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ARTICLE

Relationships between human sperm protamines,DNA damage and assisted reproduction outcomes

Luke Simon a, Judit Castillo b, Rafael Oliva b, Sheena EM Lewis a,*

a Centre for Public Health, Reproductive Medicine, Institute of Clinical Science, Queens University of Belfast,Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, UK; b Human Genetics Research Group, Genetics Unit,Faculty of Medicine, University of Barcelona, Casanova 143, 08036, Barcelona, Spain, IDIBAPS,and Biochemistry and Molecular Genetics Service, Hospital Clınic i Provincial, Villarroel 170, 08036 Barcelona, Spain* Corresponding author. E-mail address: [email protected] (SEM Lewis).

Dr Luke Simon graduated in biotechnology from the University of Madras, India in 2005 with a specialization inDNA fingerprinting. From 2005 to 2007, he was an instructor and research assistant at the University ofAgricultural Sciences, Bangalore, India. In 2007 he joined the Centre for Public Health at Queen’s UniversityBelfast, UK as a doctoral research fellow. His interests are principally sperm DNA damage, protamine and theeffects of oxidative stress on male fertility. Currently he is working as a post-doctoral research fellow atUniversity of Utah, USA with special interests in the genetics of male infertility.

Abstract The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variationsin sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships betweenDNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradientcentrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included inthe study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-ureapolyacrylamide gels. Sperm DNA fragmentation and protamine content (P1–DNA, P2–DNA, P1 + P2–DNA) decreased in spermatozoaafter DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathicinfertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or pro-tamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was nocorrelation between protamine content (P1–DNA, P2–DNA, P1 + P2–DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increasedsperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo qual-

ity and reduced pregnancy rates. RBMOnline

ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: alkaline Comet assay, clinical pregnancy, embryo quality, fertilization rate, P1/P2 ratio, sperm DNA fragmentation

Introduction

Since sperm DNA has little capacity for repair, protectionagainst damage is particularly important. As part of

spermiogenesis, the final stage of sperm differentiation,sperm DNA is subjected to major restructuring to facilitatea tighter, less vulnerable packaging. This is achieved by areplacement of around 85% of the loose network of histones

1472-6483/$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.rbmo.2011.08.010

Reproductive BioMedicine Online (2011) 23, 724–734

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by the more basic protamines (Barone et al., 1994). Humansexpress two protamines, protamine 1 (P1) and protamine 2(P2) (Balhorn et al., 1988; Bianchi et al., 1992; de Yebraet al., 1993; Torregrosa et al., 2006; Yoshii et al., 2005),which exist approximately in a 1:1 ratio (Corzett et al.,2002). In addition to its role in protection, this proteinreplacement acts to silence transcription in spermatozoaand possibly imprint the male genome (Oliva and Dixon,1991) prior to fertilization. Abnormalities in protamineratios have been associated with male infertility (Aokiet al., 2005a; Balhorn et al., 1988; de Mateo et al., 2009;de Yebra et al., 1998).

A reduction in sperm DNA integrity has been demon-strated in infertile patients using a variety of assays,including TdT (terminal deoxynucleotidyl transfer-ase)-mediated dUDP nick-end labeling (TUNEL), Cometassay, sperm chromatin structure assay (SCSA) and spermchromatin dispersion (SCD) assay (Agarwal et al., 2009b;Barratt et al., 2010; Lewis et al., 2008; Sakkas et al., 2002;Zini and Sigman, 2009). In the past decade, a number ofstudies have demonstrated the presence of a correlationbetween abnormally altered protamine P1/P2 ratios, prot-amine concentration or indirect assessment of protamineswith chromomycin A3 staining and decreased DNA integrityas evaluated by Comet, SCSA or TUNEL assays (Nasr-Esfahani et al., 2004a,b, 2005; Torregrosa et al., 2006;Zubkova and Robaire, 2006; Angelopoulou et al., 2007;Domınguez-Fandos et al., 2007; Plastira et al., 2007;O’Flaherty et al., 2008; Nili et al., 2009; Tarozzi et al.,2009; Tavalaee et al., 2009; Chiamchanya et al., 2010).However, a direct proof of the correlation of the absoluteprotamine/DNA content with the DNA damage in thespermatozoa of infertile patients and their association withassisted reproduction results is limited. Elucidating therelationship between the two will add to understandingwhether defective protamination facilitates the greaterDNA fragmentation that was observed in the spermatozoaof the same infertile patients (Aitken and De Iuliis, 2007a;Aitken et al., 2009; de Mateo et al., 2007).

Sperm DNA fragmentation is also an indicator of maleinfertility as demonstrated by numerous studies (Agarwalet al., 2009a; Barratt et al., 2010; Lewis and Aitken, 2005;Lewis et al., 2008; Sakkas et al., 2002; Zini and Sigman,2009). A close relationship between abnormal sperm chro-matin protamination and oxidative DNA damage has beenreported by De Iuliis et al. (2009), supporting their beliefthat poor chromatin remodeling is a major cause of spermDNA damage (Aoki et al., 2006a; Bianchi et al., 1993; Ziniet al., 2001). Poor protamine packaging has also beenassociated with poorer assisted reproduction outcomes,affecting fertilization rate and early embryo development(Sakkas et al., 1998). Aitken et al. (2009) have proposeda hypothesis that sperm DNA damage occurs during sper-miogenesis when inefficient remodeling becomes charac-terized by abnormal P1/P2 ratios and higher histoneretention (Carrell et al., 2008; De Iuliis et al., 2009; Sakkaset al., 1998), producing an increased vulnerability tooxidative stress.

This study investigated, as far as is known for the firsttime, the relationships between direct protamine measure-ment and DNA fragmentation as assessed by the COMETassay and IVF and ICSI outcomes.

Materials and methods

This project was approved by the Office for Research EthicsCommittees in Northern Ireland and the Royal Group of Hos-pitals Trust Clinical Governance Committee. A total of 73men, undergoing IVF treatment and 24 men undergoing ICSItreatment were included in the study. Sperm samples forresearch were obtained after written consent was given byeach couple at the Regional Fertility Centre, Royal JubileeMaternity Services, Belfast, Northern Ireland, UK. Theresults are based on total numbers of 606 oocytes for IVFand 218 oocytes inseminated for ICSI.

Semen analysis and sperm preparation

Semen samples, surplus to clinical requirements, were col-lected by masturbation from the infertile men on the day ofassisted reproduction treatment after 2–5 days of recom-mended abstinence. After liquefaction, routine semenanalyses were performed according to World HealthOrganization guidelines (WHO, 1992) and subsequentlysemen was prepared using a two-step discontinuous Pure-sperm gradient (90–45%; Hunter Scientific Limited, UK).Each semen sample was layered on top of 2 ml (90%) and4 ml (45%) gradient and centrifuged at 250g for 20 min.Hence, two populations of spermatozoa were used to mea-sure single- and double-stranded DNA breaks by the alkalineComet assay and protamines: (i) the whole population(native semen); and (ii) that used for the clinical treatments(density-gradient centrifugation; DGC) of each patient. Thenative semen was used without preparation.

Assisted reproduction treatments

All IVF cycles were performed according to the routine pro-cedures (Donnelly et al., 1998). Briefly, ovulation inductionwas achieved with recombinant FSH following a long proto-col of pituitary desensitization with a gonadotrophin-releasing hormone analog. Human chorionic gonadotrophinwas administered when there were at least four folliclesof diameter >17 mm, 36 h before oocyte retrieval. Maturemetaphase-II oocytes obtained by vaginal ultrasound-guidedaspiration were cultured in media (G5 sequential mediaseries; Vitrolife) at 37�C with 6% CO2 in air. The ICSIprocedure has been described in detail previously (VanSteirteghem et al., 1993). In brief, a suspension of washedspermatozoa was placed in polyvinylpyrrolidone (Vitrolife)and a free, motile spermatozoon was immobilized. Thespermatozoon was aspirated into the injection pipettetail-first and injected into an oocyte. Fertilization wasrecorded 12–16 h after injection. In each case, one or twoembryos were transferred into the uterine cavity after anadditional 24–48 h. Luteal-phase support was provided byvaginally administered progesterone. An intrauterine preg-nancy with fetal heartbeat was confirmed by ultrasound5 weeks after embryo transfer.

Alkaline Comet assay

Sperm DNA fragmentation was assessed using single-cell gelelectrophoresis (Comet) assay, previously optimized for

Sperm DNA damage, protamines and assisted reproduction outcomes 725


human spermatozoa by the study centre (Donnelly et al.,1999; Hughes et al., 1996). A previous study reported anintra-assay coefficient of variation of 6% for this assay(Hughes et al., 1997).

Protamine extraction and quantification

Protamine extraction was performed according to the pro-tocol published by the study centre (Castillo et al., 2011)on samples from 73 IVF and 24 ICSI men, each containingat least 10 million spermatozoa after washing twice withHam F10 1X medium (Sigma–Aldrich, Madrid, Spain). Thesediment was resuspended in 200 ll of 20 mmol/l EDTA,1 mmol/l phenylmethyl sulphonylfluoride (Sigma Chemicals,St Louis, USA) and 100 mmol/l Tris HCl (pH 8), and then pro-cessed as described elsewhere (de Yebra and Oliva, 1993; deYebra et al., 1993), with the variant that no iodoacetatetreatment was performed (de Mateo et al., 2009; Mengualet al., 2003; Torregrosa et al., 2006). Each sample wasresuspended in 14.3 ll of a sample buffer containing5.5 mol/l urea, 20% 2-mercaptoethanol and 5% acetic acid.The sediment obtained after the extraction of nuclear pro-teins with 0.5 mol/l HCl was used to quantify the DNAthrough 0.5 mol/l perchloric acid hydrolysis (90�C for20 min) and the determination of the absorbance at 260 nmwith a NanoDrop ND-1000 spectrophotometer.

Nuclear proteins were analysed in acid-urea polyacrylamidegels as described previously (Torregrosa et al., 2006), with theexception that the staining of the gels was performed withEzBlue Gel Staining Reagent (Sigma) following manufacturer’sinstructions. In addition to the samples under analysis, differentquantities of a humanprotamine standard fromapool of humannormozoospermic sperm samples (0.435, 0.87, 1.74 and2.61 lg) were also loaded into four separate lanes in each ofthe gels. This standard is available at the study laboratory andhad been prepared as previously described (Mengual et al.,2003). The stained gels were then scanned and the intensityof thebandsquantifiedwith theQuantityOneSoftware (BioRad,Hercules, USA). A regression curve was obtained from the fourdifferent concentrations of protamine standard included ineach gel and the intensity of their bands to calculate thequantity of P1 and of P2 in the samples.

Statistical analysis

Data were analysed using the Statistical Package for the SocialSciences version 15 (SPSS, Chicago, USA). The test parametersDNA fragmentation, P1/P2 ratios and protamine (P1, P2 andP1 + P2) contents in the native semen and DGC spermatozoaare all presented asmean ± SE. The fertilization ratewas calcu-lated as the percentage of all inseminated oocytes. The embryoquality is presentedas the embryo cumulative score, calculatedin day-3 embryos bymultiplying the embryo grade (A = 4, B = 3,C = 2 and D = 1) by the number of blastomeres in each embryo.Where a patient had more than one embryo, a mean acrossembryos was calculated to obtain the total quality of allembryos generated. All tests were two-sided and a P-value<0.05 was regarded as significant.

Spearman’s Rank correlation coefficient was used to ana-lyse the relationship between test parameters with semenparameters. Mann–Whitney U-test was used to detect

differences between the native semen and the DGC spermato-zoa of the test parameters. Kruskal–Wallis nonparametric testwas used to analyse categories of men’s age (�34, 35–38 and�39 years) with test parameters. Duncan’s analysis of vari-ance was used to associate categories of fertilization rate(�70%, good; <70%, low), embryo quality (embryo cumulativescore: �16, good; 12–15, moderate; �11, poor), P1–DNA(0.15–0.25 lg, normal; <0.15 and >0.25 lg, abnormal),P2–DNA (0.15–0.25 lg, normal; <0.15 and >0.25 lg, abnor-mal), total P1 + P2–DNA (0.30–0.50 lg, normal; <0.30 and>0.50 lg, abnormal) and protamine ratio (0.8–1.0, normal;<0.8 and >1.0, abnormal) with the test parameters. In addi-tion, the majority of the study population showed a P1 or P2content of approximately 0.20 lg and hence protamines werealso categorized into above and below 0.20 lg concentration.Similarly, sperm DNA fragmentation was categorized intothree groups of damage (0–40%, low; 41–60%, moderate;>60%, high) based on the percentage of DNA-fragment migra-tion in the Comet tail and assessed against reproductive out-come. Nonparametric statistics were employed when thedata were not normally distributed. No transformations wereused. Logistic regression was used to compare the differencebetween couples (pregnant and nonpregnant) and couplesdiagnosed with (male, female and unexplained factor) infer-tility with the test variables.

Results

Association between men’s age, semenparameters, protamines and sperm DNAfragmentation

Of the classic semen parameters, only abnormal morphologyshowed a positive association with the P1/P2 ratio (r2 = 329,P = 0.004). Sperm DNA fragmentation was higher in sampleswith abnormally high P1/P2 ratio (Figure 1). There was anegative association between sperm DNA fragmentationand progressive motility (r2 = �306, P = 0.008) but no associ-ation with any other semen parameters. A positive associa-tion was observed between DNA fragmentation and men’sage (r2 = 266, P = 0.023; Figure 2) although there was noassociation between age and protamine (not shown).Density-gradient centrifugation resulted in a population of

Figure 1 Increase in sperm DNA fragmentation with abnormalP1/P2 ratio (normal, 0.8–1.0; abnormal, <0.8 and >1.0).Values are mean ± SEM. *P < 0.05, compared with P1/P2 ratio0.8–0.9.

726 L Simon et al.


spermatozoa with significantly decreased DNA fragmenta-tion (P < 0.001), P1–DNA content (P = 0.005), P2–DNA con-tent (P = 0.008) and total P1 + P2–DNA content (P = 0.015)compared with native semen; however, there was no differ-ence with P1/P2 ratio (Table 1). Couples diagnosed withmale infertility showed higher sperm DNA damage(P = 0.035) and higher P2–DNA content than couples witheither female (P < 0.01) or unexplained (P < 0.001) infertil-ity problems (Table 2). An abnormal P1/P2 ratio wasobserved in 62% of couples diagnosed with unexplainedinfertility (Table 2).

Association between protamine, sperm DNAfragmentation and IVF outcome

The fertilization rates after IVF were negatively associatedwith sperm DNA fragmentation in both native semen(r2 = �0.443, P < 0.001) and DGC spermatozoa (r2 = �0.365,P = 0.001) and P1/P2 ratio (r2 = 0.397, P = 0.037), althoughnot with P2–DNA content (r2 = �0.356) in native semen.Fertilization rates (�70%) were associated with lowprotamine content (P1/DNA, P2/DNA, P1+P2/DNA) andP1/P2 ratios (Table 1). Spermatozoa with 0–40% DNAfragmentation were associated with higher fertilizationrates (83.2 ± 5.1%) compared with lower fertilization rates

of 64.4 ± 3.9% (P = 0.004) when DNA damage was 41–60%and with 44.1 ± 7.7% (P < 0.001) when DNA damage was61–100% (Figure 3).

Embryoswith good quality (embryo cumulative score�16)had significantly lower sperm DNA fragmentation in nativesemen (31.4 ± 5.7) and in DGC spermatozoa (20.1 ± 5.1) thanembryos with moderate quality (native semen, 50.1 ± 3.5,P = 0.01; DGC spermatozoa, 36.1 ± 2.8, P = 0.008) and poorquality (native semen, 53.8 ± 3.4, P = 0.002; DGC spermato-zoa, 38.2 ± 3.3, P = 0.008; Figure 4). A negative associationwas observed between embryo quality and DNA fragmenta-tion in native semen (r2 = �376, P = 0.002), DGC spermatozoa(r2 = �279, P = 0.023) and protamine content, P1–DNA(r2 = �421, P = 0.045), P2–DNA (r2 = �541, P = 0.008) andP1 + P2–DNA (r2 = �513, P = 0.012).

Sperm DNA fragmentation was significantly higher in non-pregnant couples (n = 58) compared with pregnant couples(n = 15) following IVF both with native semen (51.9 ± 2.4 ver-sus 39.7 ± 5.8; P = 0.030) and DGC spermatozoa (37.1 ± 2.2versus 26.7 ± 4.6; P = 0.038; Figure 5). Using clinically prog-nostic threshold values for spermDNA fragmentation (52% fornative semen and 42% for DGC spermatozoa; Simon et al.,2011) to predict successful pregnancies after IVF, the oddsratio (95% confidence interval) was 4.50 (1.12–19.40) and2.63 (0.59–13.27), respectively (Table 3). Measurement ofsperm DNA fragmentation in native semen had a higherspecificity (62% versus 40%) and positive predictive value (33%versus 26%) to determine IVF pregnancy outcome than DGCspermatozoa; however, the sensitivity of the assay is higherusing DGC spermatozoa. Men with sperm DNA fragmentationmore than the threshold value had an increased relative risk(1.93 for native semen and 2.21 for DGC spermatozoa) of notachieving a clinical pregnancy. The sensitivity of the Cometassay is stronger than that of other assays; in both nativesemen and DGC spermatozoa, given that DNA damage isobserved in all (not just 30% of spermatozoa as in otherassays). No significant relationships were found betweenprotamine content or ratios and pregnancy rates after IVF(Figure 5).

Association between protamines, sperm DNAfragmentation and ICSI outcome

No significant difference in sperm DNA fragmentation wasobserved between pregnant couples (n = 7) and nonpregnant

Figure 2 Increase in DNA fragmentation in native semen withincrease in men’s age. Values are mean and range. *P < 0.05,**P < 0.01, compared with age �34 years.

Table 1 Effect of sperm preparation and fertilization rate on DNA and protamine parameters.

DNA fragmentation(%)

P1/P2 ratio P1–DNA (lg) P2–DNA (lg) P1 + P2–DNA (lg)

Sperm preparationNative semen 51.4 ± 2.3 0.90 ± 0.02 0.20 ± 0.01 0.22 ± 0.01 0.43 ± 0.02DGC spermatozoa 35.1 ± 2.2 0.95 ± 0.02 0.15 ± 0.01 0.16 ± 0.01 0.32 ± 0.02P-value <0.001 NS 0.005 0.008 0.015

Fertilization rate�70% 29.2 ± 2.9 0.96 ± 0.03 0.15 ± 0.01 0.15 ± 0.01 0.30 ± 0.02<70% 40.0 ± 2.7 0.87 ± 0.02 0.18 ± 0.01 0.21 ± 0.02 0.40 ± 0.03P-value 0.010 0.042 NS NS 0.035

Values are mean ± SE.DGC = density-gradient centrifugation; NS = not statistically significant.



couples (n = 17). Further, sperm DNA fragmentation was notassociated with fertilization rates or embryo quality afterICSI treatment. A negative association in fertilization rateswas observed with P1 + P2–DNA (r2 = –564, P = 0.045) butnot with P2–DNA (r2 = –542) content of DGC spermatozoa.Also a negative association was observed between embryoquality and P1 + P2–DNA (r2 = –706, P = 0.010), P1–DNA(r2 = �702, P = 0.011) and P2–DNA (r2 = �666, P = 0.018)contents of the clinically used DGC spermatozoa after ICSI

treatment. However, no significant correlations wereobserved with P1/P2 ratios.

Discussion

This study has shown that sperm DNA fragmentation, mea-sured by the alkaline Comet assay, is associated with abnor-mally high or low P1/P2 ratios. Further, this study hasidentified that couples previously diagnosed with idiopathicinfertility have abnormally high P1/P2 ratios. Thirdly, fertili-zation rates and embryo quality, although not pregnancyrates, are negatively associated with abnormal prot-amine/DNA content. Finally, sperm DNA fragmentation hada strong negative association with assisted reproductionoutcomes after IVF but not after ICSI.

Protamines are the most abundant nuclear proteins, yettheir function is poorly understood (Balhorn, 2007; Carrellet al., 2007; Martins and Krawetz, 2007; Miescher, 1874;Miller et al., 2010; Oliva and Dixon, 1991). To date, threemajor functions of protamines have been postulated: (i) con-densation of the sperm nucleus (Balhorn, 1982; Fawcettet al., 1971); (ii) protection of the paternal genome fromnuc-leases (Sotolongo et al., 2003; Szczygiel and Ward, 2002) andfree radicals (Alvarez et al., 2002; Irvine et al., 2000); and (iii)imprinting of the paternal genome (Oliva and Dixon, 1991;Oliva, 2006; Balhorn, 2007). Protamines facilitate strongintermolecular attraction between the positively chargedprotamine and the negatively charged DNA to facilitate acompact DNA–protamine structure (Aoki and Carrell, 2003).Hence it is believed that abnormalities in protamine/DNAcontent can increase spermDNA vulnerability to assault (Aokiet al., 2006a). The P1/P2 ratio is better than other measuresof protamine/DNA content because it is easy to measure, ithas a low intra-assay variability and it has been widely usedas a marker of abnormal spermiogenesis (Oliva, 2006;Balhorn, 2007; Carrell et al., 2008; Castillo et al., 2011). AP1/P2 ratio of 0.9–1.0 has been observed in fertile men(Barone et al., 1994; Carrell and Liu, 2001; Corzett et al.,2002) and is now accepted as the normal range. Lowprotamine concentrations are associated with retention ofhistones. This combinationmay be less efficient in protectingthe spermatozoa from oxidative DNA damage (Aoki et al.,2006a; Zhang et al., 2006). The present results show aprotamine ratio of 0.8–0.9 is associatedwith the lowest levelof DNA fragmentation. Balhorn et al. (1988) showed thatabnormal P1/P2 ratios affect male fertility and infertilesubjects show increased P1/P2 ratios than fertile subjects(Cho et al., 2001; Mengual et al., 2003; Nasr-Esfahani et al.,

Table 2 Effect of DNA and protamine parameters on infertility factors.

Infertility factor n DNA fragmentation (%) P1/P2 ratio P1–DNA (lg) P2–DNA (lg)

Native DGC spermatozoa <0.8 0.8–1.0 >1.0 <0.2 �0.2 <0.2 �0.2

Male (%) 13 59.9 ± 3.9 44.4 ± 4.7 23 54 23 38 62 23 77Female (%) 18 51.8 ± 4.7 38.9 ± 4.7 11 78 11 56 44 44 56Unexplained (%) 28 47.7 ± 3.1 34.8 ± 2.8 18 39 44 50 50 46 54

Values are mean or mean ± SE.DGC = density-gradient centrifugation.

Figure 3 Decrease in IVF fertilization rates with increase insperm DNA fragmentation (n = 73). Values are mean ± SEM.**P < 0.01, ***P < 0.001, compared with 0–40% DNAfragmentation.

Figure 4 Increase in DNA fragmentation with decrease in IVFembryo quality for native and density-gradient centrifugation(DGC) spermatozoa. Values are mean ± SEM. *P < 0.05,**P < 0.01, compared with good embryo quality.

728 L Simon et al.


2008). Corzett et al. (2002) reported that the P2 contentcould vary from zero to 80% in mammalian species. Also, areduction in P2 precursors is reported in spermatozoa ofinfertile men (de Yebra et al., 1998) resulting in abnormalP2 concentrations, increased P1/P2 ratios and ultimatelyincreased DNA damage (Cho et al., 2003; Nili et al., 2009).

This report is consistent with the study centre’s previousstudies (Simon et al., 2010, 2011) showing a decrease in DNAfragmentation after sperm preparation (Avendano et al.,2010; Gandini et al., 2004; Henkel et al., 2004). Such apreparation isolates a subpopulation with better motilityand is in keeping with Simon et al. (2011), showing thatthe only semen parameter associated with sperm DNA qual-ity is progressive motility. These results again conflict withseveral studies that report no differences in DNA fragmenta-tion in native compared with DGC spermatozoa (Stevanatoet al., 2008; Thomson et al., 2009) and also indeed withthose reporting an increase in DNA damage post-preparation(Zini, 2011). The rationale as to why sperm preparationshould impair DNA quality is unclear. Both protamine/DNAcontent and ratios were associated with abnormal spermmorphology. This supports previous studies showing prot-amine abnormalities were related to sperm morphologyabnormalities (Esterhuizen et al., 2002; Franken et al.,1999; Nasr-Esfahani et al., 2001). Carrell and Liu (2001)showed that a low concentration of P2 is associated withabnormal semen parameters. The two may be connectedby defective spermiogenesis, which can cause poor protami-nation resulting in the release of immature spermatozoa

and also those with abnormal morphology (Huszar et al.,1997). However, after elimination of dead and nonprogres-sive motile spermatozoa from the native semen throughDGC, the present study observed a reduction in both P1and P2 contents. This association was surprising given theearlier proposed protective role of protamine.

The increase in sperm DNA fragmentation with anincrease in paternal age observed here is consistent withBoe-Hansen et al. (2006) and Vagnini et al. (2007). However,the present results are in contrast with some studies(Larson-Cook et al., 2003; Payne et al., 2005; Sun et al.,1997) that show no effect of a man’s age on his sperm DNAfragmentation. The effect of age on DNA damage has signifi-cant social implications as couples now choose to have theirchildren later in life (Paasch et al., 2010). Studies haveshown strong correlations between paternal age with mis-carriage (Belloc et al., 2008; Nybo Andersen et al., 2004;Slama et al., 2005). Paternal age has also been associatedwith birth defects, genetic mutations and childhood cancers(Bille et al., 2005; Crow, 1997; Kuhnert and Niesehlag, 2004;Reichenberg et al., 2006). The present study has shown thatcouples diagnosed with unexplained infertility have adetectable defect; that of abnormal P1/P2 ratios. This is inagreement with studies by de Yebra et al. (1993) and Aokiet al. (2005a). Due to the critical roles played by protaminesin the differentiation of spermatids (Iguchi et al., 2005),alterations in protamine expression could be a significantcause of unexplained fertility. Abnormal P2–DNA expressionhas been shown in both mice and humans to lead to severe

Table 3 Prognostic value of sperm DNA fragmentation to determine pregnan-cies after IVF treatment.

Prognostic parameter Native semen DGC spermatozoa

Threshold value (%) 52 42Odds ratio (95% CI) 4.50 (1.12–19.40)a 2.63 (0.59–13.27)Sensitivity (%) 73.33 80.00Specificity (%) 62.07 39.66Positive predictive value (%) 33.33 25.53Negative predictive value (%) 90.00 88.46Relative risk (95% CI)b 1.93 (1.23–3.03)a 2.21 (0.69–7.14)

DGC = density-gradient centrifugation.aP < 0.05.bRelative risk of not achieving a clinical pregnancy for men above the threshold value.

Figure 5 Relationship between pregnancy and sperm DNA fragmentation in IVF cycles (n = 73). Values are mean ± SEM. *P < 0.05.DGC = density-gradient centrifugation.



infertility (Aoki and Carrell, 2003). Interestingly, abnormallevels of P2–DNA expression are also associated with spermDNA fragmentation (Cho et al., 2003) and severely dimini-shed sperm count, motility and normal morphology (Carrelland Liu, 2001). This was further confirmed by animal modelswhere disruption of protamine expression leads to severeinfertility (Zhong et al., 1999). Complete loss of P2–DNAis also seen in infertile men (Carrell and Liu, 2001; de Yebraet al., 1993). A number of studies have associated prot-amine expression and male infertility (Aoki et al., 2005a;Balhorn et al., 1988; Belokopytova et al., 1993; Carrelland Liu, 2001; Chevaillier et al., 1987; de Yebra et al., 1993;Khara et al., 1997; Mengual et al., 2003; Nasr-Esfahaniet al., 2008) and observed that infertile male populationshave abnormal P1/P2 ratios (Aoki et al., 2005a; Carrelland Liu, 2001; Chevaillier et al., 1990; de Yebra et al., 1998;Nasr-Esfahani et al., 2008).

This study observed a negative relationship betweenabnormal protamines and assisted reproduction outcomes.A decrease in fertilization rate was associated with adecrease in the protamine P1/P2 ratio after IVF treatment.The results support those of Sakkas et al. (1996), whoreported that abnormal protamines resulted in fertilizationfailure. Indirect measurement of protamines have alsoshown that fertilization rates are lower in patients with highchromomycin A3 positivity, indicating inadequate prot-amine packaging (Lolis et al., 1996; Esterhuizen et al.,2000, 2002; Razavi et al., 2003; Nasr-Esfahani et al., 2001,2004a, 2005). Replacement of histones with protaminesplays an important role in formation of the sperm nucleuscondensation (Aoki and Carrell, 2003; Oliva and Dixon,1991). However, sperm preparation in the present studyhad no effect on P1/P2 ratios yet a marked decrease in prot-amine (P1–DNA and P2–DNA) content. Similarly, fertiliza-tion rate was not influenced by P1/P2 ratio but associatedwith protamine content in terms of P1 + P2–DNA. Theseresults show that decreases in P1–DNA and P2–DNA con-tents increase fertilization rate irrespective of P1/P2 ratio.However, such effect was not reported after ICSI treatment(Carrell and Liu, 2001; Nasr-Esfahani et al., 2004a; Aokiet al., 2005a, 2006b). Abnormalities in the protamine couldresult in defective sperm decondensation, resulting in fertil-ization failure (Esterhuizen et al., 2002) and affectingembryo development (Suganuma et al., 2005).

This study also shows a negative association betweenabnormally high or low protamine content and embryo qual-ity after IVF unlike Carrell and Liu (2001), Nasr-Esfahaniet al. (2005) and Aoki et al. (2006b) who found no effectof protamine/DNA content on embryo development. Fur-ther, Cho et al. (2001, 2003) showed that protamine defi-ciency could lead to embryonic arrest. Aoki et al. (2006c)suggest that the effect of abnormal protamine concentra-tions on post-fertilization events includes pronuclear forma-tion, chromatin remodeling and interference with paternalgene expression. Therefore, from these studies it can bepostulated that protamines may play a role in the epigeneticmarking of the paternal genome which may be important forembryo development (de Mateo et al., 2007; Martınez-Heredia et al., 2006; Oliva, 2006; Oliva and Dixon, 1991).

This study found no association of protamines with clini-cal pregnancies, which is in accordance with Carrell and Liu(2001), Nasr-Esfahani et al. (2004a), Aoki et al. (2005a) and

Steger et al. (2008) who also reported no effect of prot-amine ratio on pregnancy rate. However, the present studyis in contrast with those reporting significantly reducedpregnancy rates in patients with abnormally low or highP1/P2 ratios (Aoki et al., 2006b,c). Aoki et al. (2006b)reported that the clinical pregnancies were reduced inpatients with low protamine ratios compared with normaland higher ratio after IVF and ICSI treatments. Similarly,de Mateo et al. (2009) reported that the average protamineratio in the group that did not achieve a pregnancy waslower than the average protamine ratio in the group thatachieved a pregnancy. It is difficult to explain why this isnot supported by the present study. Perhaps it is as a resultof testing with different populations from northern andsouthern Europe.

The clinical relevance of sperm DNA fragmentation hasbeen a topic of much interest recently. Sperm DNA damagehas been reported as a cause for lower fertilization ratesduring IVF treatment (Bakos et al., 2007; Borini et al., 2006;Huang et al., 2005; Muriel et al., 2006; Payne et al., 2005).These adverse effects of DNA fragmentation may beexplained by abnormal chromatin packing in spermatozoa,which is associated with high DNA damage and also with afailure of sperm DNA to decondense post fertilization (Lopeset al., 1998; Sakkas et al., 1996). This study also observed astatistically significant decrease in fertilization rate whensperm DNA fragmentation was >40%. In support of previousstudies by this study centre (Simon et al., 2010, 2011) andmany other groups, (Bakos et al., 2007; Borini et al., 2006;Bungum et al., 2007; Greco et al., 2005; Lin et al., 2008;Ozmen et al., 2007; Zini et al., 2005), there was no signifi-cant correlation between DNA fragmentation and fertiliza-tion rate after ICSI treatment. This supports thehypothesis of Ozmen et al. (2007) and Bungum et al. (2008)that ICSI is able to compensate for the effect of DNA strandbreaks in some, as yet unknown, way.

Previous studies have shown that embryo quality afterIVF treatment is closely correlated to the presence of spermDNA fragmentation (Simon et al., 2010, 2011). A number ofstudies have associated poor embryo quality with high DNAfragmentation (Host et al., 2000; Muriel et al., 2006; Seliet al., 2004; Tesarik et al., 2004; Tomsu et al., 2002; Virroet al., 2004). This is in contrast with the report of Braudeet al. (1988) showing that the impact of damaged paternalDNA became more obvious when the embryonic genomewas activated, which was later called the ‘late paternaleffect’ by Tesarik et al. (2004). However, sperm DNA frag-mentation is not associated with embryo quality after ICSItreatment (Bakos et al., 2007; Bungum et al., 2007; Grecoet al., 2005; Huang et al., 2005; Lin et al., 2008; Payneet al., 2005). Currently, semen analysis is the only routineclinical method to decide the most suitable type of treat-ment (IVF or ICSI) for a couple. However, recent studieshave shown that measurement of DNA fragmentation bythe alkaline Comet assay is a more useful diagnostic toolto determine the type of treatment for couples (Simonet al., 2010, 2011). In accordance, this study also shows thatICSI is a more efficient treatment method than IVF whenDNA fragmentation exceeded the threshold value of 52% innative semen and 42% in DGC spermatozoa. Sperm DNA frag-mentation had a high negative predictive value in bothnative semen and DGC spermatozoa after IVF treatment.

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The present results are supported by studies reporting highDNA damage has a higher negative predictive value after IVFtreatment (Adams et al., 2004; Boe-Hansen et al., 2006;Borini et al., 2006; Bungum et al., 2004; Henkel et al., 2004;Larson et al., 2000; Muriel et al., 2006; Virro et al., 2004).However, the positive predictive value remains low, indicat-ing the influence of female fertility in these cases: whencouples with known female and unexplained factors wereremoved from the study, the positive predictive valueincreases (Giwercman et al., 2009; Simon et al., 2011).The odds ratios to determine successful pregnancies afterIVF were 4.50 for native semen and 2.63 for DGC spermato-zoa, relatively higher than the overall odds ratios obtainedby the meta-analysis when all the studies were put together(Collins et al., 2008; Zini and Sigman, 2009), suggesting thegreater efficiency of the alkaline Comet assay comparedwith SCSA and TUNEL assays which were included in themeta-analysis.

In conclusion, protamines are a critical factor for properchromatin packing, and abnormal protamination increasedthe susceptibility of spermatozoa to DNA damage. Coupleswith previously unexplained infertility have a detectabledefect in the form of abnormal P1/P2 ratios. The results indi-cate the importance of spermDNA damage and adequate pro-tamination and emphasizes their usefulness as part of routineanalysis for patients presentingwith infertility in determiningthe type of treatment they should undergo.

Acknowledgements

This work was supported by grants from Hamilton ThorneBiosciences, USA and Ministerio de Ciencia y Tecnologıa(BMC2006–03479 and BFU2009–07118), funder FEDER. Theauthors are grateful to their clinical colleagues in the Regio-nal Fertility Centre, Belfast for provision of samples andacknowledge the advice and technical support in the prot-amine determinations provided by Sara de Mateo.

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Declaration: Sheena EM Lewis is Managing Director of LewisFertility Testing Ltd which has a commercial interest in thedetection of sperm DNA damage. The other authors report nofinancial or commercial conflicts of interest.

Received 29 November 2010; refereed 2 August 2011; accepted 4August 2011.

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Author's personal copy · Author's personal copy ARTICLE Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes Luke Simona, Judit Castillo b,