Thyroid function after assisted reproductive technology in women free of thyroid disease Kris Poppe, M.D., a Daniel Glinoer, M.D., Ph.D., b Herman Tournaye, M.D., Ph.D., c Johan Schiettecatte, c Patrick Haentjens, M.D., Ph.D., a and Brigitte Velkeniers, M.D., Ph.D. a a Department of Endocrinology, Vrije Universiteit Brussel, Brussels; b Department of Endocrinology, Université Libre de Bruxelles, Brussels; and c Center for Reproductive Medicine, Vrije Universiteit Brussel, Brussels, Belgium Objective: To evaluate thyroid function in women undergoing a first assisted reproductive technology (ART) procedure and to compare women with ongoing pregnancy or miscarriage. Design: Prospective cohort study. Setting: Tertiary referral center. Patient(s): Seventy-seven women free of thyroid disease. Intervention(s): Serum TSH and FT4 were determined before and 2, 4, and 6 weeks after ET. All women received the same ART protocol. Main Outcome Measure(s): Thyroid function. Result(s): Forty-five women had ongoing pregnancies, and 32 suffered a miscarriage after 6.7 weeks (range 5 11). Mean age and number of ET were similar in both groups. Compared with baseline values, TSH and FT4 increased significantly 2 weeks after ET (ongoing pregnancies group: TSH 2.5 1.3 vs. 1.6 0.8 mu/l and FT4 13.8 1.4 vs. 12.4 1.8 ng/l; miscarriage group: TSH 2.1 1.0 vs. 1.5 0.7 mu/l and FT4 14.2 2.0 vs. 12.4 1.9 ng/l). Pregnancy outcome did not affect thyroid function and its evolution over time. Conclusion(s): In women free of thyroid diseases, thyroid function changed significantly after ART, but these changes were not different between women with ongoing pregnancy and miscarriage. (Fertil Steril 2005;83: 1753 7. 2005 by American Society for Reproductive Medicine.) Key Words: Thyroid, infertility, assisted reproductive technology, miscarriage Miscarriage occurs in approximately 30% of all pregnancies and is an important psychological burden for women. Only 10% of these miscarriages are clinically obvious, most pass without the women s awareness (biochemical pregnancy). The etiology of miscarriage is diverse and encompasses genetic anomalies, uterine factors, the presence of anticardiolipin antibodies, and hormonal abnormalities (1 3). Previous work indicated that thyroid dysfunction (hypoand hyperthyroidism) and the presence of thyroid antibodies (TAI ) increases the risk of infertility and miscarriage (4 8). The pathophysiology underlying the association between TAI and miscarriage remains largely speculative. Moreover, women with spontaneous abortion may have significantly lower thyroxine levels compared with women with ongoing pregnancy (9). With the awareness that subclinical forms of thyroid dysfunction and TAI are potentially adverse contributors in pregnancy outcome, we performed the present study to investigate whether the status of thyroid function in a cohort of women free of thyroid disease and undergoing an assisted Received December 8, 2004; revised and accepted December 8, 2004. Supported with grants from the Willy Gepts Foundation, Free University Brussels. Reprint requests: K. Poppe, M.D., Department of Endocrinology, Free University Brussels, Laarbeeklaan 101, 1090 Brussels-Belgium (FAX: 32 2 4776428; E-mail: hemopek@az.vub.ac.be). reproductive technology (ART) procedure was different between women who had ongoing pregnancies and women who had spontaneous miscarriages. While doing so, we wanted to avoid the confounding effects of thyroid autoimmunity. MATERIALS AND METHODS Research Question Among women pregnant after ART, do TSH and FT4 differ between women with ongoing pregnancies and women suffering a miscarriage? Overall Study Design Women presenting at the Center of Reproductive Medicine were systematically screened for thyroid hormone function (serum TSH and FT4) and for the presence of thyroid autoimmunity (thyroperoxidase antibodies [TPO-ab] and thyroglobulin antibodies [Tg-ab]). A diagnosis of thyroid disease was further evaluated from medical history on thyroid disease, and/or the use of thyroid medication. Only euthyroid women (normal serum TSH and FT4 levels) without thyroid antibodies (TAI ) with a clinically proven pregnancy after having received a first ART cycle were included in the study. Causes of infertility were as follows: male infertility (50%), tubal diseases (19%), endometriosis (9%), ovulatory disorders (7%), and idiopathic (15%). 0015-0282/05/$30.00 Fertility and Sterility Vol. 83, No. 6, June 2005 doi:10.1016/j.fertnstert.2004.12.036 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 1753
Thyroid function tests were further determined after confirmation of pregnancy as well as 2, 4, and 6 weeks after the ET, i.e., until the moment of miscarriage. Embryo transfer took place 5 to 7 days after the end of ovulation induction (OI). The median term for miscarriage was 6.7 weeks (range, 5 11). The institutional review board at our institution approved the study protocol. Assisted Reproductive Technology Treatment All female partners received ART treatment as previously described (10, 11). Pregnancy was diagnosed at least 10 days after ET by rising hcg levels of at least 20 IU/mL in serum on two occasions. Clinical pregnancies were diagnosed by ultrasonography performed 5 weeks after embryo transfer. Serum Assay Serum TSH and FT4 were measured using a third-generation electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). The reference values were 0.27 4.2 mu/l for TSH and 9.3 18.0 ng/l (12 to 23.2 pmol/l) for FT4 (1 ng/l FT4 1.29 pmol/l). Thyroid autoimmunity (TAI ) was excluded when TPO-ab ( 34 ku/l) and/or Tg-ab ( 115 IU/mL) were absent. TPO-ab was determined using an RIA-kit (BRAHMS Diagnostica, Berlin, Germany). The reference range was 0 34 ku/l. Antithyroglobulin antibodies (Tg-ab) were measured with an automated competitive immunoassay (Modular E170; Roche Diagnostics). The reference range was 0 115 IU/mL. Statistical Analysis We performed a formal sample size calculation based on information from a previous study from our group (10). According to this analysis, a projected total sample of 62 patients is required to detect a difference in serum TSH concentration of 1.3 mu/l between ongoing and miscarrying patients with an 80% power at an overall alpha risk of.05 (two-tailed), assuming a common standard deviation of 1.8 mu/l (12). To compare changes between pre- and post-art values of TSH and FT4 in the early stage of pregnancy (i.e., between baseline values and values at week 2 after ET) the paired Student s t-test was used. The unpaired Student s t-test was used to compare differences in thyroid function between ongoing pregnancy group and miscarriage group at baseline and week 2. A one-way (single-group) repeated measures analysis of variance (ANOVA) was conducted to explore the effect of time on TSH and FT4 serum values collected at four periods during the first months of pregnancy, i.e., before ART (time 0), and at weeks 2, 4, and 6 after ET. A two-way (between-groups) repeated measures ANOVA was conducted to explore the impact of the outcome status (ongoing pregnancy vs. miscarriage) on thyroid function, as measured by serum TSH and FT4 values during the first months of pregnancy. Given the numerous comparisons, the alpha threshold of.05 should not be applied to the P values for comparisons other than the primary outcome measure (repeated measures ANOVA on thyroid function). In this regard, a Bonferroni correction is required to avoid an alpha error. We chose to regard a level of P.006 as significant for these numerous comparisons (representing a Bonferroni correction of.05 divided by eight comparisons). All data analyses were performed using SPSS version 12.0 (SPSS, Chicago, IL). RESULTS Baseline Characteristics Table 1 shows the clinical and biochemical characteristics of all women included (N 77), stratified according to the pregnancy outcome. In the entire study group, the women s mean age was 31 5 years (range 21 42), and their mean SD serum TSH and FT4 at baseline were 1.6 0.8 mu/l and 12.4 1.8 ng/l, respectively. There were no significant differences between the ongoing pregnancy and the miscarriage groups with regard to age, baseline serum TSH and FT4 values, or the number of transferred embryos. Thyroid Function Before ART and Two Weeks After ET Compared with baseline values, serum TSH and FT4 values were significantly higher 2 weeks after ET in both groups. In the ongoing pregnancy group the TSH values increased from 1.6 0.8 mu/l at baseline to 2.5 1.3 mu/l at 2 weeks (P.0001), and the FT4 values from 12.4 1.8 ng/l to 13.8 1.48 ng/l (P.0001). In the miscarriage group the TSH values increased from 1.5 0.7 mu/l at baseline to 2.1 1.0 mu/l at 2 weeks (P.0001), and the FT4 values from 12.4 1.9 ng/l to 14.2 2.0 ng/l (P.0001). There were no statistically significant differences between the two groups for serum TSH and FT4 values at baseline (P.504 and P.906, respectively) and 2 weeks after ET (P.132 and P.399, respectively). Serum hcg levels were comparable at this stage of pregnancy (Table 2). Thyroid Function During the First Weeks of Pregnancy Figure 1 shows the pattern of changes in serum TSH and FT4 values during the first weeks of pregnancy stratified according to outcome status (ongoing pregnancy vs. miscarriage). In both groups there was a statistically significant effect for time on TSH, as well as on FT4 (one-way repeated measures ANOVA for TSH and FT4: P.001). When analyzed according to outcome status, however, both the serum TSH and 1754 Poppe et al. Thyroid function after assisted reproductive technology Vol. 83, No. 6, June 2005
TABLE 1 Baseline characteristics for all patients and for patients stratified according to the pregnancy outcome. a Pregnancy outcome All women Ongoing Miscarriage P values Number of women 77 (100%) 45 (58%) 32 (42%).17 Age (y) 31 5 31 5 31 5.89 TSH (mu/l) b 1.6 0.8 1.6 0.8 1.5 0.7.50 FT4 (ng/l) c 12.4 1.8 12.2 1.8 12.4 1.9.91 ET (n) d 2.1 0.6 2.1 0.6 2.2 0.7.32 a Data represent mean SD, unless otherwise stated. b Reference value 0.27 4.2 mu/l. c Reference value 9.3 18 ng/l (1 ng/l FT4 1.29 pmol/l). d ET No. transferred embryos. FT4 curves during this time period were not different for patients with and without miscarriage (two-way repeated measures ANOVA for TSH and FT4: P.912 and P.712, respectively). The observed differences in TSH levels among ongoing and miscarrying women were 0.12 0.77 mu/l, 0.42 1.14 mu/l, 0.26 1.04 mu/l, and 0.37 1.12 mu/l, before ART, 2, 4, and 6 weeks after ART, respectively. Serum hcg levels dissociated significantly 4 weeks after ET between the ongoing pregnancy and miscarriage group (Table 2; main effect for time and for group P.001 and P.003, respectively). DISCUSSION To the best of our knowledge, this is the first study to examine thyroid function after ART in a thyroid disease free population of women from infertile couples and to compare thyroid function with pregnancy outcome. Thyroid parameters changed significantly over time compared with baseline values. However, we could not detect an association between thyroid hormonal changes during early pregnancy and the subsequent pregnancy outcome. An assessment of power is essential in studies that fail to find an expected difference in outcome measure. Our power analysis a priori was based on information from a previous study from our group (10). Specifically, we calculated the total number of patients needed to have a power of 80% to detect a difference of 1.3 mu/l in serum TSH level between ongoing and miscarrying patients at a.05 level of significance, assuming a common standard deviation of 1.8 mu/l. The estimate was 62 women, but this was based on a sample also including women with thyroid antibodies (10). In this regard, we could not avoid confounding effects of TAI. In the actual cohort of women free of thyroid autoimmunity, the differences in serum TSH level between ongoing and miscarrying patients were much smaller than anticipated in the a priori power analysis. Knowing that only 77 women free of TAI were available in the current study, and that we TABLE 2 Serum hcg values at different time points after successful ART among women with ongoing pregnancies and miscarriage. a Time after successful ART 2Wks 4Wks 6Wks Ongoing pregnancy group 203 92 734 440 24,492 12,092 Miscarriage group 181 150 532 433 13,202 15,199 Note: Main effect for time and for group: P.001 and P.003, respectively (two-way, between-groups, repeated measures ANOVA). a Data represent mean SD (IU/L). Fertility and Sterility 1755
FIGURE 1 Pattern of change over time for the TSH and FT4 serum values (mean SD) collected at four periods: before ART (time 0) and at weeks 2, 4 and 6 after ET. The pattern of change over time is comparable for patients with ongoing pregnancies (squares) and miscarriage (diamonds). 3,5 15 Serum TSH (mu/l) ------ 3 2,5 2 1,5 1 14 13 12 11 10 Serum FT4 (ng/l) 0,5 0 2 4 6 WEEKS AFTER ET 9 observed a mean difference in TSH levels among ongoing and miscarrying women of 0.34 mu/l and a common SD of 1.10 mu/l, the power of the current study is only 28%. In view of these findings, a study to detect potential differences in TSH concentration among ongoing pregnant and miscarrying women free of thyroid disease and thyroid autoimmunity would be challenging. Based on the difference in TSH concentration and the common SD currently, to detect differences in TSH concentration of 0.35 mu/l for an alpha level of.05 and 80% (or 90%) power, a total sample size of at least 314 (or 420) participants would be needed. Our data are not in accordance with one previous report, describing lower FT4 in patients experiencing spontaneous abortions compared with women with ongoing pregnancies. Unfortunately the investigators did not provide details on the presence of thyroid antibodies or other thyroidal disease or whether the women got spontaneously pregnant. In addition, their findings were based on a small number of women (only 11 miscarrying and 21 ongoing pregnancy patients) (9). Assisted reproductive technology constitutes a unique opportunity to measure changes of thyroid function in the very early phases of pregnancy after ET. We previously demonstrated that in ART, the profiles of thyroid hormonal changes over time were significantly different between women with and without TAI (10). Assisted reproductive technology preceding pregnancy is therefore an additional stress to the thyroid. In the normal population free of thyroid disease, this results in an increase of serum TSH 2 weeks after ET. Reasons for this observation probably relate to the FSHinduced high estrogen levels, higher TBG, and lower FT4 as described by Muller et al. (13). However, the hcg injection for triggering ovulation probably counteracts the drop in serum FT4 by direct thyroidal stimulation (14). After the early peak value in serum TSH a decrease in TSH and a further slightly delayed peak value in FT4 are subsequently mediated through rising hcg levels. From the 4th week after ET on, serum TSH and FT4 levels remained comparable between both groups, independent of ART outcome. Therefore, the results from Maruo et al. may relate to the confounding effects of thyroid autoimmunity, explaining the lower FT4 values in patients experiencing a miscarriage (9). Thyroid autoimmunity indeed increases the risk of miscarriage and these patients have lower serum FT4 levels, compared with women without thyroid autoimmunity (8, 10). In the present cohort, reasons for spontaneous abortions were not further analyzed, but FT4 and TSH were not 1756 Poppe et al. Thyroid function after assisted reproductive technology Vol. 83, No. 6, June 2005
associated with pregnancy outcome in patients without thyroid disease. Therefore, in women free of thyroidal disease a particular follow-up of thyroid function after ART is not warranted, in contrast to women with TAI. In conclusion, in a thyroid disease free cohort of women of infertile couples, ART and subsequent pregnancy changed thyroid function over time: an initial increase of serum TSH being followed by a progressive decline. The pattern of hormonal changes does not allow predicting pregnancy outcome in this particular population of infertile women. Acknowledgements: The authors would like to thank W. Meul for data support and I. De Wannemacker for secretarial help. REFERENCES 1. Wilcox AJ, Weinberg CR, O Connor JF, Baird DD, Schlatterer JP, Canfield RE, et al. Incidence of early loss of pregnancy. N Engl J Med 1988;28:189 94. 2. Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24 9. 3. Steer C, Campbell S, Davies M, Mason B, Collins W. Spontaneous abortion rates after natural and assisted conception. Br Med J 1989;25: 1317 8. 4. Krassas GE. Thyroid disease and female reproduction. Fertil Steril 2000;74:1063 70. 5. Poppe K, Glinoer D. Thyroid autoimmunity and hypothyroidism before and during pregnancy. Hum Reprod Update 2003;9:149 61. 6. Poppe K, Velkeniers B. Female infertility and the thyroid. Best Pract Res Clin Endocrinol Metab 2004;18:153 65. 7. Poppe K, Glinoer D, Van Steirteghem A, Tournaye H, Devroey P, Schiettecatte J, et al. Thyroid dysfunction and autoimmunity in infertile women. Thyroid 2002;12:997 1001. 8. Poppe K, Glinoer D, Tournaye H, Devroey P, Van Steirteghem A, Velkeniers B. Assisted reproduction and thyroid autoimmunity: an unfortunate combination? J Clin Endocrinol Metab 2003;88:4149 52. 9. Maruo T, Katayama K, Matuso H, Anwar M, Mochizuki M. The role of maternal thyroid hormones in maintaining early pregnancy in threatened abortion. Acta Endocrinol (Copenh) 1992;127:118 22. 10. Poppe K, Glinoer D, Tournaye H, Schiettecatte J, Devroey P, Van Steirteghem A, et al. Impact of ovarian hyperstimulation on thyroid function in women with and without thyroid autoimmunity. J Clin Endocrinol Metab 2004;89:3808 12. 11. Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993;8:1055 60. 12. Dupont WD, Plummer WD Jr. Power and sample size calculations. A review and computer program. Control Clin Trials 1990;11:116 28. 13. Muller AF, Verhoeff A, Mantel MJ, De Jong FH, Berghout A. Decrease of free thyroxine levels after controlled ovarian hyperstimulation. J Clin Endocrinol Metab 2000;85:545 8. 14. Noppen M, Velkeniers B, Buydens P, Devroey P, Van Steirteghem A, Vanhaelst L. Hyperthyroidism after gonadotrophic ovarian stimulation. Br Med J (Clin Res Ed) 1986;16:171 2. Fertility and Sterility 1757