Impact of restoring male fertility with transplantation of in vitro propagated spermatogonial stem cells on the health of their offspring throughout life

Dear Editor, Spermatogonial stem cell transplantation (SSCT) is a potential novel fertility treatment for prepubertal cancer patients that require gonadotoxic treatment. It comprises in vitro propagation of SSCs from a cryopreserved testis biopsy followed by autotransplantation into the infertile patient’s adult testis resulting in full recovery of spermatogenesis. In this blinded preclinical study, the long-term health of SSCT-derived offspring was assessed for the first time, using a systematic blueprint testing throughout life. No major differences in health outcomes between mice born after SSCT in two consecutive generations (first generation [F1] and second generation [F2]) and control were found, thereby providing crucial evidence that SSCT is a safe procedure. Due to its germline character, SSCT is the only adult stem cell-based transplantation therapy that can restore male fertility andmay not only affect the recipient, but also the offspring. Remarkably, medically assisted reproductive (MAR) therapies have been introduced in the clinic after preclinical studies focusing mainly on its efficacy instead of safety for the offspring.1,2 Unfortunately, after clinical implementation, children born after some of these MARs have increased risk for congenital abnormalities and low birthweight,3–6 developmental delays and cardiometabolic disease later in life.3,4,7 To ensure the health of future generations born from fathers that have received SSCT to restore fertility, we here studied indicators of health in the offspring throughout life that can be linked to potential risk factors of developmental problems, cardiometabolic disease, and ultimately result in increased mortality and pathologies2 (Supporting Information). Birth assessments showed that congenital abnormalities were a rare event (control n = 1/151, SSCT-F1 n = 3/125, SSCT-F2 n = 4/123; Figure 2A) with no statistically significant differences found in either SSCT generation compared to control (odds ratio [OR]: 3.78, 95% confidence


Impact of restoring male fertility with transplantation of in vitro propagated spermatogonial stem cells on the health of their offspring throughout life
Dear Editor, Spermatogonial stem cell transplantation (SSCT) is a potential novel fertility treatment for prepubertal cancer patients that require gonadotoxic treatment. It comprises in vitro propagation of SSCs from a cryopreserved testis biopsy followed by autotransplantation into the infertile patient's adult testis resulting in full recovery of spermatogenesis. In this blinded preclinical study, the long-term health of SSCT-derived offspring was assessed for the first time, using a systematic blueprint testing throughout life. No major differences in health outcomes between mice born after SSCT in two consecutive generations (first generation [F1] and second generation [F2]) and control were found, thereby providing crucial evidence that SSCT is a safe procedure.
Due to its germline character, SSCT is the only adult stem cell-based transplantation therapy that can restore male fertility and may not only affect the recipient, but also the offspring. Remarkably, medically assisted reproductive (MAR) therapies have been introduced in the clinic after preclinical studies focusing mainly on its efficacy instead of safety for the offspring. 1,2 Unfortunately, after clinical implementation, children born after some of these MARs have increased risk for congenital abnormalities and low birthweight, 3-6 developmental delays and cardiometabolic disease later in life. 3,4,7 To ensure the health of future generations born from fathers that have received SSCT to restore fertility, we here studied indicators of health in the offspring throughout life that can be linked to potential risk factors of developmental problems, cardiometabolic disease, and ultimately result in increased mortality and pathologies 2 (Supporting Information).
Birth assessments showed that congenital abnormalities were a rare event (control n = 1/151, SSCT-F1 n = 3/125, SSCT-F2 n = 4/123; Figure 2A  I G U R E 1 Study design to determine development and health of spermatogonial stem cell transplantation (SSCT) offspring compared to control offspring. Sterile W/Wv male mice received SSCT to restore fertility. Control and transplanted males were placed in breeding with control females. The control group was bred for one generation for welfare reasons, while the SSCT group was bred for two generations to determine if any developmental or health deviations at birth, childhood, and adulthood are inherited. appropriate power calculation, based on the known incidence of congenital abnormalities in other MARs, the estimated sample size (n = 116 per group) may have been a limitation in determining the effect of SSCT on congenital abnormalities in the offspring. This was probably because congenital abnormalities were rare, which is clearly shown in the broad confidence intervals. 8 During the childhood period (first 28 days of life), the timing of physical and neurodevelopment was estimated by the occurrence of essential morphological landmarks and reflexes. 1,2 No differences were found between control and SSCT-derived offspring, in both generations, for fur growth, the timing of eye and ear opening, lower incisor eruption, and development of correct righting assessment measured by weight and glucose tolerance test (GTT) in F1-control and SSCT mice at 11 months of age. The offspring was weighed before GTT and fasting glucose levels were taken. (C) Cardiovascular assessment was measured by heart rate, systolic and diastolic blood pressures at 9-11 months of age. Mean (SD) and modeled differences (mixed models) control versus SSCT 95% CI, and graphical representation of modeled mean ± SE. (D) Time-to-death Kaplan-Meier curve in days showing no differences between SSCT-F1 and control in life expectancy (log-rank test). (E) Frequency of organ pathology at 18 months of age. Only the most severe pathology is presented in cases with multiple pathologies (Videos S1-3), grasping (Videos S4,5), negative geotaxis (Videos S6-S8) reflexes (Table S1 and Figure 3) and length ( Figure S1). However, a statistically significant difference of 1-day earlier upper incisor eruption (95% CI: [-2.09, -0.14]) and decreased weight gain (95% CI: [-0.42, -0.08]) were observed between SSCT-F2 and control, but not in F1 (Figure 3).
During adulthood, cardiometabolic risk factors and fertility were assessed. 3,7,8 Weight was consistent between control and SSCT offspring, suggesting no risk of obesity in both generations (F1 at 18 months 95% CI: [-1.29, 1.1] and F2 at 6 months 95% CI: [-0.44, 1.33]; Figure 4A and Figure S2, respectively). Although, the weight of the SSCT offspring in the F2 was significantly decreased compared to control during childhood, reassuringly, after reaching adulthood, F1-control and SSCT-F1/F2 offspring presented similar weight curves through life, indicating recovery from the reduced weight found during childhood in the F2. Additionally, all F1 animals used as breeding pairs were fertile. Furthermore, to assess glucose intolerance as a risk for diabetes, we first determined weight and fasting glucose levels (Table S1) and found no statistically significant differences between SSCT offspring and control ( Figure 4B). Glucose intolerance was tested after glucose injection also revealed no differences between SSCT and control offspring ( Figure 4B). Cardiovascular assessments, including diastolic blood pressure, systolic blood pressure and heart rate showed no differences between control and SSCT-F1 ( Figure 4C), indicating no risk for cardiovascular disease.
Ultimately, we analyzed the mortality rate and general pathology of the animals. Overall, the life expectancy of the offspring up until the end of the study (18 months) was similar between control and SSCT, registering a similar incidence of premature death throughout life ( Figure 4D and Table S2). Histological abnormalities were overall comparable between control and SSCT comprising calcification in the epicardium, steatosis, cystic endometrial hyperplasia, and nephropathy ( Figure 4E, only the most severe pathology is reported). Most pathologies were associated with aging or strain-specific (DBA/2J) complications. 9 Overall, tumor incidence was similar between both groups and characteristic of DBA/2J, including adenomas (control = 1, SSCT = 2) and lymphomas (control = 5, SSCT = 3). In the SSCT group, three animals developed sarcoma against none in the control group, which may be a coincidental finding and needs further research. In future clinical trials, the occurrence of sarcomas should be monitored in detail. Regarding the weight of the organs, a significant increase in the weight of the testes, male genital fat pads, and pancreas in SSCT and decreased weight for the right ovary, liver, and stomach ( Figure S3) was found, with no corresponding phenotype. Except for testicular histology, with confirmed fertility by the occurrence of spermatoge-nesis in all the F1 offspring, showing statistically significant larger mean tubular diameter of round seminiferous tubules SSCT (192 ± 21.3 μm) compared to control (159 ± 19.8 μm, p = 0.004; Figure S4). Together with fewer germ cells inside control tubules, this may suggest a reduced aging effect in SSCT offspring.
In conclusion, finding no major impact of SSCT on multiple markers of development and general health of two consecutive generations through life, including birth characteristics, childhood development, and adult health, this preclinical study is an important step toward clinical translation of SSCT for infertile childhood cancer survivors. Together with a previous safety and efficacy study on SSCT-recipient health, 10 this study provides crucial evidence for requesting ethical approval of SSCT for a phase-1 clinical trial, which should include thorough follow-up of the transplanted men and their children.

A C K N O W L E D G M E N T S
This was a very extensive and arduous project that involved numerous experts from different fields that we would like to thank: the animal research facility (ARIA at Amsterdam UMC, location AMC), mainly Harry van Herck, Cindy Schlosser, Joost Uilenreef (consulting veterinarian), and all the animal caretakers (Nicole Janus, Sanne Hackmann, Marieke Christophe, Laura Harper, Denise de Wit, Karin van den Oever, Lex Beekman, Nienke Calandt, and Vincent van Riet), and the Animal Welfare Body (Amsterdam UMC), namely Henriette Griffioen and Wouter Florijn for advice on animal experiments. Souad Boussata and Jitske Eliveld assisted with animal experiments and Leanne Schaper performed outcome re-assessment (Center for Reproductive Medicine, Amsterdam UMC). Morgan Shehata (Amsterdam UMC) designed and developed the mouse ramp for the negative geotaxis. Judith de Vos and Erik Bakker (Biomedical Engineering & Physics Department, Amsterdam UMC) assisted with the tail-cuff system. Wayne Ritsema and Andries Kalsbeek (Department of Endocrinology and Metabolism, Amsterdam UMC) assisted in performing the glucose tolerance test analyses. We thank Tessa Roseboom (Department of Obstetrics and Gynecology, Amsterdam UMC), Sjoerd Repping, and Sefika Canan Mizrak (Center for Reproductive Medicine, Amsterdam UMC) for advice in project design and Mariëtte Goddijn for critical reading of the manuscript.

C O N F L I C T O F I N T E R E S T
All authors declare they have no conflict of interests.