Human Spermatogonial Stem Cells and Their Niche in Male (in)Fertility

Novel Concepts From Single-cell RNA-Sequencing

Sara Di Persio; Nina Neuhaus


Hum Reprod. 2023;38(1):1-13. 

In This Article

Abstract and Introduction


The amount of single-cell RNA-sequencing (scRNA-seq) data produced in the field of human male reproduction has steadily increased. Transcriptional profiles of thousands of testicular cells have been generated covering the human neonatal, prepubertal, pubertal and adult period as well as different types of male infertility; the latter include non-obstructive azoospermia, cryptozoospermia, Klinefelter syndrome and azoospermia factor deletions. In this review, we provide an overview of transcriptional changes in different testicular subpopulations during postnatal development and in cases of male infertility. Moreover, we review novel concepts regarding the existence of spermatogonial and somatic cell subtypes as well as their crosstalk and provide corresponding marker genes to facilitate their identification. We discuss the potential clinical implications of scRNA-seq findings, the need for spatial information and the necessity to corroborate findings by exploring other levels of regulation, including at the epigenetic or protein level.

Graphical Abstract


Infertility affects about 15% of couples with half of the cases arising from a male factor (Boivin et al., 2007; Mascarenhas et al., 2012). In the past decades, procedures including IVF and ICSI have been successfully implemented to enable men with very low sperm numbers to father biological children. However, medical approaches to treat the underlying causes of reduced sperm production remain elusive. This is largely because of the limited knowledge of the molecular and cellular interplays responsible for human sperm production and the alterations associated with the failure thereof (Tüttelmann et al., 2018).

One hurdle in unveiling these molecular pathways is the high complexity of human testicular tissues, consisting of different germ cells as well as somatic cell populations. The advent of high-throughput single-cell RNA-sequencing (scRNA-seq) analyses has opened a new era of reproductive research (Li et al., 2020; Tan and Wilkinson, 2020; Marečková et al., 2022), as this approach and the associated workflow bypass the issue of high tissue complexity and enables the identification of rare and transcriptionally heterogeneous cell types. Multiple scRNA-seq methods have been developed to date, each with specific advantages and disadvantages (Ziegenhain et al., 2017). Overall, the methods all share the following steps in the workflow: the testicular tissue undergoes enzymatic/mechanical digestion to obtain thousands of single cells, which are then subjected to (droplet- or plate-based) scRNA-seq following library preparation; for visualization, sequencing results are compressed into 2D space in which cells are organized in clusters based on their transcriptional profiles, with similar cells plotted in closer proximity (Figure 1A); the cell identity of individual clusters is then assigned based on the expression of known marker genes, avoiding the isolation of individual cell types prior to analysis.

Figure 1.

Schematic representation of the single-cell RNA-sequencing studies to analyze human testicular tissues. (A) Experimental design, (B) samples used. Details are provided in Supplementary Table SI. Studies on each stage of development, and different types of infertility: 0–13 months (Guo et al., 2018, 2021; Sohni et al., 2019; Voigt et al., 2022); 2–14 years (Guo et al., 2020; Zhao et al., 2020; Voigt et al., 2022); 17–76 years (Guo et al., 2017, 2018; Neuhaus et al., 2017; Hermann et al., 2018; Wang et al., 2018; Sohni et al., 2019; Shami et al., 2020; Xia et al., 2020; Zhao et al., 2020; Alfano et al., 2021; Di Persio et al., 2021; Mahyari et al., 2021; Chen et al., 2022; Nie et al., 2022); Crypto (Di Persio et al., 2021); Idiopathic NOA (Wang et al., 2018; Zhao et al., 2020; Alfano et al., 2021; Mahyari et al., 2021; Chen et al., 2022); Yq AZFa deletions (Zhao et al., 2020); Klinefelter syndrome (Laurentino et al., 2019; Zhao et al., 2020; Mahyari et al., 2021). Crypto, cryptozoospermia; NOA, non-obstructive azoospermia; AZF, azoospermia factor.

Taking advantage of scRNA-seq, the transcriptional profiles of thousands of human testicular cells have been generated covering the human neonatal, prepubertal, pubertal and adult period as well as different types of male infertility (Figure 1B). Based on this growing resource, this review highlights relevant scRNA-seq datasets (Supplementary Table SI) and novel marker genes for different testicular cell types (Table I). What is more, considering transcriptional profiles during development and in (in)fertility, this review provides an overview of novel concepts regarding the existence of subtypes of spermatogonia as well as somatic Sertoli and peritubular myoid cells (PTMs) and grants first insights into their communication pathways in male (in)fertility. Finally, this review discusses the potential clinical implications of the insights gained from scRNA-Seq studies and the limitations associated with them.