ABSTRACT: Methods to quantify germ cell number in human immature testicular tissues are essential to evaluate the impact of chemotherapy exposures and to optimise cryopreservation protocols used in fertility preservation for prepubertal boys. Established quantification methods rely on the presence of round tubules within the tissue. However, round tubular cross sections are limited in human prepubertal testicular tissues, especially when using in vitro culture. We aimed to assess whether an alternative method of germ cell quantification would provide similar results to recently established methods, without the requirement for round tubules. Human testicular samples included fetal tissue (exposed in vitro to cisplatin, carboplatin or control) or prepubertal tissue (fresh, cryopreserved, fresh in vitro cultured or cryopreserved in vitro cultured). Immunofluorescence assessed AP2γ (gonocytes) and MAGE-A4 ((pre)spermatogonia) expression. Germ cells were quantified by tubular germ cell density (Method 1), which was compared to methods that require round tubules, including spermatogonial number per round tubular cross section (S/T) (Method 2), fertility index (Method 3) and round tubular germ cell density (Method 4). A correlation analysis between methods was performed. Method 1 is strongly and significantly correlated with Method 2 (r = 0.838, P <
0.0001
r = 0.833, P <
0.0001), Method 3 (r = 0.752, P <
0.001
r = 0.802, P <
0.0001) and Method 4 (r = 0.863, P <
0.0001
r = 0.914, P <
0.0001) for fetal and prepubertal tissues, respectively. Given that Method 1 assess tubules irrespective of shape, it may increase the total number of germ cells available for quantification, validating its use for quantification of human testicular tissue samples where the amount of tissue or presence of round tubules is limited. LAY SUMMARY: Chemotherapy can damage cells in the testicles that are required to make sperm, often leading to infertility in males. While options to preserve fertility in adult males are available, there are no established methods for young boys. To investigate how chemotherapy damages these cells and to explore approaches to preserve fertility, we require methods to count the number of cells that can develop into sperm. Existing counting methods involve only counting some of the cells in the tissue, but in young boys, it is often necessary to count all of the cells because the amount of tissue is limited. To overcome this, we counted cells in small pieces of human fetal and prepubertal testicles using an alternative method, which allows all cells to be counted. We found similar results using our method compared to three existing methods, making our method useful for counting cells in fetal and prepubertal testicle samples.