Sperm preparation: state-of-the-art--physiological aspects and application of advanced sperm preparation methods

Abstract

For assisted reproduction technologies (ART), numerous techniques were developed to isolate spermatozoa capable of fertilizing oocytes. While early methodologies only focused on isolating viable, motile spermatozoa, with progress of ART, particularly intracytoplasmic sperm injection (ICSI), it became clear that these parameters are insufficient for the identification of the most suitable spermatozoon for fertilization. Conventional sperm preparation techniques, namely, swim-up, density gradient centrifugation and glass wool filtration, are not efficient enough to produce sperm populations free of DNA damage, because these techniques are not physiological and not modeled on the stringent sperm selection processes taking place in the female genital tract. These processes only allow one male germ cell out of tens of millions to fuse with the oocyte. Sites of sperm selection in the female genital tract are the cervix, uterus, uterotubal junction, oviduct, cumulus oophorus and the zona pellucida. Newer strategies of sperm preparation are founded on: (i) morphological assessment by means of 'motile sperm organelle morphological examination (MSOME)'; (ii) electrical charge; and (iii) molecular binding characteristics of the sperm cell. Whereas separation methods based on electrical charge take advantage of the sperm's adherence to a test tube surface or separate in an electrophoresis, molecular binding techniques use Annexin V or hyaluronic acid (HA) as substrates. Techniques in this category are magnet-activated cell sorting, Annexin V-activated glass wool filtration, flow cytometry and picked spermatozoa for ICSI (PICSI) from HA-coated dishes and HA-containing media. Future developments may include Raman microspectrometry, confocal light absorption and scattering spectroscopic microscopy and polarization microscopy.

Figure 1

Sites of sperm selection in the human female. Estimated numbers of spermatozoa in the respective sections of the female genital tract are given in brackets. Soon after deposition in the vagina, spermatozoa move out of the seminal plasma and enter the highly hydrated cervical mucus where sperm selection for morphology and motility takes place. Spermatozoa that are selected here are characterized by a low plasma membrane content of PUFA. Subsequently, spermatozoa are transported through the uterus where male germ cells are selected for motility and timely capacitation. Dysfunctional spermatozoa are eliminated. After passing the uterotubal junction, which has, in contrast to numerous animal species, no significant function in man, spermatozoa enter the isthmus of the fallopian tube. In the isthmus, spermatozoa can be stored for up to 5 days. In the isthmus and the cumulus, spermatozoa are selected for capacitation, thermo- and chemotaxis-responsiveness and DNA integrity. Finally, after passing through the cumulus, spermatozoa bind to the zona pellucida where another morphological selection takes place. Moreover, male germ cells are further selected for zona-binding ability and DNA integrity (figure modified with permission from Ref.). PUFA, polyunsaturated fatty acid.