Differentiated thyroid cancer (DTC) is the most common endocrine malignancy. It affects any age group and sex, and its incidence has been increasing for the last 20 years [1, 2]. Treatment of DTC includes thyroidectomy, followed by nuclear medicine therapy with radioiodine 131I for eradication of persistent disease and ablation of residual thyroid remnant, to facilitate post-treatment radiological and clinical follow-up of the patient [3]. 131I treatment can damage radiation-sensitive tissues, and it has been shown that some radiation is delivered to the testes after administration of radioiodine [4]. Some studies have reported impairment in spermatogenic functions such as elevated serum FSH levels, reduced sperm count resulting in some cases in oligozoospermia [5–8]. These effects were usually not permanent and were reversed within 18 months, although permanent damage has been reported in patients receiving repeated or high cumulative doses [9]. Radiations are known to induce double strand breaks in DNA, and it has been shown that treatment with 131I induces a rise in γH2AX and 53BP1 DNA repair foci in blood cells [10]. There are no reports on potential effects on spermatozoa DNA, although it has been shown that higher levels of DNA strand breaks are associated with infertility and miscarriages [11].
We report the case of a 32-year-old man in whom several sperm analyses were carried out after treatment with 131I for differentiated thyroid carcinoma. Each sample was analyzed for classic semen parameters. For the first time, we report the effects of radioiodine therapy on spermatozoa chromatin and DNA, evaluated by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.
Subject
A 32-year-old man presented with thyroid carcinoma classified pT3N1R1. One month after a total thyroidectomy, he was treated with 150 mCi of radioiodine (131I). A second dose of 150 mCi 131I was administered 7 months later. The patient was referred to the CECOS for sperm banking 3 months after the first dose of 131I and before the scheduled second dose. He was seen twice during the two months after his first sperm banking, and three times after his second dose of 131I, at 12, 28 and 57 months. The patient gave a total of 6 samples, 3 for sperm banking and 3 for exploration (Fig. 1). The present study was carried out in accordance with the Declaration of Helsinski and the patient gave his informed consent for the study. All samples and exposure data were provided by the GERMETHEQUE Biobank (France).
Semen analysis
Sperm samples were collected by masturbation after 3–6 days of sexual abstinence, except for sample 1 where abstinence was 30 days. Semen was allowed to liquefy 30 min at 37 °C before parameters were analyzed. Conventional semen analysis was performed according to WHO criteria [12]. Sperm count (SC), total sperm count (TSC), motility a + b (rapidly progressive—grade a, slowly progressive—grade b) (MOT) were assessed. Sperm vitality (VIT) was determined by eosin-nigrosin. Semen samples were cryopreserved within 1 h of collection according to the standard procedures used for sperm banking in our laboratory until later processing for testing DNA integrity [13].
Spermatozoa fixation
Prior to SCSA and TUNEL, cells were fixed with 1 % paraformaldehyde. Briefly, spermatozoa were collected by thawing straws of the different samples at room temperature. Cryoprotector medium was washed by adding 5 ml of PBS medium, drop by drop to 1 ml then more rapidly for the remaining 4 ml, and centrifuging at 630 g for 10 min. Cells we resuspended in 1 ml of PBS and 4 ml of 1 % paraformaldehyde and incubated 30 min at room temperature. Samples were then centrifuged 10 min at 1500 g, the supernatant discarded and cells resuspended in the appropriate volume of PBS. Samples were kept at 4 °C until processed. A minimum of 13 × 106 spermatozoa were needed for SCSA and TUNEL analyses. In case of oligozoospermia (ie 5 months after Iodotherapy) only SCSA test was possible on 4 × 106 spermatozoa.
Sperm chromatin structure assay
Sperm DFI and high DNA stainability (HDS) were measured according to conventional sperm chromatin structure assay (SCSA) as described by Evenson and Jost [14] and routinely used in our laboratory. In brief, 2 × 106 cells were centrifuged at 1500 g for 10 min and transferred into a cytometer tube. Each sample was run in duplicate. Cells were resuspended by vortexing, then 0.4 ml of acid detergent (pH 1.2) was added. The tube was gently shaken for 30 s before adding 1.2 ml of acridine orange and incubated 3 min on ice. 5,000 cells were acquired by fluorescence-activated cell sorting (FACS) on a FC500 cytometer (Beckman Coulter Inc., Fullerton, CA, USA). The DNA fragmentation index (DFI) was defined as the ratio of denatured DNA (red fluorescence) to total DNA (red + green fluorescence). High DNA stainability (HDS) was calculated as the percentage of cells presenting a high level of red fluorescence.
DNA strand break measurement
DNA strand breaks were measured using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. A detailed protocol for the TUNEL assay of human sperm has previously been described [15]. Terminal deoxynucleotidyl transferase (TdT) enzyme, reaction buffer, CoCl2 and dUTP biotin were purchased from Roche Diagnostics (Mannheim, Germany). Streptavidin FITC was purchased from Tebu-Bio (Le Perray-en-Yvelines, France), and propidium iodide from Sigma Aldrich (St Louis, MO, USA). For each specimen, a negative sample without enzyme and two positive samples with enzyme were tested. The percentage of cells presenting DNA strand breaks was calculated by subtracting the percentage of fluorescence in the control (no TdT enzyme) from the mean percentage of fluorescence of the two TdT-positive samples. 10,000 cells were acquired by FACS on a FC500 cytometer (Beckman Coulter Inc., Fullerton, CA, USA).
) and High DNA Stainability (HDS) (
) were measured on 2 x 106 cells by the SCSA assay. Each dot represents the mean of duplicates for each sample, values are reported on the graph. 
represents surgeries, 
represents 131I treatments. The Y axis represents the values in % of DFI and HDS