The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice.

BACKGROUND: Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and represent a crucial resource for male fertility restoration. It has not been well documented, however, whether the recovery of SSC population size after cytotoxic damage associates with the kinetics of male fertility restoration. We addressed this issue using the mouse as a model. METHODS: Following single injections of busulfan at 15, 30 or 45 mg/kg into male mice, we examined their ability to sire offspring at different times by natural mating and determined SSC numbers using spermatogonial transplantation. We measured testis physiological parameters (testis weights, sperm counts, serum and intratesticular testosterone levels, and histological assessments of spermatogenic recovery) and quantified the expression of glial-cell-line-derived neurotrophic factor (GDNF) transcripts. RESULTS: Regardless of busulfan doses, fertility was lost within 4 weeks after treatment, while more than 95% of SSCs were lost within 3 days. Fertility and SSC numbers gradually recovered with time, but the recoveries were delayed at higher busulfan doses. Interestingly, SSC numbers reached ∼30% of before-treatment levels by 4 weeks prior to the time of fertility restoration, across the dose groups. Sperm counts were ∼20% of before-treatment levels at the onset of fertility restoration, regardless of busulfan doses. We detected a significant increase in total GDNF mRNA per testis immediately after busulfan treatment. CONCLUSIONS: The loss and restoration of fertility after busulfan treatment are direct consequences of SSC loss and expansion. Our data suggest that there is a threshold in SSC numbers that allows for male fertility restoration and that the testicular somatic environment responds rapidly and temporarily to the loss of spermatogonia, including SSCs, by altering GDNF mRNA levels. This study provides fundamental information to clinically apply SSCs for male fertility restoration in the future.

Current aspects of blastocyst cryopreservation.

Cryopreservation of human gametes and embryos has become an essential part of assisted reproduction. Successful cryopreservation of human blastocysts is increasingly relevant as extended in-vitro culture of human embryos becomes more common, permitting routine use of blastocyst transfer in IVF programmes. This reduces the number of embryos transferred, thereby reducing multiple pregnancies and maximizing cumulative pregnancy rates per oocyte retrieval. The superiority of blastocyst freezing over earlier stage freezing in terms of implantation per thawed embryo transferred improves overall expectations for the cryopreservation programme. Therefore, a reliable procedure for the cryopreservation of blastocysts is needed because, after transfer, only a small number of supernumerary blastocysts are likely to be available for cryopreservation. Since the early 1980s, two common techniques have been used in cryopreservation: the conventional slow cooling method and the more recent rapid procedure known as vitrification. Vitrification has become an attractive alternative to slow freezing, since it appears to result in significantly higher survival and pregnancy rates. The aim of this review is to focus on the cryopreservation of human blastocysts using slow and rapid protocols and to assess the impact of the crypreservation protocol used on the survival, implantation and pregnancy rates.