Spermatogonia in the testicular tissue of prepubertal patients with SCD compared with reference values. (A-C) Immunostained section of the testicular tissue of a patient aged 6.1 years who had previously been exposed to hydroxyurea (Off-HU patient 3). (A) Selection of a round tubular cross section (yellow circle). 4′,6-Diamidino-2-phenylindole staining (blue). (B) Expression of GATA-4 nuclear protein in Sertoli cells (red staining). (C) Expression of MAGE-A4 cytoplasmic protein in spermatogonial cells (green staining). Negative images of spermatogonial cells in panel C (yellow triangle). Four spermatogonia could be counted in this selected section. Scale bar = 50 µm. (D) The number of spermatogonia per round tubular cross section (S/T) of patients exposed to hydroxyurea (light gray circle: off-HU; dark gray circle: pon-HU) or not exposed to hydroxyurea (red diamond). Individual S/T values plotted on a meta-regression fit line of S/T reference values during testis development.¹⁶  Each mark represents the mean S/T value of a patient. The S/T values of prepubertal patients with SCD were significantly lower than the reference S/T values (Wilcoxon signed-rank test, P<.0001).