hla sharing, anti-paternal cytotoxic antibodies and mlr blocking factors in women with recurrent...

/. Obstet. Gyiiaecol. Res. Vol. 22, No. 2: 177-183 1996

HLA Sharing, Anti-Paternal Cytotoxic Antibodies and MLR Blocking Factors in Women with Recurrent Spontaneous Abortion

Raj Kishore,') Suraksha Agarwal,') A. Halder,') V. Das,') B. R. K. S h ~ k l a , ~ ) and S. S. Agarwal')

1 ) 2) 3 )

Department of Medical Genetics, Sanjay Gatidhi Postgraduate Institute of Medical Sciences, Lucknozu, India Department of Obstetiics and Gynecology, KGMC, Ltrckriow, India Department of .4ntlrropology, Uiiiveisity of Lucknozt , Lucknozu, India

Abstract

Objective: T o study the prevalence of HLA sharing between spouses and its correlation with presence of antipaternal cytotoxic antibody (APCA) and mixed lymphocyte reaction (MLR) blocking factors in recurrent spontaneous aborters (RSA).

Study was carried out at Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGI), Lucknow, from 1988-1992. Hundred coupes with 3 or more consecutive recurrent spontaneous abortions and equal number of age, parity and ethinically matched normal controls were selected for studying HLA, APCA and MLR blocking factors. Meta-analysis was performed using standard formula and significance was tested by Chi-square analysis.

Significant HLA sharing was observed in couples with RSA at A and DR loci compared to normal controls (p < 0.001). Twenty-seven point eight percent of couples with RSA were positive for APCA compared to 49% of controls (p < 0.01). MLR blocking factors were detected in 26% couples with RSA compared to 78% in controls (p < 0.001). An inverse correlation between HLA sharing and APCA and MLR positivity was demonstrated.

The study supports that greater HLA sharing between spouses, associated with lack of an appropriate immune response to them could be responsible for RSA.

Design:

Results:

Conclusion:

Key words:, spontaneous aborters, antipaternal cytotoxic antibody, mixed lymphocyte reaction-blocking factor, HLA sharing

~~

Received: Apr. 27, 1995 Accepted: Jan. 12, 1996 Reprint request to: Dr. Suraksha Agarwal, Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknom, India

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Introduction

Successful reproduction continues to re- main an immunological enigma. Survival of the conceptus despite being a semiallogenic graft bearing paternal histocompatibility antigens is a mystery.'x2) It has been suggested that an appropriate maternal immune response to paternally derived HLA antigens is essential for induction of a state of immunological tolerance leading to success of pregnancy. The immunological factors contribut- ing to success of pregnancy include development of, 1) NILR blocking factors, and 2) APCA antibodies, besides production of cytokines that favour implantation p ro~ess .~ ) As a corollary it has been hypothesized that greater HLA sharing between spouses and impaired development of APCA and MLR blocking factors could be responsible for recurrent spontaneous abortion in humans.'-") Significance of HLA sharing in increasing the risk of foetal loss in humans, however, remains controversial. Initial reports indi- cated the presence of increased HLA sharing in couples with RSA. This included greater sharing at HLA-B and DR-loci as well as at A and DR Some investigators have reported greater sharing in primary aborters compared to secondary aborters.+ But there are other studies which have not found any increase in HLA

The exact interaction between the feto-placental unit and maternal immune system re- main unclear. Although the measurement of APCA may be a marker for appropriate maternal recognition of feto-paternal antigens, the absence of APCA is of unknown significance as only 20-50% of multiparous women develop the antipaternal cytotoxic antibody.23) Similarly, the role of blocking antibody is not always specific for the male partner's lymphocytes and thus difficult to interpret.'") This lead us to find out the correlation of HLA sharing, APCA and MLR blocking factor positivity.

While there have been several studies on HLA sharing between spouses, studies on the correlation of HLA sharing with APCA and MLR-blocking factor positivity have not

178

come to our attention. Our study, first on the Indian population, supports the results of other studies conducted on Caucasian, Japa- nese and Chinese populations, that increased HLA sharing amongst spouses of primary RSA may be pathophysiologically linked to the occurrence of RSA.

Material and Methods

Patients A total of 212 women with 3 or more con-

secutive abortions, without any live birth, were referred to the Genetics Clinic at SGPGI, from 1988-1993. They were investigated to exclude known causes of recurrent abortion. In 100 couples no anatomic, hor- monal, infectious, autoimmune or genetic cause could be found. These 100 couples were studied for HLA sharing. Seventy-nine of them were investigated for presence of APCA and 50 for mixed lymphocyte reaction blocking factors. The difference in the number of couples investigated for the above 3 parameters was because the study was conducted in 3 different phases. There was no selection bias. For comparison, 100 ethnically matched couples with 3 or more live born children without any abortion, foetal loss or treatment for sterility, were studied as controls. The mean number of pregnancies in the aborter group was 3.8 f 0.3 (range 3-8) and in controls 3.3 f 0.4 (range 3-5). Mean age of women at the time of study in RSA group was 23.7 f 0.4 years while in controls it was 25.6 f 0.3 years.

H L A Typing Both spouses were typed for Class-I and

Class-I1 HLA antigens using microlympho- cytotoxicity test as per NIH protocol.25) In brief mononuclear cells (PBL) were sepa- rated on Ficoll-Hypaque density gradient. The B cells were enriched by nylon wool col- umn. Typing was performed on Terasaki plates coated with HLA antisera of high "r" value obtained from various sources. Antisera for Aw 68, 69; Bw 73, 75, 77; Cw 8, 9, 11 and DR 14, 15, 16 and 17 were not available. For each specificity a minimum of 3 antisera were

ROLE O F HLA, APCA A N D RlLR I N RS.4 P A T I E N T S

included in the typing tray. Results

HLA Sharing The degree of antigen sharing at HLA-A,

B, C, and DR loci in the RSA and control couples is given in Table 1 . It shows that there was no antigen sharing in 37%, 1 antigen sharing in 53% and 2 antigen sharing in 10% of RSA couples at A-locus, compared to 75%, 24% and 1 % in controls, respectively. Statistically the sharing of HLA antigens in the RSA group at HLA-A locus was significantly higher compared to controls (p < 0.001). No significant difference was observed in antigen sharing at B and C loci. For all the Class-I loci (A, B, C) taken together the sharing was statistically significant (p < 0.05). At Class-I1 DR locus, sharing of 0, 1 and 2 antigens in the RSA group was 36%, 49% and 15% compared to 67%, 32% and 1 %, respectively in the control group. In all, 64% of the couples with RSA shared 1 or 2 antigens at DR locus compared to only 33% in the control group (p < 0.001).

A P C A Assay Anti-paternal cytotoxic antibodies (APCA)

were tested by cross-match between maternal serum and paternal PBL according to ex- tended NIH protocol.2') Serum was serially diluted upto 1:64. Cross-match test was performed at 22"C, 4°C and 37°C against total mononuclear cells, T cells and B cells. A positive result was recorded when 50% or more cell death was observed at all the three temperatures for both T and B lymphocytes.

MLR Blocking Factor Assaji One way MLR in presence of either 20%

heat inactivated maternal serum or a pooled control AB serum, employing paternal cells as stimulators and maternal cells as respond- ers was carried out to detect the presence of MLR blocking factors. Percent inhibition of MLR response in maternal serum (test serum) compared to control serum (pooled AB serum) was calculated as follows:2,s)

mean cpm in test serum 100 - x 100 mean cpm in control serum

Reduction in thymidine incorporation by 30% or more in maternal serum compared to control serum was taken as indicative of the presence of MLR blocking factor. This cut off value was derived by plotting a graph for control women under similar conditions.

Met a - A nal-y sis A total of 27 published studies on HLA

sharing were identified by literature search through Index Medicus and Medline search for the period 1979-1993 and reference to cross references. The analysis was restricted to only those studies which met the following 3 inclusion criteria; (1) primary aborters with

Table 1. HLA sharing in recurrent aboters and controls

RSA (100) Controls (100)

X' P Locus No. of antigens shared No. of antigens shared

0 1 2 0 1 2

A. 37 53 10 75 24 1

B. 48 46 6 50 1 7 3

C. 52 45 2 54 42 4

(37.0%) (53.0%) (10.0%) (75%) (24.0%) (1.0%) 11.82 < 0.001

(48.0%) (46.0",0) (6.0%) (50%) (47%) (3%) 0.075 > 0.05

(53.0%) (46.0%) (6.0%) (54%) (42.00;) (4.0%) 1.79 > 0.05

Class-I 46.0% 48.0% 6.00/0 59.6% 37.6% 2.6% 4.56 < 0.05 combined

DR 36 49 15 67 32 1 (36.0%) (49.0%) (15.0%) (67.0%) (32.0%) (1.6%) 14.69 < 0.001

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Table 2a. Meta-analysis at HLA locus in different studies

A locus B locus

Control Control Investigators RSA p k ' ) OR RSA P (x2) OR

Gerencer (1979) 20/45 20/79 < 0.02 2.36 17/45 20/79 NS 1.79

Bolis (1989) 12/28 2/28 < 0,001 9.75 9/28 5/28 < 0.05 2.17 Oksenberg (1983) 11/33 13/30 NS 0.65 7/33 9/30 NS 0.77

Beer (1983) 164/325 178/408 <0.05 1.31 102/325 99/408 NS 1.12 Ho (1991) 68/91 28/51 <0.05 2.42 24/91 14/51 NS 0.94 Lauritsen (1986) 12/29 23/35 NS 0.36 6/29 11/35 NS 0.56 Aoki (1983) 20/26 26/45 < 0.02 2.69 9/26 10/45 NS 1.85 Johnson (1977) 17/28 21/41 NS 1.47 11/28 10/41 < 0.05 2.0 Cauchi, et nl. (1988) 32/83 52/100 NS 1.0 21/88 41/100 NS 1.0 Christiansen, et nl. (1988) 20/56 20/33 NS 1.0 18/56 11/33 NS 1.0 Eroglu, et al. (1990) 24/60 26/60 NS 0.0 19/60 16/60 NS 0.0 Our study 55/79 24/60 <0.01 3.43 31/79 16/60 NS 1.5

Total 400/804 406/910 NS 1.32 243/809 246/910 NS 0.40

Table 2b. Meta-analysis at DR-locus

Investigators RSA Control p O R

is in accordance with results of the meta- analysis.

Thomas (1985) 55/10') 33/150 < 0.01 3.6 Ho (1991) 43/91 18/51 NS 1.79 Beer (1983) S S / l l S 25/114 < 0.01 3.6 Aoki (1983) 22/26 11/45 < 0.001 17.0

Total 175/341 87/360 < 0.001 6.8 Our study 60/79 10/60 < 0.001 15.7

3 or more first trimester abortions, (2) age, parity and ethinically matched controls and (3) the minimum of 25 couples in both the groups. Out of these only 11 studies were found to meet the inclusion criteria for HLA sharing at HLA-A and B loci and 4 for HLA DR locus.

The results of the meta-analysis at HLA-A and B loci are given in Table 2a. Five of the 11 studies showed significant sharing at A locus and 2 at B locus. Pooling of the data however, showed that sharing at A and B loci was nonsignificant. Results of our study, however, showed significant sharing at A locus (p < 0.01) with odd ratio of 3.43 and no significant sharing at B locus.

Results of meta-analysis at DR locus (Table 2b) showed that in 3 out of the 4 studies the odd ratios were statistically significant. In the fourth study the odd ratio was 1.79, which was in the same direction. Our results of increased DR sharing with odd ratio of 4.1

APCA Positivity and Correlation zuith H L A Sharing

APCA was tested in 79 women with RSA. Twenty-two (27.9%)) of them were found to be positive for APCA. In comparison 49 of the 100 control women (49%) were positive for APCA. The difference was statistically significant (p < 0.001). An attempt was made to correlate APCA positivity with HLA sharing both at Class-I and Class-I1 loci. The data is shown in Table 3. There was an inverse relationship betwen HLA antigen sharing and APCA positivity, for both the RSA and control groups.

MLR Blocking Factors and Correlation zuith H L A Sharing

Presence of mixed lymphocyte reaction blocking factors was studied in 50 women with RSA and 50 controls. Thirteen out of 50 women with RSA (26%) showed the presence of MLR blocking factors as defined under methods. In comparison 39 of the 50 control women (78%) were positive for the MLR blocking factors (p < 0.001).

Correlation of MLR blocking factors with HLA sharing is also shown in Table 3. It is evident from the data that absence of blocking factors correlated with higher degree of

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ROLE O F HL.A, .APCA .AND hlLR I N R S A PATIENTS

Table 3. Correlation of HLA-Class-I and Class-I1 sharing with APCA and MLR blocking factor positivity

RSA Controls

% APCA MLR 0/6 APCA MLR sharing tested + Y E tested + VE sharing tested + VE tested + V E (n-100) (n=79) (n = 50) (n= 100) ( n = 100) (n = 25)

Class-I 0 15.0 12 8 (36.36) 10 6 (12) 58.0 58 31 (65.30) 20 20 (100) 1-2 44.0 34 10 (45) 20 4 (8.0) 30.0 30 16 (32.65) 20 16 (80) 3- 4 36.0 28 4 (16.1) 15 3 (6.0) 12.0 12 2 (40) 10 3 (30.9) 2 4 5.0 5 - - - 5 - - _. - _ - _ _

Total 100 79 22 (27.8) 50 13 (26.0) 100 49 (49.0) 50 39 (78)

Class-I I 0 36.0 25 12 (54.5) 13 7 (53.0) 67.0 67 39 (79.6) 20 20 (100.0) 1 49.0 40 9 (40.9) 27 5 (18.5) 32.2 32 10 (20.4) 20 16 (80) 2 15.0 15 1 (4.5) 10 1 (10.0) 1 3 _ - 10 3 (30)

Total 100 79 22 (44.0) 50 13 (26.0) - 100 49 (49.0) SO 39 (78)

CL-I + CL-I1 79 22 (44.0) 50 13 (26.0) - 100 49 (49.0) 50 39 (78) X’ 14.49 23.64 P < 0.001 < 0.001

HLA sharing.

Discussion

The study showed that there is significantly greater sharing of HLA antigens at A and DR loci in couples with primary RSA (3 or more spontaneous abortions in first trimester without a known cause) compared to age, parity and ethinically matched controls. This correlated with lower positivity for APCA and MLR blocking factors in women with RSX.

The results of HLA sharing studies in couples with RSA have been controversial. Increased HLA sharing has been reported at HLA B and DR loci14~“) as well as at A and DR loci.’2~’’~’5) Some investigators have reported greater sharing in primary (without any live birth) aborters compared to secondary (with one or more live births along with recurrent abortions) aborters.’’ Other group of investigators have reported no significant HLA sharing in the RSA Differences in different studies have been at- tributed to following factors: (1) number of antigens tested and those present in the population, ( 2 ) inclusion criteria and sample size, (3) stratification of RSA couples and (4)

genetic and demographic background of control and study groups. It has been recom- mended in a recent study,26) that there should be atleast 25 couples in each group to achieve 90% confidence limit if the study group has a probability of HLA sharing of atleast two times that of control group.

The meta-analysis carried out by us has shown that sharing at HLA A and DR loci is statistically significant in primary aborters. The results of our study are in conformation with these findings.

Our results show that presence of APCA was significantly lower (27%) in the RSA group compared to controls (49%). R’lany other authors have also reported higher prevalence of APCA in control subjects compared to women with RSA.27,2S’ The source of allosensitization, however, is still debatable since neither the trophoblast nor the pre-implantation embryo expresses HLA antigens. It is likely that HLA sensitization is caused by passage of foetal lymphocytes into maternal circulation, an event known to occur as early as first tr ime~ter.?~) The mother seems to respond to the antigens presented by pas- senger leukocytes either by immunity or by tolerance.’,2s) Our results show that absence of APCA is correlated with greater HLA

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R . KlSHORE ET AL.

sharing. In the present study search for MLR

blocking factors was carried out in both the study and control groups. Presence of MLR blocking factor was found to be inversely correlated with HLA sharing. Our study, to the best of our knowledge is the only study that demonstrates an inverse correlation beween MLR-bf positivity and increased HLA sharing (Table 3). Our study revealed that in both RSA and control groups positivity for MLR- bf decreases with increasing number of HLA antigens shared, which suggests that lack of an appropriate immune response in RSA patients might be due to improper recognition of the semi-allogenic foetal allograft.

However, certain points are still not clear; for instance why blocking factors often do not appear until late in the first trimester of first pregnancy; why agammaglobulinemic women have normal pregnancy and how do ani- mals which fail to develop immunoglobulins or mount a humoral immune response have successful pregnancies. The antibody to the placental trophoblast that has been thought to mediate protection of the pregnancy is not anti-HLA antibody. It is directed against an allotypic cross reacting antigen of 80 kD. Testing for this antibody might provide a more specific assay for appropriate allogenic response.

Acknowledgment

Technical assistance of Mr. R. C. Mishra and Mr. K. K. Srivastava and computer assistance of Mr. Anil Kumar Srivastava is gratefully acknowledged. This study was funded by Council of Science and Technol- ogy, U.P., India, and Indian Council of Medical Research.

Grant-Aid of Equipment and Technical Cooperation by Japan International Coop- eration Agency provided the intra structure for these study.

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