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.

The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures

“…Current methods used to establish the degree of sperm DNA fragmentation may be used only as diagnostic tools, meaning that the cells analyzed in the assays cannot be used for in vitro fertilization [13]. Taking this evidence as our starting point, we posited the question as to whether there was a way to distinguish among four preparation techniques: direct swim-up (DSU), pellet swim-up (PSU), density gradient (DG), and density gradient followed by swim-up (DG-SU), also evaluating the effects of each technique on the DNA damage rate of the sperm population obtained.…”

Intracytoplasmic morphologically selected sperm injection outcomes: the role of sperm preparation techniques

“…Under in vivo conditions, spermatozoa are separated from these detrimental components in the female genital tract by migration through the cervical mucus [16]. Several semen separation techniques have been developed to separate the sperm fraction for use in assisted reproductive techniques.…”

Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid

“…Considering that intermittent sperm binding to hyaluronic acid during their journey across the female genital tract may be an important aspect of natural fertilization (Henkel, 2012), the current study was set up to investigate the dynamics of sperm binding to hyaluronic acid in vitro and to investigate the relationship between hyaluronic-acid-binding and standard methods of sperm preparation for IVF (in this case, differential density gradient centrifugation) and measures of sperm quality that included DNA fragmentation and chromatin condensation. Initially, microscopy was used to detect hyaladherins, including CD44 on human sperm, which was detected on the acrosome as has been reported elsewhere (Bains et al, 2002) and equatorial regions of washed spermatozoa.…”

“…On the basis of classical parameters such as concentration, viability, motility and morphology under WHO guidelines, these techniques do enrich for more fertile spermatozoa (WHO, 2010). Functional properties of DDGCenriched populations, such as DNA integrity and chromatin maturity that could explain the improved fertility of these populations of spermatozoa, are, however, usually not assessed.Available evidence suggests that, although spermatozoa prepared using standard routine sperm preparation methods such as DDGC or swim-up may have generally lower levels of DNA fragmentation or compromised chromatin compared with spermatozoa from unprocessed semen samples, and may therefore be suitable for use in assisted reproduction techniques, such preparations may not exclude sperm that are compromised in these regards (Zini et al, 1999;Brahem et al, 2011b;Henkel, 2012;Mortimer and Mortimer, 2013). This is a particular issue for ICSI in which sperm selection relies almost exclusively on the embryologist's judgement.…”

“…Available evidence suggests that, although spermatozoa prepared using standard routine sperm preparation methods such as DDGC or swim-up may have generally lower levels of DNA fragmentation or compromised chromatin compared with spermatozoa from unprocessed semen samples, and may therefore be suitable for use in assisted reproduction techniques, such preparations may not exclude sperm that are compromised in these regards (Zini et al, 1999;Brahem et al, 2011b;Henkel, 2012;Mortimer and Mortimer, 2013). This is a particular issue for ICSI in which sperm selection relies almost exclusively on the embryologist's judgement.…”