http://informahealthcare.com/gye ISSN: 0951-3590 (print), 1473-0766 (electronic) Gynecol Endocrinol, 2015; 31(4): 278–281 ! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2014.989980
PCO AND ENDOMETRIUM
The evaluation of endometrial sulfate glycosaminoglycans in women with polycystic ovary syndrome Mario Vicente Giordano1,2, Luiz Augusto Giordano1,2, Regina Ce´lia Teixeira Gomes1, Ricardo Santos Simo˜es3, Helena Bonciani Nader4, Mario Ga´spare Giordano2, Edmund Chada Baracat3, and Jose´ Maria Soares Ju´nior1,3 1
Gynecology Department, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Gynecology Department, Federal University of Rio de Janeiro State, Rio de Janeiro, Brazil, 3Obstetrics and Gynecology Department, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, and 4Molecular Biology Division, Department of Biochemistry, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract
Keywords
The aim of this study was to quantify the sulfated glycosaminoglycans in the endometria of women with polycystic ovary syndrome (PCOS). Of the 18 patients recruited for this study, 10 patients with PCOS comprised the PCOS group (PCOSG), and eight patients with regular and ovulatory menstrual cycles comprised the control group (CG). The clinical, biochemical, morphological and endometrial data from both groups were analyzed. Biopsies were performed during the proliferative phase of the menstrual cycle for the CG and during the persistent proliferative phase for the PCOSG (all women were amenorrheic). In the PCOSG, there was a significant increase in the endometrial concentration levels of heparan sulfate (p ¼ 0.03), but no difference in the concentrations of chondroitin sulfate was determined between the two groups (p ¼ 0.77). Period of time without menstruation (p ¼ 0.001) and body mass index (BMI) (p ¼ 0.04) correlated directly and positively with heparan sulfate concentration. There was no association between heparan sulfate levels and basal insulin values (p ¼ 0.08). High levels of endometrial heparan sulfate in women with PCOS indicate an interference with maternal–fetal recognition, which contributes to infertility; thus, endometrial heparan sulfate may be a predictive marker of future neoplasia risk.
Endometrium, glycosaminoglycans, heparan sulfate, infertility, polycystic ovary syndrome
Introduction Polycystic ovary syndrome (PCOS) is the most common endocrinopathy observed in young women and is a major cause of infertility. PCOS afflicts 5–15% of women with reproductive difficulties [1–3] and is related to menstrual irregularities and hyperandrogenism [2,4]. The syndrome is associated with high rates of embryo implantation failure and miscarriages in the first trimester. In addition to oligo-ovulation, endometrial dysfunction has also been acknowledged as a main factor in reproductive failure [5,6]. With that in mind, women with PCOS have a higher risk of developing endometrial neoplasia [7]. The proliferative phase of the menstrual cycle is associated with follicular growth and increased estrogen secretion, which leads to endometrial proliferation. After ovulation, endometrial proliferation ceases as progesterone begins to rise. In PCOS this does not occur; the continuous estrogen stimulation of the endometrium results in proliferative lesions, increased risk for endometrial cancer, and the likelihood of embryo implantation dysfunction [7,8]. The interaction between endometrial cells and the extracellular matrix (ECM) during tissue remodeling is important for endometrial organization. That interaction is involved in proliferation, motility and cell secretion control, all of which are contributory factors to embryo implantation. The ECM is a complex structure of Address for correspondence: Mario Vicente Giordano, Gynecology Department, Federal University of Rio de Janeiro State (UNIRIO), 36 Mario Barreto St. Apt 102, Rio de Janeiro – RJ, Brazil. Tel: +55 21 3281 9320. E-mail: [email protected]
History Received 22 March 2014 Accepted 17 November 2014 Published online 28 November 2014
self-assembling macromolecules, composed mainly of collagen and elastic fibers, glycoproteins and sulfated glycosaminoglycans (GAGs) (chondroitin, dermatan, heparan and keratan sulfate) [9,10]. Recent studies have shown that chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) are present in the human endometrium [11]. For a long time, GAGs have drawn clinical interest due to their important roles in cell recognition, migration, differentiation and proliferation. They are found in different tissues and are involved in multiple processes, such as tumor formation, metastasis, angiogenesis, immunologic reactions, follicular development and fertility [12,13]. However, there are no data that describe GAG behavior in the endometrium of women with PCOS. Therefore, our study aimed to evaluate such elements and compare them to those of normal women in the proliferative phase.
Patients and methods The use of human tissues was approved by the Ethics Committee of the Gaffre´e and Guinle University Hospital (report No. 38/ 2009) and by the Institutional Review Board of the Federal University of the State of Rio de Janeiro – Brazil (UNIRIO). Written informed consent was obtained from all patients. Initially, 13 women of reproductive age with regular menstrual cycles (28–30 days) were recruited by the senior author of this study from an Infertility Clinic to participate in an infertility evaluation. The women were selected for the study if their progesterone levels were greater than 3 ng/mL during the luteal phase of the menstrual cycle. Additionally, 21 women with PCOS were
Polycystic ovary syndrome
DOI: 10.3109/09513590.2014.989980
279
Figure 1. Fluxogram showing the selection of patients.
recruited from the UNIRIO Gynecological Endocrinology Department. This study was conducted from February 2011 to June 2012. PCOS was diagnosed according to the criteria of Androgen Excess – Polycystic Ovary Syndrome Society, 2006 (AE-PCOS Society) [2]. Eight patients with regular menstrual cycles were included in this study and composed the control group (CG/n ¼ 8). Biopsies were taken during the proliferative endometrial phase of the menstrual cycle (cycle days 5–11). In the PCOS patients (PCOSG/n ¼ 10), biopsies were taken at any time because of the patients’ oligomenorrhea (Figure 1). The exclusion criteria were as follows: (1) PCOS diagnostic differential with other pathological conditions, such as hypothyroidism, adrenal virilizing hyperplasia, hyperprolactinemia, ovarian androgenproducing tumors and diabetes mellitus; (2) oral hormone therapy or psychotropic drug use within three months before the study; (3) parenteral hormone use within six months prior to the study and (4) levonorgestrel intrauterine device use within one year prior to the study. We performed hysteroscopy to exclude any unsuspected pathology of the uterine cavity, such as endometritis, myomas, polyps and adhesions. The procedure was conducted in a clinical center, and the patients were under anesthesia (sedation/ PropofolÕ -2,6-diisopropilfenol). Endometrial biopsies were performed following the Bettocchi system (Karl-StorzÕ , Tuttlingen, Germany). Immediately after removal, the endometrial biopsies were divided into two fragments. One was placed in acetone for sulfate GAG quantification, and the other was fixed for 24 h in 10% formaldehyde in phosphate-buffered saline (PBS) and histologically analyzed for future inclusion criteria (proliferative endometrium). All patients left the hospital on the same day of the procedure, and no complications were observed. After fixation, tissue samples were processed for paraffin inclusion by dehydration in ethanol, clearing in xylol and impregnation with liquid paraffin in a drying oven set at 60 C. Three micrometer sections of the paraffin-embedded blocks were cut on a Minot microtome (LeikaÕ , Nussloch, Germany) and then stained with hematoxylin and eosin (H.E). Biochemical purification and glycosaminoglycan extraction The purification and extraction of glycosaminoglycans were performed according to the protocol in Gomes et al. [10]. First,
the lipid residues were removed with acetone and then powdered and dried in an oven at 60 C for one hour. Second, the dried tissue was subjected to proteolysis with maxatase (60 C, 18 h) (Biocon, Rio de Janeiro, Brazil). Third, the protein was precipitated with trichloroacetic acid (20 C, 30 min), and the supernatant was isolated after centrifugation (5000g, 15 min). Fourth, the glycosaminoglycans in the supernatant were precipitated with methanol (20 C, 24 h). After centrifugation at 5000g for 15 min, the supernatant was discarded. The precipitate was dried in an oven at 50 C for evaporation of the methanol residue and then resuspended in distilled water. Fifth, the sulfated GAGs in the samples were separated by 0.6% agarose gel electrophoresis in diaminopropane-acetate (PDA) buffer, pH 9.0. Electrophoresis was performed for approximately 1 h at 100 V, and the compounds were precipitated in the gel with 0.1% Cetavlon for at least 2 h. The gel was dried and stained with 0.1% toluidine blue in 1% acetic acid and 50% ethanol. Sixth, the gel was dried at room temperature. Finally, the quantification was carried out by densitometry at 525 nm, with a 5% error margin. The detection limit was 1 mg/mL of sulfate GAG (CS, DS and HS). The specific GAG types were confirmed based on their distinct banding patterns on the gel by comparing them to the following standards: chondroitin sulfate, dermatan sulfate (Seikagaku Kogyo Co., Tokyo, Japan) and heparan sulfate from bovine lung (extracted and purified by the Molecular Biology Division, UNIFESP – EPM, Sa˜o Paulo, Brazil). The results were expressed as mg of GAG/mg of dry tissue powder.
Statistical analyses The Kolmogorov–Smirnov test was used to assess the normality of the variables; the Student t-test was used for normally distributed variables, and the Mann–Whitney test was used for non-normally distributed variables. The Pearson correlation coefficient was used to determine linear associations between heparan sulfate and the clinic variables: age, body mass index, last menstrual bleeding and insulin. The statistical analyses were performed using SPSS for Windows, version 12.0 (SPSS Inc., Chicago, IL). The significance level was set at p50.05.
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Table 1. Patient clinical, biochemical and hormonal data.
Age (years) BMI (kg/m2) PTWM (days) GLU (mg/dl) Insulin (mU/ml) HOMA-IR LH (mUI/ml) FSH (mUI/ml) PRL (ng/dl) TSH (mU/ml) 17OHP (ng/dl) DHEAS (mg/dl) A (ng/dl) Ttotal (ng/dl) Tfree (pg/dl)
CG (n ¼ 8)
PCOSG (n ¼ 10)
p Value
30.50 ± 3.63 24.00 ± 2.58 9.27 ± 1.73 84 ± 5.2 5.6 ± 1.4 1.18 ± 0.36 6.0 ± 3.1 5.3 ± 1.4 10.9 (3.8–35.8) 2.0 ± 1.2 115 ± 64.1 209 ± 67 1.3 (0.5–2.8) 30.6 ± 15.3 1.81 ± 0.45
24.35 ± 4.12 27.92 ± 4.89 88.07 ± 48.21 87 ± 3.1 13.6 ± 8.0 2.9 ± 1.83 10.5 ± 4.7 5.2 ± 1.3 12.4 (4.2–34.0) 1.8 ± 0.7 180 ± 34.4 210 ± 110 4.5 (2.4–7.8) 67.2 ± 25.5 2.25 ± 1.20
0.02 0.03 0.001 0.40 0.01 0.05 0.001 0.92 0.52 0.63 0.15 0.97 0.001 0.001 0.25
n ¼ number of patients, CG ¼ control group; PCOSG ¼ polycystic ovary syndrome group. Statistical tests applied: Student t-test for normally distributed variables (mean ± standard deviation) and Mann–Whitney test for non-Gaussian distributed variables (median and min–max). BMI: body mass index; PTWM: period of time without menstruation; GLU: glucose; HOMA-IR: homeostatic model assessment insulin resistance; LH: lutein hormone; FSH: follicle-stimulating hormone; PRL: prolactin; TSH: thyroid-stimulating hormone; 17OHP: 17 hydroxyprogesterone; DHEAS: dehydroepiandrosterone sulfate; A: androsterone; Ttotal: total testosterone; Tfree: free testosterone. Age, BMI, LMB, GLU, insulin, HOMA-IR, LH, FSH, 17OHP, DHEAS, TSH, Ttotal and Tfree exhibited homogeneous distributions. PRL and A exhibited heterogeneous distributions.
Table 2. Quantification of sulfate glycosaminoglycans in endometrium. Sulfate GAGs (mg/mg of dry tissue)
CS HS
CG (n ¼ 8)
PCOSG (n ¼ 10)
p Value
1.12 ± 0.22 0.17 ± 0.06
1.18 ± 0.38 0.25 ± 0.04
0.77 0.03
n ¼ number of patients, CG ¼ control group; PCOSG ¼ polycystic ovary syndrome group; GAGs: glycosaminoglycans; CS: chondroitin sulfate; HS: heparan sulfate. Student t test was employed for the normally distributed variables (mean ± standard deviation).
Results The clinical, biochemical and hormonal data from the patients in the CG and PCOSG are displayed in Table 1. Based on the histopathological examination of the endometrium in the PCOSG (n ¼ 14), 78.5% of the endometria were proliferative (n ¼ 11), 14.2% were secretory (n ¼ 2) and 7.1% were hyperplastic (n ¼ 1); in the CG (n ¼ 12), 83.3% were proliferative (n ¼ 10) and 16.7% were secretory (n ¼ 2). Only the proliferative endometrial samples were included for further analysis. The chondroitin sulfate (CS) and heparan sulfate (HS) levels were identified in the endometrium of the women in the CG and PCOSG. HS was significantly higher in the PCOSG patients than in the CG patients (p ¼ 0.03). There was no significant difference in CS between the PCOSG and CG (p ¼ 0.77). The results were expressed as mg of GAG/mg of dry tissue powder (Table 2). To assess the influence of the clinical data on the results from endometrial HS quantification, the Pearson correlation coefficient was calculated using the following variables, which were different in the two groups: age (r ¼ 0.52; p ¼ 0.01), period of time without menstruation (r ¼ 0.75; p ¼ 0.001), body mass index
(r ¼ 0.52; p ¼ 0.03) and basal insulin (r ¼ 0.42; p ¼ 0.08). A significant correlation was found between the endometrial HS level and age, period of time without menstruation and body mass index. No significant association was found for basal insulin (Figure 2). CS was the major GAG in both the PCOSG and CG, with more than 1 mg/mg of dry tissue in both groups. HS had the lowest concentration of all the GAGs analyzed, but it was higher in the PCOSG than in the CG.
Discussion The present study showed that the phenomena involving endometrial transformations in women with PCOS may be associated with changes in the levels of various glycosaminoglycans (GAGs), in particular with an increase in heparan sulfate. In fact, the endometrium is exposed to estrogen in the absence of progesterone counteraction. Women with PCOS exhibit ovulatory dysfunction and infertility. Ovulation may be restored with drugs or laparoscopic surgery. However, little attention has been placed on the endometrium, which is often unresponsive to classical treatments, leading to implantation failure and an increase in proliferative lesions [2,5,14]. Furthermore, little is known about GAGs in the endometrium of women with PCOS. This study is the first to quantify endometrial sulfated GAGs in women with PCOS. The molecular structure of GAGs allows them to interact with growth factors and other proteins that act as signals for cells to block, activate or direct cell migration through the ECM [10,15]. The ECM allows the exchange of nutrients; proliferation; and the transformation of factors, such as insulin growth factor (IGF) and fibroblast growth factor (FGF), between cells. The ECM may play a more active role in tissues [16], particularly the ECM elements that influence endometrial receptivity and embryo implantation, such as glycoproteins and cell adhesion molecules (integrin, selectin, fibronectin and laminin) [10,17]. In implantation physiology, CS and HS may be potential ligands for L-selectin/CD44 [18,19], which is important in the initial stages of trophoblast adhesion and in the promotion of integrin-dependent processes leading up to the maternal–fetal interface [20]. Studies admit higher levels of implantation failure in women with PCOS, even in IVF cycles [2,4]. Through complex mechanisms, women with PCOS exhibit endometrial dysfunction, which hinders their reproductive ability. Persistent estrogen stimulation to uterine tissue may lead to changes in endometrial gene expression and biochemical processes resulting in abnormal cell proliferation and differentiation [5,21]. It also may cause an increase in the production of HS on the endometrium in women with PCOS, as was demonstrated in the present study. An increased HS concentration may interfere with endometrial receptivity and/or embryo development, which can result in blastocyst adhesion dysfunction, implantation failure and miscarriages in women with PCOS. The presence of HS is very important on the cell surface, and research has shown that its structure is specific for different tissues and species. It has been acknowledged that HS acts as a cell recognition molecule and that it may increase the coupling of fibroblast growth factor (FGF) to its receptor [22]. In fact, the targeting of cells by FGF ligands and the subsequent complex formations that they form with their receptors are under positive control from the extracellular space by heparan sulfate proteoglycans (HSPGs). Signaling by FGF is crucial in multiple cellular processes, including cell proliferation, differentiation, survival and migration during mammalian implantation and embryogenesis [23]. Our results demonstrate that PCOS women have high levels of endometrial heparan sulfate. This phenomenon may be a cause of
DOI: 10.3109/09513590.2014.989980
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Figure 2. The Pearson correlation coefficient. A significant and positive correlation was found between HS and PTWM (p ¼ 0.001) and BMI (p ¼ 0.03). A significant and negative correlation was found between HS and age (p ¼ 0.01). No significant association was found between HS and insulin (p ¼ 0.08). Abbreviations: HS: heparan sulfate; BMI: body mass index; PTWM: period of time without menstruation.
the interference with maternal–fetal recognition, which contributes to infertility, that is observed in women with PCOS.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References 1. Azziz R, Woods KS, Reyna R, et al. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745–9. 2. Azziz R, Carmina E, Dewailly D, et al. The Androgen Excess and PCOS Society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril 2009;91:456–88. 3. Cakmak H, Taylor HS. Human implantation failure: molecular mechanisms and clinical treatment. Human Reprod Update 2011;17: 242–53. 4. Fauser BC, Tarlatzis BC, Rebar RW, et al. Consensus on women’s health aspects of polycystic ovary syndrome (PCOS). The Amsterdam ESHRE/ASRM – sponsored 3rd PCOS consensus workshop group. Fertil Steril 2012;97:28–38. 5. Giudice LC. Endometrium in PCOS: implantation and predisposition to endocrine cancers. Best Pract Res Clin Endocrinol Metab 2006;20:235–44. 6. Shang K, Jia X, Qiao J, et al. Endometrium abnormality in women with polycystic ovary syndrome. Reprod Sci 2012;19:674–83. 7. Haoula Z, Salman M, Atiomo W. Evaluating the association between endometrial cancer and polycystic ovary syndrome: meta-analysis. Human Reprod 2012;27:1327–31. 8. Navaratnarajah R, Pillay OC, Hardiman P. Polycystic ovary syndrome and endometrial cancer. Semin Reprod Med 2008;26: 62–71. 9. Afratis N, Gialeli C, Nikitovic D, et al. Glycosaminoglycans: key players in cancer cell biology and treatment. FEBS J 2012;279: 1177–97. 10. Gomes RCT, Maioral GCCC, Verna C, et al. Hyperprolactinemia changes the sulfated glycosaminoglycan amount on the murine uterus during the estrous cycle. Fertil Steril 2013;100:1419–27.
11. Nasciutti LE, Ferrari R, Berardo PT, et al. Distribution of chondroitin sulfate in human endometrium. Micron 2006;37: 544–50. 12. Ueno M, Yamada S, Zako M, et al. Structural characterization of heparin sulphated and chondroitin sulphated of syndecan-1 purified from normal murine mammary glands epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence. J Biol Chem 2001;276:29134–40. 13. Volk R, Schwartz JJ, Li J, et al. The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction. J Biol Chem 1999;274:24417–24. 14. Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Hum Reprod Update 2006;12: 731–46. 15. Kresse H, Schonher E. Proteoglycans of the extracellular matrix and growth control. J Cell Physiol 2001;189:266–74. 16. Sasisekharan R, Shriver Z, Venkataraman G, Narayanasami U. Roles of heparan-sulphate glycosaminoglycans in cancer. Nat Rev Cancer 2002;2:521–8. 17. Carson DD, Tang JP, Julian J. Heparan sulfate proteoglycan (perlecan) expression by mouse embryos during acquisition of attachment competence. Dev Biol 1993;155:97–106. 18. Atarashi K, Kawashima H, Hirata T, Miyasaka M. Chondroitin sulfate proteoglycan, common ligand of L-selectin and CD44. Tanpakushitsu Kakusan Koso 2002;47:2214–20. 19. Celie JW, Keuning ED, Beelen RH, et al. Identification of L-selectin binding heparan sulfates attached to collagen type XVIII. J Biol Chem 2005;280:26965–73. 20. Lopes IMRS, Baracat MCP, Simo˜es MJ, et al. Endometrium in women with polycystic ovary syndrome during the window of implantation. Rev Assoc Med Bras 2011;57:688–95. 21. Park JC, Lim SY, Jang TK, et al. Endometrial histology and predictable clinical factors for endometrial disease in women with polycystic ovary syndrome. Clin Exp Reprod Med 2011;38:42–6. 22. Cattaruzza S, Perris R. Approaching the proteoglycome: molecular interactions of proteoglycans and their functional output. Macromol Biosci 2006;6:667–80. 23. Matsuo I, Kimura-Yoshida C. Extracellular modulation of fibroblast growth factor signaling through heparan sulfate proteoglycans in mammalian development. Curr Opin Genet Dev 2013;4: 399–407.
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PCO AND ENDOMETRIUM
The evaluation of endometrial sulfate glycosaminoglycans in women with polycystic ovary syndrome Mario Vicente Giordano1,2, Luiz Augusto Giordano1,2, Regina Ce´lia Teixeira Gomes1, Ricardo Santos Simo˜es3, Helena Bonciani Nader4, Mario Ga´spare Giordano2, Edmund Chada Baracat3, and Jose´ Maria Soares Ju´nior1,3 1
Gynecology Department, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Gynecology Department, Federal University of Rio de Janeiro State, Rio de Janeiro, Brazil, 3Obstetrics and Gynecology Department, Faculty of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, and 4Molecular Biology Division, Department of Biochemistry, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract
Keywords
The aim of this study was to quantify the sulfated glycosaminoglycans in the endometria of women with polycystic ovary syndrome (PCOS). Of the 18 patients recruited for this study, 10 patients with PCOS comprised the PCOS group (PCOSG), and eight patients with regular and ovulatory menstrual cycles comprised the control group (CG). The clinical, biochemical, morphological and endometrial data from both groups were analyzed. Biopsies were performed during the proliferative phase of the menstrual cycle for the CG and during the persistent proliferative phase for the PCOSG (all women were amenorrheic). In the PCOSG, there was a significant increase in the endometrial concentration levels of heparan sulfate (p ¼ 0.03), but no difference in the concentrations of chondroitin sulfate was determined between the two groups (p ¼ 0.77). Period of time without menstruation (p ¼ 0.001) and body mass index (BMI) (p ¼ 0.04) correlated directly and positively with heparan sulfate concentration. There was no association between heparan sulfate levels and basal insulin values (p ¼ 0.08). High levels of endometrial heparan sulfate in women with PCOS indicate an interference with maternal–fetal recognition, which contributes to infertility; thus, endometrial heparan sulfate may be a predictive marker of future neoplasia risk.
Endometrium, glycosaminoglycans, heparan sulfate, infertility, polycystic ovary syndrome
Introduction Polycystic ovary syndrome (PCOS) is the most common endocrinopathy observed in young women and is a major cause of infertility. PCOS afflicts 5–15% of women with reproductive difficulties [1–3] and is related to menstrual irregularities and hyperandrogenism [2,4]. The syndrome is associated with high rates of embryo implantation failure and miscarriages in the first trimester. In addition to oligo-ovulation, endometrial dysfunction has also been acknowledged as a main factor in reproductive failure [5,6]. With that in mind, women with PCOS have a higher risk of developing endometrial neoplasia [7]. The proliferative phase of the menstrual cycle is associated with follicular growth and increased estrogen secretion, which leads to endometrial proliferation. After ovulation, endometrial proliferation ceases as progesterone begins to rise. In PCOS this does not occur; the continuous estrogen stimulation of the endometrium results in proliferative lesions, increased risk for endometrial cancer, and the likelihood of embryo implantation dysfunction [7,8]. The interaction between endometrial cells and the extracellular matrix (ECM) during tissue remodeling is important for endometrial organization. That interaction is involved in proliferation, motility and cell secretion control, all of which are contributory factors to embryo implantation. The ECM is a complex structure of Address for correspondence: Mario Vicente Giordano, Gynecology Department, Federal University of Rio de Janeiro State (UNIRIO), 36 Mario Barreto St. Apt 102, Rio de Janeiro – RJ, Brazil. Tel: +55 21 3281 9320. E-mail: [email protected]
History Received 22 March 2014 Accepted 17 November 2014 Published online 28 November 2014
self-assembling macromolecules, composed mainly of collagen and elastic fibers, glycoproteins and sulfated glycosaminoglycans (GAGs) (chondroitin, dermatan, heparan and keratan sulfate) [9,10]. Recent studies have shown that chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) are present in the human endometrium [11]. For a long time, GAGs have drawn clinical interest due to their important roles in cell recognition, migration, differentiation and proliferation. They are found in different tissues and are involved in multiple processes, such as tumor formation, metastasis, angiogenesis, immunologic reactions, follicular development and fertility [12,13]. However, there are no data that describe GAG behavior in the endometrium of women with PCOS. Therefore, our study aimed to evaluate such elements and compare them to those of normal women in the proliferative phase.
Patients and methods The use of human tissues was approved by the Ethics Committee of the Gaffre´e and Guinle University Hospital (report No. 38/ 2009) and by the Institutional Review Board of the Federal University of the State of Rio de Janeiro – Brazil (UNIRIO). Written informed consent was obtained from all patients. Initially, 13 women of reproductive age with regular menstrual cycles (28–30 days) were recruited by the senior author of this study from an Infertility Clinic to participate in an infertility evaluation. The women were selected for the study if their progesterone levels were greater than 3 ng/mL during the luteal phase of the menstrual cycle. Additionally, 21 women with PCOS were
Polycystic ovary syndrome
DOI: 10.3109/09513590.2014.989980
279
Figure 1. Fluxogram showing the selection of patients.
recruited from the UNIRIO Gynecological Endocrinology Department. This study was conducted from February 2011 to June 2012. PCOS was diagnosed according to the criteria of Androgen Excess – Polycystic Ovary Syndrome Society, 2006 (AE-PCOS Society) [2]. Eight patients with regular menstrual cycles were included in this study and composed the control group (CG/n ¼ 8). Biopsies were taken during the proliferative endometrial phase of the menstrual cycle (cycle days 5–11). In the PCOS patients (PCOSG/n ¼ 10), biopsies were taken at any time because of the patients’ oligomenorrhea (Figure 1). The exclusion criteria were as follows: (1) PCOS diagnostic differential with other pathological conditions, such as hypothyroidism, adrenal virilizing hyperplasia, hyperprolactinemia, ovarian androgenproducing tumors and diabetes mellitus; (2) oral hormone therapy or psychotropic drug use within three months before the study; (3) parenteral hormone use within six months prior to the study and (4) levonorgestrel intrauterine device use within one year prior to the study. We performed hysteroscopy to exclude any unsuspected pathology of the uterine cavity, such as endometritis, myomas, polyps and adhesions. The procedure was conducted in a clinical center, and the patients were under anesthesia (sedation/ PropofolÕ -2,6-diisopropilfenol). Endometrial biopsies were performed following the Bettocchi system (Karl-StorzÕ , Tuttlingen, Germany). Immediately after removal, the endometrial biopsies were divided into two fragments. One was placed in acetone for sulfate GAG quantification, and the other was fixed for 24 h in 10% formaldehyde in phosphate-buffered saline (PBS) and histologically analyzed for future inclusion criteria (proliferative endometrium). All patients left the hospital on the same day of the procedure, and no complications were observed. After fixation, tissue samples were processed for paraffin inclusion by dehydration in ethanol, clearing in xylol and impregnation with liquid paraffin in a drying oven set at 60 C. Three micrometer sections of the paraffin-embedded blocks were cut on a Minot microtome (LeikaÕ , Nussloch, Germany) and then stained with hematoxylin and eosin (H.E). Biochemical purification and glycosaminoglycan extraction The purification and extraction of glycosaminoglycans were performed according to the protocol in Gomes et al. [10]. First,
the lipid residues were removed with acetone and then powdered and dried in an oven at 60 C for one hour. Second, the dried tissue was subjected to proteolysis with maxatase (60 C, 18 h) (Biocon, Rio de Janeiro, Brazil). Third, the protein was precipitated with trichloroacetic acid (20 C, 30 min), and the supernatant was isolated after centrifugation (5000g, 15 min). Fourth, the glycosaminoglycans in the supernatant were precipitated with methanol (20 C, 24 h). After centrifugation at 5000g for 15 min, the supernatant was discarded. The precipitate was dried in an oven at 50 C for evaporation of the methanol residue and then resuspended in distilled water. Fifth, the sulfated GAGs in the samples were separated by 0.6% agarose gel electrophoresis in diaminopropane-acetate (PDA) buffer, pH 9.0. Electrophoresis was performed for approximately 1 h at 100 V, and the compounds were precipitated in the gel with 0.1% Cetavlon for at least 2 h. The gel was dried and stained with 0.1% toluidine blue in 1% acetic acid and 50% ethanol. Sixth, the gel was dried at room temperature. Finally, the quantification was carried out by densitometry at 525 nm, with a 5% error margin. The detection limit was 1 mg/mL of sulfate GAG (CS, DS and HS). The specific GAG types were confirmed based on their distinct banding patterns on the gel by comparing them to the following standards: chondroitin sulfate, dermatan sulfate (Seikagaku Kogyo Co., Tokyo, Japan) and heparan sulfate from bovine lung (extracted and purified by the Molecular Biology Division, UNIFESP – EPM, Sa˜o Paulo, Brazil). The results were expressed as mg of GAG/mg of dry tissue powder.
Statistical analyses The Kolmogorov–Smirnov test was used to assess the normality of the variables; the Student t-test was used for normally distributed variables, and the Mann–Whitney test was used for non-normally distributed variables. The Pearson correlation coefficient was used to determine linear associations between heparan sulfate and the clinic variables: age, body mass index, last menstrual bleeding and insulin. The statistical analyses were performed using SPSS for Windows, version 12.0 (SPSS Inc., Chicago, IL). The significance level was set at p50.05.
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Table 1. Patient clinical, biochemical and hormonal data.
Age (years) BMI (kg/m2) PTWM (days) GLU (mg/dl) Insulin (mU/ml) HOMA-IR LH (mUI/ml) FSH (mUI/ml) PRL (ng/dl) TSH (mU/ml) 17OHP (ng/dl) DHEAS (mg/dl) A (ng/dl) Ttotal (ng/dl) Tfree (pg/dl)
CG (n ¼ 8)
PCOSG (n ¼ 10)
p Value
30.50 ± 3.63 24.00 ± 2.58 9.27 ± 1.73 84 ± 5.2 5.6 ± 1.4 1.18 ± 0.36 6.0 ± 3.1 5.3 ± 1.4 10.9 (3.8–35.8) 2.0 ± 1.2 115 ± 64.1 209 ± 67 1.3 (0.5–2.8) 30.6 ± 15.3 1.81 ± 0.45
24.35 ± 4.12 27.92 ± 4.89 88.07 ± 48.21 87 ± 3.1 13.6 ± 8.0 2.9 ± 1.83 10.5 ± 4.7 5.2 ± 1.3 12.4 (4.2–34.0) 1.8 ± 0.7 180 ± 34.4 210 ± 110 4.5 (2.4–7.8) 67.2 ± 25.5 2.25 ± 1.20
0.02 0.03 0.001 0.40 0.01 0.05 0.001 0.92 0.52 0.63 0.15 0.97 0.001 0.001 0.25
n ¼ number of patients, CG ¼ control group; PCOSG ¼ polycystic ovary syndrome group. Statistical tests applied: Student t-test for normally distributed variables (mean ± standard deviation) and Mann–Whitney test for non-Gaussian distributed variables (median and min–max). BMI: body mass index; PTWM: period of time without menstruation; GLU: glucose; HOMA-IR: homeostatic model assessment insulin resistance; LH: lutein hormone; FSH: follicle-stimulating hormone; PRL: prolactin; TSH: thyroid-stimulating hormone; 17OHP: 17 hydroxyprogesterone; DHEAS: dehydroepiandrosterone sulfate; A: androsterone; Ttotal: total testosterone; Tfree: free testosterone. Age, BMI, LMB, GLU, insulin, HOMA-IR, LH, FSH, 17OHP, DHEAS, TSH, Ttotal and Tfree exhibited homogeneous distributions. PRL and A exhibited heterogeneous distributions.
Table 2. Quantification of sulfate glycosaminoglycans in endometrium. Sulfate GAGs (mg/mg of dry tissue)
CS HS
CG (n ¼ 8)
PCOSG (n ¼ 10)
p Value
1.12 ± 0.22 0.17 ± 0.06
1.18 ± 0.38 0.25 ± 0.04
0.77 0.03
n ¼ number of patients, CG ¼ control group; PCOSG ¼ polycystic ovary syndrome group; GAGs: glycosaminoglycans; CS: chondroitin sulfate; HS: heparan sulfate. Student t test was employed for the normally distributed variables (mean ± standard deviation).
Results The clinical, biochemical and hormonal data from the patients in the CG and PCOSG are displayed in Table 1. Based on the histopathological examination of the endometrium in the PCOSG (n ¼ 14), 78.5% of the endometria were proliferative (n ¼ 11), 14.2% were secretory (n ¼ 2) and 7.1% were hyperplastic (n ¼ 1); in the CG (n ¼ 12), 83.3% were proliferative (n ¼ 10) and 16.7% were secretory (n ¼ 2). Only the proliferative endometrial samples were included for further analysis. The chondroitin sulfate (CS) and heparan sulfate (HS) levels were identified in the endometrium of the women in the CG and PCOSG. HS was significantly higher in the PCOSG patients than in the CG patients (p ¼ 0.03). There was no significant difference in CS between the PCOSG and CG (p ¼ 0.77). The results were expressed as mg of GAG/mg of dry tissue powder (Table 2). To assess the influence of the clinical data on the results from endometrial HS quantification, the Pearson correlation coefficient was calculated using the following variables, which were different in the two groups: age (r ¼ 0.52; p ¼ 0.01), period of time without menstruation (r ¼ 0.75; p ¼ 0.001), body mass index
(r ¼ 0.52; p ¼ 0.03) and basal insulin (r ¼ 0.42; p ¼ 0.08). A significant correlation was found between the endometrial HS level and age, period of time without menstruation and body mass index. No significant association was found for basal insulin (Figure 2). CS was the major GAG in both the PCOSG and CG, with more than 1 mg/mg of dry tissue in both groups. HS had the lowest concentration of all the GAGs analyzed, but it was higher in the PCOSG than in the CG.
Discussion The present study showed that the phenomena involving endometrial transformations in women with PCOS may be associated with changes in the levels of various glycosaminoglycans (GAGs), in particular with an increase in heparan sulfate. In fact, the endometrium is exposed to estrogen in the absence of progesterone counteraction. Women with PCOS exhibit ovulatory dysfunction and infertility. Ovulation may be restored with drugs or laparoscopic surgery. However, little attention has been placed on the endometrium, which is often unresponsive to classical treatments, leading to implantation failure and an increase in proliferative lesions [2,5,14]. Furthermore, little is known about GAGs in the endometrium of women with PCOS. This study is the first to quantify endometrial sulfated GAGs in women with PCOS. The molecular structure of GAGs allows them to interact with growth factors and other proteins that act as signals for cells to block, activate or direct cell migration through the ECM [10,15]. The ECM allows the exchange of nutrients; proliferation; and the transformation of factors, such as insulin growth factor (IGF) and fibroblast growth factor (FGF), between cells. The ECM may play a more active role in tissues [16], particularly the ECM elements that influence endometrial receptivity and embryo implantation, such as glycoproteins and cell adhesion molecules (integrin, selectin, fibronectin and laminin) [10,17]. In implantation physiology, CS and HS may be potential ligands for L-selectin/CD44 [18,19], which is important in the initial stages of trophoblast adhesion and in the promotion of integrin-dependent processes leading up to the maternal–fetal interface [20]. Studies admit higher levels of implantation failure in women with PCOS, even in IVF cycles [2,4]. Through complex mechanisms, women with PCOS exhibit endometrial dysfunction, which hinders their reproductive ability. Persistent estrogen stimulation to uterine tissue may lead to changes in endometrial gene expression and biochemical processes resulting in abnormal cell proliferation and differentiation [5,21]. It also may cause an increase in the production of HS on the endometrium in women with PCOS, as was demonstrated in the present study. An increased HS concentration may interfere with endometrial receptivity and/or embryo development, which can result in blastocyst adhesion dysfunction, implantation failure and miscarriages in women with PCOS. The presence of HS is very important on the cell surface, and research has shown that its structure is specific for different tissues and species. It has been acknowledged that HS acts as a cell recognition molecule and that it may increase the coupling of fibroblast growth factor (FGF) to its receptor [22]. In fact, the targeting of cells by FGF ligands and the subsequent complex formations that they form with their receptors are under positive control from the extracellular space by heparan sulfate proteoglycans (HSPGs). Signaling by FGF is crucial in multiple cellular processes, including cell proliferation, differentiation, survival and migration during mammalian implantation and embryogenesis [23]. Our results demonstrate that PCOS women have high levels of endometrial heparan sulfate. This phenomenon may be a cause of
DOI: 10.3109/09513590.2014.989980
Polycystic ovary syndrome
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Figure 2. The Pearson correlation coefficient. A significant and positive correlation was found between HS and PTWM (p ¼ 0.001) and BMI (p ¼ 0.03). A significant and negative correlation was found between HS and age (p ¼ 0.01). No significant association was found between HS and insulin (p ¼ 0.08). Abbreviations: HS: heparan sulfate; BMI: body mass index; PTWM: period of time without menstruation.
the interference with maternal–fetal recognition, which contributes to infertility, that is observed in women with PCOS.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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