Sperm Hyaluronan-binding Assay Research Articles

Use of a sperm-Hyaluronan binding assay for evaluation of sperm quality in dromedary camels

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance.

Sperm binding to hyaluronan is an excellent predictor of Nili-Ravi buffalo bull fertility.

This study reports the first evaluation of sperm hyaluronan binding assay (HBA) for predicting the fertility of Nili-Ravi buffalo bulls in relation to standard parameters of sperm quality. Cryopreserved semen doses of low (n=6), medium (n=3) and high fertility (n=8) bulls based on their respective return rates were used. Significantly, more spermatozoa bound to hyaluronan from the most fertile bulls (57.15%±1.44) compared with medium (42.46%±1.08) and low fertility bulls (29.70%±0.78). A strongly positive correlation (r=.824, p<.01) was found between HBA and fertility that predicts a 67.9% variability (r2 =.679, p<.01) in fertility. HBA was also strongly positively correlated with sperm viability (r=.679, p<.01) followed by their live/dead ratio (r=.637, p<.01), uncapacitated spermatozoa (r=.631, p<.01), normal apical ridge (r=.459, p<.01), motility (r=.434, p<.01), mature spermatozoa with low residual histones (r=.364, p<.01), high plasma membrane integrity (r=.316, p<.01) and nonfragmented DNA levels (r=.236, p<.05). It was negatively correlated with spermatozoa having reacted acrosome (r=-.654, p<.01). A fertility model built using a combination of sperm HBA and either sperm livability or viability predicts, respectively, 86.1% (r2 =.861, p<.01) and 85.9% (r2 =.859, p<.01) variability in buffalo bull fertility. In conclusion, sperm HBA may prove to be a single robust predictor of Nili-Ravi buffalo bull fertility.

A single-arm pilot study on effects of acupuncture treatment on semen parameters of subfertile Singaporean men

Abstract Objective To evaluate the effect of acupuncture treatment on semen parameters and the proportion of mature and functional sperm as measured by using sperm-hyaluronan binding assay (HBA) in subfertile Singapore men. Methods Singaporean male subjects undergoing subfertility investigation were enrolled prospectively in this pilot study. Each subject received acupuncture treatment at fixed acupoints and two acupuncture sessions per week for 10 weeks. A total of five semen samples were obtained from each subject at 5 weekly intervals up to 20 weeks of the study; at Pre-treatment, Treatment 1 (5th week), Treatment 2 (10th week), Post-treatment 1 (15th week) and Post-treatment 2 (20th week). Semen analysis was performed to assess semen parameters and HBA score (percentage of motile sperm bound to hyaluronan) to determine the proportion of mature and functional sperm. Results Mean HBA scores improved significantly from 12.0% at Pre-treatment, to 18.4% (P = 0.03) at 5th week and was 16.8% (P = 0.07) at 10th week of acupuncture treatment. After completion of acupuncture treatment, HBA scores continued to increase significantly to 25.8% (P Conclusions HBA scores increased significantly during the first 5 weeks of acupuncture treatment (Treatment 1) and 5 weeks after the completion of treatment (Post-treatment 1). Hence acupuncture treatment was effective in increasing the proportion of mature and functional sperm but had no effect on other semen parameters.

Increased Hyaluronan Acid Binding Ability of Spermatozoa Indicating a Better Maturity, Morphology, and Higher DNA Integrity After Micronutrient Supplementation

Measuring the hyaluronan-binding ability of spermatozoa is useful in predicting the ability of spermatozoa to fertilise oocytes during in vitro fertilisation (IVF). Recent publications discuss an influence of micronutrients on sperm quality. The objective of this paper was to evaluate the effect of a non-prescription nutraceutical containing eight micronutrients on sperm-hyaluronan binding assay (SHBA) values among males with idiopathic sub-/infertility, using an open comparative pilot study. The study took place at the Outpatient Fertility Centre IMI, Vienna, Austria, and involved 67 sub-/infertile males. Sub-/infertile males were invited to participate and take two daily capsules of the active compound for a 3-month period between the first and the follow-up semen analysis. Each capsule contained L-carnitine, L-arginine, zinc, vitamin E, glutathione, selenium, coenzyme Q10 (CoQ10), and folic acid (Profertil®). 40 sub-/infertile men receiving no active treatment served as controls; this was measured by change in SHBA after 3 months. It was found that SHBA values significantly increased after 3 months of treatment with the active compound, from a median baseline value of 56.0% to 74% (p&lt;0.05). This represented a 19.7% increase compared to baseline, which was significantly higher than the 2.1% decrease observed in the control group. The rate of subjects displaying an increase in SHBA values after 3 months was significantly higher in the active group (74.6% versus 30.0%, p=0.0001), which showed that sub-/infertile men treated with the active micronutrient compound displayed increased SHBA ability. However, more research is necessary to get detailed information on this specific subject.

Varicocele decreases epididymal neutral α‐glucosidase and is associated with alteration of nuclear DNA and plasma membrane in spermatozoa

To assess the relationship between a marker of epididymal function and both the fragmentation of the sperm nucleus and the integrity and maturity of the sperm membrane in patients with or without varicocele. Semen samples were obtained from men with varicocele grades II and III (n = 60) and from a control group with zoospermia defined as normal (n = 30). Samples were evaluated by a spermiogram, a hypoosmotic swelling test (HOST), neutral α-glucosidase (NAG) enzyme activity, sperm hyaluronan-binding assay (HBA) and DNA fragmentation using a sperm chromatin dispersion (SCD) test. Seminal plasma NAG levels, percentage of sperm bound to hyaluronic acid, HOST-positive cells and sperm quality were significantly lower in the varicocele compared with the control group. Higher levels of sperm DNA fragmentation, as measured by SCD, were also observed in the varicocele group compared with the control group. Seminal NAG activity levels showed a strong negative correlation with DNA fragmentation and a significant positive correlation with the HBA test and the HOST. Varicocele causes a reduction in NAG activity by the epididymis that is associated with damage to both the membrane and sperm nucleus and a reduction in the seminal parameters. NAG levels were correlated with the quality of the sperm membrane and nucleus. Data suggest that a reduction of fertilization capacity in men during varicocele can result from damage to both the testis and the epididymis.

Correlation between sperm hyaluronan binding assay (HBA), sperm DNA fragmentation and sperm high magnification

The aim of this study is to assess the correlation between HBA and two other male diagnostic tests. Observational prospective study. The study includes 41 samples analysed at CIRH, Barcelona (Spain) following the protocol suggested by the manufacturer. Mean age of male patients was 35 years old. Sperm DNA fragmentation was assessed using the Halosperm kit (Halotech DNA SL, Madrid, Spain). To assess sperm morphology by high magnification we considered spermatozoa from groups III and IV, those spermatozoa with high vacuolization measured by inverted microscopy (Leica AM6000) at more than 6000x magnification. HBA test presents a higher negative correlation with high magnification (r=0.16) being statistically significant (p=0.012). On the contrary, the results of the HBA test are correlated positively with DNA fragmentation (r=0.05) although not statistically significant (p>0.05). The obtained results show that samples enriched with spermatozoa of bad morphology present low percentage of positive HBA. Furthermore, the integrity of the DNA does not seem to be related directly to the degree of sperm maturity. The lack of correlation could be due to the sample size or, maybe because the outsourcing of the hyaluronate receptor is made regardless of the degree of DNA integrity. It would be desirable to increase the number of samples to verify this hypothesis. It is necessary to deepen the relationship between the three sperm parameters to assess their predictive power.

Relationship between Sperm Hyaluronan-Binding Assay (HBA) Scores on Embryo Development, Fertilisation, and Pregnancy Rate in Patients Undergoing Intra-Cytoplasmic Sperm Injection (ICSI)

Introduction: The novel development of the sperm hyaluronan-binding assay (HBA) has now been routinely used in some laboratories worldwide to predict sperm maturity and functionality. Hence, the purpose of this study is to evaluate if embryo development, quality, fertilisation and pregnancy rates are affected by low HBA values. Methods: A total of192 female patients who underwent intra-cytoplasmic sperm injection (ICSI) were compared retrospectively in terms of embryo development, fertilisation, and pregnancy rates with their husbands' HBA score. Patients' husbands were required to undergo a HBA test before the start of their stimulation cycle to determine if their semen was suitable to undergo ICSI or in-vitro fertilisation (IVF). P-value &lt; 0.05 was considered significant. Results: Patients were divided into four groups, group A (HBA ≤ 15%), group B (HBA &gt; 15% &lt; 35%), group C (HBA ≥ 35% &lt; 60%), and group D (HBA ≥ 60%). The fertilisation rate for groups A, B, C, and D were 67.9%, 73.1%, 72.5%, and 77.1% respectively. Group D had a fertilisation rate significantly higher than the rest of the groups ( p = 0.016). The pregnancy rate for group D was also significantly higher amongst the four groups ( p = 0.041), whereas the pregnancy rate for groups B and C was similar (42.4% versus 41.1% respectively). Day three cleavage rate (the ability to reach six cells and beyond) was highest for group D compared to the other groups ( p = 0.002). Conclusion: The higher the HBA score, the better the fertilisation, pregnancy, and cleavage rates. This shows that HBA does have the ability to select mature sperms with normal chromosome development and oocyte-binding capability.

Relationship between hyaluronic acid binding assay and outcome in ART: a pilot study

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.

Sperm–hyaluronan-binding assay: clinical value in conventional IVF under Italian law

Hyaluronan has recently been employed in the development of a commercial diagnostic kit for assessing sperm maturity, the so-called sperm–hyaluronan-binding assay (HBA). The aim of this study was to evaluate the usefulness of this test, in addition to routine semen analysis, to identify patients with poor reproductive prognosis in conventional IVF. Furthermore, the ability of hyaluronan to select spermatozoa with low DNA fragmentation was investigated. A total of 60 IVF patients were analysed with regard to reproductive outcome, sperm parameters, HBA score and sperm DNA fragmentation. The DNA fragmentation analysis was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay on the total sperm population and on the hyaluronan-bound spermatozoa obtained from the same samples. No relationship between hyaluronan binding and fertilization, cleavage, good-quality embryos, implantation, clinical pregnancy, miscarriages and biochemical pregnancy rates was found. Otherwise, correlations between spermatozoa hyaluronan binding and morphology ( P < 0.01) and a significant difference between DNA fragmentation of the total sperm population and DNA fragmentation of the hyaluronan-bound spermatozoa ( P = 0.029) were found. The results underline the ability of hyaluronan to select spermatozoa with higher DNA integrity and morphology. Nevertheless, the clinical value of the HBA in the management of male infertility seems to be limited.

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

Is sperm hyaluronan binding assay (HBATM) a possible predictor of outcomes in IVF cycles with insemination?

OBJECTIVE: The Hyaluronan Binding Assay is based on the premise that only mature sperm have the ability to bind hyaluronan which is a main component of the cumulus oophorus matrix. A manufacturer's guideline suggests >80% HBA Score (% binding) indicates normal maturity and physiological function. This study was performed to see if the HBA test could be incorporated as a screening test for IVF with insemination.

Sperm DNA Integrity From Sperm to Egg

Sperm DNA Integrity From Sperm to Egg

P-996: Relationship among HBA, sperm motility, Kruger morphology with fertilization rate and pregnancy outcome in patients treated with IVF or ICSI

Semen analysis with Kruger morphology is widely used to assess male fertility potential. More recently hyaluronan-binding protein has been established as a biomarker for mature, functional sperm. Consequently the HBA assay (Sperm-Hyaluronan Binding assay) has been developed to differentiate functional integrity and fertilization potential of sperm cells. We investigated the relationships among HBA score, sperm motility, and Kruger morphology with fertilization rate in patients undergoing in vitro fertilization (IVF) with standard insemination or intracytoplasmic sperm injection (ICSI). Prospective comparative data analysis. The Andrology and Embryology laboratories at the Kaiser Permanente Center for Reproductive Health performed HBA assay in conjunction with a standard semen analysis with Kruger morphology for patients undergoing IVF. The decision to perform standard IVF or ICSI was made by the primary physicians and the patients. Sperm HBA score, sperm motility and Kruger morphology before and after density gradient preparation were compared and correlated with fertilization rate of oocytes inseminated with standard insemination or ICSI. Linear regression analysis was performed. We studied prospectively 31 cases in which 20 cases were treated with ICSI, 7 cases were treated with IVF, and 4 cases were performed split IVF/ICSI. In the ICSI treatment group, the fertilization rate had no correlation with sperm motility, Kruger morphology, or HBA score (r = -0.15, -0.14, and 0.07 respectively). We achieved high fertilization rates (50 - 100%) in the ICSI treatment group even when the sperm HBA score was less than 10 either before or after density gradient preparation. In general, density gradient preparation of sperm improved its motility and morphology. However, it did not necessarily improve the HBA score. In fact, some sperm samples after density gradient preparation dropped in HBA score dramatically. In the IVF treatment group, there was a positive trend between fertilization rate and initial sperm motility and HBA score. The fertilization rate, as previously suggested by Kruger et al, showed little correlation (r = -0.15) with Kruger morphology when the later score was greater than or equal to 4. HBA assay, sperm motility and Kruger morphology have all been suggested as indicators for differentiating male fertility potential. Based on our data, we can not recommend any one of them individually as a discriminating factor for IVF or ICSI decision making except when Kruger morphology score is ≤ 4. Our results demonstrate a reduction of HBA score after density gradient sperm preparation of some sperm populations. This observation raises a concern about potential damage of membrane integrity and/or unintended acrosome reaction during sperm preparation, both of which may lead to compromised fertilization potential. Future studies are needed to explore the utility of HBA assay in assessing sperm functional integrity after processing.

Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization

Sperm-hyaluronan-binding assay (HBA) is one of the commercial kits being marketed for routine testing of sperm maturity and fertility. However, there is no report of whether the HBA can provide additional information over standard semen analysis for sperm-fertilizing ability. The objective of this study was to investigate the relationship between HBA and fertilization rate in conventional IVF. A total of 175 IVF patients with > or = 3 mature oocytes inseminated were included in the study. Both the standard semen analysis and the HBA were performed on the same ejaculated sperm samples used for IVF treatments. Relationships between the semen analysis and the HBA results and fertilization rate were analysed by both the Spearman test and the multivariate logistic regression analysis. Both total and progressive sperm motility and normal morphology were highly correlated with HBA scores. While both normal sperm morphology and HBA scores were statistically significantly related to fertilization rates, the HBA was less significant than normal sperm morphology. The HBA does not provide additional information for identifying patients with a poor fertilization rate. HBA is highly significantly correlated with sperm motility and morphology but is less significant than sperm morphology in relation to the fertilization rate in IVF. Thus, the clinical predictive value of HBA for sperm-fertilizing ability in vitro is limited.

Correlation between normal sperm head morphology (NSHM) and sperm binding potential to the Sperm Hyaluronan-Binding Assay (HBA™)

Hyaluronan, a high molecular weight glycosaminoglycan, is a major constituent of the cumulus oophorous matrix surrounding the human oocyte. Hyaluronan may play a critical role in the selection of functionally competent sperm during in vivo fertilization. Research has demonstrated that specific motile sperm attach to hyaluronan and that such bound sperm carry enhanced levels of developmental maturity, sperm chromatin integrity and normal morphology. The relationship between hyaluronan-bound (HB) sperm and enhanced levels of normal morphology led to the current study examining sperm binding potential and specific morphological anomalies.

Statistically significant correlation between normal sperm morphology (NSM) and sperm binding potential to an immobilized monolayer of hyaluronan using the HYDAK(r) sperm hyaluronan-binding assay (HBA)

Objectives: Hyaluronan, a high molecular weight glycosaminoglycan, is a major constituent of the cumulus oophorous matrix surrounding the human oocyte. During the process of fertilization, hyaluronan may play a critical role in the selection of functionally competent sperm. Huszar has demonstrated that specific motile sperm attach to a monolayer of hyaluronan immobilized on a surface and that such bound sperm demonstrate specific parameters of developmental maturity. Hyaluronan-bound (HB) sperm exhibit decreased levels of both cytoplasmic inclusions and residual histones, an increased expression of the HspA2 chaperone protein, and a marked reduction in the incidence of chromosomal aneuploidy. The relationship between HB sperm and enhanced levels of developmental maturity led to the current study of sperm binding potential and morphology. A pilot study was undertaken to explore the potential correlation between the ability of a seminal population to bind to the hyaluronan substrate and the Kruger Strict Criteria (KSC) morphological assessment demonstrated by the same population. Design: Excess semen samples from 31 infertility patients were assayed in the HYDAK(r) HBA chambers. Both normal and male factor patients were included. All specimens were analyzed for percent NSM using KSC. Two independent technicians performed each evaluation. Materials and Methods: Specimen collection, analysis and disposal followed a protocol approved by the Hospital Institutional Review Board. Hydak(r) HBA chambers, supplied by Biocoat, Inc., were fabricated from Cell-Vu(r) dual chamber disposable sperm counting slides. A molecular layer of hyaluronan was covalently bound to one chamber while the second counting chamber remained unmodified and served as a control. Following a 10-minute incubation period, the number of total, free motile and bound motile sperm were scored in each chamber. The HBA score was calculated as # bound motile sperm / # total motile sperm. Three morphology slides were prepared per patient and a minimum of 300 sperm were assessed using a 100X oil immersion lens and a calibrated reticle. A two-way contingency table evaluating the variables of NSM and HB was constructed to evaluate the data. Significance was tested by the Pearson Chi-squared statistic using SPSS for Windows software. Results: The analysis showed that fluctuations in NSM were statistically related to the ability of sperm to bind to hyaluronan (Chi squared = 8.16, p = 0.007, n = 31). Seminal populations demonstrating a NSM of <4% carried a mean HBA score of 44.4% (n = 13). In contrast, seminal populations carrying a NSM of >4% demonstrated a mean HBA score of 71.5% (n = 18). Conclusions: Hyaluronan may play a critical role in the selection of functionally competent sperm during in vivo fertilization. The current study demonstrated that a statistically significant relationship existed between the HBA and NSM carried by a seminal population. A seminal population carrying a higher HBA score was concomitant with increased levels of NSM. A lower HBA score was also found to be statistically consistent with poor morphology parameters. The HYDAK(r) HBA may therefore serve as a valuable diagnostic tool in assessing the level of developmental maturity, incidence of aneuploidy and morphological parameters of a seminal population. Ongoing studies are exploring the role hyaluronan may play in the selection of functionally competent sperm and the potential value of the HBA as a diagnostic tool.