RBMOnline - Vol 18. No 4. 2009 486-495 Reproductive BioMedicine Online; www.rbmonline.com/Article/3737 on web 26 February 2009

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© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

Nicoletta Tarozzi obtained her PhD in 2004 at the University of Modena, Italy, working in the Genetics Laboratory of the Animal Biology Department. In 2004 she joined the team of Tecnobios Procreazione, Centre for Reproductive Health in Bologna. Dr Tarozzi has published several papers in the field of sperm DNA damage. Current research interests focus on male infertility, especially regarding molecular biology of the male germ cell and structure of sperm chromatin.

Dr Nicoletta Tarozzi

Nicoletta Tarozzi1,3, Marco Nadalini1, Alessandra Stronati2, Davide Bizzaro2, Luca Dal Prato1, Giovanni Coticchio1, Andrea Borini11Tecnobios Procreazione, Centre for Reproductive Health, Via Dante 15, I-40125 Bologna, Italy; 2Institute of Biology and Genetic, University Polytechnic of Marche, Via Brecce Bianche, I-60131 Ancona, Italy3Correspondence: e-mail: tarozzi@tecnobiosprocreazione.it

Abstract

Sperm protamine deficiency and DNA damage were analysed employing chromomicyn A3 (CMA3) staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, respectively, in 132 patients (82 IVF, 50 intracytoplasmic sperm injection [ICSI]). The antioxidant ability of seminal plasma was analysed in 10 men, using the total oxidant scavenging capacity assay. A significant negative correlation was found between abnormal protamination and sperm parameters, including sperm DNA fragmentation (P < 0.01). A close relationship was found between sperm protamination and fertilization and pregnancy only in IVF (P = 0.004 and P < 0.04, respectively); in ICSI there was a correlation between DNA fragmentation and pregnancy (P = 0.031). Finally, there was a negative correlation between chromatin under-protamination and the antioxidant ability of seminal plasma (P < 0.01). Results of this study underline that, despite sperm abnormal protamination and DNA fragmentation being positively correlated, they affect the reproductive outcome in different ways: in particular there was good prognostic value for CMA3 analysis only in IVF, whereas DNA fragmentation analysis was prognostic only for ICSI outcome. Data are also provided to support the idea of a relationship between defective antioxidant system activity and impairment of chromatin packaging.

Keywords: antioxidant ability, CMA3 staining, DNA fragmentation, human spermatozoa, treatment outcome

During spermiogenesis the chromatin of spermatozoa undergoes a dramatic structural change (reviewed by Dadoune, 1995). At this stage, one of the most important events is the replacement of histones by transition proteins and then protamines. In the human sperm nucleus ∼85% of the histones are replaced by protamines, which are about half of the molecular weight of histones and at least 50% more basic: these proteins have a high content of positively charged amino acids, particularly arginine; they are also rich in cysteine residues, which allow the formation of disulphide cross-links between adjacent protamine molecules and inside the proteins (inter- and intra-molecular bonds), conferring a highly stable structure to chromatin (Manicardi et al., 1995). The mechanism by which protamines interact with DNA is still under

debate: they may bind to the major groove of the DNA (Pogany et al., 1981), link both major and minor grooves (D’Auria et al., 1993), or electrostatically bind to the DNA by interacting with phosphate residues (Bianchi et al., 1994). In any case, the net positive charge of protamines allows the neutralization of the DNA negative charge, conferring an extremely high level of compactness to the sperm nucleus: sperm DNA is coiled into a toroidal or doughnut-shaped structure, each containing roughly 50 kb of DNA (Hud et al., 1993, 1994), and is sixfold more highly condensed than in mitotic chromosomes (Pogany et al., 1981; Ward and Coffey, 1991).

The unique sperm chromatin organization is important for several

Article

Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques

Introduction

reasons (reviewed by Oliva, 2006): this packaging not only allows transfer of a very tightly packed genetic information to the oocyte, but also results in protection of DNA from chemical and physical damage, making it inaccessible to reactive oxygen species (ROS) or to other factors in the internal or in the external medium (Barone et al., 1994; Braun, 2001). In fact, one of the potential consequences of abnormal protamination is the greater susceptibility to DNA damage (Carrell et al., 2007), as stressed by recent observations linking defects in protamination with sperm DNA fragmentation (Aoki et al., 2005b; Nasr-Esfahani et al., 2005; Aoki et al., 2006; Torregrosa et al., 2006). This fact is of particular importance considering that both protamine deficiency and sperm DNA damage are related to decreased reproductive capacity of men, in natural as well as in assisted reproduction (reviewed by Oliva, 2006; and Tarozzi et al., 2007).

In the present paper, there has been an attempt to evaluate the clinical relevance of sperm chromatin alteration, employing chromomycin A3 (CMA3) as the procedure of choice to identify a potential condition of underprotamination: CMA3 binds as a Mg2+-coordinated dimer at the minor groove of GC-rich DNA and induces a conformational perturbation in the DNA helix resulting in a wider and shallower minor groove at its binding site (Gao and Patel, 1990; Gao et al., 1992); this fluorochrome thus competes with protamines in its binding to DNA, acting as an indirect measure of underprotamination (Ramos et al., 2004). In particular, the link between abnormal protamination and reproductive outcome in IVF and intracytoplasmic sperm injection (ICSI) patients was analysed, in an attempt to identify a cut-off value of CMA3 positivity for prediction of assisted reproduction treatment outcome. There was also an investigation of the relationship between abnormal protamination and sperm DNA fragmentation, evaluated employing the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay in order to understand whether these two conditions overlap or represent different aspects of sperm chromatin alteration, and whether they affect the reproductive outcome in distinct manners. Furthermore, considering that spermatozoa with incomplete chromatin protamination might be much more susceptible to free radicals, a possible link between chromatin packaging alteration and the antioxidant ability of seminal plasma was also examined; the latter was evaluated using the total oxidant scavenging capacity (TOSC) assay, a new technique recently developed and applied in andrology (Balercia et al., 2003, 2005).

Materials and methods

Patients

The study was carried out at Tecnobios Procreazione, Centre for Reproductive Health, in Bologna, Italy. A total of 132 assisted reproduction treatment cycles were included: 50 intracytoplasmic sperm injection (ICSI) and 82 IVF cycles.

The mean age of the women included in the study was 37.05 ± 4.19 years and the mean body mass index (BMI) was 22.17 ± 3.13. The mean age of the men was 40.24 ± 5.17 years. Only men with ejaculated spermatozoa were included in the study.

The indications for ICSI treatment were sperm concentration

<10 × 106/ml and/or sperm motility <30% and/or normal sperm morphology <4%; this was associated with oligo-ovulation in one couple (2%), endometriosis in five couples (10%) and tubal defects in 10 couples (20%); in 27 couples, the aetiology of infertility was male factor only (54%) and it was unexplained in seven couples (14%). In IVF patients the aetiology of infertility was oligo-ovulation in two couples (2.4%), endometriosis in 14 couples (17.1%), unexplained in 17 couples (20.7%), tubal defects in 39 couples (47.6%) and male factor in 10 couples (12.2%).

Sperm collection and preparation

Sperm samples from 132 patients were collected by masturbation on the day of oocyte retrieval. The samples were analysed following World Health Organization (1999) indications for sperm concentration, motility and morphology, and were prepared using a discontinuous PureSperm gradient (Nidacon, Gothemberg, Sweden). Briefly, spermatozoa were layered upon a 40:80% PureSperm density gradient, processed by centrifugation at 600 g for 15 min and resuspended in 1 ml sperm culture medium (PureSperm wash; Nidacon). Immediately a second evaluation of concentration, motility and morphology was performed. Part of the sperm suspension obtained with density gradient separation was used for oocyte insemination, while the other part of semen suspension was washed in phosphate-buffered saline (PBS; Sigma-Aldrich, Milan, Italy) and smeared on two microscope slides per patient, for CMA3 staining and TUNEL assay.

Ten donors with seminal parameters comparable to those of IVF patients (sperm concentration >10 × 106/ml, sperm motility >30% and normal sperm morphology >4%) were selected and the sperm samples were evaluated using the TOSC assay and CMA3 staining.

Ovarian stimulation, IVF, ICSI and embryo development

The ovarian stimulation protocol, oocyte retrieval, insemination and embryo transfer were carried out as previously described (Borini et al., 2004a,b). In brief, ovarian stimulation was achieved by recombinant FSH (Gonal F, Serono, Rome, Italy; Puregon, Organon, Rome, Italy) and monitored by endovaginal echography and plasma oestradiol. Thirty-six hours before oocyte retrieval 10,000 IU human chorionic gonadotrophin (Gonasi; Amsa, Rome, Italy) was administered. Oocyte retrieval was carried out under general anaesthesia by vaginal ultrasonographic-guided aspiration. At 16–18 h after microinjection or insemination, oocytes were assessed for the presence of two pronuclei. Forty-eight hours after oocyte retrieval, embryos were classified according to morphology and then transferred into the uterus. Clinical pregnancy was assessed by ultrasound detection of gestational sac with visualisation of fetal heartbeat. Miscarriage was defined as pregnancy loss after ultrasound confirmation of embryo implantation and before 12 weeks’ gestation.

CMA3 staining

CMA3 staining was performed on sperm suspension after density gradient separation using the technique first described 487

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Article - Anomalies in sperm chromatin packaging - N Tarozzi et al.

by Bianchi et al. (1993). Briefly, sperm samples were smeared on slides, air-dried, fixed in Carnoy’s solution (methanol:glacial acetic acid, 3:1) (Fluka, Milan, Italy) for 10 min at 4°C and washed in PBS for 5 min. Each slide was treated for 20 min with 100 µl CMA3 solution (0.25 mg/ml in PBS, containing 10 mmol/l MgCl2). Slides were then rinsed in PBS and mounted with buffered glycerol (Fluka). The percentage of positive spermatozoa was determined by direct observation of 500 cells per sample with an epifluorescence microscope (Nikon eclipse 80i; Nikon, Florence, Italy).

DNA fragmentation analysis

Sperm DNA damage analysis was carried out employing the TUNEL assay, as previously described (Borini et al., 2006). Briefly, the TUNEL assay was performed on sperm suspensions after density gradient separation: smears were prepared and fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS for 30 min at 4°C and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (Sigma-Aldrich). Strand breaks in DNA were detected by the TUNEL technique using a commercially available kit (In situ Cell Death Detection Kit, Fluorescein; Roche, Monza, Italy), according to the manufacturer’s instructions. A negative control was performed for each sample by using fluorescein-isothiocyanate-labelled dUTP without enzyme. The percentage of spermatozoa with fragmented DNA was determined by direct observation of 500 spermatozoa per sample with an epifluorescence microscope (Nikon eclipse 80i).

TOSC assay

The TOSC assay is based on the reaction between various forms of ROS and the substrate α-keto-γ-methiolbutyric acid (KMBA) which is oxidized to ethylene. The antioxidant efficiency of a sample is quantified by its ability to scavenge the generated oxyradicals, thus inhibiting their reaction with KMBA and ethylene formation (Balercia et al., 2004).

Ten sperm samples were centrifuged after liquefaction at 600 g for 15 min, and the supernatant plasma was immediately separated from the pellet of spermatozoa and stored at −80°C until the assay. The technical procedures for TOSC assay towards peroxyl radicals were performed according to previous reports (Balercia et al., 2003). Briefly, peroxyl radicals were generated by thermal decomposition of 20 mmol/l ABAP (2,2’-azobis (2-methylpropionamide) dihydrochloride) at 35°C in 100 mmol/l potassium phosphate buffer, pH 7.4. Reactions with 0.2 mmol/l KMBA were carried out in 10 ml vials sealed with gas-tight Mininert valves (Supelco, Bellefonte, PA, USA) in a final volume of 1 ml. Ethylene production was measured by the gas chromatographic analysis of 0.2 ml aliquots taken from the headspace of vials at regular time intervals during the reaction. Total ethylene formation towards peroxyl radicals was quantified from the area under the kinetic curve; in particular, TOSC values were quantified using the equation: TOSC = 100−(SA/CA × 100), where SA and CA are the integrated areas calculated under the least square kinetic curve produced during the reaction course for sample (SA) and control (CA) reactions, respectively.

Statistical analysis

Statistical analysis was performed with Statistics Package for

Social Sciences (SPSS) for Windows software package version 10.1 (SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was used to determine whether the data were random samples from a normal distribution. Fisher’s exact test was used to compare clinical pregnancy rates and number of previous assisted reproduction treatments in different groups of patients. A t-test was applied to analyse maternal and paternal age, female BMI and sperm parameters in the same groups of patients. Spearman’s rank correlation, valid for data that are not normally distributed, was applied to establish the correlation between chromatin underprotamination (CMA3 positivity) and sperm parameters, between chromatin underprotamination and fertilization rate, and also between chromatin underprotamination and scavenging capacity of seminal plasma towards peroxyl radicals. Receiver operating characteristic (ROC) analysis was performed in IVF patients to identify a cut-off value of CMA3 positivity to discriminate between patients with poor or good fertilization prognosis. Statistical differences were considered significant at P < 0.05 and highly significant at P < 0.01.

Results

A total of 132 couples undergoing an assisted reproduction treatment cycle were included in this study: 50 ICSI and 82 IVF cycles. The data concerning assisted reproduction treatment procedures and the clinical outcome of patients are described in Table 1.

CMA3 positivity, sperm parameters and sperm DNA fragmentation

All semen samples were analysed for DNA underprotamination (CMA3 positivity), traditional sperm evaluation parameters (concentration, motility and morphology), and DNA fragmentation (TUNEL positivity) (Table 2).

Significant negative correlations (P < 0.05) were found between CMA3 positivity and sperm concentration, motility and morphology, before and after discontinuous gradient centrifugation (Table 3). A highly significant positive correlation was observed between CMA3 positivity and sperm DNA fragmentation (P < 0.01) (Table 3).

CMA3 positivity and fertilization rate

A correlation analysis was carried out to evaluate the relationship between fertilization rate and CMA3 positivity in the overall group of 132 patients: these two parameters appear to be negatively related (r = –0.177; P = 0.043). Dividing the overall group into IVF and ICSI patients, it was found that only in the IVF group was there a highly significant negative correlation between fertilization rate and CMA3 positivity (r = –0.311; P = 0.004), whereas the fertilization rate does not appear to have been affected by sperm underprotamination in ICSI patients (r = –0.065, not statistically significant) (Table 3).

Considering the link between fertilization rate and CMA3 positivity in the IVF group, a ROC analysis was carried out for the IVF patients in an attempt to identify a cut-off value of CMA3 positivity to discriminate between patients with poor or good fertilization prognosis. Using fertilization ‘yes’ or ‘no’ as 488

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a discriminator, the curve in Figure 1 was obtained. The area under the ROC curve was 0.769, the calculated threshold value for CMA3 staining having a prognostic role was 29.25%, with a sensitivity of 80% and a specificity of 80.6%.

CMA3 positivity and pregnancy rate

The possible relationship between CMA3 positivity and clinical pregnancy rate in both IVF and ICSI patients was evaluated. First of all, patients were divided into groups according to the threshold value of CMA3 positivity (29.25%), calculated with ROC analysis (Table 4): 68 of the 82 males undergoing IVF were characterized by low CMA3 positivity (group A) and 14 were characterized by high CMA3 positivity (group B); 22 of the 50 ICSI patients were characterized by low CMA3 positivity (group C) and 28 were characterized by high CMA3 positivity (group D). In the IVF group, a statistically significant difference was found between low and high CMA3 positivity patients as regards clinical pregnancy rates (A versus B = 27.94% versus 0%; P = 0.032), with no pregnancies in the group with high CMA3 positivity. In the ICSI group, clinical pregnancy rates were not statistically significantly

different between low and high CMA3 positivity patients (C versus D = 18.18% versus 39.29%). The groups of patients were homogeneous as regards others parameters: no differences were found between patients with low and high CMA3 positivity as regards previous assisted reproduction treatments, maternal age and female BMI, in either IVF or ICSI patients.

Moreover, the patients were divided into pregnant and non-pregnant groups (Table 5): 18 of the 82 females undergoing IVF were pregnant (group E) and 64 were not (group F); in the ICSI group, 12 of the 50 females were pregnant (group G) and 38 were not (group H). Pregnant and non-pregnant patients were compared with regard to CMA3 positivity, TUNEL positivity and other parameters (maternal age, female BMI, paternal age and some sperm characteristics), in both IVF and ICSI groups. In the IVF group a statistically significant difference was found between pregnant and non-pregnant patients as regards CMA3 positivity only (group E = 13.56 ± 6.25; group F = 20.03 ± 12.2; P = 0.033); no statistically significant differences were found between pregnant and non-pregnant patients as regards TUNEL positivity or the other parameters. On the other hand, in the ICSI group,

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Table 1. Overall data concerning assisted reproduction treatment procedures and clinical outcome.

Total IVF ICSI

No. of patients 132 82 50Maternal age (years, mean ± SD) 37.05 ± 4.2 37.2 ± 3.75 36.82 ± 4.87No. of cycles 132 82 50No. of inseminated oocytes 362 235 127Fertilization rate (%) 305 (84.25) 198 (84.26) 107 (84.25)Cleavage rate (%) 301/305 (98.69) 197/198 (99.49) 104/107 (97.20)No. of transferred embryos 298 195 103No. of transfers 122 77 45No. of gestational sacs 52 32 20Implantation rate (%) 52/298 (17.45) 32/195 (16.41) 20/103 (19.42)Clinical pregnancy rate per transfer (%) 34/122 (27.87) 19/77 (24.68) 15/45 (33.33)Miscarriage rate (%) 4/34 (11.76) 1/19 (5.26) 3/15 (20.00)Biochemical pregnancy rate (per β-HCG+) (%) 6/40 (15.00) 4/23 (17.39) 2/17 (11.76)

β-HCG+ = number of β-human chorionic gonadotrophin positive patients; ICSI = intracytoplasmic sperm injection.

Table 2. Sperm parameters (before density gradient centrifugation), TUNEL positivity and CMA3 positivity.

Total IVF ICSI

Paternal age (years) 40.24 ± 5.17 40.09 ± 4.56 40.51 ± 6.11Sexual abstinence (days) 3.97 ± 2.04 3.79 ± 1.78 4.27 ± 2.39Sperm concentration (×106/ml) 49.94 ± 54.47 64.67 ± 60.34 25.78 ± 30.89Total sperm motility (%) 43.33 ± 10.31 45.67 ± 7.85 39.5 ± 12.59Rapid sperm motilitya (%) 7.61 ± 5.97 9.76 ± 5.15 4.1 ± 5.6Normal sperm morphology (%) 19.86 ± 9.58 22.34 ± 10.02 15.8 ± 7.26TUNEL positivity (%) 9.68 ± 11.24 5.56 ± 7.34 16.43 ± 13.19CMA3 positivity (%) 24.62 ± 17.5 18.61 ± 13.13 34.46 ± 19.36

Values are mean ± SD. aRapid sperm motility = grade A motility (World Health Organization, 1999). CMA3 = chromomycin A3; ICSI = intracytoplasmic sperm injection; TUNEL = terminal deoxynucleotidyl transferase- mediated dUTP nick-end labelling.

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Table 3. Correlation analysis, carried out in 132 patients, between CMA3 positivity and (i) sperm parameters, before and after density gradient centrifugation, (ii) sperm DNA fragmentation, evaluated using TUNEL assay, (iii) fertilization rate in the total group of patients and in IVF and ICSI groups.

Parameter Correlation P-value coefficient(r)

Sperm concentration before DGC –0.361 <0.01Total sperm motility before DGC –0.207 0.017Rapid sperm motility before DGCa –0.329 <0.01Normal sperm morphology before DGC –0.325 <0.01Sperm concentration after DGC –0.506 <0.01Total sperm motility after DGC –0.366 <0.01Rapid sperm motility after DGCa –0.465 <0.01Normal sperm morphology after DGC –0.376 <0.01Sperm DNA fragmentation 0.526 <0.01Fertilization rate (total) –0.177 0.043Fertilization rate (IVF; n = 82) –0.311 <0.01Fertilization rate (ICSI; n = 50) –0.065 NSTotal oxidant scavenging capacity (n = 10) –0.842 <0.01

aRapid sperm motility = grade A motility (World Health Organization, 1999). CMA3 = chromomycin A3; DGC = density gradient centrifugation; ICSI = intracytoplasmic sperm injection; NS = not statistically significant; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Correlation analysis between CMA3 positivity and total oxidant scavenging capacity values was carried out in 10 donor semen samples. Spearman’s rho value and P-value are reported.

Figure 1. Receiver operating characteristic (ROC) curve of chromomycin A3 (CMA3) staining in IVF patients, with fertilization as selection criterion: the area under the ROC curve was 0.769; the calculated threshold value for CMA3 staining, having a prognostic role, was 29.25%; the sensitivity was 80% for a specificity of 80.6%.

a statistically significant difference was found between pregnant and non-pregnant patients as regards TUNEL positivity only (group G = 9.33 ± 10.06; group H = 18.68 ± 13.37; P = 0.031); no statistically significant differences were found between pregnant and non-pregnant patients as regards CMA3 positivity or the other parameters.

CMA3 positivity and total oxidant scavenging capacity of seminal plasma

Ten donors with sperm parameters comparable with those of IVF patients (sperm concentration >10 × 106/ml, sperm motility >30% and normal sperm morphology >4%) were selected and evaluated using the TOSC assay. A correlation analysis was carried out to evaluate the relationship between CMA3 positivity and the scavenging capacity of seminal plasma of the donors: these two parameters appeared to be negatively correlated (r = –0.842; P < 0.01), (Table 3 and Figure 2).

Discussion

Radical sperm chromatin reorganization, due to the replacement of histones by protamines, is important for several reasons (reviewed by Oliva, 2006): (i) to protect DNA from chemical and physical damage, making it inaccessible to ROS, nucleases, mutagens or to other factors potentially present in the internal or in the external media; (ii) to facilitate the movement of

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Table 4. Relationship between CMA3 positivity (low CMA3 <29.25%; high CMA3 ≥29.25%) and previous assisted reproduction treatment, maternal age, female BMI and clinical pregnancy rate.

Parameter IVF ICSI A(lowCMA3) B(highCMA3) C(lowCMA3) D(highCMA3) (n=68) (n=14) (n=22) (n = 28)

Previous treatment (%) 35.29 42.86 50.00 28.57 Maternal age (years, mean ± SD) 37.34 ± 3.62 36.5 ± 4.4 36.95 ± 4.81 36.70 ± 5.01Female BMI (kg/m2, mean ± SD) 22.29 ± 3.24 23.64 ± 3.4 20.83 ± 1.57 22.53 ± 3.76Clinical pregnancy rate (%) 27.94a 0.00b 18.18 39.29

a,bP = 0.032; there were no other statistically significant differences between low and high CMA3. BMI = body mass index; CMA3= chromomycin A3.

Table 5. Relationship between pregnancy and maternal age, female BMI, paternal age, sperm parameters (before DGC), TUNEL positivity and CMA3 positivity.

Parameter IVF ICSI E(pregnant F(non-pregnant G(pregnant H(non-pregnant patients) patients) patients) patients) (n=18) (n=64) (n=12) (n = 38)

Maternal age (years) 38.39 ± 2.85 36.86 ± 3.93 37.64 ± 5.77 36.58 ± 4.64Female BMI (kg/m2) 21.83 ± 3.71 22.72 ± 3.11 21.54 ± 3.26 21.66 ± 2.81Paternal age (years) 38.5 ± 5.51 40.53 ± 4.19 41.18 ± 5.21 40.32 ± 6.4Sperm concentration (×106/ml) 89.78 ± 101.06 57.6 ± 47.17 31.25 ± 40.33 24.06 ± 27.7Total motile sperm (%) 44.44 ± 7.65 46.02 ± 7.93 35 ± 15.52 40.92 ± 11.38Rapid motile sperm (%)a 10.56 ± 5.39 9.53 ± 5.1 4.58 ± 4.5 3.95 ± 5.95Normal sperm morphology (%) 24.11 ± 9.01 21.84 ± 10.29 15.33 ± 7.4 15.95 ± 7.3TUNEL positivity (%) 4.49 ± 5.33 5.86 ± 7.82 9.33 ± 10.06b 18.68 ± 13.37c

CMA3 positivity (%) 13.56 ± 6.25d 20.03 ± 12.2e 35.31 ± 16.18 34.2 ± 20.45

aRapid motile sperm = grade A motility (World Health Organization, 1999). b,cP = 0.031; d,eP = 0.033; there were no other statistically significant differences between pregnant and non-pregnant groups. BMI = body mass index; CMA3= chromomycin A3; ICSI = intracytoplasmic sperm injection; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling.

Figure 2. Correlation analysis between chromomycin A3 (CMA3) positivity and total oxidant scavenging capacity (TOSC) assay value towards peroxyl radicals (TOSC ROO) in 10 donors semen samples. Spearman’s rho correlation (r = –0.842; P < 0.01).

Article - Anomalies in sperm chromatin packaging - N Tarozzi et al.

the sperm cell: the spermatozoa with the most compact and hydrodynamic nucleus would move faster and may fertilize the oocyte first; (iii) to remove proteins and transcription factors from the spermatid, allowing a proper post-fertilization genetic reprogramming; (iv) to confer an epigenetic mark on some regions of the sperm genome, influencing its reactivation upon fertilization. In addition, it has also been proposed that (v) protamines could be part of a checkpoint during spermiogenesis and (vi) they could have a role in the fertilized oocyte, allowing the synchronization of the cell cycle between the oocyte in metaphase II phase and spermatozoa in G1 (Braun, 2001).

CMA3 staining is a broadly employed approach, used to identify a potential condition of underprotamination: CMA3 staining inversely correlates with the protamine content of spermatozoa, because of the competition of this fluorochrome with protamine-binding sites to DNA (Gao and Patel, 1990; Gao et al., 1992; Bizzaro et al., 1998). This approach has been used in some works to evaluate the impact of sperm chromatin alteration on reproductive outcome, in natural as well as in assisted reproduction (Sakkas et al., 1996; Tomlinson et al., 2001; Nasr-Esfahani et al., 2004, 2005, 2008; Hammadeh et al., 2006). Among these works, the present paper can be included, in which there is an analysis of the clinical relevance of sperm chromatin underprotamination in IVF and ICSI patients, with particular attention to the link between alteration in chromatin packaging and sperm characteristics, including sperm DNA fragmentation, reproductive outcome and defective antioxidant system activity.

The results of this study point first to a significant negative correlation (P < 0.05) between CMA3 positivity and sperm concentration, motility and morphology, in both native and treated semen samples (Table 3). Moreover a highly significant positive correlation was observed between CMA3 positivity and sperm DNA fragmentation, evaluated using TUNEL assay (P < 0.01). In a previous study performed in the same group of patients (Borini et al., 2006), there was also a strong negative correlation between TUNEL positivity and sperm parameters (concentration, motility and morphology) before and after discontinuous gradient centrifugation. The results of the present paper are corroborated by data from other studies, analysing the relationship between abnormal protamination and traditional sperm evaluation parameters (Bach et al., 1990; Carrell and Liu, 2001; Mengual et al., 2003; Aoki et al., 2005a; Torregrosa et al., 2006) and between underprotamination and sperm DNA fragmentation (Manicardi et al., 1995; Aoki et al., 2005b, 2006; Torregrosa et al., 2006). The relationship between abnormal protamination and diminished semen quality parameters is reasonable considering that the regulation of protamine exchange is linked to a broader control of spermatogenesis (Carrell et al., 2007). In particular, Carrell and colleagues discussed two main hypothesis about this topic: (i) abnormal protamine expression may be indicative of a general alteration of spermatogenesis, possibly due to an abnormal function of a transcriptional or translational regulator; or (ii) protamines may act as a ‘checkpoint’ during spermatogenesis, and abnormal protamine expression leads to an increased level of apoptosis that results in diminished semen quality. The strong link between abnormal protamination and sperm DNA fragmentation can also account for the important function of protamines in the protection of the paternal genome from chemical and physical damage, conferring a high level of compactness to the sperm nucleus

(Barone et al., 1994; Braun, 2001); so a potential consequence of underprotamination may be the greater susceptibility to DNA damage.

In this work, the influence of underprotamination on reproductive outcome was also analysed, and evaluated in terms of fertilization and pregnancy rates. First of all there was a strong negative correlation (P = 0.004) between CMA3 positivity and fertilization rate, but only in IVF patients (Table 3). So, in this treatment group, a ROC analysis was performed (Figure 1) to identify a threshold value for CMA3 staining to discriminate between poor or good prognosis patients. Using the calculated cut-off value (29.25%), the pregnancy rates of IVF and ICSI patients were compared (Table 4), and it was found that, only in IVF, clinical pregnancy rates significantly differed between patients with high and low CMA3 positivity (0% versus 27.94%, P = 0.032). Also demonstrated was the homogeneity of the compared groups as regards several important factors that may contribute to pregnancy outcome (multiple IVF/ICSI procedures, maternal age, female BMI) (Table 4). Additionally, there was confirmation of the link between abnormal protamination and pregnancy rate in IVF, displaying a significant difference in CMA3 positivity between pregnant and non-pregnant patients (13.56 ± 6.25 versus 20.03 ± 12.2; P = 0.033), (Table 5). Also in this analysis, the homogeneity of the compared groups as regards some important factors of both maternal and paternal origin (male and female age, maternal BMI, sperm parameters and sperm DNA damage) was demonstrated (Table 5), emphasizing the important discriminating potential of CMA3 positivity in prediction of pregnancy. On the basis of these results it can therefore be stated that sperm chromatin underprotamination seems to affect both fertilization and pregnancy rate in IVF and that the CMA3 assay appears to be a good prognostic tool for the outcome of IVF cycles. The impact of abnormal protamination on assisted reproduction treatment outcome may be traced back to the above-mentioned basic roles of protamines in sperm architecture and, as a consequence, in male reproductive ability. Otherwise, an interesting finding that came from the analysis was that, despite the clear relationship between alteration in chromatin protamination and IVF outcome, and despite high levels of CMA3 positivity in ICSI patients (Table 2), there was no association between chromatin underprotamination and reproductive outcome in ICSI. It was postulated that the selection of the most morphologically normal spermatozoa for ICSI decreases the risk of using cells with alterations in chromatin packaging, often related to sperm head decondensation, although studies evaluating sperm head morphology and protamine content in a single spermatozoon have not been performed (Carrell et al., 2007). The results, however, are in line with data displayed by two studies (Carrell and Liu, 2001; Aoki et al., 2005b) performed using a different kind of protamine detection: in these works abnormal protamination, evaluated with direct extraction and characterization of protamines, was associated with decreased fertilization ability in standard IVF, although fertilization and pregnancy rates were not different when patients underwent ICSI.

The data on the clinical relevance of sperm DNA fragmentation analysis (TUNEL assay) has been reported in a previous study (Borini et al., 2006): in that paper there was analysis of the same group of patients and cycles in order to investigate the predictive value of the TUNEL assay on IVF and ICSI outcome. 492

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It was clearly demonstrated that sperm DNA fragmentation significantly affects only the reproductive outcome of ICSI patients. In particular, the close relationship between sperm DNA damage and post-implantation development was emphasized, since there was a significant difference in clinical pregnancy and pregnancy loss rates between ICSI patients with high and low sperm DNA fragmentation. This result was confirmed in the present study, showing a significant difference in DNA damage between pregnant and non-pregnant patients (9.33 ± 10.06 versus 18.68 ± 13.37; P = 0.031) in the ICSI group only (Table 5). The finding that the DNA fragmentation test is not prognostic of success in IVF is in conflict with some published studies (Saleh et al., 2003; Boe-Hansen et al., 2006; Evenson and Wixon, 2006). Moreover, there are a few papers that have found no relationship between sperm DNA fragmentation and reproductive outcome in either ICSI or IVF patients (Gandini et al., 2004; Huang et al., 2005). To clarify these apparent discrepancies the following have to be taken into account: the different technical characteristics of DNA fragmentation tests (sperm chromatin structure assay, TUNEL assay, comet assay, sperm chromatin decondensation test or in-situ nick translation) and the different information arising, the testing modalities applied (flow cytometry or microscopy), the standardization difficulties of the tests, and the type of sample analysed (raw or treated samples). For instance, it has been shown that the predictive value of sperm DNA damage tests, performed on raw samples, decreases when sperm cells are treated by density gradient centrifugation (Sakkas et al., 2000; Tomlinson et al., 2001; O’Connell et al., 2003; Seli and Sakkas, 2005); this emphasizes the need to evaluate sperm DNA fragmentation in the appropriate context, i.e. in raw or treated semen, depending on whether it is to be correlated with natural conception or assisted reproduction, respectively (Tomlinson et al., 2001). The specific clinical context in which the observations were made also has to be considered: in fact, if and to what extent sperm DNA damage affects reproductive outcome also depends on other variables, of both male and female origin (such as maternal age or the presence of other pathological conditions), that can be detailed only by the in-depth study of the couple’s history of infertility (Ménézo, 2006). For example, one critical variable is the oocyte ability to repair sperm DNA damage, which is closely linked with maternal age (Ménézo, 2006).

On the basis of the results of the current and previous paper (Borini et al., 2006), it can be stated that, despite sperm abnormal protamination and sperm DNA fragmentation being positively correlated, they affect the reproductive outcome in different ways: while sperm DNA fragmentation seems to affect ICSI outcome, sperm chromatin underprotamination affects fertilization and pregnancy in IVF. This result may be explained considering the different nature of sperm DNA damage and sperm protamine deficiency: these two conditions are distinct aspects of chromatin alteration, so they probably have a different impact on biological quality of spermatozoa; additionally, the different technical features of the laboratory procedures used to assist fertilization (IVF and ICSI) and the contribution of the operator performing assisted reproduction procedures have to be taken into account. For instance, considering that one of the potential consequences of underprotamination is an increased susceptibility to sperm DNA damage (Carrell et al., 2007), a direct relationship between protamine deficiency and DNA damage might be expected. However the contrary is not always true: spermatozoa with DNA fragmentation, which may

derive from a number of causes (reviewed in Tarozzi et al., 2007), are not necessarily cells with abnormal protamination. In fact, sperm DNA damage is multifactorial and may be due to many conditions: in addition to poor chromatin packaging, sperm DNA fragmentation may be a consequence of high levels of free radicals, produced by both spermatozoa and leukocytes, or aberrant endonuclease activity, associated with abortive apoptosis (Aitken and De Iuliis, 2007). So, protamine-deficient spermatozoa, often characterized by sperm head decondensation, may be recognized by the operator performing ICSI, while spermatozoa with DNA fragmentation and normal level of protamination, may remain undetected and may be used for ICSI, affecting the clinical outcome deleteriously. The detrimental effect of sperm DNA fragmentation on ICSI outcome was also underlined by some recent papers: it was stated that sperm DNA fragmentation seems to be related to subtle morphological malformations of the sperm nucleus, as nuclear vacuoles, which can only be detected using a higher magnification than that of conventional ICSI (Hazout et al., 2006; Franco et al., 2008).

Male germ cells are especially vulnerable to oxidative stress because of the lack of DNA repair systems and antioxidants in spermatozoa (Aitken et al., 2003; Olsen et al., 2005). The antioxidant ability of seminal plasma, due to the presence of antioxidant molecules such as glutathione peroxidase, superoxide dismutase, vitamin C, α-tocopherol, hypotaurine, albumin and pyruvate (Perry et al., 1993; Twigg et al., 1998; van Overveld et al., 2000), and the strong sperm chromatin compactness, due to the replacement of histones by protamines, are the only defence mechanisms against oxidative stress (Tarozzi et al., 2007). In the present work, the aim was to evaluate the link between chromatin underprotamination and the antioxidant ability of seminal plasma, the latter measured using the TOSC assay. The TOSC assay is a test measuring the overall capability of biological fluids or cellular antioxidants to neutralize oxyradicals (Balercia et al., 2005); this test was described for the first time by Winston et al. (1998) and has been recently utilized in andrology, in a few studies only (Balercia et al., 2003, 2005). In particular there was a correlation analysis between CMA3 positivity values and TOSC values towards peroxyl radicals in 10 donors with sperm parameters comparable with those of IVF patients. It was decided to perform the analysis in this group of men for two reasons: (i) to further the research and provide additional information about IVF patients, because of the strong link between protamine deficiency and IVF outcome that was found previously; and (ii) to investigate the link between underprotamination and the antioxidant ability of seminal plasma in a homogeneous group of samples, without severe sperm concentration, motility and morphology anomalies. The analysis showed a significant negative correlation between chromatin underprotamination and the antioxidant ability of seminal plasma (r = –0.842; P < 0.01) (Table 3 and Figure 2). This result may be explained considering the impact of defective antioxidant systems on chromatin packaging during spermiogenesis (Conrad et al., 2005). Glutathione peroxidase, for instance, is one of the most important antioxidant systems: it has been found in the testis, epididymis, ejaculatory ducts, prostate and seminal vesicles, and its key role as scavenger of peroxyl radicals has been proved (Alvarez and Storey, 1989). Some studies, mainly performed using animal models, have demonstrated the basic role of glutathione peroxidase in conditioning the structural 493

Article - Anomalies in sperm chromatin packaging - N Tarozzi et al.

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Article - Anomalies in sperm chromatin packaging - N Tarozzi et al.

stability of sperm chromatin: it is involved in disulphide cross-linking in protamines by acting as a protein thiol peroxidase in vivo (Conrad et al., 2005) and dysfunction in its activity generates an altered thiol status that is related to male infertility (Rufas et al., 1991; Zini et al., 2001; Suganuma et al., 2005). Therefore the result of the present analysis, although performed in only a few men, corroborates the idea, derived from animal studies, that a defective antioxidant system activity is involved in alteration in chromatin packaging during spermiogenesis.

In conclusion, the data indicate that anomalies in sperm chromatin packaging, evaluated using CMA3 staining, are not only related to anomalies in traditional sperm parameters, but also affect fertilization and pregnancy rate in IVF; in particular, the work demonstrates the good predictive value of CMA3 analysis in IVF outcome and the possibility of using this test as a prognostic tool for IVF patients. It is also stressed that, despite sperm abnormal protamination and sperm DNA fragmentation being positively correlated, they are distinct aspects of chromatin alteration that affect reproductive outcome in different ways; this result underlines the importance of discriminating between tests that are often indifferently used to assess sperm chromatin alteration: taking full account of the information provided by each test is essential to use them most effectively in assisted reproduction techniques. Finally preliminary data are provided to support the idea, derived from animal studies, that defective antioxidant system activity may be associated with incomplete or improper chromatin packaging during human spermiogenesis. Further investigation is necessary to confirm the link between chromatin underprotamination and defective antioxidant system activity, and improve current knowledge of this topic.

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Declaration: The authors report no financial or commercialconflictsofinterest.

Received 19 June 2008; refereed 3 July 2008; accepted 1 December 2008.

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