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A question of the chicken or the egg

      In this scientific scavenger hunt, Santana et al. (
      • Santana V.P.
      • James E.R.
      • Miranda-Furtado C.L.
      • de Souza M.F.
      • Pompeu C.P.
      • Esteves S.C.
      • et al.
      Differential DNA methylation pattern and sperm quality in men with varicocele.
      ) aim to prove the hypothesis that men with varicoceles have unique epigenetic changes not seen in normal fertile patients. The authors’ data add to the growing body of evidence that DNA methylation patterns in sperm cells will inevitably be a measure to predict and to diagnose a spectrum of men’s health conditions. Santana et al. seek to identify changes in global sperm DNA methylation among men with and without varicoceles.
      Through array-based and regional analysis, the authors report that there was a global hypomethylation in the varicocele patients vs. controls. In addition, these authors identified 59 CpG (5'—C—phosphate—G—3') sites found in the varicocele group that were not found in the control group, and these were generally hypomethylated. Moreover, the data successfully showed significant differences between the two groups of DNA methylation at 1,695 sites. The regions identified show promising overlap with previous studies looking at DNA methylation changes in sperm and within genes known to be associated with gamete generation, cell division, and metabolic function. This discovery is interesting both for clinicians as well as for basic scientists, as it paves the way for more studies to better understand the physiology, grading, and potential prediction of the varicocele effect.
      However, one of our main concerns in reviewing these data is the composition and fidelity of the study cohort. A total of 26 men with varicoceles were compared to 26 fertile controls seen for a vasectomy and who had recently established a pregnancy. Despite the small sample size, which is a common issue in early studies such as this one, a main cause of concern is the combination of infertile and fertile men in the study cohort. Whereas the control group contains fertile controls who are negative for varicocele, the study group contains men diagnosed with varicocele irrespective of their fertility status. Some of the first studies in spermatozoan DNA methylation have already identified discrete changes in methylation patterns between fertile and infertile men (
      • Filipponi D.
      • Feil R.
      Perturbation of genomic imprinting in oligozoospermia.
      ,
      • Wu W.
      • Shen O.
      • Qin Y.
      • Niu X.
      • Lu C.
      • Xia Y.
      • et al.
      Idiopathic male infertility is strongly associated with aberrant promoter methylation of methylenetetrahydrofolate reductase (MTHFR).
      ), making the magnitude of the epigenetic findings in this manuscript likely tainted by the fact that the authors are comparing fertile and infertile men, irrespective of the varicocele. The authors report that the 26 study participants were recruited only because of their diagnosis of having a varicocele. Because the study cohort was gleaned over a 3-year period of data analysis, one must question why it took so long to identify this small group of patients with such a common clinical problem. It seems probable that men seeking help from an andrology clinic, as apparently were the participants in the cohort, are more likely to have infertility concerns; therefore, the study cohort is expected to be biased toward men with fertility issues and low sperm quality. This is evidenced by a comparison of the sperm density in the two groups, which shows a mean of 30 million sperm/mL in the study cohort as compared to 90 million sperm/mL in the control group. Because identifying varicoceles, especially grades 1 and 2, is very subjective, one would hope that a single examiner identified all men in the study group; however, we are not given this information. In addition, we do not know whether the study cohort had individually ever established pregnancies in the past.
      A more appropriate analysis would have been comparing infertile men demonstrating idiopathic oligoasthenospermia with those having a varicocele and similar evidence of testicular dysfunction. Consequently, the impact of the varicocele could be more clearly identified. In addition, it is possible to assume that the existence of men with infertility in the study cohort could obscure the results of the authors’ varicocele grading analysis in which they could not show a significant difference in the epigenomic pattern of grade 3 varicoceles compared to smaller lesions. In this small sample size, men with more severe infertility could cluster in the grade 3 scoring, and therefore the authors would see a “dose-dependent” effect that is unrelated entirely to the varicocele. It is certainly not the ideal study design from which to conclude that these men’s fertility issues were “caused” by the varicocele. The authors also go on to identify using gene ontology analysis non-overlapping regions in their epigenome, which included identification of “vasculitis” as a significant associated disease ontology. One must ask, therefore, whether the authors are trying to say that the identified epigenome causes the varicocele by way of “vasculitis,” or whether the putative toxic effects of the varicocele somehow caused a unique epigenomic pattern. The authors thoughtfully recognize the need for future analysis with a larger sample size, and we encourage them to reduce the phenotype variability as well as to draw more scientifically supportive conclusions.
      As the authors themselves state, “…the data show that DNA methylation alterations associated with varicocele are as multi-faceted as the varicocele disorder itself.” If this indeed is true, what is the take-away message for the reader? Is the varicocele causing an effect on the epigenome, or is the varicocele itself a result of epigenomic changes via vasculitis? This conundrum is a scientific case of the proverbial question: is it the chicken or the egg?

      References

        • Santana V.P.
        • James E.R.
        • Miranda-Furtado C.L.
        • de Souza M.F.
        • Pompeu C.P.
        • Esteves S.C.
        • et al.
        Differential DNA methylation pattern and sperm quality in men with varicocele.
        Fertil Steril. 2020; 114: 770-778
        • Filipponi D.
        • Feil R.
        Perturbation of genomic imprinting in oligozoospermia.
        Epigenetics. 2009; 4: 27-30
        • Wu W.
        • Shen O.
        • Qin Y.
        • Niu X.
        • Lu C.
        • Xia Y.
        • et al.
        Idiopathic male infertility is strongly associated with aberrant promoter methylation of methylenetetrahydrofolate reductase (MTHFR).
        PLoS One. 2010; 5e13884

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