Effects of medical treatments on gonadal development in boys

Prepubertal boys diagnosed with cancer face a high risk of spermatogonial stem cell (SSC) loss due to the side effects of cancer treatments, leading to potential fertility issues in the future. With increasing survival rates, fertility preservation is an increasingly vital consideration for many patients and their families. Current fertility preservation strategies are centered on the cryopreservation of testicular tissue containing SSCs. When combined with functional somatic testicular cells, experimental techniques, such as testicular explant tissue culture or cell/tissue transplantation, may offer future fertility restoration options for these patients. Therefore, the studies included in this thesis focus on strategies for quantifying and maintaining spermatogonia in testicular tissue samples obtained from these patients.

To better evaluate germ cell quantity in prepubertal boys with various treatments and diagnoses. Z-scores were utilized to account for age-related variations in standardized measures of spermatogonia quantity. A total of 89 testicular samples were analyzed in Study I. We calculated the Z-scores for spermatogonia per tubular cross-section (S/T) and fertility index (FI), based on deviations from the reference mean values (expressed as standard deviations [SDs]) obtained from a meta-analysis published in 2016.

The results showed that S/T Z-score values were significantly lower among Klinefelter syndrome patients and those treated with alkylating agents, compared to the control group. Additionally, S/T Z-score values were inversely correlated with exposure to alkylating agents: the higher the cumulative cyclophosphamide equivalent dose (CED), the lower the S/T Z-score. These findings suggest that age- adjusted Z-scores for S/T are a useful metric for quantifying spermatogonia in patients with cancer and genetic disorders.

Furthermore, we examined the effects of severe hematological diseases on spermatogonia numbers prior to hematopoietic stem cell transplantation (HSCT). This study involved 43 prepubertal patients, all diagnosed with severe hematological conditions. The patients were categorized into three groups: myelodysplastic/myeloproliferative neoplasms (MDS/MPN), aplastic anemia/bone marrow failure syndromes (AA/BMFS), and immunodeficiencies (IMMUNO). Z-scores for spermatogonia per tubule (S/T) and fertility index (FI) were used to evaluate spermatogonia quantity. The results showed that before undergoing HSCT, approximately half of the patients exhibited reduced spermatogonia numbers, and 19% experienced severe spermatogonia loss. Markedly decreased spermatogonia counts were observed in samples from prepubertal patients with Fanconi anemia (FA) and others with inherited bone marrow failure syndromes. In contrast, patients with MDS/MPN showed variable spermatogonial quantity. In contrast, patients with AA and immune disorders had only slightly decreased or normal spermatogonia numbers. Our findings highlight the importance of testicular tissue cryopreservation for patients with hematological diseases prior to HSCT, following appropriate counselling about the potentially decreased spermatogonial numbers.

Finally, we cultured testicular tissue samples using an organotypic culture system supplemented with glial cell line-derived neurotrophic factor (GDNF) for 7 and 14 days. Although cryopreservation of testicular tissue is currently the only available option for potentially restoring fertility in prepubertal boys, there is still no clinical evidence demonstrating its success in preserving fertility.

Successful spermatogenesis depends not only on the presence of SSCs but also on a functional somatic testicular microenvironment. To establish a supportive in vitro environment for human germ cell differentiation, we employed an organotypic culture system supplemented with GDNF. GDNF, primarily produced by Sertoli cells in the human testis, plays a key role in the self-renewal and differentiation of SSCs. A total of 47 testicular tissue samples from prepubertal boys were cultured for 0, 7, and 14 days. Immunohistochemistry was used to evaluate the expression of DEAD-Box helicase (DDX4), insulin-like 3 (INSL3), Cytochrome P450 family 17 subfamily A member 1 (CYP17A1), actin alpha 2 (ACTA2), Sex determining region Y-box 9 (SOX9), and cluster of differentiation 14 (CD14). Overall, DDX4 expression in seminiferous cords increased with patient age, while GDNF supplementation had no significant effect at any culture time point. Analysis of CD14 and CYP17A1 expression profiles revealed that these proteins were expressed not only in the interstitial compartment but also in Sertoli cells after 7 and 14 days of culture. These findings indicate aberrant expression of CYP17A1 and CD14, which may contribute to the failure of human testicular explant cultures to support human spermatogenesis.

In conclusion, the studies included in this PhD thesis demonstrated the establishment and application of Z-scores for S/T as a tool to evaluate spermatogonia quantity in patients with severe hematological diseases prior to gonadotoxic treatment. Furthermore, Z-scores for S/T were also utilized to assess the impact of genetic conditions and cancer treatments on spermatogonial numbers. In the final part of the study, we observed altered expression of CD14 and CYP17A1 in Sertoli cells cultured in organotypic systems for 7 and 14 days, offering a novel perspective on the dysregulation of human spermatogenesis in vitro.

List of scientific papers

I. Z-scores for comparative analyses of spermatogonia numbers throughout human development. Miriam Funke*, Yifan Yang*, Atte Lahtinen, Klara Benninghoven-Frey, Sabine Kliesch, Nina Neuhaus, Jan-Bernd Stukenborg, Kirsi Jahnukainen. Fertil Steril. 2021 Sep;116(3):713-720. https://doi.org/10.1016/j.fertnstert.2021.04.019
*Both authors contributed equally to the study.

II. Decreased spermatogonial numbers in boys with severe haematological diseases. Atte K Lahtinen, Miriam Funke, Claudia Krallmann, Margot J Wyrwoll, Andrea Jarisch, Yifan Yang, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén-Nyström, Cecilia Langenskiöld, Jann-Frederik Cremers, Sabine Kliesch, Jan-Bernd Stukenborg, Nina Neuhaus, Kirsi Jahnukainen. British Journal of Haematology, May 2024, 205(1), 229-235. https://doi.org/10.1111/bjh.19572

III. Effects of GDNF on prepubertal testicular tissue samples during organotypic cultures. Yifan Yang, Yanhua Cui, Femke Harteveld, Hajar Ba Omar, Anu Haavisto, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Hartmut Vogt, Per Frisk, Kaisa Vepsäläinen, Cecilia Petersen, Kirsi Jahnukainen, Jan-Bernd Stukenborg. [Manuscript]