Best Practice & Research Clinical Endocrinology & Metabolism Vol. 20, No. 2, pp. 235–244, 2006 doi:10.1016/j.beem.2006.03.005 available online at http://www.sciencedirect.com
6 Endometrium in PCOS: Implantation and predisposition to endocrine CA Linda C. Giudice*
MD, PhD, MSc
Professor and Chair Department of Obstetrics, Gynecology and Reproductive Sciences The Robert B. Jaffe, MD Endowed Professor in the Reproductive Sciences, University of California, San Francisco, 505 Parnassus Avenue, M1496, Box 0132, San Francisco, CA 94143-0132, USA
Polycystic ovarian syndrome (PCOS) is a common endocrinopathy characterized by oligo/anovulatiaon and elevated circulating androgens or evidence of hyperandrogenism after all known potential causes have been excluded. In addition, insulin resistance and accompanying hyperinsulinemia commonly occur in women with PCOS. There is increasing evidence that the endocrinologic and metabolic abnormalities in PCOS may have complex effects on the endometrium, contributing to the infertility and endometrial disorders observed in women with this syndrome. Androgen receptors and steroid receptor co-activators are over-expressed in the endometrium of women with PCOS. Also, biomarkers of endometrial receptivity to embryonic implantation—such as avb3-integrin and glycodelin—are decreased, and epithelial expression of estrogen receptor a (ERa) abnormally persists in the window of implantation in endometrium in women with PCOS. In addition to being responsive to the steroid hormones estradiol, progesterone, and androgens, the endometrium is also a target for insulin, the receptor for which is cyclically regulated in normo-ovulatory women. In vitro, insulin inhibits the normal process of endometrial stromal differentiation (decidualization). In addition, insulin-like growth factors (IGFs) and their binding proteins are regulated in and act on endometrial cellular constituents, and hyperinsulinemia down-regulates hepatic IGFBP-1, resulting in elevated free IGF-I in the circulation. Thus, elevated estrogen (without the opposing effects of progesterone in the absence of ovulation), hyperinsulinemia, elevated free IGF-I and androgens, and obesity all likely contribute to endometrial dysfunction, infertility, increased miscarriage rate, endometrial hyperplasia, and endometrial cancer common in women with PCOS. The potential mechanisms underlying these disorders, specifically in women with PCOS, are complex and await additional transdisciplinary research for their complete elucidation. Key words: endometrium; polycystic ovarian syndrome; hyperinsulinemia; steroid receptors; co-activators; androgens; insulin; insulin-like growth factors; implantation; endometrial receptivity; miscarriage; endometrial hyperplasia; endometrial cancer.
* Tel.: C1 415 476 2564; fax: C1 415 476 1811. E-mail address: [email protected]
1521-690X/$ - see front matter Q 2006 Published by Elsevier Ltd.
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Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder, affecting approximately 5% of women of reproductive age.1,2 The clinical features include irregular menses/amenorrhea, oligo/anovulation, and elevated circulating concentrations of androgens and/or signs of hyperandrogenism. In addition, approximately 75% of women with PCOS have insulin resistance and hyperinsulinemia, and about 50% have elevated levels of circulating luteinizing hormone (LH). Infertility associated with PCOS derives from chronic anovulation, and there are increasing data to suggest that poor oocyte quality, implantation failure, and higher rates of miscarriage further complicate achieving and maintaining a pregnancy in women with this disorder.3–6 Women with PCOS are also at significantly higher risk of endometrial hyperplasia and endometrial cancer.7 While the endometrium of women with PCOS has had limited study, several lines of evidence support abnormal gene expression and that the clinical manifestations of implantation failure, miscarriage, endometrial hyperplasia and cancer may be attributed to chronic, unopposed estrogens, hyperinsulinemia, and hyperandrogenism, and effects of members of the insulin-like growth factor (IGF) family.
ENDOMETRIAL CYCLICITY AND NON-CYCLING ENDOMETRIUM IN WOMEN WITH PCOS Human endometrium is a steroid-hormone-dependent tissue whose cellular components and tissue growth and remodeling respond to changes in circulating hormones in normal ovulatory cycles. Increasing circulating estradiol (E2) levels, a consequence of ovarian follicular growth in the aptly named ‘follicular’ phase of the cycle, promote endometrial cellular proliferation and growth of the tissue, from approximately 2 mm after the postmenstrual repair phase, to 10–12 mm in the periovulatory period.8 A recent global gene expression profiling study has demonstrated that the primary gene ontology categories of this phase of the cycle are primarily dedicated to DNA replication, cell proliferation, and tissue remodeling.9 After ovulation and the production of progesterone, the endometrium undergoes a striking series of changes, including inhibition of cellular proliferation, DNA synthesis, and cellular mitotic activity, and the onset of cellular differentiation, in preparation for an implanting conceptus. Inhibition of epithelial proliferation is due to progesteroneregulated genes, including down-regulation of the estrogen receptors in the epithelium and induction of estrogen-metabolizing enzymes sulfotransferase and 17b-hydroxysteroid dehydrogenase type 2 9–11, which effectively minimize E2 action in this cell type. In addition, progesterone down-regulates the androgen receptor (AR) in both epithelium and stroma.12 Seven to ten days after ovulation, the endometrium becomes receptive to embryonic implantation in the temporal period called the ‘window of implantation’. Endometrial stromal cells undergo the process of decidualization in response to progesterone, a process that is characterized by changes in the cytoskeleton (down-regulation of smooth muscle actin) and induction or up-regulation of prolactin, insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), the insulin receptor, relaxin, and others.9,13 The process of decidualization is important to regulate trophoblast invasion and to establish a cytokine milieu and immunomodulatory network in the stroma, should implantation occur.9 In the absence of embryonic implantation, withdrawal of E2 and progesterone initiates an immunomodulatory ‘switch’ from an innate immune gene signature to an inflammatory signature, along with
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cellular apoptosis and increased production of metalloproteinases and prostaglandins,9 resulting in endometrial tissue desquamation and menstruation.13 In women with PCOS who are anovulatory or oligo-ovulatory, the regulatory roles of progesterone and progesterone withdrawal in the endometrium are suboptimal or absent. Thus, the tissue is in a state of a relatively enhanced response to E2 and does not undergo the sequential changes in gene expression and associated biochemical processes 9 that result in normal endometrial cellular proliferation, differentiation, and tissue desquamation. Endometrial bleeding patterns in women with PCOS vary from irregular menses to amenorrhea, reflecting the oligo-ovulatory and anovulatory states. Persistent anovulation in women with PCOS, who often have hyperinsulinemia and hyperandrogenemia, results in relatively constant circulating levels of E2 comparable to the early follicular phase.14 However, moderately elevated levels of E2 are encountered due to increased peripheral conversion of androstenedione to estrone in adipose tissue, and free E2 and testosterone are elevated in the circulation in the setting of hyperinsulinemia, due in part to insulin down-regulation of sex-hormone-binding globulin (SHBG).15,16 Thus, endometrial growth and differentiation in women with PCOS are influenced by androgens, insulin, and unopposed estrogens. In the absence of ovulation and the regulatory effects of progesterone, the endometrium does not undergo secretory transformation and is continuously exposed to the stimulatory and mitogenic effects of E2 that can lead to endometrial overgrowth, unpredictable bleeding patterns, hyperplasia, and cancer.17,18
STEROID HORMONE RECEPTORS AND BIOMARKERS IN ENDOMETRIUM OF WOMEN WITH PCOS Steroid hormone receptor expression in human endometrium is cycle-dependent.13,19 ERa is the main ER in cycling endometrium and is up-regulated by E2, peaking in the late proliferative phase in epithelium and stroma. ERa expression in epithelium is markedly inhibited by the mid-secretory phase, in response to progesterone, opening the window of implantation, and it is reduced, but not completely, in stroma. ERb is mainly expressed in the endometrial epithelium.13 Progesterone receptors peak at ovulation, similarly to ERa, and decrease in the secretory phase under the regulation of progesterone.13 ARs are expressed in endometrial stroma and epithelium in normo-ovulatory women and decrease steadily from the early proliferative phase to the mid-secretory phase.19 Lack of immunostaining for the AR characterizes the late luteal phase.19 In vitro studies of endometrial stoma and epithelial cells demonstrate that AR is up-regulated by estrogens and androgens and is inhibited by progestins and epidermal growth factor (EGF).20 In addition, administration of the antiprogestin, mifepristone, results in increased AR expression in human and non-human primate endometrium 12, underscoring progestin inhibition of AR expression. Women with PCOS exhibit elevated endometrial AR expression compared to normal, fertile controls 20, likely due to relatively infrequent ovulation and thus low levels of progesterone available to act on the endometrium. There is increasing evidence of dysregulated expression of some biomarkers, including secretory products and markers of uterine receptivity, in endometrial epithelium of women with PCOS. Whether these are due to an inadequate progesterone effect or excessive androgen
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(and/or insulin) signaling is not clear at this time. For example, in vitro studies demonstrate that androstenedione inhibits secretion of glycodelin, an important secretory product of endometrial epithelium during the receptive phase of implantation, and this effect is reversed by cyproterone acetate, an androgen antagonist.21 While it is tempting to speculate that such dysregulation may be present in women with PCOS, leading to lower implantation rates, currently there are no data to support this. However, another marker of uterine receptivity, avb3 integrin 22, is either delayed in its expression or is not expressed at all in PCOS endometrium.20 Down-regulation of ERa in epithelium heralds the opening of the window of implantation 22, and its persistent expression in endometrium from women with PCOS in the mid-secretory phase has been reported.22,23 These data on avb3 integrin and ERa together suggest poor endometrial development in women with PCOS with occasional spontaneous ovulatory cycles, presumably due to inadequate progesterone action, although dysregulation by androgens or insulin cannot be excluded. A lack of the normal down-regulation of AR and ERa in the window of implantation, which is associated with decreased expression of endometrial proteins such as avb3 and glycodelin, may contribute to the lower pregnancy rates and higher miscarriage rates observed in women with PCOS. Actions of steroid hormones depend on their receptors and specific co-activators and repressors. The p160 co-activators AIB1 and TIF2 are markedly elevated in both the proliferative and secretory phases of a clomiphene-citrate-induced cycle in women with PCOS, compared to controls undergoing ovulation enhancement for male factor infertility.20,23 A higher sensitivity to estrogen, as well as elevated expression of estrogen receptor co-activators, may lead to a higher risk of endometrial hyperplasia and endometrial cancer in women with PCOS.
INSULIN AND INSULIN-LIKE GROWTH FACTORS IN HUMAN ENDOMETRIUM Insulin and endometrial function The endometrium is a target for insulin, which likely acts through its own receptor and perhaps through the structurally similar IGF type I receptor.24 The insulin receptor is up-regulated in the secretory phase of the cycle, a result of progesterone action in the tissue.9 In vitro, insulin inhibits production of the endometrial stromal product IGF binding protein 1 (IGFBP-1), a biomarker of decidualization.25 A recent study by Lathi et al.26, demonstrated that inhibition of IGFBP-1 in human endometrial stromal cells by low doses of insulin is mediated via the phosphatidylinositol-3-kinase (PI3-kinase) pathway, whereas at higher doses the mitogen-activated protein kinase (MAPK) pathway is also activated. These observations suggest that at physiological levels insulin likely plays a homeostatic role for energy metabolism in the endometrium, and in hyperinsulinemic states insulin action on the endometrium may activate cellular mitosis via the MAPK pathway and thus may predispose the endometrium to hyperplasia and/or cancer. The actions of insulin on the endometrium in vivo are difficult to distinguish from the actions of androgens because hyperandrogenism and hyperinsulinemia are positively correlated in women with anovulation.27
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IGFs/receptors/IGFBPs The IGF system is an important participant in endometrial proliferation, development, and implantation.28 IGF-I is expressed primarily in the epitheliumOstroma during the proliferative phase, and is one of two major growth factors that are ‘estromedins’ in this phase of the cycle.28,29 IGF-II is expressed primarily in epitheliumOstroma in the secretory phase, is regulated by progesterone, and has been considered to be a ‘progestomedin’.29,30 IGF-I and IGF-II are mitogenic to endometrial cells in culture and can regulate secretory functions of stromal cells.30,31 In addition, IGFBP-1, produced by decidualized endometrial stromal cells, primarily inhibits IGF actions because it binds IGFs with affinities about two orders of magnitude greater than does the type I IGF receptor.32 IGFs (in addition to insulin) inhibit IGFBP-1 production and steady-state mRNA levels in human endometrial stromal cells in vitro.31 Thus, in PCOS, with hyperinsulinemia and prolonged unopposed estrogen, IGF-I may further augment the mitogenic activity of endometrial cells, leading to acceleration of hyperplasia and perhaps transformation to cancer.
FERTILITY AND MISCARRIAGE IN THE PCOS PATIENT While anovulation is an obvious cause of infertility in women with PCOS, there are emerging data that endometrial receptivity, oocyte quality and increased risk of miscarriage contribute to infertility in the setting of ovulation induction. Miscarriage rates have been reported to be between 30 and 50% of all conceptions in women with PCOS.3,33 In addition, over 30% of women with recurrent miscarriages are reported to have PCOS.34 Several conditions exist in women with unexplained recurrent pregnancy loss (without PCOS), including high circulating levels of LH and free testosterone, low luteal progesterone, and delayed endometrial development.35 Many of these findings are frequently seen in women with PCOS when treated for infertility. Women with elevated androgens have higher rates of implantation failure and increased miscarriage rates compared to women of similar age, body mass index and insulin levels.4 Women with recurrent pregnancy loss have low levels of serum glycodelin, an endometrial and ovarian product that has been postulated to mediate, in part, the maternal immune response to the implanting embryo.36 Interestingly, when ovulation is induced with clomiphene citrate, expression of the molecular markers of endometrial receptivity, avb3 integrin and glycodelin, are decreased in endometrial biopsy specimens from women with PCOS.37,38 There are conflicting data on whether egg quality and embryo quality are compromised in women with PCOS undergoing in vitro fertilization-embryo transfer (IVF-ET). For example, when oocytes were used from donors with a history of PCOS, fertilization and implantation rates were high and similar to matched donors with other forms of infertility.39 These patients were diagnosed as PCOS by ultrasound appearance and an elevated LH:FSH ratio only, whereas other studies may include information of insulin status, thus demonstrating how important subject selection and uniformity of criteria are in comparing one study with another. Another study of PCOS women undergoing IVF-ET examined the effect of insulin resistance on several IVF outcomes and found that insulin resistance alone does not predict implantation or pregnancy rates.5,6 However, obesity was found to be associated with lower oocyte yields and an increased requirement for gonadotropin stimulation, in addition to an
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increase in miscarriage rate in the insulin-resistant group, although this was not statistically significant. An earlier study by the same group showed that obesity is a significant risk factor for spontaneous abortion after IVF-ET or intracytoplasmic sperm injection and IVF-ET for male factor infertility.5 Thus, while large-scale studies are needed to determine contributors to anovulation as additional factors in infertility experienced by women with PCOS, it is likely that the poor reproductive performance of PCOS women involves compromised oocyte quality and endometrial receptivity, obesity, hyperinsulinemia, and hyperandrogenemia.
ENDOMETRIAL HYPERPLASIA AND CANCER IN WOMEN WITH PCOS Endometrial hyperplasia occurs in 35% of women with PCOS who are not receiving either contraceptive steroids or periodic or monthly progestin withdrawal. Those at highest risk of endometrial hyperplasia are women who have fewer than four menses per year and an endometrial thickness, assessed by ultrasound, of O7 mm.18 Endometrial cancer represents 8% of all cancers occurring in women, and those at highest risk are women who are obese, have type II diabetes, and PCOS, all of which may be associated with hyperinsulinemia. There has been considerable interest in the literature on the relationship between hyperinsulinemia, endometrial hyperplasia and cancer, and several potential mechanisms have been hypothesized and investigated. Insulin receptors are present in normal endometrium 9 and in endometrial cancers 40, suggesting the potential role of hyperinsulinemia in the growth and development of endometrial cancer. The findings of different signaling pathways that are activated in human endometrial stromal cells at low versus high levels of insulin in the medium, with the MAPK pathway being activated at higher levels 26, suggests that insulin may have homeostatic functions at lower doses and may predispose to cell proliferation at higher doses. While it is difficult to extrapolate these findings to endometrial hyperplasia and adenocarcinoma (involving epithelium not stroma), further studies on insulin action on human endometrial epithelium are in order. Insulin stimulates cell proliferation in ER-negative and ER-positive endometrial cancer cell lines.41 Furthermore, it may play promote tumor angiogenesis via induction of the expression of vascular endothelial growth factor (VEGF). Insulin stimulates VEGF in a biphasic manner, with early and delayed increases in VEGF mRNA, at the transcriptional and post-transcriptional levels.42 Insulin also stimulates aromatase expression and enzyme activity in endometrial glands and stroma, and since E2 is a direct product of the aromatase enzyme, this may provide another mechanism for insulin-induced augmentation of endometrial cellular proliferation.43 Insulin can also promote tumor development by inhibiting apoptosis and stimulating cell proliferation. With regard to the IGF family, IGF-I and the type I IGF receptor have been shown to play a role in the malignant growth of several cancers.44 The type I IGF receptor mRNA is over-expressed in endometrial carcinomas, and this receptor may have an important role in their growth by both ligand-dependent and ligand-independent mechanisms.45 The number of type I IGF receptors positively correlates with the histological grade of endometrial cancers.40,46 Over-expression of LH/hCG receptors at both mRNA and protein levels is also associated with endometrial carcinogenesis which occurs through
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endometrial hyperplasia.47,48 These data overall suggest that insulin, the IGF system, and LH may contribute to the pathogenesis of endometrial cancer occurring among anovulatory women with polycystic ovarian syndrome.
CONCLUSIONS PCOS is a complex disorder with metabolic and endocrine manifestations. Women with this disorder have infertility that appears to be due to more than anovulation and likely involves abnormalities in their endometrium, perhaps related to low progesterone, and/or elevated insulin, free IGF-I, androgens, and LH in the circulation. Some of the data for these associations are firm; however, some of the data are conflicting, and further research is necessary to understand the poor reproductive performance in women with this disorder. Insulin and androgens have direct effects on endometrial function in vitro, including impairment of normal decidualization. Chronic lack of progesterone and IGFBP production, accompanying anovulation, hyperinsulinemia and hyperandrogenemia can translate into a net stimulatory effect on endometrial proliferation, poor endometrial development, and endometrial hyperplasia and cancer. While preliminary studies using insulin sensitizers have shown improvement of circulating insulin and androgen levels, as well as improved reproductive performance 49, further investigation is needed to determine precisely the mechanism(s) of insulin action and the role of the IGF system in endometrial function and implantation in order to derive optimal therapies for women with PCOS in the treatment of infertility and prevention of endometrial hyperplasia and cancer.
Practice points † infertility in women with PCOS appears to have causes beyond anovulation, and patients should be counseled regarding this in setting expectations. † women with PCOS are at significant risk of developing endometrial hyperplasia and/or endometrial cancer; evaluation of the endometrum by biopsy (with or without hysteroscopy) is appropriate for endometrial thickness O4 mm. † inducing regular withdrawal bleeding, using contraceptive steroids or progestins, is recommended.
Research agenda † a comprehensive analysis of the effects of insulin and androgens on endometrial and oocyte function is warranted. † randomized controlled clinical trials on insulin-lowering agents and pregnancy outcomes, ovulatory rates, oocyte quality, and endometrial receptivity are needed.
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ACKNOWLEDGEMENTS This work was supported by NIH R01 HD 31579.
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