A recent translational study has made significant strides in understanding the formation of testicular microcalcifications, both benign and malignant. These microcalcifications are thought to develop as a consequence of changes in gonadal phosphate homeostasis, and they often coincide with an osteogenic-like differentiation of germ cells or testicular somatic cells. Notably, the prevalence of microcalcifications is markedly higher in testicular biopsies exhibiting germ cell neoplasia in situ (GCNIS) compared to those without it, as documented by Kang et al. in 1994. This observation underscores the role of GCNIS or testicular germ cell tumors (TGCTs) in releasing factors that facilitate the development of microlithiasis. However, the study emphasizes that impaired Sertoli cell function may play a more critical role than the mere presence of malignant germ cells in this context.

In this groundbreaking manuscript, researchers demonstrate that compromised Sertoli cell function in both murine models and human subjects significantly elevates the risk of developing testicular microcalcifications. This link appears to persist more robustly than the association with malignant conditions. The study identifies FGF23, the systemic master regulator of phosphate, as being highly expressed in GCNIS, embryonal carcinoma (EC), and human embryonic stem cells (hESCs), but not in classical seminoma. This finding suggests a connection between FGF23 expression and the transformation of GCNIS into invasive EC, distinguishing it from the chromosomal duplications commonly observed in classical seminomas.

The pronounced expression of FGF23 in EC and hESCs also indicates its role as an early embryonic signal. This is further supported by the strong relationship between FGF23 and the pluripotency factor NANOG within ECs, as well as high levels of FGF23 correlating with the formation of polyembryomas. Previous reports have also highlighted FGF23 transcripts during early embryonic development, as seen in studies by Cormier et al. in 2005 and Wei et al. in 2005.

Interestingly, phosphate levels within the testis are observed to be three times higher than those found in serum, as noted by Jenkins et al. in 1980 and 1983. The interactions of FGF23 and Klotho have been proposed to regulate ion metabolism in vascular and soft tissue calcification, potentially inducing the presence of bone-like cells within these tissues. The research team found that the absence of FGF23 signaling in knockout mice led to the deposition of hydroxyapatite in the epididymis, reinforcing the notion that FGF23 plays a crucial role in mineral homeostasis.

Moreover, the study explores the dynamics of FGF23 in the context of GCNIS and EC, observing that the lack of GalNAc-T3 expression in these tumor types results in rapid cleavage of FGF23 into its C-terminal form, cFGF23. This is consistent with findings that primarily detect cFGF23not its intact formin the seminal fluid of GCNIS and EC patients. Elevated intratesticular levels of cFGF23 may interact with the Klotho/FGFR1 receptor, potentially negating the effects of iFGF23. However, it is crucial to note that experiments utilizing a human testis ex vivo model indicated that neither form of FGF23 induced significant changes in phosphate transporters or bone-related factors, suggesting that short-term exposure to high cFGF23 levels does not impact these pathways.

Interestingly, the study finds that the epididymal phenotype observed in FGF23 knockout mice is not attributable to systemic hyperphosphatemia. Long-term dietary exposure to high phosphate levels did not induce microcalcifications or alter testicular phosphate transporter expression. Instead, the research previously demonstrated that a deletion of Klotho that is specific to germ cells in mice led to unusual mineral homeostasis, particularly affecting calcium transport. This might be significant, as lower calcium levels could protect against the induction of microcalcifications in environments rich in phosphate.

The local mineral levels within the testis are primarily dictated by the presence and activity of specific transporters and sensors of calcium and phosphate in reproductive organs, rather than by systemic concentrations. This finding helps explain why certain patients with loss-of-function variants in the predominant testicular phosphate transporter SLC34A2 develop testicular microcalcifications, as noted by Corut et al. in 2006. Furthermore, patients showing loss-of-function variants in GALNT3, which leads to premature degradation of iFGF23 to its C-terminal form, experience severe testicular microlithiasis and global calcifications.

Fgf23 knockout mice exhibit hypogonadism and spermatogenic arrest, characteristics that mirror conditions seen in some men with testicular dysgenesis syndrome, who occasionally present with testicular microcalcifications in the absence of malignancy. It appears that benign microcalcifications may arise from low androgen or gonadotropin levels, which can disrupt Sertoli cell function. This disruption can lead to germ cells remaining in a prepubertal, stem cell-like state, making them vulnerable to external stimuli that could trigger abnormal differentiation.

The study's findings suggest that changes in local testicular phosphate concentrations could play a crucial role in these processes. This hypothesis is reinforced by the discovery of microcalcifications in genetically modified hpg mice that exhibit global androgen receptor ablation, as these mice possess immature Sertoli cells and are unable to complete spermatogenesis. Notably, alterations in phosphate transporter expression were observed, with a nonsignificant decrease in Slc34a2 expression and a significant increase in Slc34a1 compared to wild type mice. Nevertheless, both hpg mice and those with Sertoli cell-specific androgen receptor deletion displayed similar patterns in phosphate transporter expression, indicating that additional mechanisms are likely contributing to microlithiasis development.

The osteogenic marker Bglap was exclusively detected in hpg mice with global androgen receptor deletion, hinting that some testicular cells may undergo osteogenic trans-differentiation. While the precise origin of these bone-like cells remains undetermined, previous studies have shown that vitamin D can induce osteogenic differentiation in NTera2 cells, leading them to express bone-specific proteins like Osteocalcin.

The study also discusses the potential role of peritubular cells as candidates for osteogenic-like differentiation, given their mesenchymal origin and fibroblast-like characteristics. Interestingly, human Sertoli cells have the capacity to form large-cell calcifying tumors, indicating that various gonadal cell types can adopt bone-like features. Moreover, research highlights that the ablation of Sertoli cells before puberty leads to extensive intratubular microcalcifications, whereas similar ablation in adulthood does not result in microcalcification formation. This distinction emphasizes that microcalcification development is not merely a byproduct of cell death but is critically dependent on the integrity and maturation of Sertoli cells. The study posits that spermatogonial stem cells could also undergo osteogenic-like differentiation, as they have the potential to form multiple germ layers.

Moreover, the transcription factor RUNX2, essential for osteoblast development, was researched in germ cells. Although RUNX2 is expressed in a different isoform in germ cells, it is transcribed from the same promoter as in bone. Notably, RUNX2 was undetectable in healthy seminiferous tubules but was found in GCNIS, calcified tissues, and adjacent seminiferous tubules, indicating that RUNX2 expression can occur in testicular tissues experiencing microcalcifications.

One of the most potent inhibitors of mineralization, inorganic pyrophosphate (PPi), can be degraded by alkaline phosphatase (ALP) or pyrophosphatase, highlighting its role in controlling spermatogonial mineralization. ALP activity was notably high in malignant germ cells, suggesting that men with GCNIS and TGCTs might be more susceptible to microcalcifications due to decreased PPi levels resulting from increased ALP activity. The study elucidates that while other mineralization inhibitors and promoters could be involved, the remarkable epididymal mineralization in Fgf23 knockout mice underscores how aberrant function of a single component can facilitate microcalcification formation. Immature bone formation in the testis, often observed in teratomas, can also occur without malignant conditions.

In summary, this comprehensive study reveals that the formation of testicular microcalcifications arises from both malignant and benign etiologies, encompassing various factors such as Sertoli cell dysfunction, disrupted local phosphate homeostasis, alterations in mineralization inhibitors, and abnormal germ cell activity. The findings challenge the conventional perception of microcalcifications as solely indicators of malignancy in the testis, emphasizing the complexity of these biological processes.