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Frozen testicular tissue still viable after 20 years

By: Katherine Unger Baillie | Kbaillie@Upenn.Edu | 215-898-9194 Date: May 10, 2022
After being transferred to an infertile mouse, testes tissue from a rat that had been frozen for more than two decades gave rise to sperm and germ cells. (Image: Eoin Whelan/PLOS Biology/CC-BY 4.0
After being transferred to an infertile mouse, testes tissue from a rat that had been frozen for more than two decades gave rise to sperm and germ cells.
(Image: Eoin Whelan/PLOS Biology/CC-BY 4.0

The rate of survival for childhood cancers has increased dramatically in the last several decades, but a serious side effect of treatment is diminished fertility later in life. A potential treatment for boys facing cancer treatment would be to harvest, freeze, and, after the cancer is cured, reimplant their testicular tissue, which contains stem cells that could give rise to sperm.

What happens to that tissue, however, when subjected to the long-term freezing that could be necessary, has remained unclear.

Dr. Eoin Whelan, Penn Vet
Dr. Eoin Whelan

A new study in rats led by University of Pennsylvania School of Veterinary Medicine researchers has shown that male testis tissue that is cryopreserved can be reimplanted after more than 20 years and will go on to make viable sperm. The work, led by senior research investigator Eoin C. Whelan, was published in PLOS Biology.

While the long-frozen testicular tissue could produce sperm, the team found that the long delay did come with a cost in reduced sperm production compared to tissue that is only briefly frozen. The results may have important implications for treatment of prepubertal boys with cancer, for whom chemotherapy may be preceded by harvesting and freezing of testicular tissue for eventual reimplantation.

“The glass-half-empty way of looking at this is that stem cells do seem to be compromised in their ability to regenerate sperm after a long freezing time,” Whelan says. “But the good news is that sperm can be produced, and they seem to be transcriptionally normal when we examined their RNA.”

Dr. Ralph Brinster, Penn Vet
Dr. Ralph Brinster

The study was conducted in the laboratory of Ralph L. Brinster, the Richard King Mellon Professor of Reproductive Physiology at Penn Vet, a renowned scientist of reproductive biology.

“The findings are critical in considering transplantation of stem cells from testis biopsies obtained from prepubertal boys undergoing cancer treatment for later use in reestablishing spermatogenesis following recovery,” says Brinster. “Early transplantation of these cells is clearly better than waiting a long time before reintroducing the cells into the testis.”

Challenges of reimplantation

If a boy is treated for cancer at a young age, they might go a decade or more between having their testicular tissue harvested and having it reimplanted, raising the question of how long frozen spermatogenic stem cells (SSCs) can remain viable. To find an answer, the authors thawed rat SSCs that had been cryopreserved in their laboratory for more than 23 years and implanted them in so-called nude mice, which lack an immune response that would otherwise reject the foreign tissue. They compared the ability of the long-frozen SSCs to generate sperm to SSCs frozen for only a few months, and to freshly harvested SSCs, all from a single rat colony maintained for several decades.

The authors found that the long-frozen SSCs were able to colonize the mouse testis and generate all the necessary cell types for successful sperm production, but not as robustly as SSCs from either of the more recently harvested tissue samples. While the long-frozen SSCs had similar profiles of gene expression changes compared to the other samples, they made fewer elongating spermatids, which go on to form swimming sperm.

“When we looked at the cells immediately after thawing, they looked the same as cells that had been frozen for a short period of time,” says Whelan. “It was only after we transplanted the testicular tissue that we started to see a difference in the efficiency of sperm production.”

A road map for future research and application

These results have several important implications. First, they point out the importance of testing SSCs viability by directly tracking results after reimplantation to determine the potential of cryopreserved cells. Relying on biochemical or cellular biomarkers may not reflect the actual loss of stem cell potential over time.

Extended to humans, the findings show the potential of preserving fertility of boys undergoing cancer treatment. (Image: Eoin Whelan)
Extended to humans, the findings show the potential of preserving fertility of boys undergoing cancer treatment.
(Image: James Hayden, RBP, FBCA in Brinster, R. L. Male germline stem cells: From mice to men. Science. 2007 Apr; 316: 404-405

Second, while there currently are no protocols that can expand human SSCs for reimplantation—a requirement to translate this treatment into clinical use—such protocols may need to consider time-dependent degradation of viability, assuming human SSCs mimic those of rats.

Third, the findings underscore that sperm can be produced from long-preserved testicular tissue. Further research to potentially identify and mitigate the key drivers of viability loss could improve the reproductive options of boys whose childhood cancers are successfully treated.

“A major follow-up question is ‘What’s causing this? What’s the mechanism of operation?’” says Whelan. Ongoing work is investigating some of the genes that the team’s analysis turned up as being altered by the long-term freezing.

The findings may also hold important clues for preserving other types of stem cells, which are increasingly being investigated in a range of therapeutic applications.

“I think the research has broad relevance to freezing of all types of stem cells, which could undergo similar changes in gene regulation,” says Brinster.

Ralph L. Brinster is the Richard King Mellon Professor of Reproductive Physiology at the University of Pennsylvania School of Veterinary Medicine.

Eoin C. Whelan is a senior research investigator at Penn’s School of Veterinary Medicine. 

Whelan was first author and Brinster corresponding author on the paper. Their coauthors were Fan Wang, Mary R. Avarbock, Megan C. Sullivan, and Daniel P. Beiting of Penn Vet.

This work was supported by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.

Adapted with permission from a press release by PLOS Biology.

About Penn Vet

Ranked among the top ten veterinary schools worldwide, the University of Pennsylvania School of Veterinary Medicine (Penn Vet) is a global leader in veterinary education, research, and clinical care. Founded in 1884, Penn Vet is the first veterinary school developed in association with a medical school. The school is a proud member of the One Health initiative, linking human, animal, and environmental health.

Penn Vet serves a diverse population of animals at its two campuses, which include extensive diagnostic and research laboratories. Ryan Hospital in Philadelphia provides care for dogs, cats, and other domestic/companion animals, handling more than 34,600 patient visits a year. New Bolton Center, Penn Vet’s large-animal hospital on nearly 700 acres in rural Kennett Square, PA, cares for horses and livestock/farm animals. The hospital handles more than 6,200 patient visits a year, while our Field Services have gone out on more than 5,500 farm service calls, treating some 18,700 patients at local farms. In addition, New Bolton Center’s campus includes a swine center, working dairy, and poultry unit that provide valuable research for the agriculture industry.