THYROID DISORDERS ARE more common in women

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1 X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(11): Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: /jc Thyroid Fetal Male Microchimerisms in Mothers with Thyroid Disorders: Presence of Y-Chromosomal Immunofluorescence in Thyroid-Infiltrating Lymphocytes Is More Prevalent in Hashimoto s Thyroiditis and Graves Disease Than in Follicular Adenomas CHRISTOPH RENNÉ, ELIZABETH RAMOS LOPEZ, SUSANNE A. STEIMLE-GRAUER, PIOTR ZIOLKOWSKI, MICHAEL A. PANI, CHRISTINA LUTHER, KATHARINA HOLZER, ALBRECHT ENCKE, ROBERT A. WAHL, WOLF O. BECHSTEIN, KLAUS H. USADEL, MARTIN-LEO HANSMANN, AND KLAUS BADENHOOP Institute of Pathology (C.R., S.A.S.-G., M.-L.H.), Department of Internal Medicine I, Division of Endocrinology (E.R.L., M.A.P., K.H.U., K.B.), Department of Surgery (C.L., K.H., A.E., W.O.B.), University Hospital Frankfurt, D Frankfurt am Main, Germany; Department of Surgery, Bürgerhospital (R.A.W.), D Frankfurt am Main, Germany; and Department of Pathology, Wroclaw Medical University (P.Z.), Wroclaw, Poland THYROID DISORDERS ARE more common in women than in men, an observation that extends from autoimmunity to nodular thyroid disease and even congenital hypothyroidism (1). Whereas pregnancy leads to an amelioration of autoimmunity, the postpartal period is associated with an exacerbation of Graves disease and with postpartum thyroiditis (2). These alterations of a pregnant woman s immune system have been explained by a switch to Th2-type immune response and a rebound to Th1-type immunity after childbirth (3). The immunological changes during pregnancy promote a more successful fetal development, because the mother must tolerate a semiidentical implant. Mother and fetus are in cellular contact through the syncytiotrophoblast layer, the maternal-fetal synapse. This cell layer expresses human leukocyte antigen (HLA) G, a member of the major histocompatibility complex that is thought to play an important role in the tolerance of the mother Abbreviations: FISH, Fluorescence in situ hybridization; HLA, human leukocyte antigen. JCEM is published monthly by The Endocrine Society ( endo-society.org), the foremost professional society serving the endocrine community. The presence of fetal cells in a maternal compartment is defined as fetal-maternal microchimerism, which has been detected in thyroids of mothers suffering from autoimmunity. We analyzed the immunohistology of paraffin-embedded thyroid specimen taken at surgery from 49 women with Hashimoto s thyroiditis (n 25), Graves disease (n 15), or nodular or diffuse follicular adenomas (n 9), whose childbirth history was positive for sons. By fluorescence in situ hybridization we screened for X-chromosome- and Y-chromosome-specific staining and compared the finding with human leukocyte antigen (HLA) DQ types of the mothers and, where available, their offspring. In 23 thyroids we found Y-chromosome-specific staining, which was more frequent in thyroid autoimmune disease (60% Hashimoto s thyroiditis and 40% Graves disease) than in follicular adenomas (22.2%). There was no significant difference for HLA DQ alleles among women whose thyroids showed Y-chromosome staining and those without. However, a subgroup of all investigated microchimerism-positive mother-child pairs and women with Hashimoto s thyroiditis and Graves disease more often had the susceptibility alleles HLA DQA1*0501-DQB1*0201 or DQB1*0301. In conclusion, fetal microchimerism is observed in thyroids of mothers with sons, and this is found more frequently in thyroid autoimmune diseases. (J Clin Endocrinol Metab 89: , 2004) toward the implant, because HLA G1-expressing cells appear to be directly involved in the suppression of an immune response (4). This immunoinhibitory function is exerted through the receptors ILT2/CD85j, ILT4/CD85d, and KIR2DL4/CD158d (5 7). These molecules may induce the differentiation of CD4 T lymphocytes into regulatory/suppressive cells. Previous reports about the findings of microchimerism in thyroid disease have been based on PCR employing Y-chromosome-specific primers in thyroid tissue of autoimmune and nonautoimmune disorders (8, 9). We designed a retrospective study to elucidate the possible role of fetal-maternal microchimerism in thyroids of women who had undergone thyroid surgery. We selected for the study those who had given birth to at least one son before thyroid surgery. The presence of fetal microchimers within the thyroid was assessed by fluorescence in situ hybridization (FISH) on 49 paraffin-embedded sections and was correlated with the pathological and clinical diagnoses. Furthermore, we analyzed the immunogenetic background by HLA DQ typing of the mothers and offspring, where available, because persistent fetal microchimerism in mothers with scleroderma has 5810

2 Renné et al. Thyroid Fetal Male Microchimerisms J Clin Endocrinol Metab, November 2004, 89(11): been found to be associated with HLA DQA1*0501 and DQB1*0301 (10). We present evidence of fetal microchimerism in thyroids of mothers with sons by FISH. This phenomenon appears to be more prevalent in thyroids affected by the autoimmune disorders Hashimoto s thyroiditis and Graves disease. Subjects and Methods We studied retrospectively 49 female patients after thyroid surgery from the endocrine and surgical departments of University Hospital as well as from Bürgerhospital (Frankfurt am Main, Germany). The study was confined to those women whose pathohistological diagnosis was Hashimoto s thyroiditis, Graves disease, or follicular adenoma. A letter was sent to request permission for questions about childbirth before thyroid operations and for a blood sample. Of 58 respondents with a son, we could study 49 by FISH analyses of thyroid sections. Of the total of 49 women, 16 provided a blood sample for additional HLA DQ typing. The age of the women at thyroid surgery ranged from yr. The history of childbirth included women with one son (n 12), two sons (n 12), three or more sons (n 1), and a son and a daughter (n 24). The time span between the nearest son s birth and thyroid surgery ranged from 2 51 yr (mean, 27.2 and yr) in mothers with microchimerism. Patient material Paraffin-embedded tissue samples of the thyroid gland from 49 women who underwent thyroid surgery were analyzed for diagnostic purposes. The pathological diagnoses for histological paraffin-embedded sections stained with hematoxylin-eosin were as follows: in 25 patients Hashimoto s thyroiditis was found, 15 cases had Graves disease, and nine had follicular adenomas. To study immunogenetic compatibility between maternal and fetal cells by DNA typing for HLA DQ, we obtained blood samples from 16 patients and seven offspring, which allowed us to investigate seven mother-child pairs. The study protocol was approved by the ethics committee of University Frankfurt am Main, and written consent was obtained from all participants. Immunohistochemistry Serial sections of paraffin-embedded material were used. Antibodies directed against CD3, CD20, and CD45 (leukocyte common antigen; Dako, Hamburg, Germany) were visualized with the avidin-biotin complex technique with alkaline phosphatase using Fast Red (Dako) as chromogen. All cases were analyzed for the number of CD45-positive cells. In addition, 10 patients with Hashimoto s thyroiditis (five with and five without microchimerism) were analyzed for the ratio of CD20/ CD3-positive cells. FISH Serial sections of the immunohistochemically analyzed, paraffinembedded, human tissue samples (4 m) were dewaxed and gently dehydrated through an ethanol series of increasing grade (79%, 85%, and 96%). Then the samples were pressure-cooked in 1 mm EDTA (ph 8.0) for 2 min and fixed for each 10 min in ice-cold Carnoy s solution and ice-cold 1% paraformaldehyde. The samples were dehydrated by ethanol treatment with increasing concentrations. Both X- and Y-centromeric probes were obtained from Vysis (Downers Grove, IL). FISH was performed following the manufacturer s instructions. Slides were analyzed using an Axioskop-2 fluorescence microscope (Zeiss, Gottingen, Germany) equipped with appropriate filter sets (AHF, Tubingen, Germany), and findings were documented using the ISIS imaging system (MetaSystems, Altlussheim, Germany). For each tissue sample, two slides resulting from separate hybridization experiments were analyzed. HLA DQ typing Typing for the common HLA DQA1 and DQB1 alleles was performed as described previously by sequence-specific primers and PCR (11). This was accomplished in 16 women and seven mother-child pairs, six of them mother-son. Results Y-chromosome-positive cells in thyroid sections We detected Y-chromosomal cells in 23 of 49 (47%) thyroid tissues. The number of Y-chromosome-positive cells ranged from one to six per section. Lymphatic cells stained for Y- chromosome were lying sporadically between infiltrates of maternal lymphocytes and scattered in connective tissue adjacent to follicular epithelium cells (Fig. 1). No Y-chromosome-positive thyrocytes were found. The proportion of Y- chromosome-positive thyroid sections was highest in Hashimoto s thyroiditis (15 of 25, 60%), lower in Graves disease (six of 15, 40%), and infrequent in follicular adenomas (two of nine, 20%; Table 1). The difference between autoimmune thyroid disorders (21 of 45, 53%) and follicular adenomas did not reach significance due to the low number of FIG. 1. Fetal male microchimerisms in lymphocytes infiltrating thyroid glands of mothers with thyroid disorders. A, Hybridization of X- and Y-chromosomal centromers in a thyroid gland of a patient suffering from Hashimoto s thyroiditis. The arrow points to a fetal lymphocyte showing hybridization of the X-chromosome centromeric probe (red) and the Y-chromosome centromeric probe (green), which lies in a field of maternal lymphocytes (positivity for two red X-chromosomal centromeric FISH probes). B, X-/Y-Chromosomal centromeric hybridization in a thyroid gland with Graves disease. The arrow points to a fetal lymphocyte (presence for each one X- and Y-chromosome) that lies in the connective tissue behind the maternal follicle epithelial cells (arrowheads). Autofluorescence of erythrocytes appears as bright yellow spots.

3 5812 J Clin Endocrinol Metab, November 2004, 89(11): Renné et al. Thyroid Fetal Male Microchimerisms TABLE 1. Number of Y-chromosome-positive cells in mothers with Graves disease, Hashimoto s thyroiditis, and follicular adenoma No. (%) of Y-chromosomal cells positive Diagnosis Hashimoto s thyroiditis (n 15) 1 (6.6) 5 (33.3) 5 (33.3) 2 (13.3) 1 (6.6) 1 (6.6) Graves disease (n 6) 3 (50) 3 (50) Follicular adenoma (n 2) 1 (50) 1 (50) TABLE 2. Distribution of the HLA-DQ risk alleles in 16 patients with thyroid disease Diagnosis DQ2/DQ2 DQ8/X X/DQ8 DQ2/X X/DQ X/X Hashimoto s thyroiditis (n 12) 1 (8.33) 1 (8.33) 1 (8.33) 3 (25) 3 (25) 3 (25) Graves disease (n 2) 1 (50) 1 (50) Follicular adenoma (n 2) 1 (50) 1 (50) Values are the number of subjects (percentage). follicular adenomas (by Fisher s exact test, one-sided, P 0.1). Leukocytes per section, CD20/CD3 ratio Between 631 and 56,800 CD45-positive cells (leukocytes)/ section were observed. The highest number was found in thyroids with Hashimoto s thyroiditis (mean, 35,540), whereas patients with Graves disease had a mean of 2,058 CD45 cells/section and 1,980 follicular adenomas thyroids/ section. The number of Y-chromosomal cells/cd45-positive cells ranged from one in 200 up to one in 56,800/section. The density of Y-chromosomal cells/cd45-positive cells varied and was lowest in thyroids of women with Hashimoto s thyroiditis (mean of one per 13,369/section). Because tissues from patients with Hashimoto s thyroiditis showed the highest number of infiltrating lymphocytes, we analyzed five cases each with or without presence of microchimerism. CD20/CD3 ratios ranged between 0.94 and 3.40, and no significant difference was found between samples with or without fetal lymphocytes (data not shown). HLA DQ types in women of subgroups Typing for HLA DQA1 and DQB1 alleles was performed in 16 patients whose thyroid sections had undergone analysis for microchimerism. The major HLA DQ alleles known to confer susceptibility to thyroid autoimmune disease are HLA DQA1*0501, *0301, DQB1*0201, and DQB1*0301 (12). Either of these alleles was found in nine patients of 12 (75%) with Hashimoto s thyroiditis, six of them with microchimerism, both (100%) patients with Graves disease, and one patient (50%) with follicular adenomas (Table 2), all of the latter without microchimerism. Because not all women were available for HLA DQ typing, we could not distinguish whether thyroids with microchimerism carried more often the susceptibility alleles than thyroids without microchimerism. However, seven mother-child pairs could be investigated for HLA DQ haplotypes, of whom four mothers had Y- chromosome-positive cells in their thyroids; in all mothers or children at least one HLA DQ haplotype was observed that had previously been found to be associated with thyroid autoimmunity (12): HLA DQA1*0301-DQB1*0301, TABLE 3. Distribution of the Y-chromosome-positive cells in thyroid tissues according to number of children, their gender, and birth order No. of children Cells positive X, Y S SS SD DS DSS S, Son; SS, two sons; SD, son and daughter; DS, daughter and son; DSS, daughter and two sons. DQA1*0501-DQB1*0201, DQA1*0102-DQB1*0602/DQB1*0604, or DQA1*0103-DQB1*0603. Y-Chromosome-positive thyroids compared with thyroid tissues without microchimerism Patients with microchimerism were multiparous in 19 of 23 cases; four patients each had one son. In one of the latter patients the highest cell number of microchimers (six) was observed (Table 3). Patients without detectable microchimerism were multiparous in 18 of 26, seven of them had two sons, and 11 had a son and a daughter (data no shown). Discussion To examine the possible influence of male fetal microchimerism on autoimmune thyroid disease, we assessed thyroid tissue samples obtained at surgery in female patients with Hashimoto s thyroiditis, Graves disease, and follicular adenoma; all had previously given birth to a son. This series represents the largest cohort of female thyroid tissues examined for the presence of male microchimerism to date. The presence of a Y-chromosome in thyroid tissues was taken as evidence for persistent fetal-maternal microchimerism, a phenomenon that was described in smaller studies in 2001 (8, 13) and in Graves disease in 2002 (9). We confirm those earlier findings of a higher prevalence of microchimerism in thyroid autoimmune disease, but we also observed it in follicular adenoma. Furthermore, we extended our analyses to the immunogenetic background in patients as well as the obstetric history. The presence of microchimerism was found

4 Renné et al. Thyroid Fetal Male Microchimerisms J Clin Endocrinol Metab, November 2004, 89(11): in all mother-child pairs where at least one susceptible HLA DQ haplotype was present. It is therefore conceivable that microchimerism also occurs in nonautoimmune thyroid disorders if there is a fetal-maternal constellation of immunogenetic susceptibility through HLA DQ or other genes. If microchimerism contributes to thyroid autoimmunity, a quantitative effect might be observed by the increased exposure of a woman toward fetal antigens during and after pregnancy. However, we only observed a slightly higher prevalence of microchimerism in multiparous women compared with women with one son, and this is not significant due to the small numbers. Nevertheless, we observed the highest prevalence (60%) with one or more Y-chromosome-positive cells in the thyroids of patients with Hashimoto s thyroiditis, followed by 40% in Graves disease and only 22.2% in follicular adenoma, indicating a higher degree of microchimerism in autoimmune thyroid disease than in the benign proliferative disorder. The immunogenetic susceptibility markers, HLA DQA1*0501-DQB1*0201 and DQB1*0301, that are more frequent in patients with thyroid autoimmunity are also more common in patients of mother-child pairs with microchimerism, raising the question of whether these histocompatibility alleles predispose to microchimerism per se or to thyroid autoimmunity that may occur as a secondary event. A recent observation of maternal circulating cells in the fetus during pregnancy suggests an association of maternal HLA DQB1*0301 with microchimerism (14). Therefore, an immune reaction against microchimeric cells could be targeted on the immunogenetic basis of an HLA-restricted peptide/ antigen recognition leading in some cases to cross-immunity against thyroid cells. Whether this higher prevalence of microchimerism in thyroid autoimmunity is mere coincidence or is a marker for immune-mediated disease needs to be further investigated. The frequent postpartum thyroiditis during the first year after giving birth in women without previous thyroid autoimmunity and the exacerbation of such disorders in those with previous Graves disease and Hashimoto s thyroiditis represent the most common immune deviations after pregnancy (2). This may be due to an altered state of immunity that is geared toward tolerance in pregnancy and switches back thereafter. The state of tolerance that is necessary for a successful pregnancy is achieved through a T helper cell type 2 immune response by an alternative activation of monocytes through the expression of factors such as pregnancy-specific glycoprotein 1a (15). Therefore, antigen presentation is different after childbirth, and persistent fetal-maternal microchimeric cells could cause alloimmunity derived from HLA half-mismatched cell-cell interaction. Fetal-maternal microchimerism may be detected up to 38 yr postpartum (16), a phenomenon that has been studied in scleroderma or connective tissue syndrome, where up to 26% of female patients may carry such cells compared with a lower, but detectable, frequency in normal women with sons (17). In addition to pregnancies there are other sources of microchimerism, such as missed abortions, blood transfusions, bone marrow or organ transplants, or unrecognized twins. Fetal pluripotent stem cells or T lymphocyte precursors may persist and home in organs such as the thyroid, where they could induce an autoimmune reaction similar to a graft vs. host disease. Alternatively, these cells may be recognized as partially alloimmune and thus give rise to an immune reaction. Fetal microchimeric cells may differentiate even into cells with epithelial lineage; a recent report of a small series of these found 14 60% of microchimeric cells expressing cytokeratin in three thyroid tissues derived from goiters (18). Although we did not detect microchimeric cells with thyroid epithelial features, we cannot rule out such a possibility. This course of events would indicate that these cells might process and present thyroid antigens and lead to organ-specific autoimmunity. The immunogenetic susceptibility marker, HLA DQA1*0501-DQB1*0201, would thereby enhance the likelihood of an immune reaction targeted in the thyroid, thus explaining the high risk in women with the predisposing HLA specificities of experiencing thyroid autoimmunity after childbirth. However, our pilot study can only be seen as proof of principle. It needs to be extended to larger numbers to confirm our findings and elucidate the mechanism by which microchimerism is linked to thyroid autoimmunity and immunogenetic risk. Acknowledgments Received June 3, Accepted July 28, Address all correspondence and requests for reprints to: Dr. K. Badenhoop, Department of Internal Medicine I, Division of Endocrinology, University Hospital Frankfurt, Theodor-Stern-Kai 7, D Frankfurt am Main, Germany. badenhoop@em.uni-frankfurt.de. This investigation was made possible by funding of the European Foundation for the Study of Diabetes (EFSD) and the German Diabetes Association (DDG). 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