Determining the cause of cryopreservation fertility failures microbiologystudy

According to the American Cancer Society, survivors of childhood cancer can face health problems later in their adult life. Many boys who undergo cancer treatment before puberty can lose the ability to produce sperm as chemotherapy or radiation therapy can damage their reproductive cells.

A study published in Stem Cell Reports, led by Eoin Whelan, Ph.D., and Ralph Brinster, VMD, Ph.D., and a team of researchers at the University of Pennsylvania’s School of Veterinary Medicine (Penn Vet) and the Children’s Hospital of Philadelphia, is laying the groundwork to help childhood cancer survivors become parents later in life.

Spermatogenesis, the process of sperm cell production, is essential for male fertility. But much remains unknown about how these cells mature or differentiate into specialized sperm cells. Whelan’s and Brinster’s investigation sheds light on male sperm cell formation by detailing preserved gene regulatory networks across spermatogenesis in animal models, characterizing transcription factors, their targets across genomes, and the downstream genes they affect.

Using single cell multiomic assays, researchers identified a roadmap of how the complex development of male germ cell differentiation is achieved through gene expression and epigenetic changes. Investigators explored both meiosis, the cell process that creates gametes, or reproductive cells; and spermiogenesis, the process whereby male cells in mid-development mature into sperm cells, known as spermatozoa.

“We found that each stage is associated with distinct chromatin accessibility changes,” said Brinster, Richard King Mellon Professor of Reproductive Physiology and senior author of the study. “This enhanced understanding of the regulation of spermatogenesis provides the foundation for further studies including technologies to recover fertility in prepubertal boys lost during cancer therapies.”

Brinster envisioned such therapies nearly twenty years ago, where testicular tissue is cryopreserved for long periods and transplanted back into the patient after successful cancer treatment, restoring their reproductive capabilities. These therapies are now becoming a reality. In November 2023, a 24 year-old, cancer-surviving male, underwent re-transplantation of cryopreserved tissue. University of Pittsburgh’s Kyle Orwig who led the re-implantation, was a student of Brinster’s and served as senior postdoctoral fellow in his laboratory.

Whelan’s and Brinster’s 2022 study published in PLOS Biology showed that rat testis cells could be transplanted after over 20 years of cryopreservation and allow previously-infertile testes to produce sperm. However, these cells did so at a much-reduced rate compared to those frozen for a brief period, pointing to a block in spermatogenesis after meiosis. These deficiencies persisted long after cells were transplanted, suggesting an epigenetic change in the long-frozen stem cells that were inherited by all of the germ cells in the regenerated tissue.

“This loss of precise transmission of the differentiation program in male germline stem cells after long-term cryo-storage presents a need for continued study of the medical adaptation of this technology,” said Whelan, a senior research investigator in the Brinster laboratory. “Implementation of the genetic program will require ongoing refinement for the successful cryopreservation of all stem cells.”

Whelan and his colleagues in the Brinster laboratory continue to build on this work. Their newly-published atlas of gene expression and chromatin accessibility in normal spermatogenesis can be used as a foundation to map epigenetic changes following transplantation of long-term frozen tissue to identify specific gene and chromatin changes underpinning this loss of function.

“By creating a detailed map of how sperm-forming cells develop and identifying where defects occur after long-term freezing, we hope to potentially limit or even reverse the damage caused to stem cell regenerative capacities in the first place,” said Whelan. “This undoubtedly has real-world importance for families affected by childhood cancer.”