Spermatogonial stem cells populations in boys with haematological and oncological diseases

<p dir="ltr">While survival rates for paediatric patients with malignant and non-malignant diseases have improved considerably due to advances in treatment, intensive regimens frequently impair future fertility later in life. Subfertility or permanent infertility may emerge as lifelong consequences, yet these outcomes are frequently overlooked in clinical practice. This oversight arises from the necessity of prioritising survival in acute disease management, despite the potential long- term reproductive consequences of these treatments. The paradox is evident, advances in oncology have steadily increased childhood cancer survivorship over the past decades, but the same gonadotoxic therapies alkylating agents, radiation, and high-dose chemotherapy that cure disease frequently depletes SSCs and compromise future fertility. For adult patients, fertility preservation follows established protocols involving cryopreservation sperm before treatment initiation. Pre- and peripubertal boys face substantially greater complexity. Their only alternative is the surgical extraction and cryopreservation of immature testicular tissue including SSCs; this experimental approach relies on forthcoming technological advancements to induce these cells to become viable gametes. The urgency of this challenge is reflected in rising preservation rates recent surveys document more than 3,000 young males worldwide undergoing testicular tissue cryopreservation, with numbers doubling over five years. This rapid increase illustrates the urgent need to transform experimental approaches into clinically reliable strategies. The ultimate goal involves restoring reproductive function through laboratory-based spermatogenesis or tissue/cells transplantation approaches. Nevertheless, current methodologies for achieving fertility restoration from cryopreserved immature testicular tissue remain in experimental stages, and our ability to evaluate the impact of gonadotoxic treatments in prepubertal patients is still limited.</p><p dir="ltr"><b>Study I</b>, Testicular tissue cryopreservation is increasingly offered to boys with sickle cell disease before potentially sterilizing hematopoietic stem cell transplantation HSCT. Hydroxyurea HU is the standard disease modifying therapy for sickle-cell disease SCD. It increases fetal haemoglobin, reduces vaso-occlusive crises, and improves survival, leading international guidelines to recommend its use in all infants with SCD from 9 months of age onwards. This study evaluated the effect of HU therapy, of SDC related complications. This study evaluated the effect of HU therapy, employed as a prevention of SCD related complications, on testicular function. By analysing clinical data from 29 males aged 2.8-15.1 years enrolled in fertility preservation programs Androprotect and Nordfertil, using Z- score comparisons against healthy reference values. immunostained for MAGEA4, and mean spermatogonia per round tubular cross-section (S/T) and fertility index (FI) were calculated; Z-scores were derived from age-matched reference values. Most patients (n=17) had S/T below reference, with four patients with totally depleted spermatogonia. Spermatogonial numbers showed a positive correlation with both S/T Z-score (P = 0.029, r = 0.476) and FI Z-score (P = 0.024, r = 0.490) when HU treatment initiated. Receiver operating characteristic analysis revealed that HU initiation at or before 2.4 years effectively predicted severely reduced spermatogonial numbers. There were no discernible correlations found between spermatogonial depletion and HU dosage, exposure time, iron load, or illness severity indicators. These results show that early exposure to HU may increase the risk of spermatogonial pool depletion during the first three years of a child's life, while the testicles are still developing. The results indicate that the potential risk of reduced fertility from early HU use must be weighed against its proven benefits in lowering morbidity and extending survival in SCD, while also underscoring the importance of counselling families on treatment decisions and considering fertility preservation strategies such as testicular tissue cryopreservation prior to HSCT therapy.</p><p dir="ltr">Following this we shifted our focus toward <b>study II</b>, this study provides the groundwork for this research, focusing on sub-and infertility, which are among the most common and challenging long-term adverse effects faced by male childhood cancer survivors. This study examined the effects of alkylating and non-alkylating agents on SCCs numbers. Using current single-cell RNA sequencing investigations, several human spermatogonial cell clusters were identified, suggesting greater heterogeneity of SPG than previously thought. This study also included protein markers of these gene clusters, helping us understand the functional effects of this variability. Our findings indicate that depletion of the spermatogonial pool following chemotherapy results in different protein expression patterns. This comprehensive study employed previously generated single-cell RNA sequencing from six healthy testicular samples (ages 0-17 years) and reanalysed it. Combined with immunofluorescence analysis of key spermatogonial markers (UTF1, ID4, PIWIL4, FGFR3, and KIT) in 14 control samples and 31 prepubertal testicular tissues from paediatric cancer patients to investigate spermatogonial differentiation states and their sensitivity to chemotherapy-induced depletion. The research confirmed that prepubertal testes contain exclusively undifferentiated spermatogonia in states 0-1, while more advanced differentiated states (2-4) emerge only during pubertal development, with KIT protein expression showing positive correlation with patient age (P<0.001) and being frequently detected after age 10 years. Chemotherapy exposure demonstrated significant dose-dependent effects on spermatogonial cells. There was a significant reduction in ID4-expressing spermatogonia (P=0.001) and KIT-expressing cells (P=0.005) when the cyclophosphamide equivalent dose (CED) was increased, and exposure to the doxorubicin isoequivalent dose (DIE) decreased KIT protein expression (P=0.035). Among the most significant findings were the differences in responses to chemotherapy among different subtypes of spermatogonial cells. Cells expressing UTF1 and PIWIL4 did not correlate with spermatogonial pool depletion, suggesting that these cells represent chemotherapy-resistant "reserve" stem cells critical for fertility recovery. Conversely, spermatogonial cells expressing markers of advanced differentiation states (ID4, FGFR3, and KIT) correlated with depleted spermatogonial pools (P=0.033, P=0.050, and P=0.051), suggesting they may be more susceptible to cytotoxic damage. A key clinical implication of these findings is that chemotherapy-resistant UTF1- and PIWIL4-positive cells may serve as a valuable biomarker for assessing fertility potential in cryopreserved testicular tissues collected from prepubertal boys receiving cancer treatment.</p><p dir="ltr"><b>Study III</b>, builds on the findings from study II, in which SCCs subtypes, were evaluated based on the expression of five proteins at the time of tissue collection (day 0), and their variable responses towards treatments were reported. we then investigated how testicular tissue culture methodologies could be optimised to preserve and nurture these critical SCCs subpopulations. To this end, we applied an established organotypic culture system, previously shown to support spermatogenesis in rodents, in order to evaluate its applicability for human SSCs. By modifying the in vitro environment (supplements), we aimed to assess the ability of this approach to maintain human SSCs and support their differentiation. Supportive media formulations were used for the culture of testicular organoids, and antigen selection was informed by single-cell RNA sequencing data, with immunofluorescence and immunohistochemistry applied for characterisation. Prepubertal boys undergoing gonadotoxic cancer treatments can permanently lose spermatogonial stem cells SSCs, necessitating fertility preservation strategies. In vitro SSCs culture is a promising approach for regenerating the germline, especially when reimplantation of preserved testicular tissue is unsafe due to malignancy. However, maintaining undifferentiated human SSC subpopulations in culture remains challenging. We evaluated the effect of KnockOut Serum Replacement (KSR) supplementation on the maintenance of SSC subtypes in organotypic cultures of prepubertal human testes. We cultured testicular tissue fragments from prepubertal and peripubertal boys (ages 2.9–12.4 years) in a fertility preservation program for up to 14 days, with or without 10% KSR. We quantified SSC marker expression (UTF1, PIWIL4, ID4, FGFR3, KIT) by immunohistochemistry on days 0, 7, and 14. All spermatogonial markers declined over time in culture. KSR supplementation significantly improved the preservation of undifferentiated SSC subtypes compared to KSR-free conditions. Primitive SSC populations (UTF1⁺ and PIWIL4⁺ cells) were relatively resilient, showing partial recovery by day 14 in KSR cultures, whereas intermediate progenitors (ID4⁺ and FGFR3⁺ cells) were markedly depleted without recovery. KIT⁺ spermatogonia were absent at all time points under both conditions. Greater cumulative exposure to alkylating chemotherapy correlated with a higher fold-change in PIWIL4⁺ cells, whereas higher baseline SSC abundance (Z-score) inversely correlated with UTF1⁺ cell retention in KSR cultures. KSR supplementation is essential to sustain the most primitive SSC populations during extended in vitro culture. These results suggest that culture systems augmented with KSR provide a promising strategy for the maintenance of fertility and treatments using germline stem cells. This thesis brings together three studies that address one of the most difficult challenges faced by boys undergoing medical treatments for haematological and oncological disease: the loss of their future fertility. In Study I, we showed that the timing of HU therapy for SCD can influence the spermatogonial pool, emphasising the value of early counselling and timely cryopreservation before stem cell transplantation. In study II, we uncovered that not all spermatogonial cells respond to chemotherapy in the same way. Some subtypes, marked by UTF1 and PIWIL4, appear more resistant and may act as a reserve population critical for fertility recovery, while others are far more vulnerable. Finally, in study III, we established that the incorporation of KSR into culture systems aids in the preservation of these delicate cells in vitro, therefore providing a foundation for future fertility restoration strategies. </p><p dir="ltr">Collectively, these results enhance our comprehension of how medical interventions influence the developing male germline and provide pragmatic methods for its protection. They emphasise the significance of including fertility preservation in child treatment planning and the ongoing improvement of cultures, systems, and biomarkers to inform therapeutic choices. Although much effort is still required before these methodologies achieve standardisation, this study advances us towards a future in which overcoming childhood illness does not require relinquishing the prospect of parenting.</p><h3>List of scientific papers</h3><p dir="ltr">I. Klara M. Benninghoven-Frey, Nina Neuhaus, Atte K. Lahtinen, Claudia Krallmann, Joana M.D. Portela, Andrea Jarisch, Verena Nordhoff, Armin Soave, <b>HAJAR BA OMAR</b>, Mikael Sundin, Cecilia Langenskiod, Sabine Kliesch, Jan-Bernd Stukenborg, Kirsi Jahnukainen Early testicular maturation is sensitive to depletion of spermatogonial pool in sickle cell disease. Haematologica, 2022 Apr 1;107(4): 975- 979. <a href="https://doi.org/10.3324/haematol.2021.279253" rel="noreferrer" target="_blank">https://doi.org/10.3324/haematol.2021.279253</a></p><p dir="ltr">II. <b>HAJAR BA OMAR*</b>, Justine Stevens*, Anu Haavisto, Yanhua Cui, Femke Harteveld, YifanYang, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, CeciliaLangenskiöld, Hartmut Vogt, Per Frisk, Kaisa Vepsäläinen, Cecilia PetersenLina Cui, Jingtao Guo, Kirsi Jahnukainen#, Jan Bernd Stukenborg# * and # These authors contributed equally to this work and should be considered co-first or co-last authors., respectively Early transcriptional states of spermatogonia and marker expressions in the prepubertal human testis following chemotherapy-induced depletion. Human Reproduction, 2025 Aug 1;40(8):1467-1475. <a href="https://doi.org/10.1093/humrep/deaf103">https://doi.org/10.1093/humrep/deaf103</a></p><p dir="ltr">III. <b>HAJAR BA OMAR</b>, Anu Haavisto, Yifan Yang, Yanhua Cui, Femke Harteveld, Cecilia Langenskiöld, Hartmut Vogt, Per Frisk, Cecilia Petersen, Kirsi Jahnukainen, Jan-Bernd Stukenborg Expression profile of spermatogonial subtypes during organotypic cultures. [Manuscript]</p>