J Pediatr Endocr Met 2014; aop
Maria Francesca Messina*, Tommaso Aversa, Giuseppina Salzano, Daria Costanzo, Concetta Sferlazzas, Silvestro Mirabelli, Giuseppina Zirilli and Fortunato Lombardo
Inhibin B in adolescents and young adults with Turner syndrome assessing ovarian reserve and a longitudinal marker of the possible decline of ovarian function in TS.
Abstract Objective: Primary gonadal failure may occur in most individuals with Turner syndrome (TS). Since ovaries in TS girls undergo premature apoptosis and cryopreservation of ovarian tissue is now feasible, it would be useful to identify a reliable marker of ovarian reserve in these patients. We planned to evaluate ovarian function in a group of TS patients by measuring both traditional markers and inhibin B and to compare these results with those of a control group. Study design: We enrolled 23 patients with TS and 17 agematched healthy girls. The median age of our TS patients was 17.6 years. Three out of the 23 patients (13%) showed spontaneous pubertal development and regular menstrual cycles; the remaining 20 (86.9%) presented with primary amenorrhea. Results: The median level of inhibin B in the TS patients with primary amenorrhea was 42 pg/mL and did not differ significantly among the different subgroups in relation to karyotype. The median inhibin B level in the control group was significantly higher than in the TS girls with primary amenorrhea (83 vs. 42 pg/mL, p < 0.00001). In the three patients with TS and spontaneous menstrual cycles, the inhibin B levels were significantly higher when compared to the values of the TS girls with primary amenorrhea. Conclusion: TS patients with primary amenorrhea have significantly lower levels of inhibin B than TS girls with spontaneous puberty and healthy controls. Inhibin B does not correlate with follicle-stimulating hormone/luteinizing hormone. If our results are confirmed in further studies, inhibin B could become a first-line screening test for
*Corresponding author: Dr. Maria Francesca Messina, Dipartimento di Scienze Pediatriche, Policlinico Universitario, Via Consolare Valeria 1 – 98124 Messina, Italy, Phone: +39-090-2213023, Fax: +39-090-2212143, E-mail: [email protected]; and Department of Pediatrics, University of Messina, Messina, Italy Tommaso Aversa, Giuseppina Salzano, Daria Costanzo, Concetta Sferlazzas, Silvestro Mirabelli, Giuseppina Zirilli and Fortunato Lombardo: Department of Pediatrics, University of Messina, Messina, Italy
Keywords: anti-Müllerian hormone (AMH); infertility; inhibin A; inhibin B; primary gonadal failure; Turner syndrome. DOI 10.1515/jpem-2014-0229 Received June 3, 2014; accepted September 26, 2014
Introduction Turner syndrome (TS) is a common chromosomal disorder with a prevalence of 1/2500 female live births. It results from the loss of all or part of the X-chromosome, and a wide spectrum of karyotypic abnormalities exists within TS (1, 2). From the clinical point of view it is characterized by short stature, dysmorphic features, congenital heart and renal malformations, predisposition to autoimmune disease, a peculiar neurocognitive profile, and liver dysfunction (3–5). Primary gonadal failure occurs in most individuals with TS but it is not a constant finding. TS girls with spontaneous pubertal development have been reported in the literature and it is estimated that up to 30% of these patients achieves pubertal development, and that spontaneous pregnancy can occur in 2%–5% of them (6–8). Since ovaries in TS girls undergo premature apoptosis and cryopreservation of ovarian tissue is now feasible, it would be useful to identify a reliable marker of ovarian reserve in these patients. Traditional markers include serum gonadotropins and antral follicle counts, but follicle stimulating hormone (FSH) and luteinizing hormone (LH) are rarely useful; so recently, new markers as well as inhibin A and B and anti-Müllerian hormone (AMH) have been proposed (9–11). We planned to evaluate clinical data and hormonal markers of ovarian function in a group of TS patients, measuring both traditional markers and new markers, and to compare these results with those obtained in a control group of healthy age-matched girls.
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2 Messina et al.: Inhibin B and Turner syndrome
Patients and methods Study population and design During a 12-month period (March 2012–March 2013), in our crosssectional study we enrolled 23 patients with TS who were attending the out-patient Pediatric Endocrinology Unit at the University of Messina. A diagnosis of TS was suspected due to clinical features and was confirmed by karyotyping analysis. The median age of our TS study group was 17.6 years (range, 12.0–45.3 years). Inclusion criteria were a diagnosis of TS confirmed by karyotyping and chronological age ≥ 12 years old. Among the 23 patients enrolled in this cross-sectional study, 14 (60.8%) had a monosomy X, five (21.7%) presented a mosaicism and four (17.3%) showed a structural alteration of the X chromosome, and in particular an isochromosome of X. Three out of the 23 patients (13%) showed spontaneous pubertal development and had regular menstrual cycles (numbers 21, 22 and 23 in Table 1). Eighteen out of 20 TS amenorrhoeic patients (90%) were on hormone replacement therapy (HRT) and this regimen was started in all patients who did not undergo spontaneous pubertal development at a chronological age ≥ 13 years and/or in the presence of elevated levels of gonadotropins. Initially oral ethynilestradiol 1-24-10 μg/day was administered to induce pubertal development and it was gradually increased over a period of 2–4 years. After this pubertal induction period or when a vaginal bleeding (estradiol-induced menarche) occurred, cyclic therapy with estradiol and progestin
was started. This treatment ensured regular vaginal bleeding in all the TS patients. All the patients started on the low dosage (estradiol 1 mg plus dydrogesterone 10 mg) but if vaginal bleeding and/or other signs of estrogen deficiency occurred on treatment, the dose was increased to estradiol 2 mg plus dydrogesterone 10 mg or estradiol 4 mg plus dydrogesterone 20 mg. Two TS patients with a chronological age below 14 years and a uterus longitudinal diameter of < 40 mm were diagnosed as having primary amenorrhea secondary to hypergonadotropic hypogonadism, and this diagnosis was confirmed by the detection of very high levels of FSH and LH. During a 6-month period (October 2012–March 2013) we enrolled 17 age-matched healthy girls in our study. The inclusion criterion was spontaneous menarche with regular menstrual cycles. Exclusion criteria were chronic illness and treatment with estrogen and progestogen. The median age of our control group was 15.7 years (range, 12.2–24.6 years). The study protocol was approved by the Ethics Committee of our University Hospital. All participants or their parents gave their written informed consent.
Methods Height was measured using a Harpenden stadiometer. Body weight was measured in the morning during fasting state, with the subjects in their underwear. Body mass index was calculated as body weight divided by the squared height (kg/m2). Karyotyping analysis was performed using lymphocyte cultures and at least 50 cells were karyotyped for each patient.
Table 1 Laboratory and instrumental data of the 23 Turner syndrome patients. Patients
Age, years
FSH, mlU/mL
LH, mIU/mL
E2, pg/mL
LD, mm
Inhibin B, pg/mL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
12.0 13.1 13.3 14.3 14.3 14.3 16.0 16.5 16.6 17.2 17.6 21.0 21.6 23.9 25.5 26.8 27.1 27.7 28.5 45.3 14.9 19.9 37.6
98.6 169.0 88.8 87.4 119.0 117.0 97.5 95.4 101.0 65.3 42.0 53.4 66.7 92.5 35.8 35.0 5.0 4.9 85.4 86.1 5.2 7.4 8.6
27.8 33.9 45.6 1.0 3.0 47.8 15.2 7.4 24.7 1.6 16.2 21.5 2.0 44.6 11.5 27.5 1.0 2.7 18.0 8.5 2.6 5.2 7.3
5.0 5.0 8.2 39.4 5.0 28.2 120.0 5.0 40.0 195.0 78.3 38.0 270.0 14.1 5.0 68.3 22.4 34.2 48.3 180.0 102 43.1 32.5
30 42 38 51 60 53 49 57 61 59 55 54 95 72 63 84 77 74 62 88 61 83 66
46 40 40 40 42 42 41 38 42 41 42 46 43 43 42 39 41 44 45 45 56 87 98
Karyotype
45X 45X 45X/46X X ISO 45X 45X 46XX ISO 45X 45X 46XX ISO 45X,46XX,47XXX 45X 45X 46XX ISO 45X 46XX ISO 45X/46XX 45X 45X 45X,47XXX 45X 45X/46XX 45X 45X
Treatment
None C C A A A B B A B B B B B None A A B A B Spontaneous menstrual cycles Spontaneous menstrual cycles Spontaneous menstrual cycles
E2, 17β-estradiol; FSH, follicle-stimulating hormone; LD, uterus longitudinal diameter; LH, luteinizing hormone. Treatment A = Estradiol 1 mg+dydrogesterone 10 mg; Treatment B = Estradiol 2 mg+dydrogesterone 20 mg; Treatment C = Ethynilestradiol.
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Messina et al.: Inhibin B and Turner syndrome 3
In all the patients, a fasting blood sample was taken to analyze inhibin B, FSH, LH and 17β-estradiol (E2) levels. E2 in the serum was measured using a sensitive solid-state radioimmunoassay-coated tube kit (Orion Diagnostica, Espoo, Finland) for direct quantitative determination. The detection limit for the assay was 5 pmol/L. FSH and LH levels in the serum were measured by immunometric assays (Immulite, Diagnostic Products Corporation, Los Angeles, CA, USA). The analytical sensitivity of the FSH and LH assay was 0.1 IU/L. Blood samples for the inhibin B assay were centrifuged and stored at –20°C until analysis. Serum inhibin B was measured in duplicate in a double antibody enzyme immunometric assay using a monoclonal antibody raised against the inhibin β-subunit in combination with a labeled antibody raised against the inhibin α-subunit, as previously described (12). In the inhibin B assay the detection limit was 20 pg/mL and the intra- and inter-assay coefficients of variation were 15% and 18%, respectively. During follow-up, all patients underwent a pelvic ultrasound scan to assess the morphostructural characters of the pelvic organs, in particular to assess the diameters of the uterus and ovaries when the latter were identified.
Statistical analysis All statistical calculations were performed with SPSS statistical software version 16.0 (SPSS Inc., Chicago, IL, USA). Results are expressed as median and range or interquartile range. Data were examined by Mann-Whitney tests when comparing TS and controls. Correlation analysis was performed with Spearman’s rank correlation analysis. The level of statistical significance was p < 0.05.
Results All the data from our 23 TS patients and 17 healthy controls are analytically presented in Tables 1, 2 and Figure 1. Comparison between the median level of inhibin B, FSH, LH, E2 and the longitudinal diameter of the uterus of TS amenorrhoeic patients and those of the control group are presented in Table 3. In the 20 TS patients with primary amenorrhea, the median basal level of FSH, LH and E2 were 86.8 mIU/mL (range, 0.5–169 mIU/mL), 15.7 mIU/mL (range, 0.1–47 mIU/ mL), and 36.1 pg/mL (range, 5–270 pg/mL), respectively. In the same patient group the average longitudinal uterine diameter was 59.5 mm (range, 30–95 mm), but it was not possible to provide detailed information about ovary volume because the gonads were not visualized by pelvic ultrasound in more than 80% of the patients included in the study. The median inhibin B value in the TS patients with primary amenorrhea was 42.0 pg/mL (range 38–46 pg/mL). The median level of inhibin B did not differ
significantly among the different subgroups in relation to karyotype. It was 42.0 pg/mL in monosomic patients, 41.2 pg/mL in those with mosaicism and 42.2 pg/mL in those with structural abnormalities of the X chromosome. The median level of inhibin B in the control group was significantly higher than that of the TS patients with primary amenorrhea (83.0 vs. 42.0 pg/mL, p < 0.00001). In the three patients with TS and spontaneous menstrual cycles, the levels of inhibin B were significantly higher when compared to the values of the TS girls with primary amenorrhea and were very close to those of the healthy controls (Figure 1C). No significant correlations were found between inhibin B and other biochemical and ultrasonographic parameters in the control group, in TS patients with normal menstrual cycles and in TS amenorrhoeic patients. Moreover, inhibin B levels were very similar in TS amenorrhoeic patients with normal levels of FSH/LH and in the remaining patients with very high levels of FSH/LH Table 1. Levels were also similar in TS patients on HRT and in TS patients tested before starting HRT (Table 1).
Discussion Traditionally, TS is considered one of the best-known causes of primary gonadal failure and subsequent infertility. Although, it has been reported that premature ovarian failure frequently occurs in TS girls, it is not always the case and a wide spectrum of ovarian function exists in these patients. According to the chronological age at which hypergonadotropic hypogonadism occurs, different clinical pictures may present and in particular late or absent puberty, primary amenorrhea or secondary amenorrhea (13, 14). Spontaneous pubertal development and the appearance and maintenance of regular menstrual cycles are not exceptional and can occur in approximately 20% of TS girls (6). The diagnosis of ovarian failure is not easy for a number of reasons, in particular: a) it is mainly based on the dosage of basal gonadotropin levels that can vary considerably – even when very high they are not pathognomonic of ovarian insufficiency; b) ovarian function can reactivate spontaneously, even if intermittently, in patients affected by premature ovarian failure, as evidenced by the occasional normal levels of FSH and LH in these patients; c) the presence of normal menstrual cycles does not exclude early stages of menopause, even in the presence of high blood levels of FSH and LH; and d) the absence of menstrual cycles is not absolute evidence of menopause (15, 16). According to these data, it cannot
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4 Messina et al.: Inhibin B and Turner syndrome Table 2 Laboratory and instrumental data from the 17 healthy controls. Patients
Age, years
FSH, mlU/mL
LH, mIU/mL
E2, pg/mL
LD, mm
Inhibin B, pg/mL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
12.2 12.9 13.9 14.0 14.5 14.7 15.0 15.6 15.7 16.6 16.9 18.0 18.4 19.0 20.2 24.1 24.6
3.3 3.8 2.6 2.6 3.7 1.5 3.6 2.2 3.4 4.1 3.9 2.6 4.3 5.2 2.8 4.0 1.9
2.3 2.8 1.4 1.8 1.6 2.2 1.7 1.5 2.0 3.2 2.6 3.1 3.3 3.4 3.2 3.5 2.1
134 64 58 110 78 143 82 46 98 75 99 104 55 151 180 102 67
60 68 64 62 69 68 78 73 70 80 82 71 82 74 77 74 73
83 140 43 70 102 83 130 36 55 125 46 246 95 49 98 65 97
Treatment None None None None None None None None None None None None None None None None None
E2, 17β-estradiol; FSH, follicle-stimulating hormone; LD, uterus longitudinal diameter; LH, luteinizing hormone.
Table 3 Comparison of the median FSH, LH, E2, uterus longitudinal diameter and inhibin B levels of 20 Turner syndrome amenorrhoeic patients and 17 healthy controls.
Age, years FSH, mlU/mL LH, mlU/mL E2, pg/mL LD, mm Inhibin B, pg/mL
Turner syndrome with primary amenorrhea (n = 20)
Healthy controls (n = 17)
p-Value
17.4 (14.3–26.4) 15.7 (14.3–18.7) 0.195 86.8 (45.5–98.3) 3.4 (2.6–4.0) 0.000004 15.7 (1.9–27.7) 2.3 (1.8–3.2) 0.015 36.1 (5.8–75.8) 98.0 (65.5–122.0) 0.004 59.5 (51.5–73.5) 73.0 (68–77.5) 0.012 42.0 (40.3–43.8) 83.0 (52.0–113.5) 0.00001
E2, 17β-estradiol; FSH, follicle-stimulating hormone; LD, uterus longitudinal diameter; LH, luteinizing hormone. Data are expressed as median and interquartile range.
be excluded that TS patients with secondary amenorrhea or abnormal levels of FSH and LH may have a functional reserve of oocytes and thus they could be ideal candidates to the levy of ovarian fragments for the cryopreservation. This hypothesis is supported by the finding that patients with TS and the classic karyotype, 45 X0 have active follicles between the ages of 12 and 19 years (9). Since FSH and LH do not appear to be good and sensitive markers of ovarian function, in recent years new definite markers of ovarian function have been looked for. Hypothetically, inhibin A and B and AMH could be ideal candidates to evaluate and monitor ovarian function over time (11). Inhibin A is a marker of corpus
luteum function, whereas both inhibin B and AMH are secreted from developing follicles and have been found to positively correlate with antral follicle count in several studies (17). Inhibin A and B are gonadal hormones produced in a specific pattern in response to gonadotropin stimulation in females. Inhibin B is principally secreted in the follicular phase and inhibin A is principally secreted in the luteal phase (18). Although it can generally be assumed that inhibin B levels are undetectable in TS, some authors have proved that a small percentage of these patients is able to produce both inhibin A and B (9). This evidence is in accordance with the fact spontaneous pregnancies can occur in 2%–5% of TS girls, suggesting the presence of active follicles in a proportion of TS girls that is significantly higher than previously thought (19, 20). Our results confirm what has been documented in the few previous studies regarding the levels of inhibin B in TS. In particular, we demonstrated that our TS patients with primary amenorrhea have median values of inhibin B that are significantly lower than the healthy controls, whereas patients with TS and regular menstrual cycles have hormonal values comparable to those of control girls and significantly higher than those of TS patients with gonadal insufficiency. In our study, inhibin B levels do not differ among patients with different karyotypes; thus proving the lack of a correlation genotype/phenotype in terms of ovarian function and reserve, and supporting the possibility that functional ovarian follicles may be present in patients with classical karyotype (9).
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Messina et al.: Inhibin B and Turner syndrome 5
A
B
C
200
50
250
40
200
100
Inhibin B, pg/mL
LH, mlU/mL
FSH, mlU/mL
150 30
20
150
100
50
0 A
B
C
10
50
0
0 A
B
C
A
B
C
Figure 1 Hormonal evaluation of Turner syndrome patients both with primary amenorrhea (A) and normal menstrual cycles (B), and healthy controls (C).
Our results may deserve particular attention for the following reasons: a) no correlation has been found between FSH/LH levels, longitudinal uterus diameter and inhibin B levels in TS patients and in the control group; b) inhibin B levels were very similar in the amenorrhoeic TS patients with normal and abnormal levels of FSH and LH; c) inhibin B levels were superimposable in patients treated or untreated with HRT. These observations suggest that HRT treatment can modify gonadotropin levels but cannot influence inhibin B levels. The finding of an isolated low level of inhibin B in TS patients does not necessarily mean that ovarian function is impaired. In fact, it is known that in healthy women inhibin B correlates with the phase of the menstrual cycle (follicular and luteal), and has a daily pulsatility (with peaks of 60 min) and may also be undetectable in a small percentage of healthy women (12, 21). Although this fact restricts the utility of inhibin B as a screening test of ovarian activity, consecutive measurements of undetectable inhibin B in amenorrhoeic TS women should be considered the expression of impaired ovarian activity (22). In conclusion, if our results are confirmed in further studies involving larger study populations, inhibin B could become a fist-line screening test for assessing ovarian reserve. Moreover, it can be considered a much more useful marker to monitor longitudinally the decline of ovarian function in the small percentage of TS patients with spontaneous pubertal development and normal menstrual cycles. These TS girls with active follicles in the ovaries may be ideal candidates for the newer reproductive technologies, but we should make every effort to identify
the narrow window for cryopreservation of ovarian tissue and oocytes. Inhibin B may be a more reliable marker than traditional ones including gonadotropins or morphological images of the uterus and ovaries. This is because its levels are not modified by hormonal treatment, at least according to our preliminary results.
References 1. Gravholt CH. Epidemiological, endocrine and metabolic features in Turner syndrome. Eur J Endocrinol 2004;151:657–87. 2. Ranke MB, Saenger P. Turner’s syndrome. Lancet 2001;358:309–14. 3. Davenport ML. Approach to the patient with Turner syndrome. J Clin Endocrinol Metab 2010;95:1487–95. 4. Messina MF, Zirilli G, Civa R, Rulli I, Salzano G, et al. Neurocognitive profile in Turner’s syndrome is not affected by growth impairment. J Pediatr Endocrinol Metab 2007;20:677–84. 5. Messina MF, Squadrito G, Valenzise M, Maimone S, Iannelli S, et al. Fibroscan: a new noninvasive method for evaluation of liver dysfunction in Turner syndrome. Eur J Clin Invest 2011;41:183–8. 6. Pasquino AM, Passeri F, Pucarelli I, Segni M, Municchi G. Spontaneous pubertal development in Turner’s syndrome. Italian Study Group for Turner’s Syndrome. J Clin Endocrinol Metab 1997;82:1810–3. 7. Hovatta O. Pregnancy in women with Turner’s syndrome. Ann Med 1999;31:106–10. 8. Hreinsson JG, Otala M, Fridström M, Borgström B, Rasmussen C, et al. Follicles are found in the ovaries of adolescent girls with Turner’s syndrome. J Clin Endocrinol Metab 2002;87:3618–23. 9. Gravholt CH, Naeraa RW, Andersson AM, Christiansen JS, Skakkebaek NE. Inhibin A and B in adolescents and young adults with Turner’s syndrome and no sign of spontaneous puberty. Hum Reprod 2002;17:2049–53.
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6 Messina et al.: Inhibin B and Turner syndrome 10. Purushothaman R, Lazareva O, Oktay K, Ten S. Markers of ovarian reserve in young girls with Turner’s syndrome. Fertil Steril 2010;94:1557–9. 11. Visser JA, Hokken-Koelega AC, Zandwijken GR, Limacher A, Ranke MB, et al. Anti-Mullerian hormone levels in girls and adolescents with Turner syndrome are related to karyotype, pubertal development and growth hormone treatment. Hum Reprod 2013;28:1899–907. 12. Groome NP, Illingworth PJ, O’Brien M, Pai R, Rodger FE, et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endorcrinol Metab 1996;81:1401–5. 13. Weiss L. Additional evidence of gradual loss of germ cells in the pathogenesis of streak ovaries in Turner’s syndrome. J Med Genet 1971;8:540–4. 14. Reynaud K, Cortvrindt R, Verlinde F, De Schepper J, Bourgain C, et al. Number of ovarian follicles in human fetuses with the 45,X karyotype. Fertil Steril 2004;81:1112–9. 15. Conway GS. Clinical manifestations of genetic defects affecting gonadotropins and their receptors. Clin Endocrinol (Oxf) 1996;45:657–63. 16. Bachelot A, Rouxel A, Massin N, Dulon J, Courtillot C, et al; POF-GIS Study Group. Phenotyping and genetic studies of 357 consecutive patients presenting with premature ovarian failure. Eur J Endocrinol 2009;161:179–87. 17. Nardo LG, Christodoulou D, Gould D, Roberts SA, Fitzgerald CT, et al. Anti-Müllerian hormone levels and antral follicle count
in women enrolled in vitro fertilization cycles: relationship to lifestyle factors, chronological age and reproductive history. Gynecol Endocrinol 2007;23:486–93. 18. Sehested A, Juul A, Andersson AM, Petersen JH, Jensen TK, et al. Serum Inibin A and Inibin B in healthy prepubertal, pubertal, and adolescent girls and adult women: relation to age, stage of puberty, menstrual cycle, folliclestimulating hormone, luteinizing hormone and estradiol levels. J Clin Endocrinol Metab 2000; 85:1634–40. 19. Hadnott TN, Gould HN, Gharib AM, Bondy CA. Outcomes of spontaneous and assisted pregnancies in Turner syndrome: the U.S. National Institutes of Health experience. Fertil Steril 2011;95:2251–6. 20. Bryman I, Sylvén L, Berntorp K, Innala E, Bergström I, et al. Pregnancy rate and outcome in Swedish women with Turner syndrome. Fertil Steril 2011;95:2507–10. 21. Lockwood GM, Muttukrishna S, Groome NP, Matthews DR, Ledger WL. Mid-follicular phase pulses of inhibin B are absent in polycystic ovarian syndrome and are initiated by successful laparoscopic ovarian diathermy: a possible mechanism regulating emergence of the dominant follicle. J Clin Endocrinol Metab 1998;83:1730–5. 22. Hagen CP, Main KM, Kjaergaard S, Juul A. FSH, LH, inhibin B and estradiol levels in Turner syndrome depend on age and karyotype: longitudinal study of 70 Turner girls with or without spontaneous puberty. Hum Reprod 2010;25:3134–41.
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Maria Francesca Messina*, Tommaso Aversa, Giuseppina Salzano, Daria Costanzo, Concetta Sferlazzas, Silvestro Mirabelli, Giuseppina Zirilli and Fortunato Lombardo
Inhibin B in adolescents and young adults with Turner syndrome assessing ovarian reserve and a longitudinal marker of the possible decline of ovarian function in TS.
Abstract Objective: Primary gonadal failure may occur in most individuals with Turner syndrome (TS). Since ovaries in TS girls undergo premature apoptosis and cryopreservation of ovarian tissue is now feasible, it would be useful to identify a reliable marker of ovarian reserve in these patients. We planned to evaluate ovarian function in a group of TS patients by measuring both traditional markers and inhibin B and to compare these results with those of a control group. Study design: We enrolled 23 patients with TS and 17 agematched healthy girls. The median age of our TS patients was 17.6 years. Three out of the 23 patients (13%) showed spontaneous pubertal development and regular menstrual cycles; the remaining 20 (86.9%) presented with primary amenorrhea. Results: The median level of inhibin B in the TS patients with primary amenorrhea was 42 pg/mL and did not differ significantly among the different subgroups in relation to karyotype. The median inhibin B level in the control group was significantly higher than in the TS girls with primary amenorrhea (83 vs. 42 pg/mL, p < 0.00001). In the three patients with TS and spontaneous menstrual cycles, the inhibin B levels were significantly higher when compared to the values of the TS girls with primary amenorrhea. Conclusion: TS patients with primary amenorrhea have significantly lower levels of inhibin B than TS girls with spontaneous puberty and healthy controls. Inhibin B does not correlate with follicle-stimulating hormone/luteinizing hormone. If our results are confirmed in further studies, inhibin B could become a first-line screening test for
*Corresponding author: Dr. Maria Francesca Messina, Dipartimento di Scienze Pediatriche, Policlinico Universitario, Via Consolare Valeria 1 – 98124 Messina, Italy, Phone: +39-090-2213023, Fax: +39-090-2212143, E-mail: [email protected]; and Department of Pediatrics, University of Messina, Messina, Italy Tommaso Aversa, Giuseppina Salzano, Daria Costanzo, Concetta Sferlazzas, Silvestro Mirabelli, Giuseppina Zirilli and Fortunato Lombardo: Department of Pediatrics, University of Messina, Messina, Italy
Keywords: anti-Müllerian hormone (AMH); infertility; inhibin A; inhibin B; primary gonadal failure; Turner syndrome. DOI 10.1515/jpem-2014-0229 Received June 3, 2014; accepted September 26, 2014
Introduction Turner syndrome (TS) is a common chromosomal disorder with a prevalence of 1/2500 female live births. It results from the loss of all or part of the X-chromosome, and a wide spectrum of karyotypic abnormalities exists within TS (1, 2). From the clinical point of view it is characterized by short stature, dysmorphic features, congenital heart and renal malformations, predisposition to autoimmune disease, a peculiar neurocognitive profile, and liver dysfunction (3–5). Primary gonadal failure occurs in most individuals with TS but it is not a constant finding. TS girls with spontaneous pubertal development have been reported in the literature and it is estimated that up to 30% of these patients achieves pubertal development, and that spontaneous pregnancy can occur in 2%–5% of them (6–8). Since ovaries in TS girls undergo premature apoptosis and cryopreservation of ovarian tissue is now feasible, it would be useful to identify a reliable marker of ovarian reserve in these patients. Traditional markers include serum gonadotropins and antral follicle counts, but follicle stimulating hormone (FSH) and luteinizing hormone (LH) are rarely useful; so recently, new markers as well as inhibin A and B and anti-Müllerian hormone (AMH) have been proposed (9–11). We planned to evaluate clinical data and hormonal markers of ovarian function in a group of TS patients, measuring both traditional markers and new markers, and to compare these results with those obtained in a control group of healthy age-matched girls.
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2 Messina et al.: Inhibin B and Turner syndrome
Patients and methods Study population and design During a 12-month period (March 2012–March 2013), in our crosssectional study we enrolled 23 patients with TS who were attending the out-patient Pediatric Endocrinology Unit at the University of Messina. A diagnosis of TS was suspected due to clinical features and was confirmed by karyotyping analysis. The median age of our TS study group was 17.6 years (range, 12.0–45.3 years). Inclusion criteria were a diagnosis of TS confirmed by karyotyping and chronological age ≥ 12 years old. Among the 23 patients enrolled in this cross-sectional study, 14 (60.8%) had a monosomy X, five (21.7%) presented a mosaicism and four (17.3%) showed a structural alteration of the X chromosome, and in particular an isochromosome of X. Three out of the 23 patients (13%) showed spontaneous pubertal development and had regular menstrual cycles (numbers 21, 22 and 23 in Table 1). Eighteen out of 20 TS amenorrhoeic patients (90%) were on hormone replacement therapy (HRT) and this regimen was started in all patients who did not undergo spontaneous pubertal development at a chronological age ≥ 13 years and/or in the presence of elevated levels of gonadotropins. Initially oral ethynilestradiol 1-24-10 μg/day was administered to induce pubertal development and it was gradually increased over a period of 2–4 years. After this pubertal induction period or when a vaginal bleeding (estradiol-induced menarche) occurred, cyclic therapy with estradiol and progestin
was started. This treatment ensured regular vaginal bleeding in all the TS patients. All the patients started on the low dosage (estradiol 1 mg plus dydrogesterone 10 mg) but if vaginal bleeding and/or other signs of estrogen deficiency occurred on treatment, the dose was increased to estradiol 2 mg plus dydrogesterone 10 mg or estradiol 4 mg plus dydrogesterone 20 mg. Two TS patients with a chronological age below 14 years and a uterus longitudinal diameter of < 40 mm were diagnosed as having primary amenorrhea secondary to hypergonadotropic hypogonadism, and this diagnosis was confirmed by the detection of very high levels of FSH and LH. During a 6-month period (October 2012–March 2013) we enrolled 17 age-matched healthy girls in our study. The inclusion criterion was spontaneous menarche with regular menstrual cycles. Exclusion criteria were chronic illness and treatment with estrogen and progestogen. The median age of our control group was 15.7 years (range, 12.2–24.6 years). The study protocol was approved by the Ethics Committee of our University Hospital. All participants or their parents gave their written informed consent.
Methods Height was measured using a Harpenden stadiometer. Body weight was measured in the morning during fasting state, with the subjects in their underwear. Body mass index was calculated as body weight divided by the squared height (kg/m2). Karyotyping analysis was performed using lymphocyte cultures and at least 50 cells were karyotyped for each patient.
Table 1 Laboratory and instrumental data of the 23 Turner syndrome patients. Patients
Age, years
FSH, mlU/mL
LH, mIU/mL
E2, pg/mL
LD, mm
Inhibin B, pg/mL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
12.0 13.1 13.3 14.3 14.3 14.3 16.0 16.5 16.6 17.2 17.6 21.0 21.6 23.9 25.5 26.8 27.1 27.7 28.5 45.3 14.9 19.9 37.6
98.6 169.0 88.8 87.4 119.0 117.0 97.5 95.4 101.0 65.3 42.0 53.4 66.7 92.5 35.8 35.0 5.0 4.9 85.4 86.1 5.2 7.4 8.6
27.8 33.9 45.6 1.0 3.0 47.8 15.2 7.4 24.7 1.6 16.2 21.5 2.0 44.6 11.5 27.5 1.0 2.7 18.0 8.5 2.6 5.2 7.3
5.0 5.0 8.2 39.4 5.0 28.2 120.0 5.0 40.0 195.0 78.3 38.0 270.0 14.1 5.0 68.3 22.4 34.2 48.3 180.0 102 43.1 32.5
30 42 38 51 60 53 49 57 61 59 55 54 95 72 63 84 77 74 62 88 61 83 66
46 40 40 40 42 42 41 38 42 41 42 46 43 43 42 39 41 44 45 45 56 87 98
Karyotype
45X 45X 45X/46X X ISO 45X 45X 46XX ISO 45X 45X 46XX ISO 45X,46XX,47XXX 45X 45X 46XX ISO 45X 46XX ISO 45X/46XX 45X 45X 45X,47XXX 45X 45X/46XX 45X 45X
Treatment
None C C A A A B B A B B B B B None A A B A B Spontaneous menstrual cycles Spontaneous menstrual cycles Spontaneous menstrual cycles
E2, 17β-estradiol; FSH, follicle-stimulating hormone; LD, uterus longitudinal diameter; LH, luteinizing hormone. Treatment A = Estradiol 1 mg+dydrogesterone 10 mg; Treatment B = Estradiol 2 mg+dydrogesterone 20 mg; Treatment C = Ethynilestradiol.
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Messina et al.: Inhibin B and Turner syndrome 3
In all the patients, a fasting blood sample was taken to analyze inhibin B, FSH, LH and 17β-estradiol (E2) levels. E2 in the serum was measured using a sensitive solid-state radioimmunoassay-coated tube kit (Orion Diagnostica, Espoo, Finland) for direct quantitative determination. The detection limit for the assay was 5 pmol/L. FSH and LH levels in the serum were measured by immunometric assays (Immulite, Diagnostic Products Corporation, Los Angeles, CA, USA). The analytical sensitivity of the FSH and LH assay was 0.1 IU/L. Blood samples for the inhibin B assay were centrifuged and stored at –20°C until analysis. Serum inhibin B was measured in duplicate in a double antibody enzyme immunometric assay using a monoclonal antibody raised against the inhibin β-subunit in combination with a labeled antibody raised against the inhibin α-subunit, as previously described (12). In the inhibin B assay the detection limit was 20 pg/mL and the intra- and inter-assay coefficients of variation were 15% and 18%, respectively. During follow-up, all patients underwent a pelvic ultrasound scan to assess the morphostructural characters of the pelvic organs, in particular to assess the diameters of the uterus and ovaries when the latter were identified.
Statistical analysis All statistical calculations were performed with SPSS statistical software version 16.0 (SPSS Inc., Chicago, IL, USA). Results are expressed as median and range or interquartile range. Data were examined by Mann-Whitney tests when comparing TS and controls. Correlation analysis was performed with Spearman’s rank correlation analysis. The level of statistical significance was p < 0.05.
Results All the data from our 23 TS patients and 17 healthy controls are analytically presented in Tables 1, 2 and Figure 1. Comparison between the median level of inhibin B, FSH, LH, E2 and the longitudinal diameter of the uterus of TS amenorrhoeic patients and those of the control group are presented in Table 3. In the 20 TS patients with primary amenorrhea, the median basal level of FSH, LH and E2 were 86.8 mIU/mL (range, 0.5–169 mIU/mL), 15.7 mIU/mL (range, 0.1–47 mIU/ mL), and 36.1 pg/mL (range, 5–270 pg/mL), respectively. In the same patient group the average longitudinal uterine diameter was 59.5 mm (range, 30–95 mm), but it was not possible to provide detailed information about ovary volume because the gonads were not visualized by pelvic ultrasound in more than 80% of the patients included in the study. The median inhibin B value in the TS patients with primary amenorrhea was 42.0 pg/mL (range 38–46 pg/mL). The median level of inhibin B did not differ
significantly among the different subgroups in relation to karyotype. It was 42.0 pg/mL in monosomic patients, 41.2 pg/mL in those with mosaicism and 42.2 pg/mL in those with structural abnormalities of the X chromosome. The median level of inhibin B in the control group was significantly higher than that of the TS patients with primary amenorrhea (83.0 vs. 42.0 pg/mL, p < 0.00001). In the three patients with TS and spontaneous menstrual cycles, the levels of inhibin B were significantly higher when compared to the values of the TS girls with primary amenorrhea and were very close to those of the healthy controls (Figure 1C). No significant correlations were found between inhibin B and other biochemical and ultrasonographic parameters in the control group, in TS patients with normal menstrual cycles and in TS amenorrhoeic patients. Moreover, inhibin B levels were very similar in TS amenorrhoeic patients with normal levels of FSH/LH and in the remaining patients with very high levels of FSH/LH Table 1. Levels were also similar in TS patients on HRT and in TS patients tested before starting HRT (Table 1).
Discussion Traditionally, TS is considered one of the best-known causes of primary gonadal failure and subsequent infertility. Although, it has been reported that premature ovarian failure frequently occurs in TS girls, it is not always the case and a wide spectrum of ovarian function exists in these patients. According to the chronological age at which hypergonadotropic hypogonadism occurs, different clinical pictures may present and in particular late or absent puberty, primary amenorrhea or secondary amenorrhea (13, 14). Spontaneous pubertal development and the appearance and maintenance of regular menstrual cycles are not exceptional and can occur in approximately 20% of TS girls (6). The diagnosis of ovarian failure is not easy for a number of reasons, in particular: a) it is mainly based on the dosage of basal gonadotropin levels that can vary considerably – even when very high they are not pathognomonic of ovarian insufficiency; b) ovarian function can reactivate spontaneously, even if intermittently, in patients affected by premature ovarian failure, as evidenced by the occasional normal levels of FSH and LH in these patients; c) the presence of normal menstrual cycles does not exclude early stages of menopause, even in the presence of high blood levels of FSH and LH; and d) the absence of menstrual cycles is not absolute evidence of menopause (15, 16). According to these data, it cannot
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4 Messina et al.: Inhibin B and Turner syndrome Table 2 Laboratory and instrumental data from the 17 healthy controls. Patients
Age, years
FSH, mlU/mL
LH, mIU/mL
E2, pg/mL
LD, mm
Inhibin B, pg/mL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
12.2 12.9 13.9 14.0 14.5 14.7 15.0 15.6 15.7 16.6 16.9 18.0 18.4 19.0 20.2 24.1 24.6
3.3 3.8 2.6 2.6 3.7 1.5 3.6 2.2 3.4 4.1 3.9 2.6 4.3 5.2 2.8 4.0 1.9
2.3 2.8 1.4 1.8 1.6 2.2 1.7 1.5 2.0 3.2 2.6 3.1 3.3 3.4 3.2 3.5 2.1
134 64 58 110 78 143 82 46 98 75 99 104 55 151 180 102 67
60 68 64 62 69 68 78 73 70 80 82 71 82 74 77 74 73
83 140 43 70 102 83 130 36 55 125 46 246 95 49 98 65 97
Treatment None None None None None None None None None None None None None None None None None
E2, 17β-estradiol; FSH, follicle-stimulating hormone; LD, uterus longitudinal diameter; LH, luteinizing hormone.
Table 3 Comparison of the median FSH, LH, E2, uterus longitudinal diameter and inhibin B levels of 20 Turner syndrome amenorrhoeic patients and 17 healthy controls.
Age, years FSH, mlU/mL LH, mlU/mL E2, pg/mL LD, mm Inhibin B, pg/mL
Turner syndrome with primary amenorrhea (n = 20)
Healthy controls (n = 17)
p-Value
17.4 (14.3–26.4) 15.7 (14.3–18.7) 0.195 86.8 (45.5–98.3) 3.4 (2.6–4.0) 0.000004 15.7 (1.9–27.7) 2.3 (1.8–3.2) 0.015 36.1 (5.8–75.8) 98.0 (65.5–122.0) 0.004 59.5 (51.5–73.5) 73.0 (68–77.5) 0.012 42.0 (40.3–43.8) 83.0 (52.0–113.5) 0.00001
E2, 17β-estradiol; FSH, follicle-stimulating hormone; LD, uterus longitudinal diameter; LH, luteinizing hormone. Data are expressed as median and interquartile range.
be excluded that TS patients with secondary amenorrhea or abnormal levels of FSH and LH may have a functional reserve of oocytes and thus they could be ideal candidates to the levy of ovarian fragments for the cryopreservation. This hypothesis is supported by the finding that patients with TS and the classic karyotype, 45 X0 have active follicles between the ages of 12 and 19 years (9). Since FSH and LH do not appear to be good and sensitive markers of ovarian function, in recent years new definite markers of ovarian function have been looked for. Hypothetically, inhibin A and B and AMH could be ideal candidates to evaluate and monitor ovarian function over time (11). Inhibin A is a marker of corpus
luteum function, whereas both inhibin B and AMH are secreted from developing follicles and have been found to positively correlate with antral follicle count in several studies (17). Inhibin A and B are gonadal hormones produced in a specific pattern in response to gonadotropin stimulation in females. Inhibin B is principally secreted in the follicular phase and inhibin A is principally secreted in the luteal phase (18). Although it can generally be assumed that inhibin B levels are undetectable in TS, some authors have proved that a small percentage of these patients is able to produce both inhibin A and B (9). This evidence is in accordance with the fact spontaneous pregnancies can occur in 2%–5% of TS girls, suggesting the presence of active follicles in a proportion of TS girls that is significantly higher than previously thought (19, 20). Our results confirm what has been documented in the few previous studies regarding the levels of inhibin B in TS. In particular, we demonstrated that our TS patients with primary amenorrhea have median values of inhibin B that are significantly lower than the healthy controls, whereas patients with TS and regular menstrual cycles have hormonal values comparable to those of control girls and significantly higher than those of TS patients with gonadal insufficiency. In our study, inhibin B levels do not differ among patients with different karyotypes; thus proving the lack of a correlation genotype/phenotype in terms of ovarian function and reserve, and supporting the possibility that functional ovarian follicles may be present in patients with classical karyotype (9).
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Messina et al.: Inhibin B and Turner syndrome 5
A
B
C
200
50
250
40
200
100
Inhibin B, pg/mL
LH, mlU/mL
FSH, mlU/mL
150 30
20
150
100
50
0 A
B
C
10
50
0
0 A
B
C
A
B
C
Figure 1 Hormonal evaluation of Turner syndrome patients both with primary amenorrhea (A) and normal menstrual cycles (B), and healthy controls (C).
Our results may deserve particular attention for the following reasons: a) no correlation has been found between FSH/LH levels, longitudinal uterus diameter and inhibin B levels in TS patients and in the control group; b) inhibin B levels were very similar in the amenorrhoeic TS patients with normal and abnormal levels of FSH and LH; c) inhibin B levels were superimposable in patients treated or untreated with HRT. These observations suggest that HRT treatment can modify gonadotropin levels but cannot influence inhibin B levels. The finding of an isolated low level of inhibin B in TS patients does not necessarily mean that ovarian function is impaired. In fact, it is known that in healthy women inhibin B correlates with the phase of the menstrual cycle (follicular and luteal), and has a daily pulsatility (with peaks of 60 min) and may also be undetectable in a small percentage of healthy women (12, 21). Although this fact restricts the utility of inhibin B as a screening test of ovarian activity, consecutive measurements of undetectable inhibin B in amenorrhoeic TS women should be considered the expression of impaired ovarian activity (22). In conclusion, if our results are confirmed in further studies involving larger study populations, inhibin B could become a fist-line screening test for assessing ovarian reserve. Moreover, it can be considered a much more useful marker to monitor longitudinally the decline of ovarian function in the small percentage of TS patients with spontaneous pubertal development and normal menstrual cycles. These TS girls with active follicles in the ovaries may be ideal candidates for the newer reproductive technologies, but we should make every effort to identify
the narrow window for cryopreservation of ovarian tissue and oocytes. Inhibin B may be a more reliable marker than traditional ones including gonadotropins or morphological images of the uterus and ovaries. This is because its levels are not modified by hormonal treatment, at least according to our preliminary results.
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