Background: Iron overload in association with persistent anemia is responsible for endocrine dysfunction in β-thalassemia patients, blood transfusion combined with iron-chelation can modify life quality in these children, but they tend to suffer from delayed maturity and endocrine dysfunction. Aim: This study aims to correlate degree of hypogonadism to ferritin load in regular transfused β-thalassemia patients. Methods: It was carried out on 30 β-thalassemia major (TM) patients aged 12 to 18 years, puberty was assessed clinically, blood picture on Cell-Dyne 2700, ferritin level and pattern of FSH, LH, testosterone and estradiol before and after gonadotropin (GnRH) analogue stimulation test, they were determined on ARCHITECT ABBOTT system. Results: Twenty patients had not yet achieved puberty, FSH level was 1.45 ± 1.88 mIU/ml before (GnRH) analogue and 3.78 ± 4.19 mIU/ml after 4 hours of injection. LH level was 1.91 ± 4.79 mIU/ml before (GnRH) test, while after 4 hours it was 6.52 ± 7.50 mIU/ml, 88.24% of males had low serum testosterone level, 84.6% of girls had low serum estradiol level, FSH, LH, estradiol, testosterone before and after GNRH analogue were statistically insignificant, mean ferritin level was 3344.32 ± 1142.142 ng/ml, with insignificant correlation to hormonal pattern before and after GnRH therapy. Conclusion: Iron overload and hypogonadism are the presenting data in this study, insignificant correlation between ferritin level and hormonal reserve pattern, there may be another etiology in pathophysiology of low gonadal reserve such as severe anemia, chronic disease and may be genetic predisposition underlying susceptibility to iron toxicity, which need further investigations.
Thalassemia is a hereditary chronic anemia highly prevalent in the Mediterranean countries [
It is a prospective study; done on thirty β-TM patients, seventeen boys and thirteen girls, their age ranged from 12 to 18 years old. They were selected from the Hematology Outpatient Clinic in Sohag University Hospital. Cases were recruited during the study period from July 2013 to June 2014. Because we investigate the gonadal reserve reflected by the response to GnRH analogue stimulation test to hypothalamic pituitary gonadal axis; as a therapeutic test being already done in these patients group, but in normal healthy boys and girls we can’t apply this test to avoid hormonal disturbance during this critical period. Instead control group we used the documented reference values to determine hypogonadism and consider βTM patients who reach the normal puberty for comparison.
β-TM patients with iron overload on chelating therapy, their serum ferritin more than 1000 ng/ml; aged 12 - 18 years old on regular transfusion program 1: 2 packed RBCs units at frequency/2 weeks to 3 months according to Hb level and body weight, to maintain hemoglobin level more than 9 g/dl.
Pediatric patients less than 12 years or received hormonal replacement therapy as estrogen, progesterone, testosterone or GnRH analogue or had another disease that delayed puberty. Other causes of iron over load rather than βTM like hereditary hemochromatosis
Approval of Sohag Faculty of Medicine Research Ethical Committee and consent from the parents will be obtained.
All cases included in this work will be subjected to complete history, clinical evaluation for weight, age, and sexual developmental, degree of pallor, Jaundice and general condition. Pubertal stages were determined by Marshal and Tanner scale of physical development in children, adolescents and adults [
blood samples were taken from patients at morning at least two weeks after the blood transfusion; total of 3ml blood was withdrawn from each patient delivered into K-EDTA tube for complete blood picture using electronic counter Cell Dyne 2700 (USA), and reticulocyte count was assayed, then centrifuged, the plasma was delivered to ARCHITECT i Optical System, Abbott Ireland Diagnostics Division, Chemiluminescent Microparticle Immunoassay (CMIA) for the quantitative determination of ferritin, FSH, LH and testosterone. The patients were subjected to GnRH stimulation test (subcutaneous injection of 0.1 mg decapeptyl; GnRH analogue to detect response of pituitary to GnRH), four hours later, the second sample was taken and subjected to post GnRH stimulation test hormonal assay, and then the hormonal levels were correlated with the pubertal scale of the patients and ferritin level.
Ref. No. B7K590/49-1515/R4 [
Assay procedure: Is a two-step immunoassay to determine ferritin in serum or plasma using a chemiluminescent microparticle immunoassay (CMIA) technology with flexible assay protocol, referred to as chemiflex. In the first step, sample and anti-ferritin coated paramagnetic microparticles are combined. Ferritin present in the sample binds to the ant ferritin coated micro particles. After washing, anti-ferritin acridinium labeled conjugate is added in the second step, pre-trigger and trigger solutions are then added to the reaction mixture; the resulting chemiluminescent reaction is measured as relative light units (RLUs). A direct relationship exists between the amount of ferritin in the sample and the RLUs detected by using the Architect i optical system. Before loading the ARCHITECT ferritin reagent kit on the system for the first time, the microparticles bottle was mixed to resuspend microparticles that have settled by inverting the microparticles bottle 30 times. Then we inspect the microparticles if microparticles are still adhered to the bottle, repeat mixing until the microparticles have been completely resuspended. Once the microparticles have been suspending, the cap was removed and discards, and the reagents were loaded on the ARCHITECT I System. ARCHITECT ferritin calibrators, ARCHITECT ferritin controls should be mixed by gentle inversion prior to use, hold the bottles vertically and 4 drops were dispensed of each calibrator or 3 drops of each control into each respective sample cup. Then the samples were loaded. When we press RUN the ARCHITECT i System performs the following function: moves the sample to the aspiration point, loads a reaction vessel (RV) into the process rout, aspirates and transfers sample into the RV, advances the RV one position and transfers microparticles into the RV, mixes, incubates and washes the reaction mixture, adds conjugate to the RV, mixes, incubates and washes the reaction mixture, adds pre-trigger and trigger solutions, measures chemiluminescent emission to determine the quantity of ferritin in the sample, aspirates contents of RV to liquid waste and unloads RV to solid waste. Calibration: perform an ARCHITECT ferritin calibration, test ARCHITECT ferritin calibrators 1 and 2 in duplicate. A single sample of all levels of ARCHITECT ferritin controls must be tested to evaluate the assay calibration. Calibrators should be priority loaded. The assay protocol extends the assay range to 0 - 2000 ng/ml. Once an ARCHITECT Ferritin calibration is accepted and stored, all subsequent samples may be tested without further calibration. Normal ferritin range in males is 21.81 - 274.66 ng/ml and the mean is 75.62 ng/ml, for females the range is 4.63 - 204.0 ng/ml and the mean is 39.42 ng/ml.
The following assays were processed by ARCHITECT in the same way as ferritin.
Ref. No. B7K750 G4-5970/R09 [
Ref. No. B2P400/G3-0641/R04 [
Ref. No. B2 P130/ABB L311/R07AxS [
Ref.No.B2K 3C85-20/AxS [
Z test; test of proportion was used to compare between 2 percentages, Chi- square test (x2) was used to compare between more than 2 percentages, Student “t” test to compare 2 mean and S.D of 2 groups, Paired “t” test was used to compare between 2 mean and S.D in the same group before and after treatment, ANOVA test was used to compare between different groups.
The current study was included 30 β-TM patients; seventeen boys (56.67%) and thirteen girls (43.33%). The mean age of the patients was 13.91 ± 2.14 years; the mean of weight was 31.4 ± 7.98 kg; the mean of height was 134.23 ± 15.60 cm. As regard head circumference was 53.07 ± 2.80 cm. The mean frequency of blood transfusion was 1.06 ± 0.29/month. Splenectomy was done in 23 patients (76.67%). All the cases (30 patients) had taken routine vaccination. HCV infection was detected in 7 patients (23.33%), and no one had HBsAg. The demographic, anthropometric and clinical data were represented in
Item | Statistical values |
---|---|
Age in years (mean ± S.D) | 13.91 ± 2.14 |
male/female | 17/13 |
Weight /kg (mean ± S.D) | 31.4 ± 7.98 |
Height in cm (mean ± S.D) | 134.23 ± 15.6 |
Head circumference (cm) (mean ± S.D) | 53.07 ± 2.80 |
Frequency of blood transfusion unit/month (mean ± S.D) | 1.06 ± 0.29 |
Splenectomy (case) | 23 |
Vaccination (case) | 30 |
HCV positive (case) HBsAg positive (case) | 7 0 |
RBCs ´ 109/l (mean ± S.D) | 2.62 ± 0.68 |
Hb g/dl (mean ± S.D) | 6.79 ± 1.77 |
Hct % (mean ± S.D) | 22.06 ± 4.89 |
Ferritin ng/ml (mean ± S.D) | 3344.32 ± 1142.142 |
FSH mIU/ml (mean ± S.D) | 1.45 ± 1.88 |
LH mIU/ml (mean ± S.D) | 1.91 ± 4.79 |
Estradiol pg/dl (mean ± S.D) | 11.02 ± 18.80 |
Testosterone ng/ml (mean ± S.D) | 17.0 ± 0.68 |
FSH: Follicle Stimulating Hormone, LH: Luteinizing Hormone, HCV: Hepatitis C virus, HBsAg: Hepatitis B virus surface antigen, RBCs: Red Blood Cells, Hb: Hemoglobin, Hct: Hematocrit.
gard Tanner scale, there was twenty cases (66.67%) had not attained puberty in stage (1). Seven cases (23.33%) were belonging to stage (2). Stage (3) has 2 cases (6.67%). There is only one case in stage (4). The hormonal pattern in these patients before and after GnRH analogue stimulation test showed FSH level was 1.45 ± 1.88 mIU/ml before and after 4 hours it was 3.78 ± 4.19 mIU/ml. FSH level was highly significant low in 60% of cases with p-value < 0.0001. Also LH level was 1.91 ± 4.79 mIU/ml before GnRH, while after 4 hours of GnRH therapy it was 6.52 ± 7.50 mIU/ml, LH level was highly significant low in half of the patients; with p-value <0.0001.The testosterone level was 17 ± 0.68 ng/ ml. Fifteen boys (88.24%), had low serum testosterone level, and only two had normal level. While the mean estradiol level in the girls was 11.02 ± 18.80 pg/dl, about eleven girls (84.62%) had low level of serum estradiol and only two cases (15.38%) had normal estradiol level as in
When we correlate ferritin levels to hormonal pattern, we found that; FSH level before GnRH analogue was low in eighteen patients with the mean ferritin level 3578.52 ng/ml, and twelve patients had normal FSH with the mean ferritin
Tanner Scale Number (N) | 1 N = 20 | 2 N = 7 | 3 N = 2 | 4 N = 1 | P-values |
---|---|---|---|---|---|
Pre GnRH test | |||||
FSH mIU/ml Low Normal | 15 5 | 2 5 | 0 2 | 1 0 | 0.04 S |
LH mIU/ml Low Normal High | 13 6 1 | 1 6 0 | 0 2 0 | 1 0 0 | 0.13 NS |
Estradiol pg/dl Low Normal | 6 2 | 3 0 | 2 0 | 0 0 | 0.48 NS |
Testosterone ng/ml Low Normal | 11 1 | 3 1 | 0 0 | 1 0 | 0.62 NS |
Post GnRH test | |||||
FSH mIU/ml No Mild Moderate Good | 8 9 2 1 | 1 4 1 1 | 0 0 2 0 | 0 1 0 0 | 0.13 NS |
LH mIU/ml No Mild Moderate Good | 5 11 3 1 | 1 3 3 0 | 0 0 2 0 | 0 1 0 0 | 0.42 NS |
level 2954.58 ng/ml. About the LH pattern we noticed that, before GnRH analogue, fifteen patients had low LH level with the mean ferritin level 3481.20 ng/ml, and fourteen cases had normal LH level, their ferritin level was 3027.00 ng/ml, only one case with high LH level; the ferritin was 5272.00 ng/ml. After GnRH stimulation test we found that, FSH had good response in 2 cases (6.67%), their mean ferritin was 1118.95 ng/ml, mild response in 14 cases (46.67%) and moderate response in 5 cases (16.67%), their mean ferritin level was 314,289 and 3117.24 respectively. No response was found in 9 patients (30%) with the mean ferritin level was 4227.4 ng/ml. While LH after GnRH therapy revealed the following; only one case (3.33%) had good response LH more than 20 mIU/ml with the mean ferritin level was 5272.0 ng/ml, mild response in 15 cases (50%); their ferritin level was 2712.9 ng/ml, moderate response in 8 patients (26.67%), with the mean ferritin was 3106.6 ng/ml, and no response in 6 patients (20%), their mean ferritin was 4841.6 ng/ml. These data were presented in
Items | Hormonal Pattern | Patient number and (%) | Mean ferritin ng/ml | P value | ||
---|---|---|---|---|---|---|
Pre GnRH test | ||||||
FSH mIU/ml | Low Normal | 18 12 | 60 40 | 3578.52 2954.58 | 0.46 NS | |
LH mIU/ml | Low Normal High | 15 14 1 | 50 46.67 3.33 | 3481.20 3027.00 5272.00 | 0.59 NS | |
Estradiol pg/dl | Low Normal | 11 2 | 84.62 15.38 | 2731.38 2316.55 | 0.71 NS | |
Testosterone ng/ml | Low Normal | 15 2 | 88.24 11.78 | 3945.20 3006.00 | 0.64 NS | |
Post GnRH test | ||||||
FSH mIU/ml | No Mild Moderate Good | 9 14 5 2 | 30 46.67 16.67 6.67 | 4227.40 3142.69 3117.24 1118.95 | 0.31 NS | |
LH mIU/ml | No Mild Moderate Good | 6 15 8 1 | 20 50 26.67 3.33 | 4841.60 2712.92 3106.61 5272.00 | 0.18 NS | |
FSH: Follicle Stimulating Hormone: no response: <0.95 mIU/ml, mild increase: 0.95 - 5 mIU/ml, moderate increase: 5 - 11 high increase: >11 mIU/ml, LH: Luteinizing Hormone: no response: <0.3 mIU/ml, mild increase: 0.3 - 10 mIU/ml, moderate increase: 10 - 20 mIU/ml, high increase: >20 mIU/ml, Estradiol: 15 - 350 pg/dl, Testosterone: 2.4 - 8.2 ng/ml, GnRH: Gonadotropin Releasing Hormone.
The real distribution pattern of ferritin level for each individual patient was illustrated in
Β-TM patients characterized by repeated blood transfusions with iron deposition in different organs like liver, heart, and the endocrine glands, so tissue damage and organ failure occurred [
overload and improving overall survival. Despite the use of iron chelation therapy, the pituitary gland, peripheral endocrine tissues and gonadal axis are susceptible to iron toxicity [
on eleven girls only 3 of them at eighteen years old had regular menses, six of twenty boys at nineteen years old; had the criteria for puberty and overall, 22 patients had hypogonadism [
sult, others found that; within the low gonadotropin reserves, most of the patients with delayed puberty showed normal response to exogenous GnRH [
From our data, we can conclude that there is prominent decrease in gonadal reserve with striking iron overload in these patients, but a non-significant correlation between them was detected. These findings can open the way for thinking about other etiological aspects and pathophysiological mechanisms that lead to delayed sexual maturity, such as genetic predisposition increase susceptibility to iron toxicity or increase tolerance to iron overload; other factors, the most important Hb level and synchronous endocrine affection, so further investigations are necessary for proper management.
β-TM patients during adolescence must be subjected to throughout investigations and follow up program to keep the gonadal reserve at a range that maintain normal life.
Abo-Elwafa, H.A., Hamid, S.A., Heshmat, M.M. and Ahmed, Z.S. (2017) Impact of Ferritin Load on Go- nadal Reserve among Regular Transfused β-Thalassemia. Open Journal of Blood Di- seases, 7, 65-78. https://doi.org/10.4236/ojbd.2017.72007