Testicular function in patients with regular blood transfusion for thalassemia major

Were enrolled 28 patients with thalassemia major who had received regular blood transfusions at King Chulalongkorn Memorial Hospital. They had received blood transfusion every 4–5 weeks. Patients who had received sex hormone replacement therapy or had a history of testicular surgery or cryptorchidism were excluded. A normal control group comprised 64 healthy boys who were age matched. The clinical data, age, height, weight, the age at diagnosis, the type of thalassemia, the age at initiation of blood transfusion, the age at initial chelation therapy, a history of splenectomy, the previous hemoglobin and serum ferritin levels, were retrospectively reviewed from medical records.

At the time of blood sampling, pubertal status was assessed by genital staging according to Tanner’s classification. Testicular volume was measured using a Prader orchidometer. Testicular volume <4 mL was considered as a prepubertal and ≥4 mL defined as post puberty. An absence of secondary sexual characteristics with a testicular volume <4 mL at the age of 14 years was defined as delayed puberty. Blood samples for hormonal study were collected before blood transfusion. Serum concentrations of LH, FSH, testosterone, and AMH were measured. Informed ascent was obtained from all participants and informed consent was obtained from their parents before being included in the study. The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (approval number 268/2013).

The concentrations of FSH, LH, and testosterone were measured by using a electrochemiluminescence immunoassay (Elecsys and Cobas e analyzers, Roche Diagnostics, Indianapolis, IN, USA). The detection limits of the LH, FSH, and testosterone assays were 0.10, 0.10 mIU/mL and 0.025 ng/mL, respectively. The concentrations of AMH were measured using a two-side enzyme immunoassay (AMH Gen II ELISA, Beckman Coulter, Pasadena, CA, USA).

AMH is a peptide hormone secreted primarily by Sertoli cells, which are active during prepuberty, therefore AMH levels are a useful marker of Sertoli cell function, particularly in the childhood period. The evaluation of this hormone does not require a stimulation test. Serum AMH levels have been reported useful to assess testicular function in many conditions including isolated cryptorchidism, anorchia, childhood cancer survivors, and after hematopoietic cell transplantation [17-20]. Serum AMH levels elevate and become stable during prepuberty and then decrease after the onset of puberty [15, 16]. In our thalassemia major study, they were similar to the normal controls. Furthermore, the levels of AMH in thalassemia were not significantly different from the control group. This suggested that Sertoli cell function is normal in patients with thalassemia major and regular blood transfusions.

Serum gonadotropins (LH, FSH) and testosterone levels in prepubertal patients (n = 15) were not significantly different when compared with controls. The basal levels of serum LH and FSH were not significantly different between pubertal patients (n = 13) and controls suggested that the hypothalamic-pituitary–gonadal axis is intact. Furthermore, these patients had a normal age of pubertal onset and their testicular volumes correlated with Tanner genital stage; the median testicular volume 5 mL in Tanner II, 10–12 mL in Tanner III, and 20 mL in Tanner IV–V. However, serum testosterone levels in pubertal patients were lower than in controls; especially in Tanner IV–V. This suggested that testicular function is diminished, although they have a normal pubertal onset. Rimawi et al. [5] reported that the basal levels of serum testosterone in patients with thalassemia who have a normal puberty, were lower than in normal controls, but this was not significantly different. However, significant differences were seen after a GnRH stimulation test. The basal and peak levels of LH and FSH in patients with β-thalassemia with normal puberty and normal controls were not significantly different. This suggests that Leydig cell function is impaired in β-thalassemia, although puberty is normal. A previous study showed that histological examination of the testicular tissue in thalassemia patients demonstrated varying degrees of testicular interstitial fibrosis with small heavily pigmented undifferentiated seminiferous tubules, hyalinized, and an absence of Leydig cells. This suggested end organ fibrosis secondary to iron overload [21].

Improvement of treatment regimens, consisting of regular blood transfusions and adequate iron chelation therapy, has led to decreased morbidity and mortality in patients with thalassemia major. The prevalence of hypogonadism and/or delayed puberty was reported at 40%–80% [3, 4] by previous studies. This prevalence depended on serum ferritin levels, type of thalassemia, the onset of blood transfusion and chelating therapy [4, 7, 13, 14]. We had no patients with abnormal testicular function or delayed puberty in this study. This can be attributed to adequate treatment with blood transfusion at an appropriate time (3.8 ± 2.3 years old) and effective iron chelation shown by a mean serum ferritin level during the past year of 1,575 ± 642 ng/mL. Bronspiegel-Weintrob et al. [13] reported that patients with abnormal puberty had higher serum ferritin levels before chelation and had received suboptimal chelation therapy (serum ferritin levels 4,734 ± 3,081 and 3,572 ± 1,870 ng/mL, respectively). Important factors for developing hypogonadism and/ or delayed puberty are a high serum ferritin level [10-12]. However, this study did not show such a relationship because there was no delay in puberty.

Limitations of our study include the small number of patients in the sample and that it was a crosssectional study. Our patients were relatively young when compared with other studies. We need longterm follow-up to clarify their testicular function further. Prospective studies of larger participants with other testicular markers such as serum inhibin B are needed to evaluate testicular function in more detail.

Sertoli cell functions can be preserved by regular blood-transfusions and adequate iron-chelation. Leydig cell dysfunction may occur in the late pubertal period despite a normal pubertal onset at an appropriate age. Therefore, testicular function should be regularly evaluated in the long term to detect early abnormalities and so that appropriate treatment can be initiated if necessary.