 100 ng/mL; Calbiochem Inalco), cobalt(II) chloride (CoCl2, 10 mM, chemical hypoxia), DEHP (10–6–10–9 M), DBP (10–6–10–9 M), or BBP (10–6–10–9 M) (Sigma Aldrich s.r.l.) alone or with hCG (100 ng/mL). Three different wells were used for each experimental condition. The doses we used of the phenols were not greater than the previously demonstrated serum and follicular fluid levels (26, 27). After the incubation, human luteal cells were treated for total RNA extraction while the culture media were separately collected and assayed for P4, PG, and VEGF detection.
PGs, P4, and VEGF Assays According to manufacturer's instructions, the PGE2, PGF2a, and P4 levels in the culture media were assayed with specific EIA kits (Cayman Chemical) and VEGF with a VEGF ELISA kit (R&D Systems). For P4, the EIA kit intra-assay coefficient of variation (CVw) and interassay coefficient of variation (CVb) were 7% and 9%, respectively, and the detection limit (DL) was 10 pg/mL. For PG, the EIA kit CVw and CVb were 12% and 9%, respectively, and the DL was 9 pg/mL. For VEGF, the ELISA kit CVw and CVb were 3.5% and 6.7%, respectively, and the sensitivity was 5.0 pg/mL.
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Fertility and Sterility® Total RNA Extraction, cDNA-synthesis, and Realtime PCR Analysis Total cytoplasmic RNA was extracted using the RNeasy Micro kit (Qiagen), which included a 15-minute DNAse treatment. The RNA concentration was measured using the Quant-iTTM RiboGreen RNA Assay Kit (Invitrogen). A standard curve in the range of 0–10 ng was run in each assay using 16S and 23S ribosomal RNA (rRNA) from Escherichia coli as the standard, as provided by the kit. Aliquots (0.1 mg) of RNA were converted to cDNA using random hexamer primers. Quantitative changes in mRNA levels were estimated by real-time quantitative PCR (qPCR) using the following cycling conditions: 35 cycles of denaturation at 95 C for 20 seconds; annealing and extension at 60 C for 20 seconds, using the Brilliant III Ultra Fast SYBR Green QPCR Master Mix 2X (Stratagene). The PCR reactions were performed in a 20-mL reaction volume in a MX3000P real-time PCR machine (Stratagene). Primers used for the evaluation of gene expression were a-tubulin 984 forward (50 -CCC TCG CCA TGG TAA ATA CAT-30 ) and 1093 reverse (50 -ACT GGA TGG TAC GCT TGG TCT-30 ), which yielded a 110 base pair (bp) product; human VEGFa 1045 forward (50 TTT CTG CTG TCT TGG GTG CAT TGG-30 ) and 1160 reverse (50 -ACC ACT TCG TGA TGA TTC TGC CC-30 ), which yielded a 116-bp product; and human VEGFa-165 1458 forward (50 -AAA TCC CTG TGG GCC TTG CT-30 ) and 1683 reverse (50 GCT TGT CAC ATC TGC AAG TAG CT-30 ), which yielded a 146-bp product. The relative mRNA concentrations were calculated from the take-off point of reactions (threshold cycle, Ct)
using the comparative quantitation method performed by Stratagene software and based upon the DDCt method. This analysis approximates a given sample's target mRNA (e.g., COX2) level relative to the mean of the target mRNA levels in untreated controls (‘‘calibrator’’ value), thus permitting statistical analysis of deviation from the mean even among the controls. The Ct values for a-tub expression served as a normalizing signal. In each assay, the PCR efficiency was also calculated using serial dilution of one experimental sample; efficiency values between 94% and 98% were found for each primer set and weretaken into account for the comparative quantitation analysis (28).
Data Analysis For statistical data analysis, continuous variables were expressed as mean standard deviation (SD). Statistical differences among controls and each concentration group were determined by using one-way analysis of variance (ANOVA) statistics. P< .05 was considered statistically significant. When ANOVA resulted in a statistically significant value, a post-hoc test, Tukey HSD, was performed.
RESULTS Effect of DEHP, DBP, and BBP on P4 Release from Human Luteal Cells As expected, P4 release was increased by hCG (29), the mean concentration of P4 in the control was 13.8 1.3 ng/mL, similar to previously reported values (30, 31). After 24-hour
FIGURE 1
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on basal P4 release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control), or with hGC (100 ng/mL), or DEHP (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the mean SD of nine independent experiments, each done in triplicate. Results are expressed as a percentage of the control set equal to 100. ***P<.001; **P<.01; *P<.05 vs. control values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
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ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY incubation the P4 luteal release was statistically significantly decreased by all the tested doses of DEHP (Fig. 1). Moreover, both BBP and DBP reduced P4 by human luteal cells at all the tested doses with the exception of the lowest tested concentration (10–9 M) (see Fig. 1). To evaluate the possible influence of phthalates on hCGstimulated P4 release, human luteal cells were incubated for 24 hours with medium alone (control) or hCG (100 ng/mL) with or without different concentrations of DEHP, BBP, or DBP (10–6–10–9 M). The hCG-induced P4 secretion was statistically significantly reduced by both DEHP and BBP at the concentration of 10–6 and 10–7 M, but no effect was observed for the doses of 10–8 and 10–9 M (Fig. 2). Only the highest dose of DBP (10–6 M) reduced hCG-induced P4 secretion from human luteal cells (see Fig. 2).
Effect of DEHP, DBP, and BBP on PG Release from Human Luteal Cells The mean concentration of PGE2 and PGF2a in the control was 51.5 4.4 and 30.7 2.7 pg/mL, respectively (30, 31). Both DEHP and DBP statistically significantly reduced PGE2 (Fig. 3) at the doses of 10–6 and 10–7 M, but only the highest concentration of BBP (10–6) negatively affected PGE2 release (see Fig. 3). Moreover, DEHP (10–6 and 10–7 M) statistically significantly reduced PGF2a release after 24hour incubation. No effect was observed after the incubation with BBP and DBP at any tested doses (data not shown).
Effect of DEHP, DBP, and BBP on VEGF Protein Release and VEGF mRNA Expression in Human Luteal Cells It is well known that local factors such as chemical hypoxia rather than gonadotropin may prevail in regulating VEGF production by differentiated luteal cells (32). As expected, CoCl2 increased VEGF release (32). The mean concentration of VEGF in the control was 0.27 0.04 pg/mL (30, 31). The release of VEGF protein by human luteal cells was statistically significantly decreased after 24-hour incubation by all the tested doses of DEHP and BBP (Fig. 4). Only the doses of 10–6 and 10–7 M of DBP statistically significantly reduced VEGF release by human luteal cells (see Fig. 4). The mRNA steady-state levels of total VEGF and VEGF165 isoform were detectable by qPCR under basal conditions (average Ct z 21). As reported in the literature, the basal levels were increased in response to CoCl2 treatment (32). However, no statistically significant modifications were induced by 24-hour treatment with DEHP, BBP, or DBP (data not shown).
DISCUSSION Growing evidence suggest that polluting substances, classified as endocrine disruptors, interfere with the endogenous hormone signaling system, leading to impairment of reproductive health (11, 12, 15). To our knowledge, our study provides the first evidence of a direct inhibitory effect of
FIGURE 2
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on P4-inducted hCG release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control) or with hGC (100 ng/mL), or hGC (100 ng/mL) with DEHP added after mean SD of nine 1 hour (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. P<.001 vs. C values; ***P<.001 and **P<.01 vs. hCG values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
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FIGURE 3
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on basal PGE2 release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control) or with DEHP (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the mean SD of seven independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***P<.001; **P<.01; *P<.05 vs. control values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
DEHP, DBP, and BBP on steroidogenic cells function from fully mature human corpus luteum. In particular, our results show that DEHP, DBP, and BBP negatively influence luteal steroidogenesis by reducing both basal and hCGstimulated-P4 release by highly purified human luteal cells. The data from the literature mainly focus on DEHP and its monoester form [mono-(2-ethylhexyl) phthalate, MEHP], which is considered the active and toxic metabolite of DEHP (33–36). It is interesting that previous in vitro studies have demonstrated that DEHP itself is active at the cellular level, which suggests some intrinsic activity of this phthalate in the diester form as well (10, 27). Our results are in accordance with this hypothesis as well as with the previous published data on the negative influence of DEHP on P4 secretion in animal models both in vivo and in vitro (27, 36). In contrast, MEHP has been shown to increase P4 release or exert no effect at all in both animal models and human granulosa cells cultures (27). Intriguingly, these data may suggest that DEHP itself can exert intrinsic activity that differs from that of MEHP, which needs further investigations. Besides inhibiting P4 secretion in human luteal cells, all the tested phthalates affected the balance of important intraovarian regulators such as PGs (22, 23). Indeed, PGE2 luteal secretion was negatively affected by incubation with each of the tested phthalates. However, only the greatest reproductive toxicant DEHP exerted a negative influence on PGF2a release; the others had no effect. VOL. 102 NO. 3 / SEPTEMBER 2014
The ability of phthalates to reduce the release of the luteotrophic PGE2 further supports the hypothesis that they have a negative influence on the function of corpus luteum cells. In this assumption our demonstration of DEHP's negative influence also on the release of the classically known luteolytic PGF2a might seem conflicting. Actually, it is known that prostanoids could exert different effects in the different phases of corpus luteum since the expression of specific PGs receptors is subject to many changes in luteal cells lifespan (32). Furthermore, our in vitro study of the effect of DEHP, DBP, and BBP on luteal vasculature has shown that they negatively influence VEGF protein release from highly purified human luteal cells. Very few studies have examined the possible alteration of vascular factors after phthalate exposure, and their data mainly focus on tumour angiogenesis (37). In contrast to tumour vessel neoformation, transient luteal vasculogenesis is a tightly regulated process, driven by proangiogenic and tempered by antiangiogenic factors (38, 39). In particular, VEGF is essential for normal luteal development and function—its expression in human corpus luteum is constant from the early to late luteal phases (40). In fully functional corpus luteum, when luteal vasculature is largely complete (41), VEGF seems to act as both endothelial cell survival and vascular permeability factor (42), playing a pivotal role in maintaining luteal function (41). Thus, given the pivotal role of VEGF in this process, our demonstration of reduced VEGF levels in human luteal 835
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY
FIGURE 4
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on basal VEGF release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control) or with CoCl2 (10 mM) or with DEHP (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the mean SD of 10 independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***P<.001; **P<.01 vs. control values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
cell cultures after incubation with DEHP, DBP, or BBP further supports the hypothesis of phthalates negative influence corpus luteum function. It is interesting that, in our in vitro system, DEHP, DBP, and BBP did not affect VEGF mRNA expression. Because for VEGF synthesis several levels of regulation have been reported (40), our results suggest that the influence of phthalates on luteal VEGF production might occur at a posttranscriptional level. The exact mechanism of such a posttranscriptional regulation needs further investigation. In our present in vitro study observed a direct inhibitory effect of DEHP, DBP, and BBP on the function of human steroidogenic luteal cells. In the preovulatory ovary, phthalate exposure is known to exert antiestrogenic actions, probably through a receptor-mediated signaling pathway. This effect can suppress estradiol follicular production and often lead to anovulation (43, 44). Extrapolating from our in vitro results, it is tempting to speculate that even when ovulation occurs the exposure to phthalates can result in impaired luteal function. In 1999, the European Union banned the use of DEHP, DBP, and BBP and other phthalates in children's toys and in all child-care items intended for children younger than 3 years. Subsequently, in 2005, the use of DEHP was prohibited in all toys and child-care items products (European Union 1999, 2005) (19). In agreement with previous evidence, our results suggest the need to extend the prohibition on phthalates to products in daily use by adults as well. Indeed, in our opinion, preventing reproductive failure requires the identification and removal of any causes eventually involved in alteration of hu836
man reproductive health, and the evidence suggests that phthalates interfere in wide and complex ways with the human reproductive system, which demands public attention.
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Dello Russo C, Lisi L, Tringali G, Navarra P. Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation. Biochem Pharmacol 2009;78:1242–51. Lanzone A, Di Simone N, Castellani R, Fulghesu AM, Caruso A, Mancuso S. Human growth hormone enhances progesterone production by human luteal cells in vitro: evidence of a synergistic effect with human chorionic gonadotropin. Fertil Steril 1992;57:92–6. Tropea A, Tiberi F, Minici F, Orlando M, Gangale MF, Romani F, et al. Ghrelin affects the release of luteolytic and luteotropic factors in human luteal cells. J Clin Endocrinol Metab 2007;92:3239–45. Romani F, Lanzone A, Tropea A, Familiari A, Scarinci E, Sali M, et al. In vitro effect of unacylated ghrelin and obestatin on human luteal cell function. Fertil Steril 2012;97:991–6. Tropea A, Miceli F, Minici F, Tiberi F, Orlando M, Gangale MF, et al. Regulation of vascular endothelial growth factor synthesis and release by human luteal cells in vitro. J Clin Endocrinol Metab 2006;91:2303–9. Pollack GM, Li RC, Ermer JC, Shen DD. Effects of route of administration and repetitive dosing on the disposition kinetics of di(2-ethylhexyl) phthalate and its mono-de-esterified metabolite in rats. Toxicol Appl Pharmacol 1985;79: 246–56. Davis BJ, Weaver R, Gaines LJ, Heindel JJ. Mono-(2-ethylhexyl) phthalate suppresses estradiol production independent of FSH-cAMP stimulation in rat granulosa cells. Toxicol Appl Pharmacol 1994;128:224–8. Craig ZR, Wang W, Flaws JA. Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling. Reproduction 2011;142:633–46. Svechnikova I, Svechnikov K, Soder O. The influence of di-(2-ethylhexyl) phthalate on steroidogenesis by the ovarian granulosa cells of immature female rats. J Endocrinol 2007;194:603–9. Buteau-Lozano H, Velasco G, Cristofari M, Balaguer P, Perrot-Applanat M. Xenoestrogens modulate vascular endothelial growth factor secretion in breast cancer cells through an estrogen receptor-dependent mechanism. J Endocrinol 2008;196:399–412. Neeman M, Abramovitch R, Schiffenbauer YS, Tempel C. Regulation of angiogenesis by hypoxic stress: from solid tumours to the ovarian follicle. Int J Exp Pathol 1997;78:57–70. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:353–64. Yoo PS, Mulkeen AL, Cha CH. Post-transcriptional regulation of vascular endothelial growth factor: implications for tumor angiogenesis. World J Gastroenterol 2006;12:4937–42. Dickson SE, Bicknell R, Fraser HM. Mid-luteal angiogenesis and function in the primate is dependent on vascular endothelial growth factor. J Endocrinol 2001;168:409–16. Fraser HM, Duncan WC. SRB Reproduction, Fertility and Development Award Lecture 2008. Regulation and manipulation of angiogenesis in the ovary and endometrium. Reprod Fertil Dev 2009;21:377–92. Cobellis L, Latini G, De FC, Razzi S, Paris I, Ruggieri F, et al. High plasma concentrations of di-(2-ethylhexyl)-phthalate in women with endometriosis. Hum Reprod 2003;18:1512–5. Lovekamp TN, Davis BJ. Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells. Toxicol Appl Pharmacol 2001;172:217–24.
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Objective: To evaluate the influence of phthalates on human luteal cell function. Design: Laboratory study. Setting: University hospital. Patient(s): Twenty-three normally menstruating patients in the midluteal phase. Intervention(s): Human luteal cells isolated from corpora lutea for primary cultures. Main Outcome Measure(s): Progesterone (P4) and prostaglandin release assayed by enzyme immunoassay, vascular endothelial growth factor (VEGF) secretion by enzyme-linked immunosorbent assay (ELISA), and VEGF mRNA expression by real-time polymerase chain reaction. Result(s): We investigated the effect of di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) on basal and hCG-induced progesterone (P4) release, as well as DEHP effect on the balance between prostaglandin (PG) E2, vascular endothelial growth factor (VEGF)-luteotrophic factors, and the luteolitic PGF2a in isolated human steroidogenc cells. Phthalates influence on VEGF expression has been also evaluated. DEHP, DBP, and BBP were able to reduce both basal and hCG-stimulated P4 as well as PGE2 release. PGF2a release was reduced after DEHP incubation. VEGF protein release was decreased by the incubation with the tested phthalates. VEGF mRNA expression was not affected by DEHP, DBP, and BBP. As expected, both hCG and cobalt chloride were able to induce P4 release and VEGF release and mRNA expression in human luteal cells respectively. Conclusion(s): The results show the ability of phthalates to affect luteal steroidogenesis as well as the balance between luteotrophic and luteolytic factors suggesting an interference of phthalates in human luteal function. These data may contribute to clarify the classically known impaired reproductive health observed after Use your smartphone phthalates exposure. (Fertil SterilÒ 2014;102:831–7. Ó2014 by American Society for Reproducto scan this QR code tive Medicine.) and connect to the Key Words: Corpus luteum, endocrine disruptors, luteal function Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/romanif-endocrine-disruptors-phthalates-luteal-cells/
P
hthalates are synthetic chemicals that belong to the endocrine disruptor family (1), and their high production volume has resulted in
ubiquitous human exposure (2). Globally, more than 18 billion pounds of phthalates are used each year, primarily as plasticizers in flexible poly-
Received March 6, 2014; revised May 13, 2014; accepted May 27, 2014; published online July 10, 2014. F.R. has nothing to disclose. A.T. has nothing to disclose. E.S. has nothing to disclose. A.F. has nothing to disclose. C.D.R. has nothing to disclose. L.L. has nothing to disclose. S.C. has nothing to disclose. A.L. has nothing to disclose. R.A. has nothing to disclose. Reprint requests: Federica Romani, M.D., Cattedra di Fisiopatologia della Riproduzione Umana, Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy (E-mail: romani. Universita [email protected]). Fertility and Sterility® Vol. 102, No. 3, September 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.05.041 VOL. 102 NO. 3 / SEPTEMBER 2014
discussion forum for this article now.*
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vinyl chloride (PVC) products (2, 3); used to impart flexibility to plastics, they can be found in most PVC products including vinyl upholstery, tablecloths, shower curtains, several sprays including pesticides, solvents, and soft-squeeze children's toys. The use of these compounds also is approved in several medical devices such as tubing, blood bags, and vinyl gloves, and they also can be found as additives in cosmetics products such as lotion, perfume, and nail polish. 831
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY The high production volume and common use of phthalates has resulted in humans commonly being exposed to phthalates on a daily basis through ingestion, inhalation, and dermal contact (4–6). Because these plasticizers cannot form strong molecular bonds with polymers, they are rapidly leached into the environment and have become a widespread environmental contaminate (2, 7–10). Dozens of phthalates have been developed over the years, and an association between phthalate exposure and adverse reproductive health outcomes has been suggested (11). Interestingly, phthalates have been demonstrated to reduce fertility in both humans and animal models (12), and occupational exposure to phthalates has been associated with decreased pregnancy rates, increased miscarriages, and other gestational complications (13, 14). Moreover, it has been demonstrated that these plasticizers negatively modulate oocyte growth, ovulation, and embryonic development, leading to impaired ovarian function (12, 15). Interference with ovarian steroid synthesis and metabolism could partially explain these effects. Indeed, phthalates exert antiandrogenic or estrogenic effects in vitro, impairing 17bestradiol affinity to its receptors (2, 16–18) and modulating steroid biosynthesis and metabolism by acting as a peroxisome proliferator-activated receptor (PPAR) agonist as well (11). Of note, among the phthalates, di-(2-ethylhexyl) phthalate (DEHP) is well known to be the most abundant and potent reproductive toxicant chemical (Center for the Evaluation of Risks to Human Reproduction-CERHR 2000). Nevertheless, women of reproductive age have a specific exposure risk profile to di-n-butyl phthalate (DBP), which is commonly used in many beauty products (2, 19). Finally, exposure to n-butyl benzyl phthalate (BBP) is well known to be associated with reproductive alterations in males as well as with gynecologic diseases such as endometriosis (20, 21). To expand the available data on impaired reproductivehormone balance resulting from phthalates exposure, our in vitro study investigated the potential effects of DEHP, DBP, and BBP on human luteal cell function. The corpus luteum is a unique transient reproductive gland essential for the establishment and maintenance of early pregnancy. In particular, corpus luteum function is a hormone-dependent phenomenon. Both alpha and beta estrogen receptors as well as PPAR are expressed in the corpus luteum. Through these receptors, xenoestrogens such as phthalates can exert their influence on luteal function. We investigated whether these plasticizers can affect both basal and gonadotropinstimulated luteal progesterone (P4) production in highly purified human luteal cells. Moreover, to evaluate the possible alteration of the balance between intraovarian luteotrophic and luteolytic regulators, we analyzed the effect of DEHP, DBP, and BBP on the luteal release of vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2)—both local luteotrophic factors (19, 20)—and PGF2a, a classic luteolytic modulator (22, 23). In addition, we examined the effect of DEHP, DBP, and BBP in modulating VEGF luteal mRNA expression.
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MATERIALS AND METHODS Cell Cultures Corpora lutea were obtained from 23 normally menstruating women (25 to 38 years old) in the midluteal phase (days 5 to 6 from ovulation) at the time of surgery for nonendocrine gynecologic diseases. The protocol was approved by the institutional review board of Universita Cattolica del Sacro Cuore in Rome, and all patients provided written informed consent. The corpora lutea were dated on the basis of the presumptive day of ovulation (day 0), as determined by urinary luteinizing hormone (LH) peak, ultrasound detection of corpus luteum or disappearance of the dominant follicle, and a rise in the plasma P4 concentration. The human luteal cells cultures were performed as previously reported elsewhere (24). Briefly, luteal tissue was enzymatically dissociated in nutrient mixture F-12 (Flow Laboratories)/Hepes (Sigma-Aldrich) medium containing type IV collagenase (200 U/mL) (Sigma-Aldrich). The identify of both large and small human luteal cells was confirmed by their positive staining for lipids with oil red O14 (25). Isolated human luteal cells (250,000 cells/mL) were plated on 48-well dishes for enzyme-linked immunosorbent assay (ELISA) or enzyme immuno assay (EIA) or for real-time polymerase chain reaction (PCR) and cultured for 24 hours in 5% CO2 and 95% air at 37 C. At the end of isolation and 24 hours after all the treatments, human luteal cells were counted in a hemocytometer, and their viability was determined by trypan blue exclusion test. Neither treatment nor solvent for dissolving tested substances affected either the cell count or cell viability. Human luteal cells were incubated for 24 hours with serum-free medium alone (control), human chorionic gonadotropin (hCG, 100 ng/mL; Calbiochem Inalco), cobalt(II) chloride (CoCl2, 10 mM, chemical hypoxia), DEHP (10–6–10–9 M), DBP (10–6–10–9 M), or BBP (10–6–10–9 M) (Sigma Aldrich s.r.l.) alone or with hCG (100 ng/mL). Three different wells were used for each experimental condition. The doses we used of the phenols were not greater than the previously demonstrated serum and follicular fluid levels (26, 27). After the incubation, human luteal cells were treated for total RNA extraction while the culture media were separately collected and assayed for P4, PG, and VEGF detection.
PGs, P4, and VEGF Assays According to manufacturer's instructions, the PGE2, PGF2a, and P4 levels in the culture media were assayed with specific EIA kits (Cayman Chemical) and VEGF with a VEGF ELISA kit (R&D Systems). For P4, the EIA kit intra-assay coefficient of variation (CVw) and interassay coefficient of variation (CVb) were 7% and 9%, respectively, and the detection limit (DL) was 10 pg/mL. For PG, the EIA kit CVw and CVb were 12% and 9%, respectively, and the DL was 9 pg/mL. For VEGF, the ELISA kit CVw and CVb were 3.5% and 6.7%, respectively, and the sensitivity was 5.0 pg/mL.
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Fertility and Sterility® Total RNA Extraction, cDNA-synthesis, and Realtime PCR Analysis Total cytoplasmic RNA was extracted using the RNeasy Micro kit (Qiagen), which included a 15-minute DNAse treatment. The RNA concentration was measured using the Quant-iTTM RiboGreen RNA Assay Kit (Invitrogen). A standard curve in the range of 0–10 ng was run in each assay using 16S and 23S ribosomal RNA (rRNA) from Escherichia coli as the standard, as provided by the kit. Aliquots (0.1 mg) of RNA were converted to cDNA using random hexamer primers. Quantitative changes in mRNA levels were estimated by real-time quantitative PCR (qPCR) using the following cycling conditions: 35 cycles of denaturation at 95 C for 20 seconds; annealing and extension at 60 C for 20 seconds, using the Brilliant III Ultra Fast SYBR Green QPCR Master Mix 2X (Stratagene). The PCR reactions were performed in a 20-mL reaction volume in a MX3000P real-time PCR machine (Stratagene). Primers used for the evaluation of gene expression were a-tubulin 984 forward (50 -CCC TCG CCA TGG TAA ATA CAT-30 ) and 1093 reverse (50 -ACT GGA TGG TAC GCT TGG TCT-30 ), which yielded a 110 base pair (bp) product; human VEGFa 1045 forward (50 TTT CTG CTG TCT TGG GTG CAT TGG-30 ) and 1160 reverse (50 -ACC ACT TCG TGA TGA TTC TGC CC-30 ), which yielded a 116-bp product; and human VEGFa-165 1458 forward (50 -AAA TCC CTG TGG GCC TTG CT-30 ) and 1683 reverse (50 GCT TGT CAC ATC TGC AAG TAG CT-30 ), which yielded a 146-bp product. The relative mRNA concentrations were calculated from the take-off point of reactions (threshold cycle, Ct)
using the comparative quantitation method performed by Stratagene software and based upon the DDCt method. This analysis approximates a given sample's target mRNA (e.g., COX2) level relative to the mean of the target mRNA levels in untreated controls (‘‘calibrator’’ value), thus permitting statistical analysis of deviation from the mean even among the controls. The Ct values for a-tub expression served as a normalizing signal. In each assay, the PCR efficiency was also calculated using serial dilution of one experimental sample; efficiency values between 94% and 98% were found for each primer set and weretaken into account for the comparative quantitation analysis (28).
Data Analysis For statistical data analysis, continuous variables were expressed as mean standard deviation (SD). Statistical differences among controls and each concentration group were determined by using one-way analysis of variance (ANOVA) statistics. P< .05 was considered statistically significant. When ANOVA resulted in a statistically significant value, a post-hoc test, Tukey HSD, was performed.
RESULTS Effect of DEHP, DBP, and BBP on P4 Release from Human Luteal Cells As expected, P4 release was increased by hCG (29), the mean concentration of P4 in the control was 13.8 1.3 ng/mL, similar to previously reported values (30, 31). After 24-hour
FIGURE 1
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on basal P4 release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control), or with hGC (100 ng/mL), or DEHP (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the mean SD of nine independent experiments, each done in triplicate. Results are expressed as a percentage of the control set equal to 100. ***P<.001; **P<.01; *P<.05 vs. control values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
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ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY incubation the P4 luteal release was statistically significantly decreased by all the tested doses of DEHP (Fig. 1). Moreover, both BBP and DBP reduced P4 by human luteal cells at all the tested doses with the exception of the lowest tested concentration (10–9 M) (see Fig. 1). To evaluate the possible influence of phthalates on hCGstimulated P4 release, human luteal cells were incubated for 24 hours with medium alone (control) or hCG (100 ng/mL) with or without different concentrations of DEHP, BBP, or DBP (10–6–10–9 M). The hCG-induced P4 secretion was statistically significantly reduced by both DEHP and BBP at the concentration of 10–6 and 10–7 M, but no effect was observed for the doses of 10–8 and 10–9 M (Fig. 2). Only the highest dose of DBP (10–6 M) reduced hCG-induced P4 secretion from human luteal cells (see Fig. 2).
Effect of DEHP, DBP, and BBP on PG Release from Human Luteal Cells The mean concentration of PGE2 and PGF2a in the control was 51.5 4.4 and 30.7 2.7 pg/mL, respectively (30, 31). Both DEHP and DBP statistically significantly reduced PGE2 (Fig. 3) at the doses of 10–6 and 10–7 M, but only the highest concentration of BBP (10–6) negatively affected PGE2 release (see Fig. 3). Moreover, DEHP (10–6 and 10–7 M) statistically significantly reduced PGF2a release after 24hour incubation. No effect was observed after the incubation with BBP and DBP at any tested doses (data not shown).
Effect of DEHP, DBP, and BBP on VEGF Protein Release and VEGF mRNA Expression in Human Luteal Cells It is well known that local factors such as chemical hypoxia rather than gonadotropin may prevail in regulating VEGF production by differentiated luteal cells (32). As expected, CoCl2 increased VEGF release (32). The mean concentration of VEGF in the control was 0.27 0.04 pg/mL (30, 31). The release of VEGF protein by human luteal cells was statistically significantly decreased after 24-hour incubation by all the tested doses of DEHP and BBP (Fig. 4). Only the doses of 10–6 and 10–7 M of DBP statistically significantly reduced VEGF release by human luteal cells (see Fig. 4). The mRNA steady-state levels of total VEGF and VEGF165 isoform were detectable by qPCR under basal conditions (average Ct z 21). As reported in the literature, the basal levels were increased in response to CoCl2 treatment (32). However, no statistically significant modifications were induced by 24-hour treatment with DEHP, BBP, or DBP (data not shown).
DISCUSSION Growing evidence suggest that polluting substances, classified as endocrine disruptors, interfere with the endogenous hormone signaling system, leading to impairment of reproductive health (11, 12, 15). To our knowledge, our study provides the first evidence of a direct inhibitory effect of
FIGURE 2
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on P4-inducted hCG release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control) or with hGC (100 ng/mL), or hGC (100 ng/mL) with DEHP added after mean SD of nine 1 hour (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. P<.001 vs. C values; ***P<.001 and **P<.01 vs. hCG values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
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FIGURE 3
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on basal PGE2 release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control) or with DEHP (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the mean SD of seven independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***P<.001; **P<.01; *P<.05 vs. control values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
DEHP, DBP, and BBP on steroidogenic cells function from fully mature human corpus luteum. In particular, our results show that DEHP, DBP, and BBP negatively influence luteal steroidogenesis by reducing both basal and hCGstimulated-P4 release by highly purified human luteal cells. The data from the literature mainly focus on DEHP and its monoester form [mono-(2-ethylhexyl) phthalate, MEHP], which is considered the active and toxic metabolite of DEHP (33–36). It is interesting that previous in vitro studies have demonstrated that DEHP itself is active at the cellular level, which suggests some intrinsic activity of this phthalate in the diester form as well (10, 27). Our results are in accordance with this hypothesis as well as with the previous published data on the negative influence of DEHP on P4 secretion in animal models both in vivo and in vitro (27, 36). In contrast, MEHP has been shown to increase P4 release or exert no effect at all in both animal models and human granulosa cells cultures (27). Intriguingly, these data may suggest that DEHP itself can exert intrinsic activity that differs from that of MEHP, which needs further investigations. Besides inhibiting P4 secretion in human luteal cells, all the tested phthalates affected the balance of important intraovarian regulators such as PGs (22, 23). Indeed, PGE2 luteal secretion was negatively affected by incubation with each of the tested phthalates. However, only the greatest reproductive toxicant DEHP exerted a negative influence on PGF2a release; the others had no effect. VOL. 102 NO. 3 / SEPTEMBER 2014
The ability of phthalates to reduce the release of the luteotrophic PGE2 further supports the hypothesis that they have a negative influence on the function of corpus luteum cells. In this assumption our demonstration of DEHP's negative influence also on the release of the classically known luteolytic PGF2a might seem conflicting. Actually, it is known that prostanoids could exert different effects in the different phases of corpus luteum since the expression of specific PGs receptors is subject to many changes in luteal cells lifespan (32). Furthermore, our in vitro study of the effect of DEHP, DBP, and BBP on luteal vasculature has shown that they negatively influence VEGF protein release from highly purified human luteal cells. Very few studies have examined the possible alteration of vascular factors after phthalate exposure, and their data mainly focus on tumour angiogenesis (37). In contrast to tumour vessel neoformation, transient luteal vasculogenesis is a tightly regulated process, driven by proangiogenic and tempered by antiangiogenic factors (38, 39). In particular, VEGF is essential for normal luteal development and function—its expression in human corpus luteum is constant from the early to late luteal phases (40). In fully functional corpus luteum, when luteal vasculature is largely complete (41), VEGF seems to act as both endothelial cell survival and vascular permeability factor (42), playing a pivotal role in maintaining luteal function (41). Thus, given the pivotal role of VEGF in this process, our demonstration of reduced VEGF levels in human luteal 835
ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY
FIGURE 4
Effect of di-(2-ethylhexyl) phthalate (DEHP), n-butyl benzyl phthalate (BBP), and di-n-butyl phthalate (DBP) on basal VEGF release by human luteal cells. Luteal cells were cultured for 24 hours in medium alone (control) or with CoCl2 (10 mM) or with DEHP (10–6, 10–7, 10–8, 10–9 M), or BBP (10–6, 10–7, 10–8, 10–9 M), or DBP (10–6, 10–7, 10–8, 10–9 M). Each value represents the mean SD of 10 independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***P<.001; **P<.01 vs. control values. Romani. Endocrine disruptors and luteal cells function in vitro. Fertil Steril 2014.
cell cultures after incubation with DEHP, DBP, or BBP further supports the hypothesis of phthalates negative influence corpus luteum function. It is interesting that, in our in vitro system, DEHP, DBP, and BBP did not affect VEGF mRNA expression. Because for VEGF synthesis several levels of regulation have been reported (40), our results suggest that the influence of phthalates on luteal VEGF production might occur at a posttranscriptional level. The exact mechanism of such a posttranscriptional regulation needs further investigation. In our present in vitro study observed a direct inhibitory effect of DEHP, DBP, and BBP on the function of human steroidogenic luteal cells. In the preovulatory ovary, phthalate exposure is known to exert antiestrogenic actions, probably through a receptor-mediated signaling pathway. This effect can suppress estradiol follicular production and often lead to anovulation (43, 44). Extrapolating from our in vitro results, it is tempting to speculate that even when ovulation occurs the exposure to phthalates can result in impaired luteal function. In 1999, the European Union banned the use of DEHP, DBP, and BBP and other phthalates in children's toys and in all child-care items intended for children younger than 3 years. Subsequently, in 2005, the use of DEHP was prohibited in all toys and child-care items products (European Union 1999, 2005) (19). In agreement with previous evidence, our results suggest the need to extend the prohibition on phthalates to products in daily use by adults as well. Indeed, in our opinion, preventing reproductive failure requires the identification and removal of any causes eventually involved in alteration of hu836
man reproductive health, and the evidence suggests that phthalates interfere in wide and complex ways with the human reproductive system, which demands public attention.
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