Rodent Testicular Tissue Successfully Implanted After Being Frozen for 20 Years

By Linda Carroll

May 11, 2022

NEW YORK (Reuters Health) - In a development that might one day help boys getting treatment for cancer, researchers showed that rat testicular tissue implanted in a mouse after being cryopreserved for 20 years could colonize recipient testes and generate cell types necessary for sperm production, a new study finds.

The experiment also revealed that the long-frozen rat spermatogenic stem cells, while viable, were not as robust as those from recently harvested and cryopreserved samples, researchers report in PLOS Biology.

The study shows that "spermatogonial stem cells - that is, stem cells that make sperm - frozen for over 20 years were recovered, and once transplanted into an infertile animal, successfully regenerated the ability to produce sperm," said Dr. Eoin Whelan of the University of Pennsylvania School of Veterinary Medicine, in Philadelphia.

"We expect our findings can guide cryopreservation of human spermatogonial stem cells," he told Reuters Health by email. "This has applications for the recovery of fertility in pre-pubertal boys treated for cancer."

"These patients are too young to freeze sperm for and suffer reduced or absent fertility after chemotherapy," Dr. Whelan said. "Cryopreservation of testis biopsies taken before chemotherapy could allow for repairing fertility after the cancer has been destroyed."

To learn whether long frozen testicular tissue could be thawed, transplanted, and ultimately produce new sperm cells, Dr. Whelan and his colleagues turned to rat spermatogenic stem cells (SSCs) that had been collected and frozen more than 23 years earlier. For comparison, the researchers also collected and froze rat SSCs, some for one month and some for four months.

Dr. Whelan and his colleagues thawed the SSCs and implanted them in busulfan-treated nude mice, which lack the ability to produce an immune response to foreign tissue. The busulfan treatment removed endogenous mouse germ cells, leaving empty niches available for colonization by the thawed rat germ cells.

All three batches of SSCs were able to colonize the mouse testis and to generate all the necessary cell types for sperm production, but those frozen over 23 years earlier were not as robust as the SSCs from either of the two more recently harvested samples.

"For all stem cells in tissue there is a delicate balance of making more stem cells and differentiating cells," Dr. Whelan explained. While the SSCs "still regenerated tissue and all cell types, proportionally more of the new cells were observed in the stem cell stage and less in the later, differentiating, stages. We observed evidence that the stem cells were producing signals consistent with their own replenishment as opposed to production of the later stages of germ cells, in line with the comparative lack of the sperm and a buildup of stem cells."

The long frozen SSCs resulted in only 25% of normal sperm production, Dr. Whelan said.

"Under normal conditions, after meiosis, the germ cells go through the process of transforming into sperm, and their DNA structure is dramatically changed," he added. "Proteins called protamines are used to package the DNA instead of the normal structural proteins. In tissue regenerated from long-frozen samples, after meiosis cells often lacked protamines, suggesting these cells were not able to properly package their DNA. We also observed lower numbers of elongating spermatids, the final stage right before the production of sperm. Taken together, long-frozen cells struggled to produce the final stages of sperm production."

He added, "In addition to medical applications, this study is relevant for freezing of sperm-producing stem cells for preservation of endangered species as well as use in preserving and disseminating the reproductive ability of valuable agricultural animals."

Ovarian-tissue freezing is already available to girls getting cancer treatment, said Dr. Paula Brady, director of the oncofertility program and an assistant professor of obstetrics and gynecology at Columbia University in New York City.

And there have been "over 100 live births reported internationally (usually subsequent to in vitro fertilization)," Dr. Brady told Reuters Health by email, adding that this is similar to the principle of freezing testicular tissue in boys.

"Some centers already freeze testicular tissue in patients facing fertility-threatening medical treatment (like chemotherapy), with the counseling that it is an experimental technology, but to my knowledge no human live births have been reported," said Dr. Brady, who was not involved in the study.

The new findings are "important," said Dr. Amin Herati, director of male infertility and men's health for the Brady Urological Institute and an assistant professor of urology at the Johns Hopkins University School of Medicine, in Baltimore, Maryland.

"The timing of transplantation is an area of uncertainty," Dr. Herati, who also was not involved in the research, told Reuters Health by email. "Some institutions recommend the transplantation of cryopreserved prepubescent testicular tissue once a person goes through puberty, which would make freezing more than 20 years less relevant. However, this data will give the medical field confidence to preserve some or all of the cryopreserved tissue until a date well beyond puberty. Further, studying the mechanism that differentiates short- and long-term freezing outcomes may identify genes essential to the spermatogenesis pathway."

As for when the technique will be available to boys undergoing treatment that is toxic to their fertility, Dr. Herati said, the process described in the study "would be very similar to what will be happening soon at some institutions."

The study had no commercial funding and the researchers report no conflicts of interest.

SOURCE: PLOS Biology, online May 9, 2022.