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Leukocyte Populations and Steroid Receptor Expression in Human First-Trimester Decidua; Regulation by Antiprogestin and Prostaglandin E Analog

Department of Reproductive and Developmental Sciences (S.A.M., T.A.H., D.T.B., H.O.D.C.), Medical Research Council Human Reproductive Sciences Unit (R.W.K., P.T.S.), Centre for Reproductive Biology, The University of Edinburgh, Edinburgh EH16 4SB, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Professor Hilary O. D. Critchley, Department of Reproductive and Developmental Sciences, Centre for Reproductive Biology, The University of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom. E-mail: hilary.critchley{at}ed.ac.uk.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Progesterone acting via its cognate receptor is critical to maintaining a viable endometrial environment for implantation and pregnancy. During medical termination of pregnancy, the biological effect of progesterone is pharmacologically withdrawn and prostaglandins administered exogenously. Leukocytes within the uterus are the effector cells of an inflammatory response and play important roles in both tissue breakdown and remodeling.

Objective: The aim of this study was to identify the separate and combined effects of the antiprogestin Mifepristone (single dose, 200 mg) and the prostaglandin E (PGE) analog (gemeprost) on leukocyte populations and steroid receptor expression in human first-trimester decidua.

Patients: Eighty women were recruited from the termination of pregnancy service with a gestational age of between 35 and 65 d at the time of surgical termination of pregnancy.

Main Outcome Measures: Immunohistochemistry was used to measure macrophage (CD68 +ve), neutrophil (neutrophil elastase +ve), and uterine natural killer cell (CD56 +ve) populations and progesterone (PR_A and PR_B), estrogen (ER and ERß), and androgen receptor (AR) expression.

Results: After administration of both antiprogestin and the PGE analog, macrophage and neutrophil numbers were significantly increased, whereas natural killer cell numbers were unchanged. Antiprogestin and PGE analog coadministration also significantly decreased PR and ER immunoreactivity but had no effect on androgen receptor or ERß receptor expression. PGE analog alone was also capable of reducing PR expression.

Conclusions: In this study, we demonstrate that the inflammatory response induced by antiprogestin in combination with PGE analog is accompanied by both increases in macrophages and neutrophils numbers and decreases in PR and ER expression in human first-trimester decidua.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
THE ENDOMETRIUM IS a multicellular tissue that responds during the menstrual cycle to sequential exposure of estrogen and progesterone (1). In addition to glandular and stromal cells, leukocytes are present in the endometrium of nonpregnant women. The populations of leukocytes present in the uterine environment have been characterized extensively and are outlined in many reviews (most recently, see Ref. 2). Briefly, leukocytes of all types are present in small numbers within the endometrium. After ovulation, macrophage and NK cell numbers increase during the secretory phase and peak premenstrually and at menstruation, respectively. If implantation occurs and the endometrial layer undergoes additional differentiation, resident NK cells continue to proliferate, while, in parallel, there is a continuation of the macrophage influx seen first after ovulation. In decidua parietalis at the end of the first trimester, uterine NK cells represent 30% of all stromal cells, and macrophages represent approximately 20% (3). Leukocyte populations were, until recently, thought to be directly controlled by paracrine mediators and only indirectly by hormones with no documented evidence of sex steroid receptor expression in macrophages, neutrophils, or NK cells within the uterus (4, 5). A recent study, however, now demonstrates estrogen receptor (ERß) and glucocorticoid receptor expression in uterine NK cells (6).

Progesterone and estrogen are the principal steroid hormones responsible for physiological changes in the endometrium, and, although testosterone is present in the uterus (7), its function has yet to be determined. Steroid hormones mediate their responses via their cognate receptors that belong to the steroid receptor superfamily (8). Progesterone acts via the progesterone receptor (PR) that exists as two isoforms, PR_A and PR_B, encoded by the same gene and resulting from two distinct estrogen-regulated promoters (9, 10). Estrogens can bind to two ERs, ER and ERß, that are encoded for by different genes (11, 12). Androgens, testosterone, and its derivative dihydrotestosterone act via the androgen receptor (AR), for which only one family member has been identified (13, 14, 15).

Mifepristone (RU486) is a PR antagonist developed in the early 1980s (16) that binds to PR with five times greater affinity than progesterone (17). Administered in combination with a prostaglandin (PG) analog, Mifepristone is capable of terminating more than 95% of first-trimester pregnancies and is licensed in the United Kingdom for the medical termination of pregnancy (18). After administration of Mifepristone, there is an increase in macrophages in endometrium and decidua and enhanced contractility of the myometrium (19). In first-trimester decidua, Mifepristone induces increased COX-2 expression and PG synthesis in decidua (20) and a decrease in prostaglandin dehydrogenase, the enzyme responsible for PG metabolism (21). Studies investigating the effects of Mifepristone on steroid receptor expression in animal and human decidua show varied effects of Mifepristone on the expression of both PR subtypes and ER (21, 22, 23, 24, 25) and often included the administration of prostaglandin E (PGE) analogs (23).

During medical termination, when progesterone is pharmacologically withdrawn and PGs are administered exogenously, the separate and combined effects of these treatments on leukocyte populations and steroid receptor expression has not been determined. In the present study, we investigated the roles of both progesterone (using a model of progesterone antagonism in vivo) and PGs (using a PGE analog) in the recruitment of selected leukocyte subpopulations and on expression patterns for the steroid receptors (AR, ER, ERß, PR_A, and PR_B) in early pregnancy decidua.

	Patients and Methods

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Patient recruitment and tissue collection

Eighty women were recruited from the termination of pregnancy service at the Royal Infirmary, Edinburgh, Scotland, United Kingdom. Only women with a first-trimester pregnancy were approached about participation by a dedicated clinical research nurse. All participants provided written informed consent before tissue collection, and ethical approval was obtained from Lothian Research Ethics Committee (No. 94/6/1 and 96/6/18). The gestational age, dated from the reported first day of last menstrual period, was between 35 and 65 d at the time of surgical termination of pregnancy. Women were randomized into eight groups to receive, at a fixed time, vaginal PG analog (Cervagem, 1 mg) of 0, 1, 2, or 3 h either alone or after 24 h Mifepristone (200 mg). Treatment group was randomized by use of random number tables to allocate equally subjects to one of the possible eight treatment options. All patients were entered into only one group, and no patient received repeated biopsies. After control or Mifepristone treatment, the women were administered a vaginal PGE analog pessary, and decidual tissue was collected using curettage of the uterine wall before vacuum aspiration of the products of conception at 0 (no PGE analog), 1, 2, or 3 h after vaginal administration (n = 10 for each group). Biopsies were fixed overnight in 4% paraformaldehyde/PBS before wax embedding. Decidual tissue sections were immunostained with cytokeratin to confirm absence of trophoblast cells within the biopsy (data not shown).

Immunohistochemistry protocol

All immunohistochemical assays used techniques described previously in detail by this laboratory (26). Antibody sources and specific experimental criteria are summarized in Table 1. In brief, sections were dewaxed in Histoclear and hydrated in descending alcohol solutions and washed in PBS, and endogenous peroxidase activity was quenched with H₂O₂ (3% in distilled water). After antigen retrieval, where necessary, nonimmune serum was applied for 20 min before overnight incubation at 4 C with primary antibody. A species-specific secondary antibody and avidin-biotin peroxidase detection system was applied following the guidelines of the manufacturer (Vectastain ABC kits; Vector Laboratories, Burlingame, CA), and immunoreactivity was visualized with 3,3'-diaminobenzidine (Vector Laboratories) as the chromogen (appears brown). Sections were counterstained with Harris’s hematoxylin (blue) before dehydrating and mounting.

Quantitative analysis

Twelve separate digital images were photographed from each immunostained tissue section (pilot studies performed in-house identified that, after about 8–10 fields, interimage variance was <5%). For each digital image, Openlab software (Improvision, Coventry, UK) generated a mask for each color (brown for 3,3'-diaminobenzidine and blue for hematoxylin). Using these masks, a minimum object size was selected by the user, and then the software generated a cell number for either leukocytes (brown) or total cell number (taken as the number of blue nuclei) from each image. These were averaged for each set of 12 images to provide representative data for each tissue section.

Semiquantitative analysis

Immunohistochemical localization of steroid receptors was semiquantitatively assessed as described previously (27). In brief, two separate observers ranked the intensity of immunostaining on a scale of 0–3 (0, no staining; 3, intense staining). Once the slides had been independently scored, the values were tabulated, and an overall score for each section was determined after consultation between the two observers. Results are presented as the mean ± SEM value for each sample group in the range of 0–3.

Statistics

Quantitative data for leukocyte numbers was continuous, and the parametric ANOVA test followed by Fisher’s post hoc test was used to determine statistical significance. Data from semiquantitative analysis of steroid receptor expression was, however, noncontinuous, and statistical analysis used Kruskall-Wallis nonparametric analysis in conjunction with Dunn’s multiple comparison post hoc test.

Statistical significance for both tests was accepted for P < 0.05.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Leukocytes

Neutrophils. Negligible neutrophil numbers (0.31 ± 0.07% total cells) were observed in control decidua (Figs. 1A and 2A). After Mifepristone alone, there was no change in neutrophil numbers (0.36 ± 0.11%) (Fig. 2A). Administration of PGE analog produced a significant (P < 0.05) time-dependent increase in neutrophil numbers for both control decidua (0.68 ± 0.14% after 3 h) (Figs. 1B and 2A) and decidua exposed previously to Mifepristone (0.70 ± 0.15% after 3 h) (Figs. 1C and 2A).

View larger version (109K): [in this window] [in a new window]   FIG. 1. Immunohistochemical staining in human first-trimester decidua for neutrophil elastase (A–C), CD68 (D–F), and CD56 (G–I) after treatment with vehicle alone, PGE analog for 3 h, or antiprogestin plus PGE analog for 3 h. Note the increased staining for neutrophils and macrophage (CD68) cells 3 h after the treatment with PGE analog. Scale bar, 50 µm.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Quantitative cell counts for neutrophils [neutrophil elastase (NE); A], macrophages (CD68; B), and uterine natural killer (CD56; C) cells in first-trimester decidua treated with PGE analog (gemeprost) for 0 (control), 1, 2, and 3 h alone (open bars) or after antiprogestin (filled bars). *, P < 0.05 compared with control (no antiprogestin).

Uterine NK cells. CD56-positive cells represented about 15% of total cells in first-trimester decidua (14.7 ± 2.4%) (Figs. 1G and 2C). Treatment of patients with Mifepristone alone had no significant effect on CD56-positive cell numbers (14.1 ± 2.6%) (Fig. 2C). No significant changes were observed in PGE analog treatment samples (10.2 ± 5.6% after 3 h) (Figs. 1H and 2C) or tissue that had also been exposed to Mifepristone (11.2 ± 2.6% after 3 h) (Figs. 1I and 2C).

Steroid receptors

PR. Immunolocalization of PR with an antibody recognizing both PR_A+B isoforms demonstrated immunoreactivity mainly localized to nuclei of stromal cells (Figs. 3A and 4A). Occasional expression of PR_A+B was also seen in glandular epithelial and perivascular stromal cells but not in endothelial cells (Figs. 3A, arrow, and 5A). Mifepristone treatment alone for 24 h had no significant effect on PR_A+B expression (Figs. 4A and 5A), whereas, after local treatment with PGE analog alone, there was a significant time-dependent decrease of PR_A+B expression in all tissue compartments (P < 0.05) (Figs. 4A and 5A). Treatment with Mifepristone 24 h before local administration of PGE analog also resulted in a significant time-dependent decrease in PR_A+B expression (P < 0.05) (Figs. 3B, 4A, and 5A).

View larger version (102K): [in this window] [in a new window]   FIG. 3. Positive immunoreactivity (brown) in human first-trimester decidua for PRA+B (A, B), PRB (C, D), ER (E, F), ERß (G, H), and AR (I, J). Sections are nontreated control decidua (A, C, E, G, I) and decidua exposed to antiprogestin and PGE analog for 3 h (B, D, F, H, J). Glandular epithelium (G) and endothelial cells (arrows) are indicated. Note the decrease in staining for PRA+B, PRB, and ER in stroma and glands (P < 0.05) after 3 h treatment with PGE analog but no change in ERß or AR. Scale bar, 50 µm.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Semiquantitative assessment of stromal immunoreactivity for PRA+B, PRB, ER, ERß, and AR in first-trimester decidua treated with PGE analog (gemeprost) for 0 (control), 1, 2, and 3 h alone (open bars) or after antiprogestin (filled bars). *, P < 0.05 compared with control (no antiprogestin).

View larger version (19K): [in this window] [in a new window]   FIG. 5. Semiquantitative assessment of glandular immunoreactivity for PRA+B, PRB, ER, ERß, and AR in first-trimester decidua treated with PGE analog (gemeprost) for 0 (control), 1, 2, and 3 h alone (open bars) or after antiprogestin (filled bars). *, P < 0.05 compared with control (no antiprogestin).

ER. ER protein expression was minimal in first-trimester decidua and, when expressed, was primarily located in nuclei of stromal and epithelial cells (Fig. 3E). ER protein was not detected in endothelial cells (Fig 3E, arrow), consistent with a previous report from our group (28). Treatment with Mifepristone for 24 h before termination or exposure to PGE analog alone had no significant effect on ER expression patterns (Figs. 4C and 5C). However, after administration of Mifepristone for 24 h plus PGE analog for 3 h, a significant reduction in the ER immunoreactivity was observed in stromal cells (P < 0.05) (Figs. 3F and 4C).

ERß protein, in contrast to ER protein, was expressed in the cell nucleus in all compartments of first-trimester deciduas, including endothelial cells (Fig. 3G, arrow). Administration of only Mifepristone, only PGE analog, or both Mifepristone and PGE analog had no significant effect on ERß immunoreactivity (Figs. 3H, 4D, and 5D).

AR. AR immunoreactivity was localized primarily to the stromal compartment of first-trimester deciduas, with little perivascular but pronounced endothelial nuclear immunoreactivity (Fig. 3I, arrow). In contrast, glandular epithelial cells were only occasionally immunopositive for AR (Figs. 3I and 5E). Administration of only Mifepristone, only PGE analog, or both Mifepristone and PGE analog had no significant effect on AR immunoreactivity (Figs. 3J, 4E, and 5E).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Administration of a PG analog in conjunction with the antiprogestin Mifepristone is an effective abortifacient regimen (18). The exact local cellular mechanisms evoked with the addition a PGE analog to the antiprogestin-"primed" uterus have not been studied in depth. Although the administration of a PGE analog alone is capable of increasing local decidual macrophage numbers, only the administration to an Mifepristone-primed uterine environment induced a statistical increase in both neutrophil and macrophage cell numbers. The PGE analog gemeprost, in combination with Mifepristone, decreased expression of both isoforms of the PR, thereby potentially producing a "hormone-insensitive" state in the decidua.

The presence in the endometrium of increased numbers of leukocytes, specifically neutrophils and macrophages, only 3 h after vaginal administration of a PGE analog suggest a direct chemotactic effect. PGE₂ acts via four G protein-coupled receptors termed EP₁–EP₄. PGE₂ has been documented to enhance chemotaxis in neutrophils and macrophages, with this response being mediated via the EP₃ receptor subtype (29, 30), and the PGE analog, gemeprost, has been demonstrated to possess EP₃ receptor agonist activity (31). Additionally, PGE₂ can enhance ingress of neutrophils into tissues by a synergistic action with chemotactic agents, such as IL-8 (32, 33, 34). Such effects are likely mediated through the EP₂/EP₄ pathway, which is implicated in the vasoactive, rather than chemotactic, actions of PGE₂ (35).

A previous study has reported that PG treatment during termination of pregnancy induces loss of fetal heart rate, an effect not seen with antiprogestin (36). The loss of fetal heart rate was attributed to loss of maternal blood supply as a result of profound vasoconstriction. Hypoxia has been shown to induce chemokine and cytokine expression (37), and hypoxia has been hypothesized to induce the tissue breakdown and shedding at menstruation (38). Myometrial tissue from many species express EP₃ receptors, and PGE₂ induces myometrial contractility via this receptor. The potential physiological processes induced after vaginal administration of a PGE analog include a hypoxic insult to the decidua via myometrial contraction, resulting in chemokine and cytokine release that are then synergistically enhanced by the PGE analog.

Chemokine and cytokine release from the stromal and vascular cells in the endometrium and decidua are important for the regulation of leukocyte influx. Decreases in circulating progesterone levels premenstrually stimulate high levels of chemokine and cytokine expression, which leads to leukocyte influx and an associated inflammatory reaction (39). Administration of progesterone antagonist together with decreases in PR expression results in withdrawal of a progesterone-dominated environment and might be another way in which a hormone-insensitive state is induced within the uterus. Down-regulation of PR after progesterone binding has been suggested already as a method by which a cell can reset its response to progesterone (40). Moreover, in PR knock-out mice, there is a dramatic influx of leukocytes that cannot be prevented by administration of exogenous progesterone (41). The observed decrease in expression of PR after exposure to a PGE analog demonstrates that this same hormone-insensitive state may exist in the deciduas, although progesterone levels remain unaffected.

A recent publication describes a modification of PR receptor ratio by PGE₂ and PGF₂ (42), but, to date, no publications describe only PG-induced down-regulation in PR expression occurring in vivo. Other groups have, however, observed ER subtype modulation by G protein-coupled heptahelical receptor ligands. Human chorionic gonadotrophin and GnRH can both down-regulate expression of ER and ERß mRNA and protein in rat and human ovaries (43, 44, 45). Additional support for the findings presented here is a study by Hill and colleagues (23) demonstrating a decrease in PR concentrations in human decidua after both Mifepristone and PGE analog administration. Although the authors conclude that antiprogestin mediated the observed decrease in PR, the data obtained during the current study would suggest that the administration of PG, and not Mifepristone, was responsible for the decrease in PR expression.

PR proteins are synthesized via ribosomes situated on the endoplasmic reticulum and released into the cytoplasm before being transported into the nucleus. The transport of PR through the nuclear pore complex is an active process requiring ATP (46, 47). One explanation for the observed decrease in nuclear PR that occurred after administration of the PGE analog is that the cells were unable to transport PR into the nuclei. The net result would be reduced nuclear immunoreactivity for each receptor but a net increase in cytoplasmic immunoreactivity. In this study, however, no cytoplasmic immunoreactivity was observed with either PR antibody.

Another explanation for the decrease in PR immunoreactivity after PGE analog is increased degradation of the PR. The PR is continuously trafficked from the cell nucleus and degraded via the ubiquitin/26S proteasome pathway. R2050 (a progesterone analog) has been shown to induce ERK-mediated PR phosphorylation and enhance proteasome degradation of PR (40). PGE₂ alone can also directly phosphorylate ERK (48), and, in this study, gemeprost may be acting synergistically with endogenous progesterone to enhance PR degradation.

AR and ERß immunoreactivity in human first-trimester decidua is reported here, to our knowledge, for the first time. The immunohistochemical data in this study have identified that both stromal and endothelial cells express AR and ERß in human decidua. Previous studies have identified expression of AR in stromal and endothelial cells within the endometrium (28, 49, 50, 51), with recent data suggesting an antiproliferative role for the AR in the uterus (52). ERß has also been identified in endothelial cells within the endometrium (28) and more recently in uterine NK cells (6). Additional studies need to be performed to identify the specific role of ERß in the human uterine environment during early pregnancy.

In conclusion, we reported an increase in the numbers of neutrophils and macrophages and a loss of PR and ER expression in human first-trimester decidua after systemic antiprogestin and local (vaginal) administration of a PGE analog. Additional investigations are required to elucidate the exact signal transduction pathways linking PG receptor activation to steroid receptor down-regulation and to determine whether PR down-regulation alters progesterone signaling in vivo.

	Acknowledgments

 
We thank Ms. Cathy Hall for assistance with patient recruitment throughout the study.

	Footnotes

 
Present address for S.A.M.: Fujisawa Institute of Neuroscience in Edinburgh, Department of Neuroscience, The University of Edinburgh, 6th Floor Appleton Tower, Crichton Street, Edinburgh EH8 9ET, United Kingdom.

This work was supported by Medical Research Council Project Grant G9620138 and by Grant G9523250 to the Contraceptive Development Network from the Department for International Development and the Medical Research Council, United Kingdom.

First Published Online April 6, 2005

Abbreviations: AR, Androgen receptor; ER, estrogen receptor; PG, prostaglandin; PGE, prostaglandin E; PR, progesterone receptor.

Received December 1, 2004.

Accepted March 29, 2005.

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