Joana Margarida Bispo Serrano

Paediatric cancer treatments have substantially improved in recent years, leading to increased life expectancy. However, severe therapy-related late effects, such as infertility, affect the quality of life of male cancer survivors. A proposed therapy to restore fertility is spermatogonial stem cell autotransplantation (SSCT) into the survivor’s adult testis after in vitro propagation of the SSCs, which are retrieved from a cryopreserved testis biopsy. SSCs repopulate the testes allowing natural conception. SSCT is not yet clinically implemented since the long-term health risks of offspring are unknown. Therefore, health assessment of SSCT derived offspring in a preclinical model is essential before advancing to clinical trials.

In chapter 1 we provided a rationale for preclinical safety testing of novel medically assisted reproductive techniques (MAR) including SSCT. It provides background information and an outline of the thesis’ aims.

In chapter 2 we developed a blueprint to study the impact of future stem cell technologies and/or MAR on the health of the offspring. Previous research suggests that MAR-offspring are at increased risk for, amongst others, cardiometabolic disease later in life. Therefore, we propose a series of tests to perform in multiple generations of mice that can follow the potential appearance of pathologies throughout their lifetime.

In chapter 3 we used this blueprint to perform a blinded longitudinal preclinical study to investigate the health of two generations born after SSCT (intervention) or without SSCT (control) in a mouse model. This is the first study that thoroughly assessed birth characteristics, childhood development and adult health in offspring derived from a novel MAR using a systematic approach, prior to clinical implementation. Our results indicate that, through their lifetime, mice naturally conceived after SSCT do not have an increased rate of congenital abnormalities, developmental deviations and cardiometabolic problems compared to naturally conceived control mice. The life expectancy and incidence of pathologies are also similar between groups. Therefore, culture and autotransplantation of SSCs into sterile males do not seem to impact the health of their offspring.

In chapter 4, we took a fundamental approach and investigated if the DNA methylation status of sperm from SSCT offspring was comparable to control offspring, in order to uncover any putative epigenetic differences caused by this therapy, using the above-described mouse model. Overall, the DNA methylation patterns were similar between both groups. Although, one gene (Tal2) was hypomethylated and showed higher gene expression in the SSCT offspring in organs that normally express this gene. Nonetheless, we did not find any related pathologies in these organs of SSCT mice offspring.

Finally in chapter 5, we examined the state-of-the-art to ascertain if SSCT is sufficiently safe to proceed to clinical trials, while considering the recent developments in the topic, including the entire process from cryopreservation of the biopsy till SSCT and health of the offspring. Multiple advances were made in recent years from both the technical and clinical standpoints. The procedure of the testicular biopsy has been studied and deemed safe concerning the endocrine development and long-term growth of the biopsied testis compared to contralateral untouched testis. Multiple cryopreservation methods were also tested, establishing that cryopreservation of the testicular biopsy safeguards the viability and longevity of the SSCs over cryopreservation of isolated testicular cells. As these stem cells are quite rare in the testis, their expansion in vitro is required to increase the efficiency and success of SSCT. However, in vitro propagation holds the risk that putative genetic or epigenetic injury in the SSCs occurs and could lead to pathologies in the recipient and the offspring. Reassuringly, safety studies (including the work in this thesis) in the cultured SSCs and in preclinical models has determined that SSCs’ genetic and epigenetic status remains stable, lacking carcinogenic features. However, one hurdle still to overcome is the development of an efficient culturing method to expand human SSCs number in vitro. The protocol now established is inefficient and could still harbour original malignant cells that might be reintroduced in the adult recovered patient. Still, after optimization of the culturing method, patients with solid tumours or with non-malignant hematologic diseases are great candidates for fertility treatment with SSCT in a first clinical trial.

In chapter 6, we reflect and discuss the necessity of safety studies in reproductive medicine and put the outcomes of this thesis in perspective for SSCT and other future MARs.