1 Department of Obstetrics and Gynecology and 2 Department of Internal Medicine and Laboratory Medicine, St Marianna University School of Medicine, 2161, Sugao, Miyamae-Ku, Kawasaki, Kanagawa, 216-8511 and 3 Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-Ku, Tokyo, 160-0016, Japan.
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Abstract |
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Key words: anti-nuclear antibodies/autoimmunity/premature ovarian failure/X-chromosome aberrations
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Introduction |
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An estimated 5 to 45% of premature ovarian failure patients have an abnormal X chromosome (La Barbara et al., 1988). Studies of 45,X individuals have demonstrated that two intact X chromosomes are required for the maintenance but not for the differentiation of the oocyte (La Barbara et al., 1988
; Ogata and Matsuo, 1995
). Familial susceptibility to karyotypically normal premature ovarian failure (Coulam et al., 1983
) suggests that submicroscopic deletions of the X chromosome or alterations of the genes critical to the maintenance of ovarian function may be involved in premature ovarian failure. However, the relationship between aberrations of the X chromosome and altered autoimmunity in the aetiology of premature ovarian failure has not been investigated in detail. Most studies of autoimmunity in patients with premature ovarian failure have excluded those with chromosomal aberrations (Irvine et al., 1968
; Sotsiou, 1980; Elder et al., 1981
; Pekonen et al., 1986
; Ahonen et al., 1987
; Miyake et al., 1987
; Ho et al., 1988
; La Barbara et al., 1988
; Mignot et al., 1989
; Nelson et al., 1992
; Belvisi et al., 1993
; Blumenfeld et al., 1993
; Hoek et al., 1997
).
Our objective was to compare the incidence of ANA in patients with premature ovarian failure without known autoimmune disease with that of ANA in hypogonadotrophic patients with secondary amenorrhoea. We also investigated the genetic abnormalities and differences in the clinical course between premature ovarian failure patients with and without ANA.
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Materials and methods |
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ANA were assayed by an indirect immunofluorescence test using a human epidermal carcinoma cell line (HEp-2) as the substrate and fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin G (IgG) was used as the second antibody. ANA were considered positive at a serum dilution of 1/40 or above. Anti-adrenocortical antibody (AAA) was assayed by immunofluorescence using monkey adrenal gland as the substrate. Anti-thyroglobulin antibody and anti-thyroid microsomal antibody (AMA) concentrations were measured by indirect agglutination using artificial gelatin particle carriers sensitized with thyroglobulin or with thyroid microsomal antigen, extracted and purified from human thyroid tissue. Sera from patients positive for ANA by indirect immunofluorescence were further investigated for the presence of specific ANA. Anti-double-stranded DNA antibody (aDNA) was measured by radioimmunoassay with plasmid DNA derived from Escherichia coli as an antigen. Antibodies (Abs) against extractable nuclear antigens (anti-nuclear ribonucleoprotein Ab, anti-Sm Ab, anti-SS-A Ab, anti-SS-B Ab, anti-Jo-1 Ab, anti-SRP Ab, anti-ribosome Ab) were measured by immunodiffusion using lyophilized rabbit thymus acetone extract as the source of nuclear antigens. Anti-histone and anti-centromere Abs were measured by enzyme-linked immunosorbent assay (ELISA).
To test for cryptic X-chromosome deletions, the genomic DNAs of six patients with premature ovarian failure (three with ANA and three without ANA) and of normal controls were digested with EcoRI and hybridized with the following probes: 68A defining DXYS59 located at about 600 kb from the Xp telomere (Petit et al., 1988); pG15 defining DXYS61 located about 300 kb from the Xq telomere (Bickmore and Cooke, 1987
); and cDNA probes for the autosomal thymidine kinase gene as an internal band intensity control (Lau and Kan, 1984
).
Data are expressed as mean ± SD. The significance of autoantibody incidence and age differences between groups was determined by Student's t-test and the 2 test with Yates' correction for continuity. A level of P < 0.05 was considered statistically significant.
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Results |
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Discussion |
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The results of the present study show that the frequency of ANA in patients with premature ovarian failure is higher than that in age-matched hypogonadotrophic patients with secondary amenorrhoea, refuting the potential role of hypo-oestrogenism in the formation of ANA. ANA were detected exclusively in karyotypically normal individuals with premature ovarian failure who became amenorrhoeic before 31 years of age. Follicles spontaneously grew in 63% (15/24) of karyotypically normal premature ovarian failure patients regardless of the presence of ANA. These results are consistent with the notion that a factor other than chromosomal abnormalities that facilitates the early onset of ovarian failure but not hypogonadism itself is responsible for the tendency of patients with premature ovarian failure to produce ANA.
None of our patients who were positive for ANA by immunofluorescence on HEp-2 cells had specific Abs to DNA, histone, centromeres or any of the extractable nuclear antigens tested. Immunoblotting of the sera of ANA-positive patients failed to reveal specific reactivity between the 14- and 94-kDa proteins. Thus, the molecular identity of ANA in these patients is currently unknown.
Several studies have documented ANA in healthy individuals. The frequency increases with age, so that ANA are found in 15 to 25% of individuals over the age of 60 years, but in 1 to 3% in the group aged 20 to 40 years (Selgmann et al., 1965; Svec and Veit, 1967
; Anderson, 1977
). In general, the antibody titre is low (<1:40) and of a homogeneous pattern among normal controls. The cut-off titre for different ANA patterns needs to be evaluated separately. For example, although a cut-off titre of 1:40 for a homogeneous ANA is indicated in a given system, screening for ANA should be performed at a dilution of 1:20 for other staining patterns (Beutner et al., 1985
). In the present study, all subjects who showed ANA positivity did so at a 1:40 dilution or higher.
On the other hand, the present study showed that 41% of karyotypically normal patients with premature ovarian failure are ANA positive. Most of them exhibited a higher titre and a speckled pattern (Table I). Furthermore, ANA were positive in 77% of patients who developed premature ovarian failure at or before the age of 30 years, but in none of the patients who developed premature ovarian failure over the age of 30 years. The close association of positive ANA with the earlier development of premature ovarian failure in karyotypically normal patients suggests that some autoimmune mechanisms are involved in pathogenesis in these patients with premature ovarian failure. Wheatcroft et al. (1997a) reported that regularly menstruating infertile women with elevated serum FSH concentrations aged between 27 and 38 years, who could be considered as patients with premature ovarian failure with later onset than our patients with positive ANA, did not show any evidence of an autoimmune cause. Further studies including the identification of specific autoantibodies are required to confirm this hypothesis.
Anti-ovarian Abs have been associated almost exclusively with a group of patients with Addison's disease or polyendocrine insufficiency (Irvine et al., 1968; Sotsiou, 1980; Elder et al., 1981
; Ahonen et al., 1987
). Morphological studies using indirect immunofluorescence allow the Ab staining pattern to be detected in ovarian theca cells, corpus luteum, granulosa cells and oocytes (Irvine et al., 1968
; Sotsiou et al., 1980
; Elder et al., 1981
; Ahonen et al., 1987
). Lambert et al. (1996) recently demonstrated immunoglobulin-blocking gonadotrophin receptors in some premature ovarian failure patients using a bioassay that employs rat Sertoli cells and mouse Leydig cells. Abs detected are not specific to premature ovarian failure, and the specific factors that induce these anti-ovarian Abs or their influence on the aetiology of premature ovarian failure are obscure. Muechler et al. (1991) proposed that anti-ovarian Abs are detected during the acute phase of the disease. Results of anti-ovarian Ab detection by indirect immunofluorescence vary according the source of ovarian tissue and maturation state.
A simplified ELISA has recently been developed as a quicker method of anti-ovarian Ab detection (Luborsky et al. 1990, Wheatcroft et al. 1994
; Fenichel et al. 1997
). Wheatcroft et al. (1997b) compared the results of indirect immunofluorescence and ELISA and reported no consistent pattern of binding in premature ovarian failure patients, suggesting that neither is a robust test for the presence of these antibodies. On the other hand, Fenichel et al. (1997), using an ELISA against whole tissue homogenate from human ovaries at different ages as antigen, reported positive anti-ovarian Abs in 59% of idiopathic premature ovarian failure patients. The ratio of anti-ovarian Abs among idiopathic premature ovarian failure patients was significantly higher than that of post-menopausal women or women with a normal cycle. In addition, Fenichel et al. (1997) reported that the ratio of the IgA subtype of anti-ovarian Abs in women with positive ANA was comparable to that in premature ovarian failure patients, although whether these women with positive ANA had ovarian failure or not is unclear. These findings, in addition to the present data, suggest that circulating anti-ovarian Abs detected by ELISA are causally related to circulating ANA. This possibility must be further evaluated.
AAA, detected in 5060% of patients with Addison's disease (Sotsiou et al., 1980), was identified in only one of 24 individuals in the present study. This frequency is comparable with reported values from a study which identified one patient with AAA in 45 karyotypically normal patients with premature ovarian failure, four of whom were positive for ANA (Ho et al., 1988
). Thus, these data suggest that karyotypically normal patients with early onset of symptoms may have an increased tendency to develop autoimmunity without developing adrenal autoimmunity.
Ovarian biopsy does not usually determine conclusively the presence of viable follicles in premature ovarian failure patients because these patients usually have few follicles, and only small amounts of ovarian tissue are obtained by biopsy. Moreover, the ovaries of patients with premature ovarian failure are atrophic, adding to the difficulty of obtaining sufficient tissue. Rebar et al. (1982) and Nelson et al. (1994) reported that 50% of karyotypically normal patients with premature ovarian failure show hormonal evidence of functional follicles, which is consistent with the results of the present study. Although a longer observation period does not necessarily reduce the false-negative rate, 8 weeks without oestrogen replacement is probably the longest period that would be practical, considering clinical management of the patients. During the 8-week observation period, the incidence of follicular growth did not significantly differ between patients with and without ANA.
In addition, the present data demonstrate that premature ovarian failure patients with chromosomal abnormalities also have spontaneous follicular activity. However, follicular growth was not observed in patients who developed symptoms at or before the age of 20 years, suggesting that the age at onset of amenorrhoea, not the presence of chromosomal abnormalities or ANA, is the factor that determines the ability of premature ovarian failure patients to maintain follicular reserves sufficient to respond to gonadotrophins. The prevalence of ANA was very low in the subjects with chromosomal abnormalities, suggesting that the increased incidence of ANA in premature ovarian failure patients is not causally related to X-chromosome abnormalities and that an altered autoimmunity is involved in the aetiology of karyotypically normal premature ovarian failure.
Normal gonadal development and function are thought to depend on the integrity of a critical region existing on the long arm of the X chromosome. In terminal X-chromosome deletions, the degree of gonadal dysfunction seems to be roughly correlated with the deletion size (Wyss et al., 1982; Ogata and Matsuo, 1995
). A chromosomal imbalance may reduce the buffering effects against genetic and environmental forces, leading to a disruption of homeostasis and causing non-specific disorders such as tissue dysplasia (Ogata and Matsuo, 1995
). In this regard, the age at onset of amenorrhoea of the individuals with X-chromosome aberrations in the present study roughly correlated with the size of the X-chromosome deletion. It is likely that the oocytes of these women with X-chromosome aberrations underwent accelerated atresia because of meiotic pairing failures and that secondary amenorrhoea occurred when most oocytes had been lost. In fact, three out of the eight premature ovarian failure patients with X-chromosome aberrations whose karyotypes were 46,XX/47,XXX, 46,X,del(X)(q22,3) and 45X/46,XX/48,XXXX showed evidence of follicular activity based on serial measurements of serum oestradiol concentrations at 26, 28 and 38 years of age respectively, which suggests that a considerable proportion of premature ovarian failure patients with X-chromosome abnormalities with less significant pairing failure retains follicular reserve after the onset of amenorrhoea. Moreover, the patient with 46X,del(q22,3) ovulated in response to human menopausal gonadotrophin administration at the age of 27 years (Ishizuka et al. 1997
). None of our patients with chromosomal abnormalities was ANA positive, suggesting that the ANA found in the premature ovarian failure patients were not the result of X-chromosomal pairing failure or resultant tissue dysplasia. Thus, apparently karyotypically normal patients with premature ovarian failure and without ANA who manifest ovarian failure relatively late in their childbearing years may have submicroscopic X-chromosome deletions, although Southern blot analysis of a limited number of patients failed to reveal terminal X-chromosome microdeletions. Thus, we were unable to distinguish between premature ovarian failure patients with and without ANA based on the presence of X-terminal microdeletions.
In summary, the results of the present study indicate an increased incidence of ANA positivity in karyotypically normal patients with premature ovarian failure who manifest ovarian failure relatively early in life. All of the patients with ANA were free of symptoms of autoimmune diseases. The participants in this study did not appear to have any of the specific classes of ANA tested, suggesting that the ANA were reactive with nuclear antigen(s) that remain to be identified. The degree of follicular activity was similar in the ANA-positive and ANA-negative patients with premature ovarian failure. These data suggest that the autoimmunity seen in premature ovarian failure patients in the absence of adrenal autoimmunity is not a consequence of X-chromosomal abnormality and that premature ovarian failure patients who are karyotypically normal with early onset of symptoms are of autoimmune origin. The characteristics of the circulating ANA are currently being investigated in our laboratory.
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Notes |
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References |
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Submitted on April 14, 1998; accepted on October 2, 1998.