Thyroid autoimmunity and abortion: a prospective study in women undergoing in vitro fertilization

FERTILITY AND STERILITY VOL. 71, NO. 1, JANUARY 1999 Copyright 1998 American Society for Reproductive Medicine ublished by Elsevier Science Inc. rinted on acid-free paper in U.S.A. Thyroid autoimmunity and abortion: a prospective study in women undergoing in vitro fertilization A. F. Muller, M.D.,* A. Verhoeff, M.D., M. J. Mantel, M.Sc., and A. Berghout, M.D.* Zuiderziekenhuis Rotterdam, Rotterdam, the Netherlands Received April 27, 1998; revised and accepted August 18, 1998. resented in part at the XXIII Annual Meeting of the European Thyroid Association, Amsterdam, the Netherlands, August 31 September 4, 1996. Reprint requests and present address: A. F. Muller, M.D., Department of Internal Medicine III, Dijkzigt University Hospital Rotterdam, Dr. Molewaterplein 40, Room D438, 3015 GD, Rotterdam, the Netherlands (FAX: 0031-10-463-3268; E-mail: muller@inw3.azr.nl). * Department of Internal Medicine. Department of Obstetrics and Gynaecology. Department of Clinical Chemistry. 0015-0282/98/$19.00 II S0015-0282(98)00394-X Objective: To determine whether an association exists between the presence of thyroid peroxidase () antibodies before pregnancy and miscarriage in women without a history of habitual abortion. Design: rospective study and nested case-control study. Setting: Inner-city teaching hospital. atient(s): Four hundred eighty-nine women in an IVF program. Intervention(s): In the prospective study, we measured levels of antibodies and TSH. In the nested case-control study, we also measured levels of anticardiolipin antibodies. Main Outcome Measure: Miscarriage. Result(s): One hundred seventy-three women were observed, of whom 31% (54/173) became pregnant. regnancy occurred in 48% (12/25) of the antibody-positive women and in 28% (42/148) of the antibodynegative women. Among those who became pregnant, miscarriage occurred in 33% (4/12) of antibody positive women and in 19% (8/42) of antibody negative women. The TSH level was abnormal ( 0.2 IU/mL) in only one of the antibody positive women who miscarried. The presence of anticardiolipin antibodies was not associated with miscarriage. Conclusion(s): No association was found between the presence of antibodies before pregnancy and miscarriage in women without a history of habitual abortion. The presence of antibodies did not adversely affect a woman s chances of becoming pregnant. (Fertil Steril 1999;71:30 4. 1998 by American Society for Reproductive Medicine.) Key Words: Thyroid antibodies, miscarriage, anticardiolipin antibodies, thyroid function, IVF, pregnancy Spontaneous pregnancy loss is common. With the use of a sensitive immunoradiometric assay that measured urinary hcg, it was shown that the total rate of spontaneous pregnancy loss, including clinically recognized spontaneous abortions, was 31%. Most of these losses occurred before pregnancy was clinically recognized (1). In women with a history of habitual abortion ( 3 consecutive abortions), extensive diagnostic screening identifies abnormalities in only 55% 65% (2, 3). The presence of nonorgan-specific antibodies, notably phospholipid antibodies, has been associated with spontaneous pregnancy loss (4 7). Stagnaro-Green et al. (8) found that the presence of thyroid peroxidase () or thyroglobulin antibodies in the first trimester of pregnancy is a risk factor for subsequent spontaneous pregnancy loss. These results were confirmed by other investigators (9 12). ratt et al. (13) studied the significance of the presence of thyroid antibodies before pregnancy in women with a history of habitual abortion and found these antibodies to increase the risk for yet another miscarriage significantly. The aim of the present study was to determine the relationship between the presence of antibodies before pregnancy and the occurrence of spontaneous abortion in women without a history of pregnancy loss. We therefore designed a prospective study of women undergoing IVF. Furthermore, we performed a nested casecontrol study in which we measured levels of anticardiolipin antibodies in those who miscarried and in matched controls with ongoing pregnancies. Finally, levels of antibodies 30

were determined in those who had a miscarriage, after the event. MATERIALS AND METHODS Between March 1994 and March 1996, 489 unselected women who were eligible for an IVF procedure were invited to participate in a follow-up study. One hundred seventyseven women agreed to participate. The study was approved by the hospital s ethics committee, and all subjects gave informed consent at initial presentation. Our IVF program has been described in detail previously (14). Blood was drawn from all women at the initial visit, and serum was stored at 70 C for future assay of TSH levels and for determining the presence of and anticardiolipin antibodies. In four women, one of whom became pregnant and had an uneventful pregnancy, it was not determined whether antibodies were present. These four women were excluded from the analysis. regnancy was determined by immunoassay of urinary hcg on day 18 after embryo transfer. When findings were positive, vaginal ultrasonography was performed 2 weeks later. Women were characterized as having ongoing pregnancies (pregnancies continuing into the third trimester) or spontaneous abortions (miscarriages before the third trimester). We determined antibody and TSH levels in all serum samples. Furthermore, in the case of patients who miscarried, levels of anticardiolipin antibodies were determined in the sera obtained before pregnancy. For comparison with s obtained before pregnancy, we determined antibody levels again after miscarriage. Levels of antibodies were again determined in the first trimester of pregnancy in a random subgroup of women with ongoing pregnancies. Assays Urinary hcg levels were determined by immunoassay (acific Biotech, San Diego, CA); s of 25 U/L were considered to indicate positivity. Levels of TSH were determined with use of an immunoluminometric assay (ACS, Medfield, MA), with a reference range of 0.2 4.5 IU/mL. Levels of antibodies were determined with use of an RIA (Henning Berlin, Berlin, Germany), using a cutoff of 80 U/mL. The level of anticardiolipin antibodies was determined with use of an ELISA (Orgentec, Mainz, Germany); using a cutoff of 7 U/mL for IgM and 10 U/mL for IgG. Statistics Qualitative data between groups were tested using the 2 test. Data not normally distributed were analyzed by the Mann-Whitney U test and Wilcoxon s rank sum test. s of.05 were considered statistically significant. RESULTS General data Between participants and nonparticipants and between antibody positive and antibody negative women, there were no differences in age, cause of infertility (Table 1), number of previous pregnancies and deliveries, or pregnancy or spontaneous abortion rate. Levels of antibodies and TSH Of 173 women who were screened, 14% (25 of 173) had antibody levels of 80 U/mL. regnancy occurred in 48% (12 of 25) of antibody positive women and in 28% (42 of 148) of antibody negative women (.05). Among all women who became pregnant, miscarriage occurred in 33% (4 of 12) of antibody positive women and in 19% (8 of 42) of antibody negative women (.29) (Table 1); mean antibody titers were not significantly different. Figure 1 shows the distribution of titers in the different study groups. The antibody levels in 10 of the 12 women who had a miscarriage (two subjects who did not consent to repeated venipuncture had levels of 80 U/mL and normal TSH levels) were unchanged after the event. Levels of antibodies were again determined in the first trimester of pregnancy in a subgroup of 10 women with ongoing pregnancies who did not differ from the study population as a whole with regard to previously mentioned characteristics. No change in the presence (3 of 10 women) or absence (7 of 10 women) of antibodies was observed. Levels of TSH were not significantly different in those women who became pregnant compared with those who did not (1.9 1.2 versus 2.2 2.4 IU/mL); TSH levels were comparable in women with ongoing pregnancies and in women who miscarried (2.3 2.6 versus 1.8 1.6 IU/ ml). Among pregnant women (both those who miscarried and those who did not), TSH levels were not significantly different between those with and those without antibodies (3.8 4.7 versus 1.8 1.0 IU/mL); among women who miscarried, antibody positive and antibody negative women had similar TSH levels (1.3 1.1 versus 2.0 1.9 IU/mL) (Table 1). TSH levels in positive women were not different in nonpregnant women, pregnant women who delivered, and women who miscarried. Figure 2 shows the distribution of TSH levels in antibody positive women. In the study population as a whole, subjects with antibodies had significantly higher TSH levels than did subjects without antibodies (3.5 3.6 versus 1.7 0.9 IU/mL,.05). Elevated TSH levels ( 4.5 IU/mL) were found in three antibody positive women with ongoing pregnancies (range, 6.4 15.7 IU/mL), in four antibody positive FERTILITY & STERILITY 31

TABLE 1 Thyroid peroxidase antibodies, TSH levels, and general data. Study group All participants (n 173) regnant women (n 54) Women who did not miscarry (n 42) Women who miscarried (n 12) No. of patients with detectable antibodies 25 (14) 148 (86) 8 (19) 34 (81) 4 (33) 8 (67) Mean TSH level ( IU/mL) 3.5 3.6 1.7 0.9.05 5.0 5.3 1.7 0.6 NS 1.3 1.1 2.0 1.9 NS No. of patients with indicated TSH level* 0.2 1 (4) 5 (3) 0 1 (3) 1 (25) 0 0.2 4.5 17 (68) 140 (95) 5 (63) 33 (97) 3 (75) 7 (88) 4.5 7 (28) 1 (1) 3 (38) 0 0 1 (13) Mean age (y) 32.4 3.3 32.4 4.4 NS 31.5 2.7 31.7 4.2 NS 31.3 4.4 32.6 4.3 NS No. of patients with indicated cause of infertility Male infertility or fallopian tube obstruction 15 (60) 97 (66) NS 6 (75) 20 (59) NS 2 (50) 6 (75) NS Endometriosis 0 11 (7) NS 0 1 (3) NS 0 1 (13) NS COS 5 (20) 9 (6) NS 2 (25) 3 (9) NS 1 (25) 0 NS Idiopathic 5 (20) 31 (21) NS 0 10 (29) NS 1 (25) 1 (13) NS No. of patients with family history of thyroid disease 2 (8) 16 (11) NS 0 3 (9) NS 0 2 (25) NS No. of patients who smoked 5 (20) 55 (37) NS 1 (13) 15 (44) NS 2 (50) 1 (13) NS Note: Values are n (%) or means SD. thyroid peroxidase, NS not significant, COS polycystic ovary syndrome. * Level of TSH not determined in two antibody negative women, neither of whom became pregnant. Family history of thyroid disease unknown in two women who were positive for antibodies, one of whom subsequently miscarried. Smoking behavior unknown in four women who were negative for antibodies, one of whom subsequently miscarried. women who did not become pregnant (range, 4.9 9.5 IU/ ml), and in one antibody negative woman who subsequently miscarried (6.0 IU/mL). In four antibody negative women who did not become pregnant and in two women who did become pregnant, one of whom was antibody positive and subsequently miscarried, TSH levels of 0.20 IU/mL were found (Table 1). Nested case-control study Women who had had a first-trimester miscarriage (n 12) were compared with women who had ongoing pregnancies (n 23). No differences in previously mentioned characteristics between case subjects or controls were found. No increased prevalence of anticardiolipin antibodies was observed in case subjects compared with controls. DISCUSSION By studying patients entering an IVF program, we were able to determine the levels of and anticardiolipin antibodies before pregnancy in women without a history of habitual abortion and correlate these results with pregnancy outcome. In those women who became pregnant, no significant relationship between the presence of antibodies and subsequent miscarriage was found. One could argue that by setting the cutoff in our study at 80 U/mL, we included some women with milder thyroid autoimmunity. To investigate this possibility, we recalculated the correlation between miscarriage and the presence of antibodies using a cutoff of 100 U/mL, a used in the studies by Glinoer et al. (9, 10), in which a similar assay system was used. Again no correlation was found. In previous studies describing an association between presence of thyroid antibodies and miscarriage, antibody levels were determined during pregnancy (8 10, 12) or after a history of three or more consecutive miscarriages (13). Stagnaro-Green et al. (8) studied 552 consecutive unselected women in the first trimester of pregnancy and found the miscarriage rate in thyroid autoantibody positive women to be significantly higher than in antibody-negative women (17% versus 8.4%). These results were confirmed by Glinoer et al. (9, 10), who found an increased rate of spontaneous abortion in 45 women with detectable thyroid autoantibodies compared with 603 controls (13.3% versus 3.3%) (9). From the data of ratt et al. (13), it can be concluded that the presence of antibodies before pregnancy is associated with first-trimester pregnancy loss. However, the 42 women in this study population had histories of three or more consecutive abortions and were therefore already at high risk for yet another abortion. These results are in 32 Muller et al. Thyroid autoimmunity and abortion Vol. 71, No. 1, January 1999

FIGURE 1 Box-and-whisker plot of levels (after log-transformation). The boxes contain 50% of s between the 25th and 75th percentiles; the whiskers indicate the highest and lowest s. The horizontal line inside each box represents the median. E cases with s between 1.5 and 3 box lengths from the upper or lower edge of the box. cases with s more than 3 box lengths from the upper or lower edge of the box. contrast to those recently reported by Esplin et al. (15), who did not find an association between the presence of thyroid autoantibodies and recurrent pregnancy loss. Singh et al. (12) studied 487 women who conceived with assisted reproductive techniques. These investigators (12) found that determining the presence of thyroid autoantibodies, that were measured 14 days after embryo transfer, was useful in identifying women at risk for miscarriage after the pregnancy was clinically recognized (i.e., after visualization of a gestation sac) but not in identifying women at risk for pregnancy loss before the pregnancy was clinically recognized (i.e., hcg-detected). This could indicate that autoimmunity, as reflected by the presence of thyroid autoantibodies, may not be involved in the earliest stages of pregnancy loss. However, exclusion of these cases of biochemical pregnancy loss (n 4) (pregnancy loss before visualization of a gestation sac), did not change our findings. It could be that unexplained infertility is simply occurrence of preclinical pregnancy loss, which would mean that there were some unrecognized cases of recurrent pregnancy loss in our study population. However, Wilcox et al. (1) found that women with an early pregnancy loss proved to be highly fertile in subsequent cycles. Furthermore, only a minority of our patients had unexplained infertility; in the majority of cases there was male infertility or tubal dysfunction. It might be argued that selection bias may have been present in our study, our entire population having a higherthan-expected prevalence of antibodies, resulting in infertility. However, a borderline significant correlation (.05) between the presence of antibodies (at cutoff s of 80 and 100 U/mL) and successful IVF treatment (i.e., resulting in pregnancy) was found. It is therefore unlikely that the presence of antibodies adversely affects the chances of becoming pregnant. Alternatively, it could be argued that with the use of IVF, (thyroid) autoimmune processes are bypassed. However, if this were true, one would not expect to find a higher pregnancy rate in the antibody positive group. In addition, using only data from women referred for IVF because of male infertility or tubal obstruction, we found a borderline significant correlation (.06) between the presence of antibodies and successful IVF treatment. The TSH level was abnormal (suppressed) in only one of the women with antibodies who miscarried. Elevated TSH levels in women with antibodies were not observed in women who miscarried (Figure 2). We therefore found no evidence in this study for an association between FERTILITY & STERILITY 33

FIGURE 2 Box-and-whisker plot of TSH levels in antibody positive women. The boxes contain 50% of s between the 25th and 75th percentiles, and the whiskers indicate the highest and lowest s. The horizontal line inside each box represents the median. E case with a between 1.5 and 3 box lengths from the upper edge of the box. thyroid autoimmunity, (subclinical) hypothyroidism, and miscarriage. Moreover, TSH levels in the women in this study are comparable to those previously described in nonpregnant women of the same age and living in the same area (16). Thus, antibodies that were present before pregnancy did not increase the risk of miscarriage in a subsequent pregnancy for women who did not have a history of habitual abortion and did not reduce a woman s chance of becoming pregnant. References 1. Wilcox AJ, Weinberg CR, O Connor JF, Baird DD, Schlatterer J, Canfield RE, et al. Incidence of early loss of pregnancy. N Engl J Med 1988;319:189 94. 2. Stray-edersen B, Stray-edersen S. Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol 1984;148:140 6. 3. Harger JH, Archer DF, Marchese SG, Muracca-Clemens M, Garver L. Etiology of recurrent pregnancy losses and outcome of subsequent pregnancies. Obstet Gynecol 1983;62:574 81. 4. Cowchock S, Smith JB, Gocial B. Antibodies to phospholipids and nuclear antigens in patients with repeated abortions. Am J Obstet Gynecol 1986;155:1002 10. 5. Maier DB, arke A. Subclinical autoimmunity in recurrent aborters. Fertil Steril 1989;280:280 5. 6. Unander AM, Norberg R, Hahn L, Årfors L. Anticardiolipin antibodies and complement in ninety-nine women with habitual abortion. Am J Obstet Gynecol 1987;156:114 9. 7. Tulppala M, Ailus K, alosuo T, Ylikokala O. Antibodies to oxidized low-density lipoprotein and to cardiolipin in nonpregnant and pregnant women with habitual abortion. Fertil Steril 1995;64:947 50. 8. Stagnaro-Green A, Roman SH, Cobin RH, El-Harazy E, Alvarez- Marfany M, Davies TF. Detection of at-risk pregnancy by means of highly sensitive assays for thyroid autoantibodies. JAMA 1990;264: 1422 5. 9. Glinoer D, Fernandez Soto M, Bourdoux, Lejeune B, Delange F, Lemone M, et al. regnancy in patients with mild thyroid abnormalities: maternal and neonatal repercussions. J Clin Endocrinol Metab 1991;73:421 7. 10. Lejeune B, Grun J, De Nayer h, Servais G, Glinoer D. Antithyroid antibodies underlying thyroid abnormalities and miscarriage or pregnancy induced hypertension. Br J Obstet Gynaecol 1993;100: 669 72. 11. Gleicher N. Autoantibodies and pregnancy loss. Lancet 1994;343: 747 8. 12. Singh A, Nery Dantas Z, Stone SC, Asch R. resence of thyroid antibodies in early reproductive failure: biochemical versus clinical pregnancies. Fertil Steril 1995;63:277 81. 13. ratt DE, Kaberlein G, Dudkiewicz A, Karandi V, Gleicher N. The association of antithyroid antibodies in euthyroid nonpregnant women with recurrent first trimester abortions in the next pregnancy. Fertil Steril 1993;60:1001 5. 14. Roest J, Verhoeff A, van Lent M, Huisman GJ, Zeilmaker GH. Results of decentralized in-vitro fertilisation treatment with transport and satellite clinics. Hum Reprod 1995;10:563 7. 15. Esplin S, Branch DW, Silver R, Stagnaro-Green A. Thyroid antibodies are not associated with recurrent pregnancy loss. Thyroid 1997;7(1 Suppl):92S. (Abstract). 16. Berghout A, Endert E, Ross A, Hogerzeil HV, Smits NJ, Wiersinga WM. Thyroid function and thyroid size in normal pregnant women living in an iodine replete area. Clin Endocrinol (Oxf) 1994;41:375 9. 34 Muller et al. Thyroid autoimmunity and abortion Vol. 71, No. 1, January 1999