The endometrium is a dynamic tissue that undergoes morphological and biochemical changes during the menstrual cycle. Human endometrium contains a variety of cell types: surface and glandular epithelium associated with extracellular matrix and stromal components, including vascular and neuronal elements. The endometrium is a target tissue for hormones. The proliferative and secretory changes are primarily controlled by ovarian steroid hormones, estrogen and progesterone. Estrogen produced in the endometrial follicular phase initiates proliferation of endometrial epithelium, whereas secretory progesterone modulates endometrial transformation to create a suitable environment for blastocyst implantation.

The effects of these hormones on human endometrium are considered to be mediated by estrogen (ER) and progesterone receptors (PR). These receptors are involved in autocrine and paracrine regulation processes that respond to estrogen and progesterone. In a number of studies, expression of both receptors have been analysed in paraffin-fixed human endometrium and isolated glandular epithelial cells using immunohistochemistry.

Until recently, it was generally accepted that only one ER existed. However, the discovery of a new nuclear receptor with specificity for estrogens has provided new insights in the estrogen signalling system. The novel receptor ER beta (ER?) is highly homologous to the classical ER (ER alpha (ER)), which bind estradiol with high affinity and stimulate transcription of ER target genes. Expression of ER? has been primarily investigated in animal species, including female reproductive tissues of rat, mouse and pig. So far, expression of ER? has only been investigated at the mRNA level. Immunohistochemical detection in human endometrium was always difficult due to lack of specific antibodies against human ER?. Recently, immunohistochemical localization of the expression of ER? in human endometrium has been demonstrated using a polyclonal antibody.

ER expression and its distribution patterns is important for the understanding of normal endometrial function and its pathogenesis. Expression patterns of steroid receptors may have essential clinical implications. Furthermore, synchronization of expression of ER? in relation with ER expression may be disrupted in neoplastic endometrium, and thus playing an important role in endometrial pathogenesis. Therefore, the aims of the present study were the analysis of distribution patterns of ER, ER? and PR using monoclonal antibodies that have recently become commercially available and the assessment of relationships between receptor status and phases of endometrial specimens analysed.

Samples of human endometrium were obtained from 17 premenopausal, non-pregnant patients undergoing gynaecological surgery either by dilatation and curettage (D&C) or hysterectomy for benign diseases. All women had a normal and regular menstrual cycle with no hormonal treatment in 3 months prior to surgery. All pathological endometria were excluded from this study. Endometrium samples were classified on the basis of anamnestical and histological data into proliferative (day 1–14, n=7), early secretory (day 15–22, n=5) and late secretory phase (day 23–28, n=5) as described previously.

Immunohistochemistry was performed using a combination of microwave oven heating and the standard streptavidin–biotin–peroxidase complex using the anti-mouse-IgG-Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). Mouse monoclonal antibodies as were used for the experiments are specified in Table 1. For positive controls, sections of human breast cancer tissue and normal colon were used, whereas human ileum served as negative control tissue.

ER, estrogen receptor; PR, progesterone receptor.

Briefly, paraffin-fixed tissue sections were dewaxed using xylol for 15 min, rehydrated in ascending series of alcohol (70%, 96% and 100%), and subjected to antigen retrieval for 10 min in a pressure cooker using sodium citrate buffer (pH 6.0), containing 0.1 M citric acid and 0.1 M sodium citrate in distillated water. After cooling, sections were washed twice in phosphate-buffered saline (PBS). Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide (Merck, Darmstadt, Germany) in methanol for 20 min. Non-specific binding of the primary antibodies was blocked by incubating the sections with “diluted normal serum” (10 ml PBS containing 150 ?l horse serum; provided by Vector Laboratories) for 20 min at room temperature. Sections were then incubated at room temperature for 60 min with the primary antibodies. Anti-ER and anti-ER? antibodies were diluted in “dilution medium” (Dako, Glostrup, Denmark), whereas anti-PR antibodies were diluted in PBS. After washing with PBS, sections were incubated in “diluted biotinylated serum” (10 ml PBS containing 50 ?l horse serum; provided by Vector Laboratories) for 30 min at room temp. After incubation with the avidin–biotin–peroxidase complex (diluted in 10 ml PBS; Vector Laboratories) for 30 min and repeated washing steps with PBS, visualization was performed with substrate and chromagen 3,3?-diaminobenzidine (DAB; Dako) for 8–10 min. Sections were counterstained with Mayer’s acidic hematoxylin and dehydrated in an ascending series of alcohol (50–98%). After xylol treatment, sections were covered. Negative controls were performed by replacing the primary antibody with normal horse serum in the same dilution. Positive cells showed a brownish colour and negative controls as well as unstained cells were blue.

The intensity and distribution patterns of the specific immunohistochemical staining was evaluated using a semi-quantitative method (IRS score) as was previously described and was used in the evaluation of endometrial steroid receptor expression and cathepsin D and CA-125 expression in human endometrium. Sections were examined by two independent observers. The IRS score was calculated as follows: IRS=SI×PP, where SI is the optical staining intensity (graded as 0, no staining; 1, weak staining; 2, moderate staining and 3, strong staining) and PP the percentage of positively stained cells. The PP was estimated by counting approx. 200 cells and it was defined as 0, no staining; 1, <10% staining; 2, 11–50% staining; 3, 51–80% staining; and 4, >81% staining. The Mann–Whitney rank-sum test was used to compare the means of the different IRS scores. The Spearman factor and linear regression analysis (SPSS; Chicago, IL, USA) were used to assess the relationship between steroid receptor expression and phases of the endometrial samples. Significance of differences was assumed at p0.05.

Staining of ER in biopsies in the proliferative phase was intense, it decreased gradually up to the late secretory phase. Immunohistochemical staining of PR showed a similar decreasing pattern between proliferative phase and late secretory phase. PR appeared to be expressed as well in stromal cells. The ER? antibody was tested to give positive results on human colon tissue, whereas human ileum was used as negative control. ER? immunostaining was also present in endometrial glandular and stromal cells. However, intensity of immunostaining of ER? was lower as compared with that of ER staining. The intensity of staining in epithelial cells decreased gradually as well from the proliferative phase up to the late secretory phase.