Premature menopause: monoclonal antibody defined T lymphocyte abnormalities and antiovarian antibodies

FERTILITY AND STERILITY Copyright " 1989 The American Fertility Society Vol. 51, No.3, March 1989 Printed in U.S.A. Premature menopause: monoclonal antibody defined T lymphocyte abnormalities and antiovarian antibodies Steven L. Rabinowe, M.D.*t Veronica A. Ravnikar, M.D. Sergio A. Dib, M.D. Katherine L. George, M.S. Robert G. Dluhy, M.D. Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts The presence of other organ-specific autoimmune disorders in some patients with premature menopause has supported the concept of an autoimmune etiology. The authors analyzed the peripheral blood of 23 women with the diagnosis of premature menopause to detect the presence of monoclonal antibody-defined T-lymphocyte abnormalities and/ or antiovarian antibodies. All subjects were <40 years of age with the duration of menopause ranging from <1 year to 11 years at the time of study. Thirty-five percent of the subjects had an elevated percentage of Ia+ (Dr-activated) T cells using monoclonal antibody L243. The percent T 4 (helper) T 8 (suppressor/cytotoxic) T cells and T 4/T 8 ratio were normal in the study group. Four subjects (approximately 17%) had elevated percentages of the age-related 3G5+ T cell subset. Two of the subjects with increased 3G5+ T cells also exhibited increased Ia+ T cells. Antiovarian steroid cell antibodies and antiadrenal cortical antibodies were present in approximately 9% of subjects. Anti-islet cell antibodies were not present. Thyroid antimicrosomal antibodies were present in 17% of subjects. Study subjects exhibited immunologic abnormalities that the authors hypothesize may play a role in the development of premature menopause in a larger percentage of patients than was previously suspected. Fertil Steril51:450, 1989 The presence of autoimmune endocrine disease in some patients with premature menopause has supported the concept of an immune causation. Evidence for an autoimmune causation of some cases includes: (1) lymphocytic and plasma cell infiltration of the ovary; (2) autoantibodies to ovarian antigens; (3) association with other organ-specific "autoimmune" disorders of the type I and type II polyglandular autoimmune syndromes; (4) the thymectomized mouse model in which circulating antiovarian antibodies and T cell requirement for adoptive transfer of the disease is found; and ( 5) a possible effect of "immune" therapy. 1-5 The purpose of this investigation is to detect the Received July 5, 1988; revised and accepted October 27, 1988. * Supported by a Research and Development Award from the American Diabetes Association and Biomedical Research Support grant RR05673 from the National Institutes of Health, Bethesda, Maryland. t Reprint requests: Steven L. Rabinowe, M.D., Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215. presence of monoclonal antibody defined T-lymphocyte abnormalities and/or antiovarian antibodies in subjects with premature menopause. Subjects MATERIALS AND METHODS Twenty-three women with secondary hypergonadotropic hypoestrogenic gonadal failure who were <40 years of age at the time of initial diagnosis are the study group. Characteristics of the subjects were: age at study, 32.7 ± 1.3 years (range, 16 to 45 years); last menstrual period, 4.2 ± 0. 7 years (range, perimenopausal to 11 years). Three subjects had a clinical history of another organ-specific autoimmune disease (2 with a combination of Hashimoto's thyroiditis and Addison's disease and 1 with Graves' disease). Control groups for the T cell studies consisted of: (1) 25 women <40 years of age (mean age, 32 ± 1.1 years; range, 19 to 39 450 Rabin owe et al. Lymphocyte abnormalities in premature menopause Fertility and Sterility

years); and (2) 5 women > 40 years of age (mean, 57.8 ± 22 years; range, 50 to 63 years). Control groups for the autoantibody studies consisted of 15 of the normal women <40 years of age and 111 Red Cross blood donor controls. T Cell Studies Monoclonal antibody studies used purified T cells prepared as previously reported. 5 The percent of cells with fluorescence was determined using an Epics 752 (Coulter, Miami Lakes, FL) fluorescence-activated cell sorter with the exception of Ia+ T cells, which was determined on a Leitz microscope equipped for epifluorescence. Control antibody P3X63, which does not bind to human T cells, was used to subtract nonspecific fluorescence. Ten thousand cells were counted in each assay. Monoclonal antibodies OKT4 and OKT8 (Ortho Pharmaceuticals, Rahway, NJ), L243 (a murine monoclonal directed against the nonpolymorphic region of the Ia antigen), and 3G5 were used. Monoclonal antibody 3G5 is a mouse monoclonal antibody produced following immunization of mice with fetal rat brain. The antibody reacts with a complex membrane ganglioside expressed by neuronal cells, pancreatic islet cells, adrenal medullary but not cortical cells, a subset of pituitary cells, and thyroid follicular cells. 6 7 We have recently discovered that 3G5 reacts with a subset of circulating OKT4+ and OKT8+ T cells and approximately 8% of human thymocytes. Age-related normative data was previously published. 8 Autoantibody Studies Anti-islet antibodies were detected by indirect immunofluorescence on cryostat sections of human pancreas using fluoresceinated protein A and monoclonal antibody BISL-32 for islet identification, as previously described. 9 Antiovarian Antibodies Five-micron cryostat sections of guinea pig ovary were overlayed with 40 l of undiluted human sera and incubated for 30 minutes at room temperature. The sections were washed three times with phosphate-buffered saline (PBS) and overlayed with 40 l of 1:1000 dilution of protein A (1 mg/ml in PBS, 1% bovine serum albumin slides). After a 30-minute incubation at room temperature, the sections were washed three times in PBS. Slides were mounted with one drop of PBS 30% U) j j w 0 1- + 0 H ;! 10 8 6 4 ;:> - PREMATURE MENOPAUSE CONTROLS (AGE 40 CONTROLS >AGE 40 Figure 1 Percent Ia+ T cells (y-axis) in the peripheral blood of subjects with premature menopause, control < age 40 and controls > age 40. Note the elevated percentage in a subset of the premature menopause subjects. glycerol and read in a microscope equipped for epifluorescence. Antiadrenal Cortex Antibodies The assay was performed as for the antiovarian antibodies above, except that 5 m cryostat sections of human adrenal gland were used as the test substrate. Thyroid Antimicrosomal Antibodies Determinations were performed by a quantitative hemagglutination technique previously described.10 Statistical references were tested by the Student's t-test, Wilcoxon rank sum test, or Fisher's exact test. RESULTS The percent Ia (Or-activated) T cells are shown in Figure 1 for subjects with premature menopause, controls <40 years of age, and those >40 years of age. An elevation in Ia+ T cells was present in seven of the subjects with premature menopause ( 35%), but none of either control group. The elevation for the premature menopause group was statistically significant when compared with controls under age 40 (P < 0.05). Subjects aged 32 or younger at study date were more often positive for Ia+ Vol. 51, No.3, March 1989 Rabinowe et al. Lymphocyte abnormalities in premature menopause 451

5 3 i 2 t.. i.. t i BL---------+----+---- PREMATURE CONTROLS CONTROLS MENOPAUSE (AGE 40 ) AGE 40 I. 9.:':_ 0. 2 2.3.:':_ 0.26 2. 3.:':_ 0. 6 Figure 2 The T 4/T 8 (helper/suppressor-cytotoxic) T cell ratio (y-axis) is shown for subjects with premature menopause and controls <> age 40. T cells (P < 0.02, Fisher's exact test) than subjects older than age 32. The monoclonal antibody 3G5+ T cell subset is age-dependent in normal subjects. Four premature menopause subjects (17%) exceeded normal levels. As would be expected, none of the age-matched normal controls exceeded the 95% confidence limit previously established for normal subjects. 8 This difference was statistically significant (P < 0.05, Fisher's exact test). In addition, two of the four (3G5 elevated subjects) also had an elevated percentage of Ia+ T cells. The OKT 4 (CD4)/0KT 8 (CDS) "helper/"suppressor-cytotoxic" T cell data are shown in Figure 2. There was no statistically significant difference for the groups (1.9 ± 0.2% for premature menopause, 2.3 ± 3% controls <age 40, 2.3 ± 0.6% controls >age 40). The percentage of T 4- and T 8-positive T cells for each premature menopause subject are shown in Table 1. Steroid cell antiovarian antibodies were present in 9% of the premature menopause subjects. The same subject had antibodies that reacted with the adrenal cortex. Control women (<age 40) and blood donors did not have antibodies to either ovarian steroid cells or adrenal cortex. Seventeen percent of premature menopause subjects had antimicrosomal (thyroid) autoantibodies (Table 1). DISCUSSION Multiple causes of premature menopause exist. However, it is increasingly evident, through pathologic studies showing lymphocytic infiltrates of the ovaries and the presence of circulating antiovarian antibodies, that a subset of subjects have an autoimmune causology. Premature menopause also is associated with both the type I and type II (Dr3 associated) autoimmune polyglandular syndromes. This association suggests a possible relationship to the genetic and immunologic defects previously described in these syndromes. The thymectomized mouse model of autoimmune oophoritis is associated with both autoantibody (antiovarian) and T cell defects. In this model, the T cell is required for adoptive transfer of the disease, suggesting an important role for the T cell in causation of the disease. An elevated percentage of Ia (Dr-activated) T cells in peripheral blood has been reported in a number of organ-specific autoimmune diseases of the type II polyglandular autoimmune syndrome including Type I diabetes mellitus, Graves' disease, and Addison's disease.11-13 An increase in peripheral blood Ia+ T cells was documented in one biopsy proven case of autoimmune oophoritis. 5 The current report documents an elevated percentage of Ia + T cells in 35% of premature menopause subjects tested. The percentage of positive patients might be expected to vary depending on the duration of menopause in the subjects under study. This study with patients as long as 11 years after clinical premature menopause may in fact under-represent the percentage of patients positive with new onset disease. The relatively small number of subjects who were perimenopausal at study precludes an exact determination of that percentage in this study. Nevertheless, the percentage of subjects exhibiting the T cell activation is strikingly high, and younger subjects at study date were more likely to have Ia+ T cells. Antineuroendocrine monoclonal antibody 3G5 defines an age-related T cell subset that is prematurely increased in subjects on the cardiac drug amiodarone (associated with the development of Ia+ T cells and thyroid autoimmunity) and in a subset of subjects with Trisomy 21 (Down's syndrome subjects have an increased prevalence of thyroid autoimmunity and Type I diabetes mellitus. 14-16 The previously described subject with biopsy proven autoimmune oophoritis and Ia+ T cells also had an elevated percentage of 3G5+ T cells in the peripheral blood. Four premature menopause subjects (2 with increased Ia+ T cells) also exhibited this T cell defect. The helper/suppressor-cytotoxic (T 4/T 8) T cell ratios were not abnormal in the study group. In our 452 Rabinowe et al. Lymphocyte abnormalities in premature menopause Fertility and Sterility

Table 1 Immunologic Profile of Premature Menopause Subjects Anti-thyroid Percent Percent Anti-ovarian microsomal Anti-adrenal Patient Percent Percent 3G5 I a steroid cell antibodies cortex Autoimmune no. Age T4 Ts (NI/W (Nl < 3%) antibodies (titer) Anti-ICA antibodies disease yrs 1 24 64.8 21.7 NLb 0 1:1600 2 30 54.4 23.3 NL 5 3 33 41.6 12.4 NL 0 4 38 39.4 16.0 NL 4 5 38 50.8 26.0 NL 0 6 29 35.5 22.1 NL 6 7 32 62.8 22.4 NL 0 8 37 51.0 21.8 NL 2 9 32 ND' ND NL 6 + + 10 30 15.3 34.1 NL 5 Hashimoto's, Addison's 11 35 63.1 23.1 2 12 36 62.8 36.6 0 13 29 56.5 27.7 t 6 14 40 24.6 35.8 NL 0 15 36 30.6 33.3 NL 2 16 16 50.5 22.4 t 6 17 33 49.9 29.9 NL ND 1:102400 Graves' 18 45 56.3 37.5 NL ND 19 35 44.8 35.4 NL 0 1:6400 20 23 51.5 29.8 NL 0 21 34 51.9 35.2 NL 1 22 38 40.1 40.5 NL 0 23 33 ND ND ND ND + 1:6400 + Hashimoto's Addison's a Age-related T cell subset (reported as normal or increased for age). b Nl, normal. 'ND, not done. experience, T 4/T 8 ratios also are normal in recent onset Type I diabetes mellitus. The presence of autoantibodies to the thyroid (17%) and adrenal cortex (9%) serves to emphasize the polyglandular nature of the autoimmune disorder in some subjects. Several noncontrolled attempts at the therapy of "autoimmune" premature menopause have been performed. Corticosteroids have been associated with resumption of menses in a small number of cases. 5 Resumption of menses and conception have been reported with estrogen treatment in other studies. 17 18 A complete review of these potential therapies is beyond the scope ofthis article. Nevertheless, a means of identifying subsets of patients who may be immunologically and hormonally studied through the peripheral blood in a controlled clinical trial is necessary for significant advance in this field. The immunologic defects defined in this paper may help define and follow such a study group. In addition, we hypothesize that autoimmunity may play a role in a larger percentage of patients than was previously reported. Acknowledgments. Data was analyzed using the CLINFO system of the Brigham and Women's Hospital, Boston, Massachusetts. We wish to thank Ms. Patricia A. Cronin-Sevigny for expert secretarial assistance. REFERENCES 1. Gloor E, Hurlimann J: Autoimmune oophoritis. Am J Clin Pathol81:105, 1984 2. Coulam CB, Ryan RJ: Prevalence of circulating antibodies directed toward ovaries among women with premature ovarian failure. Am J Reprod Immunol Microbiol9:23, 1985 3. Rabinowe SL, Eisenbarth GS: Polyglandular autoimmunity. In Advances in Internal Medicine. Chicago, Year Book Medical Publishers, 1986, p 293 4. Sakagushi S, Fukuma K, Kuribayashi K, Masuca T: Organ specific autoimmune diseases induced in mice by elimination oft cell subset. I. Evidence for the active participation oft cells in natural self-tolerance; deficit of at cell subset as a possible cause of autoimmune disease. J Exp Med 161: 72, 1985 5. Rabinowe SL, Berger MJ, Welch WR, Dluhy RG: Lymphocyte dysfunction in autoimmune oophoritis: resumption of menses with corticosteroids. Am J Med 81:34 7, 1986 6. Rabinowe SL, Larsen PR, Antman EM, George KL, Friedman P, Jackson RA, Eisenbarth GS: Amiodarone therapy and autoimmune thyroid disease: evaluation of a new Vol. 51, No.3, March 1989 Rabinowe et al. Lymphocyte abnormalities in premature menopause 453

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