Impact of Ionizing Treatment on the Nuclear Structure of Human Spermatozoa.

Impact of Ionizing Treatment on the Nuclear Structure of Human Spermatozoa.

Differentiated thyroid cancer is the third cause of cancer in young men of childbearing age. Its treatment by irradiation with Radioactive Iodine 131 therapy (RAT) could alter spermatogenesis and result in azoospermia and permanent infertility. A preventive gametes cryopreservation was recommended before RAT, but without mentioning a period of teratogenic risk transmissible to the offspring. To date, RAT impact on human sperm nucleus is poorly known or even unknown, notably on telomere length.

Our objective is to define RAT effects on human sperm nucleus by in vitro irradiation exposure of human spermatozoa to mimicking that of the gonads in the context of irradiation with iodine131 used for thyroid cancer. We will analyze standard sperm parameters, major DNA alterations and telomere length using molecular and cellular assays. Nucleus morphology and chromatin organization will also be analyzed using 3D bio-imaging. This study will permit to optimize the indications for the preservation of fertility.

Our main objective is to measure the in vitro impact of irradiation treatment on sperm nuclear quality such as DNA fragmentation and oxidation, chromatin condensation and organisation, nucleus morphology and notably sperm telomere length (STL).

The secondary objectives are :

• to measure RAT impact on sperm parameters (vitality, motility and morphology)
• to measure the impact of cryopreservation on chromatin organisation and nucleus morphology
• to evaluate RAT impact on human sperm cells in comparison with cryopreservation
• to evaluate the impact of different doses and types of irradiation on human sperm cells
• to establish relations between potential alterations of standards sperm parameters (vitality, motility and morphology) and nuclear sperm parameters (DNA fragmentation and oxidation, chromatin condensation and organisation, nucleus morphology and STL.

Our final goal is to provide a significant improvement of men fertility diagnosis and optimize our fertility preservation practices.

To this end, we will expose ejaculated human spermatozoa (n = 90) at different (low and moderate) doses of gamma or X photons to mimic gonads irradiation. Absorbed doses by the samples were calculated using the GATE Monte Carlo platform (version 8.2). Experiment geometry settings were modelled as three dimension voxelized volumes inside the software by assigning a shape, size, distance and density for all the volumes created. All measurements will be made on surplus samples from men undergoing routine semen analysis at the Center for Reproductive Medicine, after receiving their written informed consent. Semen samples are collected and subdivided into 3 arms to analyse sperm quality after:

• irradiation exposure (3 conditions);
• a freezing- thawing cycle;
• fresh state: negative control without treatment.

Before (fresh state) and after each treatment we will analyse:

• standard semen parameters (vitality, motility and morphology) in accordance with WHO, 2010;
• STL using Flow FISH, q-PCR and q-FISH;
• chromatin condensation (chromomycin A3);
• DNA oxidization (8-OHdG residues);
• DNA fragmentation (TUNEL);
• 3D nucleus structure using 3D bio-imaging.

Inclusion Criteria:

• Man less than 45 years
• Man undergoing routine semen analysis at the Center for Reproductive Medicine, accepting the research protocol and signing the associated consent will be included in the protocol without distinction of physical criteria.

Exclusion Criteria:

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