Women with polycystic ovary syndrome present with altered endometrial expression of stanniocalcin-1†

Abstract

Stanniocalcin-1 (STC-1) is a pro-survival factor that protects tissues against stressors, such as hypoxia and inflammation. STC-1 is co-expressed with the endometrial receptivity markers, and recently endometrial STC-1 was reported to be dysregulated in endometriosis, a condition linked with endometrial progesterone resistance and inflammation. These features are also common in the endometrium in women with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. Given that women with PCOS present with subfertility, pregnancy complications, and increased risk for endometrial cancer, we investigated endometrial STC-1 expression in affected women. Endometrial biopsy samples were obtained from women with PCOS and controls, including samples from overweight/obese women with PCOS before and after a 3-month lifestyle intervention. A total of 98 PCOS and 85 control samples were used in immunohistochemistry, reverse-transcription polymerase chain reaction, or in vitro cell culture. STC-1 expression was analyzed at different cycle phases and in endometrial stromal cells (eSCs) after steroid hormone exposure. The eSCs were also challenged with 8-bromo-cAMP and hypoxia for STC-1 expression. The findings indicate that STC-1 expression is not steroid hormone mediated although secretory-phase STC-1 expression was blunted in PCOS. Lower expression seems to be related to attenuated STC-1 response to stressors in PCOS eSCs, shown as downregulation of protein kinase A activity. The 3-month lifestyle intervention did not restore STC-1 expression in PCOS endometrium. More studies are warranted to further elucidate the mechanisms behind the altered endometrial STC-1 expression and rescue mechanism in the PCOS endometrium.

Keywords: endometrial stromal cells; human endometrium; hypoxia; lifestyle intervention; polycystic ovary syndrome; primary cell culture; stanniocalcin-1; stress.

Figure 1

Endometrial tissue samples used in each experiment. The samples obtained from the Karolinska Institutet (n_total = 50) comprised 21 samples from controls and 29 samples from women with PCOS. These samples were obtained during cd 6–8 and 21–23. Fourteen of the PCOS samples were obtained before and after 3-month lifestyle intervention. Samples obtained from Oulu University Hospital were taken at the various cd of the menstrual cycle based on LH testing (n_total = 133). There were 64 control samples and 69 from women with PCOS.

Figure 2

Expression of STC-1 in human endometrium. (A) Quantitative reverse-transcription polymerase chain reaction analysis of endometrial samples from healthy control women (gray bar) and women with PCOS (black bar) collected at cd 7–10 (n_ctrl = 7, n_PCOS = 7), LH + 2–4 (n_ctrl = 5, n_PCOS = 5), LH + 7 (n_ctrl = 7, n_PCOS = 7), and LH + 10–12 (n_ctrl = 5, n_PCOS = 7) revealed a significant increase in endometrial STC-1 gene expression from the PE (cd 7–10) toward the LSE (LH + 10–12) in both control samples (P = 0.003) and samples from women with PCOS (P = 0.001). Data are presented as mean ± standard error of the mean (SEM). (B) Semi-quantitative immunohistochemistry data on STC-1 protein expression in different endometrial cell compartments (stromal cells (stroma), luminal epithelium (LE), and GE) from control samples assessed at cd 8–10 (n = 6), LH + 2–4 (n = 7), LH + 7–9 (n = 6), and LH + 10–12 (n = 7) revealed the highest expression in the LSE (LH + 10–12) compared with the PE (cd 8–10; P = 0.022) and MSE (LH + 7–9; P = 0.001) in stromal cells, whereas (C) there was no significant difference between the cycle phases in cells from women with PCOS. (D) STC-1 expression in different cycle phases studied by immunohistochemistry in control women (a–h) and in women with PCOS (i–l). (a) PE (cd 8–10); (b) ESE (LH + 2–4); (c) MSE (LH + 7–9); (d) LSE (LH + 10–12). Higher magnification images from control women (40×) of GE shows that in PE, STC-1 is localized to the GE cells (e), and in ESE it is concentrated in the apical parts (f) from which it is gradually secreted into the lumen during the MSE (g) and LSE (h). The stromal STC-1 staining was weak in PE (m) and significantly stronger in LSE (n), but in PCOS, the staining intensity was similar across the cycle phases. (o) LSE stroma of a PCOS case; (p) negative control staining.

Figure 3

Cyclic expression of STC-1 in women with BMI ≤ 27 kg/m² (N) and BMI > 27 kg/m² (O) in controls and in women with PCOS. (A) STC-1 expression during cd 6–8 and 21–23 in controls with BMI ≤ 27 kg/m² (N-Ctrl, n = 11 and n = 7, respectively) and in women with PCOS and BMI ≤ 27 kg/m² (N-PCOS, n = 5 and n = 5, respectively). The control women presented with elevated STC-1 expression at cd 21–23 compared with cd 6–8 (P = 0.044), whereas there was no significant increase in STC-1 expression toward cd 21–23 in women with PCOS. (B) STC-1 expression at cd 6–8 and 21–23 in controls with BMI > 27 kg/m² (O-Ctrl, n = 5 and n = 5, respectively) and in women with PCOS and BMI > 27 kg/m² (O-PCOS, n = 8 and n = 15, respectively). Control women presented with elevated STC-1 expression at cd 21–23 compared with cd 6–8 (P = 0.001), but in women with PCOS the effect was again blunted.

Figure 4

(A) STC-1 mRNA expression, (B) protein expression, and (C) PKA activity analysis in response to 8-Br-cAMP challenge in cultured eSCs obtained from control women and from women with PCOS. (A) Expression of STC-1 mRNA in stromal cells from control women (n = 10, gray bars) and women with PCOS (n = 12, black bars) after treatment with 0.5 mM 8-Br-cAMP for 96 h. STC-1 expression was blunted in the stromal cells obtained from women with PCOS (P = 0.001) compared with the controls. (B) Similarly, STC-1 protein secretion from stromal cells into culture media after 96 h of 0.5 mM 8-Br-cAMP administration was lower (P = 0.01) in women with PCOS (n = 12, black bars) compared with controls (n = 10, gray bars). Basal STC-1 secretion, however, did not differ between the study groups. (C) PKA activity was significantly higher in stromal cells from control women (n = 4, gray bars) compared to women with PCOS (n = 4, black bars; P = 0.006) after 96 h of 0.5 mM 8-Br-cAMP treatment. Interestingly, stromal cells from control women also presented with higher basal PKA activity than the stromal cells from women with PCOS (P = 0.001). Data are shown as mean values ± SEM.

Figure 5

Expression of (A) STC-1 mRNA and (B) protein in cultured eSCs during hypoxia challenge (1% oxygen). (A) STC-1 mRNA expression of eSCs obtained from control women (n = 12, gray bars) and from women with PCOS (n = 13, black bars) after 24 hand 48 h of hypoxic challenge. The stromal cells from women with PCOS showed lower STC-1 mRNA expression at both 24 h (P < 0.0001) and 48 h (P = 0.001). (B) STC-1 protein secretion into eSC culture supernatant under normoxic (24 h) and hypoxic (24 and 48 h) conditions in cells from healthy controls (n = 12, gray bars) and from women with PCOS (n = 13, black bars). No significant changes were observed between the study groups under normoxic or hypoxic conditions after 24 h. However, at 48 h, the STC-1 response to hypoxia was blunted in stromal cells obtained from women with PCOS compared with the cells from controls (P = 0.001). Data are shown as mean ± SEM.

Figure 6

Effects of weight and lifestyle intervention on endometrial STC-1 expression. (A) Endometrial STC-1 mRNA expression at cd 21–23 in control women with BMI ≤ 27 kg/m² (N-Ctrl, n = 11) and in controls with BMI > 27 kg/m² (O-Ctrl, n = 5), showing increased endometrial STC-1 expression in O-Ctrl women (P = 0.047). No significant difference in STC-1 mRNA expression was found between the two weight groups of women with PCOS (N-PCOS, n = 5 vs O-PCOS, n = 15). (B) Relative expression of STC-1 on cd 21–23 in women with PCOS and BMI > 27 kg/m² before (O-PCOS, before intervention, n = 14) and after (O-PCOS, after intervention, n = 14) 3-month lifestyle intervention. Lifestyle intervention failed to restore endometrial STC-1 expression. All values are means ± SEM.