Premature ovarian insufficiency (POI) is characterized
by hypergonadotropic amenorrhea due to
cessation of ovarian function before the age of 40
years. The diagnosis is based on amenorrhea before
the age of 40 associated with follicle stimulating
hormone (FSH) levels >40 IU/l, detected on
two occasions at least one month apart (
Although there are multiple etiologies of POI
(genetic, chromosomal, infectious, and iatrogenic
causes), the etiology cannot be identified in most
patients and this is referred to as idiopathic POI;
up to 30% of idiopathic cases may have an autoimmune
The presence of pathogenic factors might accelerate
the process of apoptosis and atresia of ovarian
follicles during the fetal and post-natal period
Our study was a prospective randomized controlled trial. The study group consisted of 20 women with POI (mean age 31.8 years, range 20-39 years) and no use of medications or oral contraceptives for at least 4 months prior to the study. The diagnosis was based on the presence of amenorrhea before the age of 40, associated with two serum FSH levels above 40 IU/l at least one month apart. All women with POI underwent karyotyping and genetic testing of the fragile X mental retardation 1 (FMR1). Women with infectious or iatrogenic causes were excluded from the study. The control group consisted of 17 healthy women volunteers. Inclusion criteria were a regular menstrual cycle, age between 18 and 39 years (mean 30.8 years), and no use of medications or oral contraceptives for at least 4 months prior to the study. They also had to be exempt from autoimmune disease or infertility problems. All women provided a complete personal and family history, with a stress on possible immune-mediated, particularly autoimmune processes (allergy, asthma, diabetes, thyroiditis, rheumatoid arthritis, andatopiceczema), and all underwent physical and vaginal ultrasound examinations.
Serum levels of luteinizing hormone (LH), FSH, estradiol (E2), prolactin (PRL), inhibin B, thyroid- stimulating hormone (TSH), anti-Müllerian hormone (AMH), antithyroglobulin (aTG) and antithyroid peroxidase antibodies (aTPO) were measured, and immunological investigations at cellular and humoral levels were performed. The adrenocorticotropic hormone (ACTH) stimulation test was performed in the study group, only. The standard Synacthen stimulation test is clinically widely used as a sensitive screening method for symptomatic adrenal insufficiency. Each ampoule of Synacthen contains 250 μg of the active ingredient, tetracosactrin (Novartis Pharmaceuticals, North Ryde NSW, Australia). Thirty minutes after 250 μg Synacthen I.M. (Alliance Pharmaceutical Wiltshire, UK), blood cortisol was measured by electro-chemiluminescence immunoassay. A normal cortisol response to Synacthen was defined as a post-stimulation peak cortisol value of >500 nmol/l at 30 minutes.
Serum AMH in peripheral blood was determined by a Personal Lab analyser using the enzyme linked immunosorbent assay (ELISA) method with Beckman Coulter reagent. The normal range of AMH levels is 0.7-3.5 mg/l. Values below 0.3 mg/l indicate a reduced ovarian reserve. Hormones were determined on a LIAISON analyser by quantitative direct competitive chemiluminescence immunoassays (CLIA). Each test is a modified two-step process, in which the specific antibodies of a certain hormone bind to magnetic cells. Normal ranges for the follicular phase of the cycle are as follows: FSH: 3.5-9.2 IU/l; LH: 1.1-11.6 IU/l; LH around ovulation: 17-77 IU/l; PRL: 6.2-23.5 μg/l; E2: on day 3 up to 310 pmol/L (0.31 nmol/L); E2 postmenopause: 0-110 pmol/L (0-0.11 nmol/l); and TSH: 0.3-3.6 mE/l. Serum Inhibin-B cut-off level on day 3 was 45 pg/mL. Serum aTG and aTPO concentrations measured by immunoassay using direct chemiluminometric technology on an ADVIA CENTAUR analyser (Siemens Medical Solutions Diagnostics, Tarrytown, USA). The normal range for serum aTG and aTPO is <60 KE/l. Detection of AOA was by indirect immunofluorescence (IIF) on cryosections of normal human ovarian tissue.
Non-human primate ovaries for the detection
of AOA are not commercially available, while
normal human ovarian tissue is available at
the Institute of Pathology, Ljubljana, Slovenia,
when ovaries are removed due to various pathological
processes, particularly tumors, and are
sent for pathohistological examination. Ovarian
tissue was incubated with patient serum diluted
1:10. The second incubation was with fluorescein
isothiocyanate-labelled anti-human IgG antibody
(Dakopatts, Copenhagen, Denmark). Positive reactions
were semi-quantitatively evaluated (on a
scale of 1– 4+). Negative control omitting the patient’s
serum was regularly included (
Normal data distribution was tested with the Kolmogorov-Smirnov test. Where variables were normally distributed, we used the Pearson’s chisquare test. If variables were not normally distributed, we used a nonparametric Mann-Whitney test. Statistical analysis was done using Statistical Package for the Social Sciences, version 18 (SPSS Inc., Chicago, IL, USA). The results were considered statistically significant at p<0.05.
The study protocol was approved by the National Ethics Committee, and all patients gave written informed consent.
The subjects were comparable with controls by
age. One patient presenting a mosaic 45X0/46XX
was excluded from the final analysis. Other patients
had a 46XX karyotype. Anamnestic data of
patients showed that four had had mumps during
their childhood; they were thus excluded from the
final analysis. Table 1 shows the clinical and endocrine
characteristics of patients and of healthy
women. All endocrine parameters in controls were
within the normal range (
Hormone and ovarian peptide levels in patients and in healthy controls
|Mean value+/- SD||Study group (n=20)||Control group (n=17)|
|88.04 +/- 47.93||6.25 +/- 2,77|
|37.85 +/- 18.71||11.64 +/- 31.1|
|180 +/- 200 (0.18 +/- 0.2nmol/L)||210+/- 130 (0.21 +/- 0.13nmol/L)|
|11.14 +/- 6.42||8.74 +/- 5.51|
|13.36 +/- 10.66||32.63 +/- 24.05|
|0.36 +/- 0.37||3.54 +/- 1.58|
POI; Premature ovarian insufficiency, FSH; Follicle stimulating hormone, LH; Luteinizing hormone, E2; Estradiol, PRL; Prolactine and AMH; Anti-Müllerian hormone.
We collected targeted history information on
personal and familial autoimmune disorders (
Personal and family history on autoimmune diseases in patients and healthy controls
|Study group (n=20)||Control group (n=17)|
|Family history||Personal history||Family history||Personal history|
POI ; Premature ovarian insufficiency.
Associated autoimmune abnormalities (some participants showed more than one condition)
|Associated autoimmune findings||Study group||Control group|
|6 (30%)||2 (11.1%)|
|11 (55%)||4 (22.2%)|
POI; premature ovarian insufficiency.
Analysis of AOA in serum was performed for all
Prevalence of serum anti-ovarian antibodies (AOA) in patients.
Cellular autoimmune reaction occurring due to
a changed T cell function was analysed as a potential
cause of POI. Cell abnormalities were more
frequent in women with POI than in healthy women
Peripheral T cell count is expressed as a percentage of various cell surface markers. In patients with POI, peripheral regulatory T lymphocytes (CD25+high, p=0.015) were low and peripheral blood B cells (CD19+) were high (p=0.014); T lymphocyte parameters were normal in the control group.
Prevalence (in %) of analysed peripheral blood lymphocyte samples for various cell surface markers in patients and in controls
|Various cell surface markers||Study group (%)||Control group (%)||P value|
Markers for peripheral blood lymphocytes: T lymphocytes (CD3+), helper T lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), natural killer cells (CD56+CD16+), regulatory T lymphocytes (CD25+high) and B cells (CD19+).
To present POI as a possible autoimmune abnormality, we focused on three potentially interconnected factors: personal and familial history of autoimmune disorders, peripheral blood T-lymphocytes levels and presence of AOA.
We performed a targeted history of personal and
familial autoimmune disorders and found associated
autoimmune disorders in our patients. Thyroid
disorders were common in personal histories and
diabetes type 1 in familial histories, confirming the
findings of previous studies (
Hoek et al. (
Thyroid disorders and the presence of antithyroid
antibodies are often mentioned in association
with POI. Thyroid disorders are the most common
of the autoimmune diseases associated with
POI, found in 12-39% of women with POI (
Genetic predisposition is known to be one of the
primary causes of autoimmunity and it is generally
observed that patients with autoimmune diseases
have several types of antibodies, as also confirmed
in our study. We found AOA in 20% of patients;
these results are consistent with other studies (
Infertile women with AOA also have a decreased
response to gonadotropin stimulation and reduced
pregnancy rate after treatment (
Abnormalities of cellular immunity of T lymphocytes,
macrophages and dendritic cells play
an important role in autoimmune events. Some of
these abnormalities have been seen in women with
POI, confirming the potential existence of an autoimmune
mechanism of the disease. Mignot et al.
In agreement with studies in patients with systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis and autoimmune vasculitis, we confirmed a reduced number of CD4+CD25+high T cells in the peripheral blood of our patients. High expressions of CD25 and CD4 surface markers have classically been used for identification of regulatory T cells. This may be problematic since CD25 is also expressed on antigen-responding activated non-regulatory T cells. The additional measurement of cellular expression of Foxp3 protein allowed a more specific analysis of Treg cells (CD4+CD25+Foxp3+ cells). However, Foxp3 is also transiently expressed in activated human effector T cells, thus complicating a correct Treg analysis. The large majority of Foxp3-expressing regulatory T cells express high levels of the interleukin-2 receptor alpha chain (CD25). Since there are no cell surface markers that are uniquely and specifically expressed on all Foxp3-expressing regulatory T cells, the measurement of CD4+CD25+high T cells is still in use in clinical studies of peripheral blood lymphocytes.
We interpreted the reduced number of
CD4+CD25+high T cells as a possible mechanism
contributing to the formation of an autoimmune
response in association with the presence of AOA
and aTG. We also found an increased number of
B cells in peripheral blood. A similar picture has
been observed with other autoimmune endocrinopathies;
therefore, we interpreted the elevated
B cell count as activation of the humoral immune
system, crucial for autoantibody production. Some
authors, though, have tried estrogen substitution in
women with POI without any effect on peripheral
B cell count (
The hormones inhibin B, FSH and AMH have
been proposed as potential markers for determining
the functional ovarian reserve (
Clinical and biological characteristics of women without known causes of disease suggest a possibility of autoimmune pathogenesis. In some patients, a combination of various autoimmune processes has been found. The presence of AOA and anti-thyroid antibodies, together with abnormalities of cellular immunity, potentially represent an autoimmune mechanism of POI. There is thus an increasing need to find a reliable and simple diagnostic procedure to determine the true prevalence of autoimmune ovarian disease. In women with POI, more attention should be paid to evaluation of associated autoimmune disorders.