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Article| Volume 35, ISSUE 4, P475-479, October 2017

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Identification of patients with primary ovarian insufficiency caused by autoimmunity

  • Author Footnotes
    1 These authors contributed equally to this work.
    Jing Gao
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China

    Center for Reproductive Medicine, Qingdao Municipal Hospital, Qingdao, 266071, Shandong, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Xue Jiao
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China

    Suzhou Institute of Shandong University, Suzhou, 215123, Jiangsu, China
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  • Yujie Dang
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China
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  • Jing Li
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China
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  • Guiyu Li
    Affiliations
    Department of Obstetrics, Qingdao University Affiliated Hospital, Qingdao, 266003, Shandong, China
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  • Ting Han
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China
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  • Yixun Liu
    Affiliations
    State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
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  • Yingying Qin
    Correspondence
    Corresponding author.
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China
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  • Zi-Jiang Chen
    Affiliations
    Center for Reproductive Medicine, Shandong University, Jinan, 250001, Shandong, China

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Jinan, 250001, Shandong, China

    The Key laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, 250001, Shandong, China

    Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200135, China

    Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, 200135, China
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  • Author Footnotes
    1 These authors contributed equally to this work.

      Abstract

      Autoimmune pathogenesis is responsible for a subset of primary ovarian insufficiency (POI) cases. The significance of autoantibodies for POI, however, remains unclear. A total of 250 women with idiopathic POI and 256 age-matched healthy women were enrolled. The presence in serum of adrenal cortex autoantibody (AAA), detected by indirect immunofluorescence and non-organ-specific antibodies, including antinuclear antibody, anti-cardiolipin antibody, and anti-double stranded DNA antibody, detected by enzyme-linked immunosorbent assay, was compared. Ovarian biopsy was carried out for histology assessment. Adrenal function was followed-up in 15 women with POI who were positive for AAA. Higher frequency of positive AAA was observed in women with POI (19.2%) compared with controls (5.9%, P < 0.01). No difference in anti-cardiolipin antibody, antinuclear antibody and anti-double stranded DNA antibody was found between the two groups. Ovarian biopsies in 13 women with POI (six AAA positive and seven negative) showed atrophic ovaries devoid of follicles. One out of fifteen women positive for AAA had symptoms of adrenal insufficiency 3 years after POI diagnosis. Significantly higher positive frequency of AAA in POI patients suggests the role of autoimmune disturbance in pathogenesis. Therefore, AAA may serve as a biomarker for ovarian autoimmunity.

      Keywords

      Introduction

      Primary ovarian insufficiency (POI), previously termed premature ovarian failure, is characterized by cessation of menstruation before the age of 40 years. It occurs in about 1% of women (
      • Coulam C.B.
      • Adamson S.C.
      • Annegers J.F.
      Incidence of premature ovarian failure.
      ). It is highly heterogeneous, with a wide spectrum of causes, including genetic, autoimmune, infectious and iatrogenic factors. Yet most cases of sporadic POI remain idiopathic (
      • Qin Y.
      • Jiao X.
      • Simpson J.L.
      • Chen Z.J.
      Genetics of primary ovarian insufficiency, new developments and opportunities.
      ). Given its activities of folliculogenesis, ovulation, luteum formation and dysgenesis, the ovary might be a target of autoimmune attacks. Once the autoimmune destruction initiates, ovarian follicles will be attacked and depleted prematurely, and consequently POI occurs. An autoimmune origin has long been recognized to contribute to a subset of women with POI (5–30%) (
      • Dragojevic-Dikic S.
      • Marisavljevic D.
      • Mitrovic A.
      • Dikic S.
      • Jovanovic T.
      • Jankovic-Raznatovic S.
      An immunological insight into premature ovarian failure (POF).
      ,
      • La Marca A.
      • Brozzetti A.
      • Sighinolfi G.
      • Marzotti S.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency, autoimmune causes.
      ,
      • Silva C.A.
      • Yamakami L.Y.
      • Aikawa N.E.
      • Araujo D.B.
      • Carvalho J.F.
      • Bonfa E.
      Autoimmune primary ovarian insufficiency.
      ,
      • Yldrm G.
      • Tokmak A.
      • Kokanal M.K.
      • Sarkaya E.
      • Zungun C.
      • Inal H.A.
      • Ylmaz F.M.
      • Ylmaz N.
      Association between some inflammatory markers and primary ovarian insufficiency.
      ). Reliable diagnosis of autoimmune POI (aPOI), however, remains elusive.
      Ascertainment of ovarian autoimmunity presents a particular challenge in clinical practice, as its diagnosis requires ovarian biopsy, which is invasive and not commonly recommended (
      • Silva C.A.
      • Yamakami L.Y.
      • Aikawa N.E.
      • Araujo D.B.
      • Carvalho J.F.
      • Bonfa E.
      Autoimmune primary ovarian insufficiency.
      ,
      • Warren B.D.
      • Kinsey W.K.
      • McGinnis L.K.
      • Christenson L.K.
      • Jasti S.
      • Stevens A.M.
      • Petroff B.K.
      • Petroff M.G.
      Ovarian autoimmune disease, clinical concepts and animal models.
      ). Previous studies have shown that aPOI may be isolated or associated with other autoimmune diseases, most frequently with thyroiditis, Addison's disease, autoimmune polyendocrine syndrome, systemic lupus erythematosus and rheumatoid arthritis (
      • Bakalov V.K.
      • Gutin L.
      • Cheng C.M.
      • Zhou J.
      • Sheth P.
      • Shah K.
      • Arepalli S.
      • Vanderhoof V.
      • Nelson L.M.
      • Bondy C.A.
      Autoimmune disorders in women with turner syndrome and women with karyotypically normal primary ovarian insufficiency.
      ,
      • Dragojevic-Dikic S.
      • Marisavljevic D.
      • Mitrovic A.
      • Dikic S.
      • Jovanovic T.
      • Jankovic-Raznatovic S.
      An immunological insight into premature ovarian failure (POF).
      ,
      • Silva C.A.
      • Yamakami L.Y.
      • Aikawa N.E.
      • Araujo D.B.
      • Carvalho J.F.
      • Bonfa E.
      Autoimmune primary ovarian insufficiency.
      ). Women with concurrent autoimmune disorders or at preclinical status of autoimmune dysregulation, are also at a higher risk to develop POI. Presence of corresponding serum autoantibodies represent markers to predict the associated risk of POI (
      • Gleicher N.
      • Weghofer A.
      • Okay K.
      • Barad D.H.
      Is the immunological noise of abnormal autoimmunity an independent risk factor for premature ovarian aging?.
      ). The search for anti-ovarian antibodies has been undertaken in numerous studies; however, results are conflicting with controversial specificity and sensitivity (
      • Forges T.
      • Monnier-Barbarino P.
      • Faure G.C.
      • Bene M.C.
      Autoimmunity and antigenic targets in ovarian pathology.
      ,
      • Otsuka N.
      • Tong Z.B.
      • Vanevski K.
      • Tu W.
      • Cheng M.H.
      • Nelson L.M.
      Autoimmune oophoritis with multiple molecular targets mitigated by transgenic expression of mater.
      ). The most promising indicators of aPOI are steroid-cell autoantibodies (StCA) and adrenal cortex autoantibodies (AAA) (
      • Falorni A.
      • Laureti S.
      Steroid cell autoantibodies are preferentially expressed in women with premature ovarian faiure who have adrenal autoimmunity.
      ). The presence of StCA can be detected almost exclusively in patients with POI who are positive for AAA, which is also the immune marker for Addison's disease (
      • Brozzetti A.
      • Alimohammadi M.
      • Morelli S.
      • Minarelli V.
      • Hallgren A.
      • Giordano R.
      • De Bellis A.
      • Perniola R.
      • Kampe O.
      • Falorni A.
      Autoantibody response against NALP5/MATER in primary ovarian insufficiency and in autoimmune Addison's disease.
      ,
      • La Marca A.
      • Brozzetti A.
      • Sighinolfi G.
      • Marzotti S.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency, autoimmune causes.
      ). Furthermore, all women identified with histological evidence of autoimmune oophoritis by ovarian biopsy had positive AAA (
      • Bakalov V.K.
      • Anasti J.N.
      • Calis K.A.
      • Vanderhoof V.H.
      • Premkumar A.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Merino M.J.
      • Nelson L.M.
      Autoimmune oophoritis as a mechanism of follicular dysfunction in women with 46,XX spontaneous premature ovarian failure.
      ). As such, AAA may function as a marker at high diagnostic sensitivity for aPOI. It is found in many patients with POI and Addison's disease, but rarely in isolated POI (
      • Bakalov V.K.
      • Vanderhoof V.H.
      • Bondy C.A.
      • Nelson L.M.
      Adrenal antibodies detect asymptomatic autoimmune adrenal insufficiency in young women with sponstaneous premature ovarian failure.
      ,
      • Falorni A.
      • Laureti S.
      Steroid cell autoantibodies are preferentially expressed in women with premature ovarian faiure who have adrenal autoimmunity.
      ,
      • Reato G.
      • Morlin L.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Masiero S.
      • Albergoni M.P.
      • Cervato S.
      • Zanchetta R.
      • Betterle C.
      Premature ovarian failure in patients with autoimmune Addison's disease, clinical, genetic, and immunological evaluation.
      ). It is also suggested that POI with positive AAA might be secondary to adrenal insufficiency (
      • Gleicher N.
      • Kushnir V.A.
      • Weghofer A.
      • Barad D.H.
      The importance of adrenal hypoandrogenism in infertile women with low functional ovarian reserve: a case study of associated adrenal insufficiency.
      ). Hence, the significance and role of AAA in isolated sporadic POI as an autoimmune origin remains to be determined.
      In this study, the prevalence of AAA and other non-specific autoantibodies, such as antinuclear antibody (ANA), anti-cardiolipin antibody (ACA), and anti-double stranded DNA antibody (anti-dsDNA), and their association with reproductive characteristics are examined in isolated idiopathic patients with POI.

      Materials and methods

      Patients and controls

      This study recruited 250 patients with POI and 256 age-matched healthy women from the Center for Reproductive Medicine, Shandong University, between 2011 and 2014. Written consent was obtained from all partiicpants, and the study was approved by the Ethical Committee of Reproductive Medicine of Shandong University (reference number 1657).
      The criteria of POI recruitment included secondary amenorrhea for at least 4 months before the age of 40 years, and two serum FSH over 40 IU/l (drawn more than 1 month apart). The healthy controls manifested with regular menstrual cycle and normal levels of serum FSH. Women with abnormal karyotype, history of ovarian surgery, radiotherapy or chemotherapy, history of recurrent spontaneous abortion, known autoimmune disease (autoimmune polyendocrine syndrome, thyroiditis, AD, systemic lupus erythematosus, Crohn's disease, rheumatoid arthritis, type I diabetes mellitus), concomitant systemic diseases and steroid hormones treatment in the previous 3 months, were excluded.

      Clinical characteristics

      Demographic and reproductive characteristics were recorded in all participants. The baseline endocrine hormone (FSH, LH, oestradiol and progesterone) were measured by the electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). The intra- and inter-assay coefficients of variation were less than 10%. Ovarian and uterine parameters were assessed by transvaginal ultrasound. Serum were collected and stored at −80°C for analysis.

      Indirect immunofluorescence assay

      Detection of AAA was by indirect immunofluorescence assay on monkey adrenal gland sections (Euroimmun, Lübeck, Germany). Slides were incubated with 25 µl diluted sera (1:10) for 30 min at room temperature. After washing with phosphate-buffered saline-Tween for at least 5 min, each slide was stained with 20 µl fluorescein isothiocyanate-conjugated anti-human globulin for 30 min and then washed with phosphate-buffered saline-Tween. Subsequently, the coded slides were embedded with cover glasses and visualized with a fluorescence microscope (Leica, Wetzlar, Germany) by two independent assessors. The fluorescent intensity was compared with positive control (human serum positive for autoantibodies to adrenal cortex) and negative control (human serum negative for antoantibodies to adrenal cortex) included in the kits (cut-off titre 1:10). Positive control showed a granular to smooth cytoplastic staining pattern, and the negative control showed no specific fluorescence pattern.

      Enzyme-linked immunosorbent assay

      Non-organ-specific antibodies (ANA, ACA, and anti-dsDNA) were determined by commercially available enzyme-linked immunosorbent assays (Orgentec, Mainz, Germany). Microplates coated with the specific antigen were incubated with 100 µl diluted sera (1:100) for 30 min at room temperature, and then washed three times with wash solution. Enzyme conjugates (100 µl) were added and incubated for 15 min. After a second washing step, 3,3′,5,5′-Tetramethylbenzidine substrates solutions (100 µl) were applied for 15 min. The reaction was stopped by the stop solution for 5 min. The optical intensity was read at 450 nm using an automated microplate reader (Tecan, Switzerland) and the concentration was calculated from the calibration curve. The samples were run in duplicates. Positivity cut-offs were set in compliance with the manufacturer's instructions, being 20 IU/ml or more for anti-dsDNA, ≥0 IU/ml or more for ACA, and index more than 1.2 for ANA. The intra-assay and inter-assay coefficient of variation were less than 10%.

      Oophoritis determination

      Ovarian biopsy was conducted to confirm the presence of histologically autoimmune oophoritis. Small pieces of ovarian cortex distant from the hilum were obtained by bilateral biopsy under laparoscope, and fixed in 4% paraformaldehyde. After embedded and serially sectioned (5 mm), 3–4 (one out of every five) sections were stained with haematoxylin and eosin and examined. Autoimmune oophoritis is characterized by mononuclear inflammatory cell infiltration in the theca cells of growing follicles and corpus luteum, with primordial follicles spared.

      Statistical analysis

      The Statistical Package for the Social Sciences (SPSS) version 22.0 (IBM Corp., USA) were used for analysis. Continuous and normally distributed variables were expressed as mean ± SD, and differences were determined by the Student's t-test. Continuous variables in non-normal distribution were presented as median (interquartile ranges), and differences were compared using Mann–Whitney U test. Categorical variables were presented as percentage, and differences in the prevalence of positive antibodies were evaluated by chi-square test. P < 0.05 was considered to be statistically significant.

      Results

      In our cohort, no difference was found at the age of presentation and age at menarche between patients with POI and control women. Among women with idiopathic POI, amenorrhea occurred at 24.96 ± 5.45 years, 2 years after the menses irregularity (22.03 ± 6.28 years). In 48 out of 250 women with POI (19.2%) AAA was found positive, which is significantly higher than controls (15/256, 5.9%) (P < 0.01) (Supplementary Figure S1). For non-specific antibodies-ACA, ANA and anti-dsDNA, no differences were found between the POI and control groups (Table 1).
      Table 1Demographic and immunological data for women with primary ovarian insufficiency and controls.
      POI (n = 250)Controls (n = 256)P-value
      Age (years)29.37 ± 4.7230.32 ± 4.36NS
      Age at menarche (yrs)14.51 ± 1.7214.23 ± 1.50NS
      FSH (IU/L)76.19 ± 25.366.83 ± 2.12<0.0001
      LH (IU/L)36.02 ± 15.474.71 ± 1.51<0.01
      E2 (pg/mL)17.78 ± 19.8233.58 ± 8.36<0.01
      AAA, % (n)19.2 (48/250)5.9 (15/256)<0.01
      ACA, % (n)0.8 (2/250)0.4 (1/256)NS
      ANA, % (n)3.6 (9/250)1.2 (3/256)NS
      ds-DNA, % (n)10.0 (25/250)5.9 (15/256)NS
      NS, not statistically significant.
      Among women with POI, no significant differences were found between patients with positive and negative AAA, in terms of age, FSH, age at irregularity, age at amenorrhoea, duration of amenorrhoea, time from irregularity to amenorrhoea and average ovarian volume (Table 2). Thirteen women with POI (six AAA positive and seven negative) underwent ovarian biopsy, and all of them presented with small and hard-textured ovaries by laparoscope. The histology showed atrophy or fibrosis, with connective tissues completely devoid of follicles (Figure 1).
      Table 2Demographic and clinical characteristics in women who are AAA positive and negative.
      CharacterizationAAA Positive (n = 48)AAA negative (n = 202)
      Age (years)29.18 ± 4.5528.86 ± 4.57
      FSH (IU/L)75.31 ± 24.0677.30 ± 26.49
      Age at menarche (years)14.20 ± 1.6514.69 ± 1.90
      Age at Irregularity (years)22.03 ± 6.2221.91 ± 6.28
      Age at amenorrhea (years)25.00 ± 5.6424.91 ± 5.21
      Duration of amenorrhea (years)4.38 ± 3.303.45 ± 3.08
      From irregularity to amenorrhea (years)2.49 ± 3.443.44 ± 5.04
      Ovarian volume (ml)0.44 (0.22–0.81)0.42 (0.00–0.72)
      There were no statistically significant differences between the two groups.
      Figure 1
      Figure 1Histology of the ovarian biopsies from two women with primary ovarian insufficiency with positive AAA. With haematoxylin and eosin staining, the ovary showed atrophy or fibrosis, with connective tissues completely devoid of follicles, bar = 50 µm.
      Fifteen women with POI who positive for AAA positive were followed-up to assess the association of AAA presence with later onset of Addison's disease. Irregular menstrual cycles and amenorrhea continued in all patients. Only one patient showed symptoms of Addison's disease (chronic fatigue, muscle weakness, weight loss, nausea, dizziness and skin hyperpigmentation) 3 years after POI diagnosis.

      Discussion

      Ovarian autoimmunity has been implicated in the cause of POI. Identification of autoimmune disturbance may permit early interventions before ovarian function fails. Currently, no consensus of aPOI diagnosis exists. In this study, we found that the positive frequency of AAA was significantly higher in patients with POI, suggesting its role as a potential marker for aPOI.
      The ovary and adrenal cortex share common autoantigens, especially those associated with steroid synthesis, which may result in accompaning attacks once autoimmune disturbance occurs in either organ. aPOI has been associated with clinical or preclinical Addison's disease, and typically positive for autoantibodies directed against steroidogenic cells or enzymes (StCA) (
      • Gleicher N.
      • Kushnir V.A.
      • Weghofer A.
      • Barad D.H.
      The importance of adrenal hypoandrogenism in infertile women with low functional ovarian reserve: a case study of associated adrenal insufficiency.
      ,
      • Reato G.
      • Morlin L.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Masiero S.
      • Albergoni M.P.
      • Cervato S.
      • Zanchetta R.
      • Betterle C.
      Premature ovarian failure in patients with autoimmune Addison's disease, clinical, genetic, and immunological evaluation.
      ). The presence of StCA can be detected almost exclusively in women with POI positive for AAA (
      • Falorni A.
      • Laureti S.
      Steroid cell autoantibodies are preferentially expressed in women with premature ovarian faiure who have adrenal autoimmunity.
      ,
      • La Marca A.
      • Marzotti S.
      • Brozzetti A.
      • Stabile G.
      • Artenisio A.C.
      • Bini V.
      • Giordano R.
      • De Bellis A.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency due to steroidogenic cell autoimmunity is associated with a preserved pool of functioning follicles.
      ,
      • La Marca A.
      • Brozzetti A.
      • Sighinolfi G.
      • Marzotti S.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency, autoimmune causes.
      ,
      • Reato G.
      • Morlin L.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Masiero S.
      • Albergoni M.P.
      • Cervato S.
      • Zanchetta R.
      • Betterle C.
      Premature ovarian failure in patients with autoimmune Addison's disease, clinical, genetic, and immunological evaluation.
      ). Therefore, AAA is recognized as a best marker of POI associated with steroid cell autoimmunity. Nonetheless,
      • Dal Pra C.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Pedini B.
      • Moscon A.
      • Zanchetta R.
      • Betterle C.
      Autoantibodies to steroidogenic enzymes in patients with premature ovarian failure with and without Addison's disease.
      detected 93% AAA positivity in patients with POI and Addison's disease, but only 8% in patients with POI and non-adrenal autoimmune disease and 10% in isolated POI. A 3.8% (10/266) positivity for AAA was also reported in 46,XX women with spontaneous POI (
      • Bakalov V.K.
      • Anasti J.N.
      • Calis K.A.
      • Vanderhoof V.H.
      • Premkumar A.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Merino M.J.
      • Nelson L.M.
      Autoimmune oophoritis as a mechanism of follicular dysfunction in women with 46,XX spontaneous premature ovarian failure.
      ). Significant higher frequency of AAA (19.2%) was observed in the POI patients than controls (5.9%) in the present study, suggesting that autoimmune disturbance is involved in POI in Chinese population.
      Adrenal antibody is a sensitive marker that implies current presence of or high risk for developing Addison's disease later in life. Women with spontaneous POI are at increased risks for Addison's disease, and vice versa (
      • Reato G.
      • Morlin L.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Masiero S.
      • Albergoni M.P.
      • Cervato S.
      • Zanchetta R.
      • Betterle C.
      Premature ovarian failure in patients with autoimmune Addison's disease, clinical, genetic, and immunological evaluation.
      ). It has been suggested that POI with hypoandrogenism actually represent secondary ovarian insufficiency, primarily driven by adrenal autoimmunity (
      • Gleicher N.
      • Kushnir V.A.
      • Weghofer A.
      • Barad D.H.
      The importance of adrenal hypoandrogenism in infertile women with low functional ovarian reserve: a case study of associated adrenal insufficiency.
      ). In this study, POI patients do not have history or symptoms of adrenal insufficiency, and detailed adrenal function evaluation was not available, and therfore we cannot distinguish whether or not POI is secondary to adrenal insufficiency. Six patients with positive AAA have affected mothers with early menopause (<45 years). We did not test for antibodies in the mothers, however, because they did not show any symptoms of adrenal disorders. Fifteen women with POI positive or AAA were followed-up to assess the association of AAA presence with later onset of Addison's disease. Most did not develop clinical Addison's disease 3 years after diagnosis of POI. Detection of detailed adrenal function, however, was not available, and the potential to develop subclinical adrenal insufficiency still could not be ruled out. In the future, women with POI who are AAA positive are recommended to evaluate adrenal cortex function, including the basal determination of adrenocorticotropic hormone, cortisol, aldosterone, plasma renin activity and cortisol after intravenous stimulation with synthetic adrenocorticotropic hormone. Symptoms, e.g., fatigue, skin hyperpigmentation, anorexia, nausea and faintness, should also be noticed.
      Isolated autoimmune oophoritis is rare, and can explain 5% of POI cases (
      • La Marca A.
      • Brozzetti A.
      • Sighinolfi G.
      • Marzotti S.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency, autoimmune causes.
      ). It is characterized by enlarged ovaries with a selective mononuclear cell infiltration into the theca layer of large, antral follicles, with earlier stage follicles consistently free of lymphocytic infiltration (
      • Welt C.K.
      • Falorni A.
      • Taylor A.E.
      • Martin K.A.
      • Hall J.E.
      Selective theca cell dysfunction in autoimmune oophoritis results in multifollicular development, decreased estradiol, and elevated inhibin B levels.
      ). Ovarian biopsy has been indicated as gold standard, but not routinely suggested, in patients with circulating autoantibodies to confirm autoimmune oophoritis. Several studies have demonstrated that an autoimmune oophoritis can only be found in women positive for StCA/AAA antibodies (
      • La Marca A.
      • Marzotti S.
      • Brozzetti A.
      • Stabile G.
      • Artenisio A.C.
      • Bini V.
      • Giordano R.
      • De Bellis A.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency due to steroidogenic cell autoimmunity is associated with a preserved pool of functioning follicles.
      ,
      • La Marca A.
      • Brozzetti A.
      • Sighinolfi G.
      • Marzotti S.
      • Volpe A.
      • Falorni A.
      Primary ovarian insufficiency, autoimmune causes.
      ).
      • Bakalov V.K.
      • Anasti J.N.
      • Calis K.A.
      • Vanderhoof V.H.
      • Premkumar A.
      • Chen S.
      • Furmaniak J.
      • Smith B.R.
      • Merino M.J.
      • Nelson L.M.
      Autoimmune oophoritis as a mechanism of follicular dysfunction in women with 46,XX spontaneous premature ovarian failure.
      found all four AAA positive POI patients presented with autoimmune oophoritis in histology. In our studies, 13 women with POI underwent bilateral ovarian biopsy. Inconsistent with previous reports, the ovarian histology, either AAA positive or negative, is characterized by atrophy without follicles or lymphocytic infiltration. Furthermore, no differences of reproductive characteristics or total ovarian volume were observed between patients with positive or negative AAA. The atrophic ovaries indicated that patients were already at the end stage of ovarian failure, in accordant with the postmenopausal level of FSH (76.19 ± 25.36 IU/l) and long duration of amenorrhea (4.40 ± 2.99 years). For most women with POI, ovarian inflammation might be transient and not present by the time when the patients' ovaries were biopsied. Consistent with the biphasic nature of the inflammation, when the acute phase of the disease subsides, inflammation rapidly disappears and an atrophic ovary remains (
      • Silva C.A.
      • Yamakami L.Y.
      • Aikawa N.E.
      • Araujo D.B.
      • Carvalho J.F.
      • Bonfa E.
      Autoimmune primary ovarian insufficiency.
      ,
      • Warren B.D.
      • Kinsey W.K.
      • McGinnis L.K.
      • Christenson L.K.
      • Jasti S.
      • Stevens A.M.
      • Petroff B.K.
      • Petroff M.G.
      Ovarian autoimmune disease, clinical concepts and animal models.
      ).
      As a continuum of ovarian dysfunction, POI may proceed through occult, biochemical, and overt ovarian failure stage (
      • Nelson L.M.
      Clinical practice. Primary ovarian insufficiency.
      ,
      • Welt C.K.
      Primary ovarian insufficiency, a more accurate term for premature ovarian failure.
      ). The autoimmune disturbance tends to precede the occurrence of clinical symptoms, and may not be detectable retrospectively after a period of time. Further study in earlier, yet progressive or late-stage, women with POI are warranted to elucidate the plausible autoimmune causes and make an earlier identification of women at high risk.
      In women affected by autoimmune disease, POI more frequently occurs, and vice versa. The concomitant association suggests an overall dysregulated autoimmune response might exist in patients with POI (
      • Bakalov V.K.
      • Gutin L.
      • Cheng C.M.
      • Zhou J.
      • Sheth P.
      • Shah K.
      • Arepalli S.
      • Vanderhoof V.
      • Nelson L.M.
      • Bondy C.A.
      Autoimmune disorders in women with turner syndrome and women with karyotypically normal primary ovarian insufficiency.
      ,
      • Silva C.A.
      • Yamakami L.Y.
      • Aikawa N.E.
      • Araujo D.B.
      • Carvalho J.F.
      • Bonfa E.
      Autoimmune primary ovarian insufficiency.
      ). Non-organ specific antibodies may also recognize and attack common antigens in ovary by mistake. Increased prevalence of ANA (42%) and anti-dsDNA (25%) were found in women with POI (24 cases) (
      • Ishizuka B.
      • Kudo Y.
      • Amemiya A.
      • Yamada H.
      • Matsuda T.
      • Ogata T.
      Anti-nuclear antibodies in patients with premature ovarian failure.
      ).
      • Chernyshov V.P.
      • Radysh T.V.
      • Gura I.V.
      • Tatarchuk T.P.
      • Khominskaya Z.B.
      Immune disorders in women with premature ovarian failure in initial period.
      reported that 34% of women with POI were positive for ACA, which is significantly higher than control women (7%). Inconsistent with previous reports, we did not found statistical differences in the positive frequency of ANA, anti-dsDNA and ACA. Limited sample size or different detection assay used in previous studies might explain the discrepancy. The possibility of random associations cannot be excluded either.
      In conclusion, significantly higher positive frequency of AAA in patients with POI confirmed the role of autoimmune disturbance in POI pathogenesis. This suggests that AAA may serve as a marker for ovarian autoimmunity. Further longitudinal studies are warranted to uncover the changes during the progress of POI and the underlying mechanisms in autoimmune POI.

      Acknowledgement

      The authors are grateful to Professor Lei Li for English correction. This research was supported in part by the National Natural Science Foundation of China (31471352, 81270662, 81471509, 81522018, 81601245) and Natural Science Foundation of Jiangsu Province (BK20160372) and Shandong Province (ZR2016HQ47).

      Appendix. Supplementary material

      The following is the supplementary data to this article:
      • Fig. S1

        Representative pictures of indirect immunofluorescence assay of anti-adrenal cortex antibody. Adrenal cortex antibody-immunoglobulin-G was detected by fluorescein isothiocyanate conjugated with polyclonal antibodies against human immunoglobulin, bar = 100 µm. (A) Negative control; (B) positive control; (C) negative sample; (D) positive sample. Arrow, granular to smooth cytoplastic staining pattern of adrenal cortex antibody.

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      Biography

      Dr Jing Gao will receive her MD from Shandong University, Shandong, China in 2017. She has received her clinical training at the Center for Reproductive Medicine of Shandong University, which is one of the largest IVF centres in China and is a pioneer in IVF, oocyte cryopreservation, reproductive endocrinology and genetic diseases. She is especially interested in reproductive endocrinology and premature ovarian insufficiency.
      Key message
      Autoimmune pathogenesis is responsible for a subset of primary ovarian insufficiency cases. Significantly higher positive frequency of adrenal cortex autoantibody in patients with primary ovarian insufficiency was found, which suggests adrenal cortex autoantibody could serve as a promising marker for ovarian autoimmunity.