A novel sorting technology allows for highly efficient selection of sperm without chromatin damage

Abstract

A novel sorting technology allows for highly efficient selection of sperm without chromatin damage, Systems Biology in Reproductive Medicine, 59:3, 172-177, DOI: 10.3109/19396368.2013 Sperm chromatin damage has been associated with male infertility, increased risk for spontaneous abortion, and poor embryo development. Available methods for detecting chromatin damage render the sperm no longer suitable for clinical use. Early apoptotic events resulting in chromatin damage are associated with increased permeability of the cell membrane to large ions. We propose the use of a large fluorescent organic cation, proprietary fluorochrome (PF-1), for fluorescence-activated cell sorting (FACS) for negative selection of sperm without chromatin damage. Sperm with chromatin damage are PF-1 positive. Performance of cell sorting by PF-1 was verified with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) after FACS on PF-1(+) and PF-1(-) subpopulations. Whereas 19.5% of PF-1 positive sperm were TUNEL positive only 1.5% sperm in the PF-1(-) fraction were TUNEL positive ( p < 0.00001). TUNEL values below 1.9% were considered background fluorescence. Post-sorting motility and vitality were 49.4% (SD: 12.5) and 65.0% (SD: 14.99), respectively. Proprietary fluorochrome activated sperm sorting may decrease or most likely eliminate all of TUNEL positive sperm without adverse effects on viability, providing a new therapeutic avenue for men with a high percentage of TUNEL positive sperm. Further research is needed to determine if the reduction in TUNEL positive sperm using PF-1 will improve in vitro fertilization (IVF) outcomes.

Novel Techniques of Sperm Selection for Improving IVF and ICSI Outcomes

“…However, the use of the properties of the plasma membrane for sperm selection could really improve ARTs if markers with higher potential for discriminating high-quality spermatozoa are discovered. Thus, new antigens could be used as targets for selection by MACS, by coated plates or even by fluorescence-activated cell sorting (Funaro et al, 2013). However, this research direction is currently poorly explored, possibly because of the lack of basic knowledge about markers discriminating the various sperm populations contained in the ejaculate.…”

An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II

“…The use of a stain or dye targeting indicators of poor sperm quality results in the negative selection of the high-quality population which can then be used for a variety of in vitro procedures. Live sperm have been flow-sorted based on membrane permeability and apoptotic markers [ 105 , 106 , 107 ], mitochondrial membrane potential [ 108 ], and even sex chromosomes [ 109 ].…”

“…A method to enrich the population of non-apoptotic spermatozoa in a sample is to tag and remove apoptotic spermatozoa from the population by FACS with fluorescently labeled Annexin-V; the antigen of phosphatidylserine [ 105 , 106 ]. Sperm negatively selected via Annexin-V FACS have been shown to possess intact chromatin [ 107 ]. Additionally, in a human clinical trial, spermatozoa from the population that did not bind Annexin-V during FACS were used for ICSI resulted in improved pregnancy and live birth rates, as well as reduced miscarriage rates over embryos generated with sperm selected from SU [ 112 ].…”