ORIGINAL ARTICLE
Relationship between hyaluronic acid binding assay and outcome in ART: a pilot study M. Nijs1, E. Creemers1, A. Cox1, M. Janssen1, E. Vanheusden1, J. Van der Elst2 & W. Ombelet1 1 Genk Institute for Fertility Technology, Ziekenhuis Oost Limburg, Genk, Belgium; 2 Vrije Universiteit Brussel and Universiteit Ghent, Ghent, Belgium
Keywords Hyaluronic acid—IVF/ICSI—sperm maturity—sperm morphology—sperm quality Correspondence Martine Nijs, M.Sc., Genk Institute for Fertility Technology, Ziekenhuis Oost Limburg, Schiepse Bos 6, 3600 Genk, Belgium. Tel.: 3289327721; E-mail:
[email protected];
[email protected] Accepted: July 22, 2009
Summary The sperm–hyaluronan binding assay (HBA) is a diagnostic kit for assessing sperm maturity, function and fertility. The aim of this prospective cohort pilot study was to evaluate the relationship between HBA and WHO sperm parameters (motility, concentration and detailed morphology) and possible influence of sperm processing on hyaluronic acid binding. A cohort of 68 patients undergoing a first combo in vitro fertilisation/intracytoplasmic sperm injection treatment after failure of three or more intrauterine insemination cycles were included in the study. Outcome measures studied were fertilisation rate, embryo quality, ongoing pregnancy rate and cumulative pregnancy rate. HBA outcome improved after sperm preparation and culture, but was not correlated to detailed sperm morphology, concentration or motility. HBA did not provide additional information for identifying patients with poor or absent fertilisation, although the latter had more immature sperm cells and cells with cytoplasmic retention present in their semen. HBA outcome in the neat sample was significantly correlated with embryo quality, with miscarriage rates and ongoing pregnancy rates in the fresh cycles, but not with the cumulative ongoing pregnancy rate. No threshold value for HBA and outcome in combo IVF/ICSI treatment could be established. The clinical value for HBA in addition to routine semen analysis for this patient population seems limited.
Introduction The human fertilisation process is a complex sequence of biological events. For human spermatozoa, it involves recognition and penetration of the cumulus oophorus complex (COC), acquisition and completion of capacitation, recognition and binding to zona pellucida (ZP) receptors as well as induction of the acrosome reaction. Different sperm tests have been developed to analyse these functionalities and include the sperm-ZP binding test, the ZP-induced acrosome reaction, the hyaluronate migration test or the hyaluronic acid (HA) penetration test (Burkman et al., 1988; Mortimer et al., 1990; Aitken et al., 1992; Oehninger et al., 2000, 2007; Liu et al., 2004; Sifer et al., 2005). A simple test, the sperm–hyaluronan binding assay (HBA), has been developed as a diagnostic kit for assessing sperm maturity and function (Cayli et al., 2003, 2004; Huszar et al., 2003): mature spermatozoa ª 2010 Blackwell Verlag GmbH
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with intact acrosomes, low degree of cytoplasmic retention and better morphology will selectively bind to a microlayer of HA (Huszar et al., 1997, 2006, 2007; Prinosilova et al., 2008). HA, a mucopolysacharide, is the main component of the COC surrounding the oocyte and the ZP (Aitken et al., 1992; Cayli et al., 2003). The aim of this pilot study was first to determine the relationship between HBA and World Health Organization (WHO) sperm characteristics; second, to determine whether sperm processing and culture would alter HA binding properties and third, in a prospective cohort way to analyse the relationship between HBA and outcome of a first combo in vitro fertilisation (IVF)/ intracytoplasmic sperm injection (ICSI) treatment for patients with three or more failures in intrauterine insemination (IUI). Main outcome measures were fertilisation rate, embryo quality, ongoing pregnancy and cumulative pregnancy rate. 291
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Materials and methods Patients This pilot study was based on a 15-month prospective cohort of 68 consecutive infertile patients (female age 0.03). Presence of detailed morphological defects had no significant impact on the cumulative ongoing pregnancy rate (logistic regression; all P > 0.11). This pilot study could not define a HBA threshold value for fertilisation, embryo quality or cumulative pregnancy chance2 (all P > 0.05). Discussion This prospective pilot study is based on a cohort of 68 patients undergoing a first combo IVF/ICSI cycle for the first time after failure of three or more IUI. To the best of our knowledge, this study demonstrated for the first time that sperm binding to a HA microlayer improved after sperm preparation and prolonged culture. Since spermatozoa are transcriptionally silent cells, it is highly unlikely that new HA receptor proteins have been produced and added to the sperm membrane during culture. Sperm preparation and especially culture, however, could have enhanced removal and/or remodelling of masking proteins that are normally present on the sperm head to stabilise membranes up to the onset of capacitation (De Jonge, 2005; Nixon et al., 2007). No intrapatient variation in HBA outcome was observed (with an interval of 5–7 days), suggesting that HA binding properties could be typical for each spermatogenic cycle and/or for each individual patient. This pilot study could not confirm a significant positive correlation between HA binding and normal overall morphology or specific sperm head abnormalities of spermatozoa (Cayli et al., 2003; Ghosh et al., 2007; Prinosilova et al., 2008). Our data did not reveal any correlation with concentration or motility of the fresh or prepared semen samples, in contrast to Ye et al. (2006) who studied semen from IVF patients. However, this study could confirm that HBA did not clearly discriminate between patients with low or high fertilisation rates: patients with fertilisation rate £50% had marginally (although significantly) lower HBA scores than those with fertilisation rate >50% (mean HBA difference 11%). Interestingly, patients with low fertilising potential had significantly more immature cells present in their fresh ª 2010 Blackwell Verlag GmbH
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neat ejaculates (P = 0.003), whereas those with total absence of fertilisation (n = 10) had significantly more spermatozoa with retained cytoplasmic droplets, although this was not reflected in their HBA outcome. This study demonstrated for the first time that HBA outcome was correlated to both embryo quality and higher miscarriage rates in a fresh transfer. Previously, Nijs et al. (2009b) demonstrated a negative correlation between HBA outcome and sperm DNA condensation (analysed by the sperm chromatin structure assay), a parameter linked to nuclear immaturity and poorer fertility outcome. Jakab et al. (2005) found that non-HA bound spermatozoa have a higher frequency of chromosomal disomy compared with HA bound spermatozoa. This could explain poorer embryo quality as well as increased risk of miscarriages for those patients with a lower mean HBA score in our study. Although logistic regression showed a clear influence of HBA2, HBA3 and HBA4 on the ongoing pregnancy rate of the fresh cycles and HBA outcome for patients with a pregnancy in a fresh cycle was significantly lower than HBA in non-pregnant patients, this influence was lost when combining the outcomes of the fresh and frozen/thawed cycles to calculate the cumulative ongoing pregnancy rate. This observation indicates that other factors besides sperm functionalities (like embryo survival after freeze/thawing or endometrial receptivity) play an important role in the overall success of a fertility treatment. In conclusion, although this pilot study on influence of HBA outcome included a limited number of selected patients (patients with three or more failed IUI cycles), it could not clearly distinguish between patients with failed, low or high rates of fertilisation, nor could HBA scores give a predictive value in obtaining an ongoing pregnancy after combination of fresh and frozen/thawed embryo transfer. Therefore, the useful clinical value for HBA in addition to the routine semen analysis for this patient population seems to be rather limited. Sources of support The HBA binding slides for this study were provided by MidAtlantic Diagnostics, Marlton, NJ, USA. References Aitken RJ, Bowie H, Buckingham D, Harkiss D, Richardson DW, West KM (1992) Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence. J Androl 13:44–54. Burkman L, Coddington C, Franken D, Kruger T, Rosenwaks Z, Hogen G (1988) The hemizona assay (HZA):
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