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Taylor A. Johnson
Over the past few months, we have repeatedly discussed ways in which cancer may intersect with reproduction. On the female side, we have seen some of the challenges women face with fertility as well as links between bacterial infections and the development of ovarian cancer. On the male side, we highlighted some foundational questions pertaining to testicular cancer, such as why testicular cancer has high remission rates and how these cancers develop. There is growing interest in studying the connection between cancer (and cancer treatments) with female and male fertility in the short-term and long-term. In this article, we will bridge these topics and highlight a recent article published from the Geyer Lab at East Carolina University, focusing on commonplace compounds used in the treatment of cancers and their effects on male fertility.
Sirolimus (AKA rapamycin) and Everolimus (a water-soluble rapamycin analog, known as a rapalog) are both known to inhibit mTORC1, a complex critical to cell growth and proliferation. Currently, rapalogs are approved by the FDA in treating aggressive cancers and minimizing organ transplant rejection. It has been reported that Sirolimus can result in reversible male infertility; however, prior to this study, to the best of our knowledge, the effects of Everolimus on long-term fertility were unknown. We designed a series of experiments using both juvenile and adult mice. We incorporated a combination of histological techniques as well as breeding trials in order to determine any temporary or permanent consequences of these compounds on reproductive health. Our primary focus was on the effects of Everolimus, but we did include some comparisons with Sirolimus.
When mice were treated with these compounds, several observations were noted. First, the number of sperm within the epididymis (the structure that houses maturing sperm outside the testicle) was significantly lower compared to untreated treatment mice. In addition, the seminiferous tubules (which form a tubular network within the testis that house maturing germ cells) were narrower and contained areas with visible germ cell loss. We also noted that these tubules also contained fewer germ cells advancing through spermatogenesis (e.g. differentiated spermatogonia, spermatocytes, spermatids) and more germ cells found in early stages of spermatogenesis (specifically undifferentiated spermatogonia). Lastly, we identified an accumulation of dying or dead cells in the treatment group testes compared to untreated testes. It is worth noting that these trends were identified in both ages of mice.
These initial experiments were conducted with a higher dose than those normally used to treat patients (when calculating for body mass) in order to determine a mode of action. Therefore, we repeated these experiments by treating a different cohort with Everolimus at a concentration 6 times lower than the clinical dose. Our results show that the same events occur, albeit to a milder degree (e.g. sperm count and tubule diameter not as reduced); however, the same histological defects were noted. Collectively, we now know that treatment with Everolimus, like Sirolimus, can have reproductive side effects, even at low doses. However, a big clinical question looms: can we reverse this?
Fortunately, our results show that there is improvement over time after the conclusion of the treatment! When adult mice were allowed 30 days to recover, treated mice had improved tubule diameter as well as improved morphological and cellular organization. In the low Everolimus dose group, not only did we see these improvements, but we also quantified sperm counts that were similar to untreated mice! We repeated these experiments in juvenile mice, and allowed them 70 days to recover. Whereas Sirolimus treated mice had lower sperm counts compared to untreated mice, Everolimus treated mice had sperm counts very similar to untreated mice, suggesting a return to normalcy. While examining tissue histology, we saw similarities between the Sirolimus- and Everolimus-treated mice with untreated mice, which is very encouraging!
Finally, we tested to see whether treated males, when paired with wild type non-treated females, could produce offspring. We felt that this was the ultimate determinant for the recovery from the treatments, and the results would be very translatable for human health. For this breeding trial, treated mice were paired with the females 10 days after the cessation of their treatment, and were allowed for breed litters over a 60 day fertility trial. The untreated pairings yielded several litters with large pup sizes overall (6 litters; average of ~ 12 pups/litter). Sirolimus treated males yielded the fewest litters (3 total) and the lowest pup/litter ratio (10). Interestingly, Everolimus treated males (both cohorts) had more litters (5/6) and had a higher pup/litter ratio (~11.5 pups/litter) when compared to the Sirolimus mice. Overall, this data does show that there are impairments to spermatogenesis at the onset of treatment with Everolimus; however if given ample time after the treatment, these impairments can be resolved. In addition, Everolimus, compared to Sirolimus, had less-severe effects on the testis overall. Lastly, Everolimus treated mice, when given time post treatment, can have litter numbers and sizes comparable to untreated mice; which is strong evidence for overall improvement of reproductive capacity over time.
These results are very exciting (from both a cancer and a reproductive standpoint); however, there are a few considerations when interpreting this data. First, we are unaware of cumulative effects of multiple years of chronic rapalog treatment on human fertility; it is certainly possible that the testes of humans have different thresholds. In addition, we are unaware of the speed of the reversibility (if this occurs) and to what degree the recovery to normalcy is. We used mice in our study due to their high fecundity, highly structured spermatogenesis, and quick turnaround between breeding of litters. In addition, due to the structure of this study (mice age up to 120 days), we are unsure of which side effects are permanent over time. From a cancer standpoint, this data reinforces the notion that patients can return to a normal life after the conclusion of their treatments, which may include the starting of, or the growing of, a family. From a reproductive standpoint, it adds to the growing literature that mTORC complex could be an avenue for targeting male contraception; it is an attractive option due to its safety (already FDA approved), reversibility, and effectiveness in limiting spermatogenesis (both of which are shown here).
Taylor Johnson, M.S. is a doctoral candidate in the department of Anatomy & Cell Biology at East Carolina University at Greenville, NC.
Edited by Sara Musetti
Header image is by marian anbu Juwan from Pixabay
Sirolimus and Everolimus images adapted from Skotnicki JS and Huryn DM. Treatment of Transplantation Rejection and Multiple Sclerosis. Chemistry, Molecular Sciences and Chemical Engineering 2007
Kirsanov O, Renegar RH, Busada JT, Serra ND, Harrington EV, Johnson TA, Geyer CB. The rapamycin analog Everolimus reversibly impairs male germ cell differentiation and fertility in the mouse. Biology of Reproduction 2020.