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EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

April 2000
Volume IV, Number 2
ISSN: 1537-6583
Pages: 154-164

Joanne Y.H. Kwak, F. Meeyoung Kwak, Alice Gilman-Sachs, Kenneth D. Beaman, Daniel D. Cho and Alan E. Beer

Reproductive Medicine, Department of Obstetrics and Gynecology, Department of Microbiology and Immunology, Finch University of Health Sciences/The Chicago Medical School, North Chicago, IL, 60064, USA

Short Title: IVIg treatment in recurrent aborters with elevated NK cells

Key Words: anticoagulation, CD56+ NK Cells, immunoglobulin G infusion, lymphocyte immunization, recurrent spontaneous abortion

Correspondence: Joanne Y.H. Kwak, MD, Reproductive Medicine, Department of Obstetrics and Gynecology, Department of Microbiology and Immunology, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, N. Chicago, IL 60064, Tel: 847-578-3233 Fax: 847-578-3339 Email: kwakj@mis.finchcms.edu

Abstract

We aimed to investigate the clinical effect of intravenous immunoglobulin G (IVIg) treatment in recurrent aborters with elevated peripheral blood CD56+ NK cell levels while on lymphocyte immunization, anticoagulation and prednisone treatment, with respect to subsequent live birth and reproductive outcome. Thirty-three women with recurrent abortions achieved alloimmune recognition after lymphocyte immunizations. All had autoimmune abnormalities and received preconception anticoagulation and prednisone treatment. At the time of positive pregnancy testing, 18 women with normal NK cell levels (<12%) and 6 with elevated NK cell levels (>12%) continued anticoagulation and prednisone treatment, and 9 with elevated NK cell level initiated additional IVIg treatment. The live birth rates of women with elevated NK cell level (>12%) who initiated post-conception IVIg treatment in addition to anticoagulation and prednisone (100.0%), women with normal NK cell levels (<12%) who continued anticoagulation and prednisone (83.3%) and women with elevated NK cell level (>12%) who continued anticoagulation and prednisone (33.3%) are significantly different (P=0.0065). Prevalence of intrauterine growth retardation and preterm delivery among 3 study groups were not different. In conclusion, post-conception IVIg treatment significantly improves reproductive outcome in women with elevated CD56+ NK cells with pregnancy who received preconception lymphocyte immunization, anticoagulation and prednisone treatment.

Introduction

When the immunophenotype of women with recurrent spontaneous abortions (RSA) of immune etiology were determined, a significant number of women demonstrated a markedly increased peripheral blood CD56+, CD56+/16+ natural killer (NK) cell levels, and CD19+ B cell levels . Indeed, 14.7% of this cohort demonstrated elevated CD56+ NK cell levels greater than 18% of peripheral lymphocytes . Down regulation of CD56+ NK cells during early pregnancy is associated with successful pregnancy outcome in women with RSA . Women who abort while receiving immunotherapy do not show a decrease in CD56+ NK cells . Among the mechanisms suggested to down regulate CD56+ NK cells are apoptosis induced by progesterone, induced pregnancy protein TJ6 , cytokines secreted by monocytes or trophoblasts and other factors present in pregnant serum .

NK cells can recognize trophoblast cells. Indeed, peripheral blood NK cells from women with RSA demonstrate cytotoxicity to autologous placental cells . In women with RSA, NK cell activity is significantly elevated when compared with that of normal pregnant women . Lymphocyte immunotherapy induces suppression of NK cell activity, possibly through the induction of prostaglandin E2 secretion from monocytes . In women with RSA and elevated CD56+ NK cells, peripheral blood CD56+, CD56+/16+ NK cells are effectively suppressed after intravenous immunoglobulin G infusion (IVIg) therapy . In vivo and in vitro NK cytotoxicity is also significantly suppressed 7 days after IVIg infusion treatment . It has been reported and suggested that systemic regulation of lymphokine activated natural killer (LAK) cells determine reproductive outcome .

In this study, we aimed to study clinical effectiveness of IVIg treatment in recurrent aborters with elevated CD56+ NK cells (>12%) at the time of positive pregnancy test who have alloimmune recognition and preconception anticoagulation and prednisone treatment. The live birth rate following treatment of these women was analyzed to that of women with normal or elevated CD56+ NK cells who were treated with anticoagulation and prednisone treatment only.

Materials and Methods

Population
A total of 33 women with a history of 2 or more unexplained pregnancy losses and lack of alloimmune recognition comprised the study groups. Women with an ectopic pregnancy, hydatidiform mole or chromosomally abnormal pregnancy as an index pregnancy were excluded from this study. Women with a chemical pregnancy loss as an index pregnancy were considered as having spontaneous abortion. Age, gravidity and number of spontaneous abortions of the 3 study groups are listed in Table 1.

Alloimmune recognition was defined as presence of maternal antipaternal IgG T and B cell antibodies greater than 20% by flow cytometric analysis. Women with 2 or more live born infants, active autoimmune disease or a history of autoimmune disease were excluded from the study. Women with positive results of IgG or IgM antiphospholipid antibodies (autoantibodies to cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol), antibodies to nuclear components (autoantibodies to dsDNA, ssDNA, polynucleotides, and histones) or ANA which were repeatedly positive on at least two occasions of 6 weeks interval, were included in the study. These antibodies were detected by enzyme linked immunosorbent assay as previously reported .

All received lymphocyte immunizations and achieved alloimmune recognition prior to index pregnancy as previously reported . All started preconception anticoagulation and prednisone treatment. Peripheral blood CD56+, CD56+/16+ NK cell levels, CD19+ and CD19+/5+ B cell levels were studied at the time of positive pregnancy testing (4 weeks gestation) by flow cytometric analysis (Table 2). Eighteen women with normal NK cell levels continued anticoagulation and prednisone treatment, 9 women with elevated NK cell levels initiated IVIg treatment with continuation of anticoagulation and prednisone treatment and 6 women with elevated NK cell levels continued anticoagulation and prednisone treatment. Following are treatment protocols for 3 study groups for conception cycle after alloimmune recognition.

Women with normal NK cells (<12%) treated with anticoagulation and prednisone
Eighteen women with RSA and normal NK cell level were treated with pre-conception anticoagulation and prednisone. Low dose aspirin (81mg/day) was taken orally from day 1 of cycle of planned conception and continued throughout pregnancy. Heparin was initiated 48 hours after ovulation at a dose of 5000 Units, self-administered, every 12 hours subcutaneously. Heparin was continued through 34 weeks gestation. Prednisone was given 5mg, orally every 12 hours starting 48 hours after ovulation and increased to 10 mg orally, every 12 hours at the time of positive pregnancy test. Prednisone was tapered off at 12 weeks gestation.

Women with elevated NK cells (>12%) treated with anticoagulation, prednisone, and additional post conception IVIg
Nine women with elevated NK cells at the time of positive pregnancy were included. Preconception anticoagulation and prednisone treatment was started 48 hours after the ovulation as described above. IVIg treatment was initiated after the confirmation of positive pregnancy test and elevated peripheral blood CD56+ NK cell levels. Immunoglobulin G 400mg/Kg was infused for 1 day intravenously and repeated every 3 weeks until 28 weeks gestation. Prednisone was tapered to 10 mg daily with the initiation of IVIg treatment and tapered off at 12 weeks gestation.

Women with elevated NK cells (>12%) treated with anticoagulation and prednisone
Six women with elevated peripheral blood NK cells at the time of positive pregnancy test received preconception anticoagulation and prednisone treatment as described previously.

Peripheral Blood Immunophenotype Assay
Two color immunophenotype analyses of lymphocyte markers were performed on a XL Flow Cytometer (Coulter Diagnostics, Miami Lakes, FL). Peripheral blood was stained directly with FITC or PE-labeled monoclonal antibodies (Ortho Diagnostic, Raritan, NJ). Antibodies to CD3 and CD25, CD4 and CD8, CD5 and CD19, CD56 and CD16 were used. Cells within the lymphocyte cell gate, as determined by light scatter, were evaluated for fluorescence after reaction with the antibodies. Mouse immunoglobulin isotype controls were run concurrently with lymphocytes from all individuals. The data was displayed on a four decade log scale .

Statistical Analysis
Reproductive outcomes of 3 study groups were analyzed. Statistical differences between groups were evaluated by c2 and Fisher’s exact test for dichotomous variables. Continuous variables of study groups were analyzed by one way analysis of variance with Tukey multiple comparison test (honestly significant difference test).

In each analysis, women or infants for whose information is missing on any variable were excluded from the analysis of that variable. All tests were two-sided, and a P value of less than 0.05 was considered to indicate statistical significance. Calculations were performed with the statistical package for social sciences/ PC+ StatisticsTM 4.0 version.

Results

Table 2 demonstrates levels of peripheral blood NK and B cell sub-populations in each study group. CD19+ B cell and CD19+/5+ B-1 cell levels were not different in 3 study groups. Since each study group was defined based on peripheral blood CD56+ NK cell level, women with normal NK cell levels have significantly lower level of CD56+ and CD56+/16+ NK cell levels as compared with those of women with elevated NK cell levels.

Pregnancy success rates of study groups are plotted in Figure 1. Fifteen of eighteen (83.3%) women with normal NK cell level treated with preconception anticoagulation and prednisone, 9 of 9 (100.0%) women with elevated NK cell level treated with IVIg in addition to preconception anticoagulation and prednisone and 2 of 6 (33.3%) women with elevated NK cell level treated with anticoagulation and prednisone delivered a live born infant (P=0.0065). Women with elevated NK cell level treated with IVIg in addition to anticoagulation and prednisone (P=0.010) and women with normal NK cell level treated with anticoagulation and prednisone (P=0.038) demonstrated significantly higher pregnancy success rates as compared to women with elevated NK cell level treated with anticoagulation and prednisone only.

Birth weight, incidence of small for gestational age, and incidence of large for gestational age were not different among 3 study groups. Length of gestation and incidence of preterm delivery were not different in 3 study groups (Table 3).

Table 4 lists obstetrical complications and medication side effects in study groups. In women with normal NK cell level treated by anticoagulation and prednisone, two experienced vaginal spotting prior to 6 weeks gestation, however, none developed ultrasonographically detectable subchorionic hematoma. One experienced insomnia and palpitation while on prednisone treatment. One developed premature rupture of membrane (37 weeks) and gestational diabetes mellitus (24 weeks gestation). In women with elevated NK cell level treated with additional postconception IVIg, one woman developed mild polyhydramnios during third trimester and one developed abruptio placentae at 37 weeks gestation. In women with elevated NK cell level treated by anticoagulation and prednisone, one had subchorionic hematoma during first trimester.

Discussion

In normal non-autoimmune pregnant women, levels of CD56+ NK cells are significantly suppressed at the time of positive pregnancy test as compared to non-pregnant NK cell level . In contrast, pregnant women with a history of RSA demonstrate significantly higher peripheral blood NK cell levels, NK cell cytotoxicity and B cell levels as compared with those of normal pregnant women . In addition, NK cytotoxicity has been reported to be significantly elevated as compared to previous levels when women miscarried while on anticoagulation and prednisone treatment .

IVIg infusion strongly suppresses NK cytotoxicity in vivo and in vitro. This effect may be considered due to a shifting of immune response to TH-2 response and weak stimulation of antibody dependent cell mediated cytotoxicity (ADCC), which may occur as an enhancement of a TH-1 response . Suppression of NK cytotoxicity by IVIg was dose dependent and through F(ab)’2 portion of immunoglobulin . Previously we reported reproductive outcome of women with elevated CD56+ NK cells who were treated with anticoagulation and IVIg . Peripheral blood NK cell (CD56+, CD56+/16+) levels are effectively suppressed after IVIg treatment (p<0.0005). Down regulation of NK cells with IVIg treatment in women with RSA significantly correlates with a favorable pregnancy outcome . CD19+ B cell levels during pregnancy were significantly lower in women with anticoagulation and IVIg treatment as compared with those of women with anticoagulation therapy only . In this study, CD19+ and CD19+/5+ B cells were not different at the time of positive pregnancy test regardless of their peripheral blood NK cell levels. These results could be due to preconception prednisone treatment.

Previously we reported pregnancy outcome of women without NK assessment who had alloimmune recognition after lymphocyte immunization and were treated with preconception anticoagulation and prednisone . Incidence of intrauterine growth retardation (IUGR) of this group was 22.6 % (7/31) and incidence of preterm delivery was 64.5% (20/31). In this study, IUGR incidence of women without elevated CD56+ NK cell levels who had alloimmune recognition and treated with anticoagulation and prednisone is 13.3% (2/15) and 0% in women with elevated CD56+ NK cells who received IVIg treatment in addition to anticoagulation and prednisone treatment. Incidence of preterm delivery was 13.3% (2/15) in women without NK elevation who were treated with anticoagulation and prednisone and 22.2% (2/9) in women with elevated CD56+ NK cells who were treated with postconception IVIg in addition to anticoagulation and prednisone. Significant reduction of preterm delivery and IUGR incidences of this study is noticed as compared to those of previous study without NK cell assessment . This finding leads to speculate that proper suppression of CD56+ NK cells during pregnancy, either by spontaneous down regulation or induced, may contribute favorable reproductive outcome.

Prednisone treatment has been reported to be related with a greater incidence of preterm delivery and a high frequency of premature rupture of membrane and associated preterm delivery . Total dose and duration of prednisone treatment in this study is lesser and shorter than previous reports of prednisone treatment. In our study, first trimester low dose prednisone treatment did not increase incidence of premature rupture of membrane. It is noteworthy that regardless of treatment protocol, preterm incidence of women with elevated NK cell level was higher than that of women with normal NK cell level.

Recently we reported that paternal lymphocyte immunization significantly suppresses peripheral blood NK cell cytotoxicity and its cell level . In this study, all study subjects received paternal lymphocyte immunization, however some proportion of women who received lymphocyte immunization, preconception anticoagulation and prednisone treatment still demonstrated elevated peripheral blood NK cell levels at the time of positive pregnancy test. It is speculated that insufficient immuno-suppression or maternal fetal histocompatibility could be related with elevated CD56+ NK cells.

In conclusion, women with elevated CD56+ NK cells while on anticoagulation and prednisone treatment after lymphocyte immunization, with the addition of IVIg treatment significantly improves reproductive outcome. Women with normal CD56+ NK cells and alloimmune recognition benefit from anticoagulation and prednisone treatment only. We speculate that NK cells or their products may play a major role in the pathogenesis of recurrent spontaneous abortions, or preterm delivery. Further study is needed to identify the mechanisms of IVIg action on down regulating NK cell numbers and NK cell cytotoxicity.

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Figure 1
The live birth rates of women with elevated NK cell level (>12%) who initiated post-conception IVIg treatment in addition to anticoagulation and prednisone (100.0%), women with normal NK cell levels (<12%) who continued anticoagulation and prednisone (83.3%) and women with elevated NK cell level (>12%) who continued anticoagulation and prednisone (33.3%) are plotted. These are significantly different (P=0.0065).

Table 1

Table 3

Table 4