Longitudinal trends in testicular volume z scores from puberty to adulthood, sperm quality, and paternity outcomes after childhood cancer
Testicular volume can serve as a fertility marker because it correlates positively with sperm output and concentration. Testicular enlargement during puberty is mainly caused by an increase in germ cell numbers in the seminiferous tubular compartment, reflecting the onset of spermatogenesis; the process is supported by the release of androgens from the Leydig cells after being activated by the hypothalamic‐pituitary‐testicular axis. , In addition to Leydig cells, the testicular microenvironment also includes other somatic cell populations, such as Sertoli cells and peritubular myoid cells, all of which fulfill essential roles during puberty and in adulthood. Cancer therapy in childhood may cause disturbances in any of these structures, causing infertility. Patients and their parents rank fertility‐related issues as one of the major concerns after childhood cancer. , To improve fertility, PanCareLIFE recently published guidelines on fertility preservation in male childhood cancer survivors (CCS), identifying treatments involving alkylating agents, radiotherapy to the testes, cranial radiotherapy, and hematopoietic stem cell transplantation (HSCT) as modalities associated with a risk of infertility and thus warranting counselling on fertility‐preservation options. However, only a few studies have examined the impact of cancer treatment on semen variables in cohorts involving large numbers of CCS. , In addition, little is known about the impact of cancer therapy on pubertal testicular growth and its association with sperm quality and reproductive outcomes in adulthood. In this study, our objective was to evaluate the influence of cancer therapy exposures on testicular volumes from puberty to adulthood, spermatogenesis in adulthood, and paternity outcomes. Testicular volumes in CCS were compared with previously published reference values, with the aim of assessing whether impaired testicular growth during puberty is associated with azoospermia and future infertility, which would allow for early infertility counseling. We also analyzed the predictive value of serum hormonal markers in identifying patients with azoospermia and low adult testicular volume.   Study cohort characteristics Hospital patient register provided identification of 255 males with childhood (age at diagnosis younger than 17 years) cancer treated at the Children’s Hospital in Helsinki during 1964–2000 and who had survived at least 5 years after diagnosis. Patients who had brain tumors were not included because their treatment occurred in another hospital. From the same patient cohort, we previously reported patterns of psychosocial development and paternity. The current study was approved by the Research Ethics Committee of Helsinki University Hospital and the Finnish Institute for Health and Welfare. The study used only registry data and medical records data; no patient consent was required. Data collection Medical records provided data on testicular volume measurements, Tanner stages, sperm samples, serum hormone levels, the use of testosterone replacement therapy (TRT), and cancer treatment characteristics. Testicular volumes were measured at ages 12, 14, 16, and 18 years and in adulthood (aged 20 years and older). Testicular dimensions were measured with a ruler, and testicular volume was calculated using the following formula: π/6 × length × width 2 . Testicular volumes were transformed into z scores using pubertal testicular volume growth references ; testicular volumes measured at age 18 years and in adulthood were converted into z scores using the reference values for age 17 years. Ruler‐based references for testicular volume closely correspond to those derived from an orchidometer. All testicular volume measurements occurred after the end of cancer treatment, with the end of therapy defined as the date of the last cancer treatment or the date of HSCT. Analyses included semen samples and serum hormone measurements that were obtained in adulthood (aged 20 years and older). Testicular volume measurements, semen samples, and serum hormone measurements obtained during ongoing TRT were excluded from all analyses. None of the patients had discontinued TRT before the analyses. The mean cumulative cyclophosphamide equivalent dose (CED) was calculated to represent alkylating agent exposure. Organ‐specific radiation doses for the pituitary gland and the testes were calculated by the Late Effects Group at the University of Texas MD Anderson Cancer Center, as previously described. The Digital and Population Data Services Agency contains information on all Finnish citizens and includes personal data, such as family relations; by using personal identification numbers, CCS were linked to their registered children. Adopted children were not included in the analyses of paternity. The register data were assessed on December 31, 2014. The register data on offspring and data on adult (aged 20 years and older) testicular volumes were also included in our previous study, in which we analyzed factors affecting the probability of paternity and marriage in male CCS. Statistical analyses Continuous data with normal distribution are reported as means and standard deviations (SDs), and data with nonnormal distribution are reported as medians and interquartile ranges (IQRs); between‐group differences were analyzed using the Mann–Whitney U test. Categorical data are presented as numbers and percentages; differences between groups were analyzed using the Fisher's exact test. The number of patients who had Tanner P stage 1 and P stage ≥2 at ages 12, 14, and 16 years were compared between CCS and previously reported references. 11 Pearson correlation was used to study the associations between two continuous variables. Associations between cancer treatment exposures and pubertal testicular volume z scores were analyzed with linear regression; the results are reported as regression coefficients (β) and their 95% confidence intervals (95% CIs). Logistic regression analyses served as a method for assessing associations between cancer treatment exposures and low testicular volume in adulthood (z score < −2) and azoospermia (total absence of sperm in the ejaculate) in adulthood (if multiple samples were available, the latest was included in the analyses); the results are reported as odds ratios (ORs) and their 95% CIs. Paternity rates were assessed using a Cox proportional hazards model with age as the time scale; patients were censored at the date of death or on December 31, 2014. Results are reported as hazard ratios (HRs) and their 95% CIs. Multivariable analyses included the following predictors: cumulative testicular radiation dose, CED, cumulative pituitary radiation dose, age at diagnosis (9 years and older vs. younger than 9 years; serving as a surrogate for pubertal status), and the time elapsed since the end of cancer therapy. Receiver operating characteristic (ROC) analyses were used to calculate areas under the curves (AUCs) and to identify optimal cutoff points (the Youden index) for testicular volume z scores in identifying patients with nonazoospermia and registered children. R version 4.2.1 for Windows (R Foundation for Statistical Computing) served as statistical software. All p values < .05 denoted statistical significance.   Hospital patient register provided identification of 255 males with childhood (age at diagnosis younger than 17 years) cancer treated at the Children’s Hospital in Helsinki during 1964–2000 and who had survived at least 5 years after diagnosis. Patients who had brain tumors were not included because their treatment occurred in another hospital. From the same patient cohort, we previously reported patterns of psychosocial development and paternity. The current study was approved by the Research Ethics Committee of Helsinki University Hospital and the Finnish Institute for Health and Welfare. The study used only registry data and medical records data; no patient consent was required.   Medical records provided data on testicular volume measurements, Tanner stages, sperm samples, serum hormone levels, the use of testosterone replacement therapy (TRT), and cancer treatment characteristics. Testicular volumes were measured at ages 12, 14, 16, and 18 years and in adulthood (aged 20 years and older). Testicular dimensions were measured with a ruler, and testicular volume was calculated using the following formula: π/6 × length × width 2 . Testicular volumes were transformed into z scores using pubertal testicular volume growth references ; testicular volumes measured at age 18 years and in adulthood were converted into z scores using the reference values for age 17 years. Ruler‐based references for testicular volume closely correspond to those derived from an orchidometer. All testicular volume measurements occurred after the end of cancer treatment, with the end of therapy defined as the date of the last cancer treatment or the date of HSCT. Analyses included semen samples and serum hormone measurements that were obtained in adulthood (aged 20 years and older). Testicular volume measurements, semen samples, and serum hormone measurements obtained during ongoing TRT were excluded from all analyses. None of the patients had discontinued TRT before the analyses. The mean cumulative cyclophosphamide equivalent dose (CED) was calculated to represent alkylating agent exposure. Organ‐specific radiation doses for the pituitary gland and the testes were calculated by the Late Effects Group at the University of Texas MD Anderson Cancer Center, as previously described. The Digital and Population Data Services Agency contains information on all Finnish citizens and includes personal data, such as family relations; by using personal identification numbers, CCS were linked to their registered children. Adopted children were not included in the analyses of paternity. The register data were assessed on December 31, 2014. The register data on offspring and data on adult (aged 20 years and older) testicular volumes were also included in our previous study, in which we analyzed factors affecting the probability of paternity and marriage in male CCS.   Continuous data with normal distribution are reported as means and standard deviations (SDs), and data with nonnormal distribution are reported as medians and interquartile ranges (IQRs); between‐group differences were analyzed using the Mann–Whitney U test. Categorical data are presented as numbers and percentages; differences between groups were analyzed using the Fisher's exact test. The number of patients who had Tanner P stage 1 and P stage ≥2 at ages 12, 14, and 16 years were compared between CCS and previously reported references. 11 Pearson correlation was used to study the associations between two continuous variables. Associations between cancer treatment exposures and pubertal testicular volume z scores were analyzed with linear regression; the results are reported as regression coefficients (β) and their 95% confidence intervals (95% CIs). Logistic regression analyses served as a method for assessing associations between cancer treatment exposures and low testicular volume in adulthood (z score < −2) and azoospermia (total absence of sperm in the ejaculate) in adulthood (if multiple samples were available, the latest was included in the analyses); the results are reported as odds ratios (ORs) and their 95% CIs. Paternity rates were assessed using a Cox proportional hazards model with age as the time scale; patients were censored at the date of death or on December 31, 2014. Results are reported as hazard ratios (HRs) and their 95% CIs. Multivariable analyses included the following predictors: cumulative testicular radiation dose, CED, cumulative pituitary radiation dose, age at diagnosis (9 years and older vs. younger than 9 years; serving as a surrogate for pubertal status), and the time elapsed since the end of cancer therapy. Receiver operating characteristic (ROC) analyses were used to calculate areas under the curves (AUCs) and to identify optimal cutoff points (the Youden index) for testicular volume z scores in identifying patients with nonazoospermia and registered children. R version 4.2.1 for Windows (R Foundation for Statistical Computing) served as statistical software. All p values < .05 denoted statistical significance.   Study cohort characteristics Table summarizes patient characteristics and cumulative exposures to cancer treatment for all 255 male CCS; the median patient age at the time of cancer diagnosis was 6.1 years, and diagnoses included hematologic malignancies (69%) and solid tumors (31%). Figure shows a flow chart of the patient selection process; testicular volume measurements were available for 38–105 CCS at ages 12–18 years and for 43 individuals in adulthood (median age, 27 years; median time from the end of cancer therapy, 18 years). Most CCS (84%) were prepubertal at age 12 years, whereas the majority (83%) had started pubertal development by age 14 years (Table ). The proportion of prepubertal patients at ages 12 and 16 years was comparable to the reference population ( p > .05); whereas, in CCS, the proportion who were prepubertal was greater at age 14 years (17% vs. 4%; p = .020). Semen samples (the last available sample obtained at a median age of 25 years; 17 years after the end of cancer therapy) were available from 92 CCS (36%); azoospermia occurred in 31 samples (34%; Table ). Repeated semen samples were available from 33 CCS (13%). Ninety‐seven CCS (39%) who were analyzed at a mean age of 37.6 years had registered children (Table ). Table shows cancer treatment exposures in the CCS who had testicular measurements available at the different time points and semen sample in adulthood. Distribution of the exposures at each time point was mostly comparable to that in the whole cohort. However, none of the patients who received testicular radiation at doses from >0 to <1 grays (Gy) had testicular volume measurements available in adulthood. Testicular volume z scores during puberty and in adulthood Figure illustrates the development of testicular volumes z scores from puberty to adulthood stratified by cumulative cancer treatment exposures (testicular radiation dose and CED). Compared with age‐specific reference values, CCS generally exhibited low testicular volume z scores at ages 12–18 years. CCS who were exposed only to chemotherapy (alkylating or nonalkylating) experienced late growth in testicular size, with z scores shifting to the reference range in adulthood (Figure ). The late testicular growth also was observed in CCS who had repeated measurements available at age 18 years and in adulthood (Figure ). In contrast, no late testicular growth occurred in CCS who were exposed to testicular radiation ≥1 Gy (median dose, 12 Gy; Figures ). In multivariable analyses, only testicular radiation ≥1 Gy (median dose, 12 Gy) had an association with testicular volume z scores < −2 SD in adulthood (Table ). Two patients who received testicular radiation ≥1 Gy had z scores > −2 SD (>15.6 mL); both had received HSCT with total body irradiation conditioning (10 and 12 Gy) at age 15 years. When the testicular volume z score was analyzed as a continuous variable, CED ≥12 g/m 2 was independently associated with reduced testicular volume z scores at ages 16 and 18 years, whereas CED from ≥4 to <12 g/m 2 and testicular radiation ≥1 Gy also demonstrated an association at age 18 years. In adulthood, only testicular radiation ≥1 Gy (median dose, 12 Gy) was associated with decreased z scores. Age at diagnosis and time elapsed from the end of cancer therapy were not associated with testicular volume z scores in linear regression analyses (Table ). Semen samples and paternity Figure displays semen sample findings stratified by cancer treatment exposures; the highest sperm counts typically occurred in samples obtained 10–30 years post‐therapy (Figure ). An increasing trend in total sperm counts also occurred in repeated semen samples from patients treated exclusively with chemotherapy (Figure ). Three CCS with azoospermia in their first semen sample (obtained 0.1–5.9 years post‐therapy) had observable amounts of sperm in a repeated sample (obtained 10.7–15.8 years post‐therapy); none had been treated with testicular radiation, and all three had received alkylating agents (at CEDs of 3.0, 3.1, and 11.6 g/m 2 ). Of 21 CCS who received testicular radiation doses ≥1 Gy, five (24%) had detectable sperm in their seminal plasma after the end of cancer treatment (median testicular radiation dose, 10 Gy; range, 10–12 Gy; median time post‐therapy, 21.0 years; range, 10.0–23.0 years; Figure ). In multivariable analyses, testicular radiation ≥1 Gy (median dose, 12 Gy) and a CED of ≥12 g/m 2 were independent risk factors for having azoospermia in adulthood, whereas a longer interval since the completion of cancer therapy approached significance as a protective factor ( p = .078). Testicular radiation ≥1 Gy (median dose, 12 Gy) and a CED ≥4 g/m 2 were associated with decreased paternity rates (Table ). Associations between testicular volume z scores and azoospermia and paternity Compared with their counterparts, CCS with nonazoospermia and with registered children had significantly higher testicular volume z scores from ages 16 and 18 years onward, respectively (see Figure ). Thirty‐seven patients had both testicular volume measurement and semen sample available in adulthood; none of the 28 patients with testicular volume z scores ≥ −2 (≥15.6 mL) had azoospermia, whereas six of nine patients (67%) who had testicular volume z scores < −2 had azoospermia ( p < .001). In ROC curve analyses, the AUCs to predict nonazoospermia (see Figure ) were 0.91 for testicular volume z scores at age 16 years (optimal cutoff point at z score −4.2) and 1.0 in adulthood (optimal cutoff point at z score −2.5). AUCs to predict registered children (see Figure ) were 0.72 for z scores at age 18 years (optimal cutoff point at z score −1.2) and 0.73 in adulthood (optimal cutoff point at z score −1.2). Associations between serum hormone levels and azoospermia and adult testicular volume Compared with their counterparts, CCS who had azoospermia and low adult testicular volume (z score < −2) demonstrated elevated serum levels of follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) and decreased levels of inhibin B. No difference occurred in serum testosterone levels between the groups (see Figure ). In ROC curve analyses, the AUCs for identifying patients with azoospermia were 0.93 for FSH (optimal cutoff at 12.0 IU/L), 0.79 for LH (optimal cutoff at 4.7 IU/L), and 0.94 for inhibin B (optimal cutoff at 44 ng/L; see Figure S5). For identifying patients who had low adult testicular volume, the AUCs were 0.96 for FSH (optimal cutoff at 10.8 IU/L), 0.90 for LH (optimal cutoff at 5.5 IU/L), and 0.94 for inhibin B (optimal cutoff at 47 ng/L; see Figure S6).   Table summarizes patient characteristics and cumulative exposures to cancer treatment for all 255 male CCS; the median patient age at the time of cancer diagnosis was 6.1 years, and diagnoses included hematologic malignancies (69%) and solid tumors (31%). Figure shows a flow chart of the patient selection process; testicular volume measurements were available for 38–105 CCS at ages 12–18 years and for 43 individuals in adulthood (median age, 27 years; median time from the end of cancer therapy, 18 years). Most CCS (84%) were prepubertal at age 12 years, whereas the majority (83%) had started pubertal development by age 14 years (Table ). The proportion of prepubertal patients at ages 12 and 16 years was comparable to the reference population ( p > .05); whereas, in CCS, the proportion who were prepubertal was greater at age 14 years (17% vs. 4%; p = .020). Semen samples (the last available sample obtained at a median age of 25 years; 17 years after the end of cancer therapy) were available from 92 CCS (36%); azoospermia occurred in 31 samples (34%; Table ). Repeated semen samples were available from 33 CCS (13%). Ninety‐seven CCS (39%) who were analyzed at a mean age of 37.6 years had registered children (Table ). Table shows cancer treatment exposures in the CCS who had testicular measurements available at the different time points and semen sample in adulthood. Distribution of the exposures at each time point was mostly comparable to that in the whole cohort. However, none of the patients who received testicular radiation at doses from >0 to <1 grays (Gy) had testicular volume measurements available in adulthood.   Figure illustrates the development of testicular volumes z scores from puberty to adulthood stratified by cumulative cancer treatment exposures (testicular radiation dose and CED). Compared with age‐specific reference values, CCS generally exhibited low testicular volume z scores at ages 12–18 years. CCS who were exposed only to chemotherapy (alkylating or nonalkylating) experienced late growth in testicular size, with z scores shifting to the reference range in adulthood (Figure ). The late testicular growth also was observed in CCS who had repeated measurements available at age 18 years and in adulthood (Figure ). In contrast, no late testicular growth occurred in CCS who were exposed to testicular radiation ≥1 Gy (median dose, 12 Gy; Figures ). In multivariable analyses, only testicular radiation ≥1 Gy (median dose, 12 Gy) had an association with testicular volume z scores < −2 SD in adulthood (Table ). Two patients who received testicular radiation ≥1 Gy had z scores > −2 SD (>15.6 mL); both had received HSCT with total body irradiation conditioning (10 and 12 Gy) at age 15 years. When the testicular volume z score was analyzed as a continuous variable, CED ≥12 g/m 2 was independently associated with reduced testicular volume z scores at ages 16 and 18 years, whereas CED from ≥4 to <12 g/m 2 and testicular radiation ≥1 Gy also demonstrated an association at age 18 years. In adulthood, only testicular radiation ≥1 Gy (median dose, 12 Gy) was associated with decreased z scores. Age at diagnosis and time elapsed from the end of cancer therapy were not associated with testicular volume z scores in linear regression analyses (Table ).   Figure displays semen sample findings stratified by cancer treatment exposures; the highest sperm counts typically occurred in samples obtained 10–30 years post‐therapy (Figure ). An increasing trend in total sperm counts also occurred in repeated semen samples from patients treated exclusively with chemotherapy (Figure ). Three CCS with azoospermia in their first semen sample (obtained 0.1–5.9 years post‐therapy) had observable amounts of sperm in a repeated sample (obtained 10.7–15.8 years post‐therapy); none had been treated with testicular radiation, and all three had received alkylating agents (at CEDs of 3.0, 3.1, and 11.6 g/m 2 ). Of 21 CCS who received testicular radiation doses ≥1 Gy, five (24%) had detectable sperm in their seminal plasma after the end of cancer treatment (median testicular radiation dose, 10 Gy; range, 10–12 Gy; median time post‐therapy, 21.0 years; range, 10.0–23.0 years; Figure ). In multivariable analyses, testicular radiation ≥1 Gy (median dose, 12 Gy) and a CED of ≥12 g/m 2 were independent risk factors for having azoospermia in adulthood, whereas a longer interval since the completion of cancer therapy approached significance as a protective factor ( p = .078). Testicular radiation ≥1 Gy (median dose, 12 Gy) and a CED ≥4 g/m 2 were associated with decreased paternity rates (Table ).   Compared with their counterparts, CCS with nonazoospermia and with registered children had significantly higher testicular volume z scores from ages 16 and 18 years onward, respectively (see Figure ). Thirty‐seven patients had both testicular volume measurement and semen sample available in adulthood; none of the 28 patients with testicular volume z scores ≥ −2 (≥15.6 mL) had azoospermia, whereas six of nine patients (67%) who had testicular volume z scores < −2 had azoospermia ( p < .001). In ROC curve analyses, the AUCs to predict nonazoospermia (see Figure ) were 0.91 for testicular volume z scores at age 16 years (optimal cutoff point at z score −4.2) and 1.0 in adulthood (optimal cutoff point at z score −2.5). AUCs to predict registered children (see Figure ) were 0.72 for z scores at age 18 years (optimal cutoff point at z score −1.2) and 0.73 in adulthood (optimal cutoff point at z score −1.2).   Compared with their counterparts, CCS who had azoospermia and low adult testicular volume (z score < −2) demonstrated elevated serum levels of follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) and decreased levels of inhibin B. No difference occurred in serum testosterone levels between the groups (see Figure ). In ROC curve analyses, the AUCs for identifying patients with azoospermia were 0.93 for FSH (optimal cutoff at 12.0 IU/L), 0.79 for LH (optimal cutoff at 4.7 IU/L), and 0.94 for inhibin B (optimal cutoff at 44 ng/L; see Figure S5). For identifying patients who had low adult testicular volume, the AUCs were 0.96 for FSH (optimal cutoff at 10.8 IU/L), 0.90 for LH (optimal cutoff at 5.5 IU/L), and 0.94 for inhibin B (optimal cutoff at 47 ng/L; see Figure S6).   In this study, we evaluated the associations between cancer therapy exposures and the pubertal and postpubertal growth of testicular size, long‐term spermatogenic recovery, and paternity outcomes. Late testicular growth and spermatogenic recovery occurred in CCS who exclusively received chemotherapy but not in those who received ≥1 Gy (median dose, 12 Gy) testicular radiation. However, both alkylating agents and high‐dose testicular radiation were associated with having prolonged azoospermia and decreased paternity rates. These diverse long‐term outcomes indicate that gonadal damage from chemotherapy may be reversible, whereas high‐dose testicular radiation inflicts damage that likely exceeds the regenerative capacity of the spermatogenic epithelium. The evidence for delayed spermatogenic recovery indicates a need for extended follow‐up beyond the pediatric years when evaluating male fertility after childhood cancer. All CCS, regardless of treatment received, exhibited low median testicular volume z scores during puberty. A delay in progression of Tanner stages was also observed at age 14 years, but nearly all CCS had reached Tanner P stage ≥2 by the age of 16. The delayed pubertal testicular growth, normalization of testicular volume z scores in adulthood, and increasing sperm counts over time in CCS treated solely with chemotherapy support the hypothesis that spermatogenic tissue can regenerate after sublethal cytotoxic insult. In the current study, recovery of testicular volume z scores was observed across all levels of alkylating agent exposure. However, CED ≥12 g/m 2 was identified as a risk factor for azoospermia. These observations are consistent with reports indicating that cumulative cyclophosphamide doses greater than 8–10 g/m 2 , analyzed after a median follow‐up of 9–26 years, are associated with azoospermia and oligospermia. , , However, even after high alkylating agent doses, our results suggest that recovery of spermatogenesis may possibly occur with extended follow‐up time, with permanent sterility potentially occurring after cumulative cyclophosphamide doses exceeding 20–25 g/m 2 . , , , The spermatogenetic recovery may be caused by the preservation of a critical population of spermatogonial stem cells, which, given sufficient recovery time, can re‐establish spermatogenesis. The variation in spermatogenetic recovery suggests the involvement of additional risk factors. These may include genetic predisposition to greater cytotoxic damage or a slower recovery rate, potentially leading to extended azoospermia. In contrast to the findings in chemotherapy‐treated patients, our data indicate that testicular radiation ≥1 Gy (median dose, 12 Gy) results in persistently reduced adult testicular volume and azoospermia, consistent with irreversible gonadal damage. Surprisingly, ≥1 Gy radiation was not associated with lower testicular volume z scores during puberty because comparably low scores were also observed in nonirradiated patients. Most CCS exposed to low testicular radiation doses (<1 Gy) had detectable sperm counts, with the highest counts occurring 10–30 years post‐therapy. These findings are consistent with prior research indicating that spermatogenesis is sensitive to scattered doses as low as 0.1 Gy, causing short‐term cessation of spermatogenesis, whereas only doses exceeding 6 Gy are sufficient to deplete the spermatogonia pool and lead to permanent infertility. Such findings underscore the necessity for early intervention strategies, such as cryopreservation of testicular tissue or sperm, when high‐dose testicular radiation therapy is planned for prepubertal and adolescent males. Moreover, consistent with previous findings, pituitary radiation given primarily as prophylactic treatment to patients with leukemia did not affect the risk of azoospermia in adulthood. We previously reported that high‐dose testicular radiation is the most significant risk factor for nonpaternity. In the current analysis of the same cohort, after adjusting for testicular and pituitary radiation and age at diagnosis, a CED ≥4 g/m 2 was identified as an independent risk factor for reduced paternity rates. This aligns with previous findings on the detrimental effect of alkylating agents on paternity after childhood cancer. The ability to have a biological child is strongly associated with the ability to find a partner among CCS. , Because psychosexual development is crucial for later paternity outcomes, the challenging early life circumstances associated with childhood cancer diagnosis and treatment may influence intimate relationships and sexual functions , and reduce paternity rates, independent of spermatogenic capacity. The current findings indicate that, although late testicular growth and recovery of spermatogenesis can occur, alkylating agents may have additional long‐term implications on reproductive outcomes. Testicular volume z scores at ages 12 and 14 years were not associated with azoospermia, indicating that future sperm production after childhood cancer cannot be reliably predicted by testicular size during early puberty. The z scores from age 16 years onward demonstrated a clearer relationship to azoospermia, with the optimal predictive thresholds for testicular volume z scores < −2. These findings suggest that testicular growth within age‐adjusted references in late puberty is predictive of the absence of azoospermia in adulthood. The association was most robust in adults because none of the patients who had testicular volume z scores > −2 had azoospermia. Among the serum markers, FSH and inhibin B demonstrated comparable efficacy in identifying azoospermic patients. The optimal sensitivity and specificity were 0.84 and 0.90 for FSH, respectively, and 0.93 and 0.85 for inhibin B, respectively. These values are consistent with , or exceed those reported in prior studies. Because some young adult patients may be reluctant to provide a semen sample, testicular volume, FSH, and inhibin B may serve as surrogate measures for azoospermia in fertility evaluations. Compared with azoospermia analyses, the associations between testicular volume z scores and paternity were less robust, aligning with our previous findings of limited associations between serum hormone markers and paternity in the same patient cohort. This is likely because of the complex and multifactorial nature of fertility and parenthood. These thresholds nonetheless provide clinicians with practical tools for identifying individuals at higher risk for long‐term reproductive problems, enabling proactive fertility counseling and potential intervention. Our study has limitations. The retrospective design and data attrition because of incomplete follow‐up must be acknowledged. Notably, CCS who received low scattered testicular radiation doses (<1 Gy) had no testicular volume measurements in adulthood, precluding analyses of late testicular growth in these patients. We considered testicular volume references at age 17 years as indicative of final adult testicular size. In other cohorts of healthy men, significant testicular growth has not been reported beyond this age, and adult testicular volume remains stable after completion of pubertal development. , The reliance on registered paternity as a surrogate marker for fertility capacity does not account for the use of assisted reproductive technologies, nor does it fully capture male fertility potential because becoming a father is also influenced by other social and individual factors. Total sperm count and azoospermia were evaluated using a single semen sample at each time point, although repeated samples were available for some patients over a longer time interval. Because of the retrospective study design, we were unable to exclude other potential causes of azoospermia in all patients (e.g., recent febrile illness, obesity). Only seven patients had received testicular radiation at doses between 1 and 9 Gy; thus, those who received testicular radiation ≥1 Gy predominantly represent patients who received ≥10 Gy radiation. Compared with CCS populations treated with contemporary protocols, our study includes a larger proportion of patients who received ≥10 Gy of testicular radiation. This is likely a contributing factor to a higher prevalence of azoospermia and nonpaternity in our cohort. The strengths of this study include the comprehensive data on treatment exposures, specifically, the precise chemotherapy and radiation doses, enabling detailed analyses of the effects of different alkylating agent and radiation doses. Testicular volume was measured at multiple time points during puberty, supplemented with testicular volumes and semen samples in adulthood, as well as registry‐based paternity data. The longitudinally assessed data allowed the investigation of associations between pubertal testicular volumes and reproductive outcomes in adulthood. In conclusion, the potential for delayed spermatogenic recovery and normalization of adult testicular volume, despite initially low testicular volumes during puberty, underscores the need for an extended follow‐up period until young adulthood to fully understand the long‐term impact of cancer therapy. Developing predictive models that include testicular volume z scores could enhance fertility counseling and intervention strategies. Prospective studies involving larger cohorts of CCS and incorporating longitudinal assessments of testicular volume, direct measures of spermatogenic activity, and detailed hormonal profiles are necessary to substantiate spermatogenic recovery after chemotherapy.   Melanie Korhonen : Conceptualization, data curation, methodology, investigation, visualization, writing–original draft, and writing–review and editing. Kirsi Jahnukainen : Conceptualization, data curation, methodology, investigation, visualization, supervision, project administration, and writing–review and editing. Mikael Koskela : Conceptualization, data curation, methodology, investigation, visualization, formal analysis, and writing–review and editing.   The authors disclosed no conflicts of interest.   Supplementary Material   