Subpopulation of leucocytes in the semen from fertile and infertile men--immunohistochemical and flow-cytometric analysis

Abstract

It is hard to differentiate leucocytes from immature germ cells among many round cells in ejaculated semen. Recently monoclonal antibodies (MoAbs) which recognize surface antigen of various subtypes of leucocytes are available. In this study we applied a panel of MoAbs for leucocyte subpopulations (granulocytes, T-lymphocytes, B-lymphocytes, Monocytes/Macrophages and pan-leucocytes). Semen smears from 64 mild oligozoospermic patients and 5 fertile volunteers were stained by immunohistological technique (avidin-biotin-immunoperoxidase method). We found highly varying total leucocyte numbers ranging from 1200 to 2.2 x 10(8) per ejaculate. When comparing semen from infertile patients with those from fertile donors, statistically significant higher medians of total leucocytes, total granulocytes and monocytes/macrophages were seen in infertile group. We compared a total number of leucocytes obtained from conventional Papanicolaou staining with that from immunohistochemical staining. Both were almost identical but the former gave a little bit higher value, because of the difficulty in differentiating leucocytes from immature germ cell by Papanicolaou staining. Moreover we performed an experiment to evaluate the applicability of flow-cytometry (FCM) to count leucocytes in semen. Cells in the semen smears were stained with MoAbs recognizing the surface antigen shared by every leucocyte, and percentage of the leucocytes which were thus stained with MoAbs was compared with that determined by FCM. Results from FCM constantly gave lower value than these from immunostaining. This is probably because FCM could not differentiate leucocytes from contaminated non cell substances (e.g. cell debris). From the present study it is not adequate to use FCM for the detection of leucocytes in semen as applied in haematology. Further techniques to remove non-cellular substances should be established for its application in semen analysis.