Analysis of AEC and AFMSC mechanisms in maintaining fertility in induced Varicocele rat model

Objectives: Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques (1,2), although they have never been tested on male infertility diseases such as varicocele (VAR). Despite the encouraging paracrine, antiinflammatory, and immune regulatory therapeutic role of these stem cell source (1,2,4), no evidence on male fertility has been collected to date. The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a validated rat induced VAR model (3) selected for its high translational value due to its capability of replicating several aspects of the human pathology. Methods: hAEC and hAFMSC were marked with PKH26 vital cell membrane dye for in vivo cell tracking and then transplanted in left testis with intratesticular injection in VAR rats groups: +hAEC, +hAFMSC, respectively. Healthy (CTR) and VAR rats (+vehicle alone) groups were considered. The influence of cells transplantation was first assessed considering the long-term impact on rat fertility by recording the newborn number after two sequential mating carried out 120 days from surgical procedures. Then, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment upon transplantation. Results: Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages recruitment and a favorable antiinflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. Conclusions: These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.