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Decreased reproductive capacity in transfusion dependent thalassemia (TDT) men is common but addressed infrequently. Previous reports estimate that more than one half of men with TDT are affected by oligospermia and asthenospermia and have abnormal sperm quality due to transfusion-induced iron overload 1, 2. Hypogonadotrphic hypogonadism and subfertility due to increased iron in the pituitary gland has been reported 3. However, additional mechanisms affecting reproduction in TDT men and progression to infertility as it relates to systemic and pituitary iron load are not fully understood, delaying efforts for effective intervention. Oxidative stress, through increased production of reactive oxygen species (ROS), is considered a major contributory factor to male infertility causing damage to the sperm membrane, nucleus, and proteins, thereby impairing sperm quality 4. Normal seminal plasma contains defense mechanisms against ROS consisting primarily of glutathione (GSH) as well as carnitine, folate, vitamins E, C, and A, zinc, and selenium 5. In iron-overload thalassemia patients, excess non-transferrin bound iron (NTBI) and labile plasma iron (LPI) can act as pro-oxidants, resulting in increased generation of ROS. This coupled with low antioxidant defense molecules was suggested as a basis of tissue damage and impaired organ function in thalassemia 6, 7. Whether abnormalities involving these biochemical pathways are the basis for abnormal spermatogenesis and subfertility in thalassemia men with a high iron burden is not well studied. Additionally, the extent of iron-induced pituitary damage resulting in low gonadotropins and affecting reproduction capacity is not known. Thus, we investigated the pituitary-gonadal axis in a subset of TDT patients in the context of their total iron burden. All TDT men, 18 years and older, at our thalassemia center were approached about the study, there was no preselection. Seven TDT men and two normal controls were studied. Anterior pituitary volume and iron accumulation (R2) were measured by pituitary MRI and corresponding Z scores calculated. Sperm count and motility were determined and sperm DNA integrity (DNA fragmentation Index; DFI) was assessed using Sperm Chromatin Structure Assay. Semen plasma elemental content was analyzed by inductively coupled plasma optimal emission spectrometry and GSH was quantified using a liquid chromatography linked tandem mass-spectrometry (LC/MS/MS) assay. NTBI/LPI were analyzed (London, England) and liver iron concentration (LIC), cardiac T2* MRI, ferritin, vitamins C and E, and plasma zinc levels reviewed. All patients (median 26, range 21–30 years) went through spontaneous puberty at age 13–16. Retrospective analysis showed consistent low iron burden in two patients (1 and 4) and variable iron overload in the others (Table 1). Mean pituitary iron (R2) was increased and mean anterior pituitary volume was decreased compare to normal age-matched male controls: R2 16.2 ± 2 vs. 11.2 Hz (Z = 6.0); 329.6 ± 83.3 vs. 596.1 ± 94.4 mm3 (Z = −3.0), respectively. Anterior pituitary volume and LH level correlated with sperm count. Five of the seven patients (70%) had either low sperm count or complete azoospermia and variable sperm motility (Table 2), an important measure of fertilization capacity. DFI was normal indicating no increase in sperm DNA fragmentation. Seminal plasma analysis revealed elevated iron concentration in 5/6 patients, highest in the three patients with azoospermia while iron was undetected in the normal controls. Seminal iron was inversely associated with sperm count and motility (Table 2). The three patients with azoospermia, also exhibited low seminal plasma zinc levels despite a normal plasma range, as well as the lowest GSH concentration, inversely correlating with systemic iron overload. Plasma vitamins C and E were within the low-normal range (not shown) and zinc levels were in low or low-normal range (62–97; nl 70–120 mcg/dL). Our results elucidate causes of infertility in NTDT men; we found high seminal iron in most patients, associated with azoospermia or oligospermia that to the best of our knowledge was not demonstrated before. High seminal plasma iron can lessen sperm motility to 10% of normal, thus significantly affecting reproductive potential 8. Further, the elevated systemic NTBI and LPI and low seminal GSH in our patients, correlating with sperm motility, imply that iron-induced oxidative stress may have a major role in the subfertility often detected in TDT men, beyond the hypogonadism caused by iron in the pituitary gland. Indirect evidence for seminal oxidative stress in TDT men was suggested 9. In nonthalassemia subfertile men low seminal GSH levels were present, while normal GSH levels were associated with better sperm qualities 10. Our results of normal sperm DNA fragmentation, an important measure for fertilization rate, contradict previous findings of increased sperm DNA fragmentation in TDT men 1, 11. It is possible that the relative low iron burden and normal seminal GSH levels found in our three patients were protective against increase DNA fragmentation. If confirmed in a larger study, it could suggest better preserved sperm quality in patients with consistent good iron chelation habits. Alternatively, diminished sperm DNA integrity was attributed to the use of the iron chelator desferroxamine 11, 12, while our three patients were not using this chelation agent. Additionally, low seminal plasma zinc levels detected in most our patients also affects fertility, as zinc is an important micronutrient for DNA repair during spermatogenesis. In males, low zinc levels adversely affected fertilization ability that improved with increase in dietary zinc intake 5, 13. Since zinc deficiency is common in TDT 14 examining the effect of increase zinc intake in TDT men, even with apparent normal plasma levels may be warranted. This study suggests that individuals with high iron burden have decreased pituitary volume and low gonadotropin levels; additionally their seminal environment has a detrimental effect on sperm vitality and quality. In patients with consistent adherence to iron chelation the pituitary size and fertility measures are better preserved. The value of serial measures of pituitary volume and iron concentration needs further study but may prove to have a better predictive value for fertility potential than gonadotropin levels. Aggressive iron reduction along with antioxidant and zinc supplementation could assist with fertility preservation. The use of antioxidants supplementation was shown to improve sperm qualities, though the overall effectiveness remains controversial 4, 15. Clarifying the risk thresholds of these causative factors in larger studies can assist in treatment interventions and fertility preservation. ST Singer, JF Smith, DW Killilela, and J Suh served as investigators on this trial, involved in the study design, data acquisition, interpretation, and reporting as well as drafting and critically reviewing the manuscript. K Ivani and P Evans participated in data acquisition and analysis. J Wang, Q Yuan, and R Fischer performed and interpreted pituitary iron measurements and contributed to the analysis and reporting of the trial data. E Vichinsky participated in interpreting the data and critically reviewing this manuscript. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Sylvia T. Singer1*, David W. Killilea2, Jung H. Suh2, Zhiyue Jerry Wang3, Qing Yuan3, Kristen Ivani4, Patricia Evans5, Elliott Vichinsky1, Roland Fischer6, James F. Smith7 1Department of Hematology/Oncology, Children's Hospital and Research Center at Oakland, California; 2Children's Hospital Oakland Research Institute, Oakland, California; 3Department of Radiology, Children's Medical Center of Dallas and UT Southwestern Medical, Dallas, Texas; 4Reproductive Science Center (RSC) of the SF Bay Area, San Ramon, California; 5Research Department of Haematology, University College London, London, WC1E 6BT, United Kingdom; 6Pediatric Hematology/Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Delaware; 7Department of Urology, University of California, San Francisco, California Additional Supporting Information may be found in the online version of this article. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.