Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME).

With the motile sperm organelle morphology examination (MSOME), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (up to 6600×). This procedure describes more precisely spermatozoa abnormalities, especially head vacuoles. However, no consensus has been established concerning normal or abnormal MSOME criteria. The aim of our study was to define MSOME vacuole criteria assessed objectively with a digital imaging system software to establish a potential relationship between conventional semen parameters. A total of 440 semen samples were obtained from males consulting in Rouen University Hospital Reproductive Biology Laboratory. Conventional semen analysis (volume, sperm concentration, progressive motility, vitality and morphology) and MSOME assessment {sperm head length, width and area as well as vacuole number, vacuole area and relative vacuole area to sperm head [RVA (%) = [vacuole area (µm(2))/head area (µm(2))] × 100)]} were performed for each semen sample. Among our 440 males, 109 presented normal conventional semen parameters and 331 abnormal ones. Sperm head vacuoles were significantly larger in abnormal semen samples (p < 0.0001). RVA was the most discriminative MSOME criterion between normal and abnormal semen samples according to ROC curves analysis, and was negatively correlated with poor sperm morphology (r = -0.53, p < 0.0001). We concluded to (i) the normal occurrence of vacuoles in sperm head whatever the normality or abnormality of semen parameters, (ii) the discriminative function of the RVA to distinguish semen samples with normal and abnormal parameters, and (iii) the strong correlation between high RVA and poor sperm morphology.

Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining.

Numerous parameters have to be tested to identify optimal conditions for prepubertal testicular tissue banking. Our study evaluated 19 different cryopreservation conditions for immature testicular tissue using a rapid screening method. Immature mice testes were cryopreserved using either 1,2-propanediol (PROH) or dimethyl sulfoxide (DMSO) at a concentration of 0.75 or 1.5 M using a controlled slow-cooling rate protocol with (S+) or without seeding (S+). Equilibration was performed either at room temperature or at 4°C for 15 or 30 minutes. Seminiferous cord cryodamage was determined by scoring morphologic alterations. Cell proliferation ability was evaluated using a proliferating cell nuclear antigen (PCNA) antibody. Testes cryopreserved with optimal conditions were grafted into immunodeficient mice. The highest proportions of PCNA-positive nuclei and lowest morphologic alterations were observed with 1.5 M DMSO. Tissues were more altered with 0.75 M DMSO or PROH. Complete germ cell maturation was observed after allografting of testicular pieces previously frozen with 1.5 M DMSO, S+, 30 minutes. The 1.5 M DMSO, S+ or S+ protocol preserved prepubertal mice testicular tissue architecture and germ cell and Sertoli cell proliferation potential. Allografting of thawed testis fragments into immunodeficient mice confirmed that the 1.5 M DMSO, S+, 30 minutes protocol maintained testicular somatic and germ cell functions. Postthaw histologic evaluation and PCNA immunostaining are useful to rapidly test numerous freeze-thaw parameters. They may also be efficient tools to control human prepubertal frozen testis quality, within the context of a clinical application.

Comparison of conditions for cryopreservation of testicular tissue from immature mice.

BACKGROUND: Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. METHODS: Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. RESULTS: DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. CONCLUSIONS: Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.