Improving ICSI: A review from the spermatozoon perspective

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1 Systems Biology in Reproductive Medicine ISSN: (Print) (Online) Journal homepage: Improving ICSI: A review from the spermatozoon perspective Mara Simopoulou, Laertis Gkoles, Panagiotis Bakas, Polina Giannelou, Theodoros Kalampokas, Konstantinos Pantos & Michael Koutsilieris To cite this article: Mara Simopoulou, Laertis Gkoles, Panagiotis Bakas, Polina Giannelou, Theodoros Kalampokas, Konstantinos Pantos & Michael Koutsilieris (2016) Improving ICSI: A review from the spermatozoon perspective, Systems Biology in Reproductive Medicine, 62:6, , DOI: / To link to this article: View supplementary material Published online: 20 Sep Submit your article to this journal Article views: 1180 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 05 December 2017, At: 23:44

2 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 2016, VOL. 62, NO. 6, REVIEW ARTICLE Improving ICSI: A review from the spermatozoon perspective Mara Simopoulou a,b, Laertis Gkoles b, Panagiotis Bakas b, Polina Giannelou b, Theodoros Kalampokas b, Konstantinos Pantos c, and Michael Koutsilieris a a Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece; b Assisted Conception Unit, 2nd Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; c Centre for Human Reproduction, Genesis Hospital, Athens, Greece ABSTRACT Intracytoplasmic sperm injection (ICSI) is the most frequently applied method for fertilization making the process of identifying the perfect spermatozoon fundamental. Herein we offer a critical and thorough presentation on the techniques reported regarding (i) handling and preparing semen samples, (ii) identifying and fishing spermatozoa, and (iii) improving key factors, such as motility for a successful ICSI practice. These approaches are suggested to make the process easier and more effective especially in atypical and challenging circumstances. Furthermore, we present an epigrammatic opinion-where appropriate-based upon our collective experience. Techniques such as intracytoplasmic morphologically selected sperm injection, hyaluronic binding, polarized light microscopy, and annexin V agent identification for comparing sperm cells and their chromatin integrity are analyzed. Moreover, for the demanding cases of total sperm immotility the use of the hypoosmotic swelling test, methylxanthines, as well as the option of laser assisted immotile sperm selection are discussed. Finally, we refer to the employment of myoinositol as a way to bioreactively improve ICSI outcome for oligoasthenoteratozoospermic men. The diversity and the constant development of novel promising techniques to improve ICSI from the spermatozoon perspective, is certainly worth pursuing. The majority of the techniques discussed are still a long way from being established in routine practices of the standard IVF laboratory. In most cases an experienced embryologist could yield the same results. Although some of the techniques show great benefits, there is a need for large scale multicenter randomized control studies to be conducted in order to specify their importance before suggesting horizontal application. Taking into consideration the aprioriinvasive nature of ICSI, when clinical application becomes a possibility we need to proceed with caution and ensure that in the pursuit for innovation we are not sacrificing safety and the balance of the physiological and biological pathways of the spermatozoon s dynamic. Abbreviations: ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; PGD: reimplantation genetic diagnosis; IVM: in vitro maturation; HCV/HIV: hepatitis C virus/human immunodeficiency virus; IMSI: intracytoplasmic morphologically selected sperm injection; DGC: density gradient centrifugations; S-U: swim-up; ART: assisted reproduction technology; IUI: intrauterine insemination; PVP: polyvinylpyrrolidone; HA: hyaluronic acid; MSOME: motile sperm organelle morphology examination; ZP: zona pellucida; MACS: magnetic activation cell sorting; HOST: hypo-osmotic swelling test; TESE: testicular sperm extraction; MMP: mitochondrial membrane potential; OAT: oligoasthenoteratozoospermic ARTICLE HISTORY Received 26 February 2016 Revised 11 July 2016 Accepted 11 July 2016 KEYWORDS ART; fertilization; ICSI; infertility; spermatozoon Introduction The evolution of several techniques dedicated to assist male factor infertility in the IVF lab incidentally led to the birth of intracytoplasmic sperm injection (ICSI) [Palermo et al. 1992].ThefaceofIVFhasneverbeenthesamesince. ICSI is a powerful procedure through which an oocyte can be fertilized independently of the morphology and/or motility of the single spermatozoon injected. ICSI has taken over ART practice globally accounting for 70 80% of the cycles performed, enabling preimplantation genetic diagnosis (PGD) and serodiscordant couples treatment [Palermo et al. 2009]. The numbers alone require our attention as there is clearly a trend toward an increase of its use worldwide [Nyboe Andersen et al. 2008]. The reality of the matter is that since the early days of ICSI practice a lot has changed with respect to carrying out the technique. More particularly until recently, all male ICSI candidates were offered a chromosomal analysis before the ICSI procedure as men eligible for ICSI treatment are supposed to be at increased risk for CONTACT Mara Simopoulou marasimopoulou@hotmail.com Clinical Embryologist/Geneticist, Departments of Physiology, and Obstetrics and Gynaecology, Medical School, National and Kapodistrian University of Athens, 75 Micras Asias, Goudi, Athens , Greece. Supplemental data for this article can be accessed on the publisher s website Taylor & Francis

3 360 M. SIMOPOULOU ET AL. genetic abnormalities [Dutch Society of Obstetrics and Gynaecology (NVOG) 2010]. However, karyotyping as a prerequisite for ICSI added another level of complexity to the overall treatment from a time and cost perspective [Stegen et al. 2012]. Therefore, this practice along with the extra stress related to yet another test to be taken was slowly dropped. Even though the debate is still active on the benefits of following such a strategy, the risk of conceiving a viable child with unbalanced structural chromosomal abnormalities in men with oligozoospermia may still not justify karyotyping [Stegen et al. 2012]. Nowadays ICSI is almost offered without discrimination or prior testing, to the point that many clinics rely solely on ICSI [NVOG 2010] as the chosen insemination technique. This routine excludes traditional IVF altogether even for cases where there is no indication for ICSI and/ or no male factor involved in the diagnosis. Such a practice should be examined carefully. ICSI does not present to secure higher pregnancy rates than standard IVF, and should only be offered based on specific indications such as low number and poor morphology oocytes, thicker zona, excess polyspermia, PGD testing, treating sero-discordant hepatitis C virus/ human immunodeficiency virus (HCV/HIV) couples, in vitro maturation (IVM), and oocyte cryopreservation. The emerging novel techniques focusing on identifying the fertilization competence of the spermatozoon could perhaps shed light and help to answer the question to ICSI or not to ICSI? [Palermo et al. 2015]. When ICSI is the method of choice for oocyte fertilization there is a much higher need for selecting the perfect sperm cell. But how can someone choose the perfect candidate when the most common assessments proposed, which are swim-up and density gradient centrifugation, are based only on the motility of spermatozoa and do not include any molecular features. Other techniques that are suggested include invasive steps that could be frowned upon. The great need for non-invasive techniques led researchers/embryologists to try to find a connection between visible features on the spermatozoon and its DNA status, whether it s fragmented or not, or carries mutations or not [Hoogendijk et al. 2009]. In the following sections we provide insight into the various approaches in relation to sperm and ICSI practice and evaluate their clinical significance in improving the outcome. The aim of this review is to provide insight into the several options and evaluate their clinical significance in improving the outcome, and hence assess the necessity of incorporating them into routine clinical practice. This is expanded in the online supplemental information that also includes a description of how this critical constructive analysis was conducted. More particularly, this presentation evaluates the preparation treatment, the sperm selection methods, the spermatozoon assessment, and its overall contribution, as well as special interest techniques such as intracytoplasmic morphologically selected sperm injection (IMSI). Sperm preparation: Swim up (S-U) and density gradients centrifugation (DGC) In S-U first described by Mahadevan and Baker [1984] the most motile sperm cells swim up the culture medium while all others stay on the lower layer [Alvarez et al. 1993]. In DGC, sperm cells are separated according to their density, following centrifugation through two discrete layers of discontinuous density gradients [Pousette et al. 1986]. Moreover, the combination of DGC and S-U can also yield favorable results especially with respect to eliminating DNA damaged sperm [Ghumman et al. 2011; Jayaraman et al. 2012]. The question that usually arises is which of the two is more efficient. Several studies tried to give the answer suggesting one over the other [Sakkas et al. 2000; Zini et al. 2000] but results were not conclusive. However, Luppi and colleagues provided a proteomic comparison and concluded that there is a difference between the two and DGC may offer a better capacitation potential, but in most studies it is concluded that both methods provide sperm cell preparations of equal quality [Kim et al. 2015; Luppi et al. 2015]. According to Borges and colleagues both techniques recover an improved sperm fraction and give similar IMSI outcomes [Borges et al. 2013]. Xue and colleagues showed that both result in spermatozoa with normal morphology and intact DNA, having a higher preference for DGC [Xue et al. 2014]. Ricci and colleagues showed that there was no significant difference in the percentage of apoptotic sperm cells in each method [Ricci et al. 2009]. Tachawiwat and colleagues showed that there is no difference in scores of the hyaluronan binding assay [Tachawiwat et al. 2015]. Furthermore the combined procedure of DGC/S-U yields spermatozoa that possess the least nuclei fragmentation and are the most motile [Jayaraman et al. 2012]. In cases of sperm preparation for seropositive (HIV) patients the combined method of DGC/S-U is employed to wash sperm cells from the seminal fluid and decrease the viral load. As it was first described by Semprini and colleagues [1992] the combined method of DGC/S-U removed all HIV-1 infected cells. All babies derived from the processed semen samples and their mothers were seronegative [Semprini et al. 1992]. According to a newer study, in such cases irrespective of etiology and even in non-male factor cases ICSI is required. In ICSI the number of spermatozoa for injection are drastically fewer in comparison to classic IVF or even IUI and with fewer

4 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 361 spermatozoa there is a reduced risk of transmission [Zhang et al. 1998]; plus ICSI in such patients ensures higher pregnancy rates. Each of the methods proposed has its advantages and disadvantages and the question still remains unanswered. Based on bibliography research and the clinical protocol followed in our practice, the combined method of S-U and DGC is at most cases, and especially if ICSI is to be applied, the procedure of choice as the spermatozoa are of higher quality by morphological and motility criteria. It results in higher fertilization rates especially in cases of normal sperm concentration but motility of under 50%. It is important to note that the method of choice to be employed should be based upon the characteristics of each ICSI case. The importance of sperm chromatin integrity The mature spermatozoon is a cell of characteristic morphology and a highly compacted and condensed chromatin structure that is derived through a very specialized biological pathway, spermatogenesis. Faults caused from intrinsic factors (oxidative stress, endogenous endonuclease and caspase activation) or extrinsic factors (radiation, environmental toxicants) in any of the steps of spermatogenesis could affect the chromatin integrity of the sperm cell [Evenson 2016; Ioannou et al. 2016]. A study published by Ioannou and colleagues [2016] highlighted the importance of chromatin packaging in establishing and maintaining a viable pregnancy. Avendano and Oehninger [2011] demonstrated that a high DNA fragmentation index (DFI) can be associated negatively with embryo quality and risk of pregnancy loss, in agreement with the work of Zini and colleagues [2008]. Moreover a high DFI seems to be a contributing factor to recurrent miscarriages [Leach et al. 2015]. On the contrary, in a metanalyses published by Zhang and colleagues [2015] current data do not support the prediction value of DFI for IVF or ICSI outcome and further studies are needed. In addition, Avendano and colleagues showed that spermatozoa processed by the swim-up technique that appear to be morphologically normal may have fragmented DNA in their nucleus [Avendano et al. 2009]. Various sperm DNA fragmentation tests have been developed such as the TUNEL test that can measure thousands of spermatozoa by flow cytometry but could take up to several hours,theacridineorange(ot)test,thecomet test, and the sperm chromatin structure assay (SCSA) that require a flow cytometer [Evenson 2016]. There is also the sperm chromatin dispersion test (SCD) or Halosperm that requires solely a light microscope as laboratory equipment. In Halosperm the deproteinized nuclei create a halo of dispersed DNA. Nuclei with intact DNA produce either small halos or no halos of dispersed DNA. Big or medium halos are observed in nuclei without or slight DNA fragmentation. Tandara and colleagues [2014] in their work stated that there was a positive correlation between high DFI and significant big halo ratio with embryo quality and pregnancy rate in conventional IVF. The major setback of these tests is that they can only be used prior to ICSI in determining the sample s profile in order to separate samples that could be more suitable for routine IVF than ICSI [Pregl Breznik et al. 2013] and not used to detect and indicate the right spermatozoon for injection. The purpose of this review was to present methods that can be employed during ICSI in order to identify the best viable spermatozoa. Advances in micromanipulation such as IMSI [Bartoov et al. 2001], or maturation markers such as hyaluronic acid [Parmegiani et al. 2012] that are discussed thoroughly below can be employed during ICSI in order to select living spermatozoa with intact DNA and minimize the risk of poor embryo quality and low pregnancy rates. Choosing the right spermatozoon to inject Polyvinylpyrrolidone (PVP)The most commonly used medium for immobilization of spermatozoa for ICSI is PVP. PVP media has very high viscosity that decreases sperm motility which helps selection, immobilization, and handling of spermatozoa before injection. It is inevitable that some traces of PVP are found in embryos [Van Steirteghem et al. 1993] and although millions of healthy ICSI children are born its detrimental effects should be investigated. According to Strehler and colleagues it may cause changes in sperm structure, sperm nucleus, and membranes [Strehler et al. 1998]. In an interesting animal model study Kato and Nagao [2009] suggested that PVP seems to induce the acrosome reaction as demonstrated in bovine spermatozoa. In a more recent study Kato and Nagao [2012] suggests that PVP exposure seems to lead to lower fertilization rates as it damages the mitochondria structure and sperm membranes. Moreover, defects in chromosomal integrity of mouse spermatozoa and embryos were reported to be associated with PVP exposure [Feichtinger et al. 1995; Mizuno et al. 2002]. These disadvantages led to the use of sperm immobilizing media without PVP such as hyaluronic acid (HA) for performing ICSI which is discussed below in detail [Fraser et al. 1997]. In an effort to avoid PVP usage and the issues related to it, Kato and colleagues

5 362 M. SIMOPOULOU ET AL. [2012] support the simple approach of ICSI application in culture and/or ph buffered low viscosity media. The same authors concluded that although performing ICSI without PVP is far more challenging for immobilizing spermatozoa, it does lead to better blastocyst development and increased pregnancy rates [Kato and Nagao 2012]. Hyaluronic acid (HA) HA, which surrounds human oocytes, has been described as a physiologic selector because only mature spermatozoa with a low level of chromosomal aneuploidy and DNA fragmentation, if any can attach [Jakab et al. 2005]. HA has a wide range of applications in the field of ART. Two of the most commonly used techniques are the physiological intracytoplasmic sperm injection (PICSI) dish and the HA rich medium SpermSlow TM that are of similar efficiency as described by Parmegiani and colleagues [2012]. Principally, the PICSI dish is a Petri dish that has spots of immobilized HA on it. An alternate approach to the PICSI dish is the viscous medium containing HA, commercially available as SpermSlow TM (Origio, Denmark). Spermatozoa bound to HA are selected with an ICSI pipette [Parmegiani et al. 2012]. The benefits of using HA as a method of selecting spermatozoa were described by Parmegiani and colleagues in 2010 who found a significant difference in embryo quality and implantation rate, although no difference in fertilization rate was noted [Parmegiani et al. 2010]. In 2007 Huszar reported that sperm cells selected through this method can increase the overall ICSI efficiency as the use on HA can remarkably decrease the percentage of abortions in ICSI cases from 18% to an impressive 10% [Huszar et al. 2007]. With respect to the actual technique, HA has been shown to have similar efficiency to PVP in slowing spermatozoa and ensuring control during the procedure, but without the detrimental effects of PVP in embryo development [Balaban et al. 2003]. Another issue during ICSI is the time consumed in the pursuit for the morphologically perfect spermatozoon, which can become injurious especially during IMSI a highly time consuming technique. HA can not only optimize the ICSI outcome, but can further decrease the time needed in selecting the morphologically appropriate spermatozoon in IMSI [Parmegiani et al. 2010]. On one hand, in the comparison between PVP and SpermSlow TM the latter seems to ensure benefit and no detriment. Nasr-Esfahani and colleagues [2002] as well as Parmegiani and colleagues (2010) showed a significant difference in the percentages of spermatozoa containing intact DNA between the PVP-sperm and HA-sperm groups. On the other hand, in an article published in 2015 by Huang and colleagues it was demonstrated that there was no significant difference between these two groups. According to the study by Huang and colleagues, a well-trained embryologist during ICSI should have the same ability to choose sperm with intact DNA as with HA-bound spermatozoa selection [Huang et al. 2015c]. Our understanding of this investigation is that they refer to plain morphological criteria by conventional microscopy for sperm selection, although this study does not specify the exact morphological characteristics evaluated or the magnification employed. However, the same study specifies that even though both approaches select sperm of similar DNA integrity levels, their screening efficiency is limited with respect to choosing sperm containing intact DNA for ICSI. The assessment of sperm DNA status with AO fluorescence staining may prove beneficial for selecting sperm prior to ICSI procedures [Huang et al. 2016]. According to the clinical protocol followed in our practice, the use of HA in the form of media and not in the form of the PICSI dish - the latter being an option which adds substantial cost and time to the procedure - is an excellent choice for the ICSI practitioner. To conclude, literature supports that HA is certainly less harmful and less toxic when compared to PVP, but at the same time confers substantial control due to its viscous nature coupled by the assurance of selecting superior spermatozoa for ICSI. Its added cost in comparison to PVP is neutralized by the above mentioned advantages. Polarized light microscopy Centrosomes, a structure of utmost importance for successful fertilization, are formed by centrioles and centrosome proteins. During fertilization the spermatozoon gives its centrioles, amongst other centrosomal components to the fertilized oocyte as they serve as a mitotic center for the first and all the following cell divisions [Schatten and Sun 2009]. Centrioles are cylindrical shaped cellular organelles made of microtubules that are longitudinally aligned offering to the spermatozoon head anisotropic properties that cause its birefringence [Gianaroli et al. 2008]. Advances in microscopy have made the visualization of sperm heads birefringence feasible [Baccetti 2004]. Furthermore, one could even detect spermatozoa that have completed their acrosomal reaction adding further value, as those are associated with higher fertilization potential [Gianaroli et al. 2010]. Moreover, Petersen and colleagues concluded in their study that there is a positive correlation between the sperm head s birefringence and DNA fragmentation [Petersen et al. 2011]. The results of a recent study by

6 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 363 Vermey and colleagues support the results of Gianaroli s group, indicating that when sperm cells were selected based on their birefringence, a higher fertilization rate and embryo quality could be expected [Vermey et al. 2015]. This method shows great potential in cases of couples undergoing ICSI cycles as most studies show that it can select spermatozoa with a highly ordered nucleus, low DNA fragmentation index, and completion of their acrosomal reaction which is positively correlated with higher fertilization, implantation rate, and embryo quality. Further studies are required to secure its role in routine practice. Intracytoplasmic morphologically selected sperm injection (IMSI) The selection of spermatozoa to inject is solely based on motility and morphological criteria by observing under x200 magnification. Teratozoospermia can affect the ICSI outcome, implantation, and pregnancy rates as it is associated with aneuploidies [De Vos et al. 2013; Vicari et al. 2003]. The need for a more detailed morphologically based selection led Bartoov and colleagues to first describe the motile sperm organelle morphology examination (MSOME) [Bartoov et al. 2001], which is based on observation under higher magnification (x 6,000) and gave birth to the IMSI procedure. Ultrastructural studies employing transmission electron microscopy showed that morphological anomalies in the head-neck attachment are linked to abnormal sperm centriolar function, leading to defective fertilization and subsequent centriolar derivation in the embryo. In fact, even following successful fertilization, abnormal centriolar function in sperm that is related to pathological morphology may lead to a lack of syngamy and cleavage [Rawe et al. 2002]. Proper centriolar function in morphologically healthy sperm should secure an active zygotic centrosome. In cases of dysfunction this could lead to post-fertilization developmental arrests [Chemes and Rawe 2003]. To emphasize, the same group published a very interesting study linking spermatozoa of pathological morphology with defective centriolar function to decreased proteasome activity leading to disrupted sperm aster formation and pronuclear and zygote development following ICSI [Rawe et al. 2008] confirming a series of molecular studies [Platts et al. 2007]. Such studies make the option of highly detailed, thorough, careful morphological evaluation of sperm during ICSI a requirement in cases of the above mentioned sperm pathology [Rawe et al. 2002]. Several studies support the effectiveness of IMSI while others play down its significance. There are two major opposing schools to the question ICSI or IMSI. The first is well described by the work patterned by the group of Seti, who reported that when spermatozoa with large vacuoles are selected for injection then the possibility of those embryos to develop into blastocysts is lower [Setti et al. 2013b]. This study supports the work of Vanderzwalmen and colleagues and Knez and colleagues who described the significant difference in the rate of blastocyst development when IMSI was applied [Knez et al. 2011; Vanderzwalmen et al. 2008]. Setti s data also supported the notion that IMSI increases the implantation rate in couples with male factor infertility and should be the method of choice for these cases [Setti et al. 2013a; Setti et al. 2013b]. This is in accord with data of others suggesting that IMSI should have a clear role as a routine procedure in every IVF laboratory [Berkovitz et al. 2006]. On the opposing side represented by the group of Boitrelle it is suggested that there are not enough randomized studies to positively confirm the effectiveness of IMSI and therefore its use should be limited strictly in cases of severe male factor infertility and repeated implantation failures following ICSI [Boitrelle et al. 2014]. The same view is shared and supported by Klement and colleagues, and Ebner s group stating that IMSI should only be applied in cases when there was at least one previous failed ICSI cycle [Ebner et al. 2014; Klement et al. 2013]. Undoubtedly, IMSI is a promising technique enabling the selection of the most morphologically suitable sperm during ICSI, and therefore of major benefit for the targeted group of patients that require it. It does however come hand in hand with an added cost and the disadvantage that its application is time consuming. Purchasing special equipment to perform IMSI is an expensive endeavor, as is the time required by the practitioner to perform it. ICSI s time sensitive nature mainly refers to the extra time that the oocytes remain exposed in order for the morphological evaluation of the sperm to be completed. It should be clear that as a first step IMSI includes a sperm pre-selection procedure prior to the microinjection hence extended time does not apply to the oocytes. It is well known that sperm physiology is more robust than the oocyte s. However, according to Antinori and colleagues selecting sperm cells for IMSI based on MSOME criteria could take up to 120 minutes [Antinori et al. 2008]. This prolonged exposure is certainly a considerable disadvantage, since it could understandably affect sperm morphology [Peer et al. 2007]. It has the potential to make the application of the technique a race between identifying the right spermatozoon in a timely fashion, and ensuring the safety of the procedure with respect to time exposure. Compromising conditions like temperature can have a

7 364 M. SIMOPOULOU ET AL. negative effect on sperm physiology [Peer et al. 2007]. This study suggests that extended handling with spermatozoa should be performed at a temperature of 21 C on the grounds that prolonged sperm cell manipulations at 37 C is detrimental to sperm morphology [Peer et al. 2007]. It is clear that the benefits of IMSI,when required, certainly outweigh the potential detriment to the sperm physiology even in cases were sperm selection exceeds the time anticipated. IMSI has a very clear role in the IVF laboratory, but the equipment required is not standard for the average IVF laboratory. IMSI should come with strict criteria, such as recurrent implantation failure following ICSI and severe male factor infertility [Ebner et al. 2014], to select the cases that can in fact benefit from it. Zona binding Several interesting articles propose one more method of selecting spermatozoa for ICSI: the zona pellucida (ZP) [Yuzpe et al. 2000] binding assay, in which a zona from a patient s immature sibling oocytes is incubated with motile sperm (previously prepared by density gradient centrifugation) for two hours. Following suitable sperm preparation and subsequent exposure to ZP, the spermatozoa bound to the ZP can be selected for ICSI [Black et al. 2010]. As demonstrated by various studies the ZP binding assay although not superior to conventional ICSI, does however seem to play a role in improving embryo quality and implantation rate through improving the selection of sperm to enhance ICSI outcomes [Liu 2011; Paes Almeida Ferreira de Braga et al. 2009]. It is extrapolated that when the spermatozoon s ability to bind to the ZP is affected then even ICSI outcomes can be compromised [Casciani et al. 2014]. Perhaps a horizontal application is not yet advised or feasible, especially in consideration of the heavy workload and the strict timelines of embryology laboratories. However, this approach can be of major benefit to certain patient groups, such as patients presenting with previous poor outcomes of conventional ICSI, or diagnosed with severe abnormal morphology, or DNA damaged sperm. Nonetheless, controlled clinical trials in larger numbers of subjects are required to confirm the advantages prior to employing it routinely for a wider group of ICSI patients. Zeta method The zeta method is a simple technique for the isolation of mature spermatozoa by allowing them to adhere to the surface of a positively charged test tube. The benefits of this procedure rely on its ability to isolate charged cells from a suspension so that the resulting population of spermatozoa is of better quality. It is suggested that they have normal DNA integrity, normal morphology, and normal chromatin condensation. The method has been shown to select acrosome and DNAintact spermatozoa from populations obtained after cryopreservation and increase fertilization rates after ICSI [Kheirollahi-Kouhestani et al. 2009]. This promising new method presents to be easy, straightforward, with no added cost or burdening the already hectic timeline of a busy IVF laboratory. It does not seem to exert any negative effect on the physiology of the spermatozoa. It does however present with certain limitations such as the narrow window of performance straight after seminal plasma separation and a low recovery rate reported to be 8.8% by Chan and colleagues [Chan et al. 2006]. Therefore, its use for ICSI where samples are oligozoospermic in their majority is somewhat questioned. Further studies will delineate its use for routine ICSI cases. Annexin V: Annexin V-positive and annexin V-negative selection Annexin V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine (an early marker of apoptosis) [Arends and Wyllie 1991]. By conjugating annexin V with a fluorophore it is possible to recognize apoptotic sperm cells (annexin V-positive) and separate them from the other spermatozoa of higher fertilization potential (annexin V-negative) [Hoogendijk et al. 2009]. Hoogendijk and team concluded that the annexin V-negative sperm has less nucleus DNA fragmentation and is of morphologically higher quality compared to the annexin V-positive sperm. According to Tygerberg s criteria, this visible feature could potentially be used in every IVF laboratory - especially when ICSI is to be applied - and may lead to higher implantation and pregnancy rates. Said and colleagues [2008] proposed and described a twostep sperm preparation protocol that combined double density gradient centrifugation and magnetic activation cell sorting (MACS) separating apoptotic from nonapoptotic spermatozoa. This resulted in a suspension of spermatozoa of higher quality with enhanced spermoocyte penetration potential [Said et al. 2008]. Lee and colleagues [2010] reported that the above protocol reduced the level of apoptotic markers in patients with unexplained infertility and suggested that these spermatozoa have a higher fertilization potential confirming the work of Said and colleagues [2008]. Such results are confirmed by others [Grunewald and Paasch 2013; Lukaszuk et al. 2015]. A recent study by

8 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 365 Romany and colleagues [2014] assessed the presence of annexin V as an apoptotic marker and reported that the annexin V-negative spermatozoa also happen to be of higher morphological assessment during ICSI. However, if the laboratory personnel has adequate experience on the common preparation techniques (S-U and DGC) and on the morphological selection criteria then although there are innovative technologies, such as the MACS-annexin V protocol, there is not a statistically significant difference in the ICSI outcome [Romany et al. 2014]. This is a view that is consistent with the work of Nadalini and colleagues [2014] who reported that the combined method of DGC/S-U can provide a sample of spermatozoa with better motility, higher morphology, and less DNA fragmentation compared to the DGC/MACS-annexin V protocol [Nadalini et al. 2014]. Identifying a viable spermatozoon In many occasions infertility is due to male factor, obstructive or non-obstructive azoospermia for example. The progress of the ICSI technique helped to overcome this obstacle by utilizing sperm cells extracted from testicular tissue (TESE) [Ezeh et al. 1998]. Unfortunately, for such samples isolating spermatozoa is not an easy task because of their inability to move, as well as because of the presence of various cell types that are in abundance. Methods that are proposed include the use of collagenase to eliminate all other cells except spermatozoa [Hammitt et al. 2002], or the addition of agents that induce motility like caffeine [Hammitt et al. 1989], relaxin [Essig et al. 1982], deoxyadenosine [Aitken et al. 1986], pentoxifylline [Sharma and Agarwal 1997; Stanic et al. 2002], and theophylline [Ebner et al. 2011]. Hypo-osmotic swelling test (HOST) Studies have shown that although spermatozoa may pass the morphological criteria men with severe male infertility will most likely present with a high percentage of DNA fragmentation [Avendano and Oehninger 2011; Bassiri et al. 2013]. HOST is employed to provide an estimate of chromatin integrity [Charehjooy et al. 2014]. As first introduced by Jeyendran and colleagues [1984] and later demonstrated by Rossato and colleagues spermatozoa expand when exposed to hypo-osmotic conditions with functional membranes. This tends to be highlighted especially in their tails [Rossato et al. 1996]. Different patterns of swelling have been reported that correspond to different levels of chromatin integrity (a-g pattern) suggesting that those among the c and especially the d pattern are of the best quality and should be chosen when performing ICSI [Bassiri et al. 2012]. HOST can give information on viability and functionality of spermatozoa [Nasr-Esfahani et al. 2002; Stanger et al. 2010] that morphological criteria or other tests like sperm tail flexibility or single laser shot test cannot. According to the World Health Organization [WHO 2010] laboratory manual for the examination and processing of human semen, HOST can be employed in cases of total asthenozoospermia in order to choose viable spermatozoa for ICSI. This procedure was first described by Jeyendran and colleagues [1984] and later Casper s group used it to choose immotile but viable spermatozoa [Casper et al. 1996] and reported an increase in fertilization rates from 26%, when spermatozoa were chosen randomly, to 43%, and from 23% to 39% in cleavage rates, respectively. The above is in agreement with the work of Sallam and colleagues [2005], who following HOST prior to ICSI ensured higher fertilization and pregnancy rates. They also demonstrated that in cycles with testicular samples that feature solely immotile spermatozoa, HOST increased fertilization rates and deliveries. Several studies including those of Charehjooy and colleagues [2014] showed that the application of HOST lead to embryos with higher developmental potential and implantation rates as well as higher pregnancy rates. Furthermore, Goksan Pabuccu and colleagues suggested that HOST should be employed in a subsequent ICSI cycle following total fertilization failure in order to acquire better quality sperm [Goksan Pabuccu et al. 2015]. The majority of the studies support the effectiveness of HOST and propose its use as routine practice prior to ICSI. In spite of the numerous benefits claimed that are associated with HOST, it can be argued that even sperm with a low HOST grade has the same fertility potential when the method for fertilization is ICSI, and therefore HOST is moot for ICSI cycles [Check et al. 2012]. Since there is no study proving a detrimental effect of HOST, its use should be a decision for each IVF laboratory taking into account its practice, setting, and protocols. Methylxanthines: Pentoxifylline and theophylline A very promising motility induction technique, involves the use of culture media enriched with pentoxifylline (PF) [Kovacic et al. 2006; Morales et al. 1993]. PF is a phosphodiesterase inhibitor of the methylxantine group inhibiting the breakdown of cyclic adenosine monophosphate [Nomikos et al. 2013] which is required for sperm motility. Its beneficial properties for assisted reproductive technologies have been mentioned in various studies

9 366 M. SIMOPOULOU ET AL. [Tarlatzis et al. 1995] as well as its deleterious qualities when it was added in oocyte culture media [Tournaye et al. 1993]. Kovacic and his team [2006] investigated the dangers that lurk in the use of pentoxifylline where caution must be exercised to avoid any exposure of the oocyte. They concluded that when a thawed testicular sample is used PF could be used for short periods of time. This triggers sperm motility leading to quicker identification and selection of vital sperm for ICSI and consequently resulting in higher fertilization rates. Contrary to PF, properties of theophylline, another member of the methylxanthine group, have not been as deeply studied. It is less soluble to water and has a much higher half-life (bioavailability) compared to PF, thus making it easier to use [Ebner et al. 2011]. Its utility was somewhat resolved by the prospective study of Ebner and colleagues [2011]. This group showed that theophylline had the same stimulatory effect as PF. It induces movement in immotile but alive spermatozoa within minutes, making the process of identification and isolation far easier. This results in reducing the time required by embryologists to perform micromanipulation, also reducing the length of time of oocyte exposure to room conditions. The above mentioned benefits of theophylline led to higher fertilization rates, developmental potential, and superior blastocyst morphology. These results are in agreement with a later study performed by Wober and colleagues, where the addition of theophylline enhanced motility in 24 out of 28 samples and led to better fertilization rates, blastocyst formation, implantation, and clinical pregnancy rates [Wober et al. 2015]. It is important to note that all necessary precautions must be taken to avoid the oocyte s and/or embryo s exposure to methylxanthines (PF and theophylline), due to the well documented deleterious side effects they might exert equally on oocyte culture and embryo development [Tarlatzis et al. 1995]. Their use should be restricted strictly in cases of 100% immotile spermatozoa mainly from testicular samples. In challenging cases where the issues arising reflect problems on both the oocyte and the sperm the technique of artificial oocyte activation (AOA) in conjunction with methylxanthines treatment has been shown to ensure fertilization. However, it seems that the results are mostly attributed to the AOA rather than the actual use of methylxanthines treatment [Kang et al. 2015]. ATP/MgSO 4 The use of ATP/MgSO 4 was also proposed in order to distinguish viable spermatozoa in samples of only immotile spermatozoa. Following the addition of ATP, a considerable percentage of spermatozoa regained movement (a twitch of the flagellum was noticed) as Neri and colleagues [2014] reported. Specifically, when a frozen epididymal specimen were exposed to ATP motility was induced and noted for 59.8% of the sperm cells. Laser assisted immotile sperm selection (LAISS) In this novel technique originally introduced by Aktan and colleagues for the purpose of identifying viability in immotile samples, a single laser beam of 1.48 μm diode is shot near the tip of the tail of the spermatozoon [Aktan et al. 2004]. In vital spermatozoa the tail starts to curl almost immediately after the shot is applied. If there is no reaction recorded, then the spermatozoon is considered nonviable. This method can be used in any culture media and the sperm cell that is selected can be used directly for injection. Thus, it reduces the time required for performing ICSI, and hence the time that the gametes are exposed to unfavorable conditions. It has been shown that the laser shot does not damage the sperm membrane [Montag et al. 2000] nor increase DNA fragmentation [Ebner et al. 2005]. Gerber and colleagues [2008] reported that a singleton pregnancy was achieved by LAISS combined with HOST. They noted the usefulness of the LAISS procedure especially in cases where HOST doesn t give much information. The LAISS method is slowly gaining ground as the method of choice for identifying a viable spermatozoon. It is faster, safer, easier, does not require prior treatment, and can be performed during ICSI. This is in comparison to the above mentioned chemical treatments that present with known possible detriments to the dynamic of the future embryo and that induce motility. Unfortunately, due to its cost, the laser is not yet a must have equipment for the standard IVF laboratory. Bioreactively improving quality in OAT samples for ICSI: Myoinositol Myoinositol is the most abundant steroisomer of inositol which modulates the intracellular Ca 2+ concentration. It is synthesized by the enzymes myo-1-phosphate synthase and myo-monophosphatase-1 which are found in high concentration in the testes [Chiu et al. 2002; Eisenberg and Parthasarathy 1987; Foskett2010].In arecentstudy Carlomagno and colleagues demonstrated that incubating spermatozoa of oligoasthenoteratozoospermic (OAT) samples with myoinositol significantly improved their motility [Carlomagno et al. 2011]. The rise of inner sperm mitochondrial Ca 2+ that is inferred by their exposure to myoinositol probably results in a higher percentage of sperm cells with high inner mitochondrial membrane potential (MMP), an apoptotic marker. When

10 SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 367 there is an apoptotic stimulus then MMP is reduced [Condorelli et al. 2012]. Within this context, Rubino and colleagues [2015] set out to find the outcome of ICSI cycles where spermatozoa were incubated with myoinositol. Their results were very encouraging as they showed that in OAT men, myoinositol improved motility and there was a higher fertilization rate in the myoinositol spermatozoa treated group. Marchetti s group also demonstrated that MMP, which is increased by myoinositol, was positively correlated with fertilization rates [Marchetti et al. 2002]. The studies of Condorelli and colleagues as well as Rubino and colleagues suggest the use of myoinositol for in vitro assisted reproductive techniques [Condorelli et al. 2012; Rubino et al. 2015]. With respect to the actual ICSI procedure we assume that applying myoinositol to OAT samples can also shorten the timeline of the procedure, adding another benefit to this approach. Our aim in the IVF laboratory is to reproduce conditions that mimic those in vivo, and therefore avoid additional treatments, or exposure to biochemical agents. As alteration of MMP is biochemically invasive for the spermatozoa, further investigation is required on the physiology of the spermatozoon following exposure to myoinositol before promoting this approach for standard clinical practice. Conclusion When applying ICSI one of the major concerns is the time consumed in trying to choose the right spermatozoon and to determine what are the suitable criteria. Unfortunately, there is a lack of large multicenter randomized control studies identifying the highly sought after universal protocol that should be adopted by all laboratories worldwide. The value of some of the above analyzed approaches is sometimes showcased mostly in cases of unfortunate scenarios such as strictly immotile spermatozoa in which the application of pentoxifylline, theophylline, or even LAISS can help in detecting a viable spermatozoon so that a cycle continues. The most common criterion is based on spermatozoon morphology and its motility when exposed to PVP. Although PVP is the most common medium in which spermatozoa are exposed for ICSI, it is demonstrated that it can cause embryotoxicity that affects embryo development and ultimately pregnancy rates [Kato and Nagao 2009; Mizuno et al. 2002]. To avoid an unfortunate outcome, the use of approaches such as the PICSI dish or SpermSlow TM have been used with very promising results but still there are no randomized control trials to confirm their use in everyday practice. Other proposed methods such as the HOST, do not solely rely on morphology. All available approaches analyzed in this article are not part of a routine practice within the standard IVF laboratory. IMSI is certainly gaining ground when it presents with strict application criteria and benefits when employed to the right patients. However, it remains controversial. We need to rely on evidence based studies before rushing to adopt novel promising techniques and define the Holy Grail of ICSI. With respect to ICSI the literature survey suggests aiming to improve sperm preparation, and/or select the right spermatozoon. On one hand, a well-trained embryologist in an adequately equipped laboratory may not require usage of any of these techniques in order to ensure satisfactory results with ICSI. On the other hand, exemption for cases with previous failed fertilization [Huang et al. 2015a; Huang et al. 2015b; Nadalini et al. 2014; Romany et al. 2014] may be required. The field of IVF and the practice of ICSI would certainly benefit from standardization, at several levels, rather than relying on highly promising techniques of the future that increase the cost and adds further levels of complexity to the already invasive nature of the technique. With our mission being to ensure safety and added effectiveness, we need to responsibly assess how the biology and physiology of the sperm could be affected by the potential application of novel techniques and weigh that against our desire to improve the outcome. Acknowledgments We are very appreciative to all embryologists, clinicians, and scientists at the Assisted Conception Unit of the 2nd Department of Obstetrics and Gynecology at Aretaieion Hospital, at the Department of Physiology of the National and Kapodistrian University of Athens Medical School, and at the Centre for Human Reproduction at Genesis Hospital. Declaration of interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. The authors report no declaration of interest. Notes on contributors Concept, analysis, format, revision, and editing: MS; Literature search, extraction analysis, and drafted the manuscript: MS, LG;. Contributed to literature search, proofread the manuscript, and contributed with comments: PG. All remaining authors contributed to this review equally each including their input on their respective expertise. Insights from the IVF specialist/clinician point of view: PB, TK, KP; Oversaw the sections involving the physiology aspects of the human spermatozoon and embryo: MK. All authors approved the final version of the manuscript.

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