21 Stephen Harbottle Since the pioneering work of Sir Robert Edwards, Patrick Steptoe, and Jean Purdy (Edwards et al.1969) in the latter half of the twentieth century, which culminated in the birth of the world’s first test‐tube baby, Louise Joy Brown on 25 July 1978, it has been clear that methodology must be developed and refined to prepare sperm for use in assisted reproduction technology (ART). The need to separate spermatozoa from seminal plasma is fundamental to the in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or intrauterine insemination (IUI) processes as it allows sperm to capacitate and exhibit a hyperactivated pattern of activity which is an intrinsic expression of their fertilization ability (Mortimer 1991). Since a simple one stage ‘washing’ process was first described for human fertilization (Edwards et al. 1969), the process has been further refined through the additions of ‘swim‐up’ (Lopata et al. 1978), two washes (Edwards et al. 1980), ‘density gradient separation’ (Pertoft et al. 1978), and a wash combined with a swim‐up (Mahadevan and Baker 1984). What is both noticeable and interesting is that all of these techniques as described still make a valuable contribution to our laboratory processes today. Despite the methodology adopted, the aim is consistent throughout our most modern laboratories; to remove the seminal plasma and other nonspematozoal seminal components and be left with a highly purified seminal fraction containing the highest possible population of highly motile and morphologically normal sperm for use in insemination or injection. This chapter aims to provide an overview of the techniques currently available to clinical scientists in their tool kit of sperm preparation. Before we consider what we must do to prepare sperm we must consider why we must do it. Pivotal to this understanding is a realization that, under in vivo conditions, human semen would not find itself in the vicinity of an ovulated egg in the vestments of the Fallopian tubes. During its journey from the site of insemination (the vagina) through the challenging and hostile environment of the female reproductive tract, those sperm with the greatest fertilization potential are actively selected from those sperm with poor potential and the other nonsperm cellular and molecular components of the semen. Supported in their journey by cervical mucus, potentially fertile spermatozoa migrate to the site of conception and undergo the processes of capacitation, gaining a hyperactivated state of motility and the acrosome reaction, prior to finally making contact with the oocyte itself (Mortimer 1989). The aim of any sperm preparation procedure is multifactorial: The widespread introduction of IVF procedures during the 1980s led to a rapid development of a range of deployable sperm preparation techniques as described previously. In general, the range of techniques available today can be summarized into four broad categories (for the purpose of this text, the author has given focus to those techniques most commonly practiced in ART laboratories today; it is accepted that other more ‘niche’ techniques may be encountered, particularly outside the UK by more adventurous readers): These techniques are interchangeable to some extent and their main applications and benefits are briefly summarized in Table 21.1. The savvy clinical scientist will approach each sperm preparation without prejudice and will adapt their preparation strategy based on the quality of the sample observed to ensure that they take every step possible to maximize the quality of the resultant yield. A summary of the relative advantages and disadvantages of each technique is listed later in Table 21.3. Table 21.1 A summary of the four most commonly adopted sperm preparation techniques. Sperm cells are highly sensitive to changes in temperature so great measures are taken to ensure that temperature control (and particularly prevention of temperature elevation) is maintained throughout the sperm preparation process. Historically, little attention has been afforded to the consumables and ‘plastic ware’ used during sperm preparation procedures and the direct effect this may have on the resultant sperm yield. ‘Reprotoxicity’, or the release of reprotoxic components or molecules into the IVF culture system (Nijs et al. 2009) can result in a reduction in the efficacy of treatment or, in its most extreme case, a complete culture system failure. You may be further alarmed to discover that in an audit of IVF laboratory plastic ware of the 36 common types of laboratory consumable tested, 16 (36%) of them were reprotoxic to the IVF culture system (Nijs et al. 2009). It is for this reason that it is essential that our starting point for any ART procedure, including the preparation of sperm for treatment, is the production of the sample into a known nonreprotoxic container and its subsequent processing through a validated reprotoxin free chain of consumables to its final use in ART or cryopreservation. This process represents the most basic of sperm preparation techniques and has changed very little since it was first described by Edwards et al. for ART use in 1969 (Figure 21.1). Its usual application is when a sperm sample appears severely oligozoospermic or even cryptozoospermic. Working with consumables known to be nonreprotoxic, the strategy is to concentrate any spermatozoa present in the sample into a smaller and more workable volume, usually for ICSI treatment. The sample is pipetted into a nonreprotoxic conical tube and centrifuged at 300 g for 10–20 min. At the end of the centrifugation step the seminal supernatant is carefully removed and the pellet is resuspended in 2–3 mL of a suitable culture media designed to sustain spermatozoal metabolism for an extended period of time (most if not all IVF culture media manufacturers produce such a medium which clinics choose based on personal preference). The tube is centrifuged for a further 10 min at 300 g, the supernatant is again discarded and the small final volume of culture medium (usually 0.1–0.25 mL) is added prior to assessment for use in treatment. Figure 21.1 Sperm washing. Source: Author’s own design. The main disadvantage of this technique is, due to it being a ‘concentrate everything you have’ approach, the resultant sperm preparation is often hard to work with as all of the nonsperm cells and cellular debris are also collected. Allowing motile sperm time to swim out of the debris in an ICSI dish for 15–30 min prior to an injection procedure is highly recommended. Separation by swim‐up is in essence a sperm race, in that the principle is based entirely upon the selection of sperm based on their motility and nothing else. Progressive motility is clearly an essential trait a sperm capable of fertilizing an egg in vivo must possess, so this technique is firmly based on the functionality of reproduction and as such can be regarded as a more natural technique by comparison. That said, despite promising fertilization rates in early ART procedures in the tubal infertility population, case reports soon followed suggesting that more complex cases involving male factor, multifactorial, or idiopathic infertility could result in a failure of fertilization when using swim‐up prepared sperm (Trounson et al. 1980; Yates et al. 1987). The technique is clearly of little value in cases of significant oligo‐ or asthenozoospermia as it will not serve to concentrate a final yield in the same way as the density gradient centrifugation or direct washing preparatory procedures, and therefore its role is somewhat niche in modern IVF practice with most clinics adopting the density gradient procedure as their front‐line methodology. Swim‐up may be deployed following density gradient as a further step to refine the final yield for use in IVF treatment. This is, however, time consuming and is not common practice, particularly in large and high‐throughput ART service laboratories. Working with neat semen rather than precentrifuged and pelleted semen is recommended to reduce the risk of process‐induced ROS accumulation (Aitken and Clarkson 1988). Working with consumables and media known to be nonreprotoxic, the swim‐up column can be prepared by either under‐laying or over‐laying 1.0 mL of semen against 1.0 mL of sperm culture medium, although most experienced operators would agree that the best differentiation is obtained by under‐laying the semen once competence has been obtained. The preparation is then incubated at 35–37 °C for a period of 60 min before the top (culture medium) fraction is harvested which, in theory, should contain the most motile fraction of the sample or at least a fraction with enhanced motility (Figure 21.2). Figure 21.2 The swim‐up procedure. Source: Author’s own design. Care should be taken when harvesting the swim‐up, particularly if the swim‐up is performed with semen rather than prepared sperm post density gradient, as it is entirely possible to compromise the whole procedure with an accidental slip of the wrist by accidentally contaminating the yield fraction with seminal plasma and all the nonsperm cells and potential sources of ROS it contains. Harvesting should be performed with a clean pipette starting at the upper meniscus and aspirating gradually downwards until around 75% of the upper layer has been collected. This highly motile seminal fraction can then be resuspended in culture medium to adjust the concentration to the recommended range for IVF (5–20 million sperm per mL) or to a lower or higher concentration for ICSI or IUI. A more advanced sperm separation technique based on the principles of the swim‐up is the migration sedimentation chamber (MSC, Research Instruments, UK). The MSC, as first described comprises of ‘…two built‐in concentric tubes in which the progressive sperm ‘jump over’ the edge of the central tube. They then sediment at the bottom of the central conical tube where they can be collected by aspiration’ (Tea et al. 1984). The MSC is, by comparison to other methodologies, noninvasive, particularly when compared with methodology dependent upon the use of centrifugation (Yener et al. 1990). Essentially the MSC method constitutes a swim‐up technique combined with a sedimentation step. The external well of the MSC device is loaded with 400 μL of semen and 2 mL of culture medium is added to the device gallery. The device can then be incubated at 35–37 °C for 60 min before the motile fraction is carefully aspirated from the central gallery taking care not to disturb or accidentally harvest any of the semen from the outer well (Figure 21.3). Figure 21.3 The migration sedimentation chamber (MSC). Reproduced with permission of Research Instruments. MSC sperm preparation, when compared to swim‐up of neat or centrifuged and pelleted semen, demonstrated an improvement in the yield and the motility of the recovered sperm whilst the morphology remained unchanged (Ramos et al. 2015). Zavos et al. (2000) evaluated an alternate device, the Multi‐ZSC (Biogen, Turkey) which deploys four tiered levels within a single MSC device for sperm preparation. Their study demonstrated that the device was effective in compartmentalizing sperm populations based on their motility and morphology and proposed that the device had a bright future by facilitating the selection of specific subpopulations of sperm for different clinical and research applications. A further study on the efficacy of the Multi‐ZSC refuted some of these claims, showing that the yield was much reduced when compared to that published by Zavos and that the morphometry of the sperm heads declined in the populations harvested from higher up the device (Lampiao and Plessis 2006). Limited further detail is available on the efficacy of MSC devices and they have not been integrated into routine clinical practice despite the early promise shown. Density gradient separation of semen is the most commonly deployed method of sperm preparation used in the field of ART today (Figure 21.4). Two methods were developed, continuous and discontinuous density gradient centrifugation, both of which have been demonstrated to be effective in enhancing resultant motility and fertilization capacity as judged by the enhanced ability to penetrate zona‐free hamster ova (Berger et al. 1985) as well as the morphology of the resultant sperm preparation (Pousette et al. 1986
Sperm Preparation: Strategy and Methodology
Introduction
Sperm Separation Techniques: Why Do We Need Them?
What Separation Techniques Are Available?
Sperm Preparation Method
Description and Principle
Outline of Procedure
Direct sperm washing
Simple means of removing seminal plasma without materially altering the characteristics of the sperm
Add of a volume of sperm wash culture media, centrifuge, remove supernatant and resuspend
Sperm challenge separation (swim‐up)
Separates sperm based on motility with migration of highly motile sperm into a ‘clean’ and harvestable layer of culture media for insemination
Layer sperm wash media onto semen or sperm pellet. Incubate for 60 min. Harvest motile sperm from wash media layer
Density gradient centrifugation (DGC)
Selection by density using colloidal silica to trap low density cells whereas high density sperm with improved sperm function penetrate the column and form a pellet
Gradient media formed into two layers of different densities (high/low). Set up differential gradient column, usually either 90%/45% or 80%/40%. Centrifuge for 20 min at 300 g followed by removal and washing of the pellet
Adaptive filtration
Pass prepared sperm exposed to a selection solution through an adaptive filter (e.g. magnetic) to further enhance yield quality
Using MACS as an example, coat with magnetic particles, pass through magnetic filtration field, harvest enhanced yield for treatment
The Importance of Consideration of ‘Reprotoxicity’
Direct Sperm Washing
Sperm Challenge Separation (The ‘Swim‐Up’)
Use of the Migration Sedimentation Chamber for Sperm Preparation
Density Gradient Separation
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