AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 28:269-273 © 1992 MUNKSGAARD
Autoantibodies in Normal and Abnormal Pregnancy NORBERT GLEICHER Center for Human Reproduction and Foundation for Reproductive Medicine, Inc. Chicago, Illinois
Abnormal autoimmune function has been associated with reproductive failure for decades. In fact, all medical conditions historically associated with pregnancy loss (and on occasion infertility) are now recognized to have an autoimmune etiology. It is therefore quite surprising that only less than a decade ago a correlation between the presence of abnormal autoantibodies and pregnancy loss was reported for the first time. Since reproductive failure, similar to other abnormal autoimmune states, is not a monoclonal event, we have established that affected patients demonstrate polyclonal autoantibody abnormalities including a variety of nonorganospecific as well as organspecific autoantibody groupings. For example, patients with repeated pregnancy loss will exhibit abnormal levels of anti-phospholipid antibodies (PA). In normal pregnancy natural autoantibodies do not follow the standard immunoglobulin (Ig) pattern, characterized by a decrease in levels despite a probably mild increase in production, which is excessively compensated by the vasodilatation of pregnancy. PA, especially, demonstrate a mild increase during pregnancy, though levels only in the peripartal period may reach abnormally high titers (in comparison to nonpregnant controls). This dichotomy between total Ig and natural autoantibodies is interesting since it suggests that autoantibodies may be under distinct control. In fact, we have suggested that this observed elevation of autoantibodies may be the result of an antigenic stimulus by self-like antigen, represented by the maternal growth of the parasitic fetus. In abnormal pregnancies, especially those associated with maternal hypertension and fetal growth retardation (IUGR), autoantibody levels do reach highly abnormal levels. We have reported that in both of these conditions significant abnormalities especially in IgG-PA can be detected, which stand in direct correlation with severity of disease (though autoimmune hemolytic anemia (AHA) can also be found). This is an interesting observation because both of these conditions have been associated with an imbalance between thromboxane and prostacycline in favor of the platelet aggregating and vasoconstrictive thromboxane. This is exactly the imbalance reported in women with habitual abortions, which is assumed to be PA-induced. Aspirin, recently widely reported as being successful in the prevention of pregnancy-induced hypertension (PIH) and IUGR, has also been recommended as treatment for ABSTRACT:
Submitted for publication June 30, 1992; accepted July 6, 1992. Address reprint requests to Norbert Gleicher, Center for Human Reproduction and Foundation for Reproductive Medicine, Inc., 750 N. Orleans Street, Chicago, IL60610.
habitual aborters. Lastly, habitual aborters who are successfully treated and reach viability in a subsequent pregnancy, experience a highly increased incidence of PIH and IUGR. All of these observations strongly suggest that PIH, IUGR, and repeated pregnancy loss may have a common pathophysiology. It is tempting to speculate that it is autoantibody-and especially PA- related. (Am J Reprod Immunol. 1992; 28:269-273.) Key words: Anti-phospholipid, intrauterine growth regardation, pre-eclampsia autoantibody, cardiolipin INTRODUCTION
The fetus is a semi-allograft and semi-autograft, As feto-maternal cell traffic increases with advancing gestational age, one can presume that the maternal immune system sees not only increased loads of alloantigens but also epitopes identical, or at least similar, to autoantigens. In fact, such an autoantigen load would seem almost unprecedented, especially late in pregnancy and during delivery, when cell traffic is the largest. A compensatory mechanism to neutralize this antigen load during normal pregnancy can therefore be expected and probably, in fact, exists. NORMAL PREGNANCY
Total immunoglobulin levels in pregnancy decrease, despite an increase in total immunoglobulin production. This is due to the considerable expansion in plasma volume during pregnancy, which exceeds the increase in immunoglobulin production and thus results in a dilutional hypogammaglobulinemia.! Autoantibody levels in pregnancy do not follow this pattern. In a longitudinal study involving 43 patients, we were able to demonstrate that neither phospholipid antibodies nor antibodies to histones or to polynucleotides decrease in parallel to total immunoglobulin levels (Tables 1III) and, in fact, IgM autoantibodies increase significantly at time of delivery. 2 The same study also revealed no change in lupus anticoagulant level over the length of gestation. This dichotomy between total immunoglobulin and autoantibody levels strongly suggests that the regulation of autoantibody levels during pregnancy is under separate control from that of other antibodies. It is therefore tempting to speculate that the apparently selectively increased autoantibody levels during gestation represent the previously noted compensatory mechanism for the excessive autoantigen load faced by the pregnant woman. One could further speculate that this specifically female need to compensate for an autoantibody overload during pregnancy is also related to the fact that women almost universally demonstrate higher autoantibody levels than normal males 3 ,4 and experience many times their incidence of autoimmune diseases.V'
270
GLEICHER
TABLE I. Immunoglobulin (lg) G Autoantibodies Throughout Gestation" Nonpregnant controls Total IgG (mg/dl.) Phospholipids Cardiolipin Phosphatidylserine PhosphatidyIglycerol Phosphatidylethanolamine Phosphatidylinositol Phosphatidic acid Histones HI H2A H2B H3 H4 Polynucleotides ssDNA dsDNA Poly(l) Poly(dT)
1220 ± 350
Week of pregnancy 24
16
20
930 ± 280 b
970 ± 195
916 ± 195b
28
Delivery (wk39 + 1.9)
32
1010 ± 276
974 ± 195 b
939 ± 280 b
24 16 26 5 18 15
± ± ± ± ± ±
25 19 26 13 18 19
11 10 19 9 45 15
± ± ± ± ± ±
13 43 20 17 37 b 9
8 ± 13 6±8 10 ± 6 3±4 10 ± 40 11 ± 13
16 ± 18 8 ± 10 16 ± 23 4±4 31 ± 20 13 ± 20
14 7 11 5 12 15
13 10 10 18 15 11
7±9 6±8 25 ± 36 11 ± 33 10 ± 2 21 ± 24
7 ± 10 7±9 34 ± 40 c,d 12 ± 36 8±2 22 ± 27 c ,d
17 13 9 11 32
± ± ± ± ±
17 17 10 13 39
13 8 36 19 31
± ± ± ± ±
14 13 40b 22 33
4±4 7 ± 10 16 ± 43 18 ± 62 28 ± 32
11±11 7 ± 11 17 ± 13 11 ± 17 27 ± 17
4±4 5 ± 12 14 ± 21 15 ± 25 21 ± 13
5±5 8 ± 14 18 ± 43 22 ± 34 31 ± 23
6±7 9 ± 15 20 ± 46 24 ± 38 34 ± 25
55 120 60 166
± ± ± ±
51 80 44 90
31 84 10 100
± ± ± ±
50 44 15 66
25 ± 48 75 ± 51 5±5 62 ± 62
38 51 17 82
22 ± 30 62 ± 75 22 ± 16 53 + 50
24 ± 22 59 ± 44 11 ± 10 74 + 67
27 ± 25 79 ± 39 12 ± 11 81 + 74
± ± ± ±
42 67 20 43
± ± ± ± ± ±
"From EI-Roeiy et al.,2 with permission. Data are presented as optical density x 103 at 405 nm. "Statistically increased level compared with nonpregnant controls (P < .05). 'Statistically increased level compared with levels at 16 wk (before normalization) (P < .05). dStatistically increased level compared with levels at 16 wk (after normalization) (P < .05).
since basically all medical conditions historically associated with excessive pregnancy loss are now understood or believed to have an autoimmune etiology (Table IV). Our early work with lupus anticoagulant-positive habitual aborters suggested to us that their B lymphocyte hyperactivity was not restricted to the production oflupus anticoagulant." In fact, we quickly became convinced that their B cell function was similar to that of such classical autoimmune diseases as systemic lupus erythematosus (SLE) in that they produced a wide variety of abnormal autoantibody specificities." It was this observation that led us to investigate patients with suspected pregnancy abnormalities due to abnormal B cell activity with a broadly based autoantibody profile characterized by six antibodies to phospholipid antigens, five
ABNORMAL PREGNANCY
While autoantibody levels (especially of IgM isotype) rise during late normal pregnancy, they only rarely reach abnormal levels, whether defined by a 95 or 99 confidence interval of a normal population.f This kind of statistical evaluation is important whenever autoantibody levels are evaluated because of their nonparametrical distribution, which results in an inordinate number of false positive results if, instead, multiples of means are utilized." Abnormal levels of autoantibodies have repeatedly been implicated in pregnancy abnormalities after Lubbe et al. reported their initial observation of an association between lupus anticoagulant and repeated pregnancy 10ss.7 It is actually quite surprising that the association between pregnancy wastage and abnormal autoimmune function has not been recognized earlier
TABLE II. Immunoglobulin (lg) M Autoantibodies Throughout Gestation" Nonpregnant controls Total IgM (rng/dl.) Phospholipids Cardiolipin Phosphatidylserine Phosphatidylglycerol Phosphatidylethanolamine Phosphatidy!inositol Phosphatidic acid Histones HI H2A H2B H3 H4 Polynucleotides ssDNA dsDNA Poly(l) Poly(dTl
180 ± 70 24 ± 19 29 ± 31 32 ± 38 5±8 26 ± 26 20 ± 24
16 155 ± 40
20
Week of pregnancy 24
28
32
151 ± 64
161 ± 66
153 ± 54
158 ± 62
Delivery (wk39 + 1.9) 151 ± 55
23 ± 19 14 ± 13 33 ± 26 2±3 38 ± 22 d 32 ± 27 d
11 ± 8 8 ± 11 34 ± 18 3±3 13 ± 8 19 ± 13
15 14 23 3 39 14
10 11 22 4 22 10
29 14 16 3 41 15
36 13 22 5 24 d 11
22 35 39 4 20 20
13 22 45 4 17 24
24 39 42 5 22 22
14b 24 49 b ,c 4 18 27
56 46 48 47 139
± ± ± ± ±
38 36 35 33 79
44 61 12 39 80
± ± ± ± ±
30 31 18 21 72
45 37 29 29 73
± ± ± ± ±
25 25 20 20 56
22 26 36 25 29
25 23 22 22 23
27 38 34 25 46
20 21 21 19 34
54 63 59 50 96
32 39 38 27 60
60 70 65 55 105
35 c 43 c 41 b ,c 30 66
115 169 143 171
± ± ± ±
76 84 86 102
12. ± 85 ± 79 ± 68 ±
25 75 41 46
33 69 106 78
± ± ± ±
18 97 80 48
36 87 80 75
27 91 80 95
18 75 89 95
20 77 44 77
45 119 127 117
29 71 59 60
50 100 140 128
32 81 64 b ,c 66 b ,c
"From EI-Roeiy et al.,2 with permission. Data are presented as optical density x 103 at 405 nm. bStatistically increased level compared with levels at 16 wk (before normalization) (J' < .05). 'Statistically increased level compared with levels at 16 wk (after normalization) (P < .05). dStatistically increased level compared with nonpregnant controls (P < .05).
AUTOANTIBODIES IN NORMAL AND ABNORMAL PREGNANCY
271
TABLE III. Immunoglobulin (Ig) A Autoantibodies Throughout Gestation" Nonpregnant controls Total IgA (mg/dl.) Phospholipids Cardiolipin Phosphatidylserine Phosphatidylglycerol Phosphatidylethanolamine Phosphatidylinositol Phosphatidic acid Histones HI H2A H2B H3 H4 Polynucleotides ssDNA dsDNA Poly(l) Poly(dT)
165 ± 75
165 ± 51
Week of pregnancy 24
20
16
28
32
Delivery (wk39 ± 1.9)
164 ± 55
160 ± 42
167 ± 40
158 ± 60
159 ± 59
6±9 25 ± 45 8 ± 21 4±8 7±8 9 ± 11
9±6 4±8 11 ± 14 9 ± 11 13 ± 14b 10 ± 8
4±3 4±3 16 ± 5 2± 1 16 ± 11 10 ± 9
4±6 3±2 15 ± 13 12 ± 16 14 ± 9 11 ± 10
6±3 5±4 3±4 3±2 25 ± 19 10 ± 9
4±4 8±8 7±6 3 ± 2 17 ± 14 10 ± 6
4±5 9±9 8±7 3±3 19 ± 16 11 ± 17
10 ± 15 6±7 8 ± 10 11 ± 21 20 ± 19
9±9 11 ± 13 13 ± 15 11 ± 23 41 ± 28b
8±9 6±6 7±8 5±6 13 ± 11
5±6 7±7 4±4 8 ± 10 8±8
4±4 5±8 4±4 4±3 10 ± 10
6±4 7±8 10 ± 7 9±8 13 ± 10
7± 5 8±8 11 ± 8 10 ± 9 15 ± 11
19 90 34 49
9 50 24 11
10 48 17 7
11 ± 9 56 ± 38 44 ± 48 8±7
13 ± 13 59 ± 56 21 ± 16 9±5
14 63 36 15
15 60 40 17
± ± ± ±
22 87 87 36
± ± ± ±
14 34 17 13
± 8 ± 35
± 22 ±5
± ± ± ±
11 39 27 8
± ± ± ±
13 40 29 8
"From El -Roeiy et al.: with permission. Data are presented as optical density x 103 at 405 nm. bStatistically increased level compared with nonpregnant controls (P < .05).
to histones and their subfractions, and four to polynucleotides, all separately for IgG, IgA, and IgM isotypes. The economic performance of 45 assays on a single blood sample was possible through the establishment of an automated enzyme-linked immunosorbent assay (ELISA) system for that purpose. More recently, we have added the investigation of antithyroid antibodies by radioimmunoassay to this profile in view of a recent report by Stagnaro-Green et al., who reported a high predictive value of elevated antithyroid antibodies in patients with pregnancy 10SS.1O Moreover, similar to other autoantibody specificities, antithyroid antibodies are detectable in a large number ofSLE patients. 11 Therefore, we can anticipate a similar incidence in pregnant patients with autoantibody-associated pregnancy abnormalities. Successfully treated habitual aborters who reach fetal viability suffer from an abnormally high incidence of pregnancy-associated hypertensive conditions'P and from fetal intrauterine growth retardation and fetal distress." This observation led us to expand our antibody studies from patients with pregnancy wasta.re to women with hypertensive diseases of pregnancy.' In a statistically highly complex study, we demonstrated convincingly that hypertensive diseases of pregnancy were statistically
related to the severity of autoantibody abnormalities, defined by the number of abnormally elevated autoantibodies (Fig. 1). Moreover, the incidence of autoantibody abnormalities was also closely related to the risk of peri-
TABLE IV. Conditions Characterized by Abnormal Autoimmune Function That Have Been Historically Associated With an Increased Risk in Pregnancy Collagen vasular diseases Systemic lupus erythematosus Mixed connective tissue disease Systemic sclerosis Dermatitis herpetiformis Celiac disease Herpes gestationis Ulcerative colitis Crohn's disease Chronic active hepatitis Thyroid disease Diabetes mellitus Endometriosis
Fig.!. The distribution between growth retarded (lUGR) and normal size (AGA)infants correlated significantly at "P < 0.05, **p < 0.01, or ***p < 0.001 in each patient group, whether normotensive or hypertensive, with autoantibody positivity. The horizontal line indicates the number of expected positives in view of 45 assays performed for each patient. Moreover, hypertensive pregnancies show significantly more positive autoantibodies than normotensive controls. From El-Roeiy et al.,14with permission.
272
GLEICHER
•
Nor-mal pr-egnant contr-ol n-49
[T"/iMlld pr-eeclampsia lL.l n-1B rv'!Chr-onic hyper-tension ~ without SPE n-15 ~
Sever-e pr-eec lamps ia
~
Chr-onic hyper-tens ion with SPE n-3
~n-11 ~
Fig. 2. Correlation between incidence of antibody positivity and hypertensive condition of pregnancy. Antibody positivity increases in paral-
lei to the increasing perinatal mortality reported for various hypertensive conditions of pregnancy.
natal mortality from the underlying hypertensive condition (Fig. 2). The incidence of autoantibody abnormalities also correlated directly to the occurrence of intrauterine growth retardation. This correlation was even evident among normotensive control patients who did not express an excessive incidence of autoantibody abnormalities (Fig. 1). In an attempt to identify the most likely antibody grouping responsible for all these findings, it became apparent that the antiphospholipid antibodies of IgG isotype were the most likely culprits (Figs. 3 ,4). PATHOPHYSIOLOGY
Fig. 3. Distribution of autoantibody isotypes in normotensive and hypertensive groups. (*) Significantly (P < 0.05) different from normotensive (AGAand IUGR) and hypertensive (AGA) groups. Most reactivity can be seen among IgG autoantibodies. From EI-Roeiy et al.,14 with permission.
Phospholipid antibody specificities have been closely associated with the presence of lupus anticoagulant. Lupus anticoagulant affects the normal balance of thromboxane and prostacyclin in favor of the vasoconstrictive and platelet aggregating thromboxane. The result is a clinical risk for thromboembolic phenomena. 15 It has furthermore been suggested that the hypercoagulable state in these patients may lead to microvascular thrombotic events within the placenta, which in turn may be a contributing factor to autoantibody induced pregnancy complications.F Whether the latter mechanism in fact plays a role has to be questioned because histologic studies have so far been unable to demonstrate such microthrombi. Some investigators have demonstrated anecdotally a special affinity of monoclonal phospholipid antibodies to placental vessels. Others have suggested that antibodies may exert their effect (at least in habitual aborters) by affecting the coagulation cascade and, thus, result in excessive fibrin deposits. Our thinking has developed in a different direction. Impressed by the multitude of untoward effects of autoantibodies on reproductivefrocesses, which are not only restricted to pregnancy.P'! ,17 we felt that autoantibod-
AUTOANTIBODIES IN NORMAL AND ABNORMAL PREGNANCY
273
Fig. 4. Autoantibody positivity is primarily concentrated among phospholipid antibodies. Significant differences are denoted by (*). Antihistone antibody findings are difficult to interpret because of the
fact that many hypertensive patients were receiving treatment with methylolopa, which can induce such autoantibodies. From El Roeiy et al.,t4 with permission.
ies had to exert their effects at a very basic, possibly cellular, level. Since many second messengers of classical signal transduction processes are phospholipids, antibodies to phospholipids could, thus, theoretically interfere with different and essential cellular processes and could do so at greatly varying degrees. Therefore, we investigated this concept in a classical signal transduction model, utilizing hCG production by trophoblast cells from term pregnancies at the end product of the signaling process. Studies with polyclonal sera from patients with a multitude of phospholipid antibodies supported our hypothesis by demonstrating a reduction of signal with the addition of these sera. Moreover,phospholipid antibodypositive sera also demonstrated a considerable reactivity to IP3, a classical second messenger. IS While at this point these studies have to be considered as preliminary, their confirmation with monoclonal antibodies (rather than polyclonal sera) would suggest the fascinating possibility that autoantibodies at normal levels may serve as regulators of physiologic signal transduction processes. At abnormal levels, they would then obviously interfere with those same physiological processes, which they are teleologically meant to protect. This hypothesis could also explain why normal individuals express autoantibodies at low levels.
Hutterites: 1: Sex differences in antibody levels. Am J Reprod Immunol, 1990; 22:85. (Abstract) Shoenfeld Y, Isenberg D. The mosaic of autoimmunity. Research Monographs in Immunology, Vol 12; Elsevier, Amsterdam, New York, Oxford, 1989. El-Roeiy A, Gleicher N. Autoantibodies in a healthy normal population. Obstet Gynecol. 1988; 72:596-602. Lubbe WF, Butler WS, Palmer SJ, Liggins GC. Lupus anticoagulant in pregnancy. Br J Obstet Gynaecol. 1984; 91:359-364. Gleicher N, Friberg J. IpM gammopathy and the lupus anticoagulant syndrome in habitual aborters. JAMA. 1985; 253:3278-3281. Gleicher N, El-Roeiy A, Confino E. Reproductive failure because of autoantibodies: Unexplained infertility and pregnancy wastage. Am J Obstet Gynecol. 1989; 160:1376-1380. Stagnaro-Green A, Roman SH, Cobin RH, El-Harazy E, AlvarezMarfany M, Davies TF. Detection of at-risk pregnancy by means of highly sensitive assays for thyroid autoantibodies. JAMA. 1990; 264:1422-1425. Magaro M, Zoll A, Altomonte L, Mirone L, La Sala L, Barini A, Scuder F. The association of silent thyroiditis with active systemic lupus erythematosus. Clin Exp Rheumatol. 1992; 10:67-70. Branch DW, Scott Jr, Kochenour NK, Hershgold E. Obstetric complications associated with the lupus anticoagulant. N Engl J Med. 1986; 313:1322-1326. Lockshin MD, Druzin MC, Goei S, Gamar T, Magid MS, Jovanovic L, Ferenc M. Antibody to cardiolipin as a predictor of fetal distress or death in pregnant patients with systemic lupus erythematosus. N Engl J Med. 1985; 313:152-155. El-Roeiy A, Myers SA, Gleicher N. The relationship between autoantibodies and intrauterine growth retardation in hypertensive disorders of pregnancy. Am J Obstet Gynecol. 1991; 164: 1253-1261. Editorial. Lupus anticoagulant. Lancet. 1984;'1:1157-1158. El-Roeiy A, Valesini G, Friberg J, Shoenfeld Y, Kennedy R, Gleicher N. Autoantibodies and common idiotypes in men and women with sperm antibodies. Am J Obstet Gynecol. 1988; 158:596-603. El-Roeiy A, Gleicher N, Friberg J, Confino E, Dudkiewicz A. Correlation between peripheral blood and follicular autoantibodies and impact on in vitro fertilization. Obstet Gynecol. 1987; 70:163-170. Gleicher N, Harlow L, Zilberstein M. The regulatory effect of antiphospholipid antibodies (APA) on signal transduction: A possible model for autoantibody induced reproductive failure. Am J Obstet Gynecol. 1992; 167:637 -642.
REFERENCES 1. Myers SA, Gleicher N. Physiologic changes in normal pregnancy. In: Gleicher N, ed. Principles and Practice of Medical Therapy in Pregnancy, 2nd Ed; Appleton & Lange, 1991:35-:-52. 2. El-Roeiy A, Myers SA, Gleicher N. The prevalence of autoantibodies and lupus anticoagulant in healthy pregnant women. Obstet Gynecol. 1990; 75:390-396. 3. Theofilopoulos AN. Autoimmunity. In: Stites DP, Stobo JD, Fudenberg HH, Wells JV, eds. Basic and Clinical Immunology, 5th Ed., Los Altos, CA, Lange Medical Publications, 1982:152-186. 4. Ober C, Gleicher N, Elias S, Harlow G. Natural autoantibodies in
5. 6. 7. 8. 9. 10.
11. 12. 13.
14.
15. 16. 17. 18.
Autoantibodies in Normal and Abnormal Pregnancy NORBERT GLEICHER Center for Human Reproduction and Foundation for Reproductive Medicine, Inc. Chicago, Illinois
Abnormal autoimmune function has been associated with reproductive failure for decades. In fact, all medical conditions historically associated with pregnancy loss (and on occasion infertility) are now recognized to have an autoimmune etiology. It is therefore quite surprising that only less than a decade ago a correlation between the presence of abnormal autoantibodies and pregnancy loss was reported for the first time. Since reproductive failure, similar to other abnormal autoimmune states, is not a monoclonal event, we have established that affected patients demonstrate polyclonal autoantibody abnormalities including a variety of nonorganospecific as well as organspecific autoantibody groupings. For example, patients with repeated pregnancy loss will exhibit abnormal levels of anti-phospholipid antibodies (PA). In normal pregnancy natural autoantibodies do not follow the standard immunoglobulin (Ig) pattern, characterized by a decrease in levels despite a probably mild increase in production, which is excessively compensated by the vasodilatation of pregnancy. PA, especially, demonstrate a mild increase during pregnancy, though levels only in the peripartal period may reach abnormally high titers (in comparison to nonpregnant controls). This dichotomy between total Ig and natural autoantibodies is interesting since it suggests that autoantibodies may be under distinct control. In fact, we have suggested that this observed elevation of autoantibodies may be the result of an antigenic stimulus by self-like antigen, represented by the maternal growth of the parasitic fetus. In abnormal pregnancies, especially those associated with maternal hypertension and fetal growth retardation (IUGR), autoantibody levels do reach highly abnormal levels. We have reported that in both of these conditions significant abnormalities especially in IgG-PA can be detected, which stand in direct correlation with severity of disease (though autoimmune hemolytic anemia (AHA) can also be found). This is an interesting observation because both of these conditions have been associated with an imbalance between thromboxane and prostacycline in favor of the platelet aggregating and vasoconstrictive thromboxane. This is exactly the imbalance reported in women with habitual abortions, which is assumed to be PA-induced. Aspirin, recently widely reported as being successful in the prevention of pregnancy-induced hypertension (PIH) and IUGR, has also been recommended as treatment for ABSTRACT:
Submitted for publication June 30, 1992; accepted July 6, 1992. Address reprint requests to Norbert Gleicher, Center for Human Reproduction and Foundation for Reproductive Medicine, Inc., 750 N. Orleans Street, Chicago, IL60610.
habitual aborters. Lastly, habitual aborters who are successfully treated and reach viability in a subsequent pregnancy, experience a highly increased incidence of PIH and IUGR. All of these observations strongly suggest that PIH, IUGR, and repeated pregnancy loss may have a common pathophysiology. It is tempting to speculate that it is autoantibody-and especially PA- related. (Am J Reprod Immunol. 1992; 28:269-273.) Key words: Anti-phospholipid, intrauterine growth regardation, pre-eclampsia autoantibody, cardiolipin INTRODUCTION
The fetus is a semi-allograft and semi-autograft, As feto-maternal cell traffic increases with advancing gestational age, one can presume that the maternal immune system sees not only increased loads of alloantigens but also epitopes identical, or at least similar, to autoantigens. In fact, such an autoantigen load would seem almost unprecedented, especially late in pregnancy and during delivery, when cell traffic is the largest. A compensatory mechanism to neutralize this antigen load during normal pregnancy can therefore be expected and probably, in fact, exists. NORMAL PREGNANCY
Total immunoglobulin levels in pregnancy decrease, despite an increase in total immunoglobulin production. This is due to the considerable expansion in plasma volume during pregnancy, which exceeds the increase in immunoglobulin production and thus results in a dilutional hypogammaglobulinemia.! Autoantibody levels in pregnancy do not follow this pattern. In a longitudinal study involving 43 patients, we were able to demonstrate that neither phospholipid antibodies nor antibodies to histones or to polynucleotides decrease in parallel to total immunoglobulin levels (Tables 1III) and, in fact, IgM autoantibodies increase significantly at time of delivery. 2 The same study also revealed no change in lupus anticoagulant level over the length of gestation. This dichotomy between total immunoglobulin and autoantibody levels strongly suggests that the regulation of autoantibody levels during pregnancy is under separate control from that of other antibodies. It is therefore tempting to speculate that the apparently selectively increased autoantibody levels during gestation represent the previously noted compensatory mechanism for the excessive autoantigen load faced by the pregnant woman. One could further speculate that this specifically female need to compensate for an autoantibody overload during pregnancy is also related to the fact that women almost universally demonstrate higher autoantibody levels than normal males 3 ,4 and experience many times their incidence of autoimmune diseases.V'
270
GLEICHER
TABLE I. Immunoglobulin (lg) G Autoantibodies Throughout Gestation" Nonpregnant controls Total IgG (mg/dl.) Phospholipids Cardiolipin Phosphatidylserine PhosphatidyIglycerol Phosphatidylethanolamine Phosphatidylinositol Phosphatidic acid Histones HI H2A H2B H3 H4 Polynucleotides ssDNA dsDNA Poly(l) Poly(dT)
1220 ± 350
Week of pregnancy 24
16
20
930 ± 280 b
970 ± 195
916 ± 195b
28
Delivery (wk39 + 1.9)
32
1010 ± 276
974 ± 195 b
939 ± 280 b
24 16 26 5 18 15
± ± ± ± ± ±
25 19 26 13 18 19
11 10 19 9 45 15
± ± ± ± ± ±
13 43 20 17 37 b 9
8 ± 13 6±8 10 ± 6 3±4 10 ± 40 11 ± 13
16 ± 18 8 ± 10 16 ± 23 4±4 31 ± 20 13 ± 20
14 7 11 5 12 15
13 10 10 18 15 11
7±9 6±8 25 ± 36 11 ± 33 10 ± 2 21 ± 24
7 ± 10 7±9 34 ± 40 c,d 12 ± 36 8±2 22 ± 27 c ,d
17 13 9 11 32
± ± ± ± ±
17 17 10 13 39
13 8 36 19 31
± ± ± ± ±
14 13 40b 22 33
4±4 7 ± 10 16 ± 43 18 ± 62 28 ± 32
11±11 7 ± 11 17 ± 13 11 ± 17 27 ± 17
4±4 5 ± 12 14 ± 21 15 ± 25 21 ± 13
5±5 8 ± 14 18 ± 43 22 ± 34 31 ± 23
6±7 9 ± 15 20 ± 46 24 ± 38 34 ± 25
55 120 60 166
± ± ± ±
51 80 44 90
31 84 10 100
± ± ± ±
50 44 15 66
25 ± 48 75 ± 51 5±5 62 ± 62
38 51 17 82
22 ± 30 62 ± 75 22 ± 16 53 + 50
24 ± 22 59 ± 44 11 ± 10 74 + 67
27 ± 25 79 ± 39 12 ± 11 81 + 74
± ± ± ±
42 67 20 43
± ± ± ± ± ±
"From EI-Roeiy et al.,2 with permission. Data are presented as optical density x 103 at 405 nm. "Statistically increased level compared with nonpregnant controls (P < .05). 'Statistically increased level compared with levels at 16 wk (before normalization) (P < .05). dStatistically increased level compared with levels at 16 wk (after normalization) (P < .05).
since basically all medical conditions historically associated with excessive pregnancy loss are now understood or believed to have an autoimmune etiology (Table IV). Our early work with lupus anticoagulant-positive habitual aborters suggested to us that their B lymphocyte hyperactivity was not restricted to the production oflupus anticoagulant." In fact, we quickly became convinced that their B cell function was similar to that of such classical autoimmune diseases as systemic lupus erythematosus (SLE) in that they produced a wide variety of abnormal autoantibody specificities." It was this observation that led us to investigate patients with suspected pregnancy abnormalities due to abnormal B cell activity with a broadly based autoantibody profile characterized by six antibodies to phospholipid antigens, five
ABNORMAL PREGNANCY
While autoantibody levels (especially of IgM isotype) rise during late normal pregnancy, they only rarely reach abnormal levels, whether defined by a 95 or 99 confidence interval of a normal population.f This kind of statistical evaluation is important whenever autoantibody levels are evaluated because of their nonparametrical distribution, which results in an inordinate number of false positive results if, instead, multiples of means are utilized." Abnormal levels of autoantibodies have repeatedly been implicated in pregnancy abnormalities after Lubbe et al. reported their initial observation of an association between lupus anticoagulant and repeated pregnancy 10ss.7 It is actually quite surprising that the association between pregnancy wastage and abnormal autoimmune function has not been recognized earlier
TABLE II. Immunoglobulin (lg) M Autoantibodies Throughout Gestation" Nonpregnant controls Total IgM (rng/dl.) Phospholipids Cardiolipin Phosphatidylserine Phosphatidylglycerol Phosphatidylethanolamine Phosphatidy!inositol Phosphatidic acid Histones HI H2A H2B H3 H4 Polynucleotides ssDNA dsDNA Poly(l) Poly(dTl
180 ± 70 24 ± 19 29 ± 31 32 ± 38 5±8 26 ± 26 20 ± 24
16 155 ± 40
20
Week of pregnancy 24
28
32
151 ± 64
161 ± 66
153 ± 54
158 ± 62
Delivery (wk39 + 1.9) 151 ± 55
23 ± 19 14 ± 13 33 ± 26 2±3 38 ± 22 d 32 ± 27 d
11 ± 8 8 ± 11 34 ± 18 3±3 13 ± 8 19 ± 13
15 14 23 3 39 14
10 11 22 4 22 10
29 14 16 3 41 15
36 13 22 5 24 d 11
22 35 39 4 20 20
13 22 45 4 17 24
24 39 42 5 22 22
14b 24 49 b ,c 4 18 27
56 46 48 47 139
± ± ± ± ±
38 36 35 33 79
44 61 12 39 80
± ± ± ± ±
30 31 18 21 72
45 37 29 29 73
± ± ± ± ±
25 25 20 20 56
22 26 36 25 29
25 23 22 22 23
27 38 34 25 46
20 21 21 19 34
54 63 59 50 96
32 39 38 27 60
60 70 65 55 105
35 c 43 c 41 b ,c 30 66
115 169 143 171
± ± ± ±
76 84 86 102
12. ± 85 ± 79 ± 68 ±
25 75 41 46
33 69 106 78
± ± ± ±
18 97 80 48
36 87 80 75
27 91 80 95
18 75 89 95
20 77 44 77
45 119 127 117
29 71 59 60
50 100 140 128
32 81 64 b ,c 66 b ,c
"From EI-Roeiy et al.,2 with permission. Data are presented as optical density x 103 at 405 nm. bStatistically increased level compared with levels at 16 wk (before normalization) (J' < .05). 'Statistically increased level compared with levels at 16 wk (after normalization) (P < .05). dStatistically increased level compared with nonpregnant controls (P < .05).
AUTOANTIBODIES IN NORMAL AND ABNORMAL PREGNANCY
271
TABLE III. Immunoglobulin (Ig) A Autoantibodies Throughout Gestation" Nonpregnant controls Total IgA (mg/dl.) Phospholipids Cardiolipin Phosphatidylserine Phosphatidylglycerol Phosphatidylethanolamine Phosphatidylinositol Phosphatidic acid Histones HI H2A H2B H3 H4 Polynucleotides ssDNA dsDNA Poly(l) Poly(dT)
165 ± 75
165 ± 51
Week of pregnancy 24
20
16
28
32
Delivery (wk39 ± 1.9)
164 ± 55
160 ± 42
167 ± 40
158 ± 60
159 ± 59
6±9 25 ± 45 8 ± 21 4±8 7±8 9 ± 11
9±6 4±8 11 ± 14 9 ± 11 13 ± 14b 10 ± 8
4±3 4±3 16 ± 5 2± 1 16 ± 11 10 ± 9
4±6 3±2 15 ± 13 12 ± 16 14 ± 9 11 ± 10
6±3 5±4 3±4 3±2 25 ± 19 10 ± 9
4±4 8±8 7±6 3 ± 2 17 ± 14 10 ± 6
4±5 9±9 8±7 3±3 19 ± 16 11 ± 17
10 ± 15 6±7 8 ± 10 11 ± 21 20 ± 19
9±9 11 ± 13 13 ± 15 11 ± 23 41 ± 28b
8±9 6±6 7±8 5±6 13 ± 11
5±6 7±7 4±4 8 ± 10 8±8
4±4 5±8 4±4 4±3 10 ± 10
6±4 7±8 10 ± 7 9±8 13 ± 10
7± 5 8±8 11 ± 8 10 ± 9 15 ± 11
19 90 34 49
9 50 24 11
10 48 17 7
11 ± 9 56 ± 38 44 ± 48 8±7
13 ± 13 59 ± 56 21 ± 16 9±5
14 63 36 15
15 60 40 17
± ± ± ±
22 87 87 36
± ± ± ±
14 34 17 13
± 8 ± 35
± 22 ±5
± ± ± ±
11 39 27 8
± ± ± ±
13 40 29 8
"From El -Roeiy et al.: with permission. Data are presented as optical density x 103 at 405 nm. bStatistically increased level compared with nonpregnant controls (P < .05).
to histones and their subfractions, and four to polynucleotides, all separately for IgG, IgA, and IgM isotypes. The economic performance of 45 assays on a single blood sample was possible through the establishment of an automated enzyme-linked immunosorbent assay (ELISA) system for that purpose. More recently, we have added the investigation of antithyroid antibodies by radioimmunoassay to this profile in view of a recent report by Stagnaro-Green et al., who reported a high predictive value of elevated antithyroid antibodies in patients with pregnancy 10SS.1O Moreover, similar to other autoantibody specificities, antithyroid antibodies are detectable in a large number ofSLE patients. 11 Therefore, we can anticipate a similar incidence in pregnant patients with autoantibody-associated pregnancy abnormalities. Successfully treated habitual aborters who reach fetal viability suffer from an abnormally high incidence of pregnancy-associated hypertensive conditions'P and from fetal intrauterine growth retardation and fetal distress." This observation led us to expand our antibody studies from patients with pregnancy wasta.re to women with hypertensive diseases of pregnancy.' In a statistically highly complex study, we demonstrated convincingly that hypertensive diseases of pregnancy were statistically
related to the severity of autoantibody abnormalities, defined by the number of abnormally elevated autoantibodies (Fig. 1). Moreover, the incidence of autoantibody abnormalities was also closely related to the risk of peri-
TABLE IV. Conditions Characterized by Abnormal Autoimmune Function That Have Been Historically Associated With an Increased Risk in Pregnancy Collagen vasular diseases Systemic lupus erythematosus Mixed connective tissue disease Systemic sclerosis Dermatitis herpetiformis Celiac disease Herpes gestationis Ulcerative colitis Crohn's disease Chronic active hepatitis Thyroid disease Diabetes mellitus Endometriosis
Fig.!. The distribution between growth retarded (lUGR) and normal size (AGA)infants correlated significantly at "P < 0.05, **p < 0.01, or ***p < 0.001 in each patient group, whether normotensive or hypertensive, with autoantibody positivity. The horizontal line indicates the number of expected positives in view of 45 assays performed for each patient. Moreover, hypertensive pregnancies show significantly more positive autoantibodies than normotensive controls. From El-Roeiy et al.,14with permission.
272
GLEICHER
•
Nor-mal pr-egnant contr-ol n-49
[T"/iMlld pr-eeclampsia lL.l n-1B rv'!Chr-onic hyper-tension ~ without SPE n-15 ~
Sever-e pr-eec lamps ia
~
Chr-onic hyper-tens ion with SPE n-3
~n-11 ~
Fig. 2. Correlation between incidence of antibody positivity and hypertensive condition of pregnancy. Antibody positivity increases in paral-
lei to the increasing perinatal mortality reported for various hypertensive conditions of pregnancy.
natal mortality from the underlying hypertensive condition (Fig. 2). The incidence of autoantibody abnormalities also correlated directly to the occurrence of intrauterine growth retardation. This correlation was even evident among normotensive control patients who did not express an excessive incidence of autoantibody abnormalities (Fig. 1). In an attempt to identify the most likely antibody grouping responsible for all these findings, it became apparent that the antiphospholipid antibodies of IgG isotype were the most likely culprits (Figs. 3 ,4). PATHOPHYSIOLOGY
Fig. 3. Distribution of autoantibody isotypes in normotensive and hypertensive groups. (*) Significantly (P < 0.05) different from normotensive (AGAand IUGR) and hypertensive (AGA) groups. Most reactivity can be seen among IgG autoantibodies. From EI-Roeiy et al.,14 with permission.
Phospholipid antibody specificities have been closely associated with the presence of lupus anticoagulant. Lupus anticoagulant affects the normal balance of thromboxane and prostacyclin in favor of the vasoconstrictive and platelet aggregating thromboxane. The result is a clinical risk for thromboembolic phenomena. 15 It has furthermore been suggested that the hypercoagulable state in these patients may lead to microvascular thrombotic events within the placenta, which in turn may be a contributing factor to autoantibody induced pregnancy complications.F Whether the latter mechanism in fact plays a role has to be questioned because histologic studies have so far been unable to demonstrate such microthrombi. Some investigators have demonstrated anecdotally a special affinity of monoclonal phospholipid antibodies to placental vessels. Others have suggested that antibodies may exert their effect (at least in habitual aborters) by affecting the coagulation cascade and, thus, result in excessive fibrin deposits. Our thinking has developed in a different direction. Impressed by the multitude of untoward effects of autoantibodies on reproductivefrocesses, which are not only restricted to pregnancy.P'! ,17 we felt that autoantibod-
AUTOANTIBODIES IN NORMAL AND ABNORMAL PREGNANCY
273
Fig. 4. Autoantibody positivity is primarily concentrated among phospholipid antibodies. Significant differences are denoted by (*). Antihistone antibody findings are difficult to interpret because of the
fact that many hypertensive patients were receiving treatment with methylolopa, which can induce such autoantibodies. From El Roeiy et al.,t4 with permission.
ies had to exert their effects at a very basic, possibly cellular, level. Since many second messengers of classical signal transduction processes are phospholipids, antibodies to phospholipids could, thus, theoretically interfere with different and essential cellular processes and could do so at greatly varying degrees. Therefore, we investigated this concept in a classical signal transduction model, utilizing hCG production by trophoblast cells from term pregnancies at the end product of the signaling process. Studies with polyclonal sera from patients with a multitude of phospholipid antibodies supported our hypothesis by demonstrating a reduction of signal with the addition of these sera. Moreover,phospholipid antibodypositive sera also demonstrated a considerable reactivity to IP3, a classical second messenger. IS While at this point these studies have to be considered as preliminary, their confirmation with monoclonal antibodies (rather than polyclonal sera) would suggest the fascinating possibility that autoantibodies at normal levels may serve as regulators of physiologic signal transduction processes. At abnormal levels, they would then obviously interfere with those same physiological processes, which they are teleologically meant to protect. This hypothesis could also explain why normal individuals express autoantibodies at low levels.
Hutterites: 1: Sex differences in antibody levels. Am J Reprod Immunol, 1990; 22:85. (Abstract) Shoenfeld Y, Isenberg D. The mosaic of autoimmunity. Research Monographs in Immunology, Vol 12; Elsevier, Amsterdam, New York, Oxford, 1989. El-Roeiy A, Gleicher N. Autoantibodies in a healthy normal population. Obstet Gynecol. 1988; 72:596-602. Lubbe WF, Butler WS, Palmer SJ, Liggins GC. Lupus anticoagulant in pregnancy. Br J Obstet Gynaecol. 1984; 91:359-364. Gleicher N, Friberg J. IpM gammopathy and the lupus anticoagulant syndrome in habitual aborters. JAMA. 1985; 253:3278-3281. Gleicher N, El-Roeiy A, Confino E. Reproductive failure because of autoantibodies: Unexplained infertility and pregnancy wastage. Am J Obstet Gynecol. 1989; 160:1376-1380. Stagnaro-Green A, Roman SH, Cobin RH, El-Harazy E, AlvarezMarfany M, Davies TF. Detection of at-risk pregnancy by means of highly sensitive assays for thyroid autoantibodies. JAMA. 1990; 264:1422-1425. Magaro M, Zoll A, Altomonte L, Mirone L, La Sala L, Barini A, Scuder F. The association of silent thyroiditis with active systemic lupus erythematosus. Clin Exp Rheumatol. 1992; 10:67-70. Branch DW, Scott Jr, Kochenour NK, Hershgold E. Obstetric complications associated with the lupus anticoagulant. N Engl J Med. 1986; 313:1322-1326. Lockshin MD, Druzin MC, Goei S, Gamar T, Magid MS, Jovanovic L, Ferenc M. Antibody to cardiolipin as a predictor of fetal distress or death in pregnant patients with systemic lupus erythematosus. N Engl J Med. 1985; 313:152-155. El-Roeiy A, Myers SA, Gleicher N. The relationship between autoantibodies and intrauterine growth retardation in hypertensive disorders of pregnancy. Am J Obstet Gynecol. 1991; 164: 1253-1261. Editorial. Lupus anticoagulant. Lancet. 1984;'1:1157-1158. El-Roeiy A, Valesini G, Friberg J, Shoenfeld Y, Kennedy R, Gleicher N. Autoantibodies and common idiotypes in men and women with sperm antibodies. Am J Obstet Gynecol. 1988; 158:596-603. El-Roeiy A, Gleicher N, Friberg J, Confino E, Dudkiewicz A. Correlation between peripheral blood and follicular autoantibodies and impact on in vitro fertilization. Obstet Gynecol. 1987; 70:163-170. Gleicher N, Harlow L, Zilberstein M. The regulatory effect of antiphospholipid antibodies (APA) on signal transduction: A possible model for autoantibody induced reproductive failure. Am J Obstet Gynecol. 1992; 167:637 -642.
REFERENCES 1. Myers SA, Gleicher N. Physiologic changes in normal pregnancy. In: Gleicher N, ed. Principles and Practice of Medical Therapy in Pregnancy, 2nd Ed; Appleton & Lange, 1991:35-:-52. 2. El-Roeiy A, Myers SA, Gleicher N. The prevalence of autoantibodies and lupus anticoagulant in healthy pregnant women. Obstet Gynecol. 1990; 75:390-396. 3. Theofilopoulos AN. Autoimmunity. In: Stites DP, Stobo JD, Fudenberg HH, Wells JV, eds. Basic and Clinical Immunology, 5th Ed., Los Altos, CA, Lange Medical Publications, 1982:152-186. 4. Ober C, Gleicher N, Elias S, Harlow G. Natural autoantibodies in
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