Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation

Abstract

One concern during intracytoplasmic sperm injection (ICSI) is that selected spermatozoa may have increased levels of DNA damage; however, the available testing for this is largely destructive in nature and therefore unsuitable as a tool for sperm selection. One alternative selection process that has previously achieved pregnancies is the hypo-osmotic swelling test (HOST). This study reports that low HOST values of neat semen samples were significantly (P < 0.001) associated with increased DNA damage identified by the DNA fragmentation index (DFI) from the sperm chromatin structure assay as well as the TdT-mediated dUTP nick-end labelling (TUNEL) assay. The HOST value was highly predictive of an abnormal DFI value by receiver operating characteristic curve analysis (P < 0.001). Furthermore, when individual spermatozoa were assessed for both HOST status and DNA fragmentation by TUNEL, the key HOST-induced tail-swelling grades D, E and F were most commonly associated with high HOST values and were significantly (P < 0.001) associated with minimal DNA damage regardless of the DNA status of the ejaculate. The application of HOST may be a valuable tool in the routine identification and selection of viable, DNA-intact individual spermatozoa for ICSI after further research to demonstrate its efficacy and safety.

The hypo-osmotic resistance by spermatozoa, as measured by the hypo-osmotic swelling test, was shown to strongly predict the degree of sperm DNA damage when assessed by the sperm chromatin structure assay. The spermatozoa that displayed only distal swelling were shown to strongly correlate to spermatozoa with minimal DNA damage. This study suggests that such spermatozoa may be suitable candidates for selection of viable spermatozoa for microinjection in intracytoplasmic sperm injection cycles.

Introduction

The development of intracytoplasmic sperm injection (ICSI) after many years of IVF (Yovich and Stanger, 1984) revolutionized the management of male factor infertility (Palermo et al., 1992). The use of minimal ovarian stimulation and low egg numbers (Borini et al., 2006), the use of preimplantation genetic screening and surgically collected spermatozoa in IVF has further increased the incidence of ICSI over and above its original application for severe male-factor patients. Despite the current debate over whether ICSI should be used routinely (Aitken, 2008), in time, ICSI conceptions may overtake IVF conceptions as the primary vehicle for insemination (Wang et al., 2009). One of the hurdles for its acceptance is that ICSI methodology has not changed since first described by Palermo et al. (1992) and there appears to be minimal imperative to explore alternative approaches to the technique and in particular to sperm selection (Van Voorhis, 2007).There is no standardized methodology for sperm selection that has been defined and validated and ensuring that spermatozoa of similar quality are being selected by each embryologist is difficult from a quality management issue but in time may be an essential requirement.

Sperm selection for ICSI is visually based primarily upon motility and, to a lesser extent, morphology. Recent reports aimed at improving sperm selection have centred either on increasing the visual selection process (Berkovitz et al., 2005) or functional criteria such as hyaluronic acid binding capabilities (Huszar et al., 2007), the net surface charge (Ainsworth et al., 2007) or the identification of apoptotic markers (Said et al., 2008). Yet spermatozoa used in ICSI are not necessarily the same as those that achieve fertilization in IVF since the latter have been shown to conform to very specific morphological criteria associated with binding to the zona pellucida and that these have minimal DNA damage (Liu and Baker, 2007). The aim for sperm selection for ICSI should be to emulate the in-vivo process whereby the spermatozoon that facilitates fertilization has minimal DNA damage.

Recent interest in the integrity of sperm DNA (Aitken, 2008) has raised concerns that, while spermatozoa may appear motile, those with fragmented or incomplete protamination of DNA may still be selected in ICSI and these have been linked to poor embryonic development, reduced conception rates and increased possibility of miscarriage (Borini et al., 2006, Evenson and Wixon, 2006, Evenson and Wixon, 2008, Gandini et al., 2004, Sakkas et al., 1996) While both the sperm chromatin structure assay (SCSA) and the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay are standard methods for assessing sperm DNA integrity (Evenson et al., 1999), they nevertheless require a high degree of skill and they are expensive, time consuming and, above all, destructive to spermatozoa. This means it is not possible to use any of the spermatozoa processed by these tests for the purpose of ICSI and therefore the tests are of minimal clinical value.

An alternative approach is to use a non-destructive test for sperm viability where spermatozoa are self-selecting. The hypo-osmotic swelling test (HOST) is one sperm function test that maintains sperm viability (Jeyendran et al., 1984). Initially introduced for the diagnosis of male infertility, it has consequently been applied as a useful additional test for sperm cell membrane vitality (Rogers and Parker, 1991, Rossato et al., 2004). This technique is simple, cost effective, quick and, above all, non-invasive (Hossain et al., 1998, Tartagni et al., 2002), which commends it as potential routine method to select individual healthy spermatozoa for ICSI (Sallam et al., 2001) particularly since the degree of swelling of spermatozoa is associated with sperm viability, fertilization and pregnancy rates (Check et al., 2001, Tartagni et al., 2002). There is less information on the status of the HOST score and on differences between individual categories of tail swelling to DNA fragmentation. The current report sought to clarify whether the HOST value may predict the degree of sperm DNA damage in an ejaculate and whether the degree of tail swelling may predict the likelihood of DNA damage in individual spermatozoa, such that their identification by HOST may facilitate their routine selection for ICSI.