Alkylation, DNA leads to Decrease, Sperm count

The biological plausibility of this KER is strong. DNA alkylation refers to the covalent addition of alkyl groups (e.g., methyl, ethyl, propyl) to nucleotides at noncanonical positions in DNA, generating lesions that interfere with DNA replication, transcription, and chromosome integrity (Soll et al., 2017). These lesions can occur across all stages of spermatogenesis and trigger downstream signalling cascades that lead to apoptosis. Across species, actively proliferating cells have long been shown to be particularly susceptible to chemotherapeutic alkylating drugs due to ongoing DNA replication; this provides a conserved biological basis for alkylation-induced impairment of spermatogenesis (Drewinko et al., 1981; Meistrich, 1982a).

Unrepaired alkylation damage in male germ cells can block DNA replication and induce base mispairing and strand breaks, which activate DNA damage response pathways and lead to cell cycle arrest and selective elimination of damaged cells through programmed cell death (Kaina, 2003). This stringent quality control mechanism is important to preserve germline genomic integrity; however, it inevitably reduces the supply of cells progressing through spermatogenesis (Agarwal et al., 2020; Li et al., 2025). Therefore, DNA alkylation-induced apoptosis in germ cells can plausibly lead to reduced sperm counts.

The consequences of alkylation-induced apoptosis on sperm counts vary depending on the developmental stage at which the damage occurs. Alkylation of DNA in proliferating or differentiating germ cells at early (pre-meiotic) stages compromises the ability of the testis to replenish the pool of developing germ cells and can cause sustained or irreversible reductions in sperm counts (Meistrich et al., 2013).

Meiosis gives rise to spermatocytes, which have tightly regulated meiotic DNA damage checkpoints to coordinate repair of endogenous programmed DNA strand breaks (Hunter, 2015). Alkylation-induced DNA lesions in meiotic cells can increase the overall repair burden and interfere with the tightly regulated repair of programmed meiotic breaks. When lesions persist, checkpoint signaling promotes the elimination of defective meiotic cells (Roeder and Bailis, 2000; Li et al., 2025). However, this effect is typically temporary because surviving SSCs can repopulate the seminiferous epithelium over time (Meistrich, 1982a).

In contrast, post-meiotic germ cells in late spermatogenesis are largely DNA repair deficient; thus, DNA damage at this stage can only be repaired after fertilization through maternal repair mechanisms (Olsen et al., 2005; Newman et al., 2021). Alkylation of DNA in post-meiotic germ cells does not typically drive germ cell death (and thus reduced sperm counts) contributing instead to sperm DNA damage and reduced sperm quality.

In addition to germ cells, alkylation damage in testicular Sertoli and Leydig cells may also indirectly contribute to reduced sperm counts through disruption of structural support and hormonal regulation required for spermatogenesis. However, differentiating germ cells are considered particularly sensitive targets of alkylating agents due to ongoing DNA replication, and are likely the primary drivers of alkylation-induced impairment of sperm production.

In addition to impacts on adult spermatogenesis, clinical and experimental studies demonstrate that exposure to alkylating chemotherapeutic agents during prepubertal or adolescent ages can impair germ cell populations and lead to persistent reductions in sperm production detectable in adulthood (reviewed by Delessard et al., 2019). Alterations in Sertoli cells and Leydig cells have also been reported following chemotherapy exposure, albeit the effects are inconsistent across studies and do not yet allow definitive conclusions (Delessard et al., 2019). These observations in cancer survivors and experimental models support the relevance of early-life stages and the delayed manifestation of impaired spermatogenesis following chemotherapy.

Together, these well-established biological processes support strong plausibility for a causal relationship between DNA alkylation damage and reduced sperm counts. While alkylation damage can occur across all stages of spermatogenesis, effects on sperm counts are primarily driven by damage to proliferating and meiotic germ cells and have typically been observed following exposure in sexually mature individuals. However, exposures during early developmental stages may result in more persistent or severe impairments due to disruption of the germ cell pool, with effects becoming apparent later in adulthood.

Strong empirical evidence is available from experimental animal models and human clinical observations. Although direct measurement of both DNA adducts and sperm counts in the same study is limited, a substantial body of evidence demonstrates consistent patterns of temporal and dose concordance across studies examining well-characterized DNA alkylating agents. Human data further support the biological relevance of this relationship.

Altakroni et al. (2021) analyzed 105 semen samples from men undergoing assisted reproduction treatment (ART)and quantified the levels of N7-methyldeoxyguanosine (N7-MedG), a biomarker of exposure to methylating agents, in sperm genomic DNA using an immunoslotblot assay. A negative correlation was noted between N7-MedG concentrations and sperm concentration (Spearman's rank correlation coefficient= -0.32, p<0.001). Logistic regression analyses demonstrated that the elevated N7-MedG level was significantly associated with low sperm concentration (odds ratio [OR] = 6.46, 95% CI: 1.51-27.7); this association remained strong after adjustment for other DNA damage markers, including the percentage of sperm with high DNA damage and median % tail DNA measured by the neutral comet assay (adjusted OR = 6.42, 95% CI: 1.47-28.0) (Altakroni et al., 2021). This study provides evidence linking DNA alkylation to reduced sperm counts in humans.

It is well characterized that various DNA adducts rapidly form in mouse male germ cells within two hours after exposure to alkylating agents: N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The frequency of adduct formation increases with dose, supporting dose-dependent activation of the upstream KE. Data for ENU exposure are shown as examples (Figure 1) (van Zeeland et al., 1990).

Accordingly, DNA adduct formation can be reasonably inferred following exposures to well-characterized DNA alkylating agents. Empirical evidence supporting this KER primally comes from studies in animals and humans. Below, we summarize human clinical observations and selected data on several alkylating medications as examples to illustrate consistent patterns of reduced sperm counts following exposure.

Evidence in human

Azoospermia (absence of sperm in semen, < 0.1 million cells/mL) and oligozoospermia (semen with a low sperm count, 0.1-10 million cells/mL) are well-recognized side effects of cancer treatment with alkylating agents in men of reproductive age (reviewed by Howell and Shalet, 2005; Okada and Fujisawa, 2018). Dramatic declines in ejaculated sperm counts can occur within 1-2 months after receiving the treatment, while azoospermia usually occur after 2 months (Meistrich et al., 2013). The duration and reversibility of treatment-induced azoospermia is largely dependent on the dose of the drugs administered (Okada and Fujisawa, 2018).

Clinical studies provide evidence linking treatment with alkylating agents to reduced sperm counts in humans. More than 50% of patients with malignant lymphomas developed azoospermia after receiving chlorambucil treatment at a cumulative dose of more than 400 mg (Richter et al., 1970; Cheviakoff et al., 1973). Similarly, in 35 patients treated with cyclophosphamide, sperm production in 22 patients declined to azoospermic levels, and 6 declined to oligozoospermic levels within a few months after treatment initiation (Meistrich et al., 1992). In this cohort, patients received less than 7.5 g/m² (median dose 4.1 g/m²) of cyclophosphamide restored sperm production to a normal range within 5 years, whereas only 10% of patients recovered when treated with higher doses (> 7.5 g/m²), likely reflecting SSC depletion due to cumulative cytotoxicity (Meistrich et al., 1992).

Consistent patterns have also been reported for chemotherapy regimens containing alkylating agents. For instance, MVPP (mustine, vinblastine, procarbazine, and prednisolone) and COPP (cyclophosphamide, vincristine, procarbazine, and prednisolone) caused azoospermia in more than 90% of adult patients (reviewed by Howell and Shalet, 2005). When the doses of alkylating agents were reduced in these regimens, gonadotoxicity was also reduced, suggesting the role of DNA alkylation in causing reduced sperm counts and a dose-dependent relationship (Okada and Fujisawa, 2018).

Long-term reproductive effects of alkylating drugs and irradiation during early life stages have been reported. Green et al. (2014) evaluated semen parameters in 214 adult male survivors of childhood cancer who had received alkylating chemotherapeutic drugs. Azoospermia was diagnosed in 53 (25%) of participants and oligospermia in 59 (28%); the mean cumulative alkylating agent dose was higher in both groups than in participants with normospermia. Moreover, alkylating agent exposure was negatively correlated with sperm concentrations (r=-0.37, p<0.0001); specifically, each 1000 mg/m² increase in cumulative dose increased the odds of azoospermia by 22% (Green et al., 2014).

Beaud et al. (2019) revealed correlations between sperm parameters and treatment characteristics in 13 childhood cancer survivors. The participants received various doses of Mustargen®, procarbazine, or cyclophosphamide around puberty. Despite the small sample size, a strong link was observed between lower sperm counts and the use of alkylating agents (Spearman's rank correlation coefficient r=-0.62, p≤0.05), but not the use of other two non-alkylating drug classes, vinca alkaloids (r=0.07, p>0.05) and anthracyclines (r=-0.02, p>0.05) (Beaud et al., 2019).

Evidence in animal models in vivo

Meistrich (1982a) examined the effects of triethylenethiophosphoramide (thio-TEPA) on testicular cell survival and sperm production in C₃H mice. Male mice were injected i.p. with eight doses of thio-TEPA (0-35 mg/kg), and the number of sperm heads was counted in testis homogenates. Thio-TEPA exerts its anticancer activity through the formation of alkyl DNA adducts (Musser et al., 1989; Grigorii et al., 1991). A clear dose-dependent decrease in sperm head numbers was observed 8 weeks post-treatment. At doses ≥ 10 mg/kg, sperm counts were reduced to less than 10% of the control level, while the highest dose (35 mg/kg) reduced the number to ~1% of control (Meistrich, 1982a). These findings demonstrate dose concordance, with increasing exposure to this known alkylating agents associated with progressively greater reductions in sperm counts, and these decreases occur subsequent to treatment with alkylating agents, consistent with temporal concordance.

The same research group further examined spermatogenesis in C₃H mice following single i.p. injections of 14 chemotherapeutic drugs, including 7 alkylating agents (Meistrich et al., 1982b). Eleven days post-treatment, histological analyses showed that spermatogonial cells were the most sensitive germ cell population to cell death, whereas the spermatocytes and spermatids were relatively resistant at this time point, consistent with known biology. Dose-dependent loss of differentiating spermatogonia was observed following treatment with lomustine (40 mg/kg), mustargen (3 mg/kg), carmustine (10 and 60 mg/kg), mitomycin C (1.5 and 5 mg/kg), chlorambucil (20 mg/kg), thio-TEPA (3 and 26 mg/kg), and procarbazine (120 and 800 mg/kg). Assessment of sperm production at later time points supports temporal concordance between alkylation-induced germ cell loss and downstream reductions in sperm counts. Testicular sperm counts in testis homogenates measured 29 days post-treatment confirmed moderate to strong killing effects of alkylating agents on differentiating spermatogonia; all drugs reduced sperm counts to less than 10% of control within the dose range tested. Sperm counts measured 56 days after treatment reflect stem cell survival and recovery capacity of sperm production. At this timepoint, lomustine and mustargen induced up to a 25% decrease in sperm head numbers, whereas carmustine, chlorambucil, mitomycin C, and procarbazine reduced sperm counts to 30-60% of control levels. Nearly no sperm were counted in testes treated with thio-TEPA at 26 mg/kg, indicating minimal stem cell survival and recovery. Thus, a large number of alkylating agents lead to spermatogonial cell death and decline in sperm counts.

Busulfan, a potent bifunctional alkylating agent, produced similar effects. Bucci and Meistrich (1987) reported clear dose-dependent decreases in testicular sperm counts in C₃H mice exposed to busulfan (6, 13, 20, 28, 40 mg/kg) via i.p. injection. The dose required to reduce sperm head numbers by 50% of controls was estimated at 10 mg/kg at 29 days and 8 mg/kg at 56 days post-treatment. At 44 weeks, testicular and epididymal sperm counts remained significantly decreased by more than 50% in mice treated with busulfan ≥ 20 mg/kg compared to controls, while sperm counts returned to normal ranges in the lower dose groups (Bucci and Meistrich, 1987). These data demonstrate delayed onset and persistence of reduced sperm counts following exposure to a potent alkylating agent, supporting strong dose and temporal concordance and limited recovery of SSCs at higher doses.

Similar findings have been reported in a non-human primate model, supporting the conservation of this KER across species. Hermann et al. (2009) reported the effects of a single intravenous exposure to busulfan (4, 8, 12 mg/kg) exposure on spermatogenesis in adult rhesus macaques. Semen samples were collected weekly and total sperm counts per ejaculate were monitored for up to 52 weeks. Exposure to all three doses of busulfan caused dose-dependent and progressive decreases in sperm counts, which declined to zero by 7 weeks in animals treated with 12 mg/kg busulfan, and by 10 weeks following treatment with 4 mg/kg and 8 mg/kg busulfan. Testis volumes were also decreased correspondingly. Animals treated with 4 mg/kg busulfan restored normal spermatogenesis by 24 weeks following the exposure, albeit the sperm count is still lower than those in the controls. In contrast, sperm counts remained undetectable 52 weeks following exposure to 8 mg/kg and 12 mg/kg busulfan, suggesting SSC depletion and a longe-term loss of fertility. This study provides strong evidence for temporal and dose concordance, as reductions in sperm counts occur after exposure with a delay consistent with spermatogenic progression, with higher doses leading to more rapid and sustained depletion of sperm.

Repeated administration of a potent alkylating agent, ENU (100 mg/kg/week for three weeks), in C57BL/6J mice caused a delayed but significant reduction in sperm counts (Yin et al., 2014). No significant change in sperm count was observed during the first two weeks following the initial exposure, confirming that lack of apoptotic response in post-meiotic germ cells; however, sperm counts declined markedly from weeks 3-8 and reached near-zero levels by week 8. Sperm counts then gradually recovered, reaching approximately 85% of control levels by week 12, indicating profound but reversible impairment of spermatogenesis following ENU exposure (Figure 2). This study provides indirect support for essentiality through a stop-start (stressor removal) design.

In another Balb/c mouse study examining germline chromatin damage and sperm counts, ENU was used as a positive control. A single i.p. injection of 75 mg/kg of ENU at 8 weeks of age resulted in a delayed ~43% decrease in cauda epididymal sperm counts at 18 weeks of age, relative to control, accompanied by increased sperm DNA fragmentation and elevated germline mutation frequencies (Swayne et al., 2012). These studies provide strong temporal evidence.

Prenatal exposure studies provide evidence that early life exposure to alkylating agents and ionizing radiation can cause long-term effects on spermatogenesis. Comish et al. (2014) exposed pregnant 129 mice to either whole-body irradiated (0.8 Gy) or cyclophosphamide (7.5 mg/kg, i.p.) on embryonic days E10.5 and E11.5, corresponding to a critical period of primordial germ cell development. Male offspring were evaluated for testicular development and sperm production. At 4 weeks of age, male offspring from both treatment groups presented significantly reduced testicular weights (~70% of control), indicative of significant germ cell loss. The average number of atrophic Sertoli cell-only seminiferous tubules increased from 1.7 per testis section in controls to 4.8-5.8 tubules per section in irradiated and cyclophosphamide-treated animals. At 8 weeks of age, testicular weight remained reduced to 78% of control values, while testicular head counts and epididymal sperm counts were reduced to 62%-70% of control levels. These findings demonstrate that alkylation damage during fetal life can result in long-lasting germ cell depletion and reductions in sperm production in adulthood.

Together, these findings demonstrate that alkylation-induced genomic damage in male germ cells is consistently associated with impaired spermatogenesis and reduced sperm output. Although direct measurement of DNA adduct formation and sperm counts within the same study is uncommon, parallel evidence from studies of well-characterized alkylating agents shows consistent, dose-dependent changes in both KEs, supporting dose concordance. Significant reductions in sperm output are typically observed weeks to months following exposure, reflecting the time required for species-specific spermatogenic transit and confirming temporal concordance. The extent, duration, and reversibility of these effects highly depend on exposure dose, survival of SSCs, and spermatogenic kinetics in the test species.

Several uncertainties remain in this KER. First, DNA alkyl adducts in germ cells and sperm output are rarely measured in the same study, and many studies rely on surrogate markers of DNA damage, such as DNA strand breaks, making it difficult to attribute downstream effects specifically to DNA alkylation.

Second, reduced sperm count can arise through multiple mechanisms. In addition to direct alkylation-induced DNA damage in germ cells, other defects such as spermiation failure (i.e., retention of mature spermatozoa in late stage tubules) (Meistrich et al., 1982b), Sertoli cell dysfunction (Cui et al. 2024), and hormonal disruption (Sriram et al., 2024) may co-occur with alkylation damage and contribute to reduced testicular or epididymal sperm output. Moreover, alkylating agents have been shown to induce epigenetic alterations in sperm, including changes in DNA methylation at specific fertility-related genes (Altakroni et al., 2021). These alterations are associated with sperm DNA damage and reduced sperm counts (Jenkins et al., 2016); however, reported correlations between DNA damage markers and methylation are gene-specific and inconsistent across studies (Altakroni et al., 2021). As such, the relative contribution of epigenetic dysregulation to reduced sperm counts in this context remains uncertain.

Although Altakroni et al. (2021) reported an association between N7-MedG levels and decreased sperm concentration in 105 patients, this result was not replicated in a smaller follow-up study (n=16) (Altakroni et al., 2025). The latter study was based on a limited number of samples with N7-MedG measurements, which may have reduced statistical power. No significant correlations between N7-MedG levels, sperm concentration, or neutral comet assay measures (% tail DNA), despite observing a significant association with reduced fertilization rates.