Age- and dose-dependent gonadotoxicity

The harmful effects that therapy may have on the gonads and the subsequent implications for fertility are among the most typical concerns for both young and adult patients. Permanent or temporary infertility may result after oncological chemotherapy, depending on whether single agents or combination regimens were used [22]. The extent of gonadotoxicity is influenced by several factors, including the specific drug, its dosage, frequency of administration, the type of malignancy being treated, and the concurrent use of other medications such as immunosuppressants. Furthermore, while the gonadotoxic profiles of traditional chemotherapies are well-documented, the reproductive impacts of emerging targeted treatments like cytokine-based immunotherapies designed to modulate the tumor microenvironment [23]. To help develop preventive methods, it is crucial to understand the precise processes by which various types of chemotherapeutic drugs directly target and harm the prepubertal testis. Recent clinical data highlight the variability in gonadotoxicity across different drug classes and patient ages. As detailed in Table 1, specific agents exhibit distinct toxicity profiles based on cumulative dosages. For instance, in adolescents and adults treated for Chronic Myeloid Leukemia, Imatinib at doses of 240 mg/m² has been linked to decreased sperm density and survival rates. Similarly, in colorectal cancer patients, Oxaliplatin and 5-fluorouracil regimens have resulted in subnormal sperm counts. High-dose chemotherapy in Ewing sarcoma and osteosarcoma often results in permanent spermatogenic failure.

Alkylating agents like procarbazine and cyclophosphamide are frequently associated with an increased risk of permanent infertility [33]. Chemotherapy regimens exhibit different levels of gonadotoxicity; for example, mustine, oncovin, procarbazine, and prednisone are more likely to cause azoospermia than regimens such as BEP (bleomycin, etoposide, and cisplatin) for testicular cancer or adriamycin, bleomycin, vinblastine, and dacarbazine for Hodgkin’s disease [34]. For up to two years after the treatment, males who received bleomycin, etoposide, and cisplatin (BEP) for testicular cancer have shown signs of chemotherapy-induced sperm diploidy, which may indicate that the chemotherapy directly contributes to sperm genetic damage [25]. Procarbazine, which is presently considered to be one of the most gonadotoxic alkylating substances, induce severe gonadal damage and long-term sterility by forming covalent bonds that break DNA double strands [35]. Additionally, mice’s seminiferous tubules were nearly devoid of germ cells 30 days after receiving a single 400 mg/kg treatment [36]. This was not a long-lasting impact for two months after treatment; testes weight was fully recovered in parallel with repopulation. The risk of gonadotoxicity varies substantially by developmental stage. In prepubertal children, the testis contains a developing SSC, so injury at this early stage can limit sperm-producing capacity later in life. In adolescents, the testes grow rapidly, which makes both developing germ cells and hormone regulation vulnerable to treatment [37]. In young adults, spermatogenesis is established, and treatment mainly affects actively dividing germ cells; the chance of recovery depends largely on whether SSCs survive and the cumulative dose exposure.

When administering cyclophosphamide alone, dosages of 19 g/m² are necessary to produce prolonged azoospermia. After completing cyclophosphamide, a follow-up of 26 male patients with azoospermia, 12 individuals resumed spermatogenesis within a period ranging from 15–49 months, with a mean recovery time of 31 months [38]. A preclinical study on prepubertal mice showed that cyclophosphamide exposure triggers rapid transcriptomic reprogramming, including the perturbation of apoptotic and developmental pathways, as early as 16 hours post-treatment [39]. The cumulative dosage of cyclophosphamide was associated with the occurrence of gonadal dysfunction. Busulfan is used in a hematopoietic cell transplant preparation regimen. One dosage of 40 mg/kg was sufficient to damage germ, Sertoli, and Leydig cells in prepubertal rats for up to 10 weeks, after treatment there was a mild recovery [40]. Clinical reports of busulfan in male patients on a cumulative exposure level between < 14 mg/kg and ≥ 16 mg/kg revealed no significant differences in the onset of infertility. Melphalan also functions as an alkylating agent and, when given to juvenile rats, it has been shown to decrease testosterone levels and induce the loss of germ cells. When alkylating agents are used together with platinum-based drugs, such as cisplatin or carboplatin, it has been found that patients had a higher incidence of impaired spermatogenesis, as indicated by elevated follicular-stimulating hormone (FSH) or low inhibin B, compared to those exposed to alkylators alone [41]. According to childhood medulloblastoma cancer survivor male patients, even moderate dose and cumulative doses of cisplatin have been linked to lower livebirth rates [42]. There was no discernible link between cisplatin exposure and infertility in other investigations. Although there were no laboratory studies specifically involving humans, preclinical models demonstrate that cisplatin can decrease germ cells. Also, seminiferous tubules maintained a degenerated appearance with a less severe phenotype over the same recovery period.

Disruption of the HPG axis

An integrated system including the testis, pituitary, and hypothalamus is responsible for maintaining normal spermatogenic processes and regulating the proper release of male hormones. The neuroendocrine activities along the HPG axis play a major role in the endocrine regulation of spermatogenesis [43]. Normal reproductive competence requires the proper development and organization of the HPG axis. Gonadotropin-releasing hormone (GnRH) is a key molecule that controls how the HPG axis functions. Primary or peripheral gonadal failure is most predominantly arisen by gonadal or pelvic irradiation or direct gonadotoxic effects of chemotherapy, especially alkylating drugs. Such interventions have the potential to permanently damage the gonads’ germ cells and somatic support cells, which might result in decreased sex steroids (testosterone), hampered gametogenesis, and either premature gonadal insufficiency or pubertal failure [44]. FSH and luteinizing hormone (LH), two important endocrine signals secreted by the pituitary gland, are controlled by a strong and pulsating release of GnRH from the hypothalamus [45]. When any of these functions are dysregulated, puberty may be delayed or may not present at all, which may lead to infertility, as shown in Figure 2. Reduced FSH and LH production from low GnRH leads to hypogonadotropic hypogonadism, which in turn causes reduced androgen secretion and compromised spermatogenesis [46].

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Figure 2. Hormonal and cellular pathways interrupt spermatogenesis in male reproduction. The figure details the feedback loop between the anterior pituitary and the testis. Disruption leads to the inhibition of Sertoli cells and the production of abnormal sperm cells (head, chromosomal, or tail defects).

Numerous variables, including as the patient’s age at therapy onset, sex, cumulative dosage, and kind of chemotherapeutic drugs, radiation dose and field, and time since treatment conclusion, influence the likelihood and degree of HPG axis dysfunction. GnRH secretion by the hypothalamus or LH and FSH secretion by the anterior pituitary might be affected by cranial irradiation, particularly when it targets the hypothalamic or pituitary regions [47]. This condition, known as central (or secondary) hypogonadism, causes the gonads to not be sufficiently stimulated, which impairs spermatogenesis and results in abnormal sex-steroid synthesis (testosterone) [48]. On the other hand, primary hypogonadism, characterized by impaired testicular responsiveness to gonadotropin stimulation due to germ cell and Leydig cell damage, frequently results from direct gonadal injury. This injury is frequently associated with exposure to alkylating agents (e.g., cyclophosphamide, busulfan) or pelvic/testicular irradiation [49]. It is known that survivors of hematopoietic stem cell transplantation with total body irradiation are more vulnerable to primary and central hypogonadism, with a high prevalence of gonadal failure and pubertal arrest observed in a subgroup [50]. Additionally, even while advances in radiotherapy techniques such as proton beam therapy try to reduce collateral damage, there is still a possibility that the HPG axis might be disrupted, when multimodal regimens are employed. Beyond reproductive impairment, the long-term impacts of HPG axis dysfunction include poor linear development, inadequate bone mineral density accumulation, and adverse psychosocial outcomes [51]. Consequently, prolonged surveillance coupled with prompt endocrine intervention constitutes a critical aspect of survivorship care in this patient population, aiming to reduce morbidity and enhance the overall quality of life. Importantly, HPG-axis vulnerability is age-dependent. In prepubertal children, cranial irradiation or chemotherapy may prevent normal pubertal activation of the axis. In adolescents, during the establishment of GnRH pulsatility, treatment may interrupt pubertal progression or result in delayed/secondary hypogonadism [52]. In young adults, the HPG axis is already developed, so damage is more likely to appear as low testosterone levels, increased gonadotropins, and reduced sperm production.

Synergistic effects with radiation and surgery

Radiation has been shown to decrease spermatogenesis, alter hormone production, and result in infertility. At 0.1–1.2 grey (Gy), radiation dosages start to negatively impact spermatogenesis, and at 4 Gy the damage is permanent [53]. Permanent azoospermia results from fractionated testicular irradiation at target doses of 20 Gy [54]. This therapy is often administered to individuals who have testicular carcinoma in situ (CIS) or as a follow-up procedure after testis-conserving surgery for a minor testicular cancer. Leydig cells can survive up to 30 Gy of radiation, making them more resilient to damage from radiation. However, it may take up to 9 years after therapy for spermatogenesis to recover [55]. There is still uncertainty about the maximum dosage levels at which persistent azoospermia becomes unavoidable. In leukaemia, less than 20% of individuals who get this therapy have been found to recover their gonadal function. Radioiodine ¹³¹I may interfere with spermatogenesis when given to individuals with thyroid cancer in a single standard dosage [56]. However, excessive cumulative dosages may cause permanent damage, thus patients who are expected to need multiple ¹³¹I treatments ought to think about semen cryopreservation. There is reason to be concerned about the extent of genetic damage induced to the spermatozoa during the treatment as radiation and the majority of cytotoxic chemotherapies harm genomic material. Even potential genotoxic cancer treatments have been shown to cause relatively few or no detectable mutations in human SSCs, at least according to the current methodologies [57, 58]. This is supported by a large number of research on offspring conceived often a year or more after therapy when the sperm cells would have been produced from living stem cells. The children of male survivors of radiation exposure from the atomic bomb in Japan and the children of survivors of childhood cancer treated with radiation and/or possibly mutagenic chemotherapy are two important studies. Specifically, alkylating agents and radiation may induce mutations in spermatozoa and spermatids, whereas topoisomerase inhibitors can alter spermatocytes, and some nucleoside analogs can alter early spermatocytes and spermatogonia. Chemotherapy and radiation therapy together will cause higher gonad toxicity than either treatment alone [59]. Apart from the effects on sperm concentration, radiation therapy has been shown to increase sperm DNA fragmentation, with these effects potentially lasting for up to two years after treatment [60]. As a result, even if spermatogenesis recovers, it still influences fertilization rates. Higher degrees of spermatocytic damage are caused when the testes receive an increasing dosage of dispersed radiation during radiotherapy for Hodgkin’s disease or retroperitoneal lymph node metastases of testicular cancer [61]. The radiation dose will be reduced by shielding the gonads, but dispersed radiation may still be quite harmful.

Epigenetic alterations and transgenerational concerns

In spermatogonia, radiation and alkylating agents can induce single gene mutations as well as chromosomal translocations [62]. As a result, it has become standard practice to advise patients to postpone pregnancy for 3–4 months following treatment to allow for the restoration of healthy sperm [63]. Chromosome abnormalities are 10–15 times more prevalent in fertile patients, and they have been on the rise in infertile patients, accounting for 4.3% of oligozoospermic patients and 20.6% of azoospermic patients [64, 65]. According to new research, epigenetic modifications that do not involve modifications to the DNA sequence itself can be passed down via the male germline in both human and animal models, which may exert potential consequences in the health outcomes of subsequent generations [66]. Epigenetic inheritance mechanisms include DNA methylation, retained histone alterations, and small non-coding RNAs. Among these, the most studied inheritance mechanism is DNA methylation particularly when germ cells are exposed to chemotherapy agents. DNA methyltransferases (DNMTs) regulate DNA methylation patterns, and they may be hindered by substances like 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine), the latter of which is used therapeutically to treat cancer. A longitudinal study of a testicular germ cell tumor patient treated with BEP chemotherapy identified 179 early differentially methylated regions, 43 of which remained epigenetically altered even 24 months post-therapy [67]. Exposure at the spermatid stage seemed to have no discernible effect, but exposure during the spermatocyte stage had milder effects. Moreover, chemotherapeutic regimens not specifically formulated to inhibit DNMTs have nonetheless been shown to induce alterations in sperm DNA methylation patterns when administered continuously throughout the entire spermatogenic cycle. These epigenetic alterations have been correlated with early postnatal mortality in offspring; the precise causal mechanisms underlying the relationship between DNA methylation changes and the observed outcomes have yet to be clearly established. Even though azoospermia is often the most visible clinical concern, a risk exists in survivors retaining sperm production while carrying chemotherapy-induced DNA methylation abnormalities [68]. This ‘Epigenetic Legacy’ challenges the current reliance on standard semen analysis. Survivorship guidelines should differentiate between reproductive recovery (the ability to achieve conception) and genomic recovery (the production of epigenetically intact sperm). Radiation and chemotherapy cause genetic instability in rodents, which may be passed on to offspring and later generations. In some cases, this can lead to reduced growth, learning, and reproductive potential as well as an increased susceptibility to cancer [69]. Instead of genetic transmission the inheritance patterns of these transgenerational abnormalities suggest epigenetic transmission. Evidence from various studies indicates that both spermatogonia and spermatids exhibit susceptibility to the induction of transgenerational effects, although none of these studies have determined if there is a dependency on the stage of germ cells [70]. Chemotherapy-induced damage may significantly affect a patient’s subsequent reproductive result, potentially affecting fertility and impairing the development of sexual traits. SSCs, a kind of male germ cell, and functional supporting somatic cells must continue to survive for long-term fertility. Chemotherapeutic drugs may harm SSCs and may cause genetically aberrant sperm precursors in addition to acute consequences [71]. Although preclinical models provide consistent evidence that chemotherapy and radiation can induce epigenetic alterations in germ cells, the corresponding evidence in humans remains comparatively limited. In the offspring of cancer survivors, most of the available studies are observational, and the associations reported between parental cancer treatment, epigenetic modifications, and health outcomes do not establish definitive germline epigenetic inheritance [72]. Human research in this area is difficult because many other factors can influence outcomes, including parental age, lifestyle, environment, and the direct impact of the original cancer. It is also challenging to separate epigenetic changes that are genuinely inherited from those that arise after birth. For these reasons, while the mechanistic data from preclinical models are strong and biologically credible, current human evidence should be interpreted cautiously and regarded as largely associative rather than confirmatory.

Fertility outcomes in long-term cancer survivors

Male gonads tend to be protected from radiation damage. However, there are two exceptions, whole body radiation prior to bone marrow transplantation and in application of irradiation as a therapeutic modality in case of tumor cell involvement in the testis. Testicular irradiation therapy of administered doses ranging from 16–18 Gy for the treatment of testicular CIS is strongly associated with a high incidence of irreversible sterility [73]. Numerous studies have been conducted on post-treatment fertility. Apart from the oncological treatment that the patient has received, his pre-treatment fertility condition appears to be a significant predictor of post-therapy spermatogenic recovery. For example, males with stage 1 seminomas who had just undergone para-aortic irradiation showed a high rate of sperm recovery than in patients treated for testicular germ cell tumor (TGCT) still, no cases of azoospermia were witnessed [74]. In contrast, 55–80% of males who had chemotherapy based on cisplatin were spotted sperm recovery. Chemotherapy based on carboplatin was linked with lower spermatogonial damage [75]. Cancer therapy has the potential to induce genetic mutations in germ cells, which may elevate the risk of developmental abnormalities and paediatric disorders, including congenital malformations [76]. Crucially, there was no documented elevated risk for congenital abnormalities. Also, children of males who had previously had chemotherapy or radiation treatment for cancer did not have an increased incidence of juvenile malignancies, except for some types of hereditary cancers.

Cohort studies and meta-analyses

A comprehensive assessment of reproductive health in cancer survivors should include a detailed history of cancer treatment. In male patients aged 13 years and older, this assessment should also include a comprehensive sexual history. For survivors of childhood, adolescent, and young adult cancers, exposure to increasing doses of cyclophosphamide, procarbazine, or ifosfamide exceeding 50g/m² is associated with a lower probability of achieving future paternity, though current supporting data is limited in quality [77]. Despite their limited participation in formal studies, several substances often used in conditioning regimens for hematopoietic stem cell transplants, including busulfan, melphalan, fludarabine, and ifosfamide, have also been implicated based on expert consent. A dose-dependent association between exposure to alkylating agents and paternity outcomes was observed in a childhood cancer survivor study [78]. Male survivors who were treated with cyclophosphamide, procarbazine, ifosfamide, or cisplatin exhibited significantly lower rates of fatherhood compared to their sibling controls [79]. However, individual reproductive outcomes are not always predicted by cumulative medication dosage. The St. Jude Lifetime Cohort Study found that sperm concentration and cyclophosphamide equivalent dosage were negatively correlated [80]. However, since some patients showed total germ cell loss even at low doses, it was difficult to determine a precise threshold for azoospermia. These findings strengthen the idea that all male survivors exposed to alkylating agents should be considered at risk for altered spermatogenesis, regardless of the severity of treatment, and highlight the significant interindividual variations in gonadal vulnerability. The use of serum inhibin B and FSH as surrogate indicators for azoospermia is debatable. In limited cohorts, several studies showed a direct relationship between sperm concentration and inhibin B level [81]. It was found that inhibin B had a 91% sensitivity and 90% specificity for detecting azoospermia, but in a study of 275 male survivors of childhood or teenage cancer, the positive predictive value was only 66%. Inhibin B’s specificity for detecting azoospermia was 45.0%, and its positive predictive value was 52.1% [82]. FSH had a positive predictive value of 65.1% and a specificity of 74.1%. Therefore, neither inhibin B nor FSH is a satisfactory substitute for semen analysis.

Emerging biomarkers for predictive assessment

Sperm molecular components may serve as key indicators of reproductive damage. This is particularly crucial for long-term exposures such as those that occur during chemotherapy regimens in which cytotoxic medications are often administered for months rather than days [83]. The mature sperm in the ejaculate mimic the testicular conditions throughout the human spermatogenesis stage. The simplest way to measure this could be to measure alterations in mRNA [84]. Glutathione S-transferase and aldehyde dehydrogenase transcripts were found to be elevated in round spermatids in a recent study conducted on rats to examine the effects of BEP chemotherapy on drug-metabolizing enzymes [85]. These results imply that phase II metabolizing enzymes, including the glutathione S-transferase and aldehyde dehydrogenases, might be predictive biomarkers for chemotherapy-induced testicular toxicity. A number of techniques have been developed to quantify the damage to sperm DNA. The frequently used assays are the Sperm Chromatin Structure Assay (SCSA), the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL), and the Sperm Chromatin Dispersion Test (SCD) [86]. Because SCSA depends on flow cytometry, it requires expensive equipment and central laboratory analysis. TUNEL and SCD, on the other hand, are easier for in-house implementation because of their simpler instrumentation needs and comparatively lower cost. In research, SCD and TUNEL in males were compared to unexplained infertility [87]. Based on chromatin structural susceptibility, SCD works via a two-step process that includes regulated DNA denaturation and protamine depletion, whereas TUNEL uses tagged nucleotides at damage regions to directly measure single- and double-stranded DNA breaks. Despite this, SCD proved to be more user-friendly, sensitive, and rapid than TUNEL. The United States Environmental Protection Agency has investigated SP22, a sperm-specific protein that declines in abundance following exposure to toxicants affecting both the epididymis and the testes. Recent human clinical updates shows that while established as a fertility biomarker across mammalian species, its expression pattern varies significantly between fertile and infertile cohorts [88]. Young males (20 to 34 years old) are most likely to develop TGCTs. Chemotherapy based on cisplatin is effective in treating about 90% of TGCTs [89]. On the other hand, this treatment increases the risk of developing secondary cancers. Several novel biomarkers, including CIS or Intratubular Germ Cell Neoplasia that represent innovative molecular targeted therapies, have been discovered to effectively distinguish between TGCT subtypes. A comprehensive classification of these markers, highlighting the differential expression between seminomas and non-seminomas. While OCT3/4 is a well-established transcription factor expressed in CIS, seminoma, and embryonal carcinoma, it is notably absent in yolk sac tumors and choriocarcinoma. Meanwhile, SOX17 offers a distinct profile, being expressed in CIS and seminoma but variable in yolk sac tumors, aiding in differential diagnosis. Other markers provide insight into chromatin structure and cell cycle regulation. For example, chromatin-associated proteins like HMGA1 are expressed across CIS, seminoma, and embryonal carcinoma, whereas HMGA2 is absent in seminoma, making it a specific marker for embryonal carcinoma and yolk sac tumors. About 40% of testicular mRNAs are carried by sperm cells, which makes the sperm transcriptome a useful, non-invasive proxy for tracking gene expression during spermatogenesis [90, 91]. A range of mRNA transcripts, such as motility-related mRNAs that exhibit reduced abundance in males with disrupted sperm motility, have been recognized as potential biomarkers of male fertility [92]. Infertile males have an unusual amount of protamine mRNAs, which are essential for chromatin packing. The anti-apoptotic marker Bcl-2 mRNA is upregulated in infertile individuals’ sperm [93]. Sperm RNA is positioned as a promising molecular biomarker for fertility assessment because these RNAs can either be retained passively or functionally contribute to processes like chromatin remodelling, gene imprinting, and early embryogenesis. Spermatogenic integrity will reflect in the epigenetic biomarkers of sperm DNA methylation patterns. Germline DNA dynamically reprogrammes during spermatogenesis, and the functioning of the testicles will be associated with abnormal methylation patterns. For instance, infertile men have abnormal methylation in both imprinted and non-imprinted genes (H19, IGF2) [94]. Extensive research in adult rodent models has explored toxicant exposure effects on sperm DNA methylation, including detailed analyses of epigenetic alterations following chemotherapy and tamoxifen-induced changes in the methylation patterns of imprinted genes. The tumor biomarkers listed in Table 2 (e.g., OCT3/4, c-Kit, SOX17, NANOG) are not only diagnostic markers for TGCT, but they also serve for long-term male fertility prognosis. These markers identify neoplastic or premalignant germ cells that frequently require gonadotoxic treatments such as radiotherapy or platinum-based chemotherapy. As a result, the presence of such biomarker-positive lesions is linked with the intensity of treatment exposure and spermatogenic loss. Furthermore, biomarkers such as OCT3/4 and c-Kit are directly involved in germ cell self-renewal and differentiation pathways; persistent expression after therapy may indicate impaired spermatogonial stem-cell recovery and, consequently, reduced fertility restoration potential [95]. A shift is needed from isolated fertility markers to a multimodal assessment model. Instead of relying on FSH alone, Inhibin B, a direct hormone product secreted by the Sertoli cells inside the testes. It acts as a direct molecular proxy for how healthy the seminiferous tubules which act as a “non-invasive biopsy” of testicular function [96]. Such an approach enables clearer differentiation between maturation arrest and Sertoli-cell-only patterns.