Changes in local mineral homeostasis facilitate the formation of benign and malignant testicular microcalcifications

Reviewer #1 (Public Review):

Summary:

Despite the study being a collation of important results likely to have an overall positive effect on the field, methodological weaknesses and suboptimal use of statistics make it difficult to give confidence to the study's message.

Strengths:

Relevant human and mouse models approached with in vivo and in vitro techniques.

Weaknesses:

The methodology, statistics, reagents, analyses, and manuscripts' language all lack rigour.

(1) The authors used statistics to generate P-values and Rsquare values to evaluate the strength of their findings.

However, it is unclear how stats were used and/or whether stats were used correctly. For instance, the authors write: "Gaussian distribution of all numerical variables was evaluated by QQ plots". But why? For statistical tests that fall under the umbrella of General Linear Models (line ANOVA, t-tests, and correlations (Pearson's)), there are several assumptions that ought to be checked, including typically:

(a) Gaussian distribution of residuals.

(b) Homoskedasticity of the residuals.

(c) Independence of Y, but that's assumed to be valid due to experimental design.

So what is the point of evaluating the Gaussian distribution of the data themselves? It is not necessary. In this reviewer's opinion, it is irrelevant, not a good use of statistics, and we ought to be leading by example here.

Additionally, it is not clear whether the homoscedasticity of the residuals was checked. Many of the data appear to have particularly heteroskedastic residuals. In many respects, homoscedasticity matters more than the normal distribution of the residuals. In Graphpad analyses if ANOVA is used but equal variances are assumed (when variances among groups are unequal then standard deviations assigned in each group will be wrong and thus incorrect p values are being calculated.

Based on the incomplete and/or wrong statistical analyses it is difficult to evaluate the study in greater depth.

While on the subject of stats, it is worth mentioning this misuse of statistics in Figure 3D, where the authors added the Slc34a1 transcript levels from controls in the correlation analyses, thereby driving the intercept down. Without the Control data there does not appear to be a correlation between the Slc34a1 levels and tumor size.

There is more. The authors make statements (e.g. in the figure levels as: "Correlations indicated by R2.". What does that mean? In a simple correlation, the P value is used to evaluate the strength of the slope being different from zero. The authors also give R2 values for the correlations but they do not provide R2 values for the other stats (like ANOVAs). Why not?

(2) The authors used antibodies for immunos and WBs. I checked those antibodies online and it was concerning:

(a) Many are discontinued.

(b) Many are not validated.

(c) Many performed poorly in the Immunos, e.g. FGF23, FGFR1, and Kotho are not really convincing. PO5F1 (gene: OCT4) is the one that looks convincing as it is expressed at the correct cell types.

(d) Others like NPT2A (product of gene SLC34A1) are equally unconvincing. Shouldn't the immuno show them to be in the plasma membrane?

If there is some brown staining, this does not mean the antibodies are working. If your antibodies are not validated then you ought to omit the immunos from the manuscript.

Reviewer #2 (Public Review):

Summary:

This study set out to examine microlithiasis associated with an increased risk of testicular germ cell tumors (TGCT). This reviewer considers this to be an excellent study. It raises questions regarding exactly how aberrant Sertoli cell function could induce osteogenic-like differentiation of germ cells but then all research should raise more questions than it answers.

Strengths:

Data showing the link between a disruption in testicular mineral (phosphate) homeostasis, FGF23 expression, and Sertoli cell dysfunction, are compelling.

Weaknesses:

Not sure I see any weaknesses here, as this study advances this area of inquiry and ends with a hypothesis for future testing.