In a groundbreaking translational study, researchers have unveiled significant findings regarding testicular microcalcificationsboth benign and malignantthat may arise as a consequence of altered gonadal phosphate homeostasis. It has been established that the presence of microcalcifications typically correlates with a disruption in Sertoli cell function, a crucial component in male reproductive health. This discovery underscores the importance of Sertoli cells, particularly in the context of testicular germ cell neoplasia in situ (GCNIS) and testicular germ cell tumors (TGCTs).

Evidence indicates that microcalcifications are more prevalent in testicular biopsies associated with GCNIS compared to those without it, further enhanced by findings from Kang et al. (1994). The research highlights that GCNIS and TGCTs release specific factors that contribute to the development of microlithiasis, a condition characterized by the formation of small calcified deposits. Interestingly, it appears that the impairment of Sertoli cell function may play an even more pivotal role than the mere presence of malignant germ cells in the pathogenesis of these microcalcifications. Our study has shown that disrupted Sertoli cell function in both murine models and human subjects significantly increases the likelihood of developing testicular microcalcifications, suggesting a more enduring association than that with malignant conditions.

A key molecular player identified in this context is FGF23, the systemic master regulator of phosphate, which is found to be highly expressed in GCNIS and embryonic stem cells (EC) but absent in classical seminoma. This observation raises intriguing questions regarding the transformation of GCNIS to invasive EC, as the expression of FGF23 does not appear to be linked to a duplication of chromosome 12p, often seen in classical seminoma. The heightened expression of FGF23 in embryonic contexts highlights its role as an early developmental signal, supported by correlations with pluripotency factors such as NANOG in ECs and the formation of polyembryomas.

Research has shown that phosphate levels in the testis are three times higher than those found in serum. Enhanced FGF23-Klotho activity has been implicated in regulating ion metabolism, which could lead to bone-like cell formations within vascular and soft tissues, according to studies by Memon et al. (2008) and Leszczynska and Murphy (2018). Observations of hydroxyapatite accumulation in the cauda epididymis of Fgf23-deficient mice indicate that the absence of FGF23 signaling triggers hydroxyapatite deposition. Notably, GCNIS and EC lack expression of GalNAc-T3, which leads to rapid cleavage of FGF23 into its C-terminal form (cFGF23). The predominance of cFGF23 over iFGF23 in the seminal fluid of patients diagnosed with GCNIS and EC further underscores the complexities of FGF23s role in testicular physiology.

Our findings suggest that high levels of intratesticular cFGF23 might interact with the Klotho/FGFR1 receptor to negate the effects of iFGF23, thus inducing a phenotype reminiscent of Fgf23-deficient mice, notably with elevated expression of the bone marker Bglap. However, when we examined human testis ex vivo, neither form of FGF23 induced observable changes in phosphate transporters or BGLAP, indicating that short-term exposure (24 hours) to elevated cFGF23 does not alter these parameters.

The study also sheds light on the mechanisms underlying testicular microcalcifications, noting that the epididymal phenotype observed in Fgf23-deficient mice does not appear to be driven by systemic hyperphosphatemia. In fact, a prolonged high-phosphate diet did not induce testicular or epididymal microcalcifications nor did it affect the expression of testicular phosphate transporters. Our previous research indicated that specific deletion of Klotho in germ cells results in abnormal mineral homeostasis, particularly impacting calcium transport through TRPV5, as revealed by Bllehuus Hansen et al. (2020). This suggests that lower calcium levels may provide a protective effect against microcalcifications in phosphate-rich environments.

Local mineral levels within the testis are predominantly influenced by the presence and activity of specialized transporters and sensors for calcium and phosphate, rather than systemic concentrations. This is further exemplified by patients with loss-of-function variants in the predominant testicular phosphate transporter, SLC34A2, who present with testicular microcalcifications, as noted by Corut et al. (2006). Additionally, mutations in GALNT3, which lead to premature degradation of iFGF23, have been associated with severe testicular microlithiasis and global calcifications, as discussed in studies by Garringer et al. (2007) and Campagnoli et al. (2006).

Interestingly, Fgf23-deficient mice exhibit symptoms of hypogonadism and spermatogenic arrest, which mirrors conditions in some males with testicular dysgenesis syndrome, who may also have microcalcifications without malignancy, according to Pedersen et al. (2016) and Rebourcet et al. (2014). The presence of benign microcalcifications could be exacerbated by hormonal imbalances, including low androgens or gonadotropins, which can disrupt Sertoli cell function, causing germ cells to remain in a prepubertal stem cell state rather than maturing appropriately.

In light of these findings, the local concentration of testicular phosphatethree times higher than in serumcould serve as a significant stimulus for microcalcification development, as supported by previous studies (Jenkins et al., 1980; Sharpe et al., 2003; OShaughnessy et al., 2010b). The presence of microcalcifications in hpg mice, which exhibit global androgen receptor ablation and have immature Sertoli cells unable to complete spermatogenesis, further substantiates this hypothesis.

Moreover, the study reveals the role of osteogenic-like differentiation in non-malignant germ cells. Although the specific cellular origins remain to be determined, previous research has indicated that vitamin D can induce osteogenic-like differentiation of NTera2 cells, leading to the expression of bone-specific proteins such as Osteocalcin. This differentiation may be facilitated by peritubular cells, which are mesenchymal-derived and can undergo osteogenic-like differentiation during vascular calcification. In humans, Sertoli cells have also been known to create large-cell calcifying tumors. This indicates that various gonadal cell types may possess the capability for such transformations.

The findings also indicate that extensive intratubular microcalcifications result from Sertoli cell ablation before puberty, whereas similar ablation in adulthood does not yield the same results, highlighting the critical role of Sertoli cell maturation and function in preventing the osteogenic-like differentiation of germ cells or other testicular cells. The relationship between Sertoli cell function and the propensity for benign microcalcifications appears to hinge on prepubertal disturbances.

Additionally, spermatogonial stem cells may also be susceptible to osteogenic-like differentiation, as demonstrated by their ability to develop into various germ layers when isolated and injected into blastocysts. The essential transcription factor RUNX2, vital for osteoblast development, is expressed in different isoforms by germ cells. The presence of bone-specific RUNX2 isoforms in GCNIS and calcified tissues adjacent to bone indicates that these cells can express features associated with osteogenesis under certain conditions.

To further elucidate mineralization mechanisms, we identified that pyrophosphate (PPi) serves as a potent inhibitor of mineralization, with its degradation influenced by alkaline phosphatase (ALP) activity, particularly in primordial and malignant germ cells. Our results show that malignant germ cells such as TCam2 exhibit high ALP activity, suggesting that they may promote mineralization through reductions in PPi levels. This finding implies that individuals with GCNIS and TGCTs could be more susceptible to microcalcifications due to heightened ALP activity.

In conclusion, our study reveals that testicular microcalcifications can form due to benign or malignant etiologies, driven by various factors such as Sertoli cell dysfunction, alterations in local phosphate homeostasis, changes in mineralization inhibitors, and aberrant germ cell function. These interactions can lead to osteogenic-like differentiation of germ cells, culminating in the deposition of microcalcifications composed of hydroxyapatite. Therefore, it is crucial to reconsider the interpretation of microcalcifications in the testis, as they should not be solely viewed as markers for malignancy.