High throughput, cell type-specific analysis of key proteins in human endometrial biopsies of women from fertile and infertile couples

Abstract

Background: Although histological dating of endometrial biopsies provides little help for prediction or diagnosis of infertility, analysis of individual endometrial proteins, proteomic profiling and transcriptome analysis have suggested several biomarkers with altered expression arising from intrinsic abnormalities, inadequate stimulation by or in response to gonadal steroids or altered function due to systemic disorders. The objective of this study was to delineate the developmental dynamics of potentially important proteins in the secretory phase of the menstrual cycle, utilizing a collection of endometrial biopsies from women of fertile (n = 89) and infertile (n = 89) couples.

Methods and results: Progesterone receptor-B (PGR-B), leukemia inhibitory factor, glycodelin/progestagen-associated endometrial protein (PAEP), homeobox A10, heparin-binding EGF-like growth factor, calcitonin and chemokine ligand 14 (CXCL14) were measured using a high-throughput, quantitative immunohistochemical method. Significant cyclic and tissue-specific regulation was documented for each protein, as well as their dysregulation in women of infertile couples. Infertile patients demonstrated a delay early in the secretory phase in the decline of PGR-B (P < 0.05) and premature mid-secretory increases in PAEP (P < 0.05) and CXCL14 (P < 0.05), suggesting that the implantation interval could be closing early. Correlation analysis identified potential interactions among certain proteins that were disrupted by infertility.

Conclusions: This approach overcomes the limitations of a small sample number. Protein expression and localization provided important insights into the potential roles of these proteins in normal and pathological development of the endometrium that is not attainable from transcriptome analysis, establishing a basis for biomarker, diagnostic and targeted drug development for women with infertility.

Figure 1

Localization of PGR-B and LIF in endometrial biopsies of women from fertile and infertile couples as assessed by IHC. (A–D) Representative images of PGR-B in fertile controls (A, B) and in women from infertile couples (C, D) during the early (A, C) and mid-secretory (B, D) phases. (E–J) LIF in fertile controls (E–G) and in women from infertile couples (H–J) during the early (E, H), mid (F, I) and late (G, J) secretory phases. Images were not counterstained. Glands (GE), stroma (STR) and luminal epithelium (LE) are indicated in each image. Magnification, ×200.

Figure 2

IHC localization of PAEP, HOXA10, CALCA, HBEGF and CXCL14 in endometrial biopsies of women from fertile and infertile couples. Representative images of (A) PAEP, (B) HOXA10, (C) CALCA, (D) HBEGF and (E) CXCL14. Pictures F and G represent control staining where a primary antibody for LIF or HBEGF was excluded. Magnification, ×200.

Figure 3

Quantitative IHC of PGR-B, LIF, PAEP and HOXA10 in endometrial biopsies of women from fertile and infertile couples. The mean gray levels (arbitrary units; y-axis) were determined for PGR-B (A; fertile, n = 89; infertile, n = 86), LIF (B; fertile, n = 87; infertile, n = 86), PAEP (C; fertile, n = 89; infertile, n = 89) and HOXA10 (D; fertile, n = 86; infertile, n = 87) in each tissue type, as described in the section Materials and Methods, showing temporal changes in each cell type (lines), and cell-type differences (bars) during the ESP (n = 57), MSP (n = 80) and LSP (n = 41). Fertile controls are represented by a solid line (triangles) or open bar, and samples from women of infertile couples by a dashed line (squares) or solid bar. *P < 0.05 for temporal changes (lines) according to Student–Newman–Keuls post hoc comparisons, with indication of changes specific to the fertile controls (fer), infertile couples (inf) or both (both) groups; cell-type-specific differences (bars), or differences with fertility status (bars) according to Student's t-test. Error bars indicate the SD.

Figure 4

Analysis of PGR-B levels in endometrial biopsies of women from fertile and infertile couples at different stages of the cycle. Mean gray levels were grouped according to histological dating. Tissue type-specific data are shown for LE (A), GE (B) and STR (C), and for fertile controls (open bars) and women of infertile couples (solid bars). The number of patients in each group is indicated above each bar in the first panel. The decrease in expression seen in fertile controls during the early secretory phase appears to be delayed in women of infertile couples. Error bars indicate the SD.

Figure 5

Quantitative IHC of HBEGF, CALCA and CXCL14 in endometrial biopsies of women from fertile and infertile couples. The mean gray levels (arbitrary units; y-axis) were determined for HBEGF (A; fertile, n = 86; infertile, n = 87), CALCA (B; fertile, n= 82; infertile, n = 83) and CXCL14 (C; fertile, n = 82; infertile, n = 83), and are displayed as in Fig. 3. Error bars indicate the SD.