1234 Results T.S.H. values from the dried blood spot were determined after varying periods of extraction, and the maximum values were obtained after two hours of extraction. Stability of the blood spotted onto filter-paper was determined by measuring the T.s.H. values from two days to fifty-five days after spotting, virtually no change being noted over this period of time. Fig.1 shows the correlation between the T.s.H. values obtained by the dried blood spot method and those obtained by measuring T.S.H. in serum samples from the same subjects. The detection limit for blood T.S.H. was 5-10 µU/ml by the dried blood spot method. Fig. 2 shows the correlation between two values of T.S.H. in two blood discs obtained from same individuals. Using this method, adult hypothyroid patients showed high concentrations of T.S.H., and T.S.H. concentrations rose after the injection of 500 µg synthetic T.R.H. (table). Of BLOOD T.S.H. CONCENTRATION MEASURED ON DRIED BLOOD SPOTS FROM PATIENTS WITH HYPOTHYROIDISM, NORMAL SUBJECTS, AND IN CORD
well as T.s.H. would be necessary, the microdetermination of T. S. II. has substantial value since primary hypothyroidism seems to be much more common than secondary or tertiary hypothyroidism. Klein et al.have shown that treatment before the age of three months could prevent mental retardation among hypothyroid infants. With the method we have described patients with congenital or juvenile primary hypothyroidism could be detected during mass-screening for metabolic disorders, and treatment could begin during the first days or weeks of life.
iodothyronine
as
We thank Dr H. L. Levy of the State Laboratory Institute, Boston, Massachusetts, U.S.A., for his help in the preparation of this paper.
This study was partly supported by a grant from the Ministry of Health of Japan. The materials to measure T.s.H. were supplied by the National Institutes of Health, Bethesda, Maryland, U.S.A., and the Daiichi Radioisotope Company, Tokyo, Japan. We also thank Dr H. Enomoto, Dr K. Inoue, Dr T. Nukui, Dr M. Kuroda, Dr S. Takeda, Dr M. Mizotam, Dr Y. Ito, and Ms S. Oshima for their help; Dr T. Aoki, Dr T. Kumagai, Dr K. Nakayama of the Pædiatric Department of Toho University for their help with the paediatric patients, and Ms Y. litaka and M. Katakura for secretarial work.
BLOOD
Requests for reprints should be addressed to M.l. REFERENCES
1. 2.
*245
µU/ml in the case of hypothyroidism (see text).
227 newborn infants tested none had a raised T.S.H., and values in 5 cord-blood specimens were also normal. From the dried blood spots obtained from outpatients, 1 case of primary hypothyroidism was detected-in a four-year-old girl with clinical signs of hypothyroidism and a T.S.H. of 245 uU/ml.
Dussault, J. H , Laberge, L. Union Meed Can. 1973, 102, 2062 Dussault, J. H., Coulombe, P., Laberge, L., Guyda, H , Khoury, K J. Pediat. 1975, 86, 670. 3. Klein, A. H., Augustin, A. V., Foley, Jr. T. P. Lancet, 1974, ii, 77 4. Guthrie, R., Susi, A., Pediatrics, 1963, 32, 338. 5. Odell, W. D., Wliber, J. F , Utiger, R. D. Rec. Progr Hormone Res 1967, 23, 47. 6. Hershman, J. M., Pittman, Jr. J. A. Ann. intern Med 1971, 74, 481 7. Fisher, D. A., Odell, W D. J. clin Invest 1969, 48, 1670 8. Klein, A. H., Meltzer, S., Kenny, F. M. J Pediat. 1972, 81, 912
NATURAL COURSE OF SYMPTOMLESS AUTOIMMUNE THYROIDITIS
A. GORDIN Discussion We have shown that T.s.H. can be measured in 20 µl of whole blood dried on filter-paper. T.S.H. in blood collected in this manner is stable and extraction is simple. Most importantly T.S.H. values obtained in this way correlate well with those obtained on serum. Though the values of T.S.H. from the dried blood spot are a little lower than T.S.H. values obtained from serum, this method is quite acceptable for use in the detection of primary hypothyroidism by mass screening of the newborne because this method can detect approximately 10 µU/ml Of T.S.H., a value far below that of most cases of primary hypothyroidism.5 Furthermore, the assay does not have to be duplicated, a factor which greatly enhances its value for mass screening. Blood should be obtained three or more days after birth, because serum-T.s.H. levels rise sharply in the postnatal period.67However, a postnatal T.S.H. rise is not a consistent feature because we found normal blood-T.s.H. values in umbilical cord blood and in specimens obtained on the first and second days of life. In patients with secondary or tertiary hypothyroidism, the bloodt. S. H. should be reduced, but such lower than normal levels cannot yet be measured. Although for the diagnosis of all types of hypothyroidism the measurement of blood thyroxine and tri-
B.-A. LAMBERG
Endocrine Research Unit, University of Helsinki; Minerva Foundation Institute for Medical Research, Helsinki; and Third and Fourth Departments of Medicine, University of Helsinki
Summary
Out of 18 subjects with symptomless autoimmune thyroiditis (S.A.T.) 5 (28%) became hypothyroid within 4 to 39 months of observation, whereas 13 (72%) remained euthyroid up to at least 28 to 50 months. In all those who developed hypothyroidism the basal serum level of thyroidstimulating hormone (T.S.H.) was already initially above normal (normal range 1·6-6·9 µU/ml) and 4 had markedly elevated concentrations (>19 µU/ml). All those subjects developing hypothyroidism also had
initially an exaggerated response to thyrotropinreleasing hormone (T.R.H.) (upper normal limit, &Dgr; T.S.H. 30 µU/ml), and in 4 the response was much exaggerated (&Dgr; T.S.H. >70 µU/ml). In 3 of these subjects the basal T.S.H. and the response to T.R.H. were reassessed before starting the substitution therapy and in all there was a further increase in both values. The basal serum T.S.H. was initially also slightly increased in 2 and the response to T.R.H. slightly above normal in 4 subjects who remained euthyroid. The basal T.S.H. level became normal in both cases with
1235 elevated values; and the response to T.R.H. declined to a normal level in 3 of the latter 4 subjects, but showed a further increase in 1. The thyroglobulin
TABLE I-DATA ON PATIENTS WITH SYMPTOMLESS AUTOIMMUNE THYROIDITIS
antibody (TgA) titres were initially significantly elevated in 15 subjects and the thyroid microsomal antibodies (MsA) in 1. The TgA titres decreased during the observation period in all but 1 subject and a similar trend was observed as regards the MsA titres. It is concluded that within a few years of observation a substantial number of subjects with s.A.T. will be hypothyroid. A definitively increased basal serum T.S.H. level and a markedly exaggerated response to T.R.H. in the symptomless stage of the disease is connected with a high risk of late hypothyroidism. Introduction AUTOIMMUNE thyroiditis may occur in two different forms: Hashimoto’s goitrous thyroiditis and atrophic thyroiditis, the latter being much more common,22 In both forms overt hypothyroidism may ensue, but this is not an all-or-none phenomenon and probably all patients go through a stage of subclinical hypothyroidism before developing clinical hypothyroidism)4 The clinical equivalent to the latent stage of atrophic autoimmune thyroiditis is called asymptomatic atrophic thyroiditis1 or symptomless autoimmune thyroiditis (S.A.T.).4 Subjects with s.A.T. are clinically euthyroid; they have a normal thyroid on palpation and normal thyroid-function tests, high titres of circulating thyroid antibodies, and lymphocytic infiltration in the thyroid gland.4 During the course of the disease the thyroid gland may atrophy and hypo.
thyroidism ensues. Fairly little is known about the natural course of S.A.T.1 5 During the preparation of this manuscript a larger report appeared on chronic thyroiditis in children.6 The present report may contribute to the solution of the problem of how often and how soon hypothyroidism, will develop in adults after the diagnosis of S.A.T. has been established.
Subjects
and methods
Subjects The study comprised 18 subjects with S.A.T. (table l) of whom were included in our original report. The time of follow-up varied from 28 to 50 months (mean 36 months). During this period they were subjected to clinical and laboratory examinations at intervals. most
Syrnptomless Autoimmune Thyroiditis According to definition, the subjects with S.A.T. were clinically euthyroid; they had a thyroid of normal size and no history of thyroid disease. The thyroid-function tests were normal. Autoimmune thyroiditis was considered to be present when the titre of circulating thyroglobulin
antibodies (TgA)
was
1/25 000
or
more
and/or that of
n.r. = not
representative.
n.d. =not done.
thyroid microsomal antibodies (MsA) 1/324 or more. When the thyroid antibody titres were below these limits, positive cytological evidence from fine-needle biopsy was required for the diagnosis. Owing to the small thyroid size, biopsies were not performed in all patients. The findings on biopsy were positive in 10 cases, but in 3 cases the specimens obtained were not representative. The subjects with S.A.T. originally came to be investigated either because of a family history of thyroiditis or because they had some other autoimmune disease. In some cases markedly elevated thyroid antibody titres were found on routine examination for unspecified symptoms or for increase in the erythrocyte-sedimentation rate. Clinical Hypothyroidism The diagnosis of clinical hypothyroidism was based on typical symptoms and signs of hypothyroidism and subnormal thyroid function tests.4 The diagnosis was confirmed in all cases by the disappearance of symptoms and signs of hypothyroidism and a definite and lasting improvement in the clinical condition following adequate substitution .
therapy. Laboratory Tests Serum protein-bound-iodine (P.B.I.) was measured with the ’Autoanalyzer’ Technicon N-56 method (normal range 4.0-8.0 ug/100 ml).. A free-thyroxine index (F.T.I.)7 was calculated by multiplying the P.B.I. by the values for the uptake of triiodothyronine by ’Sephadex’ (T3U) (normal range 80-120 % and dividing the results (for convenience) by ten (normal range 40-90).’ Serum thyrotrophin (T.S.H.) was determined by doubleantibody radioimmunoassay9. (normal range 1.6-6.9 uU// ml). The thyrotrophin-releasing hormone (T.R.H.) stimulation test was performed as follows 4 10 200 ug of synthetic T.R.H. (Hoffmann-La Roche, Basle) was given intravenously as a rapid injection. The serum T.s.H. was determined before and 20 and 60 minutes after the injection. The normal values (means.D.) in 25 euthyroid control subjects were
TABLE II-THYROID PARAMETERS BEFORE AND AT END OF OBSERVATION PERIOD IN PATIENTS WHO BECAME HYPOTHYROID
*
Inverse
figure.
n.d.
not done.
1236 TABLE III-DATA ON PATIENTS WHO REMAINED EUTHYROID
*
Inverse
figure..
z
n.d. - not done.
2-8±1’2, 15’4_L8’0, and ll’O .6-7 1tU/ml, respectively. A maxi-nal rise (T.S.H.) at 20 minutes of more than 30.0 uU// ml was regarded as abnormally high and a response of less than 3-0 U/ml as abnormally low.4 The presence of TgA in the circulation was determined by the tanned-red-cell technique and of MsA by the complement-fixation techniqueTitres of 1/25 for TgA and 1/2 for MsA were regarded as normal, of between 1/250 and 1/2500 and 1/4 and 1/16 as low, and of 1/25 000 or more and 1/32 or more as significantly increased. The cytological diagnosis was based on fine-needle biopsy according to Persson. 12 Abundant lymphocytes and Hurthle cells were regarded as indicative of autoimmune
thyroiditis. Results Clinical Status 5 of the 18
subjects (28°o) with S.A.T. became hypothyroid during the follow-up period (table 11). The time elapsed from establishing the diagnosis of S.A.T. to the moment when hypothyroidism was diagnosed ranged from 4 to 39 months (mean 26 months). 13 subjects (72%) with S.A.T. were still euthyroid after a
Fig. 1-Serum T.S.H. at first examination and at follow-up study in patients with symptomless autoimmune thyroiditis who remained euthyroid (8) and in those who became hypothyroid ([]). Control level indicated by horizontal broken lines. &mid ot;
follow-up period of 28 to 50 months (mean 36 months) (table in). Serum T.S.H. and its Response to T.R.H. The basal serum T.s.H. level was initially increased in 7 subjects 5 of whom later developed hypothyroidism (fig. 1). The basal serum T.S.H. increased in 3 of the hypothyroid patients during the follow-up period, but in 2 the determination was not repeated before the substitution therapy was instituted. 4 of the 5 patients who later developed hypothyroidism had initially a markedly elevated T.S.H. level (above 19-5jjLU/ml) (fig. 1). In the 2 subjects remaining euthyroid the T.S.H. level was only slightly elevated initially and in one it became normal during the
follow-up period (fig. 1). The response in
serum T.S.H. to T.R.H. was
initially
Fig. 2-Increment in serum T.S.H. after injection of 200 g of T.S.H.( T.S.H.) at first examination and at follow-up study in patients with symptomless autoimmune thyroiditis who remained euthyroid (8) and in those who became hypothyroid (l ]). Level of normal response indicated by horizontal broken hnes.
1237
exaggerated in 9 subjects of whom 5 developed hypothyroidism. The T.R.H. test was repeated in 3 of those becoming hypothyroid before the substitution therapy was instituted, and in all cases the T.s.H. had increased further (fig. 2). In 4 of the 5 subjects who developed hypothyroidism the A T.S.H. was initially markedly above the normal upper limit of response (above 70 U/ml). In 4 subjects who remained euthyroid the T.S.H. was initially only slightly elevated. In 3 subjects it became normal during the follow-up period, but in. 1 it increased greatly (fig. 2). Circulating Thyroid Antibodies TgA were significantly elevated (=1/25 000) in 15 of the 18 subjects at the start of the study and 3 had a low TgA titre. The MsA were intially measured in 12 subjects and were significantly elevated (1/32) in 1 and low in 4 subjects, whereas 7 were MsA-negative (tables 11 and III). At the end of the total follow-up period only 2 subjects had a signfiicantly elevated TgA titre; the titres were low in 8 and normal in 8 subjects. MsA were measured at this stage only in 5 subjects, none of them had significantly elevated titres; 1 had a low titre and 4 normal titres (tables i and II). Discussion The present study may contribute to the information about the incidence of spontaneous hypothyroidism in symptomless autoimmune thyroiditis (S.A.T.). Hypothyroidism ensued in 5 subjects outof 18 within 4 to 39 months of observation. On the contrary, in juvenile autoimmune thyroiditis none of the 32 subjects followed for about 3 years became hypothyroid.6 All 5 patients in our series who became hypothyroid had initially an elevated basal serum T.s.H. level and an exaggerated response to T.R.H., and in 4 cases these values were indeed markedly above the upper limit of normal in both respects. On the other hand, among those subjects who remained euthyroid there were 2 who initially had an elevated basal T.S.H. level and 4 with an exaggerated response to T.R.H. ; but in all these cases the values were close to the normal upper limit. In 1 euthyroid subject the response to T.R.H. had increased markedly at follow-up, which is probably indicative of development of hypothyroidism within a near future. The present study seems to indicate that a definitely elevated basal serum T.s.H. and a markedly exaggerated response to T.R.H. in S.A.T. is connected with a high risk of later hypothyroidism. It has been repeatedly recognised that the basal and the response to T.R.H. may be elevated in S.A.T. and in euthyroid subjects after subtotal thyroidectomy or radioiodine treatment for hyperthyroidism.4 13-19. In our own- studies we found in S.A.T. an elevated basal T.s.H. in about 1/4 of the patients and an exaggerated response to T.R.H. in about 1/3 of the subjects.4 19 However, the value of these changes as parameters in predicting ensuing hypothyroidism has not been well documented. Toft et al.20 studied patients 2-18 months after treatment of hyperthyroidism with radioiodine. In that study T.s.H. seemed not to be a reliable indicator of future thyroid failure developing in the early months after serum T.S.H.
the treatment. In another study these workers2I 22 found that a raised serum T.s.H. level in patients treated years earlier for hyperthyroidism did not alter over a period of 15 months. However, 3 out of 69 patients in this series with a raised basal T.S.H. level became hypothyroid while none of the 61 with a normal T.S.H. level developed hypothyroidism during this
period. A recent report from the same centre23 showed that from the pool of patients with elevated T.S.H. levels some are transferred each year to the group with hypothyroidism. The situation is, however, somewhat different from S.A.T., since in treated hyperthyroidism one has to consider unresponsiveness on the pituitary level for various reasons, of which one is a continuing block after prolonged depression of the T.S.H. secretion and another one is a block due to an increased level of T3 although the patients are euthyroid. Furthermore, the situation is also different at the thyroid level, since in treated hyperthyroidism, in addition to the tissue damage in the thyroid, circulating thyroid stimulators may also be present and make the situation more complex.24 25 In S.A.T. the situation is different, and it seems that serum T.S.H. really in a way is a measure of the damage on the thyroid.
There appears to be a quantitative correlation between lymphocytic infiltration in the thyroid gland and the presence of circulating thyroid antibodies.2 However, definitely elevated titres are evidently indicative of active thyroiditis. The highest titres are found in Hashimoto’s goitrous thyroiditis. Thyroid antibodies are present in about 70-80% of cases of spontaneous hypothyroidism and are significantly elevated in 10-20%. In s.A.T., by our definition, the antibody titres are significantly elevated, but in some cases with proven lymphocytic infiltration of the thyroid the titres have been low. In the present study there was a distinct tendency for TgA titres to decline during the follow-up both in patients becoming hypothyroid and those still euthyroid after follow-up. MsA were not routinely determined, but there was also a tendency for these to decrease in titre. Thus it seems that probably all patients with spontaneous hypothyroidism at some stage of the disease may have circulating thyroid antibodies, probably in high titres. Later on, when atrophy of the gland has developed, the antibodies may disappear, which may be why in hypothyroidism high titres are less common. Determination of thyroid antibodies seems to have no prognostic value in these cases. The gift of human T.s.H. and T.S.H. antiserum from the Pituitary Agency of the National Institutes of Health, Bethesda, Maryland, U.S.A., and of human reference Standard A (68/38) from the National Institute of Medical Research, London, Great Britain, are gratefully acknowledged. Synthetic T.R.H. was generously supplied by Hoffman-La Roche, Basle, Switzerland. Our thanks
due to Mrs M.-L. Kuhanen for skilful technical assistance. This study was aided by grants from the National Research Council for Medical Sciences in Finland, the Sigrid Juselius Foundation, Medicinska Understodsforeningen Liv och Halsa, and the Finska are
Lakaresallskapet. Requests for reprints should be addressed to B.-A. Lamberg, Endocrine Research Unit, University of Helsinki, Minerva Foundation, P.O. Box 819, SF-00101 Helsinki 10, Finland.
1238 SMOOTH-MUSCLE ANTIBODIES IN INFERTILITY O. E. EADE C. WILSON R. LLOYD MAX ELSTEIN RALPH WRIGHT
Southampton University Hospitals, Royal South Hants Hospital, Southampton, SO9 4PE Sera from 77 infertile women and 77 post-partum controls were examined for autoantibodies by indirect immunofluorescence. 27 (35%) of the infertile group had smooth-muscle antibody compared with 2 (3%) controls. Significantly more infertile patients had anti-nuclear factor, and reticulin antibodies were also more commonly found in this group. The differences for organ-specific autoantibodies between the two groups were not significant. There was no correlation between the occurrence of smooth-muscle antibodies and immunofluorescent anti-sperm antibodies. The high frequency of smooth-muscle antibodies in infertile women requires further study to determine whether an underlying viral infection is involved.
with autoimmune disease, notably autoimmune adrenalitis, ovarian antibodies are often present and may be associated with organ-specific autoantibodies to thyroid and gastric parietal cells, particularly in those patients with premature menopause., We found a high frequency of antibodies to smoothmuscle and tissue nuclei in a group of women attending an infertility clinic.
tility associated
Patients
Summary
Infertile Patients This group comprised 77 women attending the infertility clinic in Southampton who were being screened for evidence of latent coeliac disease. Ovarian dysfunction had been diagnosed in 74 using basal temperature chart measurements,’ and three had amenorrhoea; 49 had primary infertility and 28 secondary infertility. 2 of these patients had bicornuate uteri, but in all the remainder uterine or tubal pathology had been excluded. Apart from 1 patient who had been treated for hyperthyroidism, none had evidence of systemic, endocrine, or autoimmune disease. 66 had already been treated at some stage with drugs which stimulate ovarian function; 65 had received clomiphene, and 10 of these had received human chorionic gonadotrophins as well. 6 had been given human menopausal gonadotrophins.
Introduction human spermatozoa 12 in the sera of some infertile women has suggested that immunological mechanisms may be involved in reproductive failure, but women who conceive normally often have anti-sperm antibodies. In the rare cases of inferTHE detection of antibodies
to
DR GORDON AND PROFESSOR LAMBERG: REFERENCES 1. Bastenie, P. A., Bonnyns, M., Vanhaelst, L. in Thyroiditis and Thyroid Function (edited by P. A. Bastenie and A. M. Ermans); p. 211. Oxford, 1972. 2. Bastenie, P. A., Bonnyns, M., Ermans, A. M., Nève, P., Vanhaelst, L. ibid. p. 229. 3. Evered, D. C., Ormston, B. J., Smith, P. A., Hall, R., Bird, T. Br. med. J. 1973, i, 657. 4. Gordin, A., Saarinen, P., Pelkonen, R., Lamberg, B.-A. Acta endocr., Copenh. 1974, 75, 274. 5. Bastenie, P. A., Nève, P., Bonnyns, M., Vanhaelst, L., Chailly, M. Lancet, 1967, i, 915. 6. Rallison, M. L., Dobyns, B. M., Keating, F. R., Rail, J. E., Tyler, F. H. J. Pediat. 1975, 86, 675. 7. Clark, F., Horn, D. B. J. clin. Endocr. Metab. 1966, 26, 352. 8. Lamberg, B.-A., Heinonen, O. P., Viherkoski, M., Aro, A., Liewendahl, K., Kvist, G., Laitinen, O., Knekt, P. Acta endocr., Copenh. 1970, suppl. 146. 9. Gordin, A., Saarinen, P. ibid. 1972, 71, 24. 10. Ormston, B. J., Garry, R., Cryer, E. J., Besser, G. M., Hall, R. Lancet, 1971, ii, 10. 11. Roitt, I. M., Doniach, D. ibid. 1958, ii, 1027. 12. Persson, P. S. Acta med. scand. 1968, suppl. 483, p. 1. 13. Hedley, A. J., Hall, R., Amos, J., Michie, W., Crooks, J. Lancet,
1971, i, 455. Slingerland, D. W., Dell, E. S., Burrows, B. A. in Further Advances in Thyroid Research (edited by K. Fellinger and R. Höfer) p. 993. Vienna, 1971. 15. Slingerland, D. W., Hershman, J. M., Dell, E. S., Burrows, B. A J. clin. Endocr. Metab. 1972, 35, 912. 16. Bellabarba, D.. Bénard, B., Langlois, M. Clin. Endocr. 1972, 1, 345. 17. Gordin, A. Acta endocr., Copenh. 1973, 74, 283. 18. Gordin, A., Wägar, G., Hernberg, C. A. Acta med. scand. 1973, 194 14.
Controls 77 fertile patients of similar age from whom blood had been taken during the early post-partum period as part of another study were used as controls.
Methods 20 ml of blood was taken by venepuncture and the stored at -20°C until testing.
serum
Immunofluorescence and Serological Studies Immunofluorescence ().F.) studies for the detection of antibodies to tissue antigens were carried out as described elsewhere."Sera were used initially at a dilution of 1 in 10, and the fluorescein-conjugated polyvalent and monospecific antihuman sera were obtained from Wellcome Reagents Ltd. I. F. studies for the detection of anti-sperm antibodies were carried out as described by Tung,6 and thyroglobulin titres were determined by haemagglutination. Results The autoantibodies detected in both groups are listed in the accompanying table. Differences between the groups for organ-specific antibodies do not reach statistical significance. However, significantly more infertile AUTOANTIBODIES AND ANTI-SPERM ANTIBODIES IN SERA OF INFERTH I WOMEN AND CONTROLS
335. 19. Gordin, A., Heinonen, O. P., Saarinen, P.,
Lamberg, B.-A. Lancet 1972, i, 551. 20. Toft, A. D., Barnes, E. W., Hunter, W. M., Seth, J., Irvine, W. J ibid. 1973, ii, 644. 21. Toft, A. D., Smith, J., Hunter, W. M., Irvine, W. J. ibid., 1974 i. 704.
Toft, A. D., Irvine, W. J., Hunter, W. M., Seth, J. Br. med. J. 1974 iii, 152. 23. Toft, A. D., Irvine, W. J., Seth, J., Hunter, W. M., Cameron E. H. D. Lancet, 1975, ii, 576. 25. Volpé, R., Farid, N. R., von Westarp, C., Row, V. V. Clin. Endocr 1974, 3, 239. 25. Calder, E. A., Irvine, W. J. Ibid. 1975, 4, 287.
22.
*I’ernicious
anaemia
type.
f)’
well as T.s.H. would be necessary, the microdetermination of T. S. II. has substantial value since primary hypothyroidism seems to be much more common than secondary or tertiary hypothyroidism. Klein et al.have shown that treatment before the age of three months could prevent mental retardation among hypothyroid infants. With the method we have described patients with congenital or juvenile primary hypothyroidism could be detected during mass-screening for metabolic disorders, and treatment could begin during the first days or weeks of life.
iodothyronine
as
We thank Dr H. L. Levy of the State Laboratory Institute, Boston, Massachusetts, U.S.A., for his help in the preparation of this paper.
This study was partly supported by a grant from the Ministry of Health of Japan. The materials to measure T.s.H. were supplied by the National Institutes of Health, Bethesda, Maryland, U.S.A., and the Daiichi Radioisotope Company, Tokyo, Japan. We also thank Dr H. Enomoto, Dr K. Inoue, Dr T. Nukui, Dr M. Kuroda, Dr S. Takeda, Dr M. Mizotam, Dr Y. Ito, and Ms S. Oshima for their help; Dr T. Aoki, Dr T. Kumagai, Dr K. Nakayama of the Pædiatric Department of Toho University for their help with the paediatric patients, and Ms Y. litaka and M. Katakura for secretarial work.
BLOOD
Requests for reprints should be addressed to M.l. REFERENCES
1. 2.
*245
µU/ml in the case of hypothyroidism (see text).
227 newborn infants tested none had a raised T.S.H., and values in 5 cord-blood specimens were also normal. From the dried blood spots obtained from outpatients, 1 case of primary hypothyroidism was detected-in a four-year-old girl with clinical signs of hypothyroidism and a T.S.H. of 245 uU/ml.
Dussault, J. H , Laberge, L. Union Meed Can. 1973, 102, 2062 Dussault, J. H., Coulombe, P., Laberge, L., Guyda, H , Khoury, K J. Pediat. 1975, 86, 670. 3. Klein, A. H., Augustin, A. V., Foley, Jr. T. P. Lancet, 1974, ii, 77 4. Guthrie, R., Susi, A., Pediatrics, 1963, 32, 338. 5. Odell, W. D., Wliber, J. F , Utiger, R. D. Rec. Progr Hormone Res 1967, 23, 47. 6. Hershman, J. M., Pittman, Jr. J. A. Ann. intern Med 1971, 74, 481 7. Fisher, D. A., Odell, W D. J. clin Invest 1969, 48, 1670 8. Klein, A. H., Meltzer, S., Kenny, F. M. J Pediat. 1972, 81, 912
NATURAL COURSE OF SYMPTOMLESS AUTOIMMUNE THYROIDITIS
A. GORDIN Discussion We have shown that T.s.H. can be measured in 20 µl of whole blood dried on filter-paper. T.S.H. in blood collected in this manner is stable and extraction is simple. Most importantly T.S.H. values obtained in this way correlate well with those obtained on serum. Though the values of T.S.H. from the dried blood spot are a little lower than T.S.H. values obtained from serum, this method is quite acceptable for use in the detection of primary hypothyroidism by mass screening of the newborne because this method can detect approximately 10 µU/ml Of T.S.H., a value far below that of most cases of primary hypothyroidism.5 Furthermore, the assay does not have to be duplicated, a factor which greatly enhances its value for mass screening. Blood should be obtained three or more days after birth, because serum-T.s.H. levels rise sharply in the postnatal period.67However, a postnatal T.S.H. rise is not a consistent feature because we found normal blood-T.s.H. values in umbilical cord blood and in specimens obtained on the first and second days of life. In patients with secondary or tertiary hypothyroidism, the bloodt. S. H. should be reduced, but such lower than normal levels cannot yet be measured. Although for the diagnosis of all types of hypothyroidism the measurement of blood thyroxine and tri-
B.-A. LAMBERG
Endocrine Research Unit, University of Helsinki; Minerva Foundation Institute for Medical Research, Helsinki; and Third and Fourth Departments of Medicine, University of Helsinki
Summary
Out of 18 subjects with symptomless autoimmune thyroiditis (S.A.T.) 5 (28%) became hypothyroid within 4 to 39 months of observation, whereas 13 (72%) remained euthyroid up to at least 28 to 50 months. In all those who developed hypothyroidism the basal serum level of thyroidstimulating hormone (T.S.H.) was already initially above normal (normal range 1·6-6·9 µU/ml) and 4 had markedly elevated concentrations (>19 µU/ml). All those subjects developing hypothyroidism also had
initially an exaggerated response to thyrotropinreleasing hormone (T.R.H.) (upper normal limit, &Dgr; T.S.H. 30 µU/ml), and in 4 the response was much exaggerated (&Dgr; T.S.H. >70 µU/ml). In 3 of these subjects the basal T.S.H. and the response to T.R.H. were reassessed before starting the substitution therapy and in all there was a further increase in both values. The basal serum T.S.H. was initially also slightly increased in 2 and the response to T.R.H. slightly above normal in 4 subjects who remained euthyroid. The basal T.S.H. level became normal in both cases with
1235 elevated values; and the response to T.R.H. declined to a normal level in 3 of the latter 4 subjects, but showed a further increase in 1. The thyroglobulin
TABLE I-DATA ON PATIENTS WITH SYMPTOMLESS AUTOIMMUNE THYROIDITIS
antibody (TgA) titres were initially significantly elevated in 15 subjects and the thyroid microsomal antibodies (MsA) in 1. The TgA titres decreased during the observation period in all but 1 subject and a similar trend was observed as regards the MsA titres. It is concluded that within a few years of observation a substantial number of subjects with s.A.T. will be hypothyroid. A definitively increased basal serum T.S.H. level and a markedly exaggerated response to T.R.H. in the symptomless stage of the disease is connected with a high risk of late hypothyroidism. Introduction AUTOIMMUNE thyroiditis may occur in two different forms: Hashimoto’s goitrous thyroiditis and atrophic thyroiditis, the latter being much more common,22 In both forms overt hypothyroidism may ensue, but this is not an all-or-none phenomenon and probably all patients go through a stage of subclinical hypothyroidism before developing clinical hypothyroidism)4 The clinical equivalent to the latent stage of atrophic autoimmune thyroiditis is called asymptomatic atrophic thyroiditis1 or symptomless autoimmune thyroiditis (S.A.T.).4 Subjects with s.A.T. are clinically euthyroid; they have a normal thyroid on palpation and normal thyroid-function tests, high titres of circulating thyroid antibodies, and lymphocytic infiltration in the thyroid gland.4 During the course of the disease the thyroid gland may atrophy and hypo.
thyroidism ensues. Fairly little is known about the natural course of S.A.T.1 5 During the preparation of this manuscript a larger report appeared on chronic thyroiditis in children.6 The present report may contribute to the solution of the problem of how often and how soon hypothyroidism, will develop in adults after the diagnosis of S.A.T. has been established.
Subjects
and methods
Subjects The study comprised 18 subjects with S.A.T. (table l) of whom were included in our original report. The time of follow-up varied from 28 to 50 months (mean 36 months). During this period they were subjected to clinical and laboratory examinations at intervals. most
Syrnptomless Autoimmune Thyroiditis According to definition, the subjects with S.A.T. were clinically euthyroid; they had a thyroid of normal size and no history of thyroid disease. The thyroid-function tests were normal. Autoimmune thyroiditis was considered to be present when the titre of circulating thyroglobulin
antibodies (TgA)
was
1/25 000
or
more
and/or that of
n.r. = not
representative.
n.d. =not done.
thyroid microsomal antibodies (MsA) 1/324 or more. When the thyroid antibody titres were below these limits, positive cytological evidence from fine-needle biopsy was required for the diagnosis. Owing to the small thyroid size, biopsies were not performed in all patients. The findings on biopsy were positive in 10 cases, but in 3 cases the specimens obtained were not representative. The subjects with S.A.T. originally came to be investigated either because of a family history of thyroiditis or because they had some other autoimmune disease. In some cases markedly elevated thyroid antibody titres were found on routine examination for unspecified symptoms or for increase in the erythrocyte-sedimentation rate. Clinical Hypothyroidism The diagnosis of clinical hypothyroidism was based on typical symptoms and signs of hypothyroidism and subnormal thyroid function tests.4 The diagnosis was confirmed in all cases by the disappearance of symptoms and signs of hypothyroidism and a definite and lasting improvement in the clinical condition following adequate substitution .
therapy. Laboratory Tests Serum protein-bound-iodine (P.B.I.) was measured with the ’Autoanalyzer’ Technicon N-56 method (normal range 4.0-8.0 ug/100 ml).. A free-thyroxine index (F.T.I.)7 was calculated by multiplying the P.B.I. by the values for the uptake of triiodothyronine by ’Sephadex’ (T3U) (normal range 80-120 % and dividing the results (for convenience) by ten (normal range 40-90).’ Serum thyrotrophin (T.S.H.) was determined by doubleantibody radioimmunoassay9. (normal range 1.6-6.9 uU// ml). The thyrotrophin-releasing hormone (T.R.H.) stimulation test was performed as follows 4 10 200 ug of synthetic T.R.H. (Hoffmann-La Roche, Basle) was given intravenously as a rapid injection. The serum T.s.H. was determined before and 20 and 60 minutes after the injection. The normal values (means.D.) in 25 euthyroid control subjects were
TABLE II-THYROID PARAMETERS BEFORE AND AT END OF OBSERVATION PERIOD IN PATIENTS WHO BECAME HYPOTHYROID
*
Inverse
figure.
n.d.
not done.
1236 TABLE III-DATA ON PATIENTS WHO REMAINED EUTHYROID
*
Inverse
figure..
z
n.d. - not done.
2-8±1’2, 15’4_L8’0, and ll’O .6-7 1tU/ml, respectively. A maxi-nal rise (T.S.H.) at 20 minutes of more than 30.0 uU// ml was regarded as abnormally high and a response of less than 3-0 U/ml as abnormally low.4 The presence of TgA in the circulation was determined by the tanned-red-cell technique and of MsA by the complement-fixation techniqueTitres of 1/25 for TgA and 1/2 for MsA were regarded as normal, of between 1/250 and 1/2500 and 1/4 and 1/16 as low, and of 1/25 000 or more and 1/32 or more as significantly increased. The cytological diagnosis was based on fine-needle biopsy according to Persson. 12 Abundant lymphocytes and Hurthle cells were regarded as indicative of autoimmune
thyroiditis. Results Clinical Status 5 of the 18
subjects (28°o) with S.A.T. became hypothyroid during the follow-up period (table 11). The time elapsed from establishing the diagnosis of S.A.T. to the moment when hypothyroidism was diagnosed ranged from 4 to 39 months (mean 26 months). 13 subjects (72%) with S.A.T. were still euthyroid after a
Fig. 1-Serum T.S.H. at first examination and at follow-up study in patients with symptomless autoimmune thyroiditis who remained euthyroid (8) and in those who became hypothyroid ([]). Control level indicated by horizontal broken lines. &mid ot;
follow-up period of 28 to 50 months (mean 36 months) (table in). Serum T.S.H. and its Response to T.R.H. The basal serum T.s.H. level was initially increased in 7 subjects 5 of whom later developed hypothyroidism (fig. 1). The basal serum T.S.H. increased in 3 of the hypothyroid patients during the follow-up period, but in 2 the determination was not repeated before the substitution therapy was instituted. 4 of the 5 patients who later developed hypothyroidism had initially a markedly elevated T.S.H. level (above 19-5jjLU/ml) (fig. 1). In the 2 subjects remaining euthyroid the T.S.H. level was only slightly elevated initially and in one it became normal during the
follow-up period (fig. 1). The response in
serum T.S.H. to T.R.H. was
initially
Fig. 2-Increment in serum T.S.H. after injection of 200 g of T.S.H.( T.S.H.) at first examination and at follow-up study in patients with symptomless autoimmune thyroiditis who remained euthyroid (8) and in those who became hypothyroid (l ]). Level of normal response indicated by horizontal broken hnes.
1237
exaggerated in 9 subjects of whom 5 developed hypothyroidism. The T.R.H. test was repeated in 3 of those becoming hypothyroid before the substitution therapy was instituted, and in all cases the T.s.H. had increased further (fig. 2). In 4 of the 5 subjects who developed hypothyroidism the A T.S.H. was initially markedly above the normal upper limit of response (above 70 U/ml). In 4 subjects who remained euthyroid the T.S.H. was initially only slightly elevated. In 3 subjects it became normal during the follow-up period, but in. 1 it increased greatly (fig. 2). Circulating Thyroid Antibodies TgA were significantly elevated (=1/25 000) in 15 of the 18 subjects at the start of the study and 3 had a low TgA titre. The MsA were intially measured in 12 subjects and were significantly elevated (1/32) in 1 and low in 4 subjects, whereas 7 were MsA-negative (tables 11 and III). At the end of the total follow-up period only 2 subjects had a signfiicantly elevated TgA titre; the titres were low in 8 and normal in 8 subjects. MsA were measured at this stage only in 5 subjects, none of them had significantly elevated titres; 1 had a low titre and 4 normal titres (tables i and II). Discussion The present study may contribute to the information about the incidence of spontaneous hypothyroidism in symptomless autoimmune thyroiditis (S.A.T.). Hypothyroidism ensued in 5 subjects outof 18 within 4 to 39 months of observation. On the contrary, in juvenile autoimmune thyroiditis none of the 32 subjects followed for about 3 years became hypothyroid.6 All 5 patients in our series who became hypothyroid had initially an elevated basal serum T.s.H. level and an exaggerated response to T.R.H., and in 4 cases these values were indeed markedly above the upper limit of normal in both respects. On the other hand, among those subjects who remained euthyroid there were 2 who initially had an elevated basal T.S.H. level and 4 with an exaggerated response to T.R.H. ; but in all these cases the values were close to the normal upper limit. In 1 euthyroid subject the response to T.R.H. had increased markedly at follow-up, which is probably indicative of development of hypothyroidism within a near future. The present study seems to indicate that a definitely elevated basal serum T.s.H. and a markedly exaggerated response to T.R.H. in S.A.T. is connected with a high risk of later hypothyroidism. It has been repeatedly recognised that the basal and the response to T.R.H. may be elevated in S.A.T. and in euthyroid subjects after subtotal thyroidectomy or radioiodine treatment for hyperthyroidism.4 13-19. In our own- studies we found in S.A.T. an elevated basal T.s.H. in about 1/4 of the patients and an exaggerated response to T.R.H. in about 1/3 of the subjects.4 19 However, the value of these changes as parameters in predicting ensuing hypothyroidism has not been well documented. Toft et al.20 studied patients 2-18 months after treatment of hyperthyroidism with radioiodine. In that study T.s.H. seemed not to be a reliable indicator of future thyroid failure developing in the early months after serum T.S.H.
the treatment. In another study these workers2I 22 found that a raised serum T.s.H. level in patients treated years earlier for hyperthyroidism did not alter over a period of 15 months. However, 3 out of 69 patients in this series with a raised basal T.S.H. level became hypothyroid while none of the 61 with a normal T.S.H. level developed hypothyroidism during this
period. A recent report from the same centre23 showed that from the pool of patients with elevated T.S.H. levels some are transferred each year to the group with hypothyroidism. The situation is, however, somewhat different from S.A.T., since in treated hyperthyroidism one has to consider unresponsiveness on the pituitary level for various reasons, of which one is a continuing block after prolonged depression of the T.S.H. secretion and another one is a block due to an increased level of T3 although the patients are euthyroid. Furthermore, the situation is also different at the thyroid level, since in treated hyperthyroidism, in addition to the tissue damage in the thyroid, circulating thyroid stimulators may also be present and make the situation more complex.24 25 In S.A.T. the situation is different, and it seems that serum T.S.H. really in a way is a measure of the damage on the thyroid.
There appears to be a quantitative correlation between lymphocytic infiltration in the thyroid gland and the presence of circulating thyroid antibodies.2 However, definitely elevated titres are evidently indicative of active thyroiditis. The highest titres are found in Hashimoto’s goitrous thyroiditis. Thyroid antibodies are present in about 70-80% of cases of spontaneous hypothyroidism and are significantly elevated in 10-20%. In s.A.T., by our definition, the antibody titres are significantly elevated, but in some cases with proven lymphocytic infiltration of the thyroid the titres have been low. In the present study there was a distinct tendency for TgA titres to decline during the follow-up both in patients becoming hypothyroid and those still euthyroid after follow-up. MsA were not routinely determined, but there was also a tendency for these to decrease in titre. Thus it seems that probably all patients with spontaneous hypothyroidism at some stage of the disease may have circulating thyroid antibodies, probably in high titres. Later on, when atrophy of the gland has developed, the antibodies may disappear, which may be why in hypothyroidism high titres are less common. Determination of thyroid antibodies seems to have no prognostic value in these cases. The gift of human T.s.H. and T.S.H. antiserum from the Pituitary Agency of the National Institutes of Health, Bethesda, Maryland, U.S.A., and of human reference Standard A (68/38) from the National Institute of Medical Research, London, Great Britain, are gratefully acknowledged. Synthetic T.R.H. was generously supplied by Hoffman-La Roche, Basle, Switzerland. Our thanks
due to Mrs M.-L. Kuhanen for skilful technical assistance. This study was aided by grants from the National Research Council for Medical Sciences in Finland, the Sigrid Juselius Foundation, Medicinska Understodsforeningen Liv och Halsa, and the Finska are
Lakaresallskapet. Requests for reprints should be addressed to B.-A. Lamberg, Endocrine Research Unit, University of Helsinki, Minerva Foundation, P.O. Box 819, SF-00101 Helsinki 10, Finland.
1238 SMOOTH-MUSCLE ANTIBODIES IN INFERTILITY O. E. EADE C. WILSON R. LLOYD MAX ELSTEIN RALPH WRIGHT
Southampton University Hospitals, Royal South Hants Hospital, Southampton, SO9 4PE Sera from 77 infertile women and 77 post-partum controls were examined for autoantibodies by indirect immunofluorescence. 27 (35%) of the infertile group had smooth-muscle antibody compared with 2 (3%) controls. Significantly more infertile patients had anti-nuclear factor, and reticulin antibodies were also more commonly found in this group. The differences for organ-specific autoantibodies between the two groups were not significant. There was no correlation between the occurrence of smooth-muscle antibodies and immunofluorescent anti-sperm antibodies. The high frequency of smooth-muscle antibodies in infertile women requires further study to determine whether an underlying viral infection is involved.
with autoimmune disease, notably autoimmune adrenalitis, ovarian antibodies are often present and may be associated with organ-specific autoantibodies to thyroid and gastric parietal cells, particularly in those patients with premature menopause., We found a high frequency of antibodies to smoothmuscle and tissue nuclei in a group of women attending an infertility clinic.
tility associated
Patients
Summary
Infertile Patients This group comprised 77 women attending the infertility clinic in Southampton who were being screened for evidence of latent coeliac disease. Ovarian dysfunction had been diagnosed in 74 using basal temperature chart measurements,’ and three had amenorrhoea; 49 had primary infertility and 28 secondary infertility. 2 of these patients had bicornuate uteri, but in all the remainder uterine or tubal pathology had been excluded. Apart from 1 patient who had been treated for hyperthyroidism, none had evidence of systemic, endocrine, or autoimmune disease. 66 had already been treated at some stage with drugs which stimulate ovarian function; 65 had received clomiphene, and 10 of these had received human chorionic gonadotrophins as well. 6 had been given human menopausal gonadotrophins.
Introduction human spermatozoa 12 in the sera of some infertile women has suggested that immunological mechanisms may be involved in reproductive failure, but women who conceive normally often have anti-sperm antibodies. In the rare cases of inferTHE detection of antibodies
to
DR GORDON AND PROFESSOR LAMBERG: REFERENCES 1. Bastenie, P. A., Bonnyns, M., Vanhaelst, L. in Thyroiditis and Thyroid Function (edited by P. A. Bastenie and A. M. Ermans); p. 211. Oxford, 1972. 2. Bastenie, P. A., Bonnyns, M., Ermans, A. M., Nève, P., Vanhaelst, L. ibid. p. 229. 3. Evered, D. C., Ormston, B. J., Smith, P. A., Hall, R., Bird, T. Br. med. J. 1973, i, 657. 4. Gordin, A., Saarinen, P., Pelkonen, R., Lamberg, B.-A. Acta endocr., Copenh. 1974, 75, 274. 5. Bastenie, P. A., Nève, P., Bonnyns, M., Vanhaelst, L., Chailly, M. Lancet, 1967, i, 915. 6. Rallison, M. L., Dobyns, B. M., Keating, F. R., Rail, J. E., Tyler, F. H. J. Pediat. 1975, 86, 675. 7. Clark, F., Horn, D. B. J. clin. Endocr. Metab. 1966, 26, 352. 8. Lamberg, B.-A., Heinonen, O. P., Viherkoski, M., Aro, A., Liewendahl, K., Kvist, G., Laitinen, O., Knekt, P. Acta endocr., Copenh. 1970, suppl. 146. 9. Gordin, A., Saarinen, P. ibid. 1972, 71, 24. 10. Ormston, B. J., Garry, R., Cryer, E. J., Besser, G. M., Hall, R. Lancet, 1971, ii, 10. 11. Roitt, I. M., Doniach, D. ibid. 1958, ii, 1027. 12. Persson, P. S. Acta med. scand. 1968, suppl. 483, p. 1. 13. Hedley, A. J., Hall, R., Amos, J., Michie, W., Crooks, J. Lancet,
1971, i, 455. Slingerland, D. W., Dell, E. S., Burrows, B. A. in Further Advances in Thyroid Research (edited by K. Fellinger and R. Höfer) p. 993. Vienna, 1971. 15. Slingerland, D. W., Hershman, J. M., Dell, E. S., Burrows, B. A J. clin. Endocr. Metab. 1972, 35, 912. 16. Bellabarba, D.. Bénard, B., Langlois, M. Clin. Endocr. 1972, 1, 345. 17. Gordin, A. Acta endocr., Copenh. 1973, 74, 283. 18. Gordin, A., Wägar, G., Hernberg, C. A. Acta med. scand. 1973, 194 14.
Controls 77 fertile patients of similar age from whom blood had been taken during the early post-partum period as part of another study were used as controls.
Methods 20 ml of blood was taken by venepuncture and the stored at -20°C until testing.
serum
Immunofluorescence and Serological Studies Immunofluorescence ().F.) studies for the detection of antibodies to tissue antigens were carried out as described elsewhere."Sera were used initially at a dilution of 1 in 10, and the fluorescein-conjugated polyvalent and monospecific antihuman sera were obtained from Wellcome Reagents Ltd. I. F. studies for the detection of anti-sperm antibodies were carried out as described by Tung,6 and thyroglobulin titres were determined by haemagglutination. Results The autoantibodies detected in both groups are listed in the accompanying table. Differences between the groups for organ-specific antibodies do not reach statistical significance. However, significantly more infertile AUTOANTIBODIES AND ANTI-SPERM ANTIBODIES IN SERA OF INFERTH I WOMEN AND CONTROLS
335. 19. Gordin, A., Heinonen, O. P., Saarinen, P.,
Lamberg, B.-A. Lancet 1972, i, 551. 20. Toft, A. D., Barnes, E. W., Hunter, W. M., Seth, J., Irvine, W. J ibid. 1973, ii, 644. 21. Toft, A. D., Smith, J., Hunter, W. M., Irvine, W. J. ibid., 1974 i. 704.
Toft, A. D., Irvine, W. J., Hunter, W. M., Seth, J. Br. med. J. 1974 iii, 152. 23. Toft, A. D., Irvine, W. J., Seth, J., Hunter, W. M., Cameron E. H. D. Lancet, 1975, ii, 576. 25. Volpé, R., Farid, N. R., von Westarp, C., Row, V. V. Clin. Endocr 1974, 3, 239. 25. Calder, E. A., Irvine, W. J. Ibid. 1975, 4, 287.
22.
*I’ernicious
anaemia
type.
f)’
