Keywords

Introduction

Sperm DNA quality plays a major role in male fertility. Many couples failing to achieve pregnancy may be affected by male genomic factors, including sperm DNA damage (Siffroi et al. 1997; Joly-Helas et al. 2007; Kim et al. 2011; Sakkas and Alvarez 2010). It has been demonstrated that when the male partner has a high rate of DNA-damaged spermatozoa, the couple has a high probability of waiting a long time before conceiving (Loft et al. 2003) and should be considered of low potential for natural fertility (Spanò et al. 2000; Zini et al. 2001). Furthermore, if these couples finally achieve pregnancy, they have a higher risk of miscarriage (Spanò et al. 2000). Finally, it should also be pointed out that multiple studies have demonstrated that sperm DNA damage increases with advancing male age (Humm and Sakkas 2013).

For these reasons, the selection of spermatozoa without DNA fragmentation and chromosomal diseases prior to assisted reproductive (AR) techniques helps to optimize the outcome of the treatment, in particular, in the case of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (Ebner et al. 2012). In fact, ICSI of aneuploid spermatozoa seems to be the cause of the vast majority of genetic deviations in ICSI newborns (Bonduelle et al. 2002), and the insemination of DNA-damaged spermatozoa seems to be involved in an increased abortion rate (Zini et al. 2008). Furthermore, the injection of DNA-damaged spermatozoa also seems to have long-term side effects in adult animals, such as aberrant growth, premature aging, abnormal behavior, and mesenchymal tumors (Fernandez-Gonzalez et al. 2008). In addition, it should be pointed out that the widespread use of ICSI increases not only the chances of injecting spermatozoa that are defective in their genetic constitution (Sakkas et al. 1997; Marchesi and Feng 2007) but also for centrosome integrity (Schatten and Sun 2009), phospholipase C zeta content (Heytens et al. 2009), and DNA methylation (Navarro-Costa et al. 2010).

For all these reasons sperm selection prior to in vitro insemination by IVF and ICSI is crucial. This selection should be performed keeping to a minimum any additional risk that might increase sperm damage in the laboratory, and – at least for those processes that might be influenced by the embryologist – should be performed in a safe and preferably physiological manner (Parmegiani et al. 2010a, b, c).

In the case of DNA damage, before IVF–ICSI, the basal sperm DNA fragmentation rate can be significantly reduced by some sperm processing procedures such as swim-up (Spanò et al. 2000; Parmegiani et al. 2010a), density gradient (Sakkas et al. 2000), selection by motility without centrifugation (Ebner et al. 2011), and magnetic cell sorting (MACS) with annexin V (Rawe et al. 2010).

These semen selection procedures improve the percentage of spermatozoa with a normal chromatin structure by filtering out DNA-damaged spermatozoa. After this first step, new advances in micromanipulation can be performed to chose the “ideal” mature spermatozoa for ICSI by the restoration of natural fertilization checkpoints such as sperm-hyaluronic acid binding (Parmegiani et al. 2010a) or sperm-zona pellucida binding (Paes de Almeida Ferreira Braga et al. 2009) or by using high magnification microscopy, i.e., intracytoplasmic morphologically selected sperm injection (IMSI) (Bartoov et al. 2003).

Sperm DNA Damage

DNA fragmentation can be induced by numerous factors, such as apoptosis, oxygen radicals, radiation and chemotherapy, or environmental toxicants. It has been hypothesized that DNA damage in sperm may occur during production or transport by the testes (Sakkas and Alvarez 2010). During spermatogenesis, specific screening mechanisms in the testes mark individual spermatozoa with apoptotic markers, which cause phagocytosis of these cells (Billig et al. 1996). Furthermore, testicular endogenous nuclease activity, assisting protamination, increases the percentage of DNA-damaged spermatozoa (McPherson and Longo 1993). Since testicular spermatozoa show lower levels of DNA damage than epididymal or ejaculated sperm, it could be hypothesized that sperm DNA fragmentation is even more frequently generated during sperm transport through the epididymis (Ollero et al. 2001; Greco et al. 2005). In this scenario, if intratesticular and intraepididymal mechanisms for the removal of defective spermatozoa do not function properly, these gametes will end up in the ejaculate. In fact, sperm DNA fragmentation is high in oligoasthenoteratozoospermic men and correlates with failed fertilization, impaired preimplantation development, and altered pregnancy outcome (Evenson et al. 1999; Carrell et al. 2003; Seli et al. 2004; Borini et al. 2006; Velez de la Calle et al. 2008; Zini et al. 2008). Sperm DNA integrity, and in particular DNA fragmentation, is commonly checked in assisted reproduction (AR) laboratories by specific tests:

  • Sperm chromatin structure assay (SCSA) (Evenson et al. 2002)

  • TdT-mediated-dUTP nick-end labeling (TUNEL) (Gorczyca et al. 1993)

  • Sperm chromatin dispersion (SCD) test (Fernandez et al. 2003)

  • Comet assay (Hughes et al. 1996; Enciso et al. 2009).

These assays are based on two different approaches – directly detecting DNA damage (direct tests, e.g., TUNEL) or measuring DNA fragmentation after a rather mild denaturation process (indirect tests, e.g., SCSA, SCD). Both direct and indirect assays seem to be reliable in predicting the levels of DNA fragmentation (Chohan et al. 2006; Zhang et al. 2010). DNA breaks can be single- or double-stranded; single defects are probably easier to repair compared with double breaks (Sakkas and Alvarez 2010), which may generate reciprocal translocations (Richardson and Jasin 2000).

DNA Damage and the Outcome of Assisted Reproduction Techniques

Animal models can be useful in investigating the effect DNA-damaged spermatozoa (Ahmadi and Ng 1999a, b; Fernandez-Gonzales et al. 2008; Upadhya et al. 2010). It is now known that DNA-damaged spermatozoa have the ability to fertilize an oocyte, but embryo development seems to be related to the degree of DNA damage (Upadhya et al. 2010; Ahmadi and Ng 1999a, b). Furthermore, it can be hypothesized that the oocyte can repair the sperm DNA damage. However, this repairing mechanism seems to be efficient only if sperm DNA damage is less than 8 %; damage beyond this level could result in a low rate of embryonic development and high early pregnancy loss (Ahmadi and Ng 1999a).

In humans, the correlation between sperm DNA damage and low fertility is evident in natural conception and when performing a “low-technology” AR procedure such as intrauterine insemination (IUI). One prospective study revealed that above a threshold of 12 % sperm having strand breaks, no clinical pregnancy was achieved when performing IUI (Duran et al. 2002). Others studies on IUI confirmed that the best results in terms of pregnancy rate are obtainable when sperm DNA fragmentation index (DFI) is lower than 27–30 % (Bungum et al. 2004, 2007).

When analyzing in vitro insemination techniques, IVF and, particularly, ICSI, the correlation between sperm DNA damage and clinical outcome is more controversial (Table 10.1). Some studies – five original papers (Bungum et al. 2004, 2007, 2008; Gandini et al. 2004; Lin et al. 2008) and two metaanalyses (Li et al. 2006; Collins et al. 2008) -found no correlation between sperm DNA fragmentation in the neat semen and fertilization rate, embryo quality, or clinical pregnancy. Other studies demonstrated that ICSI allowed a better clinical outcome than IVF in the case of high sperm DNA fragmentation rate (Bungum et al. 2004, 2007; Huang et al. 2005). Furthermore DNA damage is inversely correlated with IVF fertilization rates (Sun et al. 1997; Høst et al. 2000a, b; Huang et al. 2005; Borini et al. 2006). Simon et al. observed that increased sperm DNA fragmentation resulted in lower fertilization rates, poorer embryo quality, and reduced pregnancy rates after IVF and ICSI (2011) and in reduced live birth rates in IVF, but not in ICSI (Simon et al. 2013).

Table 10.1 Effect of sperm DNA damage on IVF/ICSI outcomea
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In a very recent study on sibling oocytes, Pregl Breznik et al. (2013) found that sperm DNA damage was inversely related to fertilization and embryo quality in IVF, but the DFI had no relation to these parameters in the case of ICSI. According to these findings, ICSI should be the method of choice when the DFI is near or exceeds the threshold value of 30 % (Bungum et al. 2004, 2008).

DNA Damage and IVF/ICSI; Fertilization, Embryo Quality-Development Rate

Some authors have found a negative correlation between sperm DNA fragmentation and fertilization rate (Lopes et al. 1998; Benchaib et al. 2003; Huang et al. 2005; Borini et al. 2006) and zygote formation (Muriel et al. 2006) in both IVF and ICSI. However, this seems to be balanced in ICSI by a good subsequent development of embryos (Payne et al. 2005). Other studies reported an inverse correlation between DFI and embryo quality (Tomsu et al. 2002; Virant-Klun et al. 2002; Avendaño et al. 2010; Seli et al. 2004; Virro et al. 2004; Muriel et al. 2006).

DNA Damage and IVF/ICSI; Pregnancy-Implantation Rate

Meseguer et al. (2011) measured the effect of the DFI on subsequent pregnancy independent of the fertilization procedure (IVF or ICSI) or sperm origin (ejaculate or testicular) and calculated that for every 10 % increase in the DFI, the probability of not achieving pregnancy increased by 1.31. Avendaño et al. (2010) found a 3.5 times higher likelihood of pregnancy when the DFI was less than or equal to 17.6 % after sperm treatment. The DFI threshold in raw semen is still controversial: in a 2003 study, no clinical pregnancy was noted in the case of a DFI greater than or equal to 27 % (Larson-Cook et al. 2003), whereas Henkel et al. (2004) stated that the probability of pregnancy is higher when DFI is less than 36.5 %. Correspondingly, Spanò et al. (2000) observed that under a threshold of 40 % DFI, the pregnancy rate was significantly higher than in patients with a higher DFI. Similarly, a prospective analysis on 360 couples (Simon et al. 2010) showed significantly lower pregnancy rates above a threshold of 44 % DFI.

In terms of implantation, ICSI with a DFI greater than or equal to 19 % was associated with a lower implantation rate (Speyer et al. 2010). It has been hypothesized that “healthy/young” oocytes may compensate for the potential adverse effects on pregnancy and implantation of high sperm DNA fragmentation, but this has been refuted by some studies (Borini et al. 2006; Frydman et al. 2008). Nevertheless, some refinements of ICSI that allow us to minimize the risk of injection of DNA damaged spermatozoa seem to improve the clinical outcome, especially in the case of oocytes of older women (Souza Setti et al. 2013); this may suggest that sperm DNA damage plays a critical role in the case of advanced age of the female partner.

DNA Damage and IVF/ICSI Miscarriage Rate

It has been hypothesized that, although DNA-damaged spermatozoa can fertilize an oocyte, they are often associated with failed pregnancy or pregnancy loss (Henkel et al. 2004). Borini et al. (2006) observed a higher miscarriage rate in ICSI patients with a DFI greater than 10 %. Kennedy et al. (2011) reported a significant correlation between DFI and spontaneous abortion; furthermore, they observed that couples having triplet pregnancies had a lower DFI than those with pregnancy loss. Conversely, Bellever et al. (2010) found no correlation between a high DFI and spontaneous abortion. In a recent large-scale metaanalysis (1,549 cycles: 640 pregnancies and 122 pregnancy losses) Zini et al. (2008) concluded that sperm DNA damage is associated with a significantly increased risk of pregnancy loss after both IVF and ICSI (2008).

Remarks

All these studies seem to indicate a correlation between DNA damage and outcome of AR technologies, including IVF and ICSI. Since the outcome of AR treatments depends on many more factors than just sperm quality (above all female factors, but also environmental factors, patient selection, choice of appropriate AR treatment, and others), this makes it extremely difficult to compare the studies and to find a general agreement on the role of sperm DNA damage. Furthermore, it should be pointed out that sperm DNA fragmentation is not a static seminal parameter and the timing for assessing the DNA damage is critical: the in vitro aging and spermatozoa or exposure to nonphysiological conditions before DNA testing have a detrimental effect on DNA (Santiso et al. 2012). Unfortunately, information on the timing of DNA testing and DNA fragmentation dynamics is very often absent in the literature. In conclusion, the various factors influencing treatment outcome, the timing in performing the DNA fragmentation test, and the use of different DNA tests contribute to jeopardizing the reliability of the majority of the studies in the literature.

Sperm Selection for Assisted Reproduction Techniques: Effect on Sperm DNA Quality

DNA fragmentation rate in the raw semen can be reduced by sperm processing procedures, which improve the percentage of spermatozoa with a normal chromatin structure by filtering out apoptotic spermatozoa. In the case of in vitro insemination techniques (IVF/ICSI) following sperm preparation, high-magnification microscopy and selection by some sperm markers are valid tools in selecting spermatozoa with a reduced risk of being DNA-damaged.

Relationship Between Sperm Motility and DNA Integrity

It is accepted that oligozoospermia (Høst et al. 1999; Burrello et al. 2004), teratozoospermia (Høst et al. 1999; Muratori et al. 2003), and asthenozoospermia (Irvine et al. 2000; Giwercman et al. 2003; Varghese et al. 2009; Mahfouz et al. 2010) are associated with a higher percentage of sperm DNA aberrations.

Conversely, it seems that good sperm motility is related to sperm DNA integrity (Van den Bergh et al. 1998; Ramos and Wetzels 2001; Avendaño and Oehninger 2011). This has been highlighted by Ebner and colleagues (2011), who showed that fast progressive spermatozoa have intact DNA. This is probably due to the fact that not only nuclear but also mitochondrial DNA can be affected by DNA fragmentation (Alvarez 2005); in the latter case, the spermatozoa may have alterations in the production of ATP, which is the “fuel” for sperm motility. This hypothesis is confirmed by various studies. Kasai et al. (2002) observed a direct association between motility, mitochondrial volume, and membrane potential within the sperm midpiece; mutations or deletions within mitochondrial DNA were shown to be associated with reduced sperm motility (Ozmen et al. 2007); and Speyer et al. (2010) noted a positive correlation between DFI and sperm midpiece.

Sperm Preparation Techniques Before IVF/ICSI

In the case of DNA damage, prior to IVF–ICSI, basal sperm DNA fragmentation rate can be significantly reduced by some sperm processing procedures. These allow us to select motile spermatozoa, filtering out the low-motile apoptotic ones. During sperm preparation special care should be taken because it should be kept in mind that these procedures themselves might cause DNA damage (Twigg et al. 1998; Donelly et al. 2001; Dalzell et al. 2004; Santiso et al. 2012; Toro et al. 2009). First of all, the sperm must be processed immediately after liquefaction in order to avoid in vitro aging and degeneration of nuclear proteins (Sakkas and Alvarez 2010); then the procedure must be performed so as to avoid spermatozoa exposure to stressful conditions, such as, for example, too high/low temperature and repeated high-speed centrifugation.

Although some articles reported no significant reduction in basal DFI following sperm preparation (Zini et al. 2000a; Gandini et al. 2004; Stevanato et al. 2008; Ebner et al. 2011; Boe-Hansen et al. 2005; Zini et al. 2000b), the vast majority of studies in this field support the fact that the following techniques improve the yield of DNA-intact male gametes:

  • “Swim-up” (Zini et al. 2000a; Younglai et al. 2001; Jackson et al. 2010; Marchesi et al. 2010)

  • Density gradient centrifugation (Donnelly et al. 2000, 2001; Morrell et al. 2004; Ahmad et al. 2007; Jackson et al. 2010; Marchesi et al. 2010; Castillo et al. 2011)

  • Sperm selection based on membrane charge (Chan et al. 2006; Razawi et al. 2010)

  • Cell sorting using a membrane-based electrophoretic filtration system (Fleming et al. 2008)

  • Magnetic-activated cell sorting (Said et al. 2006; Lee et al. 2010; Polak de Fried and Denaday 2010; Rawe et al. 2010)

  • Selection by motility without centrifugation (Ebner et al. 2011).

    These studies are summarized in Table 10.2.

    Table 10.2 Sperm processing techniques and DNA fragmentation index (DFI) reductiona
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Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)

The introduction of ICSI (Palermo et al. 1992; Van Steirteghem et al. 1993) made possible pregnancies also in couples affected by severe male factor, in which the male partner had low sperm motility, poor zona binding, poor acrosome reaction, or abnormal sperm morphology. The observation of sperm morphology (Kruger et al. 1986, 1988) at the conventional magnification used for sperm evaluation (400×) has controversial prognostic value in ICSI cycle outcomes (Svalander et al. 1996; De Vos et al. 2003; French et al. 2010) and does not seem to influence embryo development or morphology (French et al. 2010; De Vos et al. 2003). However, at this magnification, sperm dimension and shape are not reliable attributes for predicting chromatin integrity or the presence of numerical chromosomal aberrations (Celik-Ozenci et al. 2004).

When using an unstained, real-time, high-magnification examination of spermatozoa called motile sperm organelle morphology examination (MSOME), it is possible to overcome the limitations of conventional magnification (Bartoov et al. 1994, 2001, 2002). MSOME is performed using an inverted light microscope equipped with high-power Nomarski optic enhanced by digital imaging to achieve a magnification of up to 6300× (Fig. 10.1). “The ICSI procedure using MSOME criteria is defined as IMSI: intracytoplasmic morphologically selected sperm injection” (Bartoov et al. 2003). Some authors have demonstrated that ICSI outcomes can be significantly improved by the exclusive microinjection of spermatozoa with a strictly defined, morphologically normal nucleus, especially in couples with previous ICSI failures (Bartoov et al. 2003; Berkovitz et al. 2005; Hazout et al. 2006; Antinori et al. 2008; Franco et al. 2008; Souza Setti et al. 2010), with severe male factor (Balaban et al. 2011; Souza Setti et al. 2011), or with advanced maternal age (Souza Setti et al. 2013). Conversely, the injection of spermatozoa with abnormal sperm head or with nuclear vacuoles negatively affects embryo development (Vanderzwalmen et al. 2008; Cassuto et al. 2009) and ICSI outcome (Berkovitz et al. 2006a, b; Cassuto et al. 2009; Nadalini et al. 2009). The positive effect of the injection of spermatozoa without nuclear vacuoles is probably related to their significantly better mitochondrial function and chromatin status, and reduced aneuploidy rate as compared with vacuolized spermatozoa (Garolla et al. 2008; Boitrelle, et al. 2011). Furthermore, spermatozoa free of nuclear morphological malformations were found to be significantly associated with a lower incidence of aneuploidy in derived embryos (Figueira et al. 2011). However, in a few studies ICSI had the same performance as IMSI in terms of fertilization and early embryo development (Nadalini et al. 2009; Mauri et al. 2010; De Vos et al. 2013);

Fig. 10.1
figure 00101

Spermatozoon at high magnification (>6300×) for IMSI

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It should be pointed out that the large majority of articles published on IMSI were based on weak study designs or performed on a small number of patients. However, in a recent strict prospective sibling-oocyte study comparing ICSI and IMSI, performed on 350 treatments, De Vos and colleagues (2013) observed comparable results in terms of fertilization, embryo quality, and clinical outcome between the two groups. They concluded that a routine application of IMSI in unselected AR technology patients cannot be advocated. Furthermore, it is true that IMSI is undoubtedly a time-consuming procedure; selecting a normal spermatozoon according to MSOME criteria may require 60–120 min (Antinori et al. 2008). In addition, the process of searching for spermatozoa at high magnification may itself damage sperm cytoplasm: after 2 h on the microscope’s heated stage, sperm nucleus vacuolization significantly increases (Peer et al. 2007). Despite these observations, IMSI can be considered a valid tool for safe, noninvasive selection of spermatozoa that have a reduced risk of DNA damage (Garolla et al. 2008; Ebner et al. 2012).

Restoration of Natural Fertilization Checkpoints: Hyaluronic Acid and Zona Pellucida Sperm Binding

In humans during the natural fertilization process, spermatozoa must pass two barriers before fusing with the oocyte membrane. The first is the cumulus oophorus–corona radiata complex, made up of cells and an extracellular matrix of polymerized hyaluronic acid (HA) and proteins. The second is the zona pellucida (ZP), a thick elastic coat of glycoproteins located right next to the oocyte (Yanagimachi 1994). In addition, in the testes during spermiogenesis, still immature spermatozoa (elongated spermatids) undergo cytoplasmic extrusion and plasma membrane remodeling to prepare themselves for oocyte fertilization (Kovanci et al. 2001). This sperm membrane remodeling determines the formation of the HA and ZP receptors. In human spermatozoa, two specific proteins are related to maturity, DNA integrity, chromosomal aneuploidy frequency, and fertilizing potential: the heat shock protein HspA2 chaperone – involved in meiosis – and creatine kinase (CK) – abundant in the sperm cytoplasm (Cayli et al. 2003). Mature spermatozoa have a high expression of HspA2 (Huszar et al. 2000) and a low expression of CK (Cayli et al. 2003). In contrast, spermatozoa with arrested maturity have a low HspA2 and a high CK expression. Low levels of HspA2 can cause meiotic defects and chromosomal aneuploidies, whereas high retention of CK and other cytoplasmic enzymes can cause lipid peroxidation and, consequently, DNA fragmentation and abnormal sperm morphology (Huszar and Vigue 1993; Cayli et al. 2003).

Due to a lack of or partial membrane remodeling, immature spermatozoa have deficiency in the ZP and HA binding sites, and for this reason they are not able to fertilize oocytes naturally.

Hyaluronic Acid Binding

Since in natural fertilization human oocytes are surrounded by HA, this glycoprotein has a pivotal role in the mechanism of sperm selection. In fact, only mature spermatozoa that have extruded their specific receptors to bind to HA are able to reach and fertilize the oocyte. The role of HA as a physiological selector is also well recognized in vitro. In fact, spermatozoa that can bind to HA in vitro have completed their plasma membrane remodeling, cytoplasmic extrusion, and nuclear maturation (Cayli et al. 2003; Huszar et al. 2003, 2007).

Based on this selective ability of HA in binding only to mature sperm (Huszar et al. 2003; Prinosilova et al. 2009), a very simple diagnostic tool for assessing sperm maturity and function, the so-called sperm-hyaluronan-binding assay (HBA), has been developed as a commercial kit (Cayli et al. 2003; Huszar et al. 2003). HBA may be useful in predicting sperm-fertilizing ability in IVF or ICSI. In fact, HBA was found to be correlated with morphology (Ye et al. 2006; Tarozzi et al. 2009) but to a less significant extent than the association between sperm morphology and fertilization rate in IVF (Ye et al. 2006). At present, HBA does not seem to provide information for identifying patients with poor or absent fertilization (Ye et al. 2006; Nijs et al. 2010) or a threshold value for outcome in conventional IVF/ICSI treatment (Nijs et al. 2010; Kovacs et al. 2011). On the other hand, HBA seems to be significantly correlated with good embryo quality, miscarriage rate, and ongoing pregnancy rate after ICSI (Nijs et al. 2010) and with fertilization rate after IVF (Pregl Breznik et al. 2013). Recently, Worrilow et al. (2010, 2013) demonstrated that in patients in whom less than 65 % of total spermatozoa were bound to HA (HBA ≤ 65 %), a modified ICSI procedure with HA-bound spermatozoa – physiological HA-ICSI (see next paragraph) – produced a significant improvement in clinical outcome compared with conventional ICSI.

Physiological HA-ICSI

The selection of mature spermatozoa by HA represents a straightforward refinement of the ICSI procedure (Parmegiani et al. 2010a, b). It is known that HA-containing products have no negative effects on postinjection zygote development and can be metabolized by the oocyte, and HA-bound spermatozoa can be easily recovered using an injecting pipette (Balaban et al. 2003; Barak et al. 2001; Van den Bergh et al. 2009). A “homemade” HA–sperm selection system can be produced in any IVF lab; however, at the present time two ready-to-use systems specially designed for sperm–HA binding selection are currently available: (1) PICSI, a plastic culture dish with microdots of HA hydrogel attached to the bottom of the dish, and (2) SpermSlow, a viscous medium containing HA that can be used instead of polyvinylpyrrolidone (PVP), the viscous medium commonly used to slow spermatozoa prior to ICSI.

This new approach to ICSI with HA-bound spermatozoa, when using a HA-viscous medium or HA-culture dishes, has been called physiological ICSI (Parmegiani et al. 2010a). It has been demonstrated that the injection of HA-bound spermatozoa improves embryo quality and development by favoring selection of spermatozoa with normal nucleus and intact DNA (Parmegiani et al. 2010a). Furthermore, improved implantation rates were observed when injecting HA-bound sperm as compared to PVP sperm (Parmegiani et al. 2010b). Nasr-Esfahani et al. (2008) found a higher fertilization rate when injecting oocytes with HA-selected spermatozoa using homemade HA–ICSI dishes. An improvement in fertilization rate and embryo quality and a reduction in the number of miscarriages were found by Worrilow et al. (2007) with PICSI versus conventional ICSI. In subsequent studies, the same authors confirmed that PICSI significantly improved clinical results in ICSI, particularly in patients with an HBA score less than or equal to 65 % (Worrilow et al. 2010, 2013). In contrast, some authors found no differences in ICSI outcome parameters when injecting HA-bound sperm compared with PVP sperm (Mènèzo and Nicollet 2004; Sanchez et al. 2005; Mènèzo et al. 2010; Van den Berg et al. 2009).

At the very least, in none of the studies did physiological HA-ICSI cause a detrimental effect on ICSI outcome parameters. If larger, multicenter, prospective-randomized studies confirm the suggested beneficial effects on ICSI outcome, HA should be considered the first choice for physiologic sperm selection prior to ICSI because of its capacity to reduce genetic complications and for its total lack of toxicity (Parmegiani et al. 2010c). In addition, the use of HA-ICSI avoids PVP, which seems to be toxic and to generate sperm DNA fragmentation (Salian et al. 2012). Since both the sperm–HA binding selection systems (PICSI and SpermSlow) are easily available, efficient, and approved for human IVF use, AR centers can choose the one best suited to their needs (Parmegiani et al. 2012a).

Zona-Bound Spermatozoa

As well as binding for HA, the spermatozoa–ZP binding process also plays a crucial role in the natural selection of spermatozoa. Immature spermatozoa show a low density of ZP binding sites as well as HA receptors (Huszar et al. 2003). Sperm binding to ZP exhibit attributes similar to those of HA-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies (Yagci et al. 2010). Furthermore, the same sperm membrane protein seems to be involved, firstly, in hyaluronidase activity and, subsequently, in ZP binding (Hunnicutt et al. 1996). The spermatozoa–ZP binding test can be performed by culturing spermatozoa for a short time with immature metaphase I oocytes; in this way, ZP-bound spermatozoa can be recovered and used for ICSI (Paes de Almeida Ferreira Braga et al. 2009). An increased embryo quality was observed when ZP-bound ICSI was performed compared with conventional ICSI (Paes de Almeida Ferreira Braga et al. 2009; Liu et al. 2011). In a small study, Black et al. (2010) observed a slightly better trend in pregnancy and implantation comparing 39 ZP-ICSI versus 39 conventional ICSI. At the present time, there is little information regarding all the factors involved in sperm-ZP binding and its mechanism, but the spermatozoa–ZP binding test could be an efficient method for identifying competent spermatozoa for ICSI. ZP selection could then be coupled with HA selection in order to replicate the natural path of the spermatozoa toward the oocyte.

Conclusions

Several factors contribute to the outcome of AR treatments, sperm DNA quality being just one factor. Although the role of sperm DNA damage in the clinical outcome of high-technology AR procedures like ICSI could still be considered debatable, it is undoubtedly wise to use all precautions to limit any potential long-term side effects of these techniques (Ebner et al. 2012).

At present, in the literature, there are still few data on the selection of the ideal sperm for ICSI, perhaps because for many years it was not possible to identify definitively a DNA-intact spermatozoon (Nijs et al. 2009). A potentially worrying aspect of the ICSI of DNA-damaged spermatozoa has been suggested by studies performed on animals that showed not only a negative effect on pregnancy and birth, but also later side effects on the health of adult animals such as aberrant growth, premature aging, abnormal behavior, and mesenchymal tumors (Fernandez-Gonzales et al. 2008). Fortunately, in humans, the risk of injecting DNA-damaged spermatozoa seems to be minimized by conventional sperm preparation techniques prior to ICSI (Zini et al. 2000a; Younglai et al. 2001; Donnelly et al. 2000, 2001; Morrell et al. 2004, Ahmad et al. 2007; Jackson et al. 2010; Marchesi et al. 2010; Castillo et al. 2011), and follow-up studies on ICSI children have demonstrated the basic safety of this revolutionary technique (Van Steirteghem et al. 2002; Leunens et al. 2008; Belva et al. 2011; Woldringh et al. 2011), although a slight increase in chromosome aberration seems to be caused by the injection of aneuploid spermatozoa (Bonduelle et al. 2002).

Given the need to reduce all potential long-term risks on ICSI adults, the search for the ideal spermatozoa is a stimulating challenge for embryologists. A final precise sperm selection is performed at the micromanipulator via IMSI, HA, or ZP sperm binding to minimize the chance of injecting DNA-damaged sperm. There is evidence that HA-bound mature spermatozoa show better morphology than immature sperm, both at conventional (Tygerberg criteria) and at high magnification (MSOME criteria) (Prinosilova et al. 2009; Nasr-Esfahani et al. 2008; Oliveira et al. 2009; Parmegiani et al. 2010a). Furthermore, HA-bound sperm show a low DFI and a reduced incidence of aneuploidies (Nasr-Esfahani et al. 2008; Parmegiani et al. 2010a; Jakab et al. 2005; Sanchez et al. 2005; Yagci et al. 2010). HA-bound mature spermatozoa showed no cytoplasmic retentions, persistent histones, or DNA fragmentation (Huszar et al. 2007). It should be hypothesized that ZP-bound spermatozoa have characteristics similar to those of HA-bound sperm (Huszar et al. 2007; Yagci et al. 2010; Hunnicutt et al. 1996). In addition, sperm–HA binding seems to be related to DNA methylation, which is important for correct chromatin packaging; in fact, in patients with a total lack of HA binding this feature is restored with supplementation of a methylation effector (Junca et al. 2012). Since during normal spermiogenesis and plasma membrane remodeling, along with formation of the ZP binding site, formation of the HA binding site also occurs, this should form the basis of ideal ICSI sperm selection (Huszar et al. 2007; Ebner et al. 2012).

Regarding sperm morphology, modern technology allows us to obtain very high magnification, and the introduction of IMSI should be considered an exciting chapter in IVF history, whether or not its clinical usefulness is confirmed with studies on a large scale. IMSI technology has enabled exploration of the relationship between the morphology of spermatozoa and their DNA constitution, revealing that vacuole-free spermatozoa have a lower rate of DNA-fragmentation (Franco Jr. et al. 2008), MSOME abnormal spermatozoa show a high level of DNA fragmentation (Wilding et al. 2011), and, in particular, spermatozoa with large vacuoles are characterized by aneuploidies and chromatin condensation defects (Perdrix et al. 2011).

IMSI’s lengthy procedure can be circumvented by preselecting HA sperm merging high-magnification microscopy with HA-sperm selection. This procedure has been called physiological IMSI (Parmegiani et al 2012b). In fact, HA helps to select a subpopulation of spermatozoa with normal nuclei according to MSOME criteria (Parmegiani et al. 2010a).

In conclusion, most of the alternative possible refinements of the ICSI procedure described in this chapter are easily reproducible and straightforward; for this reason, they could be offered to ICSI patients not only to optimize clinical results but, at the very least, to prevent fertilization by DNA-damaged and chromosomally unbalanced spermatozoa.