Testicular torsion is defined by the twisting of the testis around the vertical axis of the spermatic cord (Hamed et al., 2011). The incidence of this disorder is estimated at 1/4000 of men younger than 25 years (Mansbach et al., 2005). Ischemic injury induced by testicular torsion give rise to the interruption of testicular blood flow and consequently testicular atrophy (Cox et al., 2012). Detorsion operation is widely used for the recovery of disrupted blood flow induced by testicular torsion but this procedure usually lead to testicular reperfusion injury (Ahmed et al., 2000). Torsion/detorsion-induced ischemia/reperfusion injury is accompanied by enhanced ROS production, inflammatory responses, apoptosis, impaired spermatogenesis, reduced sperm counts, and infertility (Lysiak et al., 2001, Vakili et al., 2011). In rodent testes, A single (As) spermatogonia are the foundation of spermatogenesis and necessary for male reproduction (Yoshida, 2010). As spermatogonia are located close to the basement membrane of seminiferous tubules (Nakata, 2019). As can undergo a symmetric cell division to generate two unconnected self-renewing As. Also, they can experience an asymmetric cell division to procreate a progenitor state destined for differentiation and a self- renewing As (Fayomi, 2018). Progenitor As then further divided to produce two A paired (Apr) spermatogonia, which remain connected by an intercellular cytoplasmic bridge (Li et al., 2019). Apr spermatogonia give rise to chains of A aligned (Aal) spermatogonia that differentiate to A1 spermatogonia without a cellular division (Lord and Oatley, 2017). A1 spermatogonia begin organized subsequent mitotic divisions to create A2, A3, A4, intermediate, and B spermatogonia (Teletin et al., 2019). Type B spermatogonia is divided to generate primary spermatocytes (Endo et al., 2015) which in turn go through two meiotic cell divisions to produce secondary spermatocytes and round spermatids (Nebel et al., 1961). The round spermatids will undergo an intricate morphological differentiation and generate haploid spermatozoa (Johnson et al., 2008). Spermatogenesis is associated with precise regulation of gene expression (Ibtisham et al., 2017). c-kit is detected in A1–A4, intermediate, B spermatogonia, and preleptotene spermatocytes in adult testis (Tang and Fan, 2019). Expression of c-kit is necessary for the migration, proliferation and maturation of the germ cells. The expression of c-kit continues until the beginning of meiosis and c-kit is a marker for loss of SSCs pluripotency (Gu et al., 2009, Zhang et al., 2013). Protamine 1 (PRM1) and protamine 2 (PRM2) are used as an alternative to histones in the chromatin of elongated spermatids during later stages of spermiogenesis (Schneider et al., 2016). The replacement of histones by protamines compact sperm DNA into an extraordinarily stable and inactive structure (Le Blévec et al., 2020).

Mesenchymal stem cells (MSCs) derived from the umbilical cord, bone marrow, amniotic membrane, other fetal sources and adipose tissue have been proved for clinical cell therapy (Barberini et al., 2014, Hu et al., 2020, Munoz-Perez et al., 2021). Amniotic membrane is simply obtained after the newborn birth, it demonstrates a noninvasive source of stem cells because it is considered an inconsequential medical waste (Kimura et al., 2012). No-tumorigenicity, immunomodulatory effects and multi-differentiation potential have made human amniotic mesenchymal stem cells (hAMSCs) a promising origin of stem cells for cell therapy (Liu et al., 2021). Avoid the ethical issues of embryonic stem cells and low immunogenicity are other advantageous of them (García-López et al., 2015). MSCs have regulated acrosomal activity (Qamar et al., 2020), mitochondrial metabolism (Hsu et al., 2016), enhanced sperm motility, capacitation, restore spermatogenesis (Zickri et al., 2021) and fertilization (Liu et al., 2020). They have enhanced the antioxidant defense system of testicular tissue and reduced ROS production by scavenging free radicals (Stavely and Nurgali, 2020). MSCs secrete high levels of paracrine bioactive molecules in response to their local microenvironment (Kusuma et al., 2017). The MSCs-derived cell-free secretome was demonstrated to create many features of MSCs (Bader et al., 2019). It is considered a non-cell-based therapy that avoids tumorigenic potential and uncontrolled cell divisions (Qamar et al., 2021). MSCs secreted factors are introduced with many of biologically active molecules such as immunomodulatory agents, cytokines, chemokines and growth factors such as epidermal growth factor (EGF), stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), angiopoietin-1 and keratinocyte growth factor (KGF) (Hu et al., 2020, Saheli et al., 2020). Inhibition of apoptosis (Hsiao et al., 2019), increasing cell division and stimulating tissue remodeling are their pro-regenerative effects (Sagaradze et al., 2020).

Since the application of human amniotic membrane-derived mesenchymal stem cell (hAMSCs) secreted factors on sperm chromatin condensation and spermatogenesis criteria has not been reported. In the present study, we decided to evaluate the protective effects of secreted factors prepared from human amniotic membrane-derived MSCs after torsion/detorsion-induced ischemia/reperfusion injury.