Ocena przydatności oznaczeń stężeń hormonu antymüllerowskiego i inhibny B jako markerów czynności jajników u pacjentek z zespołem Turnera – wyniki wstępne

Introduction

Primary ovarian insufficiency means diminished functional activity of ovaries when defect is inherent within the gonads. One of the chromosomal abnormalities which is characterized by primary ovarian failure is Turner syndrome (TS). Although TS is in most patients associated with pubertal delay or failure and infertility, hypogonadism may occur at different age and clinically manifest as late or absent puberty, primary or secondary amenorrhea [1, 2]. According to literature up to 30% of TS patients present with spontaneous pubertal development, 2–5% have regular menstrual cycles and 2% may get pregnant spontaneously [3]. As an ovarian insufficiency in TS pa-tients is caused by accelerated loss of follicles that may start already into fetal life, it is important to identify right patients and time point for potential cryopreservation of ovarian tissue before follicles begin to disappear [4–6]. Moreover, estrogens are secreted from the he-althy ovaries already in the prepubertal period and they seem to improve cognition and bone mineral density, optimize response to growth hormone treatment and bring some beneficial metabolic effect [7–14]. Gonadal axis in prepubertal children with ZT is difficult to be diagnosed, because it is centrally inhibited [15]. In that age, there is an overlap of the gonadotropin levels values between healthy girls and TS patients, therefore they may not reflect ovarian reserve in this period of life [15–17]. Some authors tried to define cut off value of serum FSH concentration in prepubertal girls with TS as an index for predicting spontaneous puberty and menarche, but to date there are no concrete guidelines in this field [18, 19]. To date no reliable markers of spontaneous puberty have been defined. Novel can-didates for ovarian function markers in TS patients are: Antymüllerian hormone (AMH) and inhibin B, that are secreted from developing follicles and have been found to positively correlate with antral follicle count [20]. It is believed that serum AMH and inhibin B levels reflects ovarian reserve independent from the hypothalamo-pituitary-gonadal axis. Moreover the decline in serum AMH levels precedes the changes in another markers for ovarian reserve (FSH, estradiol and inhibin B) [21, 22].

Aim

The present study aimed to evaluate the usefulness of AMH and inhibin B assessment in predicting ovarian function and sponta-neous puberty in patients with TS.

Material and methods

The study conducted in the Department of Pediatric and Adolescent Endocrinology in Children’s University Hospital in Krakow inc-luded 35 girls with TS (12 with monosomic karyotype [45,X] and 23 with non-monosomic karyotype [non 45, X]) treated with rhGH. Evaluation of gonadal axis function was performed at the age of physiological puberty (10–12 years, mean 10.5 years), before introduc-tion of hormonal replacement therapy (HRT). Additionally AMH and inhibin B levels were assessed. In follow up patients were divided into 2 groups: with spontaneous puberty (SP) and without (WP). Spontaneous puberty was defined as Tanner stage 2 or higher breast development. In patients without spontaneous puberty induction of puberty using oral estrogen (Estrofem mite Novo Nordisk pills con-taining 1 mg of 17-estradiol) was started in accordance with present standards after the age of 12.
Serum FSH and LH levels were measured by chemiluminometric assay with an ADVIA CENTAUR XPT machine. Serum es-tradiol was measured with radioimmunoassay. Serum AMH was measured by electrochemiluminescence immunoassay (ECLIA) with a COBAS machine. Serum Inhibin B was measured by enzyme-linked immunosorbent assay (ELISA) with an EUROIMMUN analy-zer. Karyotypes results were obtained from patients records. All karyotyping analysis was performed using lymphocyte cultures.
To compare the two sets of data, the Student’s t test or two-sided Mann–Whitney U test was used. For a correlation analysis, the correlation coefficient (R) and regression analysis were used. The purpose of determining the odds ratio (OR) was to use the logistic regression analysis. Statistically significant results were assumed for which the probability value was less than 0.05.

Results

Patients without spontaneous puberty were observed until the mean age of 16 years. Spontaneous puberty occurred in 16 patients at the mean age of 10 years (9–12 years old). Patients with SP presented with significantly lower mean FSH level (1.14–91.1 mIU/ml, mean value 24.5 mIU/ml vs. 7.7–196.4 mIU/ml, mean value 66.5 mIU/ml, p = 0.002), significantly higher mean estradiol (10.5–68.8 pg/ml, mean value 28.4 pg/ml vs. 6.1–26.0 pg/ml, mean value 14.9 pg/ml, p = 0.005), AMH (0.0–3.11 ng/ml, mean value 0.8 ng/ml vs. 0.0–0.002 ng/ml, mean value 0.003 ng/ml, p = 0.001) and inhibin B (0.0–110.0 pg/ml, mean value 29.1 pg/ml vs. 0.0–11.0 pg/ml, mean value 1.06 pg/ml, p = 0.026) levels. Interestingly, in three SP patients with not elevated FSH level (FSH < 35 mIU/ml) we found zero concentration levels of AMH and inhibin B. Patients with SP had mosaic (non 45, X) karyotype in 87.5% (14/16) and monosomy (45, X) only in 12.5% (2/16). Patients without spontaneous puberty had mosaic (non 45, X) karyotype in 47 % (9/19) and monosomy in 53% (10/19).

Discussion

Primary hypogonadism is stated as one of the main features of TS. Although most patients demand estrogen replacement therapy, secretion of endogenous estrogens may be sufficient in some patients to finish the process of puberty, maintain menstrual cycles and even lead to not assisted pregnancy [3].
In our study 45% of TS patients presented with spontaneous puberty before the age of 12. Our results remain in accordance with another studies with the frequency of spontaneous puberty in TS patients of about 50% [4, 19]. In contrast there are also authors noticing lower percentage of spontaneous puberty in this group of patients [23–25]. Undoubtedly recognition of non-full-blown forms of TS will be growing thanks to the availability of karyotyping, so it may reveal noticeably increased prevalence of spontaneous puberty.
Correlation between karyotype and ovarian function in TS patients has been widely discussed and many authors stated that monosomic TS patients (45,X) are less likely to develop spontaneous puberty than patients with non-45,X karyotype [4, 15, 19, 21, 23]. Also in our study we observed that patients with non-monosomic karyotype are more likely to start puberty spontaneously than mono-somic ones (14/23 61% vs. 2/12 16%). Nevertheless, definitive prediction of the occurrence of spontaneous puberty on the basis of karyotype is not possible. Therefore, there is no reason to wait longer than until 11–12 years of age for the first symptoms of puberty in non-45, X karyotype TS girls [19].
Diagnosis of hypergonadotropic hypogonadism in adult post-menstrual women is defined as FSH level above 40 µIU/ml. In prepubertal children there are no specific guidelines for predicting hypogonadism, although some authors suggested limit of FSH (6.7 mIU/ml and 10 mIU/ml respectively) above which spontaneous puberty is less probable [18, 19].
In big cross-sectional study Visser et al. found negative correlation between FSH and AMH as well as between LH and AMH. For the subgroup of girls before the age of 12 with FSH level > 10 mIU/ml the odds of measurable AMH was 19 times lower. Strong relationship was observed for measurable serum AMH and signs of spontaneous puberty [4]. The chance of spontaneous pubertal onset was increased 19 times if AMH was detectable [4]. It was also showed by Lunding et al. that any of girls with AMH < 4 pmol/l entered puberty spontaneously [26]. In Hagen’s et al. [15] longitudinal study ovarian failure was predicted in all patients with exclusively unde-tectable inhibin B.
In our study we also found significantly lower concentration of FSH and higher AMH and inhibin B levels in patients with spontaneous puberty. Interestingly in three SP patients without elevated FSH AMH and inhibin B concentrations were zero. This stays in compliance with Hagen’s and Kalsey’s observations that decline in serum AMH levels precedes the changes in another markers for ovarian reserve (FSH, estradiol and inhibin B) [21, 22].
Limitation of our study may be observation, that 37% of healthy girls have undetectable inhibin B levels [27]. Another weak point of our study may be also the fact that we used cut-off age of 12 when about 75–90% of TS girls enter puberty. At this age, accor-ding to the present guidelines induction of puberty is usually started. Nevertheless, some of the patients might enter spontaneous puberty later [28–31]. Also about 50% of TS patents who entered puberty spontaneously would not be able to complete puberty so longitudinal monitoring of puberty and AMH and Inhibin B levels would be needed [32]. What is more in our study we did not differentiate patients according to growth hormone treatment whilst Visser et al. claimed that GH therapy increases the odds of having measurable AMH in TS so this aspect also needs further examination [4].
Despite these limitations, results of the study provide next argument supporting consideration of monitoring AMH and inhibin B levels to predict spontaneous puberty in TS patients and start estrogen replacement in most suitable moment. Further investigations in this field are needed for development of better standards of comprehensive care for patients with TS.

Conclusions

AMH and inhibin B assessment may be a valuable complement to the diagnosis of ovarian function in patients with TS. Low levels of these parameters may indicate a risk of ovarian failure even in patients with spontaneous puberty and without hypergonadotropic hypogonadism.

Acknowledgements

Source of founding: Jagiellonian University Grant No N41/DBS000256.

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