Sperm DNA fragmentation after radioiodine treatment for differentiated thyroid cancer

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).

Treatment by ¹³¹I induces perturbations in semen parameters

TSC was lower than normal values 12 months after the second dose of radioiodine (32 × 10⁶ spermatozoa). At twenty-eight months, SC, TSC, MOT and VIT returned to normal values. By 57 months post-treatment, all semen parameters were normal.

DNA fragmentation

The DNA fragmentation index (DFI) measured by SCSA was under the threshold value of 20 %, defined in our laboratory [16] as the 90th percentiles of SCSA values of 51 fertile men, for all samples except for specimen 1 collected 3.25 months after the first dose of ¹³¹I, where DFI was highest (24.5 %) (Fig. 2).

Values decreased to 12.9 % and 16.5 % at 5 and 5.25 months after the first dose. Twelve months after the second dose of ¹³¹I the value remained stable at 16.1 %. The lowest values were found at 28 and 57 months after the second dose of radioiodine (6.55 % and 11.8 %, respectively). HDS values remained constant during the whole study period (around 4 %), under the threshold value of 7.5 %, with a slight elevation to 6.45 % at 5.25 months after the first dose (Fig. 2).

The percentage of cells with fragmented DNA assessed by TUNEL was in the normal range during the study, i.e. <16.5 % [16] (Table 1), although the highest value was found 3.25 months after the first dose of ¹³¹I (10.5 %). The technique could not be performed in samples at 5 and 5.25 months after the first dose of radioiodine due to an insufficient number of spermatozoa.