sperm chromatin || sperm chromatin and art (iui, ivf and icsi) pregnancy

441A. Zini and A. Agarwal (eds.), Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction, DOI 10.1007/978-1-4419-6857-9_31, © Springer Science+Business Media, LLC 2011

Abstract

Diagnosis of male infertility has been based mainly on the traditional semen parameters, namely, concentration, motility and morphology. In assisted reproductive technologies (ART), sperm samples are prepared by methods such as swim-up or density-gradient centrifugation to sort out sperm populations believed to have the highest fertilization potential. Traditionally, results of semen analysis and sperm preparation have been the fundaments on which clinicians could base their decision of what ART method should be used for a given couple. It has, however, become appar-ent that none of these procedures are sufficient for the determination of male fertility capacity. Owing to the lack of adequate methods to evaluate the fertility potential of a couple, the choice of ART method is made more or less blindly. A continuous search for better markers of male fertility has led to an increased focus on sperm chromatin integrity testing in fertility workup and ART. Numerous sperm DNA integrity tests have been devel-oped. In the context of fertility, the comet, TUNEL, and Sperm Chromatin Structure assays are the most frequently used. Sperm DNA fragmentation has shown to be an independent predictor of success in couples undergo-ing intrauterine insemination. More contrasting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracy-toplasmic sperm injection (ICSI).

Keywords

Male infertility • Assisted reproductive technology • Intracytoplasmic sperm injection • Intrauterine insemination • In vitro fertilization

Mona Bungum

M. Bungum () Reproductive Medicine Centre, Skåne University Hospital Malmö, Lund University, Sodra Forstadsgatan, Malmö 20502, Sweden e-mail: [email protected]

Sperm Chromatin and ART (IUI, IVF and ICSI) Pregnancy 31

In Western countries, 17–25% of couples in reproductive age are seeking medical care for problems of conception [1, 2]. Thanks to the introduction of assisted reproductive technolo-gies (ART), now, almost every involuntarily

442 M. Bungum

childless couple has a realistic hope of parenting. In particular, the introduction of intracytoplasmic sperm injection (ICSI) has revolutionized the area of fertility [3]. The number of ART treatments, in particular ICSI cycles, is steadily increasing [4]. While in the beginning of the era of ICSI the indi-cation for this type of treatment was severe male infertility, now also couples whose male partners are without sperm defects request and are treated with ICSI. However, by ICSI all natural biologi-cal barriers that prevent fertilization with defec-tive sperm are bypassed, and its increasing use has led to a growing concern of transmission of genetic and epigenetic diseases.

Although the development of ART has brought us further and led to a vast increase in our under-standing of early reproductive function, ART performances have been stable and we have wit-nessed no net improvement in healthy term preg-nancy rate during the last two decades [5]. One reason for this can be a lack of adequate methods to evaluate the fertility potential of a couple and also a lack of methods to identify the most effec-tive type of ART treatment for a given couple.

So far, the traditional semen analysis has been a cornerstone in the diagnosis of male fertility and also used as a tool to decide which ART method to use. The sperm parameters, namely, concentration, motility and morphology, are, however, claimed to be poorly standardized, subjective [6] and not powerful predictors of fertility [7, 8]. A search for better predictors of fertility has contributed to a growing focus on the genomic integrity of the male gametes used for ART [9, 10]. During the last few decades, several methods to assess sperm DNA damage have been developed. Although still many questions remain to be answered, it is evident that sperm DNA integrity is a valuable marker of male fertility, alone or in combination with the conven-tional semen parameters, in natural conception as well as in ART. This chapter reviews the role of sperm chromatin integrity in ART.

Assisted Reproductive Technologies

The term ART covers all reproductive technolo-gies that involve the handling of gametes outside the body, either sperm alone as in intrauterine

insemination (IUI), or oocytes, sperm and embryos as in in vitro fertilization (IVF) and ICSI [11]. ART is primarily used as a treatment of infertility/subfertility and also, to some extent, in establishing pregnancy in couples carrying inher-ited genetic diseases. The very first documented successful use of ART in humans was in 1978 when the world first IVF-baby was born [12]. Now, about 30 years later, ART is applied world-wide, and it is estimated that more than three million babies have been born as a result of ART since then [13]. The number of ART treatments is rising every year [14].

The first choice of treatment used in ovulatory dysfunction, minimal endometriosis, unexplained subfertility and milder forms of male subfertility is the relatively simple IUI. Following a mild con-trolled ovarian stimulation, prepared semen is inseminated into the woman’s uterus. In tubal fac-tors, IVF is used [11]. In IVF, oocytes are fertil-ized by sperm in vitro. Two to five days later the pre-embryo is replaced into the woman’s uterus. In ICSI, nearly the same principles are followed, but one single spermatozoon is selected and injected directly into the cytoplasm of the oocyte.

Traditional Markers of ART Fertility Potential

Prediction of the fertility potential of a couple has never been more crucial than now. We are facing delayed childbearing and falling sperm counts as possible threats to fertility. Various predictors of fertility have been suggested; however, none is shown to be ideal. While in the female age is the only parameter that has been shown to have the potential to predict ART outcome [15], for long it was thought that the traditional sperm parameters could predict male fertilization capability. In ART, sperm samples are prepared by methods such as swim-up or density gradient centrifugation to sort out populations of sperm believed to have the highest fertilization potential. Traditionally, con-centration and motility after sperm preparation have been one of the fundaments driving clini-cians decisions about the choice of the specific ART method recommended for a given couple.

44331 Sperm Chromatin and ART (IUI, IVF and ICSI) Pregnancy

It has, however, not shown to be sufficient for assessing of the fertilizing capacity of a sperm.

Several other laboratory tests of sperm function have been suggested, such as antisperm antibody test, vital staining, biochemical analysis of semen, hypoosmotic swelling test, sperm penetration assay, hemizona assay, creatine kinase, reactive oxygen species (ROS) tests and computer-assisted sperm analysis (CASA) [16]; however, the clini-cal value of these tests has been questioned [17], and only a few of them have been implemented in routine clinical use.

Owing to the lack of tools to predict sperm fertilizating capacity, the criteria for choosing ICSI and, as a consequence, the ratio between IVF and ICSI vary from clinic to clinic. Despite the fact that, in unexplained infertility, fertiliza-tion rates are as good in IVF as in ICSI [18], many clinics now perform ICSI as their primary, if not the only, ART technique [4, 19].

Sperm Chromatin Integrity Testing

The evidence that infertile men in general pos-sess substantially more sperm DNA damage than fertile men [20–28] has led to a growing focus on sperm chromatin integrity testing as an adjunct tool to the traditional sperm parameters in pre-diction of fertility. During the past three decades, a variety of techniques to assess sperm chromatin integrity have been developed. Principles, proce-dures and other aspects of the different tests are reviewed in detail in other chapters of this book. Briefly, mainly four tests assessing sperm DNA damage are used in ART, namely, the comet assay (single cell gel electrophoresis) [29], the TUNEL (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) assay [30], the Sperm Chromatin Structure assay (SCSA) [31, 32] and the Sperm Chromatin Dispersion (SCD) test [33].

Comet assay is a fluorescence-microscopy-based test. In this assay, spermatozoa are mixed with melted agarose and then placed on a glass slide. Thereafter, the cells are lysed and subjected to horizontal electrophoresis. DNA is visualized with the help of DNA-specific fluorescent dyes, and DNA damage is quantified by measuring the

displacement between the nuclear genetic mate-rial of the comet head and the broken DNA migrated in the tail.

TUNEL assay can be run using both bright-field/fluorescence microscopy and flow cytometry. In the TUNEL assay, terminal deoxynucleotidyl transferase (TdT) incorporates labelled nucle-otides to 3¢-OH at single- and double-strand DNA breaks to create a signal, which increases with the number of DNA breaks. On a microscope slide, sperm are scored and classified as positive or neg-ative depending whether they are labelled or not. In flow cytometry, the fraction of positive sperm is represented by the cells above a threshold channel value on a relative fluorescence intensity scale.

SCSA is a flow-cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ, followed by staining with acridine orange [31, 32]. The level of DNA denaturation is determined by measuring the shift from green fluorescence (double-stranded, native DNA) to red fluorescence (single-stranded, dena-tured DNA) in a flow cytometer, followed by fur-ther analysis by a specific SCSA software. The extent of DNA denaturation is expressed as DNA fragmentation index (DFI) [32]. The fraction of high DNA stainable (HDS) cells, thought to repre-sent immature spermatozoa, is also recorded [32].

Similar to the SCSA, the fluorescence/light microscopic SCD test determines the susceptibil-ity of sperm DNA to acid denaturation [33, 34]. Briefly, intact spermatozoa are immersed in an agarose matrix on a slide, treated with an acid solution to denature DNA that contains breaks and then treated with lysis buffer to remove mem-branes and proteins. Removal of nuclear proteins results in nucleoids with a central core and a periph-eric halo of dispersed DNA loops. Sperm nuclei with elevated DNA fragmentation produce very small or no halos of DNA dispersion, whereas those sperm with low levels of DNA fragmentation release their DNA loops forming large halos. The sperm nucleoids may be visualized using fluores-cence microscopy, after staining with a DNA-specific fluorochrome, or bright-field microscopy.

Moderate-to-high correlations between these different tests have been reported [30, 34–36], indicating that, very likely, these tests are not addressing identical aspects of the complex

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processes underlying sperm nuclear packaging potentially resulting in DNA breaks [37]. The test that has been most extensively tested clinically and found to have the most stable threshold values is the SCSA [20, 24, 31, 38], and this chapter focuses mainly on the results from SCSA-based studies.

Several reports have demonstrated that the association between sperm DNA damage and the traditional semen parameters is only weak-to-moderate [39, 40]. It is also shown that infertile men may have normal standard sperm character-istics according to WHO criteria, but a high number of sperm DNA defects.

In a recent case–control study on infertile vs. fertile men, the risk of being infertile resulted increased when DFI, as measured by SCSA, was above 20% in men with normal standard semen parameters, with an odds ratio (OR) of 5.1 (CI: 1.2–23). If any one of the WHO parameters were abnormal, the OR for infertility was increased already at DFI above 10% (OR 16, CI: 4.2–60). DFI above 20% was found in 40% of men with otherwise normal standard parameters [41]. In another study of 350 Latvian men from infertile couples [42], 20% of the men with otherwise nor-mal WHO sperm parameters had a SCSA-DFI above 20%. This is clinically relevant in counsel-ling couples during the fertility workup, and also in couples seeking ART where the choice of treatment most often is based upon the traditional sperm parameters and where an underlying high DFI can hinder a pregnancy.

Sperm Chromatin Integrity Testing in ART

Intrauterine InseminationThe first study indicating an association between sperm DNA damage and reduced pregnancy chances after IUI was published by Duran et al. [43]. In a retrospective study of 154 IUI cycles, they found that pregnancy could not be achieved when DFI, as measured by the TUNEL assay, was above 12%. Similar findings have been reported by Saleh’s group [28] who performed a small study where 12 of 19 couples had a DFI value as measured by SCSA above 28% and

none of these couples achieved a pregnancy. Boe-Hansen et al. [44] used SCSA in a study on 48 IUI couples. Only two of the couples had a DFI value above 30%, and none of the couples achieved a pregnancy. Recently, in a study of 387 IUI cycles, it shown that the SCSA param-eter DFI can be used as an independent predic-tor of fertility [38]. While the proportion of children born per cycle was 19.0% when the DFI value was below 30%, those with a DFI value above 30% only had a take-home-baby rate of 1.5%. These IUI results are in good accordance with those results obtained from natural conception. In fact, both Evenson et al. [20] and Spanò et al. [24] demonstrated that, after unprotected intercourse, time-to-pregnancy increased (fertile couples took longer to con-ceive) as a function of the proportion of sperm with abnormal chromatin measured by the SCSA [20, 24]. By contrast, no correlation was found between SCD results and pregnancy outcome in 100 Spanish IUI patients [45].

Normal sperm DNA integrity seems to be particularly important when the contact between the two gametes occurs in a natural way as in natural conception and IUI. It has been suggested that selective pressures operate to avoid the development of an embryo derived from sperm with a high load of genetic damage in a natural environment [29]. Additionally, spermatozoa with damaged DNA could be more prone to undergo apoptosis during the transport through the genital tract than spermatozoa with normal DNA integrity. For an overview of IUI-papers, see Table 31.1.

In Vitro Fertilization and Intracytoplasmic Sperm InjectionNumerous of retrospective studies have exam-ined the role of sperm chromatin damage in IVF and ICSI. In Table 31.2, an overview of studies using SCSA, TUNEL, comet or SCD assays is presented.

Sperm DNA Damage in Relation to Pregnancy OutcomeSome of the first studies relating outcome of ART to sperm DNA damage suggested that a DFI


Table 31.1 Influence of sperm DNA damage on pregnancy rates in IUI treatment

References Patients (n)Pregnancy rates impaired Test applied

DNA fragmentation index (DFI)-threshold suggested (%)

Duran et al. [43] 154 Yes TUNEL 12Saleh et al. [28] 19 Yes SCSA 30Bungum et al. [48] 131 Yes SCSA 27Muriel et al. [45] 100 No SCD –Bungum et al. [38] 387 Yes SCSA 30

IUI Intrauterine insemination; SCSA Sperm Chromatin Structure assay; TUNEL terminal deoxynucleotidyl transferase dUTP nick-end labelling; SCD Sperm Chromatin Dispersion test

Table 31.2 Influence of sperm DNA damage on fertilization, embryo development and pregnancy rates in IVF and ICSI

References IVF (n) ICSI (n)Fertilization rates impaired

Embryo development impaired

Pregnancy rates impaired

Test applied

Tomsu et al. [62] 40 0 No Yes Yes CometMorris et al. [29] 20 40 No Yes NA CometCaglar et al. [116] 0 56 No No No CometLewis et al. [64] 0 77 No NA Yes CometNasr-Esfahani et al. [66]

0 28 No No NA Comet

Larson-Cook et al. [47]

55 34 No No Yes SCSA

Larson et al. [46] 24 IVF/ICSI NA No No Yes SCSASaleh et al. [28] 10 4 Yes Yes Yes SCSABungum et al. [48] 109 66 No No Yes SCSAGandini et al. [49] 12 24 No Yes

(blastocysts)Yes SCSA

Virro et al. [50] 249 IVF/ICSI NA No No Yes SCSACheck et al. [117] 0 106 No No Yes SCSAPayne et al. [52] 46 54 No No No SCSABoe-Hansen et al. [44]

139 47 No No Yes SCSA

Bungum et al. [38] 388 223 No No Yes SCSASun et al. [67] 143 0 Yes Yes NA TUNELLopes et al. [68] 0 150 Yes No NA TUNELHost et al. [22] 50 61 Yes NA NA TUNELTomlinson et al. [61] 140 0 No No Yes TUNELBenchaib et al. [85] 50 54 Yes No Yes TUNELHenkel et al. [63] 208 54 No No No TUNELHuang et al. [65] 217 86 Yes No No TUNELSeli et al. [75] 49 NA NA Yes No TUNELHenkel et al. [118] 208 54 No No No TUNELHammadeh et al. [87]

26 22 NA NA No TUNEL

Borini et al. [88] 82 50 NA NA Only for ICSI TUNELBenchaib et al. [86] 88 234 Only for ICSI Only for

ICSINo TUNEL

Bakos et al. [119] 45 68 Only for IVF No Only for ICSI TUNELFrydman et al. [120] 117 0 NA NA Yes TUNEL

(continued)

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above 27% as measured by SCSA could be used as a cut-off value for infertility. The authors reported that in couples with a DFI above 27%, no pregnancy could be obtained, regardless of the type of ART applied [46, 47]. However, in 2004 when three independent SCSA reports demon-strated that a DFI level above 27% was indeed compatible with pregnancy and delivery after both IVF and ICSI [48–50], it became evident that ART can compensate poor sperm chromatin quality.

Gandini et al. [49], in a study involving 34 couples (12 IVF and 22 ICSI), did not note any difference between patients initiating pregnan-cies or not. They reported healthy full-term preg-nancies with levels of DFI up to 66.3%. Bungum et al. [48] investigated 109 consecutive couples undergoing IVF and 66 couples undergoing ICSI. No statistically significant difference in the pregnancy outcome was noted by dividing patients according to the DFI level of 27%. However, in the group with a DFI above 27%, the results of ICSI were significantly better than those of IVF, clinical pregnancy (52.9 vs. 22.2%), implantation (37.5 vs. 19.4%) and delivery (47.1 vs. 22.2%). Virro et al. [50] studied 249 couples undergoing IVF/ICSI and noted that men with DFI below 33% had a significantly greater chance of initiating a pregnancy, lower rate of spontaneous abortions and an increased rate of ongoing pregnancies at 12 weeks (47 vs. 28%) than those with a DFI above 33%.

These data were in agreement with other pre-vious smaller reports using TUNEL or comet assays, showing that sperm DNA damage is

more predictive in IVF and, less in ICSI [22, 51]. This was later confirmed in a larger data set including nearly 1,000 men in IUI, IVF or ICSI treatment using DFI 30% as threshold level. No statistically significant difference between the outcomes of ICSI vs. IVF in the group with DFI £30% was seen. In the DFI >30% group, how-ever, the results of ICSI were significantly better than those of IVF. The odds ratios (ORs) for biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D) were 3.0 (95% CI: 1.4–6.2), 2.3 (5% CI: 1.1–4.6) and 2.2 (95% CI: 1.0–4.5), respectively. For ICSI, there was even a tendency towards higher rates of BP, CP and D with a DFI >30% vs. a DFI £30%, however, not reaching a statistically significant difference. Moreover, the implantation rate in the ICSI group with DFI >30% seemed to be higher than in any other subgroup. The other SCSA param-eter, HDS did, however, not predict the outcome of IVF or ICSI, neither alone nor in combination with DFI [38]. By contrast, one single study had, however, reported that DFI and HDS threshold values were not valid [52]. The authors found that the poorer the integrity of sperm nuclear DNA, the better is the pregnancy outcome and suggested to “redefine the relationship between SCSA data and ART outcomes”. The study was, however, based on only 100 IVF/ICSI treatments where female factor infertility not was taken into consideration.

Despite convincing data from several authors, some reports have challenged the predictive value of the SCSA test [53]. One example is a position paper from the Practice Committee of

References IVF (n) ICSI (n)Fertilization rates impaired

Embryo development impaired

Pregnancy rates impaired

Test applied

Tarozzi et al. [121] 82 50 NA NA Only for ICSI TUNELMuriel et al. [45] 85 IVF/ICSI NA NA NA No SCDVelez de la Calle et al. [122]

622 IVF/ICSI NA No Yes No SCD

Tavalaee et al. [123] 92 IVF/ICSI NA Only for ICSI NA No SCD

IVF In vitro fertilization; ICSI intracytoplasmic sperm injection; SCSA Sperm Chromatin Structure assay; TUNEL terminal deoxynucleotidyl transferase dUTP nick-end labelling; SCD Sperm Chromatin Dispersion test; NA not applicable

Table 31.2 (continued)


the American Society for Reproductive Medicine [54]. Although ASRM, after a meta-analysis on 14 published studies, stated that fragmented sperm DNA is more frequent in infertile than in fertile and may contribute to poor reproductive performance, but concluded that, so far, there was no proven role for routine DNA integrity testing in the evaluation of infertility. Other examples are two meta-analyses including stud-ies using either TUNEL and SCSA assays. Both Collins et al. [55], who considered 13 IVF/ICSI studies (9 carried by SCSA and 4 by the TUNEL assay), and Zini et al. [56], who considered 9 IVF (6 carried out by TUNEL assay and 3 by SCSA) and 11 ICSI studies (6 carried by SCSA and 5 by the TUNEL assay) found only small associations between sperm DNA integrity test results and pregnancy in IVF and ICSI. Two other meta-analysis including only SCSA-studies have been performed. Based on 14 papers, Evenson and Wixon [57] reported that in IVF and ICSI, CP was closely related to DFI as mea-sured by SCSA. By contrast, based on three papers, Li et al. [58] found that neither DFI nor HDS had an effect on the chance of CP after IVF or ICSI treatment.

Sperm DNA Damage in Relation to FertilizationThere is conflicting evidence about the relation-ship between sperm DNA fragmentation and fer-tilization rates after IVF and ICSI. Ahmadi and Ng [59] in a mouse model demonstrated that, despite a high DNA damage load, sperm were able to fertilize an oocyte. Also, several studies in the human have shown that men with high num-ber of sperm with damaged DNA can have the same ability to fertilize in vitro as men with a lower fraction of sperm with DNA damage as measured by SCSA [38, 46–50, 58] or by other sperm DNA integrity assays [29, 60–66].

On the contrary, the presence of damaged sperm DNA was shown to have a significant inverse relationship with fertilization in other studies [22, 67] and to contribute to a failure of fertilization even in ICSI [68]. Host et al. [22] found a negative correlation between the propor-tion of spermatozoa with DNA strand breaks and

the fertilization rates in all groups except for those undergoing ICSI.

Also, the SCSA parameter HDS, thought to represent immature spermatozoa with incomplete protamination, was found to be related to IVF fertilization rates, but not in ICSI [50]. Conse-quently, the authors suggested that men with HDS >15% should be treated with ICSI. This finding has, however, not been confirmed by oth-ers, and thus, HDS does not seem to have any clinical impact.

Sperm DNA Damage in Relation to Pre-Embryo DevelopmentAlthough fertilization may be independent of sperm DNA integrity, the post-fertilization devel-opment of the pre-embryo can be impaired by sperm DNA damage.

It has been speculated in if and how sperm DNA damage has impact on human embryo and foetal development as well as on offspring health [69]. Incomplete or aberrant sperm DNA repair by the oocyte is hypothesized to create mutations in the genome of the zygote, which potentially could lead to implantation failure, early miscar-riages or, in worst cases, diseases in the offspring [9, 70, 71]. While the mature spermatozoon itself does not have the capability to repair DNA damage, oocytes and early embryos may have this capacity [72] to a certain degree [73].

Among the first reports to indicate that sperm DNA damage is related to poor embryo develop-ment was studies in mice by Ahmadi and Ng [73]. The human data regarding pre-embryo development in relation to sperm DNA damage is somewhat conflicting. While some authors have reported similar cleavage stage embryo developmental rates between high and low DFI groups as measured by SCSA [44, 46, 47, 52, 74], others have shown that sperm DNA damage is negatively correlated with embryo quality after IVF and ICSI [28, 29, 67]. Two studies have also reported that men with high levels of DNA fragmentation are at increased risk of low blastocyst formation compared to men with a low DFI [50, 75], and consequently, it has been suggested to practice blastocyst culture as a rou-tine in ART.

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Raw vs. Prepared Semen

In a vast majority of cases, spermatozoa used for ART are prepared by density-gradient centrifuga-tion or swim-up methods. Both approaches aim at separating normal sperm from lymphocytes, epithelial cells, abnormal or immature sperm, cell debris, bacteria and seminal fluid. Several previous reports have shown an improvement in the sperm chromatin parameters comparing neat semen samples and samples prepared for ART [39, 49, 61, 76–82]. On the contrary, other reports showed unchanged or worse results [29, 45, 62, 65, 75, 83–87].

One study has analyzed the same semen sam-ples before and after density gradient centrifuged considering 510 ART cycles. In contrast to what has been seen for raw semen, no predictive value of the SCSA parameters DFI and HDS, evalu-ated on the prepared semen, emerged in relation to pregnancy outcome [82]. These data sup-ported the two first SCSA-ART studies where the SCSA parameters were assessed also on prepared semen, even if on a more limited number of patients, 24 and 34, respectively [46, 49]. Using the TUNEL assay, Borini et al. [88] in ICSI patients found DFI >10% in density- gradient-centrifuged semen to be discriminative for pregnancy. Also, Duran et al. [43], in a study on IUI couples, used washed semen samples and found no pregnancy if DFI, as measured by the TUNEL assay, exceeded the level of 12%. Larson et al. [46] suggested that elevated DFI in neat semen may reflect chromatin or other abnor-malities within the entire sperm population interfering with the ability of the sperm to fertil-ize, but not completely eliminated by the sperm preparation procedure.

Incubation of Sperm

Temperature and pH are known to influence on stability and developmental potential of gametes [89, 90], but as yet there is no developed suffi-cient good laboratory standards for incubation of sperm during the period between sperm prepara-tion and fertilization. The duration and environ-

ment for sperm incubation vary from clinic to clinic. Peer et al. [91] found that a 2-h incubation of density-gradient-prepared ejaculates at 37°C led to increased nuclear degradation in terms of vacuolated nuclei in comparison to that at 21°C. Testicular sperm appear to be more susceptible to damage than ejaculated sperm, yet they are subjected to conditions under the assumption that they have similar resistance to injury. For example, incubation under aerobic conditions for 4 or 24 h at 37°C leads to marked sperm DNA damage [92, 93].

Testicular vs. Ejaculated Sperm in ART

Previous reports have shown that sperm DNA damage is significantly lower in the seminiferous tubules compared with the epididymis [94] or in ejaculated sperm [95]. Use of testicular sperm in couples with repeated pregnancy failure in ART and high sperm DNA fragmentation resulted in significant better pregnancy rates [94, 95]. Although use of testicular sperm may only have a potential of solving ROS-induced sperm DNA damage, these findings should be followed up by larger prospective, randomized studies. In the majority of cases, sperm DNA damage is believed to be ROS-induced [96].

The Use of Cryopreserved Sperm in ART

Some studies of cryopreservation of sperm have demonstrated that freezing–thawing has a nega-tive effect on sperm DNA integrity [74], espe-cially in infertile men [93, 97]. Cryopreservation can induce an increased rate of lipid peroxida-tion in the sperm plasma membrane, causing an overall increase in the concentration of oxygen radicals in the sample. Exposure to high ROS concentrations can result in the disruption of mitochondrial and plasma membranes, causing DNA fragmentation and a reduction in sperm motility [98]. Adding antioxidants to the cryo-protection media [99] have shown to be a prom-ising ameliorating procedure. Another strategy shown to cause less chromatin damage to sperm


is freezing of density-gradient-prepared semen instead of raw semen [97]. However, larger studies are needed to clarify whether these are more effective and gentle methods compared to those in use.

Intraindividual Variation of DFI in Relation to ART

One of the drawbacks by the conventional sperm analysis is the huge intraindividual variation reported for concentration, motility and morphol-ogy [100]. By contrast, the first SCSA reports found a lower intraindividual variation for DFI [101]. A more recent study of infertile men in ART treatment has, however, demonstrated a significant day-to-day variation of DFI with a mean coefficient of variation (CV) of 29% [102]. Data from a so far unpublished study has shown that among 616 men who had their semen ana-lyzed by SCSA both in infertility workup and in the actual ART cycle, 85% of the men remained in the same DFI category; £30% or >30% from mea-surement 1 to 2. This implies that only 15% had a clinical effect of repeating the SCSA measurement (Oleszczuk et al., unpublished). Also, data from Giwercman et al. [41] demonstrated that a single SCSA analysis is a strong predictor of infertility.

Future Perspectives

Despite the growing knowledge in the field of sperm chromatin integrity testing in fertility, fun-damental questions remain to be answered as part of a more detailed understanding of sperm chro-matin and its packaging during spermatogenesis, sperm maturation, ejaculation and unpackaging in the oocyte. For further clinical relevance, we need to know more about the following: (1) the type of DNA damage, (2) the percentage of sperm with DNA damage, (3) the extent of DNA damage per spermatozoon, (4) whether there is combined nucleotide damage and DNA fragmentation, (5) whether DNA damage affects introns or exons and (6) the ability of the oocyte to repair sperm DNA damage in the fertilizing sperm [103]. Developing

standardized sperm DNA integrity assays provid-ing such information is of highest value.

We also know too little about the origins of the damage and what can be done to prevent or cure sperm DNA damage. Cause-related therapy in the form of antioxidants has been attempted to reduce DNA damage caused by oxidative stress [95, 104–110]. However, such studies have been rather limited in size and the data are conflicting. Further large-scale studies are needed to investi-gate the type, role and mode of antioxidant ther-apy, as well as other types of causal treatment.

Another important issue for the future should be the development of new sperm separation or sorting techniques where individual or popula-tions of sperm with intact DNA are isolated. Currently, a number of new techniques to favour sperm with normal sperm DNA integrity have been suggested and used; however, none of them are implemented into clinical practice. These include the so-called high-magnification ICSI, a method where spermatozoa with surface vacu-oles are discarded [111] and the recently intro-duced confocal light absorption scattering spectroscopy (CLASS) technology, which allows for the non-invasive visualization of subcellular structures [112]. Also, the use of Annexin-V col-umns has shown to reduce the number of sperm with DNA fragmentation [113].

Another strategy suggested to follow the role of sperm DNA damage on pre-implantation development is to assess whether a quantity of known DNA damage has been repaired by the oocyte or the embryo by analyzing DNA damage in the trophoblast cells obtained by blastocyst biopsy [103].

Data from mice show links between DNA damage in spermatozoa and defects in embryonic development as well as the long-term health of the offspring [114]. However, knowledge on if and how sperm DNA defects may influence the human offspring is lacking, and it is urgent to ini-tiate such studies.

Lastly, the question whether sperm DNA integrity tests can be used as a tool in ART to find the most effective treatment type in a given cou-ple is only partly solved. Although it is clearly shown that men with a SCSA-DFI above 30%

450 M. Bungum

should benefit from being referred directly to IVF/ICSI, it is still questionable whether there is, in these men, a clear difference in efficacy between IVF and ICSI [38]. As all available IVF/ICSI data come from retrospective studies, pro-spective randomized controlled trials should be conducted.

Conclusions and Clinical Recommendations

ART fertility is a multifactorial issue and involves factors from both partners. Sperm DNA integrity status is only one piece in this puzzle. However, it covers an important aspect of sperm quality and function and should be routinely imple-mented as an adjunct to the conventional sperm parameters in fertility workup and ART, espe-cially in unexplained subfertility. Among the sperm DNA integrity tests currently available, SCSA has provided the most stabile clinical threshold values in relation to infertility.

Based on existing data, it is evident that the relevance of sperm DNA integrity testing con-cerns, first of all, in vivo fertilization. In addition to its role as a predictor of natural conception, the SCSA parameter DFI, as measured in raw semen, can be used as an independent predictor of success in couples undergoing IUI. The pre-dictive role of SCSA in IVF and ICSI are, how-ever, more doubtful and needs to be further investigated by prospective randomized studies. In IVF and ICSI, it seems clear that no associa-tion between sperm DNA damage and fertiliza-tion rates exist. The same seems to be the case for embryo development until day 3. This has, however, indicated that blastocyst development is impaired in patients with high numbers of sperm with DNA damage.

In men having standard sperm parameters that indicate ICSI, there are no therapeutic conse-quences of performing SCSA [38, 48–50]. Men with high numbers of DNA-fragmented sperm have similar chances of obtaining pregnancy by IVF and ICSI as men with low sperm DNA frag-mentation. However, the group of men who, first of all, will benefit from SCSA assessment would

be unexplained subfertile men. Roughly, 20–25% of subfertile men, one out of four, with normal WHO sperm parameters have a SCSA-DFI above 20–30%, which is the DFI level where the chance of giving rise to a spontaneous or IUI-induced pregnancy reduces significantly. In order to find men with sperm DNA damage as a hidden cause to their childlessness, where the traditional semen analysis shows one or no abnormality, a SCSA analysis should be offered [41]. In men where all standard parameters are normal, chances of in vivo pregnancy starts to reduce for DFI above 20%. In the presence of one abnormal semen quality parameter, the chance of spontaneous pregnancy is significantly reduced already at DFI above 10%. Thus, in such couples DFI should be taken into consideration and the couples should be referred directly to IVF/ICSI [38].

The SCSA parameter DFI is more stable than the conventional WHO parameters.[115] In most men, a single analysis is enough to be of clinical value for the choice of ART treatment. However, when DFI is above 20% it is recommended to repeat the test prior to the actual ART treatment. Unfortunately, couples seeking ART are only to a limited degree counselled in regard to the impact of lifestyle factors on fertility. Existing knowl-edge on factors contributing to sperm DNA dam-age as for instance smoking and obesity should to a higher degree be communicated to the couples.

Laboratory procedures can harm sperm DNA integrity. In order to prevent further sperm DNA damage and to sort out sperm with fragmented DNA, density-gradient preparation is a good choice for sperm preparation. However, one should be aware that repeated centrifugations as well as the speed of centrifugation could have negative effects on sperm chromatin. Also, to prevent further DNA damage, semen samples should be processed as close to the fertilization procedure as possible. Cryopreserved and thawed semen should be tested in regard to sperm DNA damage prior to use in ART, and the fertilization method chosen according to the DFI should be assessed post thawing.

In conclusion, more research is needed to improve our current knowledge on DNA anoma-lies in spermatozoa. It is necessary to standardize


better the methods of DNA damage evaluation and the time to apply them as pregnancy predic-tors in assisted reproduction. Moreover, a greater insight into the causes of sperm DNA damage is needed to develop appropriate treatment strate-gies and to enhance the genomic integrity of spermatozoa, thus contributing to optimize assisted reproduction outcome. So far, available data has shown that DFI as measured by SCSA can be used as a valuable tool in ART treatment and adds to the clinical management of subfer-tile/infertile couples. DFI has also shown to be an independent predictor of fertility in IUI and can be used to decide which type of ART treatment is needed for a couple.

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sperm chromatin || sperm chromatin and art (iui, ivf and icsi) pregnancy

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