Expression Patterns of Progesterone Receptor Membrane Components 1 and 2 in Endometria From Women With and Without Endometriosis

Abstract

Endometriosis is a hormone-dependent inflammatory condition associated with pain and infertility. A growing body of evidence supports attenuated secretory-phase progesterone responsiveness in women with this disease. Herein, we compare the expression of progesterone receptor membrane components (PGRMC) 1 and 2 in eutopic endometrium from 11 women with laparoscopically and/or histologically proven stage III/IV endometriosis and 23 disease-free women. Menstrual cycle phase was determined using a combination of reported cycle day, serum hormone profile, and endometrial histologic dating. The PGRMC-1 (fold change −3.3; P < .05) and PGRMC-2 (fold-change −8.8; P < .05) gene expression were significantly downregulated in secretory phase, eutopic endometrium from women with endometriosis. Immunohistochemistry demonstrated decreased PGRMC-1 and PGRMC-2 protein expression in the secretory phase endometrial stroma cells of women with endometriosis. Consistent with the preclinical work of others, our results reflect downregulation of endometrial PGRMC-1 and PGRMC-2 expression in secretory phase endometrium from women with advanced stage endometriosis. Understanding the molecular mechanisms of attenuated progesterone action in endometriosis has important diagnostic and therapeutic implications.

Keywords

progesterone receptor membrane component 1 progesterone receptor membrane component 2 PGRMC-1 PGRMC-2 endometriosis progesterone

Introduction

Endometriosis is an estrogen-dependent disorder associated with infertility and pelvic pain. Endometriosis affects up to 10% of the general female population and is currently diagnosed by direct and/or microscopic observation of ectopic endometrial tissue outside the uterine cavity.¹ Several theories offer explanations of the pathophysiology, with a leading theory positing that retrograde menstruation leads to ectopic tissue implantation and growth in women with a biological predisposition.² The biology of clinically significant endometriosis remains unclear, but it is likely dependent on a combination of circulating hormones and other factors. Although the estrodiol (E2) dependence of endometriosis is well documented, recent studies support progesterone (P) resistance in the pathophysiology of the disease.^2–4

The P resistance implies a decreased responsiveness of target tissue to bioavailable hormone.² Studies in women with endometriosis revealed attenuated P action in the endometrium from women with endometriosis.^2,5 Although P resistance could be explained by decreased P production, recent data indicate that a decrease in the ratio of P receptor (PR) PR-B and PR-A isoforms promotes P resistance in endometriosis implants.⁶

Classically, sex steroid hormones bind with high affinity to specific cytoplasmic and nuclear hormone response elements and alter cellular metabolism through changes in gene transcription, which can take hours or days for their biological effects to be fully realized.^7,8 However, studies in mice lacking classical P receptors demonstrate that not all actions of P are mediated through cytoplasmic and nuclear receptors.⁹ Rather than a single steroid hormone pathway, the effects of sex steroids may be mediated through other novel pathways, such as PR membrane components (PGRMCs), PGRMC-1 and PGRMC-2.^10–12

Both PGRMC-1 and PGRMC-2 are small adapter-like proteins thought to mediate fast-acting membrane-associated nongenomic P signaling. Although recent evidence suggests these receptor components do not bind P directly, they do bind cholesterol and may act as regulators of sterol synthesis.¹³ In murine endometrium, Pgrmc-2 is upregulated during the P-dominant metestrus phase of the estrous cycle.¹⁴ In human endometrium, PGRMC-1 expression decreases in the P-dominant secretory endometrium.^15,16 And in nonhuman primates, PGRMC-2 expression is significantly decreased in animals with advanced endometriosis.¹⁷

The PGRMC-1 protein is overexpressed in several cancers,¹⁸ suggesting that these receptors are active during rapid growth and proliferation. Conversely, PGRMC proteins enhance ligand-dependent apoptosis in normal granulosa and luteal cells exposed to physiologic levels of circulating sex steroids,¹⁹ and 1 study has correlated expression and phosphorylation of PGRMC-1 to estrogen receptor (ER) expression.²⁰ Thus, PGRMC-1 expression may be linked to both E2- and P-mediated signaling and might be particularly important during the E2-/P-driven menstrual cycle or the growth of ectopic endometrial implants. Although less is known about PGRMC-2, sequence homology studies suggests that, like PGRMC-1, PGRMC-2 should have trophic effects on steroid-sensitive tissues²¹; indeed, recent work in our laboratory has demonstrated that PGRMC-2 transcript positively correlated with ER-α transcript in patients with ER-positive breast cancer.²²

Given the compelling evidence implicating attenuated P response in endometriosis, we measured the differential expression of PGRMC-1 and PGRMC-2 in endometrial tissue from women with and without endometriosis. We hypothesized that there is reduced expression of novel P membrane complexes in women with endometriosis. Indeed, our results demonstrate reduced expression of PGRMC-1 and PGRMC-2 in secretory phase in women with advanced stage endometriosis, which may contribute to the phenomena of P resistance in the disease.

Materials and Methods

Study Population

This retrospective study involved 34 endometrial samples (11 with and 23 without endometriosis) procured from the Madigan Healthcare System ENDOPATH tissue bank. All women gave informed consent prior to enrolling in the study that was approved by the Madigan Healthcare System Institutional Review Board.

Participants were premenopausal women between 25 and 40 years with regular menstrual cycles undergoing laparoscopy for infertility and/or pelvic pain (see Table 1). Women with pelvic inflammatory disease or hydrosalpinx, recent abdominal surgery, or hysterosalpingography in the 3 months prior to laparoscopy were excluded from the study. Additional exclusion criteria included uterine leiomyomata, hormone therapy, being pregnant or breast-feeding, and intrauterine device use 3 months prior to laparoscopy. Although all women underwent laparoscopy for infertility and/or pelvic pain, for the purposes of this study, only women with no prior history of endometriosis and no detectable endometriotic lesions at the time of laparoscopy were considered control.

Table 1.

Patient Characteristics.^a

Patient ID	Age	Ethnicity	Endo Present	Cycle Phase	E2 (pg/mL)	P4 (ng/mL)
3	35	A	N	M	20	1.14
23	29	C	N	M	22	0.27
26	30	AA	N	M	20	1.27
16	27	C	Y	M	20	1.18
17	28	C	Y	M	28	0.42
45	28	C	Y	M	56	0.59
4	38	C	N	P	178	1.21
9	37	C	N	P	72	0.69
29	29	C	N	P	142	1.42
47	28	C	N	P	103	0.72
48	31	C	N	P	78	2.91
49	25	C	N	P	NT	NT
52	31	C	N	P	147	0.62
5	36	C	Y	P	335	0.89
10	39	C	Y	P	119	0.54
41	40	A	Y	P	NT	NT
53	35	A	Y	P	128	1.63
13	33	AA	N	S	50	2.02
19	29	C	N	S	101	12.81
20	31	C	N	S	353	1.25
21	28	C	N	S	37	1.43
24	31	AA	N	S	41	6.63
27	29	C	N	S	36	2.31
30	32	C	N	S	53	1.96
34	33	C	N	S	86	2.83
35	30	C	N	S	393	5.22
36	35	AA	N	S	114	11.22
38	36	C	N	S	136	9.34
42	37	C	N	S	139	11.36
50	35	C	N	S	181	12.75
12	28	A	Y	S	54	3.72
18	27	C	Y	S	23	2.69
32	32	C	Y	S	388	12.6
44	30	A	Y	S	67	4.78

Abbreviations: A, Asian; AA, African-American; C, Caucasian; N, no; Y, yes; M, menstrual; P, proliferative, S, secretory; E2, estradiol; P4, progesterone; NT, not tested.

^a Serum hormone levels were obtained on the day of surgery.

The revised staging system designed by the former American Fertility Society (rAFS—now the American Society for Reproductive Medicine) was used to stage patients with endometriosis. Laparoscopic peritoneal biopsies taken from all suspicious lesions were fixed in formalin and processed using standard histologic methods. All specimens were examined by pathologists experienced in evaluating endometrial and peritoneal biopsies.

Tissue Specimens

Endometrial pipelle biopsies were collected from patients at the time of laparoscopic surgery. Menstrual cycle phase was determined by a combination of cycle history, endometrial dating according to the criteria of Noyes,²³ and serum hormonal profile (E2 and P). A portion of the fresh endometrial biopsy was preserved in RNAlater (Ambion, Grand Island, New York), snap frozen, and stored at −70°C, and another portion was preserved in formalin for histology. Frozen specimens were homogenized in ice-cold guanidine thiocyanate buffer (RLT) buffer (RNeasy kit) using a dounce and pestle. Gel and spectral analysis of 28S and 18S RNA confirmed that quality intact RNA was (10-50 μg total RNA per extraction) used for polymerase chain reaction (PCR) experiments (data not shown).

Polymerase Chain Reaction

As described previously, the following primers and amplicons were used to amplify transcripts of PGRMC-1 and PGRMC-2: PGRMC-1: forward 5’-tgacctttctgacctcactgc-3’, reverse 5’-gcccacgtgatgatacttga-3’, probe #3, 85 nucleotide amplicon; PGRMC-2: forward 5’-tcgagaatgggaaatgcag -3’, reverse 5’-ttgtgatccttggtatcttcttca-3’, probe #1, 111 nucleotide amplicon.²² Neither PGRMC-1 nor PGRMC-2 is known to have alternative splicing products. Further, a search by gene name using the Web site for Alternative Splicing Prediction was conducted.²⁴ Neither PGRMC-1 nor PGRMC-2 was found to harbor exons with known splicing alternative, nor was any de novo predictions determined from sequence data.

Transcript levels were determined by relative quantitative PCR (RelqPCR) with the 96-well Roche LightCycler 480 platform and the 1-shot method LightCycler 480 RNA Master Hydrolysis kit according to manufacturer’s instructions (Roche Pharmaceuticals, Mannheim, Germany). Reagent requirements of the RelqPCR experiments included 100 ng total RNA template and a final concentration of 0.75 μmol/L primers and 0.05 μmol/L hydrolysis probe. Thermocycling parameters included 10-minute reverse transcription at 63°C; 30-second denaturation at 95°C; 45 cycles of 10-second denaturation at 95°C, 30-second anneal at 57°C, 10-second extension at 72°C; and a final cooling step at 40°C. The use of intron spanning primers and a real-time analysis of product avoids nonspecific by-products typically associated with product degradation and/or high cycles. As anticipated, the housekeeping gene phosphoglycerate kinase 1 (PGK1) was consistently expressed in all tested endometrial specimens culled from the ENDOPATH biobank, allowing us to normalize PGRMC transcripts to PGK1 transcripts and quantify PGRMC gene expression. Amplified transcripts were separated on a 1% agarose gel (Invitrogen, Grand Island, New York) to ensure that a single amplicon was produced during each assay.

Histology and Immunohistochemistry

Formalin fixed samples were placed into cassettes for processing and paraffin embedding. Sections of 5 µm were cut from each paraffin block, placed on coated glass slides for hematoxylin and eosin staining, and coverslipped. The PGRMC-1 and PGRMC-2 immunohistochemistry (IHC) was performed on sections mounted on charged glass slides that were air-dried and heated to 60°C for 20 minutes. After cooling, the slides were deparaffinized in 3 changes of xylene (3 minutes each), 2 changes of absolute ethanol (30 seconds each), 1 change of 95% ethanol (30 seconds), and washed for 1.5 minutes under running tap water. Washed slides were stored in Tris-buffered saline (TBS) prior to antigen retrieval.

Antigen retrieval was enhanced by warming the slides in a 97°C to 99°C water bath for 24 minutes. Afterward, the slides were cooled to 85°C and returned to TBS buffer for 5 minutes. Primary and secondary antibody incubations were performed with a Dako autostainer (Dako North America, Carpinteria, California). The slides were incubated with primary antibody (PGRMC-1 and 2—1:100 dilution) at room temperature for 30 minutes. Afterward, the slides were rinsed with TBS and incubated for 5 minutes in 3% hydrogen peroxide to quench endogenous peroxidase activity. Another TBS buffer wash removed residual hydrogen peroxide before the final 30 minutes secondary antibody incubation, which used labeled polymer horseradish peroxidase (Dako). Sections were exposed to excess diaminobenzidine for 5 minutes and rinsed in distilled water. The slides were counterstained with Mayer hematoxylin (Biogenex, Freemont, California) for 1.5 minutes, rinsed under running water, dehydrated in graded alcohols and xylenes, and coverslipped.

Semiquantitative PGRMC protein expression was scored using a modified method described by Klein et al.²⁵ For each specimen, several random ×60 fields were imaged and independently scored by 3 blinded investigators. In each random field, the area fraction of labeled cells (ALC; 0 = none, 1 = <30%, 2 = 30%-60%, 3 ≥ 60%), and the immunostaining intensity (ITI_sq; 0 = none, 1 = weak, 2 = mild, 3 = strong) were determined, allowing calculation of an immunostain score (EI_sq), EI_sq = ALC*ITI_sq, and the mean EI_sq was calculated for both the stromal and the glandular compartments. The EI_sq was compared across cycle phases in specimens from women with or without endometriosis.

Statistical Analysis

Statistical calculations were performed by Roche proprietary software and SPSS (Roche Pharmaceuticals, Mannheim, Germany; SPSS Inc, Chicago, Illinois). Relative messenger RNA (mRNA) expression was calculated as the ratio of the average relative copy number of PGRMC-1 (or PGRMC-2) to PGK1. Relative copy number was computed as the average of the replicate crossing point ratios (ie, PGRMC-1: PGK1; ∑(0.5_CP)/n: ∑(0.5_CP)/n; cp = crossing point). Relative sample expression levels were averaged and shown graphically to compare with the means of the endometriosis samples and controls. The control group consisted of 23 control samples (13 proliferative, 7 secretory, and 3 menstrual), and the experimental group consisted of 11 stage III to IV endometriosis samples (4 proliferative, 4 secretory, and 3 menstrual). Mann-Whitney U nonparametric analysis (PASW; SPSS Inc, Chicago, Illinois) was used to compare means across groups. EI_sq was calculated in Excel.

Results

A total of 34 endometrial pipelle biopsy specimens from women with (n = 11) or without (n = 23) endometriosis were included in this study (see Table 1 for patient demographics). Since hormonal influences on PGRMC gene expression may vary across the menstrual cycle, the cycle phase of each specimen was carefully determined by menstrual history, histologic dating, and serum hormone profile (serum E2 and P) on the day of specimen collection. Patients with laparoscopically identified rAFS stages III and IV endometriosis were included in the study group, and 73% (8 of 11) of these patients had histologically confirmed endometriosis. There were no significant differences between the 2 groups with respect to age, ethnicity, body mass index, or E2 and P levels on the day of specimen collection (data not shown).

As expected, mean serum E2 levels were higher in proliferative and secretory phases than the menstrual phase (Figure 1A). Furthermore, the mean serum P was 5.28-fold higher in secretory phase relative to the proliferative phase specimens (Figure 1B). However, there were no significant differences in circulating E2 or P levels in women with or without endometriosis in any of the phases. When phase comparisons of PGRMC-1 and PGRMC-2 mRNA expression were made, we found (Figure 1C and D) significant decreases in both PGRMC receptor expression in secretory phase endometria taken from women with endometriosis (−3.3-fold, P = .024 and −8.8-fold; P = .027, respectively) compared to their control counterparts. In contrast, menstrual phase patients with endometriosis had a 5.5-fold increase in PGRMC-1 expression, although PGRMC-2 expression levels were found to be similar between the control and the endometriosis groups.

To determine whether PGRMC-1 and PGRMC-2 transcript levels correlated with expressed proteins, we examined PGRMC-1 and PGRMC-2 protein expression in endometrial biopsy tissue sections using tissue immunohistochemistry (Figure 2A-F). In control tissues, PGRMC-1 (Figure 2A) and PGRMC-2 (Figure 2C) proteins were expressed by endometrial glands and stroma (black arrows indicate PGRMC staining); stromal cell protein expression was predominantly membranous but also in the cytoplasm. The PGRMC expression was quite variable in glandular cells, making generalizations about subcellular protein localization unreliable.

Qualitatively, PGRMC-1 and PGRMC-2 protein expression in secretory phase stromal cells correlated with PGRMC-1 and PGRMC-2 transcript levels measured in the same tissue. Compared to controls, however, relatively lower stromal cell PGRMC-1 (Figure 2B) and PGRMC-2 (Figure 2D) protein expression was observed in endometrial tissue from women with endometriosis. Taken together, these IHC results support our RelqPCR data, showing that PGRMC-1 and PGRMC-2 are decreased predominately in stromal cells in secretory phases of the menstrual cycle in women with endometriosis. Expression was visually evaluated and scored (see methods, Figure 2E and F). Expression levels of both PGRMC-1 and PGMRC-2 were decreased in secretory phase in women with endometriosis.

Discussion

The endometrium is a steroid responsive tissue continuously undergoing cyclic proliferation, differentiation, and sloughing in anticipation of pregnancy. Endometrial growth and development are a consequence of sequential actions of E2 and P on the endometrium, growth factors involved in cellular metabolism, and other cellular processes. Abnormal development of the endometrium or imbalances in hormonal actions between E2 and P can lead to infertility and presumably to gynecological disorders such as endometriosis.

Progesterone is a key hormone in the transition from the proliferative to secretory phase in the normal endometrium, and aberrant responsiveness to the circulating hormone could play a central role in the pathogenesis of forming endometriotic implants. Comparative gene expression analysis of P-regulated genes in the endometria of women with endometriosis demonstrates persistent patterns of gene expression that more closely mimic the pattern seen in proliferative endometrium.^5,26–28 Our results extend these findings by showing attenuated gene expression and protein production of the nonclassical P receptors, PGRMC-1 and PGRMC-2, in women with advanced stage endometriosis.

In human eutopic endometriual stromal fibroblasts cultured from women with endometriosis, however, Aghajanova and colleagues demonstrate dysregulation of select steroidogenic enzymes, which is explained through a blunted cyclic adenosine monophosphate mechanism rather than P4 resistance.²⁹ Dysregulation of steroidogenic enzymes are thought to shift the status to an E2-enriched endometrial milieu that influences proliferation and survival of endometrial cells.²⁹ Aghajanova’s results are consistent with previous studies that demonstrate a relative deficiency of the 17β hydroxysteroid dehydrogenase type II (17β-HSD)enzyme during the secretory phase, which could help to stabilize endometriotic implants by maintaining elevated levels of E2,^30,31 however, counter to the P resistance theory. Importantly, P is known to stimulate conversion of biologically active E2 to the less active estrone via 17β-HSD. Thus, it is conceivable that both P resistance and dysregulation of steroidogenic enzymes contribute to the same phenomenology, an E2-enriched endometrial milieu.

Sex steroids play important roles in regulating diverse cellular pathways, most notably to include gene expression.³² Our results demonstrate that changing E2 levels coincided with changes in PGRMC-1 gene expression in endometrial tissue taken from women without endometriosis. Although our sample number was limited and there is a significant potential for contamination by products from the blood in this phase, we measured a 5.5-fold increase in PGRMC-1 gene expression in menstruating women with endometriosis that appears to be independent of the levels of circulating sex hormones. In view of the retrograde menstruation pathogenesis theory, gene and/or protein changes that are independent of circulating sex steroids in the menstrual phase may be particularly relevant to imparting a selective survival advantage to ectopic endometrial tissue in women predisposed to endometriosis.

Stromal and glandular cells proliferate and differentiate throughout the menstral cycle, mainly under the control of oviarian steroid hormones. The stromal cells, however, play a critical role as the receptors for E2 and P. Women with endometriosis have much lower endometrial PGRMC-1 and PGRMC-2 mRNA levels than controls, which becomes statistically significant in the secretory phase. Our results are consistent with a recent study showing decreased PGRMC-2 transcript and protein expression in macaques with advanced endometriosis¹⁷; however, the decrease in expression was primarily in glandular tissue. The relative downregulation of these fast-acting PGRMC-1 and PGRMC-2 receptors in secretory phase endometrium may contribute to an attenuated P response in women with endometriosis, thereby altering appropriate proliferation and differentiation. Further research, however, is necessary to determine the functional role of these receptors in the normal and diseased endometrium.

The results of our study contribute to the burgeoning understanding of a complex disease with a multifactorial pathogenesis. Given that hormonal signals appear to be an important aspect of endometriosis, hormone receptors become attractive therapeutic targets. Ultimately, identification of molecular pathways involved in hormonal regulation of the endometrium is an essential step toward developing diagnostic tests and targeted biological therapies for this debilitating gynecologic disorder.

Footnotes

Acknowledgments

We would like to thank the physicians and surgeons who assisted with specimen collection in the assembly of the ENDOPATH endometriosis biobank.

Authors’Note

The views expressed are those of the authors and do not reflect the official policy of the Department of the Army, the Department of Defense or the US Government. This paper has been submitted solely to this journal and has not been previously published in any form in another publication of any type.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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