Changes in Progesterone-Induced-Blocking-Factor Expression Rates Following Mifepristone Administration in Termination of Pregnancy at 5 to 8 Weeks

Abstract

Objective. To study changes in the expression rate of PIBF by peripheral lymphocytes in healthy pregnant women after administration of mifepristone for non-surgical termination of pregnancy at 5-8 wks of gestation.

Methods. Patients requesting early social termination of pregnancy, in a 3-month period, were included. A first venous blood sample was taken before oral administration of 600 mg of mifepristone (day 0). A second venous blood sample was taken 2 days later. PIBF on lymphocytes was determined by immunocytochemistry using a PIBF-specific polyclonal antibody.

Results. Termination of pregnancy was successful and complete in all cases. In 17 out of 21 patients, the percentage of PIBF positive lymphocytes decreased after anti-progesterone administration. The percentage of PIBF-expressing lymphocytes significantly decreased from 52.8% 21.6% (day 0) to 39.8% 18.2% by day 2 0 = 0.001).

Conclusions. These data suggest a strong relationship between early termination of pregnancy induced with mifepristone and disturbances of progesterone-mediated immunosuppression.

Keywords: Progesterone, immunosuppression, mifepristone, elective pregnancy termination, abortion

Introduction

The conceptus may be considered as a sort of semiallogenic graft which is recognized by the maternal immune system [1]. Mechanisms protecting the fetus from rejection by the maternal immune system have been the source of a continuous debate and among potentially immunosuppressive substances present in the serum of pregnant women, progesterone seems to play a major role in reducing responsiveness of maternal lymphocytes [2] in vitro. Although the physiological importance of systemic immunosuppression is not clear, it seems likely that the target of progesterone is not restricted to the fetomaternal interface, where high local concentrations of this hormone are present [2].

Following recognition of fetus-derived antigens, gamma/delta TCR+ cells develop progesterone receptors [3]. Progesterone binding to its receptors triggers the synthesis of progesterone-induced blocking factor (PIBF), a mediator protein which has an anti- abortive effect [4]. Indeed, PIBF blocks natural killer (NK) cells activity which has been suggested to play a role in pregnancy termination [5-9] by blocking interleukin-12 (IL-12) production and reducing TNF alpha plasma levels. Alterations of this system can lead to a synergy in the expression of IL-12 and TNF alpha, leading to a dominant production of Th 1 type cytokines and pregnancy termination [10]. Furthermore, PIBF-producing cells are likely to be present in the decidua and might contribute to inhibit the cytotoxicity of NK cells by blocking their degranulation [11]. PIBF also increases expression of interleukin 10 [12] and reduces that of IL-12 via the inhibition of arachidonic acid metabolism [13]. Inhibition of cytotoxic NK cells activity is also associated with a normal outcome of pregnancy [13]. PIBF therefore affects the Th1/ Th2 cytokines balance, and contributes to a decrease in cell- mediated responses during pregnancy [14].

Mifepristone is a steroid which presents a high affinity for progesterone receptors without any progesterone activity. Mifepristone is widely used clinically for early termination of pregnancy in association with prostaglandins [15]. In vitro and animal experiments in mice suggest that mifepristone affects the immunosuppression needed for the maintenance of normal gestation [16- 19].

This study was therefore undertaken to study changes in the rate of PIBF-expressing peripheral lymphocytes in healthy pregnant women after administration of mifepristone for medically-induced early termination of pregnancy.

Material and methods

Patients

Women requesting social termination of pregnancy (TOP) at 5-8 wks of gestation in a 3-month period were offered to participate. A 7- day interval was arranged to confirm the decision for TOP, as requested by the French law at that time. Women who confirmed their request were offered medically induced TOP using a mifepristone- misoprostol regimen as published elsewhere [15]. A first venous blood sample was taken before oral administration of 600 mg of mifepristone (day 0). A second venous blood sample was taken before vaginal administration of 400 mg of misoprostol, 2 days later (day 2). Rh negative women were also given anti-Rh immunoglobulin (300 g). All patients had a follow-up ultrasound examination 7 days later to confirm completeness of abortion.

The study was approved by the local ethics committee and all patients signed informed consent.

Immunocytochemistry

Dry glass bottles with separating gel and glass bottles with lithium heparinate were used (Beckton-Dickinson Vacutainer system). Samples were taken to the laboratory for immediate processing. PIBF on lymphocytes was determined by immunocytochemistry method using a PIBF-specific polyclonal antibody [7]. Lymphocytes were isolated on a Ficoll Paque gradient (Amersham-Pharmacia-Biotech AB, Sweden) within 2 h after sampling. After washing with RPMI 1640 medium, cell counts were adjusted to a concentration of 1 million per ml. One hundred microliter aliquots of cell suspension were added to sample chambers for cytocentrifugation. Slides were air-dried at room temperature overnight, and then fixed in cold acetone. A negative control (male lymphocytes) and a positive control (MCF7 mammary carcinoma cells) were also run in each assay. Endogenous peroxydase activity was blocked with 1% H^sub 2^O^sub 2^. The cells were first incubated with 2% BSA in order to block non-specific binding sites, and then for 1 h with 1:200 dilution of a polyclonal anti-PIBF IgG. Non-specific binding was controlled by including a slide without anti-PIBF antibody for each case. The slides were then washed in PBS and incubated for 45 min with 1:100 diluted anti-rabbit peroxidase- conjugated antibody (DAKO).

After washing in PBS, the cells were incubated with amino- ethylcarbazole chromogen solution (DAKO) for 35 min; the reaction was stopped with distilled water and the cells were counterstained with hematoxylin. The slides were then coverslipped and read under oil-immersion (100 x objective). A positive reaction was indicated by a reddish precipitate at sites of specific cellular antigen localization (Figure 1). Four hundred cells were counted and the percentage of positive cells was determined.

Serum progesterone concentrations in day O and day 2 venous blood samples were determined by ACS 180 (Bayer).

Statistical analysis of the data

Statistical analysis was performed using a paired t-test and a paired Wilcoxon test. All tests were twotailed, and P values of 0.05 or less were considered to indicate statistical significance. As this was a preliminary study, sample size was not based on prespecified power calculations. The aim was to recruit all eligible women in a 3-month period.

Results

The effect of mifepristone-misoprostol treatment on PIBF expression of peripheral lymphocytes

Twenty-one patients with a mean age of 31.1 6.7 years were involved in this study. Maternal venous blood was taken between 37 and 58 (48 6 days) days of gestation. The mean parity of the patients was 1.3 1.2.

Figure 1. Lymphocytes at Day 0. Presence of PIBF on lymphocytes. Positive reaction indicated by a precipitate at sites of specific cellular antigen localization (arrows).

Termination of pregnancy was successful and complete in all cases as confirmed by ultrasound on day 7.

In 17 out of 21 patients, the percentage of PIBF positive lymphocytes decreased after anti-progesterone treatment (Figures 1 and 2). No change was seen in two cases, and in two women a slight increase was observed. The percentage of PIBFexpressing lymphocytes significantly decreased from 52.8% 21.6% (day 0) to 39.8% 18.2% by day 2 (Figures 1 and 2), p = 0.001.

The effect of mifepristone-misoprostol treatment on plasma progesterone levels

Plasma progesterone levels did not change during treatment (25.3 11.4ng/ml on day 0 and 23.8 9.8 ng/ml on day 2) (NS).

Discussion

This preliminary study, suggests a significant decrease in PIBF expression in maternal lymphocytes, 2 days after oral administration of mifepristone, in successful termination of pregnancy at 5-8 weeks of gestation. A combination of mifepristone and misoprostol is widely used for early termination of pregnancy allowing successful abortion in most cases without the need for surgical procedures [20].

Progesterone-dependent immunomodulation is one of the mechanisms that enables pregnancy to proceed to term [8]. Mifepristone has a strong affinity for progesterone receptors without any progesterone activity. The administration of mifepristone induces interruption of the luteal phase in the menstrual cycle of non-pregnant women and facilitates termination of pregnancy in pregnant women [21]. Our results suggest that PIBF could be involved in mifepristone-induced pregnancy termination. Since plasma concentrations of progesterone did not change during this period, pregnancy termination is unlikely to be related to a lack of progesterone.

Figure 2. Lymphocytes at Day 2. The percentage of PIBF positive lymphocytes has decreased after anti-progesterone treatment.

PIBF is produced followingprogesterone binding to its receptor, thus mifepristone may reduce PIBF production by blocking progesterone receptors. This could result in an increased NK activity and a ThI dominant immunological environment leading to abortion of the early pregnancy. This has not been observed in vivo but is consistent with other studies reporting in vitro inhibition of PIBF production by RU 486 [17]. Earlier studies showed that in 8 days pregnant BALB/c mice after administration of mifepristone resulted in 100% rsorption of all fetuses. Simultaneous administration of the supernatant from progesterone-treated murine pregnancy spleen cells containing PIBF corrected the high resorption rates [18]. These data therefore suggest that functional progesterone binding sites in lymphocytes are needed for the maintenance of a normal pregnancy. The fact that administration of the preformed blocking factor could counteract the effect of antiprogesterone treatment suggests that progesterone-mediated immunosuppression is needed for the maintenance of a normal gestation. It was also found that the proportion of PIBF positive cells is significantly reduced in peripheral blood of women undergoing recurrent spontaneous abortions [7].

Since PIBF seems to be a key factor in the maintenance of pregnancy, it is noticeable that changes in its rates can be detected in such a small time interval. We were able to measure a significant decrease within 2 days. Such kinetic analysis might therefore be of interest in predicting the outcome of pregnancy. Although, isolated low levels of PIBF do not seem to be related to first trimester spontaneous abortions [22], short decrease in PIBF expression rates might be informative in threatened abortion as well as in threatened preterm delivery, since initiation of labor and threatened pre-term delivery could be associated with increased natural cytotoxic activity [23]. However, the role of decreased PIBF production during early medical termination of pregnancy cannot be considered as being exclusive. Indeed, in 4 out of 21 cases, termination was successful despite normal PIBF expression. In a previous study [24], high and low responders were identified among patients treated by a progesterone antagonist and it is not known, whether mifepristone blocks A or B type or both types of progesterone receptors and the type of progesterone receptors expressed by activated lymphocytes has not been identified either.

Although preliminary, our data suggest an immunological mechanism of action of mifepristone in human pregnancy termination. Large prospective studies should be carried out to evaluate the predictive value of changes in PIBF expression rates during threatened abortion in the first trimester of pregnancy.

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LAURENT J. SALOMON1, PATRICK ROZENBERG1, JULIA SZEKERES-BARTHO2, LYDIA MALAGRIDA3, YVES GIUDICELLI3, & YVES VILLE1

1 Department of Obstetrics and Gynecology, Poissy-Saint Germain Hospital, Versailles-St Quentin University, France, 2 Department of Medical Microbiology and Immunology, Medical School, Pecs, University, Pecs, Hungary, and 3 Department of Biology, Poissy- Saint Germain Hospital, Versailles-St Quentin University, France

Correspondence: Professor Yves Ville, Centre hospitalier Poissy- Saint-Germain, 10, Rue du Champ Gaillard, 78303 Poissy Cedex, France. Tel: 33 1 39 27 52 57. Fax: 33 1 39 27 44 79. E-mail: yville@wanadoo.fr

Copyright CRC Press May 2005