Impact of histocompatibility antigens on pregnancy outcome Gungor Eroglu, MD: George Betz, MD: and Caprice Torregano, BS' N ew Orleans, Louisiana, and Denver, Colorado OBJECTIVE: The role of histocompatibility antigens in pregnancy outcome is controversial. This controversy may be because the initial studies were of small numbers or the patient groups were not homogeneous. The purpose of this study is to clarify these discrepant results by carrying out histocompatibility antigen typing and mixed lymphocyte culture on couples with idiopathic recurrent spontaneous abortion and by comparing results with those of fertile couples . STUDY DESIGN: Sixty couples with at least three spontaneous abortions and 60 normal couples with at least two successful pregnancies were included. Histocompatibility antigen typing and mixed lymphocyte culture were performed by using the standard techniques. The data were analyzed statistically with the Fisher exact test and x 2 • RESULTS: Our results failed to show any difference between normal and aborting couples with regard to HLA-A, HLA-B, and HLA-DR distribution or sharing or to mixed lymphocyte culture responsiveness . CONCLUSION: Our study, along with other studies, emphasizes that the histocompatibility antigen system does not have an impact on pregnancy outcome. (AM J OasTET GVNECOl 1992;166:1364-9.)
Key words: Recurrent spontaneous abortion , human leuk ocyte antigen sharing, reproductive immunology Successful reproduction in humans continues to be an immunologic enigma as the conceptus is a semiallogcnic graft with partially foreign , paternal histocompatibility antigens. There are studies':' in the literature suggesting that reproductive success is due to antigenic disparity between mating partners. This disparity is necessary so that the con ceptus will be immunogenic and stimulate recognition to evoke a protective mechanism against expected immunologic destruction. The role of antigenic disparity, or incompatibility, has been examined by determining sharing of histocompatibility antigens (HLA) between wife and husband. The majority of these studies were done on couples with idiopathic recurrent spontaneous abortion. Several studies reported increased sharing of HLA-A and HLA-B antigens between the partners of these couples.":" Furthermore , findings of hyporesponsiveness in maternalpaternal mixed lymphocyte culture implied that compatibility in the HLA-D locus may be the cause of recurrent spontaneous abortion. " These findings were supported by studies showing increased HLA-DR (serodefined antigens of the D locus) sharing in such couples.": 16.17Consequently, antigenic similarity (increased HLA sharing in any of the HLA loci) between husband and wife has also been proposed as a cause of recurrent
From the Departm ent of Med icine, Louisiana State University M edical Center: and the Department of Obstetrics and Gynecology, Colorad o Health Sciences Center and Kaiser Foundation.' R eceived for publication Ma» 15, 1991.. revised No vember 7, 1991 .. accepted November 30,1991. Reprint requests: Gungor Eroglu, MD, Department ofMedicine, LSU Medical Center, 1542 Tulane A ve., N ew Orleans, LA 70112-2822 . 6/1/35369
1364
spontaneous abortion. In recent literature, however, the number of studies refuting those findings has been increasing. l B·. ' Therefore the role of the HLA system in the outcome of pregnancy is co ntroversial. These incon sistencies ma y be explained by studies of patients with heterogeneous causes of recurrent spontaneous abortion or because of the limited size of some groups. The current study was undertaken to carry out HLAA, HLA-B, and HLA-DR typing and mixed lymphocyte culture on a large and homogeneous group with recurrent spontaneous abortion . These results were compared with those of normal couples with no history of spontaneous abortion. A compilation of studies from the literature is also presented .
Material and methods Patients. Couples with at least three spontaneous abortions and no living children in both Colorado and Louisiana were investigated thoroughly to rule out anatomic abnormalities of the uterus and genetic, endocrinologic, infectious, and autoimmune diseases. Fiftyfive white couples, four black couples, and one mixed (wife white, husband black) couple met the criteria and were included in the study. Controls. The same number of couples were matched with the aborting couples for age and race. These couples had at least two successful pregnancies and no history of spontaneous abor tion . Histocompatibility testing. HLA-A, HLA-B , and HLA-DR typing was performed with the standard National Institutes of Health technique described by Terasaki et al}6 with tissue typing tr ays obtained from One Lambda (Los Angeles). Gen Trak (Plymouth Meeting,
Histocompatibility antigens and pregnancy 1365
Volume 166 Number 5
Table I. HLA-A and HLA-B antigen frequencies (percent) in aborting and normal couples HLA type in A and B loci
HLA-A I 2 3 II 23 24 25 26 28 29 30 31 32 Blank HLA-B 7 8 13 14 18 27 35 37 38 39 41 44 49 50 51 52 53 55 57 60 61 62 Blank
Table II. HLA-DR antigen frequencies (percent) in aborting and normal couples
Aborting (60 couples) (% frequency)
Normal (60 couples) (% frequency)
27.9 46.7 27.5 16.4 4.9 22.1 8.2 5.7 4.9 5.3 7.3 4.1 8.9 12.3
30.7 44.3 35.2 6.8 2.3 17.5 3.4 3.4 6.8 8.0 9.1 5.7 10.2 9.8
22.1 22.1 5.7 7.4 8.2 8.2 22.1 2.5 4.1 5.7 0.0 22.1 3.3 2.5 4.9 0.8 3.3 2.5 4.1 6.6 4.6 16.4 9.0
20.5 20.5 5.7 6.8 11.4 9.1 23.9 2.3 2.3 4.5 2.3 26.1 l.l 2.3 5.7 2.3 4.5 2.3 6.8 9.1 4.5 13.6 7.5
HLA type in DR locus
HLA-DR I 2 3 4 5 6 7 8 9 10 II 13
Aborting (60 couples) (% frequency)
Normal (60 couples) (% frequency)
22.1 26.2 25.4 38.6 14.8 11.5 18.0 7.4 4.9 2.5 11.4 2.8
18.2 23.9 21.6 39.8 18.2 12.5 25.0 6.5 0.4 l.l 11.4 3.4
Chemical Company, New Haven, Conn.) with 100,000 responder and 100,000 irradiated stimulator cells in a volume of 200 f.L1. Media and autologous and pooled lymphocytes were used to be negative and positive controls. Cultures were incubated at 37° C with 100% humidity in a 5% carbon dioxide and air atmosphere for 5 days. Tritiated thymidine, specific activity 6.7 Ci/mmol (New England Nuclear, Boston) was then added, 1 u.Ci per well. After 12 hours of additional incubation, plates were harvested in a multiple harvester. The samples were then placed in vials containing scintillation fluid and the amount of radioactivity that was incorporated into the deoxyribonucleic acid of proliferating cells was determined by liquid scintillation counting. The results of mixed lymphocyte culture were expressed as relative response and were calculated by Relative response =
Pa.), and Pel-Freeze (Brown Deer, Wis.). B Lymphocytes for DR typing were prepared with the nylon-wool method." Mixed lymphocyte culture. One-way mixed lymphocyte culture was performed with traditional methods. Two couples were tested at a time, one from the aborting group and one from the cntrol group. A total of 15 different experiments were performed. Briefly, after purification of peripheral blood mononuclear cells, the samples were suspended at I x 106 in RPMI 1640 (GIBCO, Grand Island, N.Y.) containing 100 V/ml penicillin, 100 f.Lg/ml streptomycin, 2 mmol/L glutamine, and 10% pooled male serum. Stimulating cells were irradiated with a dose of 2000 rads to abolish their capacity to proliferate. Cultures were carried out in triplicate in round-bottomed microtiter trays (Linbro
Test counts/min Autologous counts/min Median response counts/min Autologous counts/min When the relative response ~20, mixed lymphocyte culture was accepted to be positive in another way, stimulation occurred. Statistical analysis. The data were analyzed with the Fisher exact test and X2 analysis. Results
HLA class I and II antigens (HLA-A, HLA-B and HLA-DR). The distribution of HLA-A, HLA-B, and HLA-DR antigens was analyzed in both aborting and normal couples. The frequency of antigens is shown in Tables I and II. There was no significant difference between the two groups. Also, no association between recurrent spontaneous abortion and a particular HLA
1366 Eroglu, Betz, and Torregano
May 1992 Am J Obstel Gynecol
160140,Q) (f)
0
120 -
0
c
o
0..
100'-
(f)
Q)
0:::: --+--J
o
Q)
0::::
00
60 40 -
8
8
00 0
00
8
00
000
00
00
00
000
8
00 0
20
8
§
0
80 -
0
0
0
Q)
>
0 0 0
00
8
0 0
0
000 ,...
00
0
o
Wx vs H Hx vs W Wx vs H Hx vs W
Aborting Couples
Normal Couples
Fig. 1. Mixed lymphocyte culture in 15 aborting and 15 normal couples. Reactivity was expressed as relative response. Positive response was acce pted when relative res ponse 2:20. Wife ( Wx ) versus husband (H); wife's irrad iated cells as stimulator, husband's cells as responder; husband (JIx) versus wife (W); hu sband's irradiated cells as stimulator, wife's cells as re sponder.
Table III. Number of H LA-A, HLA-B , and H LA-Dr antigens shared by 60 normal and 60 abo r ting cou ples Aborting couples No. of antigens shared
A,B loci 0 1 2 3 4 DR locus 0 I
2 A,B, and DR loci 0 I
2 3 4 5 6
No. and %
sharing
I
Norma l couples
No. and % not sharing
(60.0%) (58.3 %) (83.3%) (98.3%) (100.0 %)
24 (40 .0%) 25 (41.7 %) 10 (16.7%) 1 (1.7 %) 0 (0.0%)
36 35 50 59 60
45 (75.0%) 15 (25.0%) 0 (0.0% )
15 (25.0%) 45 (75.0%) 60 (100.0%)
18 24 15 3 0 0 0
42 36 45 57 60 60 60
(30.0%) (40.0%) (25.0%) (5.0%) (0.0%) (0.0%) (0.0%)
(70.0%) (60.0%) (75 .0%) (95 .0%) (100.0%) (100 .0%) (100 .0%)
No. and % sharing
22 34 4 0 0
(36.7%) (56 .7%) (6.7%) (0.0%) (0.0%)
I
N o. and % not sharing
(63.3%) (13.3%) (93.3%) (100 .0%) (100 .0%)
0.851 0.144 0.153 1.000 1.000
36 (60.0%) 23 (38.3%) I (1.7%)
24 (40 .0%) 37 (61.7 %) 59 (98 .3%)
0. /18 0.169 1.000
12 33 12 2 0 0 0
48 27 48 58 60 60 10
0.292 0.143 0.663 0.493
(20.0%) (55.0 %) (20.0%) (3.3%) (0.0%) (0.0%) (0.0%)
38 26 56 60 60
p*
(80 .0%) (45 .0%) (80.0%) (96. 7%) (100.0%) (100 .0%) (16.0%)
*F is her's exact te s t.
antigen was fou nd. Furthermore, no excess of materna l or pa te rnal homozygosity was observed. The number of sh ared H LA antige ns in both groups is show n in Table III. To accentua te th e distinction be tween abo rtin g and nor m al couples, the data were a nalyzed se parately fo r each locus and for all the loci
together. As illustrated , the results for eac h locu s were not di fferent fro m th ose fou nd with normal couples. In a co mbined analysis of HLA-A, HLA-B and HLADR , 30% (18/60) abortin g co u ples d id not share any antige n , 40% (24/60) sh ared o ne an tige n, 25% (15/60) two antige n s, and only 5% (3 /60) had thr ee antigen s
Histocompatibility antigens and pregnancy
Volume 16li Number 5
1367
Table IV. Prevalence of shared HLA antigens at the A. B, and DR loci in aborting and normal couples Ahorting couplr»
I
IfLA-B
Normal couples
I
Investigation
HLA-A
Group A Komlos et al.:' (1977) Ccrencer" (1979) Schacter'' (1979) Taylor and Faulk" (1981) Aoki (1982) Beer et al." (1983) Unander and Olding' (1983) Mclntyre and Faulk!" (1983) .Iohnson et al." (1984) Thomas et al. '3 (1985) Coulam et al. 14 (1987)* Group B Lauritsen et al." (1976) Caudle et al;'" (1983) Oskenberg et al." (1983) Nordlander et al." (1983) Cauchi et al." (1988) Sargent et al." (1988) Christiansen et al." (1989) Smith et al." (1988)t Eroglu et al. (1990)
11/22 (50.0',7,)
H/22 (36.4'lr)
1'\0
20/45 (44.4'/,) 12/39 (30.Wlr) 4/4 (100.0';')
17/45 (37.H'lr) 9/39 (23.I'X) :\/4 (75.0'lr)
NO l':D
20/26 (76.9%)
9110 (90.07
Key words: Recurrent spontaneous abortion , human leuk ocyte antigen sharing, reproductive immunology Successful reproduction in humans continues to be an immunologic enigma as the conceptus is a semiallogcnic graft with partially foreign , paternal histocompatibility antigens. There are studies':' in the literature suggesting that reproductive success is due to antigenic disparity between mating partners. This disparity is necessary so that the con ceptus will be immunogenic and stimulate recognition to evoke a protective mechanism against expected immunologic destruction. The role of antigenic disparity, or incompatibility, has been examined by determining sharing of histocompatibility antigens (HLA) between wife and husband. The majority of these studies were done on couples with idiopathic recurrent spontaneous abortion. Several studies reported increased sharing of HLA-A and HLA-B antigens between the partners of these couples.":" Furthermore , findings of hyporesponsiveness in maternalpaternal mixed lymphocyte culture implied that compatibility in the HLA-D locus may be the cause of recurrent spontaneous abortion. " These findings were supported by studies showing increased HLA-DR (serodefined antigens of the D locus) sharing in such couples.": 16.17Consequently, antigenic similarity (increased HLA sharing in any of the HLA loci) between husband and wife has also been proposed as a cause of recurrent
From the Departm ent of Med icine, Louisiana State University M edical Center: and the Department of Obstetrics and Gynecology, Colorad o Health Sciences Center and Kaiser Foundation.' R eceived for publication Ma» 15, 1991.. revised No vember 7, 1991 .. accepted November 30,1991. Reprint requests: Gungor Eroglu, MD, Department ofMedicine, LSU Medical Center, 1542 Tulane A ve., N ew Orleans, LA 70112-2822 . 6/1/35369
1364
spontaneous abortion. In recent literature, however, the number of studies refuting those findings has been increasing. l B·. ' Therefore the role of the HLA system in the outcome of pregnancy is co ntroversial. These incon sistencies ma y be explained by studies of patients with heterogeneous causes of recurrent spontaneous abortion or because of the limited size of some groups. The current study was undertaken to carry out HLAA, HLA-B, and HLA-DR typing and mixed lymphocyte culture on a large and homogeneous group with recurrent spontaneous abortion . These results were compared with those of normal couples with no history of spontaneous abortion. A compilation of studies from the literature is also presented .
Material and methods Patients. Couples with at least three spontaneous abortions and no living children in both Colorado and Louisiana were investigated thoroughly to rule out anatomic abnormalities of the uterus and genetic, endocrinologic, infectious, and autoimmune diseases. Fiftyfive white couples, four black couples, and one mixed (wife white, husband black) couple met the criteria and were included in the study. Controls. The same number of couples were matched with the aborting couples for age and race. These couples had at least two successful pregnancies and no history of spontaneous abor tion . Histocompatibility testing. HLA-A, HLA-B , and HLA-DR typing was performed with the standard National Institutes of Health technique described by Terasaki et al}6 with tissue typing tr ays obtained from One Lambda (Los Angeles). Gen Trak (Plymouth Meeting,
Histocompatibility antigens and pregnancy 1365
Volume 166 Number 5
Table I. HLA-A and HLA-B antigen frequencies (percent) in aborting and normal couples HLA type in A and B loci
HLA-A I 2 3 II 23 24 25 26 28 29 30 31 32 Blank HLA-B 7 8 13 14 18 27 35 37 38 39 41 44 49 50 51 52 53 55 57 60 61 62 Blank
Table II. HLA-DR antigen frequencies (percent) in aborting and normal couples
Aborting (60 couples) (% frequency)
Normal (60 couples) (% frequency)
27.9 46.7 27.5 16.4 4.9 22.1 8.2 5.7 4.9 5.3 7.3 4.1 8.9 12.3
30.7 44.3 35.2 6.8 2.3 17.5 3.4 3.4 6.8 8.0 9.1 5.7 10.2 9.8
22.1 22.1 5.7 7.4 8.2 8.2 22.1 2.5 4.1 5.7 0.0 22.1 3.3 2.5 4.9 0.8 3.3 2.5 4.1 6.6 4.6 16.4 9.0
20.5 20.5 5.7 6.8 11.4 9.1 23.9 2.3 2.3 4.5 2.3 26.1 l.l 2.3 5.7 2.3 4.5 2.3 6.8 9.1 4.5 13.6 7.5
HLA type in DR locus
HLA-DR I 2 3 4 5 6 7 8 9 10 II 13
Aborting (60 couples) (% frequency)
Normal (60 couples) (% frequency)
22.1 26.2 25.4 38.6 14.8 11.5 18.0 7.4 4.9 2.5 11.4 2.8
18.2 23.9 21.6 39.8 18.2 12.5 25.0 6.5 0.4 l.l 11.4 3.4
Chemical Company, New Haven, Conn.) with 100,000 responder and 100,000 irradiated stimulator cells in a volume of 200 f.L1. Media and autologous and pooled lymphocytes were used to be negative and positive controls. Cultures were incubated at 37° C with 100% humidity in a 5% carbon dioxide and air atmosphere for 5 days. Tritiated thymidine, specific activity 6.7 Ci/mmol (New England Nuclear, Boston) was then added, 1 u.Ci per well. After 12 hours of additional incubation, plates were harvested in a multiple harvester. The samples were then placed in vials containing scintillation fluid and the amount of radioactivity that was incorporated into the deoxyribonucleic acid of proliferating cells was determined by liquid scintillation counting. The results of mixed lymphocyte culture were expressed as relative response and were calculated by Relative response =
Pa.), and Pel-Freeze (Brown Deer, Wis.). B Lymphocytes for DR typing were prepared with the nylon-wool method." Mixed lymphocyte culture. One-way mixed lymphocyte culture was performed with traditional methods. Two couples were tested at a time, one from the aborting group and one from the cntrol group. A total of 15 different experiments were performed. Briefly, after purification of peripheral blood mononuclear cells, the samples were suspended at I x 106 in RPMI 1640 (GIBCO, Grand Island, N.Y.) containing 100 V/ml penicillin, 100 f.Lg/ml streptomycin, 2 mmol/L glutamine, and 10% pooled male serum. Stimulating cells were irradiated with a dose of 2000 rads to abolish their capacity to proliferate. Cultures were carried out in triplicate in round-bottomed microtiter trays (Linbro
Test counts/min Autologous counts/min Median response counts/min Autologous counts/min When the relative response ~20, mixed lymphocyte culture was accepted to be positive in another way, stimulation occurred. Statistical analysis. The data were analyzed with the Fisher exact test and X2 analysis. Results
HLA class I and II antigens (HLA-A, HLA-B and HLA-DR). The distribution of HLA-A, HLA-B, and HLA-DR antigens was analyzed in both aborting and normal couples. The frequency of antigens is shown in Tables I and II. There was no significant difference between the two groups. Also, no association between recurrent spontaneous abortion and a particular HLA
1366 Eroglu, Betz, and Torregano
May 1992 Am J Obstel Gynecol
160140,Q) (f)
0
120 -
0
c
o
0..
100'-
(f)
Q)
0:::: --+--J
o
Q)
0::::
00
60 40 -
8
8
00 0
00
8
00
000
00
00
00
000
8
00 0
20
8
§
0
80 -
0
0
0
Q)
>
0 0 0
00
8
0 0
0
000 ,...
00
0
o
Wx vs H Hx vs W Wx vs H Hx vs W
Aborting Couples
Normal Couples
Fig. 1. Mixed lymphocyte culture in 15 aborting and 15 normal couples. Reactivity was expressed as relative response. Positive response was acce pted when relative res ponse 2:20. Wife ( Wx ) versus husband (H); wife's irrad iated cells as stimulator, husband's cells as responder; husband (JIx) versus wife (W); hu sband's irradiated cells as stimulator, wife's cells as re sponder.
Table III. Number of H LA-A, HLA-B , and H LA-Dr antigens shared by 60 normal and 60 abo r ting cou ples Aborting couples No. of antigens shared
A,B loci 0 1 2 3 4 DR locus 0 I
2 A,B, and DR loci 0 I
2 3 4 5 6
No. and %
sharing
I
Norma l couples
No. and % not sharing
(60.0%) (58.3 %) (83.3%) (98.3%) (100.0 %)
24 (40 .0%) 25 (41.7 %) 10 (16.7%) 1 (1.7 %) 0 (0.0%)
36 35 50 59 60
45 (75.0%) 15 (25.0%) 0 (0.0% )
15 (25.0%) 45 (75.0%) 60 (100.0%)
18 24 15 3 0 0 0
42 36 45 57 60 60 60
(30.0%) (40.0%) (25.0%) (5.0%) (0.0%) (0.0%) (0.0%)
(70.0%) (60.0%) (75 .0%) (95 .0%) (100.0%) (100 .0%) (100 .0%)
No. and % sharing
22 34 4 0 0
(36.7%) (56 .7%) (6.7%) (0.0%) (0.0%)
I
N o. and % not sharing
(63.3%) (13.3%) (93.3%) (100 .0%) (100 .0%)
0.851 0.144 0.153 1.000 1.000
36 (60.0%) 23 (38.3%) I (1.7%)
24 (40 .0%) 37 (61.7 %) 59 (98 .3%)
0. /18 0.169 1.000
12 33 12 2 0 0 0
48 27 48 58 60 60 10
0.292 0.143 0.663 0.493
(20.0%) (55.0 %) (20.0%) (3.3%) (0.0%) (0.0%) (0.0%)
38 26 56 60 60
p*
(80 .0%) (45 .0%) (80.0%) (96. 7%) (100.0%) (100 .0%) (16.0%)
*F is her's exact te s t.
antigen was fou nd. Furthermore, no excess of materna l or pa te rnal homozygosity was observed. The number of sh ared H LA antige ns in both groups is show n in Table III. To accentua te th e distinction be tween abo rtin g and nor m al couples, the data were a nalyzed se parately fo r each locus and for all the loci
together. As illustrated , the results for eac h locu s were not di fferent fro m th ose fou nd with normal couples. In a co mbined analysis of HLA-A, HLA-B and HLADR , 30% (18/60) abortin g co u ples d id not share any antige n , 40% (24/60) sh ared o ne an tige n, 25% (15/60) two antige n s, and only 5% (3 /60) had thr ee antigen s
Histocompatibility antigens and pregnancy
Volume 16li Number 5
1367
Table IV. Prevalence of shared HLA antigens at the A. B, and DR loci in aborting and normal couples Ahorting couplr»
I
IfLA-B
Normal couples
I
Investigation
HLA-A
Group A Komlos et al.:' (1977) Ccrencer" (1979) Schacter'' (1979) Taylor and Faulk" (1981) Aoki (1982) Beer et al." (1983) Unander and Olding' (1983) Mclntyre and Faulk!" (1983) .Iohnson et al." (1984) Thomas et al. '3 (1985) Coulam et al. 14 (1987)* Group B Lauritsen et al." (1976) Caudle et al;'" (1983) Oskenberg et al." (1983) Nordlander et al." (1983) Cauchi et al." (1988) Sargent et al." (1988) Christiansen et al." (1989) Smith et al." (1988)t Eroglu et al. (1990)
11/22 (50.0',7,)
H/22 (36.4'lr)
1'\0
20/45 (44.4'/,) 12/39 (30.Wlr) 4/4 (100.0';')
17/45 (37.H'lr) 9/39 (23.I'X) :\/4 (75.0'lr)
NO l':D
20/26 (76.9%)
9110 (90.07
