Hum. Reprod. Advance Access originally published online on April 4, 2006
Human Reproduction 2006 21(6):1545-1550; doi:10.1093/humrep/del008
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Relationship between human sperm-hyaluronan binding assay and fertilization rate in conventional in vitro fertilization
1 Reproductive and Genetics Institute, Chongqing Obstetrics and Gynecology Hospital, Chongqing, China and 2 Department of Obstetrics and Gynecology, University of Melbourne, Royal Women’s Hospital, Carlton, Victoria, Australia
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Melbourne, Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. E-mail: dyl{at}unimelb.edu.au
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Abstract |
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BACKGROUND: 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. METHODS: A total of 175 IVF patients with
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. RESULTS: 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. CONCLUSION: 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.
Key words: clinical IVF/semen analysis/sperm-hyaluronan–binding assay
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Introduction |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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Conventional semen analysis including sperm concentration, motility and morphology is widely used as a fundamental indicator for male fertility. However, the results do not provide accurate diagnostic or prognostic information about human fertility either in vivo or in vitro (Mahadevan and Trounson, 1984
; Zaini et al., 1985; Jeulin et al., 1986; Silber, 1989; Liu and Baker, 1992a; ESHRE Andrology Special Interest Group, 1996; World Health Organization, 1999; Guzick et al., 2001). Men can be diagnosed as sterile if their ejaculates persistently contain no sperm, or all immotile sperm, or all sperm with uniform morphologically structural defects such as round-head sperm without acrosome (Lalonde et al., 1988; von Bernhardi et al., 1990; Bourne et al., 1995; Baker, 2001). But in infertility clinics, these are uncommon causes of severe male infertility. Most men seen for infertility have either normal or reduced sperm counts, motility or morphology alone or in combination. About one-third of infertile men often have consistently normal semen analysis results, and they are usually classified as having unexplained infertility. Clinically, these patients with unexplained infertility have difficulty deciding which of the assisted reproductive technologies (ART) would be the best option to assist them to achieve pregnancy with a minimum cost and less invasive procedure.
Poor predictive values of routine semen analysis for sperm-fertilizing ability are not only because of a large variation of seminal parameters between ejaculates or because of insufficient or inadequate quality controls for the semen analysis (Guzick et al., 2001; Slama et al., 2002; Alvarez et al., 2003; Jequier, 2005). It is mostly because routine semen analysis only determines sperm count, motility and morphology but cannot detect many other aspects of sperm function such as nuclear maturity, DNA normality and the ability of sperm to interact with oocytes (Liu and Baker, 1992b; Cayli et al., 2003, 2004; Huszar et al., 2003). Therefore, many other specific sperm tests have been developed such as sperm–zona pellucida binding, the zona pellucida-induced acrosome reaction (AR), tests for sperm nuclear maturity, DNA damage and sperm velocities using computer-assisted semen analysis (CASA) (Burkman et al., 1988; Liu et al., 1988; Liu and Baker, 1992a; ESHRE Andrology Special Interest Group, 1996; Oehninger et al., 1997, 2000; Agarwal and Said, 2003; Liu et al., 2004; Virro et al., 2004; Sifer et al., 2005). While these new tests can provide additional information about sperm fertility, they are time consuming, require expensive equipment or require sufficient amounts of biological material that is in short supply, such as human oocytes. Thus, only a few laboratories with large ART clinics are able to perform these tests. The majority of ART clinics mainly use routine semen analysis for diagnosis and management of male infertility.
Recently, a very simple test, the sperm-hyaluronan–binding assay (HBA), has been developed as a commercial diagnostic kit for assessing sperm maturity and function (Cayli et al., 2003, 2004; Huszar et al., 2003). This test is based on previous reports of hyaluronic acid (HA) selectively binding to mature sperm with intact acrosome and better morphology (Huszar et al., 2003). It has been suggested that HBA may be a useful test in addition to the routine semen analysis to improve clinical diagnosis of male infertility or to assist in clinical assignment of patients for treatment with either conventional IVF or ICSI (Cayli et al., 2004). Since the HBA diagnostic kit has become available in the market, it is starting to become a popular routine clinical test in many ART clinics, in particular in some south Asian countries such as China and Singapore. However, clinical value of this test has not been independently evaluated, and there are no previous reports on whether the HBA can predict sperm-fertilizing ability. The aim of this study was to determine the relationship between HBA and routine semen analysis results and fertilization rate with conventional IVF in a large number of patients.
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Subjects and methods |
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Subjects
During March–May 2005, 195 patients undergoing the first cycle of conventional IVF treatment in Chongqing Obstetrics and Gynecology Hospital were included in this study. In our clinic, most couples were treated with the standard IVF, and only those with severe oligozoospermic (<10 x 106/ml) or severe asthenozoospermic (motility <25%) semen were treated with ICSI. To minimize errors of fertilization rate with lower number of oocytes inseminated, 20 patients with <3 mature oocytes inseminated were excluded, and only 175 patients who had
3 mature oocytes inseminated were included in the final statistical analysis. The maturity of oocytes was determined at the time of checking fertilization at about 16–18 h after the insemination. The average female age was 31 years (range 22–47) and male 33 years (range 25–47). Couples had been infertile for a mean of about 5 years, with a wide range of 1–20 years and no clear diagnosis of causes of infertility. However, most couples (n = 123) presented with mild-to-moderate tubal disease and endometriosis, and the remainders were a mixture of male factor and unexplained infertility.
Semen analysis
Semen samples were obtained by masturbation after 2–5 days abstinence. The same ejaculated sperm was used for both IVF treatments and all the sperm tests in this study. All the sperm tests were performed after liquefaction of the semen within 2 h. Sperm concentration and motility in semen were determined using standard methods (World Health Organization, 1999). Sperm motility was assessed by counting 200 sperm per sample, and progressive motility was classified as a combination of the results from the grade A (fast progression) and B (slow progression) motility. Sperm morphology was assessed on smears prepared by washing of semen with 10 ml of 0.9% sodium chloride to remove seminal plasma to decrease background staining. Morphology slides were stained with the 2% hematoxylin–eosin Y after the smears were fixed in 90% ethanol for 30 min. The percentage of normal sperm morphology was assessed using strict criteria (Kruger et al., 1988; World Health Organization, 1999). For each sperm sample, 200 spermatozoa were scored from at least 10 individual fields using oil immersion with a magnification of x1000 under bright-field illumination. Normal sperm morphology was assessed by two independent observers, and an average of two results was used for statistical analysis. There was a highly significant correlation between the two morphology results obtained from the two observers (n = 175, Spearman r = 0.930, P < 0.001). The mean difference of the two results was 1.3% with an SD of 4%.
Sperm-hyaluronan–binding assay
Commercial HBA kits were purchased from Biocoat (Fort Washington, PA, USA), and the HBA test was performed following the manufacturer’s instructions. Briefly, 10 µl of semen (well mixed) was added to the centre of the HBA chamber and the Cell-Vu grid cover slip was put on without entrapping air bubbles. The cover slip provided a grid of 100 squares (each 0.1 mm x 0.1 mm) within a viewing circle. After incubation of the slide for 15 min, the unbound motile sperm and the bound motile sperm were counted in the same grid squares. For the HBA test, 400 motile sperm were counted. The percentage of hyaluronan-binding sperm was calculated using the bound motile sperm divided by the sum of bound and unbound motile sperm counted in the same squares and then multiplied by 100.
When duplicated HBA tests were performed on the same sperm samples from 30 individual men, similar results were obtained (80 ± 10 versus 80 ± 10%, not significant). The mean difference between the two results was 1.6% with an SD of 0.97. Spearman’s test also showed a highly significant correlation between the two results (n = 30, Spearman r = 0.929, P < 0.001). Thus, the HBA was highly reproducible within the same sample.
Conventional IVF procedures
For all the patients, a standard long luteal down-regulation protocol was used with GnRH agonist (Decapetyl 0.1 mg/Amp, Ferring) to achieve down-regulation (E2 < 50 pg/ml, LH < 5 mIU/ml, endometrial thickness 
5 mm and arrested follicular development), then gonadotrophin 150–450 IU/day r-FSH (Gonal-F 75 IU/Amp, Serono, Geneva, Switzerland) was followed. When patients had a leading follicle reach an average diameter of 18 mm plus two or more additional follicles with a diameter of 17 mm, they were given 10 000 IU u-HCG (Profasi, Serono, Geneva, Switzerland). Ultrasound-guided transvaginal follicular aspiration was performed 36 h after HCG injection.
Oocytes (four to five) were inseminated with 1–1.5 x 105/ml motile sperm selected by PureSperm (layers of 40 and 80%) in 0.6 ml Quinn’s advantage fertilization medium (Irvine Scientific, Irvine, CA, USA). Fertilization was checked at about 16–18 h after insemination, and normal fertilization was defined as two pronuclei (PN) which was used for all the statistical analyses. All oocytes with either one or three PN were excluded from the analysis. The embryo transfer (two to three embryos) was performed on day 3 of oocyte retrieval. In our IVF program, the current implantation rate per embryo transfer averages 28%.
Statistical analysis
The significance of correlations between HBA scores and semen analysis results and fertilization rates was examined by the Spearman (non-parametric) test. Multiple regression analysis was also used to determine which sperm characteristics were the most significantly related to the HBA scores. The difference of fertilization rate for various normal sperm morphology groups was examined by the Wilcoxon rank sum test. All the data were examined by the multivariate logistic regression analysis to determine which sperm test results were most independently related to fertilization rate in vitro.
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Results |
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All the sperm test results are summarized in Table I, and there was a wide range for all the sperm test results including the HBA. In 175 IVF patients, an average of nine mature oocytes were inseminated, and normal fertilization rate was 69% (range 0–100%). There were 21% (37 of 175) of the patients with fertilization rate
50%. While there was a statistical significance in HBA scores between patients with fertilization rate 50% (n = 37) and >50% (n = 138), the difference was very small (69 ± 12 versus 75 ± 10%, P < 0.05). In 37 patients with fertilization 50%, only 16% (six of 37) of them had low HBA scores (<60%) as recommended by the manufacturer and 84% (31 of 37) of them had normal HBA scores (>60%). Furthermore, five patients with zero fertilization rate had HBA scores 28, 56, 75, 75 and 79%, respectively, and again only two of them had HBA scores below 60%.
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There was a highly significant correlation between the HBA scores and both total and progressive sperm motility and normal sperm morphology (Figure 1). By using Spearman’s test, only sperm count, morphology (Figure 2) and HBA (Figure 3) were significantly correlated with fertilization rates (Table II). However, when all the data were analysed by multivariate logistic regression, only normal sperm morphology and HBA were independently significantly related to fertilization rate (Table III). Normal sperm morphology was more significant than the HBA in both Spearman’s test and logistic regression model (Tables II and III).
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In this study, female age was not significantly related to fertilization rates in both the Spearman test and the logistic regression analysis.
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Discussion |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAcknowledgementsReferences |
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The present study showed that HBA was significantly correlated with fertilization rate in conventional IVF. However, in comparison with strict sperm morphology, HBA was still less significant than sperm morphology in relation to fertilization rates in both the Spearman test and the multivariate logistic regression analysis. Patients with
5% normal sperm morphology had very low (average 41%) fertilization rate compared to those with normal sperm morphology >5% (average fertilization rate 72%, Figure 2). In contrast, the HBA did not clearly distinguish between patients with a low and high fertilization rate (Figure 3). Patients with fertilization rate 50% only had marginally lower HBA than those with fertilization rate >50% (69 ± 12 versus 75 ± 10%, P < 0.05). Similarly, 16 of 175 patients with HBA < 60% had an average fertilization rate of 60%. Therefore, HBA does not distinguish between patients with low and high fertilization rates in conventional IVF; thus it is unlikely to be a useful clinical value in addition to the routine semen analysis for diagnosis and management of male infertility. In this study, we found that HBA was highly correlated with the total and progressive sperm motility and normal sperm morphology, suggesting that HBA is likely to reflect the semen quality revealed by the routine semen analysis. Thus, HBA does not provide additional information about sperm-fertilizing ability over the routine semen analysis. Recently, the HBA test has been becoming a popular routine test for sperm maturity or function in many ART clinics in south Asian countries such as Singapore and China. Indeed, the HBA is very simple and relatively reproducible. The cost of each HBA slide (for two samples) is about 50–60 US dollars, which is equivalent to about two weeks’ salary of an ordinary person in China. However, data obtained from this study do not support the HBA as a useful routine clinical test for prediction of sperm-fertilizing ability in vitro. Most importantly, HBA is not superior to the routine semen analysis for predicting sperm-fertilizing ability.
The HBA is designed based on the sperm surface containing a receptor of HA or hyaluronidase, which binds to HA coated on a glass slide. In the past, HA in solution has been used as a sperm preparation medium, known as the Sperm Select (Pharmacia AB, Uppsala, Sweden, Huszar et al., 1990). It was reported that the HA could significantly improve the retention of sperm motility and velocity, possibly due to the effect of HA on sperm metabolism rather than protection of sperm membrane integrity (Huszar et al., 1990). A similar effect of HA on retention of motility of crypreserved/thawed human spermatozoa was also subsequently observed (Sbracia et al., 1997). Huszar et al. (2003) showed that HA-bound human spermatozoa had intact acrosomes, mature nuclei and better morphology. Our present data showed clearly that HBA was highly significantly correlated with sperm motility and normal sperm morphology, suggesting that motile sperm with normal morphology are superior binding to HA. In these aspects, HA has some similarities to the human zona pellucida that both can selectively bind to sperm with mature nuclei, intact acrosomes and normal morphology (Liu and Baker, 1992b; Huszar et al. l994). It was also reported that pig sperm bound to HA had low frequency of aneuploidy (Park et al., 2005). It is possible that HA-bound sperm may have better sperm quality, which is worthy of further investigation.
In this study, we further confirm that the strict sperm morphology was highly related to fertilization rates in IVF. Thus, sperm morphology is one of most useful semen analysis results for clinical diagnosis and management of patients (Liu and Baker, 1992a; Ombelet et al., 1995; Menkveld et al., 1996; Garrett et al., 1997). The results of this study in unselected patients further indicated that couples with 5% normal sperm morphology had a low fertilization rate (average 40%, Figure 2B) in IVF, and therefore, these patients should be directly assigned to treatment with ICSI to avoid low fertilization in IVF. Patients with such severe teratozoospermic semen also have high frequencies of defects of sperm binding, the zona pellucida-induced AR and sperm–zona pellucida penetration, which are major causes of failure of fertilization with conventional IVF (Liu and Baker, 2003; Liu et al., 2004).
In summary, HBA is highly correlated with sperm motility and strict normal sperm morphology, suggesting that HBA reflects the semen quality indicated by routine semen analysis. Although a statistically significant difference between HBA and fertilization rate in conventional IVF was observed, no clear threshold value can be used for prediction of sperm-fertilizing ability. Therefore, the clinical value of HBA for diagnosis and management of male infertility is limited.
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Acknowledgements |
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The authors thank Liu Hong, Wen-hong Gui and Deng-hua Li from Genetics and Reproductive Institute, Chongqing Obstetrics and Gynecology Hospital, Chongqing, China, for performing IVF procedures and Mingli Liu from Department of Obstetrics and Gynecology, University of Melbourne, Melbourne, Australia for assessment of sperm morphology.
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Submitted on October 13, 2005; resubmitted on December 19, 2005; accepted on January 5, 2006.
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