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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 1 238-243
Copyright © 2003 by The Endocrine Society

Original Article

Regulation of HOXA-10 Expression by Testosterone in Vitro and in the Endometrium of Patients with Polycystic Ovary Syndrome

Dilek Cermik, Belgin Selam and Hugh S. Taylor

Department of Obstetrics and Gynecology (D.C., B.S., H.S.T.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Hugh S. Taylor, M.D., Associate Professor, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, Connecticut 06520. E-mail: hugh.taylor{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) affects approximately 5% of reproductive-age women and is characterized by anovulation and increased androgen production. Despite the ability to correct ovulatory disorders, pregnancy rates remain paradoxically low, and spontaneous pregnancy loss rates are high. To determine whether uterine dysfunction contributed to the adverse reproductive outcomes in PCOS, we assessed the effect of the increased ovarian androgens on a well-characterized gene essential to endometrial receptivity. Up-regulation of HOXA10 in the endometrium is necessary for receptivity to embryo implantation. In vitro, HOXA10 expression was repressed by testosterone but not by dehydroepiandrosterone, dehydroepiandrosterone sulfate, or insulin. Testosterone also prevented the increased expression of HOXA10 previously reported with estradiol or progesterone. Dihydrotestosterone produced an effect similar to that of testosterone, whereas flutamide blocked the testosterone effect. Endometrial biopsies, obtained from women with PCOS, demonstrated decreased HOXA10 mRNA. Testosterone is a novel regulator of HOXA10. Diminished uterine HOXA10 expression may contribute to the diminished reproduction potential of women with PCOS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) refers to an endocrinopathy characterized by menstrual irregularity and hyperandrogenism and is the most common cause of oligoovulatory infertility (1, 2). PCOS is perhaps the most prevalent endocrine disorder in women, with incidence of 4.0–6.5% (3, 4, 5). The excess ovarian androgen production is induced by extraovarian factors, including insulin resistance and hyperinsulinemia (6, 7, 8, 9, 10, 11, 12, 13). The clinical manifestations and ramifications of this disorder vary with the degree of hyperandrogenism and include a number of metabolic disorders, including insulin resistance, diabetes mellitus, hypertension, dyslipidemia, and cardiovascular disease (14, 15, 16, 17).

Elevated serum concentrations of testosterone, as well as other androgens, may contribute to infertility in PCOS patients. The chronic anovulation and infertility associated with PCOS can usually be treated; however, overall pregnancy rates are not high (18). Successful folliculogenesis occurs in 80% of treated women, yet the ultimate pregnancy rate is only 40–50%, even when other factors are excluded. Furthermore, spontaneous miscarriages also occur frequently in this group (19). Similarly, women with recurrent spontaneous miscarriages are often diagnosed with PCOS (19, 20). Despite the correction of defects in ovulation, reproductive success is ultimately limited. These facts suggest that a disorder of endometrial development and receptivity to blastocyst implantation may contribute to the decreased fertility and poor reproductive outcomes of PCOS patients.

Endometrial development and receptivity requires transcriptional regulation by homeobox genes. HOX genes are highly conserved developmental control genes (21, 22, 23, 24, 25). The expression of HOX genes in defined locations along the paramesonephric duct leads to the development of the adult reproductive tract (26). Specifically, HOXA10 is essential for the development of the uterus during organogenesis (27).

HOXA10 expression is also essential for endometrial development in the adult, allowing uterine receptivity to implantation (28). Female mice with a targeted disruption of HOXA10 are viable but have infertility caused by uterine defects (29). These mice ovulate normally, however are unable to support implantation. The persistent expression of HOXA10 in the adult enables the endometrium to retain a developmental plasticity and allows the sequential differentiation of the endometrium during each menstrual cycle (30). In mice, blocking maternal HOXA10 expression with antisense, in the setting of normal uterine development, decreases implantation and litter size. In women, HOXA10 expression varies during the menstrual cycle, with a dramatic rise in the midluteal phase (31). This rise corresponds to the time of implantation in humans. The expression remains elevated throughout the rest of the luteal phase. Regulation of maternal HOXA10 expression is essential to blastocyst implantation and reproductive success.

Endometrial development is dependent on the cyclic influence of estrogen and progesterone, but the molecular mechanism by which sex steroids lead to implantation is poorly understood. Estrogen, progesterone, and perhaps other molecules, regulate the HOX gene expression that is necessary for implantation. There are few known regulators of HOX gene expression. Functional retinoic-acid response elements regulate expression of the 3' Hox genes. Our laboratory has demonstrated a role for the sex steroids 17ß-estradiol and progesterone in regulating the 5' Hox genes, including HOXA10 (27, 31, 32). The expression of HOXA10 is increased in response to estrogen or progesterone in the human endometrium. Maximal expression is obtained after simultaneous administration of both these hormones.

In this study, we evaluated the effect of testosterone on HOX gene expression. Androgen levels are higher in women with infertility (or recurrent miscarriages) than in normal fertile women. The androgen receptor is expressed in endometrium (33, 34). Elevated levels of androgens may have a detrimental effect on endometrial function. We postulated that hyperandrogenism may result in changes in HOXA10 expression in the endometrium. Altered levels of HOXA10 expression, attributable to elevated testosterone, may effect the molecular pathway that leads to implantation and endometrial receptivity. As a consequence, patients with PCOS may suffer from infertility and early spontaneous miscarriages, despite successful correction of anovulation. Here, we demonstrate that testosterone changes HOXA10 expression in vitro. We also show that HOXA10 expression is altered in the endometrium of hyperandrogenic PCOS patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture cell

Ishikawa cells were maintained in MEM (Life Technologies, Inc., Carlsbad, CA) with 2.0 mM L-glutamine and Earle’s salts supplemented with 10% FBS, 1% sodium pyruvate, and 1% penicillin/streptomycin. Expression estrogen receptor and progesterone receptor were confirmed by ELISA, and 80% confluent monolayers of Ishikawa cells were maintained in serum free media for 12 h. Cells were then treated for 6 h or 245 h with hormones or pharmacologic agents. To generate a dose-response curve, testosterone was used at a final concentration of 1 x 10-8-1 x 10-4 M. Estradiol (5 x 10-8 M) and progesterone (1 x 10-6 M) were used at approximately maximal physiologic concentrations, at which we have previously demonstrated alteration in HOX gene expression in this cell line (31, 32). DHA sulfate (DHAS, 1 x 10-7 M) and DHA (1 x 10-7 M) and insulin (30 u/l) were as previously described in PCOS patients (35, 36, 37, 38). Dihydrotestosterone (DHT; 1 x 10-7 M) was used at the same concentration as the minimal effective testosterone concentration. Similarly, flutamide (1 x 10-6 M) was used as an androgen receptor antagonist at a final concentration identical to testosterone. Clomiphene citrate (1 x 10-6 M) was used at approximately 10 times typical therapeutic serum levels (39).

Probe preparation

Plasmids used for probe preparation have been previously well characterized (27, 28, 32). A pGEM plasmid, containing 103 base pairs of the 3' untranslated region of human HOXA10, was linearized with Eco RI or Hind III (New England Biolabs, Inc., Beverly, MA), ethanol precipitated, and used as template for generating riboprobes. Radiolabeled RNA probes were generated by in vitro transcription using the Riboprobe Kit (Promega Corp., Madison, WI). Antisense probes were generated using the RNA polymerase (SP6) and labeled with [32]P-uridine 5'-triphosphate (Amersham, Piscataway, NJ).

Statistical analysis

The autoradiographic bands were quantified using a laser densitometer. Each HOXA10 band was normalized to the value obtained from the same lane hybridized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed using Kruskal-Wallis ANOVA on ranks. Statistical significance was defined as P < 0.05.

Tissue collection

Endometrium was collected from both normal cycling women (n = 5) and women with PCOS (n = 7), by Pipell endometrial biopsy, under an approved Human Investigations Committee protocol. PCOS was diagnosed clinically. PCOS patients were 24–32 yr old (mean, 27.8), nulliparous, and oligoovulatory and had clinical evidence of androgen excess, and a body mass index of 25.9 ± 0.5 kg/m2. PCOS patients had no evidence of Cushing disease, thyroid disease, or hormone-secreting tumors. Control patients were nulliparous, had a mean age of 26.7 yr (range, 21–34), a body mass index of 23.1 ± 3.7 kg/m2, and no evidence of PCOS, endometriosis, abnormal uterine bleeding, or hyperandrogenism. Those patients with elevated free testosterone levels (free testosterone levels > 0.2 ng/ml) and who had occasional ovulation were selected for inclusion in this study. The endometrial samples were obtained from both groups in the midsecretory phase (cycle d 21–24) of ovulatory cycles as assessed by urinary LH detection kits (Ovuquick, Quidel, San Diego, CA). The tissue was immediately placed in liquid nitrogen and stored at -72 C. Menstrual cycle dating was determined from menstrual cycle history and confirmed histologically using the criteria of Noyes et al. (40). Discrepancy between histology and endometrial dating, predicted by the time of LH detection of greater than 2 d, resulted in exclusion of the 3 PCOS patients from 10 originally volunteering for the study; no controls required exclusion.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HOXA10 expression is regulated by testosterone in endometrial cells

Endometrial HOX gene expression has been shown to be regulated by estrogen and progesterone (31, 32). To determine whether HOXA10 expression is regulated by ovarian androgens, HOXA10 expression was measured in Ishikawa cells after treatment with testosterone. Ishikawa cells are a well-differentiated human endometrial adenocarcinoma cell line that expresses estrogen, progesterone, and androgen receptors (41, 42, 43, 44, 45, 46, 47) and in which HOX gene expression has been previously characterized (27, 31, 32). Figure 1AGo shows that HOXA10 expression decreased after treatment with testosterone, in a dose-responsive manner, in Ishikawa cells. Testosterone concentrations at or less than 10-8 M produced no discernible effect; however, at 10-7 M, a significant decrease in HOXA10 mRNA expression was seen. A further decrease in HOXA10 expression was seen with supraphysiologic treatments (10-6-10-4 M). The decrease in HOXA10 mRNA expression persisted 24 h after testosterone (10-6 M) treatment.



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  		Figure 1. Effect of testosterone on HOXA10 expression. Ishikawa cells were treated with either testosterone or vehicle control (Ctl). A, Representative Northern analysis. HOXA10 expression is decreased after treatment for 6 h with testosterone at 10-5 M [T10(-5)], but not at 10-8 M [T10(-8)]. At 24 h, HOXA10 expression remained decreased after treatment with 10-6 M testosterone. B, Quantification and normalization of HOXA10 mRNA levels after testosterone treatment. Testosterone concentration raged from 10-4-10-8 M. A dose-responsive decrease in normalized HOXA10 mRNA levels was observed. All experiments were repeated in triplicate. Error bars, SEM; *, statistically different from control (P < 0.05).

 
As previously demonstrated, treatment of Ishikawa cells with estradiol and progesterone leads to increased HOXA10 mRNA expression (31, 32). Figure 2Go shows the effect of simultaneous treatment with estradiol (5 x 10-8 M) and testosterone (10-6 M). Concomitant use of estradiol and testosterone resulted in decreased HOXA10 expression (P < 0.05). Figure 3Go demonstrates the effect of simultaneous treatment with progesterone and testosterone (progesterone, 10-6 M; testosterone, 10-6 M). HOXA10 expression is decreased after simultaneous progesterone and testosterone treatment (P < 0.05). Testosterone blocked the expected estradiol- or progesterone-induced increase in HOXA10 mRNA. Combined treatment with estradiol, progesterone, and testosterone decreased expression of HOXA10, as shown in Fig. 4Go (P < 0.05). Similarly, simultaneous treatment with estradiol, progesterone, and DHT decreases HOXA10 expression in vitro. Both testosterone and DHT blocked the stimulatory effects of estradiol and/or progesterone on HOXA10 expression.



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  		Figure 2. Testosterone reverses the effects of estradiol on HOXA10 expression. Ishikawa cells were treated with either vehicle control (C), 17ß-estradiol (5 x 10-8 M), or the combination of estradiol and testosterone (10-6 M) (E+T). Testosterone prevents the estradiol-induced rise in HOXA10 mRNA. Error bars, SEM; *, statistically different from control (P < 0.05); #, statistically different from estradiol treatment (P < 0.05).

 


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  		Figure 3. Testosterone reverses the effect of progesterone on HOXA10 expression. Ishikawa cells were treated with either vehicle control (C), progesterone (P, 10-6 M), or the combination of progesterone and testosterone (P+T). Testosterone prevents the progesterone-induced rise in HOXA10 mRNA. Error bars, SEM; *, statistically different from control (P < 0.05); #, statistically different from progesterone treatment (P < 0.05).

 


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  		Figure 4. Testosterone and DHT each prevent the action of 17ß-estradiol and progesterone on HOXA10 expression. Ishikawa cells were treated with either vehicle control (C); estrogen and progesterone (E+P); estrogen, progesterone, and testosterone (E+P+T); or estrogen, progesterone, and DHT (E+P+D). Either T or DHT blocked the effect of combined E+P on HOXA10 mRNA expression. Error bars, SEM; *, statistically different from control (P < 0.05); #, statistically differs from E+P (P < 0.05).

 
To determine whether the effect of testosterone can be blocked by an androgen receptor antagonist in vitro, Ishikawa cells were treated with either testosterone (10-6 M), flutamide (10-6 M), or flutamide and testosterone together (Fig. 5Go). Whereas flutamide had no independent effect, the repressive effect of testosterone on HOXA10 expression was blocked by flutamide.



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  		Figure 5. The androgen receptor antagonist, flutamide, blocks the effect of testosterone on HOXA10 expression. Ishikawa cells were treated with either control vehicle (C), testosterone (T, 10-6 M), flutamide (F, 10-6 M), or both testosterone and flutamide (T+F). Testosterone decreased HOXA10 expression, whereas flutamide alone had no effect. The testosterone-induced decrease on HOXA10 expression was prevented by flutamide treatment. Clomiphene citrate (C1) had no direct effect on HOXA10 expression; nor did insulin (I).

 
PCOS patients also frequently have increased adrenal androgen production. HOXA10 expression was measured in Ishikawa cells after the treatment with dehydroepiandrosterone (DHA) or DHAS. There was no effect on HOXA10 expression with DHAS or DHA treatment (data not shown). Similarly, PCOS is characterized by insulin resistance and hyperinsulinemia (48, 49). Treatment with insulin at levels typical of serum levels in insulin resistant women with PCOS did not alter HOXA10 expression (Fig. 5Go).

Clomiphene citrate is often used to treat ovulatory defects in women with PCOS (50). Clomiphene has been implicated in endometrial defects seen in women treated for this condition (51, 52). We assessed the direct effect of clomiphene on HOXA10 expression in Ishikawa cells. Clomiphene at a therapeutic concentration (10-6 M) had no direct effect on HOXA10 expression, as measured by Northern analysis (Fig. 5Go). Of the known endocrinologic defects in PCOS and agents used as therapeutic treatments, only elevated testosterone was demonstrated to alter HOXA10 expression in this model.

Patients with PCOS have decreased endometrial HOXA10 mRNA levels

To investigate HOXA10 expression in the endometrium of PCOS patients, midsecretory-phase endometrial samples were obtained by Pipell biopsy, under an approved Human Investigation Committee protocol. Those who demonstrated an elevated free testosterone level and were oligoovulatory, but not anovulatory, were included in the study. The mean free testosterone in PCOS patients was 0.24 ng/ml (range, 0.02–0.36); and in controls, 0.06 ng/ml (range, 0.02–0.18). Biopsies included in testing were obtained from seven PCOS patients and from five normally cycling controls without evidence of hyperandrogenism. Samples were immediately placed in liquid nitrogen. After RNA extraction, Northern blot analysis was performed. Densitometric analysis results, normalized to glyceraldehyde-3-phosphate dehydrogenase, are shown in Fig. 6Go. Patients with PCOS have significantly decreased HOXA10 expression (P < 0.05), compared with controls.



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  		Figure 6. HOXA10 mRNA expression is diminished in hyperandrogenic PCOS patients in ovulatory cycles. Endometrial biopsies were obtained in the midsecretory phase of the menstrual cycle, based on urinary LH detection from seven PCOS patients and five controls. HOXA10 expression was assessed by Northern analysis. Endometrial HOXA10 expression was higher in control endometrium than in the endometrium of PCOS patients (P < 0.005, by t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously described a role for HOX genes in the endometrium, leading to uterine receptivity. Alterations of endometrial HOXA10 expression in both humans and animal models has demonstrated the necessary role of this gene in development of the endometrium and receptivity to embryonic implantation (30, 31). Estrogen, progesterone, and retinoic acid are the only regulators of HOX gene expression identified thus far. Here, we report that testosterone is a novel regulator of HOXA10 expression in the endometrium. Decreased HOXA10 expression in Ishikawa cells treated with testosterone, but not DHA or DHAS, suggest that this androgen is responsible for the decreased expression of HOXA10 in PCOS patients.

PCOS is perhaps the most common endocrinopathy, effecting approximately 5% of women in developed countries. It is a common cause of anovulation and infertility, characterized by elevated levels of circulating androgens or clinical manifestations of androgen excess. The chronic anovulation and infertility can often be successfully treated in PCOS; however, resultant successful pregnancy rates are less than expected (18). Elevated serum androgen concentrations could have an adverse effect on endometrium. Androgen receptors are present within the endometrium (33, 34). Little is known about the effect of high androgen concentrations on endometrial function. Androgens can act as antagonists of estrogen or progesterone. In addition to opposing the action of estrogen or progesterone at their respective receptors, androgens could affect the endometrium directly, acting through the androgen receptor. Here, we implicate testosterone as having a negative impact on the expression of a gene essential for endometrial receptivity. The molecular mechanisms that mediate these clinical observations may involve the effect of testosterone on HOXA10.

Recurrent miscarriage is also common in women with PCOS (18, 19, 20). The mechanism by which hyperandrogenemia could be linked to increased miscarriage risk is not known. Continued HOX gene expression in the endometrium is necessary after implantation, to maintain a successful pregnancy. Increased maternal HOXA10 expression in mice results in increased litter size, without changing implantation rates (30). In PCOS, elevated follicular-phase concentrations of androgens may prevent or delay the timing of HOX gene activation.

Clomiphene citrate is commonly used to treat ovulatory dysfunction in PCOS patients. Endometrial luteal-phase defects and decreased implantation have been reported in up to 50% of infertile women treated with clomiphene citrate (51, 52, 53, 54). Additionally, an increased miscarriage rate has been noted in clomiphene-treated cycles. It is, however, not clear whether these defects, noted with clomiphene, are caused by the effects of clomiphene or by the underlying condition that prompted the use of clomiphene. No direct effect of clomiphene on the endometrium has been demonstrated on histologic exam or scanning electron microscopic evaluation of normal ovulatory women (55, 56, 57, 58). Similarly, no such effect has ever been identified in normally ovulating women treated with clomiphene citrate. Our data implicates testosterone as the etiologic agent of endometrial dysfunction, decreased implantation, and increased miscarriage in these women, rather than a direct effect of the clomiphene. Clomiphene had no effect on HOXA10 expression in vitro, whereas testosterone did.

Insulin resistance is important in the pathophysiology of PCOS (6, 7, 8, 9, 10, 11, 12, 13, 51, 52). We demonstrate that insulin does not directly effect HOXA10 expression in Ishikawa cells. This suggests that the effects of insulin on the endometrium are indirect, attributable to the insulin increasing serum androgen levels rather than the effect of insulin on the endometrium. Treatment with insulin sensitizing agents improves fertility in PCOS. These agents decrease serum androgen levels. The mechanism of action of these agents may therefore include increased endometrial HOXA10 expression and improved endometrial receptivity.

Testosterone is a novel negative regulator of endometrial HOXA10 expression. Testosterone-induced endometrial dysfunction may contribute to diminished reproductive success, including a decreased implantation and increased miscarriage rate. Therapies aimed at correcting hyperandrogenism may be necessary to improve endometrial receptivity; ovulation induction alone may not provide optimal treatment. Taken together, these observations may explain the paradox of poor reproductive outcomes in women with PCOS despite correction of ovulatory defects.


   Acknowledgments
 


   Footnotes
 
This work was supported by NIH Grants HD-36887 and ES-10610.

Present address for D.C.: Department of Obstetric, Gynecology and Women’s Health, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103.

Abbreviations: DHA, Dehydroepiandrosterone; DHAS, DHA sulfate; DHT, dihydrotestosterone; PCOS, polycystic ovary syndrome.

Received July 10, 2002.

Accepted September 26, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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