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Common HLA antigens in couples with repeated abortions
Common
HLA Antigens
in Couples
with Repeated
Abortions
Received August IO. iY7h Seventy-nine couples, including cases of single and repeated abortions. hydatidiform moles, and a control group of fertile couples with no history of abortions. were tested for HLA antigens. locus A and B. A significantly higher frequency of common HLA antigens was shared by both members of the couples with repeated abortion\ a\ compared to controls. The possibility of a higher incidence of homozygotic fetuses for certain HLA antigens which may lead to abortions is discussed.
INTRODUCTION
Various hypotheses have been suggested to explain the protective mechanism involved in the survival of the allogeneic fetus (1 .2.5). Paternally derived transplantation antigens have been shown to induce maternal cellular and/or humoral immunologic reactions. The relatively high incidence of feto-maternal stimulation with respect to the HLA system could raise the possibility that these reactions may be associated with significant incompatibility selection against the fetus (3). However, maternal HLA antibodies are in general harmless to the fetus and not associated with higher rates of stillbirth or abortions. There is also not yet any evidence for a selection which favors or disfavors antigenic disparity with respect to the HLA system (3). Various investigators have used mixed wife-husband leukocyte cultures to study feto-maternal interactions with respect to the HLA system in pregnancy. This system has been used as an in isitvo model for testing histocompatibility (8-13). In the present investigation, HLA antigens for locus A and B were serologically determined in couples with disturbed pregnancies, and the results were compared to fertile couples with no history of abortion. MATERIALS
AND METHODS
Sixty-one women with single abortions. repeated abortions. or hydatidiform moles were typed together with their husbands for HLA alleles of the A and B loci. Eighteen couples with two or more children (normal deliveries) and with no history of disturbed pregnancies served as controls (Table 1). Samples of unheparinized blood for antibody determinations were taken from each woman, and heparinized blood for lymphocyte preparation was taken from both members of each couple. I Address correspondence tion and Fetal Development,
to Dr. Luise Komlos, Hasharon Hospital,
B. Gattegno Petah-Tiqva,
Research Israel.
Institute
of Human
Reproduc-
330 Copyright All
rights
0
1977
of reproduction
by
Academic in any
Press.
Inc.
form
reserved.
ISSN
,“,%,-I??‘)
HLA
ANTIGENS
IN
COUPLES
WITH
TABLE COUPLES
TYPED
Group
Number of couples
Control One abortion Repeated abortions Hydatidiform moles
18 13 23 25
Totals
79
331
ABORTIONS
I
FOR HLA
AN.TIGENS
Couples sharing common HLA antigens Number
(%)
x2 P
7 9 17 14
38 70 77 56
N.S.” N.S. p < 0.05 N.S.
47
60
” N.S., not significant.
HLA Typing
HLA typing was performed routinely by the two-stage microlympocytotoxicity method (16). For a majority of the cases the couples were typed for 24 HLA alleles, 10 for locus A and 14 for locus B. Each specificity was tested for at least three antisera, and the total number of antisera used varied between 90 and 120. The antisera were supplied by the National Institute of Health Serum Bank and a TABLE HLA Couples
TYPING
IN COUPLES
Normaf deliveries
2
WITH
REPEATED
ABORTIONS
Abortions
HLA Type”
None
2 CIP
Al. A2: BW35 (vY Al, A9; B12, BWl8
2
1
2 (LII)
Al 1; BW35 (v) Al. AW19; BW35, B7
3
None
2 (I)
Al, All; BW35 (v) Al, AIO; B5
4
I
2 (1)
AZ. A10: B12, B13 (v) A3, All; BW40. B13
3 (1.11)
Al, A2: BW35 (v) AlO. A2; BW35
4 (13)
All. A9; BW3.5 (v) Al, AZ; BW35, B5 AIO. A3; BW35, BW40 (v) AIO. A2; B12, BW40
5 6
2
7
None
2 (I)
8
2
2 (1)
AIO;
BW17. B14 (v) BW17
9
3
2 (I)
Al, Al 1; B12 A2, A9; BW5. BW17
10
None
3 (LIU
A2, A9; B5, B12 Al. All; BW35
11
None
2 (1)
A9, A2; B14 (v) A9. A10
Couples 12
Normal
deliveries
None
15
None
lh
None
IX
None None
2 (II)
i (1.11) 4 (1.11)
Type”
,410:
A::
B8 (L) BWiF
-22: 42. AIO:
BW40. BIJ
41.
None
Ii
I?
HLA
A:. A IO:
B7 IV)
H14. BW30
nw7’
.4.i.
All:
Ai.
4’:
.4’;
BW3I i\j B12. RI? Bl.3.
BI:
A IO. .4 I I 19
20
I None
2 (l,II)
A2. ,410 (\‘! A IO
x (II)
.42,
49: 71
I
22
None
13
I
A’); B7. BI1 ,\, RI:. I311
3 (I)
A I: Ai:
BI4(\! BIZ
2 ~11.111)
‘4 I : HWIT .4l. AIX: RIJ
2 II)
iio: Al.
AZ:
,\ 1
BW?’ BWl7.
BWdO
n Wife listed first. ’ Trimester of pregnancy in which abortions occurred in parenthesez. ’ Couples sharing common HLA antigens indicated by (v).
serum panel from our own laboratory. The antigen specificities typed for locus A were HLA-Al, 2, 3.9, 10. 11, 28, 29, W19, and W31 (the last could be identified in a number of cases). The following specificities were typed for locus B: HLA-BS, 7, 8, 12, 13, 14, W17, W35, W15, W27, W40, W16, W18. and W22. Crossmatch Tests Crossmatch tests between maternal serum and paternal lymphocytes formed in all cases by the two-stage cytotoxicity method (16).
were per-
RESULTS
As can be seen from Tables 1 and 2. a significantly higher percentage of common HLA antigens (~~0.05) was shared by the couples with repeated abortions when compared to the control group. A higher percentage of common HLA antigens could also be found in the groups with only one abortion and with hydatidiform moles, although the differences between the test and control groups
HLA
ANTIGENS
IN
COUPLES
WITH
TABLE DISTRIBUTION COUPLES
OF COMMON HLA IN THE CONTROL
3
ANTIGENS
AND
BY BOTH MEMBERS ABORTIONS GROUPS
HLA
antigens
Locus A One antigen 1 2 3 9 10 Total Two antigens 2. 28 Total Locus B One antigen 13 w 17 w 35 Total Locus A and B Two and three antigens Al; BW35 A2; BW35 A9: BW35 A9; B14 AIO; BW40 Al, A2; BW17 Total
Number
Repeated (No. (%)
1
(14) -
1 2
(14)
1 1
(15) (1%
(28)
1 I
(15) (15)
1
(%‘o)
(22)
4 2 I I 1 9
(12) (6) (6) (6)
(52) (6) (6) (18)
1 1 3 5
(30)
1
-
(14) -
1 3
abortions = 17)
Number
(14) -
I
OF
SHARED
REPEATED
Controls (No. = 7) Common
333
ABORTIONS
1 1
-
(14)
(4-3
3
(6) (6) (6) (18)
were not statistically significant. Fifty percent of the women with repeated abortions miscarried in the first trimester of pregnancy (Table 2), usually in the 10th or 12th week. In nine cases the abortions occurred in the first and second trimesters of pregnancy, usually in the 16th or 18th week. In only three cases did abortions occur in the second or third trimester of pregnancy (Table 2). Sixty-five percent of the women with repeated abortions have had two abortions, 30% have had three abortions, and I had eight abortions. No correlation between the number of normal deliveries, the number of repeated abortions, and the frequency of common antigens was found, except in one case (No. 20, Table 2) with eight abortions, no normal deliveries, and two common antigens (locus A and B) shared by both members of the couple. Concerning the distribution of common wifehusband HLA antigens for each locus (Table 3), the highest frequency of common antigens was observed in the group of repeated abortions at locus A (53% as compared to 28% in the control group). Similarly, for locus B the percentage of common antigens was higher in the repeated abortions group than in the controls (30% as compared to 15% in the control group). In contrast, the frequency of two
334
KOMLOS
EI .41.
and three common antigens for one or two loci was higher in the control group than in the group with repeated abortions (Table 3). By crossmatch tests, lymphocytotoxic maternal antibodies against paternal lymphocytes were found in two cases in the control group (I 19) and in four cases with repeated abortions t 17%). DISCUSSION The significantly higher percentage of couples sharing common HLA antigens in the group with repeated abortions as compared to the control group suggests the possibility of an increased incidence of homozygotic fetuses in these cases of disturbed pregnancy. Experimental work which favors the concept that strong selective pressures contribute to a superior reproductive proficiency of heterozygotes comes mainly from histocompatibility studies in animals (15). In man, the overall frequency of homozygotes for the HLA system is very low (for Caucasians, 18% for locus A and 10% for locus B). and this indicates the remarkable level of variability for the HLA system in comparison with other known polymorphisms (3. 4. 6). A comparative study of healthy and cancer patients in comparable age groups (7) suggests that the elderly healthy people had a larger number of HLA specificities than did the cancer patients of all age groups and the young people. There was an apparent correlation between heterozygosity in the HLA system. survival to old age. and decreased susceptibility to cancer. This study. however. does not take into account the possible fertility differences. That gametic selection of specific HLA haplotypes occurs was suggested by Lindblom rr al. (14). In a study on couples with unexplained infertility, the authors observed that certain haplotypes which in a control group of fertile couples showed negative association between A- and B-locus alleles seemed to OCCUI more frequently in the group of infertile couples. The results of our study suggest that gene dose effects of certain HLA antigens probably intluenced by specific haplotype associations may cause functional disturbances in pregnancy leading to abortion. The different associations of two or three common HLA antigens in the control group and in the repeated abortions group (Table 3) suggest the selective advantage ( 14) of certain antigen combinations in fertility. Further studies on HLA antigens from the offspring of couples with repeated abortions will help to clarify this problem. REFERENCES I. 2. 3. 4. 5. 6. 7.
Beer. A. S.. and Billingham, R. E.. .Sc,i. Anrer. 230. 36. 1971. Billingham. R. E.. Amer. .I. Ohstet. Gytrrc,cd. 111. 469. 1971 Bodmer. W. F.. Ntrtrfw 237, 139. 1972. But-net. F. M.. Ntrrurr 245, 3.59, 1973. Clarke, B., and Kirby. D. R. S., Ncrtrtrr 211, 999. 1966. Dausset. J.. Acta Patid. Micro&. Stand. 78. 529. 1970. Gerkins. V. R.. Ting. A.. Menek. H. T.. Casagrande. J. T.. Terasaki, P. I.. and Pike, Nut/. C’ar,c~. Inst. 52. 1909. 1974. 8. Halbrecht. I.. and Komlos. L.. Ohstrt. Gy~eco/. 31. 173. 196X. 9. Halbrecht. I.. and Komlos. L., In (A. lngelman-Sundberg and N. 0. Lunnel. Eds.). Problems in Fertility” pp. 215-221, Plenum Press. London, 1971.
M. C.. .1.
“Current
HLA
10. Il. 12. 13.
ANTIGENS
IN
COUPLES
WITH
ABORTIONS
335
Halbrecht, I., and Komlos L., Zsr. J. Med. Sci. 10, 1100, 1974. Kanazawa, K., and Takeuki, S., Acta Obstet. Gynecol. Japarl 19, 176, 1972. Komlos, L.. Halbrecht, I., and Ben-Efraim, S., Reproduction 1, 252, 1974. Lewis, J., Whang. J., Nagel, B., Oppenhein, J. J., and Perry, S., Amer. J. Obstet. Gynecd. 96, 287 1966. 14. Lindblom, J. B., Friberg. J., Hogman, C. F., and Gemze11, C., Tissue Antigens 2, 352, 1972. 15. Palm, J., Transpl. Proc. 2, 162, 1970. 16. Ray, J. G.. Jr.. Hare, D. B., Pedersen, P. D., and Kayhoe, D. E., “Manual of Tissue Typing Techniques,” pp. 20-22. Transplantation and Immunology Branch N.I.A.I.D., Bethesda, 1974.
HLA Antigens
in Couples
with Repeated
Abortions
Received August IO. iY7h Seventy-nine couples, including cases of single and repeated abortions. hydatidiform moles, and a control group of fertile couples with no history of abortions. were tested for HLA antigens. locus A and B. A significantly higher frequency of common HLA antigens was shared by both members of the couples with repeated abortion\ a\ compared to controls. The possibility of a higher incidence of homozygotic fetuses for certain HLA antigens which may lead to abortions is discussed.
INTRODUCTION
Various hypotheses have been suggested to explain the protective mechanism involved in the survival of the allogeneic fetus (1 .2.5). Paternally derived transplantation antigens have been shown to induce maternal cellular and/or humoral immunologic reactions. The relatively high incidence of feto-maternal stimulation with respect to the HLA system could raise the possibility that these reactions may be associated with significant incompatibility selection against the fetus (3). However, maternal HLA antibodies are in general harmless to the fetus and not associated with higher rates of stillbirth or abortions. There is also not yet any evidence for a selection which favors or disfavors antigenic disparity with respect to the HLA system (3). Various investigators have used mixed wife-husband leukocyte cultures to study feto-maternal interactions with respect to the HLA system in pregnancy. This system has been used as an in isitvo model for testing histocompatibility (8-13). In the present investigation, HLA antigens for locus A and B were serologically determined in couples with disturbed pregnancies, and the results were compared to fertile couples with no history of abortion. MATERIALS
AND METHODS
Sixty-one women with single abortions. repeated abortions. or hydatidiform moles were typed together with their husbands for HLA alleles of the A and B loci. Eighteen couples with two or more children (normal deliveries) and with no history of disturbed pregnancies served as controls (Table 1). Samples of unheparinized blood for antibody determinations were taken from each woman, and heparinized blood for lymphocyte preparation was taken from both members of each couple. I Address correspondence tion and Fetal Development,
to Dr. Luise Komlos, Hasharon Hospital,
B. Gattegno Petah-Tiqva,
Research Israel.
Institute
of Human
Reproduc-
330 Copyright All
rights
0
1977
of reproduction
by
Academic in any
Press.
Inc.
form
reserved.
ISSN
,“,%,-I??‘)
HLA
ANTIGENS
IN
COUPLES
WITH
TABLE COUPLES
TYPED
Group
Number of couples
Control One abortion Repeated abortions Hydatidiform moles
18 13 23 25
Totals
79
331
ABORTIONS
I
FOR HLA
AN.TIGENS
Couples sharing common HLA antigens Number
(%)
x2 P
7 9 17 14
38 70 77 56
N.S.” N.S. p < 0.05 N.S.
47
60
” N.S., not significant.
HLA Typing
HLA typing was performed routinely by the two-stage microlympocytotoxicity method (16). For a majority of the cases the couples were typed for 24 HLA alleles, 10 for locus A and 14 for locus B. Each specificity was tested for at least three antisera, and the total number of antisera used varied between 90 and 120. The antisera were supplied by the National Institute of Health Serum Bank and a TABLE HLA Couples
TYPING
IN COUPLES
Normaf deliveries
2
WITH
REPEATED
ABORTIONS
Abortions
HLA Type”
None
2 CIP
Al. A2: BW35 (vY Al, A9; B12, BWl8
2
1
2 (LII)
Al 1; BW35 (v) Al. AW19; BW35, B7
3
None
2 (I)
Al, All; BW35 (v) Al, AIO; B5
4
I
2 (1)
AZ. A10: B12, B13 (v) A3, All; BW40. B13
3 (1.11)
Al, A2: BW35 (v) AlO. A2; BW35
4 (13)
All. A9; BW3.5 (v) Al, AZ; BW35, B5 AIO. A3; BW35, BW40 (v) AIO. A2; B12, BW40
5 6
2
7
None
2 (I)
8
2
2 (1)
AIO;
BW17. B14 (v) BW17
9
3
2 (I)
Al, Al 1; B12 A2, A9; BW5. BW17
10
None
3 (LIU
A2, A9; B5, B12 Al. All; BW35
11
None
2 (1)
A9, A2; B14 (v) A9. A10
Couples 12
Normal
deliveries
None
15
None
lh
None
IX
None None
2 (II)
i (1.11) 4 (1.11)
Type”
,410:
A::
B8 (L) BWiF
-22: 42. AIO:
BW40. BIJ
41.
None
Ii
I?
HLA
A:. A IO:
B7 IV)
H14. BW30
nw7’
.4.i.
All:
Ai.
4’:
.4’;
BW3I i\j B12. RI? Bl.3.
BI:
A IO. .4 I I 19
20
I None
2 (l,II)
A2. ,410 (\‘! A IO
x (II)
.42,
49: 71
I
22
None
13
I
A’); B7. BI1 ,\, RI:. I311
3 (I)
A I: Ai:
BI4(\! BIZ
2 ~11.111)
‘4 I : HWIT .4l. AIX: RIJ
2 II)
iio: Al.
AZ:
,\ 1
BW?’ BWl7.
BWdO
n Wife listed first. ’ Trimester of pregnancy in which abortions occurred in parenthesez. ’ Couples sharing common HLA antigens indicated by (v).
serum panel from our own laboratory. The antigen specificities typed for locus A were HLA-Al, 2, 3.9, 10. 11, 28, 29, W19, and W31 (the last could be identified in a number of cases). The following specificities were typed for locus B: HLA-BS, 7, 8, 12, 13, 14, W17, W35, W15, W27, W40, W16, W18. and W22. Crossmatch Tests Crossmatch tests between maternal serum and paternal lymphocytes formed in all cases by the two-stage cytotoxicity method (16).
were per-
RESULTS
As can be seen from Tables 1 and 2. a significantly higher percentage of common HLA antigens (~~0.05) was shared by the couples with repeated abortions when compared to the control group. A higher percentage of common HLA antigens could also be found in the groups with only one abortion and with hydatidiform moles, although the differences between the test and control groups
HLA
ANTIGENS
IN
COUPLES
WITH
TABLE DISTRIBUTION COUPLES
OF COMMON HLA IN THE CONTROL
3
ANTIGENS
AND
BY BOTH MEMBERS ABORTIONS GROUPS
HLA
antigens
Locus A One antigen 1 2 3 9 10 Total Two antigens 2. 28 Total Locus B One antigen 13 w 17 w 35 Total Locus A and B Two and three antigens Al; BW35 A2; BW35 A9: BW35 A9; B14 AIO; BW40 Al, A2; BW17 Total
Number
Repeated (No. (%)
1
(14) -
1 2
(14)
1 1
(15) (1%
(28)
1 I
(15) (15)
1
(%‘o)
(22)
4 2 I I 1 9
(12) (6) (6) (6)
(52) (6) (6) (18)
1 1 3 5
(30)
1
-
(14) -
1 3
abortions = 17)
Number
(14) -
I
OF
SHARED
REPEATED
Controls (No. = 7) Common
333
ABORTIONS
1 1
-
(14)
(4-3
3
(6) (6) (6) (18)
were not statistically significant. Fifty percent of the women with repeated abortions miscarried in the first trimester of pregnancy (Table 2), usually in the 10th or 12th week. In nine cases the abortions occurred in the first and second trimesters of pregnancy, usually in the 16th or 18th week. In only three cases did abortions occur in the second or third trimester of pregnancy (Table 2). Sixty-five percent of the women with repeated abortions have had two abortions, 30% have had three abortions, and I had eight abortions. No correlation between the number of normal deliveries, the number of repeated abortions, and the frequency of common antigens was found, except in one case (No. 20, Table 2) with eight abortions, no normal deliveries, and two common antigens (locus A and B) shared by both members of the couple. Concerning the distribution of common wifehusband HLA antigens for each locus (Table 3), the highest frequency of common antigens was observed in the group of repeated abortions at locus A (53% as compared to 28% in the control group). Similarly, for locus B the percentage of common antigens was higher in the repeated abortions group than in the controls (30% as compared to 15% in the control group). In contrast, the frequency of two
334
KOMLOS
EI .41.
and three common antigens for one or two loci was higher in the control group than in the group with repeated abortions (Table 3). By crossmatch tests, lymphocytotoxic maternal antibodies against paternal lymphocytes were found in two cases in the control group (I 19) and in four cases with repeated abortions t 17%). DISCUSSION The significantly higher percentage of couples sharing common HLA antigens in the group with repeated abortions as compared to the control group suggests the possibility of an increased incidence of homozygotic fetuses in these cases of disturbed pregnancy. Experimental work which favors the concept that strong selective pressures contribute to a superior reproductive proficiency of heterozygotes comes mainly from histocompatibility studies in animals (15). In man, the overall frequency of homozygotes for the HLA system is very low (for Caucasians, 18% for locus A and 10% for locus B). and this indicates the remarkable level of variability for the HLA system in comparison with other known polymorphisms (3. 4. 6). A comparative study of healthy and cancer patients in comparable age groups (7) suggests that the elderly healthy people had a larger number of HLA specificities than did the cancer patients of all age groups and the young people. There was an apparent correlation between heterozygosity in the HLA system. survival to old age. and decreased susceptibility to cancer. This study. however. does not take into account the possible fertility differences. That gametic selection of specific HLA haplotypes occurs was suggested by Lindblom rr al. (14). In a study on couples with unexplained infertility, the authors observed that certain haplotypes which in a control group of fertile couples showed negative association between A- and B-locus alleles seemed to OCCUI more frequently in the group of infertile couples. The results of our study suggest that gene dose effects of certain HLA antigens probably intluenced by specific haplotype associations may cause functional disturbances in pregnancy leading to abortion. The different associations of two or three common HLA antigens in the control group and in the repeated abortions group (Table 3) suggest the selective advantage ( 14) of certain antigen combinations in fertility. Further studies on HLA antigens from the offspring of couples with repeated abortions will help to clarify this problem. REFERENCES I. 2. 3. 4. 5. 6. 7.
Beer. A. S.. and Billingham, R. E.. .Sc,i. Anrer. 230. 36. 1971. Billingham. R. E.. Amer. .I. Ohstet. Gytrrc,cd. 111. 469. 1971 Bodmer. W. F.. Ntrtrfw 237, 139. 1972. But-net. F. M.. Ntrrurr 245, 3.59, 1973. Clarke, B., and Kirby. D. R. S., Ncrtrtrr 211, 999. 1966. Dausset. J.. Acta Patid. Micro&. Stand. 78. 529. 1970. Gerkins. V. R.. Ting. A.. Menek. H. T.. Casagrande. J. T.. Terasaki, P. I.. and Pike, Nut/. C’ar,c~. Inst. 52. 1909. 1974. 8. Halbrecht. I.. and Komlos. L.. Ohstrt. Gy~eco/. 31. 173. 196X. 9. Halbrecht. I.. and Komlos. L., In (A. lngelman-Sundberg and N. 0. Lunnel. Eds.). Problems in Fertility” pp. 215-221, Plenum Press. London, 1971.
M. C.. .1.
“Current
HLA
10. Il. 12. 13.
ANTIGENS
IN
COUPLES
WITH
ABORTIONS
335
Halbrecht, I., and Komlos L., Zsr. J. Med. Sci. 10, 1100, 1974. Kanazawa, K., and Takeuki, S., Acta Obstet. Gynecol. Japarl 19, 176, 1972. Komlos, L.. Halbrecht, I., and Ben-Efraim, S., Reproduction 1, 252, 1974. Lewis, J., Whang. J., Nagel, B., Oppenhein, J. J., and Perry, S., Amer. J. Obstet. Gynecd. 96, 287 1966. 14. Lindblom, J. B., Friberg. J., Hogman, C. F., and Gemze11, C., Tissue Antigens 2, 352, 1972. 15. Palm, J., Transpl. Proc. 2, 162, 1970. 16. Ray, J. G.. Jr.. Hare, D. B., Pedersen, P. D., and Kayhoe, D. E., “Manual of Tissue Typing Techniques,” pp. 20-22. Transplantation and Immunology Branch N.I.A.I.D., Bethesda, 1974.