Current status of sperm cryopreservation: why isn't it better?

In the USA alone, >650 000 women will be afflicted by cancer in 2003, and 8% of these cases will be aged <40 years. Due to improvements in cancer therapy, cure rates of both adult and childhood cancers increased significantly over the past three decades. However, long-term consequences of cancer therapy and impact on quality of life are now being recognized. One of the major sequelae of cytotoxic chemotherapy is gonadal failure. Cytotoxic chemotherapy and/or radiotherapy are not only used to treat malignant diseases, but also non-malignant systemic conditions. Upon reviewing the extent and mechanism of gonadal damage due to chemo-/radiotherapy, this article discusses indications and the wide range of methods of fertility preservation in a comprehensive manner. All current, emerging, experimental as well as controversial approaches are reviewed. A comprehensive algorithm to manage fertility preservation through an individualized approach is presented.

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Fertility preservation in female patients

The progress of human genome sequencing is driving genetic approaches to define gene function. Strategies such as gene traps and chemical mutagenesis will soon generate a large mutant mouse resource. Point mutations induced by N -ethyl- N -nitrosourea (ENU) provide a unique mutant resource because they: (i) reflect the consequences of single gene change independent of position effects; (ii) provide a fine-structure dissection of protein function; (iii) display a range of mutant effects from complete or partial loss of function to exaggerated function; and (iv) discover gene functions in an unbiased manner. Phenotype-driven ENU screens in the mouse are emphasizing relevance to human clinical disease by targeting cardiology, physiology, neurology, immunity, hematopoiesis and mammalian development. Such approaches are extremely powerful in understanding complex human diseases and traits: the base-pair changes may accurately model base changes found in human diseases, and subtle mutant alleles in a standard genetic background provide the ability to analyze the consequences of compound genotypes. Ongoing mouse ENU mutagenesis experiments are generating a treasure trove of new mutations to allow an in-depth study of a single gene, a chromosomal region or a biological system.

Mouse ENU Mutagenesis

https://s9423e139345e2186.jimcontent.com/download/version/1448400568/module/10206425198/name/2011_Ohtake_JPharmaceutSci100%2C2020.pdf

Trehalose: Current Use and Future Applications

Intercellular Communication via Connexin43 Gap Junctions Is Required for Ovarian Folliculogenesis in the Mouse

Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.

In Vitro Fertilization with Cryopreserved Inbred Mouse Sperm

Mouse half ovaries were cryopreserved and orthotopically transplanted into ovariectomized recipients genetically identical to ovary donors except for the coat color gene. Fertility was reestablished in 57% of the female recipients, which became pregnant in an average of 40 days after transplantation of frozen-thawed half ovaries. These experiments demonstrate that ovary cryopreservation can be a very useful option for banking mouse germplasm, or managing subfertile animal colonies, when embryo or sperm freezing cannot be used or is not cost effective.

Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking.

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation.

Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.

Only primordial and primary follicles of frozen-thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen-thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/ 6J × SJL/J)F 1 mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen-thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen-thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.

Jane S. Farley

Papers

In Vitro Fertilization with Cryopreserved Inbred Mouse Sperm

Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking.

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation.

Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.

Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term