Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients

Reproductive BioMedicine Online (2012) 25, 620– 626

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ARTICLE

Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients ´nszki a, Zsuzsanna Molna ´r b, Aniko ´ Ujfalusi a, Erzse ´bet Balogh a, Attila Moka ´va Ola ´h a,* ´ne ´ b, Attila Varga c, Attila Jakab b, E Zsuzsa Kassai Bazsa a Clinical Genetic Center, Department of Pediatrics, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; b Department of Obstetrics and Gynecology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; c Department of Urology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary

* Corresponding author. E-mail address: [email protected] (E ´. Ola ´h). Attila Mokanszki is a third-year PhD student at the Clinical Genetic Center, Department of Pediatrics, Medical and Health Science Center, University of Debrecen, Hungary. His main research interest is in the field of human infertility, especially genetic causes associated with male infertility and recurrent spontaneous abortion.

Abstract Infertile men with low sperm concentration and/or less motile spermatozoa have an increased risk of producing aneuploid

spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding may reduce genetic risks such as chromosomal rearrangements and numerical aberrations. Fluorescence in-situ hybridization (FISH) has been used to evaluate the presence of aneuploidies. This study examined spermatozoa of 10 oligozoospermic, 9 asthenozoospermic, 9 oligoasthenozoospermic and 17 normozoospermic men by HA binding and FISH. Mean percentage of HA-bound spermatozoa in the normozoospermic group was 81%, which was significantly higher than in the oligozoospermic (P < 0.001), asthenozoospermic (P < 0.001) and oligoasthenozoospermic (P < 0.001) groups. Disomy of sex chromosomes (P = 0.014) and chromosome 17 (P = 0.0019), diploidy (P = 0.03) and estimated numerical chromosome aberrations (P = 0.004) were significantly higher in the oligoasthenozoospermic group compared with the other groups. There were statistically significant relationships (P < 0.001) between sperm concentration and HA binding (r = 0.658), between sperm concentration and estimated numerical chromosome aberrations (r = –0.668) and between HA binding and estimated numerical chromosome aberrations (r = –0.682). HA binding and aneuploidy studies of spermatozoa in individual cases allow prediction of reproductive prognosis and provision of appropriate genetic counselling. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: hyaluronic acid-binding capacity, numerical chromosome aberrations, sperm aneuploidy, sperm concentration, sperm FISH

1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.08.003

Correlation between sperm concentration, hyaluronic acid binding and aneuploidy

Introduction The proportion of immature spermatozoa and frequency of chromosomal disomies are closely related to each other, suggesting that disomies originate primarily in immature spermatozoa (Kovanci et al., 2001). The proportion of spermatozoa with arrested development is variable from man to man. In general, this proportion declines as the sperm concentration rises (Celik-Ozenci et al., 2004; Kovanci et al., 2001). Infertile men with normal karyotypes and low sperm concentrations or higher concentrations of morphologically abnormal spermatozoa have significantly increased risks of producing aneuploid spermatozoa, particularly for sex chromosomes (Shi and Martin, 2001). Aneuploid and diploid spermatozoa may occur with abnormal or normal shapes, but aberrant nuclei can be found with a higher frequency in spermatozoa with abnormal shapes (Celik-Ozenci et al., 2003). Simultaneously with cytoplasmic extrusion in spermiogenesis, there is a remodelling of the plasma membrane that facilitates the formation of the zona pellucida- and hyaluronic acid (HA)-binding sites (Huszar et al., 1997, 2003). The HA binding associated with the presence of the HA receptors on the sperm surface is related to sperm development (Huszar and Vigue, 1993). Spermatozoa with HA-binding ability are viable, having either intact or slightly capacitated acrosomal status, and HA-selected spermatozoa are devoid of DNA degradation (Huszar et al., 2007; Yagci et al., 2010). Acrosome-reacted and non-viable spermatozoa have lost their HA-binding capacity (Huszar et al., 2003). Diminished expression of HSPA2, a testis-specific chaperone protein which is part of the meiotic synaptonemal complex, causes meiotic defects such as aneuploidies (Kovanci et al., 2001). There is a relationship between diminished sperm development (associated with oligozoo/asthenozoo/teratozoospermia), low levels of HSPA2 expression, increased frequency of chromosomal aneuploidies, presence of apoptosis and fragmented DNA (Huszar and Vigue, 1993; Huszar et al., 2000, 2003, 2007; Yagci et al., 2010). Solid-state HA binding facilitates the selection of individual mature spermatozoa with low levels of chromosomal aneuploidies (Jakab et al., 2005). The inception of intracytoplasmic sperm injection (ICSI) provided a new approach in the treatment of infertile men with very low motile sperm concentrations. Spermatozoa selected for ICSI may have fragmented DNA or chromosomal impairments (Celik-Ozenci et al., 2004; Huszar et al., 2007). The issue of diminished sperm maturity has a major impact on sperm selection for ICSI, because severely oligozoo/asthenozoo/teratozoospermic men have an increased proportion of immature spermatozoa showing higher incidence of chromosomal aneuploidy compared with normospermic fertile men (Bernardini et al., 1997, 1998; In‘t Veld et al., 1997; Storeng et al., 1998; Templado et al., 2002). The HA sperm selection method for ICSI may reduce the potential genetic complications and adverse public health effects of ICSI (Jakab et al., 2005). The present study examined sperm samples of 45 different men – oligozoospermic (n = 10), asthenozoospermic (n = 9), oligoasthenozoospermic (n = 9) and normozoospermic (n = 17) – by HA-binding assay and fluorescence in-situ

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hybridization (FISH). Sperm samples were analysed by FISH to evaluate aneuploidy frequencies of chromosomes X, Y and 17. This study also determined the differences in HA-binding capacity and chromosome aberrations between groups as well as the correlation between the sperm concentration, HA-binding capacity and estimated numerical chromosome aberrations.

Materials and methods Patients Semen samples of 45 men referred for semen analysis to the Andrology Laboratory of the Department of Obstetrics and Gynecology and the Andrology Laboratory of the Department of Urology, Medical and Health Science Center, University of Debrecen were studied. Semen specimen was collected after a requested abstinence of 2–4 days. Semen analysis was performed manually according to WHO guidelines and morphology was examined using strict criteria (WHO, 2010). According to the sperm concentration and motility, patients were divided into four groups: oligozoospermic, asthenozoospermic, oligoasthenozoospermic and normozoospermic. Two sperm samples per patient taken at different times were evaluated in order to classify the patients. The oligozoospermic group consisted of 10 men with infertility (age range 31–42 years; sperm concentration 15 million/ml (range 1–15 million/ml); progressive motility >32% (range 35–80%). The asthenozoospermic group consisted of nine men with subfertility (age range 31–39 years; sperm concentration >15 million/ml (range 18–58 million/ml); progressive motility <32% (range 5–30%). The oligoasthenozoospermic group consisted of nine men (age range 27–37 years; sperm concentration <15 million/ml (range 4–12 million/ml); progressive motility 32% (range 0–32%). The control group (normozoospermic men) consisted of 17 clinically healthy sperm donors with normal sperm parameters (age range 24–42 years; sperm concentration range 40–160 million/ml; progressive motility range 40–85%). Prior to the study, all patients were given detailed information about the aim and method of investigation and their consents were obtained. All protocols must have been approved by the author‘s respective Institutional Review Board for human subjects (IRB reference number 2976/2012-EHR, granted 1 February 2012).

Sperm HA-binding assay The sperm sample maintained at room temperature (18–28C) for 30–60 min to allow it to liquefy. The HA-binding assay (MidAtlantic Diagnostics, Martlon, NJ, USA) was carried out at room temperature. Each sample was mixed and 7–10 ll was pipetted near the centre of the chamber. The CELL-VU-gridded cover slip was located over the chamber, taking care to avoid air bubble formation. The chamber was incubated at room temperature for at least 10 min, but not more than 20 min: this period proved to be necessary for spermatozoa to bind to HA (according to the test protocol). The number of bound, motile spermatozoa and total, motile spermatozoa was scored. The ratio of HA-binding motile

622 spermatozoa was calculated as follows: %Bound = 100 · bound motile/total motile. The normal distribution of the test was >80%.

FISH analysis on spermatozoa Smears of semen samples (10 ll) were fixed with methanol/ acetic acid (3:1) for 10 min, air dried, dehydrated in a series of 70, 85 and 100% ethanol for 2 min each and stored at 20C until FISH was performed. For decondensation, the sperm slides were warmed to room temperature, and in order to render the sperm chromatin accessible to DNA probes they were treated first with 10 mmol/l dithiothreitol (Sigma, St. Louis, MO, USA) in 0.1 mol/l Tris-HCl (pH 8.0) for 30 min, then with 10 mmol/l lithium diidosalicylate (Sigma) in Tris-HCl for 1–3 h. After decondensation, the sperm slides were dehydrated in a series of 70, 85 and 100% ethanol. FISH was performed using three probes: alpha-satellite sequence-specific centromeric probes for chromosome 17 (D17Z1, SpectrumAqua), X (DXZ1, SpectrumOrange) and Y (DYZ1, SpectrumGreen) (Abbott/Vysis, Des Plaines, IL, USA). Sample and probe co-denaturation was carried out at 76C for 3 min. The hybridization was carried out at 37C in a moist chamber for 16–18 h (Hybrite; Abbott/Vysis). Post-hybridization washes were performed with 50% formamide/2·SSC at 42C for 15 min. The slides were then washed with 2·SSC at room temperature for 10 min and 2·SSC/0.1% NP-40 for 5 min. After washing, the nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Abbott/Vysis).

FISH scoring criteria The sperm FISH experiment was carried out by scoring at least 5000 sperm heads. The overall hybridization efficiency was >98%. Nuclei that were overlapped or displayed no signal due to hybridization failure were omitted from the scoring. A spermatozoon was considered disomic when it showed two fluorescence signals of the same colour, comparable in size and brightness of the sperm head and at least one signal apart. Scoring was performed using Zeiss Axioplan2 (Carl Zeiss, Jena, Germany) fluorescence microscope and the images were captured and analysed by ISIS software (Metasystems, Althussheim, Germany). The sperm disomy frequencies and estimated numerical chromosome aberrations were calculated according to earlier studies (Downie et al., 1997; Egozcue et al., 1997).

Statistical analysis Statistical analyses were performed with the commercial software SigmaStat and SPSS. The sample normality were analysed using the Shapiro–Wilk test and the sample homogenity using the Barlett test. Differencies in the sperm concentration, HA-binding ability, disomy frequencies, diploidy frequencies and estimated numerical chromosome aberrations in the different groups of patients were analysed using Mann–Whitney/Wilcoxon two-sample test, Kruskal–Wallis test (when normality does not exist) and two-sample t-probe (when normality is exist). A value of P < 0.05 was considered as indicating a significant

A Moka ´nszki et al. difference. Correlation analysis between the sperm concentration, HA-binding capacity and sperm estimated numerical chromosome aberrations using all samples in the four group were examined with the Pearson correlation test. The strength of the linear relationship between each pair of variables, having corrected for other variables were studied with partial correlation analysis.

Results Sperm HA-binding assay The mean sperm HA-bound capacity of the motile spermatozoa was 81% (range 58–95%) in the normozoospermic group, 53% (range 10–68%) in the oligozoospermic group, 37% (range 0–83%) in the asthenozoospermic group and 30% (range 2–55%) in the oligoasthenozoospermic group. The HA-binding capacity of the normozoospermic men proved to be significantly higher than the oligozoospermic (P < 0.001), the asthenozoospermic (P < 0.001) and the oligoasthenozoospermic (P < 0.001) men.

Investigation of chromosome aberrations by sperm FISH The results of the FISH analysis are summarized in Table 1 and the results of the statistical analysis are summarized in Table 2. The X/Y ratios were close to 1:1 in all groups. The sex chromosome disomy frequencies were significantly higher in the oligoasthenozoospermic group compared with the normozoospermic (P = 0.001), oligozoospermic (P = 0.014) and asthenozoospermic (P = 0.004) groups. There was also a significantly higher frequency of disomy 17 in the oligoasthenozoospermic group compared with normozoospermic (P = 0.0001), oligozoospermic (P = 0.0019) and asthenozoospermic (P = 0.0011) patients. The total diploidy frequency of oligoasthenozoospermic patients was significantly higher than the three other groups (normozoospermic controls P < 0.0001; oligozoospermic patients P = 0.03; asthenozoospermic patients P = 0.001). A significant difference was observed in the frequency of estimated numerical chromosome aberrations between oligoasthenozoospermic and normozoospermic (P < 0.001), oligozoospermic (P = 0.004) and asthenozoospermic (P = 0.001) groups. Thus, disomy of sex chromosomes and chromosome 17, diploidy and estimated numerical chromosome aberration frequencies were significantly higher in the oligoasthenozoospermic group compared with the three other groups. Compared with the normozoospermic group, significantly higher disomy 17 (P = 0.0019), diploidy (P = 0.001) and estimated numerical chromosome aberrations (P = 0.002) were found in the asthenozoospermic group, and significantly higher disomy 17 was found in oligozoospermic men (P = 0.0387).

Correlation analysis between sperm concentration, HA-binding capacity and estimated numerical chromosome aberrations This study calculated the Pearson correlation (r) and the partial correlation (pr) between sperm concentration,

Correlation between sperm concentration, hyaluronic acid binding and aneuploidy

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Table 1 Frequencies of disomy and diploidy and estimated chromosome aberrations detected by FISH with probes for chromosomes X, Y and 17. Chromosome aberration X/Y ratio Sex chromosome disomy XY XX YY 17,17 disomy Sex chromosome diploidy XY XX YY Estimated numerical chromosome aberrations

Normozoospermiaa

Oligozoospermiab

Asthenozoospermiac

Oligoasthenozoospermiad

1.07 0.33

1.07 0.38

1.08 0.37

1.08 0.48

0.15 0.10 0.08 0.07 0.18

0.17 0.12 0.09 0.09 0.26

0.17 0.12 0.08 0.10 0.27

0.21 0.15 0.12 0.14 0.40

0.08 0.06 0.04 3.99

0.13 0.08 0.05 5.24

0.12 0.09 0.06 5.45

0.18 0.13 0.09 7.42

Values are mean percentages. a Seventeen patients, 89,452 sperm heads.bTen patients, 48,461 sperm heads.cNine patients, 45,860 sperm heads. d Nine patients, 43,666 sperm heads.

Table 2 Statistical analysis (P values) of the differences between oligozoospermic, asthenozoospermic, oligoasthenozoospermic and normozoospermic groups.

Normozoospermia Sex chromosome disomy 17 chromosome disomy Diploidy Estimated numerical chromosome aberrations Oligozoospermia Sex chromosome disomy 17 chromosome disomy Diploidy Estimated numerical chromosome aberrations Asthenozoospermia Sex chromosome disomy 17 chromosome disomy Diploidy Estimated numerical chromosome aberrations

Oligozoospermia

Asthenozoospermia

Oligoasthenozoospermia

NS 0.0387 NS NS

NS 0.0019 0.001 0.002

0.001 0.0001 <0.0001 <0.001

NS NS NS NS

0.014 0.0019 0.030 0.004

HA-binding capacity and estimated numerical chromosome aberrations comparing all samples in the four groups. A statistically significant positive correlation was found between sperm concentration and HA-binding capacity (Figure 1A, r = 0.658, P < 0.001) and statistically significant negative correlations were observed between sperm concentration and estimated numerical chromosome aberrations (Figure 1B, r = –0.668, P < 0.001) and between HA-binding

0.004 0.0011 0.001 0.001

ability and estimated numerical chromosome aberrations (Figure 1C, r = 0.682, P < 0.001). Using partial correlation analysis, the strength of the linear relationship between each pair of variables was statistically significant (pr = 0.372, P = 0.013 between the sperm concentration and the HA-binding capacity; pr = –0.399, P = 0.007 between the sperm concentration and the estimated numerical chromosome aberrations; pr = 0.432, P = 0.003

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Figure 1

A Moka ´nszki et al.

Correlations between sperm concentration, HA-binding capacity and estimated numerical chromosome aberrations.

between the HA-binding ability and the estimated numerical chromosome aberrations).

Discussion The present study found significantly higher disomy and diploidy of sex chromosomes, chromosome 17 disomy and estimated numerical chromosome aberration frequencies in infertile patients with abnormal semen compared with normozoospermic patients. These findings are supported by other studies, in which it has been well documented that severely oligospermic men, who are candidates for ICSI, have higher rates of aneuploidies than normospermic men (Calogero et al., 2001; Pang et al., 1999; Rives et al., 1999; Zeyneloglu et al., 2000). Infertile men with normal karyotypes and low sperm concentrations or higher concentration of morphologically abnormal spermatozoa have significantly increased risk of producing aneuploid spermatozoa, particularly for sex chromosomes (Colombero et al., 1999; In‘t Veld et al., 1997; Vegetti et al., 2000). Disomy frequencies in infertile males have been found to directly correlate with the severity of oligozoospermia (Rives et al., 1999). There is a close correlation between the proportion of immature spermatozoa and chromosomal disomies (Kovanci et al., 2001). This relationship between the frequencies of chromosomal aneuploidies and diminished sperm maturity is probably based on the finding that cytoplasmic retention and diminished maturity in spermatozoa are associated with a low expression of the HSPA2 gene (Eddy, 1999; Huszar et al., 2000). As it was shown in the mouse, HSP702, a homologue of the HSPA2 chaperone protein, is a component of the synaptonemal complex and also facilitates the intracellular movement of proteins (Dix et al., 1996; Eddy,1999). This association may explain the relationship between meiotic errors, sperm aneuploidies and diminished maturity (Jakab et al., 2003). Aneuploidies are primarily found in immature spermatozoa with cytoplasmic retention and diminished HSPA2 concentrations (Kovanci et al., 2001). In general, a higher proportion of undeveloped spermatozoa can be found in oligozoospermic samples (Huszar and Vigue, 1993; Huszar et al., 2000). The relationship between sperm zona pellucida-binding competence and maturity has been identified earlier. In the semen samples, there were spermatozoa with various

degrees of cytoplasmic retention, but all spermatozoa bound to the zona pellucida were mature as characterized with the absence of any cytoplasmic retention. Immature spermatozoa with retained cytoplasm and low expression of HSPA2 are deficient in the zona-binding site (Huszar et al., 1996). Mature spermatozoa selectively attach and remain bound to HA, similarly to the zona pellucida. The HA-receptor, similar to the sperm zona pellucida-binding site, is developmentally regulated and is not present in immature spermatozoa as identified by cytoplasmic retention and low HSPA2 expression (Huszar et al., 2003). According to the earlier studies, the mature and motile spermatozoa show >80% HA-binding capacity (Huszar et al., 2007; Yagci et al., 2010; Kovacs et al., 2011). In the present study, the mean percentage of HA-bound spermatozoa, in accordance with the normal distribution, in the normozoospermic group was 81%. The HA-binding capacity of this group was significantly higher than the oligozoospermic, asthenozoospermic and oligoasthenozoospermic groups (P < 0.001). Relationships between synaptic anomalies during meiosis, chromosomal abnormalities and male infertility have already been recognized (Colombero et al., 1999; Egozcue et al., 1983; Rives et al., 1999; Vendrell et al., 1999). Other authors, however, did not find any relationship between sperm concentrations and frequencies of aneuploidies, but the study population and the scoring was significantly different (Jakab et al., 2003). The present approach found a relationship between sperm HA binding and seminal sperm concentration and frequency of chromosome aberrations. After ICSI fertilization, there are increased rates of de-novo numerical and cytogenetically detectable structural chromosomal aberrations (Van Steirteghem et al., 2002) as well. In cases of ICSI, there is a significantly higher incidence of spontaneous abortions, about 18%, compared with 10% of normal conceptions (Simpson and Lamb, 2001). Numerical chromosomal aberrations in spermatozoa of oligozoospermic or severely oligozoospermic candidates for ICSI are most likely due to the low concentration of HSPA2 in the undeveloped spermatozoa (Huszar et al., 2007). Males participating in ICSI treatment show a higher rate of structural chromosome rearrangements, such as reciprocal and Robertsonian translocations as well. These rearrangements through interchromosomal effects may result in

Correlation between sperm concentration, hyaluronic acid binding and aneuploidy disomies and diploidies (Martini et al., 1999; Morel et al., 2004; Perrin et al., 2010). Another male-related adverse consequence of ICSI is the transmission of Y-chromosome deletions (Simpson and Lamb, 2001). HA sperm selection for ICSI may reduce the potential genetic complications in infertile men (Jakab et al., 2005). HA binding did not improve spontaneous fertilization in patients with unexplained infertility undergoing IVF. According to Kovacs et al. (2011), HA binding used for screening did not help to select the method of fertilization (Kovacs et al., 2011). Injection of HA-bound spermatozoa significantly improves embryo quality and development, but does not improve the fertilization rate in significant way. However, it is difficult to analyse the contribution of the spermatozoa in fertilization when ICSI is performed on a limited number of oocytes or the reduction of miscarriage rate with HA-selected spermatozoa when there is a legal obligation to transfer all available embryos (Parmegiani et al., 2010). FISH studies on spermatozoa before ICSI should also be useful in detection of aneuploidies. The percentage of chromosomally abnormal spermatozoa estimated by FISH may have a predictive value on the outcome of ICSI. A HAbinding assay combined with ICSI may reduce the incidence of genetic abnormalities.

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