Major histocompatibility complex antigens, maternal and paternal immune responses, and chronic habitual abortions in humans

Major histocompatibility complex antigens, maternal and paternal immune responses, and chronic habitual abortions in humans ALAN E. BEER, M.D. JAMES F. QUEBBEMAN, M.S., M.A . .JONATHAN W. T. AYERS, M.D. RICHARD F. HAINES, PH.D. Ann Arbor, Michigan Women with recurrent consecutive spontaneous abortions of unknown etiology compared with those with recurrent spontaneous abortions of known etiology had a significantly increased frequency of sharing HLA antigens at the A, 8, and D/DR loci with their spouses. This major histocompatibility complex (MHC) homozygosity was generally associated with female (responder) and male (stimuiator) hyporeactivily in mixed iymphocyte cuiture reactions. The resuits indicate that MHC homozygosity between spouses is associated with postfertilization pregnancy wastage in humans. (Atv1. J. 08STET. GYt'.JECOL.141:987, 1981.)

SINCE THE EARLIEST DAYS of tissue typing and definition of the antigenic determinants of the major histocompatibility complex (MHC) in man, it became strikingly clear that maternal sensitization to paternal MHC antigens is a common event during successful pregnancies and that the immune effectors produced, specifically antipaternal antileukocyte alloantibodies, have no detrimentai effect on pregnancy outcome.'-" These observations and others have refuted the notion that the femaie subject is immunoiogicaiiy unaware of the presence and activities of allogeneic cells or tissues within the reproductive tract. During mating, impiantation, and placentation and throughout gestation, fetal and MHC antigens are presented to the mother in a unique manner, and she does respond to these antigens.4 This evidence has allowed us to conclude that

From the Department of Obstetrics and Gynecology, Women's Hospital, and Department of Surgery, University of Michigan Medical Center. Supported in part by National Institutes of Health Grant No. HD14234-02.

Presented by invitatio"T"i at the Second Colnlli.,-utd Annual Meeting of the American Gynecological Society and American Association of Obstetricians and Gynecologists, Montreal, Quebec, Canada, May 20-23, 1981. Reprint requests: Dr. Alan E. Beer, Department of Obstetrics and Gynecology, L2122 Women's Hospital, The University of Michigan, Ann Arbor, Michigan 48109. 0002-9378/81/240987+13$01.30/0© 1981 The C. V. Mosby Co.

active immune responses on the part of the female subject following early recognitive events during pregnancy play an important if not an essential role in conferring selective benefits upon the fetoplacental unit from the time of implantation to parturition.~· 6 The mechanisms contributing to the immunologic success of the fetoplacemal unit as an allograft are outlined in Tabie 1. 7 The immune response capabilities of the human are exquisitely compiex. The effectors, both antibodies and lymphocytes, are numerous and belong to many classes and subclasses. Immune responses are under srricr genetic control. Near the centromere on the short arm of human chromosome number six is an area invoiving from 300 to I ,000 separate genetic loci with as many different alleles at some loci. 8 Genes within the MHC code for cell surbce molecules present on all nucleated cells of the body. These are called histocompatibility or HLA antigens. Loci within this polymorphic region determine erythrocyte antigens and complement components, and other loci are involved in cell-mediated lysis of virus-infected cells, cell-mediated lysis of chemically modified celils, macrophage-lymphocyte cooperation in antibody productiont T and B cell interaction in antibody production, and production ofT cell helper and suppressor t::tctor. 9 Much progress has been made in our understanding of the biologic significance of the MHC. There have been many explanations as to why it is advantageous

987

988

Beer et al.

lltTcmher

\m.

54.9

(Jb,t~!.

1:~, i~1!i~

t;,

t>e<

ol

52.7 43.0

lSI

3rdParty ~

Cells

Stimulator

Responder

J.

j+PHA

O

3

3

Fig. l. One-way MLC responses of three unrelated third-party cell populations to lymphocyte antigens of Patient B. M. Control panels indicate mitogen responses and responses to alloantigens for the same third-party cell populations.

Table I. Mechanisms or factors proposed to explain the nonrejection of allogeneic fetoplacental units l. Complete separation of maternal and fetal blOod circula-

tions 2. Afferent blockade of the immunologic reflex arc a. Impairment of lymphatic drainage from placenta by decidual tissue b. Avoidance of lymphatic vessels and lymph nodes by invading trophoblast 3. Immunologic barrier or buffer zone at maternal/fetal tissue interface resulting from a. Adaptive failure of trophoblast to synthesize or express alloantigens in immunogenetically effective manner (intrinsically determined privileged tissue) b. Modulation of antigenic expression by trophoblast in response to maternal antibodies (extrinsically determined privileged tissue) 4. Masking of surface alloantigens on the trophoblast cells a. By own synthesis of sialomucin coat that discourages or prevents interaction with maternal lymphocytes b. As consequence of the passive acquisition or absorption of agents that result in "masking" i. by transferrin that binds to specific receptors on trophoblast cells ii. by antibodies or antibody/antigen complexes that bind specifically or nonspecifically (via Fe receptors) to trophoblast surface 5. Specific binding, internalization, and inactivation (digestion) of maternal immunologic effector agents by trophoblast 6. Synthesis by syncytial trophoblast, and maintenance in high local concentration, of hormones and other agents which fulfill an immunosuppressive role, e.g., progesterone, estrogens, chorionic gonadotropin, cortisol-binding globulin 7. Production by fetus of immunosuppressive agents that enter maternal circulation, e.g., alpha fetoprotein, factor released from stimulated fetal lymphocytes 8. Production by the mother of immunoregulatory agents that confer protection upon the fetoplacental unit a. Increased synthesis of adrenal corticosteroids, special plasma proteins (pregnancy-zone or PZ proteins), early pregnancy factor, alpha-2 globulins b. Synthesis of "blocking" antibodies c. Generation of suppressor cells d. Inversion ofT and B cell numbers in bloodstream

for the species to possess extreme degrees of genetic diversity or genetic polymorphism of the MHC. What are the reproductive advantages and/or disadvantages of MHC homozygosity or heterozygosity with regard to the pregnant female and the fetoplacental unit? Results from well-designed studies in inbred animals show that the advantages are real: I. There are intense selective pressures operating during gestation against individuals that are homozygous with their mother with regard to MHC antigens~ 10 2. There is decreased reproductive efficiency when MHC homozygous young are gestated in homozygous mothers. 3. There is selective elimination of MHC homozygotes, either prenatally or early postnatally, as a result of maternal immunologic responses to fetal antigens not associated with the MHC. 11 Does this clear-cut advantage of heterozygotes in laboratory populations of inbred animals reflect an important, naturally occurring phenomenon in outbred populations including humans? Does genetic compatibility (homozygosity) with regard to serologically defined MHC antigens between the female and the male and/or alterations in immunologic responses on the part of the female to paternal MHC antigens have any relationship to pregnancy wastage? Two groups of patients were studied to address these questions: ( l) chronic habitual aborters of diagnosed etiology and (2) chronic habitual aborters of unknown etiology.

Material and methods Clinical evaluation. Twenty-six couples with a history of multiple, consecutive, spontaneous abortions were studied. Since karyotypic abnormalities are associated with spontaneous abortions, only karyotypically normal couples whose pregnancy losses were histologi-

MHC, immune responses, and habitual abortion

Volume Ill

989

:'\lumber~

34.9

30

X

Gl

"C

..: c 0

·;:

:;"' E ·;: en

10-1 I ~ I I I Stimulator Responder

8.2 5.5

t

+PHA

3rd Party

Q

Fig. 2. One-way MLC responses of patient D. W. to alloantigens presented by her spouse or by an unrelated third party. The control panel indicates mitogen responsiveness of the patient's lymphocytes.

cally documented were included. Clinical evaluation to rule out structural (miillerian). endocrinologic, and infectious etiologies is outlined in Table II. The diagnosis of cervical incompetence was a radiographic one, made at the time of hysterosalpingography. Cervical canals that measured more than 4 mm in width during the proliferative phase of the menstrual cycle were designated incompetent. This correlated consistently with the clinical problem in each patient. Couples were screened for serum and cervical mucus antispermatozoal antibodies according to the techniques of Menge, 12 since it has been shown that the presence of these antibodies is associated with an increased incidence of abortion once a pregnancy is achieved. 13 Those with antibodies were excluded from the study. Couples whose medical workup was normal by the above-descrihed standards were designated our experimental group, and those with a documented etiology for their abortions, as controls. Tissue typing. Tissue typing tor the HLA system of antigens was performed with the use of lymphocytotoxicity principles developed by Terasaki and McCielland.14 The actual procedure used is a slight modification of that known as the National Institutes of Health techniqueY' The major distinction is that the incubation times of the reactants have been increased by 50% in an attempt to minimize false negative reactions. Specifically, lymphocytes and antiserum are incubated

Table II. Clinical evaluation of couples experiencing chronic habitual abortions A. Uterine evaluation l. Ultrasound 2. Hysterosalpingogram B. Genetic evaluation 1. Karyotyping of woman and man 2. Q- and R-bandin!! assays 3. Family studies where indicated C. Microbiologic evaluation 1. A.erobic cultures, cervix 2. Anaerobic cultures, cervix 3. Chlamydia! cultures, cervix 4. Toxoplasmosis and mycoplasma serologic screen D. Endocrinologic evaluation l. 2-hour postprandial blood sugar 2. Thyroid function, triiodothyronine and thyroxine 3. Prolactin determination (midfollicular, 8 AM fasting) 4. Luteal phase evaluation a. Hormones, serum progesterone on menses days -4, -7, and -10 b. Endometrial biopsy, menses day -2 E. Serum and cervical mucus antisperm antibody testing

for 45 minutes, rather than 30, and the incubation following the addition of complement is extended from 60 to 90 minutes. Currently, the Tissue Typing Laboratory is capable of determining the following HLA specificities: HLA-A I, A2, A3, All, Aw23, Aw24, A25, A26, A28, A29, Aw30, Aw3l, Aw32, Aw33, Aw34, Aw36, HLA-Bw4. Bw6, B7,

990

Beer et al.

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Table Ill. Clinical data of control and experimental couples with two or more consecutive spontaneous abortions Age of woman

No. of abortilmS

Gestational weeks

Couple

()'r)

Control: H.C+R

32

4

18-22

M.L+S C.J+T

34 27

3 4

6-12 16-34

D.M+W H.C+P

28 33

4 3

6-10 14-20

H.D+C

31

4

14-28

S.M+M E.C+M R.B+D H.T+M A.L+T

33 30 30 31 35

4

3 3 5 3

8-16 6-20 9-ll 6-ll 16-30

P.G+R

30

5

6-14

G.L+W

33

4

9-11

Q.C+j P.C+G H.E+R

31 29 31

2 5 3

8-10 6-9 7-9

32 28 25 30 32 34 29 41 35 29

4 5 3 3 4 12 5 7 5

8-12 6-9 8-IO 6-8 7-8 5-8 7-8 6-9 6-9 7-8

Living

Diagnosis Incompetent cervix; luteal phase defect Bicornuate uterus Hypoplastic right uterine horn; cervical incompetence Luteal phase defect; anovulation Cervical laceration; cervical incomperenee Cervical laceration; incompetent cervix Hypoplastic uterus Cervical laceration with incompetence Luteal phase defect; anovulation Luteal phase defect Septate uterus; recurrent placental villi tis Elevated prolactin; uterine septum; luteal phase defect Septate uterus; luteal phase defect; elevated prolactin Anovulation; luteal phase defect Hypothyroid; luteal phase defect Endometriosis; luteal phase defect; endometrial filling defect

children 1 (First) 0 0 1 (First) I (Former spouse) 1 (First) I (First) 0 0 0 1 (Former spouse) 0 1 (Last) 0 0

Experimental: B.J+G F.J+S H.D+R W.N+M B.L+W W.D+O G.J+M S.J+J H.P+W M.D+B

5

1 (First) I (Last) 0 0 1 (First) 0 1 (First) 0 0 0

WBC: White blood cells.

B8, Bl3, Bl4, Bl8, B27, Bw35, B37, Bw4l, Bw42, Bw46, Bw49, Bw50, Bw51, Bw52, Bw53, Bw54, Bw56, Bw57, Bw58, Bw60, Bw61, Bw62, Bw63, HLA-Cw I, Cw2, Cw3, Cw4, HLA-DRl, DR2, DR3, DR4, DR5, DR6, DR7, DRS, MTI, MT2, MT3, and MT4. Not all specificities can be determined with the same degree of ease because of considerable differences in the number of different antisera available that are capable of measuring a given specificity. Mixed lymphocyte culture (MLC). One-way MLCs 16 were performed in Falcon flat-bottomed microtiter plates with the use of RPMI 1640 containing 20% (v/v) pooled human male AB serum, I ,000 UI ml of penicillin, 100 #Lg/ml of streptomycin, and 4 mM L-glutamine. White blood cells were obtained from heparinized venous blood by Ficoll-Hypaque density centrifugationP Cultures were set up in triplicate with the use of 5 X 104 total responding lymphocytes and 2 X 105 stimulator

lymphocytes per 0.2 ml culture. Stimulator cells were treated previously with mitomycin C at a concentration of 0.03 mg per 2 x 106 lymphocytes per milliliter of RPM! 1640 containing penicillin-streptomycin. Mitogen-stimulated cultures contained 1 J.Lg of phytohem~ agglutinin (PHA) per welL MLCs were incubated at 37o C in a humidified atmosphere of 5% carbon dioxide for 7 days followed by a 7-hour period during which the cells were exposed to 1 #LCi per well of 3 H-methylthyrnidine. Following this pulse, cultures were processed for liquid scintillation counting by precipitation with a mini-MASH sample harvester. The samples were then placed in minivials and the amount of radioactivity that was incorporated into each culture was determined by liquid scintilJation counting. The following stimulator-responder cell cultures were included: femalestimulator-female responder; female stimulator-female responder plus PHA; male stimulator-female responder;

Volume HI

MHC, immune responses, and habitual abortion

Nun1ber ~

Blood type

I

Blood transfusion

1'vfan

l\o

0

A,

No

0 A,

0 0

Yes

0 0

0 0

Yes

A,

A,

No No No No No

A, A,

B A,

A

A

A A,

A 0

No

0

B

No

0

0

No

A, A,

A,

Yes

No

No No

No No No No No No No No No No

B

Az

A2 0 A, AI 0

A, A, A 0

Woman

Remarks

Anti-LeA Anti-LeB Anti-Kell Anti-E Anti-LeA Anti-LeB

A, A, A, A,B B 0

A, 0

A, A, 0 A,

WBC immunized WBC immunized WBC immunized

third-party stimulator-female responder; male stimulator-male responder; male stimulator-male responder plus PHA; female stimulator-male responder; thirdparty stimulator-male responder: third-party stimulator-third-party responder; third-party stimulatorthird-party responder plus PHA; female stimulatorthird-party responder; and male stimulator-thirdparty responder. Data are expressed as a stimulation index (Sl) after subtraction of background counts according to the following formula: average counts per minute per culture SI = average counts per mmute . per contro1 cu Iture , where the control culture is the appropriate syngeneic cell combination (i.e., female stimulator-female responder). Detection of antisperm antibodies. The methods

991

used for detection of antibodies against sperm cells follow the general procedures described in a World Health Organization publication.'s Serum antibody titers were determined by the tray agglutination technique of Friberg and the complement-dependent sperm immobilization technique of Isojima modified for use in a microtiter plate. Midcyde cervical mucus was analyzed for antisperm actiYity bv the spermcervical mucus penetration technique of' Kramer with the use of rectangular-shaped glass capillaries and rhe sperm immobilization test. For the latteJ' method the cervical mucus is first solubilized by either mechanical or enzymatic means. Sperm cells of the male partners were also evaluated for penetration of' donor cervical mucus derived from estrous female bmines. w White blood cell sensitization. Patients demonstrating significant sharing of antigens between spouses at the HLA-A, B, C, and D/DR loci and female hyporesponsiveness in one-way MLC to spouse antigens were treated as follows: I . Approximately 30 ml of blood was collected from the male into Vacutainers containing sodinm heparin. White blood cells were isolated, with a sterile technique. by Ficoli-Hypaque centrifugation ' 7 and washed three or four times with RPMI 1640. 2. The cells wne counted with a hemocytometer and resuspended in a small volume of bacteriostatic sodium chloride for injection. :L Then 0.1 ml aliquots of this <:ell suspension were injected intradermally on the forearm(s) of the woman. StatisticaJ analysis of data. Evaluation of HLA antigen sharing between the experimental group and the control group was reported with the use of the data for the A locus only. Inclusion of data regarding the B locus did not alter the results obtained. A 2-by-2 contingency table was constructed to assess the probability that the antigen matching observed in the experimental group was different from that observed in the control group by chi-square analysis. In addition, the Fisher exact test was performed with the use ot this table to assess the likelihood that the observed cell frequencies could have occurred hv chance alone.

Results Clinical profile~>. The clinical data of the control and experimental couples are summarized in Table IlL The ages of the women in the control and experimental groups were not different, 31.1 ± 2.2 years and 3 I .5 ± -t.5 years. The numbers of abortions in the experimental group were significantly greater than in the control group, 5.:3 ± 2.6and 3.7 ± 0.9, respectively. Of interest are the earlier abortions in the expt·rimental

992

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Beer et al.

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Table IV. Frequency of HLA antigens in Caucasians

A

B

34 36 43 42

23 25 26

28 29 30

13

41

14

45

27 37 38

48 49 50

39

52 53

31 32 33 55 56

ll

24

18

7

51

8

62

35

2

3

47 54

57 58

44

60

59

c DR

Cl

C2 (6)

61

63 C8

C5 C6

8

9

5

C3 C4 3 4

C7 2

7

10

group, 6.6 to 8.9 weeks, in comparison to those in the control group, 9.7 to 16.7 weeks. The numbers of living children were not significantly different in the two groups and 10 of 12 were first pregnancies. All abortions were consecutive. The distribution of blood groups of couples did not appear to be skewed. The high incidence of mi.illerian (structural) abnormalities, cervical incompetence, and luteal phase defects in the control group is understandable in that these couples were referred to our tertiary center from a six-state area, many after thorough local workups. No couples with antispermatozoal antibodies in the woman or man or couples with karyotypic abnormalities were included. Couples with repeated losses of karyotypically abnormal fetuses were also excluded. Three women in the control group demonstrated red blood cell antibodies whereas none in the experimental group did. The antibodies found have not correlated with early pregnancy wastage with the exceptions of anti- Kell and anti-E; the man of couple C.J+ Tis Kelland E antigen negative, however, and the sensitization most likelv resulted from prior blood transfusions. Sharing of HLA antigens. In the population at large, although no appropriate studies are available on the frequency of shared HL-A determinants between spouses, some conclusions can be drawn from the gene frequency studies in patients typed for tissue transplantation (Table IV). It is apparent that only a few of the antigens have a frequency greater than 30%. Most have an antigen frequency of one to 10 per 100.2° Table V compares the degree of HLA A, B, C, and D/DR antigen sharing in experimental couples with no documented cause for abortion and control couples in

whom an etiology has been demonstrated. Each couple in the experimental group shares A locus antigens. Apart from A-26, all the shared antigens are highfrequency antigens. Control couples appear to share less than the anticipated number of A locus antigens. Four of the 10 experimental couples share B locus antigens, while none is shared in the control group. The gene frequency of these shared B locus antigens is less than the gene frequency of shared A locus antigens. Eight of 10 experimental couples and three of 12 control couples share D/DR locus antigens. This high degree of HLA antigen sharing at the A and B loci in experimental couples as compared to control couples is highly significant (p < 0.001). Further analysis indicates that the probability of obtaining these data on sharing at the A and B loci is less than 1110,000 by chance alone. Analysis of D/DR locus antigen sharing is complicated by the high blank rate and the lack of agreement on the specificities of the antisera used for typing. Approximately 20% of patients following one pregnancy show antipaternal antileukocyte antibodies as evidenced by a positive leukocyte cross match. Three of 12 control patients and one of l 0 experimental patients demonstrated such antibodies. These antileukocyte antibodies did not correlate with the patient's blood transfusion history. Since genes within the D/DR locus determine MLC responses, MLC responses of peripheral blood leukocytes were studied. Table VI lists the results of one-way MLC responses between spouses and third-party HLA incompatible subjects. All but one control, P.G+R, showed excellent stimulation indices when female lymphocytes were challenged by mitomycin-treated male

Volume I ~I "iumher ~

MHC, immune responses, and habitual abortion

stimulator cells. In the experimental group, by contrast, lymphocytes in six of 10 women were specifically hyporesponsive to male stimulator cells and all but one showed good responses to third-party HLA incompatible donors. The MLC responses of experimental subjects were lower than those in control subjects; however, because of wide variability in background counts, these results are difficult to evaluate statistically. The degree of HLA antigen sharing at the D/DR locus was not always predictive on a case-to-case basis of the male stimulator-female responder results. We have asked if a certain MHC phenotype of the m:1IP with lvmnhocvte cell ------- rmtrtnPr -- ----- m:1v ----; hP - - associated -- - - ' • surface determinants that are poor stimulators in MLC reactions. The data presented in Fig. 1 raise this possibility. When peripheral blood lymphocytes of male B. M. were used as stimulators with MHC and DR locus-incompatible third-party responders, the responses were consistently less than when various combinations of the MHC typed responders were placed together in culture. In another study of a couple in our experimental group, unexpected findings emerged. D. W. is a woman who experienced 12 consecutive documented spontaneous abortions with the same spouse. She and her husband share antigens at the A, B, and D/DR loci. Their cross match is negative, showing no antipaternal antileukocyte activity. In one-way MLC reactions, she showed hyporesponsiveness to her husband's lymphocytes as stimulators. In addition, her lymphocytes failed to respond when cultured with HLA-typed third party stimulators (Fig. 2), although they responded perfectly normally to PHA. We question, as with Patient B. M., if a certain HLA phenotype may result in cell surface glycoproteins (HLA antigens) that communicate inefficiently on a non-antigen-specific basis in cell cooperation involved in in vitro and in vivo immune responses to tissue antigens. Equally plausible is the possibility that there are inherent lymphocyte defects not involving the plasma membrane of her lymphocytes that account for this abnormal reactivity. These findings allow us to conclude that MHC antigen sharing between the woman and the man is not always causally related to the immune hyporeactivity seen. Three couples sharing antigens at the A, B, and D/DR loci and who were nonresponders in MLC reactions were enrolled in our white blood cell sensitization protocol. Peripheral blood leukocytes from the male partner were prepared according to the protocol in Material and methods. Approximately 40 x 10 6 viable mononuclear cells were injected intradermally on the volar aspect of the forearm of the woman at six sites. Wheal-and-Hare reactions were recorded daily for 14 1

993

Tab!e V. Distribution of common Hl. 1\ antigens shared by both members of couples in the control and experimental groups Cam,rnon H LA antigens Locus A:

One antigen I

2 5

2

I I

II 26 Two antigens 2, 3 Locus B:

One antigen fl-W6 12 40-W6 Locus C: One antigen 3 LoCJ.ls DR: One antigen I

2 3 4 MTI MT2 MT3 Two antigens L MTl C)

2 3

2 2

~J'Tl

.4, lYJ..l .l

4, MT3 Three antigens 3, MTl, MT2 Locus A and B:

A-I, B-8-W6 A-2, B-40-W6 A-II,B-12 Locr..LS A and DR: A-1, DR-MT2 A-2, DR-4, MT3 A-1, DR-3, MTI, MT2 A-2, DR-I, MTI A-2, DR-MTI A-26, DR-MT3 Locus B and DR: B-8-Wfi, DR-3, MTI, MT2 B-8-W6, DR-MT2

2 I I I

2 I I I I

Locus A, B, and DR: A

1

£""1-J.,

D.

Q

Ult::.

nD '1

.LJO-u-YTV, .LI.l'--...1,

'K'T' 1

.l.l'.l.l

\..fTC)

.t, 1Y.l .1..:;,.

A-I, B-8-W6, DR-MT2

days. All women immunized developed wheal-andflare reactions by 48 hours and ali reported reappearance in the skin test reactivity as late as 7 to 12 days. This was expected since the male lymphocvtes were not irradiated. Three to 6 months after sensitization, lymphocyte stimulation assays were repeated and the results are listed in Table VII. To date, couples H.P+W and M .D+ B failed to show any changes in mixed lyrnpho-

994

Beer et al.

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\m.

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Table VI. One-way mixed lymphocyte reactivity of women experiencing chronic habitual abortions to lvmphocyte alloantigens presented by husband and ALA-incompatible third partv Sf (cpm)

Couple

Female stimulaturfemale respcmder

Experimental: B.J+G F.J+S H.D+R W.N+M H.P+W S.J+J B.L+W M.D+B G.J.+M W.D+D Control: C.J+T D.M+W H.C+P H.D+C S.M+M E.C+M R.B+D H.T+M A.L+T Q.C+J H.C+R G.L+W H.E+R P.G+R

l.O 1.0

l.O 1.0 1.0

(393) (190) (794) (217)

Female stimulatorfemale respander plus PHA 13.0

(5,118)

39.1 (31,046) 18.3 (3,967) 30.0

1.0 (4,685) 1.0 (1,983) 1.0 (2,568) l.O (785)

6.2 20.1 5.4 34.9

(29,190) (39,793) (13,957) (27,418)

(366) (340) (1,362) (380) (1,642) (2,063) (741) (217) (1,370) (732) (688) (577) (673) (1,606)

22.9 70.1 15.1 49.0 7.6 15.2 16.7 64.4 105 106 33.1 23.4 18.2 10.9

(8,365) (23,848) (20,571) (18,626) (12,415) (31,393) (12,361) (13,967) (143,715) (77,498) (22,743) (13,521) (12,306) (17,444)

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

LO 1.0 1.0 1.0

Male stimulatorfemale responder

Third-party stimulatorfemale responder

12.0 39.5 12.0 21.4 2.1 2.47* 4.3 2.6 3.5 5.5

(4,730) (7,506) (9,505) (4,638)

16.9 31.4 9.1 10.1 14.0

(6,635) (5,970) (7,254) (2,193)

(20,215) (5,116) (9,049) (4,353)

11.2 37.9 27.2 8.2

(52,451) (75,140) (69,885) (6,451)

17.3 4l.l 18.1 41.4 18.2 6.2 34.2 126 69.0 5l.l 27.2 43.0 24.2 4.4

(6,329) (13,987) (24,592) (15,744) (29,848) (12,832) (25,358) (27,261) (94,592) (37,371) (18,683) (24,811) (16,275) (7,032)

98.3 (35,985) 23.7 (8,066) 6.1 (8,335) 103 (39,019) 16.0 (26,341) 12.8 (26,419) 30.1 (22,281) 75.9 (16,481) 33.2 (45,510) 59.0 (43, 164) 19.4 ( 13,325) 28.7 (16,531) 20.5 (13,810) 14.8 (23,816)

No. of HL-A antigens shared A

I 1

1 2 1 1

I B I c I DRIMT 2 1 1 1 ND ND 1

A1LC respmider

+ +

+ +

1

1 I 1

+ +

+ + + + + + +

+ +

+ +

*MLR done in another laboratory (Immunological Associates, Denver. Colorado).

cyte culture reactivity after immunization. Neither woman developed antileukocyte antibodies after immunization. Couple S.J+ ]. whose initial MLC reactions were done in another laboratory (Immunological Associates, Denver, Colorado), and who showed female responder-male stimulator hyporeactivity in MLC, demonstrated reactivity when retested after immunization. Following immunization, two couples have established pregnancies. Couple S.J +J established a pregnancy, their eighth, after immunization. This pregnancy was successfully carried to term, producing a healthy 6 pound, 8 ounce male infant with Apgar scores of 9 and 10. He has done well during the first 8 months of life. Detailed histologic analyses of the placenta were normal. Couple M.D+ B after five abortions established a pregnancy 4 months after immunization. The pregnancy is currently at 30 weeks' gestation without complications or additional therapy. The third couple has not yet established a pregnancy. Comment

What are the possible reproductive advantages and/ or disadvantages to the pregnant woman and fetoplacental unit of homozygosity or heterozygosity with regard to major histocompatibility antigens between the female and the male? Studies in inbred strains of ani-

mals have shown that conceptuses that are disparate with their mothers at MHC antigens appeared to enjoy a selective advantage over conceptuses that are compatible with their mothers at these loci. In studies by Michie and Anderson 10 referred to above, it appeared that there was intense selective pressure against individuals who were homozygous. Similarly, Palm 11 found from studies in rats that when homozygous young were gestated in MHC homozygous mothers, reproductive performance was greatly curtailed. She concluded that the selective elimination of homozygotes took place either prenatally or early postnatally as a result of immunologic responses on the part ofthe mother against antigens coded for by loci outside the MHC. She suggested that this clear-cut advantage of heterozygotes in laboratorv populations might reflect an important, naturally occurring phenomenon in outbred populations that could influence the degree of polymorphism at the major histocompatibility locus and preserve genetic polymorphism within the species. Much like the results of Michie, Anderson, and Palm, the present study, in which we sought evidence of an association of fertility differences with differing degrees of MHC antigen disparity between spouses, has shown a highly significant degree of MHC homozygosity in couples experiencing spontaneous habitual abortions with no

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documented etiology. In addition, certain of these couples demonstrated marked alterations in MLC reactions bet,veen spouses ,,vhen compared ¥-lith MHCtyped third-party stimulators and responders. Similarly, Lauritsen and associates, 21 in a critical analysis of couples with recurrent spontaneous abortions of karyotypically normal fetuses, showed a significantly depressed mitogenic response for MLC reaction in mothers' lymphocytes when stimulated by the respective spouses' lymphocytes but not when stimulated by unrelated donor lymphocytes. This hyporeactivity was seen only when the fetus was karyotypically normal and was not seen when the fetus was karyotypically abnormal. These results suggested a defect in the elicitation and/or the expression of a cellular immune response on the part of the mother to paternal histocompatibility antigens. Since tissue typing was not done, the effect of HLA compatibility and/or incompatibility on MLC responses was not determined. Komlos and associates 22 reported that a significantly higher percentage of women with repeated abortions shared common MHC antigens with their husbands when compared to control groups and suggested that homozygous fetuses were less capable of stimulating the mother's immune system along the lines of a protective blocking antibody response. These studies raised the possibility that maternal genetic compatibility for certain MHC coded antigens of the father may result in a situation where the mother responds to organ-specific and/or tissuespecific antigens of spermatozoa and/or early embryos that might induce cytotoxic effects during gestation. A successful pregnancy appears to involve immune responses in the mother that confer protection upon the fetoplacental unit. When these reactions were analyzed in women with chronic habitual abortion by Rocklin and associates, 23 studying the reactivity of maternal lymphocytes and the properties of maternal serum, interesting results were forthcoming. It is known that when circulating lymphocytes are sensitized against a specific antigen they produce a lymphokine, migration-inhibition factor (MIF), that prevents the migration of macrophages. This factor is produced by lymphocytes following organ transplantation and blood transfusion and during a normal pregnancy. Blocking antibodies produced during immunization can prevent the release of MiF by sensitized lymphocytes. When sera from women with chronic abonion were analyzed during pregnancy, their lymphocytes were found to produce MIF but no blocking factors were found in the sera. Studies were also conducted on patients having previous abortions who later experienced successful pregnancies. In these individuals the emergence of a blocking factor during a successful pregnancy was documented. Studies on the

nature of the blocking factor revealed it to be a immunoglobulin, most certainly IgG. The blocking activity was not directed against antigens f.lf HLA. A., B. t)r C~. Appropriate absorption studies with platelets carrying the A, B, and C locus antigens did not remove the blocking activity, indicating that the responsible antigen system is most likely associated with or determined by the HLA D locus. Women with spontaneous abortions who show a normal degree of migration inhibition in the presence of autologous antigen and in whom no blocking factor is present in the sera may possess cytotoxic lymphocytes. These unsuppressed cells may focus attention on the fetoplacental unit, inciting either direct damage or indirect dam
996

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lku.·tHbcr l.:"1, 11:.-lK! "~\r::l

. .J. Oh~1(·1. ( ~\'lh'n ,f

Table VII. One-way MLC reactivity of women experiencing chronic habitual abortions to lymphocyte alloantigens presented by husband and HLA-incompatible third party before and after white blood cell sel!sitization Sf (cpm)

Presensitization Couple

Female stimulator-female respunder

S.J+J H.P+W M.D+B

I femaleFemale stimulatorrespunder plus PHA

l.O 1.0 ( 1,983)

30 20.1 (39,793)

1Hale

I stimulator-female respunder 2.47* 2.1 2.6 (5,116)

I stimulator-female Third-party respunder 14 37.9 (75,140)

*MLR done in another laboratory (Immunological Associates, Denver, Colorado).

t Assays done when patient was 5 to 6 weeks pregnant. :j:Assays done at 12 weeks of pregnancy.

efficiency. At the present time we are not able to link this inefficiency comfortably to alterations in immune responses on the part of the woman during pregnancy since some women sharing histocompatibility antigens with their spouses are not immunologically unresponsive and the woman of one couple sharing no antigens in common demonstrated hyporesponsiveness. The recent findings regarding the androgenetic origin of hydatidiform molar pregnancies may highlight the importance of immunogenetic processes in trophoblastic behavior. By several mechanisms a human ovum is fertilized by a diploid spermatozoa or one that subsequently undergoes a second meiotic division; elimination of the female pronucleus takes place, which results in a conceptus that is XX, both X chromosomes having come from the man. 27 By definition the resulting pregnancy is more MHC incompatible with the woman than if she had contributed a haploid component of her histocompatibility antigens. Under most circumstances this results in embryonic lethality but proliferation of the trophoblastic epithelium continues. The well-known behavior of hydatidiform moles as true transplants in women may be the most dramatic example of "hybrid vigor" or heterozygote advantage. The fact that women having hydatidiform molar pregnancies do produce HLA antibodies to paternal MHC antigens highlights their immunogenicity .2 " The results described herein raise three important questions: I. Does MHC homozygosity between spouses account for the hyporeactivity in the in vitro assays and

does this lead to the absence of some pregnancy protective factor, i.e., blocking antibodies? Until the blocking factors and/or blocking antibodies can be more fully defined and their functioning during a successful pregnancy can be critically evaluated, this question cannot be answered fully. Our initial results of successful pregnancies in patients following white blood cell immunization will most assuredly assist in definingthe nature and the role of blocking antibodies during pregnancy. 2. Does MHC homozygosity between spouses increase the likelihood of homozygosity at other nonMHC loci, specifically the equivalent of the T locus~ Homozygosity with regard to T locus alleles in mice is associated with abnormalities of development and em~ bryonic lethality. 29 If this mechanism is operational, then systemic immunization should prove of no benefit: however, donor insemination with HLAincompatible spermatozoa may well lead to successful pregnancies. ~3. Is a certain HLA phenotype of a female or a male associated with unique cell surface glycoproteins or other cellular features that lead to problems of cellular cooperation in immune responses? Our present results support this possibility. If this deficiency or defect in cellular cooperation is real and involved in pregnancy losses in the female, then donor insemination with HL-A-incompatible semen may fail. On the other hand, if the pregnancy losses relate to abnormalities of antigen presentation by the male only, donor insemination should succeed.

REFERENCES 1. Jenson. K. G.: Vox Sang. 7:454, 1962. 2. Ahron, S.: Tissue Antigens 1:129, 1971. 3. Zmijewski, C. M., Zmijewski, H. E., and Huneycutt, H. C.: Int. Arch. Allergy Appl. lmmunol. 32:574, 1967.

4. Beer, A. E.: Eur. J. Obstet. Gynecol. Reprod. Bioi. 5:135, 1975. 5. Beer, A. E., and Billingham, R. E.: J. Reprod. Fertil. (Suppl.) 21:59, 1974.

MHC, immune responses, and habitual abortion

Volume 141 :\umber H

997

Sf (cpm)

Postsensitiwtion Female stimulator -female responder

Female stimulatorfemale responder plus PHA

1.0 (1,569) 1.0 (897) 1.0 (1,505) 1.0 (3,377)

19.9 (31,294) 80.0 (71,770) 29.6 (44,543) 4.5 (15,069)

6. Beer, A. E., and Billingham, R. E.: Folia Bioi. 26:225, 1980. 7. Beer, A. E., and Sio, J. 0.: In Dhindsa, D. S., and Schumacher. G. F. B .. editors: Immunolo!lical Asnects of Infertility a~d Fertility Regulation, Ne~ York', 1980. Elsevier/North-Holland, p. 143. 8. Klein, J.: Science 203:516, 1979. 9. Dausset, J., and Contu, L.: In Fougereau, M., and Dausset, j .. editors: Immunology 80, New York. 1981, Academic Press, Inc., p. 513. 10. Michie, D., and Anderson. N. F.: Ann. N. Y. Acad. Sci. 192:88, 1966. II. Palm, J.: Cancer Res. 34:2061, 1974. 12. Menge, A. F.: J. Reprod. Fertil. (Suppl.) 10:171, 1970. 13. Jones, \N. F.: In Scott, J. S., and Jones, W. R., editors: Immunology of Reproduction, New York, 1976, Academic Press. Inc., p. 375. 14. Terasaki, P. I., and lvicCieHand, j. D.: Nature 204:998, 1964. 15. Ray, J. G., editor: NIAID Manual of Tissue Typing Techniques, Department of Health, Education, and Welfare, Publications, National Institutes of Health, No. 80545, p. 39. 16. Weir, D. M., editor: Handbook of Experimental Immunology. ed. 3, Oxford, 1978, Oxford University Press. chap. 6.

Editors' notr: This manuscript was revised after these discussions were presented.

Discussion DR. EARL R.

PLUNKETT, London, Ontario, Canada. For 30 years, apart from the diagnostic application of cytogenetics, the recogmtJon of inadequate luteal phase, and certain surgical refinements in the correction of uterine anomalies, there has been little significant advance in the management of habitual abortion. Therefore, I was doubly interested in the data presented by Dr. Beer and his colleagues, which apply the technology of tissue typing used in transplant work to the spontaneous abortion problem. It has been shown that chromosome number 6 is the focal point for most of the genetic control mechanisms which determine our immunologic portrait. Between

Male stimulator -female responder

15.7 15.0 3.8 1.8

Third-party stimulator-femalr responder

30.0 84.9 21.6 15.5

(24,637:· (13,427) (5,753) (6,237)

(47 ,034) (76,189) (32,537)t (52.371):j:

17. Boyum, A.: Scand. J. Clin. Lab. Invest. (Suppl. 97) 21:7, 1968. 18. Rose, N. R., Njort, T., Rumke, P., Harper, M.J. K., and Vyazov, 0.: Clin. Exp. 1mmunol. 23:175, 1976. 19. Mangione, C. M., Medley, N. E., and Menge, A. C.: Int. .J. Fertil. 26:1981. In press. 20. Terasaki, P. I.. editor: Histocompatibility Testing 1980, Los Angeles, California, 1980. UCLA Tissue Typing Laboratorv. 21. Lauritsen,']. G., Kristensen, T., and Grunnert. N.: AM . .J. 0BSTET. GYNECOL. 125:35, 1976. 22. Komlos, L., Zamir, R.,Joshua, H., and Halbrecht, I.: Clin. Immunol. Imrnunopathol. 7:330, 1977. 23. Rocklin, R. E., Kitzmiller, J. L., Carpenter, C. B., Garovoy, M. R.. and David, J. R.: !\. Eng!. J Med. 295:1209, 1976. 24. ~au;_i,tsen;. J. G.),?~~e~~~~· J., and Kissmever-Nielsen. r.: \.....lin. venec. :t:.:>t.:>,

1';1/0.

25. Gerencer, M., Drazancic, A., Kuvaric, I. M., Tomaskovic. z. M., and Kastelan, A.: Fertil. Steril. 31:401, 1979. 26. Tho, T. P., Byrd, J. R., and McDonough. P. G.: Fertil. Steril. 32:389, 1979. 27. Wake, N., Takogi, ;\;.,and Saski, M.: JNCI 60:!11, 1978. 28. Lawler, S.: Br. Med. Bull, 34:305, 1978. 29. Bennett, D.: Science 144:263, 1964.

300 and l ,000 separate genetic loci are located on the sixth chromosome. They rigidly control the immune response. The various antigens determined by these loci on the sixth chromosome are identified as extensively as possible prior to tissue transplant. Obvwusly, a dose antigenic match between the donor tissue and that of the reCipient is sought. This ''ponrait" is known as the MHC. Keeping this transplant situation in mind. Beer and Bellingham in earlier publications referred to pregnancy as a "fetal graft." Obviously the embryo represents a combination of genetic cellular material from two separate individuals. Since most pregnancies are successful and since most marriages are at best random, it appears obvious that a good tissue match between parents as based upon MHC criteria would rarely occur. Indeed, Dr. Beer's data indicate that when matching is

998

Beer et al.

lkcemhcr L'>. .\llL

"good," with great similarity between the antigenic profiles of the parents, there appears to be an increased chance of spontaneous abortion. This is supported by the considerable biologic data that indicate that inbreeding, with its greater opportunity for immunologic similarity between mates, is associated with a significant reduction in reproductive efficiency. On the other hand, outbreeding is associated with improved efficiem:y. In this submission, Dr. Beer points out that not all habitual abortions occur on the same basis. Specific etiologic factors can be identified in many instances and these may include uterine and cervical abnormalities. infections, endocrine disorders, and parental chromosomal disorders. Of the 26 cases of habitual abortions studied, 16 were found to be in the above category. These were referred to as controls. The remaining l 0 in which no obvious etiologic factors were identified became the "experimental" group. Thus, the study design is based upon the premise that immunologic function in the control aborter group would be normal, while that of the experimental group might show a distinct difference. In view of the fact that it is often very difficult to identify posith·ely the cause of spontaneous abortion, I feel that the design would have been improved if a second control group of couples with a normal reproductive history, matched for age and parity. had been included. Increased sharing of antigens between spouses (excessive homozygosity) appeared to be a significantly greater event among the experimental group as compared with the control group. This was associated with hyporesponsiveness to lymphocyte cell surface determinants of the spouse in mixed culture. This also appears to be associated with an increased fetal wastage. I make no pretense of being particularly knowledgeable about the immunologic field, but I was curious to know if an abnormal immunologic response might be identified by the addition of a haploid cell (the sperm). In terms of the MHC antigens, would any difference be anticipated in the haploid cell? Was there any difference in the sex of the aborted embryos between the experimental and control groups of cases? In effect, was there any dominance of one sex over the other when the two groups were compared? The authors suggest that excess immunologic homozygosity between the spouses may be associated with a deficiency of certain blocking factors, which leads to fetal loss. One wonders whether this might be related to a specific genetic locus rather than the homozygosity of the whole complex. I would also ask whether increased sharing of HLA antigens may not be associated with an increased production of cytotoxic T cells, which leads eventually to a rejection-like phenomenon? It was not completely clear to me whether fetal chromosomal studies had been done on all abortuses in the two groups and whether these served as an exclusion criterion for the experimental group. In any event the immunologic (experimental) group aborted earlier

_l.

l~liil

()h'-tel. {•\-llf'{_'Ot

than the control group. Do you think there is a possibility that increased homozygosity could be associated with extremely early abortion, which may not e\en be identified on some occasions as a pregnancy? Finally, I was not sure whether all cases in the experimental group might be amenable to immunization treatment. I congratulate Dr. Beer and his co-workers on this innovative approach to a very old problem. In the broad field of medical endocrinology, immunologicallY induced disorders are relatively common. It is challenging and exciting to see this potential application in the field of obstetrics and gynecology. DR. S.J. BEHRMAN, Birmingham, Michigan. I found the presentation very interesting, yet I feel it is incomplete. If you propose the idea of nonresponsivity, can you resolve these two questions for me~ In our studies of the last 15 years of antisperm production, we found that while there is no meaningful infertility associated with increased circulating antibodies there is a 43% incidence of repeated abortion in patients with high levels of antibodies to the husband. There is a paper. not well known, by the pathologist Jorgensen from Denmark, who in routine curettage in infertility patients found remnants of trophoblastic tissue with marked cellular form, which suggests an immunologic response to early embryonic parts. In other words, I am proposing the idea that there is responsivity on the part of the patient, the mother, that may lead to abortion. Does this conflict with your concept of nonresponsivity? DR. KAMRAN MOGHISSI, Detroit, Michigan. Several days ago Dr. Lawrence Young reported what Dr. Behrman has stated, that there was approximately a 45% abortion rate among couples who had antisperm antibodies in either the man or woman. In over 200 couples that we have studied very carefully. with the assay of agglutinating and immobilizing sperm antibodies in the blood and cervical mucus, we find also that women who achieved pregnancv had an abortion rate of about ~2'k. My question to Dr. Beer is whether or not he found any relationship between the lack of the blocking antibodies in the woman and the presence of antisperm antibodies either in the circulation or in the cervical mucus? DR. BEER (Closing). Concerning Dr. Plunkett's question regarding the design of the study, we are, at the present time, studying a second control group matched for age and parity. In addition, we are prospectively studying a third control group involving assay of both the HLA type and immunoreactivity of couples prior to the establishment of a first pregnancy. Regarding the use of haploid cells instead of lymphocytes, haploid spermatozoa have been utilized in some studies to stimulate cells in culture. These experiments have been unsuccessful because there are not only immunosuppressive factors of germ cell origin but immunosup-

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pressive factors that coat spermatozoa during their passage through the efferent ducts. As a result, it is difficult w conduct in vitro experiments with spermatozoa used as stimulating antigens. Regarding the sex of the aborted embryos, I have no data as to the gender of the abortus. Regarding the question of family studies, from family histories, we were unable to "how an increased incidence of deformities in a preceding successful pregnancy or in pregnancies by other members of the family. Dr. Plunkett questioned if karyotypic analysis of the aborted fetus was accomplished; all individuals included in the present study had at least one of their abortuses karyotyped. We eliminated any patients from the study who had aborted a fetus with a karyotypic abnormality. Dr. Little gave a very erudite discussion. Many of the questions posed by Dr. Smith are very detailed, and to answer them fully would take more time than I have. I do agree that a prospective study is needed to look for immunologic deficiencies between couples sharing HLA antigens but not experiencing chronic habitual abortion. He commented on the syngeneic animal model where the mother and her conceptuses share the major histocompatibility antigens and reproductive efficiency seems not to be greatly curtailed. As addressed in the paper, there are intense selective pressures to preserve genetic heterogeneity between a mother and her offspring and the surviving syngeneic animals currently used in the laboratory may be quite artifactual. Although mothers and fetuses share major and minor transplantation antigens, there is newer evidence that during pregnancy immune responses to de\·elopmental and embryonic antigens are initiated. These responses may be benefiCial and necessary for pregnancy survival. Regarding the HLA phenotypes of the living children born in each group, at the present time we are studying the living children in both the control and the experimental groups. In our experimental group, three of the living children studied showed an HLA haplotype that was not identical to that of the mother. We plan to study the MLC dynamics between mother. father. and child at a future time. Dr. Smith questioned if the maternal hyporesponsivity was due to an effect of paritv, that is, pregnancy-induced

unresponsiveness. Since the unresponsiveness was not seen in couples aborting consecutive pregnancies for known reasons. it is unlikely that the hyporesponsiveness is due to a parity effect. Until prospective studies are accomplished, a definitive answer to 1his qm:stion will not be given. Dr. Romney asked an age-old question regarding the role of male antigens in spermatozoa and subseL]Uent cervical dysplasia and/or carcinoma of the cervix. In addition, do antigens from multiple male partners have anything to do with cervical dvsplasia and/or carcinoma? This hypothesis is quite attractive, and now that we have more sophisticated tools for tissue typing answers should be forthcoming. One ol the problems with this type of study is to get the willing trio, quartet, or octet to submit to the tissue typing. The comments from Dr. Behrman regarding antisperm antibodies and infertility are provocative. I think it is well established that women with antisperm antibodies suffer a higher incidence of spontaneous abortion if a pregnancy is established. This type of couple has been eliminated from uur study in that anv woman with cerdcal mucus or serum antibodies was excluded. :'\onetheless, there appears to be the possibility emerging from the studies in progress that M HC homozyg( >sitv between the woman and the man at certain loci may render th<" woman more responsive to organ-specific antigens of spermatozoa and/or seminal plasma. \Ve must now question if the HLA antigens on spermatozoa are "first-line" antigens that in some way prott:ct the organspecific or time-specific autoamigens from being recognized by the host. For example. it is 20 times Jess likely for a woman to become immunized to the Rh antigens if the immunizing cells also present a blood group A orB incompatibility. Dr. Moghissi asked if the iack of blocking antibodies in patients with reproductive inefficiencies may be associated with an increased incidence of antisperm antibodies. At the present time. we are studying a large number of couples wherein a11tispenn antibodies are present either in the woman or in the man or both and are seeking to determine if \'\'OI nen and men sharing DR locus antigens are more prone to develop antisperm antibodies than those not sharing these antigenic determinants.