This retrospective cohort study was conducted at a single tertiary care institution. The study population comprised male patients aged 11-25 years who were referred to the institution’s fertility preservation center between October 2014 and December 2023. Patients were referred for fertility preservation counseling immediately after being diagnosed with a malignant disease by the hematology-oncology or pediatric hematology-oncology departments, including those scheduled for radiotherapy or neurosurgical procedures. Of the 365 patients initially referred after cancer diagnosis, 67 were excluded: 39 due to non-cancer conditions (e.g., benign hematologic, metabolic, autoimmune, and soft tissue disorders) and 28 due to prior chemotherapy (e.g., post-chemotherapy referral, recurrent disease, or secondary malignancy). Thus, 298 patients diagnosed with cancer were included in the analysis.

Collected data included patient age, height, weight at diagnosis, cancer type, decision to attempt cryobanking, success of semen collection, and number of collection attempts. Reasons for not attempting cryobanking, unsuccessful semen collection, and patient consent for use of cryopreserved sperm in research were reviewed from medical records. For patients who successfully underwent cryobanking, the disposition of cryopreserved semen samples was analyzed as of December 31, 2023. The 298 patients were stratified into three age groups: 11-15 years (n=146; 49.0%), 16-20 years (n=101; 33.9%), and 21-25 years (n=51; 17.1%). Among these, 215 patients attempted sperm cryobanking; of these, 183 successfully produced semen samples, and 167 successfully completed sperm freezing. A total of 268 semen samples were analyzed, resulting in 1,272 cryopreserved straws (Fig. 1).

Semen samples were collected via masturbation with the participants’ consent. Although a minimum of 3 days of abstinence was recommended, this was not strictly enforced due to the patients’ age and the urgency of cancer treatment. Samples were obtained either during semen analysis or oocyte retrieval. After allowing the semen to liquefy for 30 minutes at room temperature, each sample was analyzed using a computer-assisted semen analysis system (FAS2011, Medical Supply Co., Seoul, Korea). Parameters assessed included volume, concentration, motility, and morphology, following the 2010 World Health Organization (WHO) criteria [27].

Several smears were prepared from each specimen to assess strict morphology and chromatin status. Hemacolor staining (MERCK, Darmstadt, Germany) was used to evaluate strict morphology, with 200 spermatozoa analyzed under light microscopy at ×1,000 magnification using oil immersion. Sperm morphology was considered normal if at least 4% of sperm exhibited normal morphology. Additional semen parameters analyzed included amplitude of lateral head displacement, velocity of the curved line, velocity of the straight line, linearity (ratio of velocity of the straight line to velocity of the curved line), and total motile sperm count (TMSC).

Semen cryobanking was performed following the manufacturer’s protocol for the CooperSurgical-Origio Sperm Freezing Medium (CooperSurgical Inc., Trumbull, CT, USA). After liquefaction, the total ejaculate volume was measured, and semen analysis was conducted. A two-step density gradient centrifugation was then performed at 300×g for 20 minutes. The supernatant was discarded, and the sperm pellet was collected. The sperm pellet was diluted with the freezing medium at a 1:1 ratio at room temperature, with the medium added dropwise and the mixture gently mixed after each addition. The mixture was then allowed to rest at room temperature for at least 10 minutes. Subsequently, the diluted sperm suspension was loaded into straws, sealed, and suspended horizontally just above the surface of liquid nitrogen vapor for 30 minutes. Finally, the straws were transferred into liquid nitrogen for storage at -196°C.

Categorical variables are presented as percentages. Continuous clinical variables are summarized using medians and ranges, while semen analysis parameters are expressed as means±standard error of the mean. Logistic regression analysis was performed to identify clinical factors associated with successful sperm cryobanking. All statistical analyses were conducted using SPSS software version 25 (IBM Corp., Armonk, NY, USA).

This study was approved by the Institutional Review Board (IRB) under approval number 2302-097-1407, granted on March 13, 2023. A waiver of written informed consent was granted as the study exclusively involved retrospective review of medical records and posed minimal risk to participants.

Among the 298 patients with cancer referred for fertility preservation counseling, 215 (72.1%) attempted sperm cryobanking. Of these, 183 (85.1%) successfully provided semen samples, and 167 (77.7%) ultimately underwent successful sperm cryopreservation. A total of 268 semen samples were analyzed, yielding 1,272 cryopreserved straws. Hematologic malignancies were the most prevalent cancer type across the study period, accounting for 14.5% to 34.5% of cases. Sarcoma incidence demonstrated an increasing trend, rising from 12.0% in 2014-2015 to 23.6% in 2022-2023. The prevalence of lymphoma fluctuated between 12.1% and 22.2%, while germ cell tumors and central nervous system tumors showed variable rates throughout the years (Supplementary Fig. 1).

The mean patient age was 16.0 years (range, 11-25), and the median body mass index (BMI) was 21.3 kg/m² (range, 12.7-45.9). Leukemia was the most common diagnosis (n=67; 22.5%), followed by sarcoma (n=66; 22.1%) and lymphoma (n=51; 17.1%) (Table 1).

The rate of attempts to cryobank sperm increased significantly with age: 59.6% (87/146) in the 11-15-year group, 82.2% (83/101) in the 16-20-year group, and 88.2% (45/51) in the 21-25-year group (Table 2). Among the 83 patients who did not attempt cryopreservation, the predominant reason was refusal by both patients and parents (43.4%; 36/83), followed by parental refusal alone (26.5%; 22/83), and pre-spermarche status (14.5%; 12/83).

Semen collection success rates among those who attempted collection also demonstrated an age-related increase: 80.5% (70/87) in the 11-15-year group, 88.0% (73/83) in the 16-20-year group, and 88.9% (40/45) in the 21-25-year group. The primary cause of collection failure was inability to ejaculate, occurring in 16.1% (14/87), 8.4% (7/83), and 8.9% (4/45) of patients in each respective group.

Similarly, successful sperm cryopreservation rates-calculated among those with successful semen collection-increased with age: 85.7% (60/70) in the 11-15-year group, 94.5% (69/73) in the 16-20-year group, and 95.0% (38/40) in the 21-25-year group. Reasons for unsuccessful cryopreservation included azoospermia (n=12), oligozoospermia (n=2), asthenozoospermia (n=1), and voluntary withdrawal from cryopreservation (n=1).

Logistic regression analysis (Table 3) identified age as a significant predictor of whether patients attempted sperm cryobanking. Specifically, patients in the 11-15-year age group were significantly less likely to attempt cryobanking compared to those aged 21-25 years (adjusted odds ratio [OR], 5.059; 95% confidence interval [CI], 2.024-12.645; P=0.001). Among those who attempted cryobanking, age-related differences in success rates were less marked and did not reach statistical significance. A BMI below 25 kg/m² was associated with a trend toward higher success rates (adjusted OR, 1.706; 95% CI, 0.728-4.000; P=0.219), whereas patients with hematologic malignancies showed a trend toward lower success rates (adjusted OR, 0.574; 95% CI, 0.283-1.163; P=0.123), though these associations were not statistically significant.

Semen quality parameters improved with increasing age (Supplementary Table 1). The mean ejaculate volume increased significantly, from 1.23±0.11 mL in the 11-15-year group to 2.35±0.19 mL in the 21-25-year group. Similarly, total sperm count and progressive motility improved with age, while strict morphology percentages remained stable across groups. TMSC was lowest in the youngest cohort (11-15 years; 51.98±8.41×10⁶), compared with the 16-20-year (115.14±18.86×10⁶), and 21-25-year (98.68±16.19×10⁶) groups. Semen abnormalities, including azoospermia and teratozoospermia, were most prevalent among the youngest patients.

By December 31, 2023, disposition data for cryopreserved sperm samples were evaluated (Table 4). The median storage duration was 1,292 days (interquartile range, 633-1,858 days). A majority (77.2%) of patients opted to continue cryobanking, while disposal was primarily due to patient request (13.2%), death (7.8%), or loss to follow-up (1.8%). Disposal rates were higher among younger patients, often reflecting family decisions. Among patients alive at the time of analysis, 14.3% elected to discard their frozen samples. Logistic regression examining predictors of sample disposal, including age at cryobanking, cancer diagnosis (hematologic vs. non-hematologic), and BMI, did not identify statistically significant associations. Compared to patients aged 21-25 years, the odds ratios for discarding samples were 0.954 (95% CI, 0.308-2.953; P=0.935) for the 11-15-year group and 0.740 (95% CI, 0.236-2.321; P=0.606) for the 16-20-year group.

Our results demonstrated clear age-related trends in sperm cryobanking behavior and outcomes. The rate of attempts at sperm cryobanking increased markedly with age-from 59.6% in the 11-15-year group to 88.2% in the 21-25-year group. Similarly, semen collection success rose from 47.9% in the youngest group to 78.4% in the oldest group. These findings align with previously published data; for example, the literature reports approximately a 19% failure rate of semen collection in the 11-14-year age group, comparable to the 19.5% (17/87) failure rate observed in a large cohort study [21].

In younger patients, the primary challenge was failure to ejaculate. van Casteren et al. [28] similarly reported that younger adolescents often experience difficulties with conventional collection methods due to illness or fatigue, resulting in insufficient or immotile samples in about 17.5% of cases. This highlights the critical need for offering alternative sperm retrieval techniques. Methods such as penile vibratory stimulation (PVS), electroejaculation, and testicular sperm extraction have shown promise in improving collection rates among younger patients who cannot provide samples by masturbation [29,30]. For instance, Adank et al. [31] reported a 27% success rate with electroejaculation in 11 adolescent patients unable to ejaculate conventionally. These findings emphasize the importance of a multimodal approach to sperm retrieval to optimize fertility preservation, especially in younger adolescents.

Our findings regarding factors influencing sperm cryobanking attempts and success rates align with those reported in previous studies. Age emerged as a significant factor affecting cryobanking attempts, with younger adolescents (11-15 years) less likely to attempt sperm banking. This is consistent with Klosky et al. [32], who identified age as a crucial determinant in sperm banking engagement. The lower attempts in younger adolescents may reflect challenges related to emotional immaturity or limited comprehension of fertility preservation procedures.

Although not statistically significant in our cohort, we observed a trend toward higher success rates in patients with a BMI <25 kg/m², consistent with Ferrari et al. [33], who reported a negative association between elevated BMI and sperm quality. Similarly, reduced success rates in patients with hematological cancers, particularly leukemia, paralleled observations by Li et al. [18], who documented diminished sperm quality in this population.

The overall sperm cryobanking success rate in our study was comparable to that reported by Daudin et al. [21], who found an 85.1% success rate in semen sample acquisition. Notably, our success rates among younger adolescents were higher than those previously reported; for example, Daudin et al. [21] documented only a 20% success rate in 10-14-year-olds. This discrepancy may reflect improvements in fertility counseling and heightened awareness of preservation options in recent years. These findings underscore the complex interplay of biological and psychosocial factors affecting fertility preservation outcomes in young male patients with cancer and highlight the necessity for individualized fertility counseling tailored to age and clinical context.

Our study revealed distinct age-related differences in semen parameters among male AYA patients with cancer, offering important insights for fertility preservation strategies. Sperm concentration peaked during late adolescence, followed by a slight decline in early adulthood, consistent with prior reports documenting age-related increases in sperm counts with a peak in older adolescents [19,21]. Improvements in sperm motility and morphology were also observed with advancing age. Total motility increased from 45.32% in the youngest group to 60.61% in patients aged 21-25 years, aligning with Hagenäs et al. [34], who correlated greater testicular volume in maturing adolescents with enhanced motility. The morphology rates followed a similar trend, with normal morphology increasing from 27.7% to 43.5%, supporting the observations of Daudin et al. [21], who reported enhanced semen quality as adolescents transitioned into young adulthood. TMSC, a key predictor of fertility potential and treatment viability such as in vitro fertilization, demonstrated peak values in the 16-20-year age group. These results corroborate Adam et al. [19], who reported that TMSC increases with age, reaching a maximum in older adolescents. Although the WHO laboratory manual for semen analysis was updated in 2021 with changes to terminology, classification, and methodology, the fundamental reference values for semen parameters have remained largely consistent since the 2010 edition. Our study primarily applied the 2010 reference criteria due to the data collection period (2014-2023). However, some updated laboratory practices from the 2021 manual-such as vitality testing when total motility is below 40% and refined morphological assessments-were implemented during later years of the study. These adjustments do not compromise the validity of our findings, as the reference thresholds remain substantially unchanged between editions.

In our study, the median storage duration for cryopreserved sperm was 1,292 days, with 77.2% of patients opting to continue storage. This aligns with findings by Menon et al. [20], who reported that most adolescent patients maintain cryobanking post-treatment, highlighting a preference for long-term preservation, particularly among younger patients with favorable prognoses. Bizet et al. [22] observed that 18.7% of patients with cancer discarded samples, mainly due to death (42.9%) or resumption of spermatogenesis (27.6%). Similarly, our data revealed patient death (7.8%) and personal request (13.2%) as the most common reasons for sample disposal. Unlike previous studies, we examined disposal frequency across age groups and found that disposal rates were highest among younger patients, particularly due to requests from patients and caregivers. These disposition patterns underscore the importance of individualized counseling in fertility preservation, especially for younger patients whose families often play a more pivotal role in decision-making compared to older patients.

Our analysis of factors influencing sample discard showed that 16.2% of patients alive at evaluation chose to discard their cryopreserved sperm. Although age-group analysis suggested younger patients were less likely to discard samples compared to those aged 21-25 years, this trend was not statistically significant. The absence of significant predictors in our logistic regression model implies that personal circumstances and psychological factors may be more influential in long-term storage decisions than demographic or clinical variables. Moreover, initial consent for cryopreservation in younger patients was typically obtained from parents; however, long-term disposition decisions may be shaped by evolving family perspectives, patient health recovery, or improvements in semen quality after treatment.

These findings highlight the need for tailored counseling and consistent follow-up within fertility preservation programs for young male patients with cancer. Future studies with larger cohorts and a focus on psychosocial determinants could further elucidate decision-making processes related to cryopreserved sperm management.

While this study offers valuable insights into sperm cryobanking outcomes among AYA male patients with cancer, several limitations warrant consideration. First, as a single-center retrospective study, the generalizability of our findings to broader populations is limited. The retrospective design also constrains control over variables and introduces potential biases inherent to observational data. Key clinical variables such as Tanner stage, detailed pubertal development, and overall health status at the time of semen collection were unavailable. These factors are critical influencers of semen quality and collection success. Due to the absence of Tanner stage data, we employed age-based groupings as proxies for pubertal status, which limited our ability to accurately assess the impact of biological maturity on cryobanking outcomes. Additionally, information regarding abstinence duration prior to sample collection was not recorded, potentially influencing sperm parameters but not analyzable within our study framework. Our protocol did not include adjunctive sperm retrieval techniques, such as electroejaculation or PVS, which may have enhanced collection success rates, particularly among younger patients unable to produce samples via masturbation. Furthermore, psychosocial variables, including family motivation, emotional status, and financial considerations, were not evaluated; these may significantly affect decisions regarding long-term sperm storage and disposition. The relatively small sample size in the 21-25-year age group could introduce selection bias, especially if certain cancer types were disproportionately represented. Moreover, the retrospective nature of this study precluded determination of reasons for sperm disposal, such as patient mortality, successful recovery of fertility, or psychosocial factors, which remains a key limitation. Finally, the short duration of follow-up limited our capacity to evaluate post-thaw sperm utilization and long-term fertility outcomes. Future research should focus on multicenter prospective studies with longer follow-up periods, incorporating comprehensive clinical, developmental, and psychosocial data to validate and expand upon these findings. Such studies will help refine fertility preservation strategies tailored to the unique needs of the AYA cancer population.

In summary, our study identifies significant age-related factors affecting sperm cryobanking success and long-term storage decisions in AYA male patients with cancer, emphasizing the necessity for individualized, age-appropriate fertility preservation approaches in this group.