State-of-the-art techniques attempt to select sperm with the best functional capacity to produce pregnancy and, subsequently, healthy offspring. A variety of approaches are now being evaluated. Future approaches may allow for selection of sperm based on sperm DNA integrity, degree of aneuploidy, or apoptosis. Other approaches involve attempting to improve the in vitro function of sperm with exposure to compounds such as pentoxifylline or platelet activating factor. In the future, we are likely to see significant improvements in the ability to select the best sperm for assisted-reproductive-technology procedures and the use of these procedures in routine clinical practice.
Key points
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State-of-the-art techniques of sperm preparation for assisted reproductive technologies (ART) aim to select sperm with the best functional capacity to produce pregnancy and, subsequently, healthy offspring.
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Each of the currently used sperm selection methods has specific advantages and disadvantages with respect to safety, feasibility, and sperm yield.
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The potential for improved ART outcomes following the use of advanced sperm selection methods must be weighed against the increases in processing time, technical expertise, and cost associated with these technologies and the possible damage to sperm from prolonged exposure to nonphysiologic conditions.
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Future approaches may allow for the selection of sperm based on sperm DNA integrity or the degree of aneuploidy or apoptosis.
Introduction
Modern assisted reproductive technologies (ART) have dramatically advanced the treatment of both male and female factor infertility. Since the birth of Louise Brown in 1978, more than 5 million babies have been born with the help of these technologies. Within the United States, the use of ART has doubled over the past decade; according to current estimates from the US Centers for Disease Control and Prevention, more than 1% of infants are conceived using ART.
The successes of assisted reproduction would not be possible without concurrent advances in the laboratory identification, manipulation, and preparation of spermatozoa for use with intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The diagnostic semen analysis remains the cornerstone for the assessment of overall semen quality. However, individual semen parameters, such as motility and morphology, are unable to reliably identify the best-quality sperm for use with ART. Instead, sperm selection strategies have come to rely increasingly on the assessment of sperm function and sperm fertilization potential in order to optimize sperm quality and improve ART outcomes.
Early sperm preparation methods were nonselective and comprised 1 or 2 simple washings, followed by resuspension of the male germ cells. Mahadevan and Baker then described a single wash followed by a swim-up procedure from the cell pellet, a simple and classic technique that continues to be used today. However, as the indications for ART have evolved from primarily gynecologic conditions to increasingly include andrological diagnoses, andrological research has attempted to better understand sperm physiology and develop more sophisticated techniques to identify motile and functionally competent spermatozoa for use with ART.
This article reviews the most common conventional and advanced sperm identification and preparation methods in use today and discusses their advantages and disadvantages regarding modern ART. The techniques to improve sperm functions in vitro are also discussed.
Introduction
Modern assisted reproductive technologies (ART) have dramatically advanced the treatment of both male and female factor infertility. Since the birth of Louise Brown in 1978, more than 5 million babies have been born with the help of these technologies. Within the United States, the use of ART has doubled over the past decade; according to current estimates from the US Centers for Disease Control and Prevention, more than 1% of infants are conceived using ART.
The successes of assisted reproduction would not be possible without concurrent advances in the laboratory identification, manipulation, and preparation of spermatozoa for use with intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The diagnostic semen analysis remains the cornerstone for the assessment of overall semen quality. However, individual semen parameters, such as motility and morphology, are unable to reliably identify the best-quality sperm for use with ART. Instead, sperm selection strategies have come to rely increasingly on the assessment of sperm function and sperm fertilization potential in order to optimize sperm quality and improve ART outcomes.
Early sperm preparation methods were nonselective and comprised 1 or 2 simple washings, followed by resuspension of the male germ cells. Mahadevan and Baker then described a single wash followed by a swim-up procedure from the cell pellet, a simple and classic technique that continues to be used today. However, as the indications for ART have evolved from primarily gynecologic conditions to increasingly include andrological diagnoses, andrological research has attempted to better understand sperm physiology and develop more sophisticated techniques to identify motile and functionally competent spermatozoa for use with ART.
This article reviews the most common conventional and advanced sperm identification and preparation methods in use today and discusses their advantages and disadvantages regarding modern ART. The techniques to improve sperm functions in vitro are also discussed.
Modern era of ART
When Leeuwenhoek and Hamm first discovered spermatozoa in 1677, they were thought to be parasites of the male genital tract. Their role in fertilization and reproduction was not appreciated until the nineteenth century, following the discovery of the mammalian oocyte in 1827 and the observation of mammalian fertilization in 1875.
Despite an imperfect understanding of sperm function and biology, the first successful attempts at artificial insemination in humans were performed as early as the 1780s, using crude and untreated semen samples. The development of IVF required an understanding of sperm capacitation and the ability to reproduce this physiologic change in vitro using appropriate sperm incubation conditions. Human IVF was introduced in 1969, with a successful birth reported less than a decade later. Normal sperm-oocyte interactions, however, were the limiting factor for the success rate of IVF. The development of direct ICSI in 1992 overcame this limitation and greatly enhanced the treatment of infertile couples.
Principles of sperm selection
The female reproductive tract naturally separates the best-quality spermatozoa for oocyte fertilization via a stringent selection process. Of the millions of sperm deposited into the vagina at ejaculation, only about 10.0% enter the cervix, 1.0% makes it to the uterus, and 0.1% into the fallopian tube. Eventually, of the 1000 to 10,000 spermatozoa that reach the cumulus-oocyte complex, only one fertilizes the egg.
The advent of ART necessitated the development of in vitro techniques for the identification and preparation of spermatozoa for fertilization. Because ART bypasses the natural selection processes taking place at various levels of the female reproductive tract, sperm selection strategies can significantly impact the quality of spermatozoa used for ART and, in turn, influence reproductive outcomes.
In the early era of ART, the primary focus of sperm separation techniques was to obtain motile spermatozoa. As experience with ART evolved, this focus shifted to the isolation of functional spermatozoa, dictated by the observations that functional sperm parameters correlate with the results of fertilization in vitro. Conventional techniques to separate sperm from seminal plasma are based on motility, adhesion, and filtration characteristics, without regard to the functional competence or genetic quality of sperm. Use of the selected sperm may, therefore, result in impaired embryo development, pregnancy loss, or other health problems in the offspring, despite acceptable fertilization and pregnancy rates. In contrast, advanced sperm selection methods aim to identify the most functional spermatozoa for oocyte fertilization, with the ultimate goal of safeguarding the health of the progeny rather than just achieving pregnancy.
Some investigators have suggested that the principles of sperm selection in vitro should, to the extent possible, mimic the natural selection processes that occur in the female reproductive tract. Additionally, the ideal sperm selection technique should be quick, easy, nontoxic, and cost-effective to perform. It should permit the separation of sperm from bacteria, leukocytes, and toxic substances like reactive oxygen species while maintaining the normal physiologic characteristics of the selected sperm and maximizing the yield of viable, motile sperm. Unfortunately, no presently available sperm selection technique displays all of these characteristics.
Conventional sperm selection methods
Conventional techniques for the separation of spermatozoa from seminal plasma can be broadly categorized as techniques based on migration, density gradient centrifugation (DGC), and filtration. Their goal is to obtain an optimal yield of normal motile spermatozoa for use with ART. Each of these techniques has distinct advantages and disadvantages, which should be considered in light of the quality of the semen samples being processed.
Sperm Washing
The simplest semen processing technique, sperm washing, removes seminal plasma and concentrates the specimen into a small volume. This technique is performed by mixing the semen with media, centrifuging the specimen, discarding the supernatant, repeating these steps, and ending with resuspending the pellet in the final desired volume of media. Sperm washing removes the seminal plasma but does not separate the sperm from other cells within the semen. In addition, both motile and nonmotile sperm are retained. The overall sperm yield using this method is approximately 50%, which is higher than most more-sophisticated techniques. Because it is simple and quick, it is one of the most common techniques used for IUI. However, this method does not separate sperm based on quality or functional capacity.
Migration Techniques
Migration methods rely on the presence of motile spermatozoa within the semen sample. The classic swim-up procedure described by Mahadevan and Baker and the migration-sedimentation procedure described by Tea and colleagues are both examples of migration techniques. In the swim-up procedure, liquefied semen is centrifuged to generate a cell pellet. Following an incubation period of approximately 60 minutes, motile sperm migrate from this pellet into supernatant medium, from where they are collected. The swim-up procedure remains the oldest and most commonly used sperm preparation method currently in use. Although its application for male factor infertility is limited, its use for couples with normozoospermia and female factor infertility is standard. The primary advantage of this method is the yield of a very high proportion (>90%) of morphologically normal motile sperm, without other cells or cellular debris. However, the absolute yield of sperm is low because many potentially motile spermatozoa in the lower levels of the pellet never reach the interface with the overlying culture medium. Additionally, pelleting of spermatozoa forces contact between spermatozoa, cellular debris, and leukocytes, which are known to produce high levels of reactive oxygen species (ROS) that can be damaging to sperm.
The migration-sedimentation procedure described by Tea and colleagues combines the swim-up procedure with an additional sedimentation step and requires a special glass or plastic tube with an inner cone. Spermatozoa swim up directly from liquefied semen into supernatant medium and subsequently sediment in the inner cone. The lack of a centrifugation step in this method reduces the exposure of sperm to ROS compared with the conventional swim-up procedure. Another advantage of migration-sedimentation over the swim-up procedure is that it selects for normally chromatic condensed spermatozoa, a parameter that is predictive of fertilization in vitro. Unfortunately, the yield of motile spermatozoa is low, limiting the utility of this method for IVF.
DGC
DGC relies on sperm motility as well as the property of sperm to collect at the border between liquid phases. Either a continuous or discontinuous density gradient is created by layering media in the order of density, with the densest layer at the bottom and the least dense layer at the top. When a semen sample is placed on the upper-most layer and centrifuged, any debris, round cells, nonmotile and poor-quality sperm remain in the upper layers, while motile sperm migrate to and concentrate in the bottom layer. Like the classic swim-up procedure, DGC yields a clean fraction of highly motile spermatozoa. Additionally, it can be used for semen samples with very low sperm density because it uses the entire volume of an ejaculated semen sample. However, DGC is more laborious than the swim-up procedure, owing in large part to the setup of a good-density gradient.
Filtration
Filtration methods rely on sperm motility and the propensity of sperm to adhere to filtration matrices. Glass wool filtration is the most commonly used filtration technique, in which motile spermatozoa are separated from immotile cells by movement through densely packed glass wool fibers. The type of glass wool used has been shown to impact the success of this method. Like DGC, glass wool filtration uses the entire volume of the ejaculate, thereby providing a good yield of motile spermatozoa, even in the setting of oligospermia or asthenozoospermia. However, the filtrate is typically not as clean as with other sperm preparation methods. Glass wool filtration effectively eliminates up to 90% of leukocytes, which, in turn, reduces ROS and ROS-induced damage to sperm. Glass wool filtration significantly selects for normally chromatic condensed sperm, which may be important for optimizing the outcomes of assisted reproduction.
Over the years, various other filtration methods, such as glass beads, cross-linked dextran columns, and micropore membranes, were introduced for the separation of motile sperm. However, sooner or later these fell out of favor because of either safety concerns or a low yield of motile sperm.
A Cochrane Database systematic review of reproductive outcomes in subfertile couples undergoing IUI using either swim-up, gradient, or wash-and-centrifugation semen preparation techniques demonstrated no difference in pregnancy and miscarriage rates, regardless of the technique used. Unfortunately, none of the randomized controlled trials included in this meta-analysis reported live births as a primary outcome. Nevertheless, in the absence of any evidence to identify one sperm selection technique as being superior to another, the choice of the technique used should be dictated by the quality of the ejaculate and the desired postprocessing yield of spermatozoa.
As previously mentioned, limitations of conventional sperm selection methods include the inability to assess sperm functional status and fertilization potential, both of which are important for IVF and ICSI. Several advanced sperm selection methods have, therefore, been developed in an effort to better identify the best-quality functional sperm for assisted reproduction.
Advanced sperm selection methods
Advanced strategies for the selection of sperm for ART, especially IVF and ICSI, include methods based on sperm maturity, sperm ultramorphology, sperm surface electrical charge, and the separation of apoptotic from nonapoptotic sperm. Like the conventional techniques described earlier, these advanced techniques also have specific advantages and disadvantages with respect to safety, feasibility, and yield. It follows that the choice of methods used for the selection and preparation of sperm must be tailored to the type of ART being used and the quality of the sperm sample at hand.
Sperm Maturity
Hyaluronic acid (HA) is a major component of the extracellular matrix of the cumulus oophorus, which sperm must penetrate to bind to the zona pellucida and fertilize the oocyte. During the maturation phase of spermatogenesis, remodeling of the sperm plasma membrane exposes HA receptors on the surface of the sperm cell, rendering it capable to binding to HA and the zona pellucida. HA-bound spermatozoa exhibit lower percentages of chromosomal aneuploidies and DNA damage and show significantly improved overall and nuclear morphology than sperm that do not bind to HA. In fact, sperm selection by HA binding has been shown to increase the proportion of morphologically normal sperm by threefold. The presence of sperm surface HA-receptors has, thus, been used as a marker of sperm maturity and a tool to select functional spermatozoa for ART.
Two commercially available methods for HA binding are currently in use. The first, the PICSI dish (Origio MidAtlantic Devices, Inc, Mt Laurel, NJ, USA), is a Petri dish containing immobilized spots of HA. Washed or DGC-prepared sperm are placed in the dish and incubated for 15 minutes, after which freely moving sperm are rinsed off, and HA-bound sperm are picked with an ICSI pipette. The alternative involves a viscous, HA-containing medium. A droplet of the medium is mixed with a droplet of DGC-prepared sperm and incubated for 15 minutes, then the sperm bound to HA at the interface of the 2 droplets are selected with an ICSI pipette.
HA-based sperm selection is highly specific with minimal safety concerns, given that HA is a naturally occurring component of cervical mucus, cumulus cells, and follicular fluid. To date, no adverse events have been reported following the use of HA-selected sperm in clinical ART settings. An obvious disadvantage of this approach, however, is the time commitment involved, particularly if the fertilization of more than one oocyte is planned. Few studies have explored the clinical impact of this selection method. With the exception of one prospective study of couples undergoing ICSI that showed higher fertilization rates using HA-selected sperm, fertilization and pregnancy rates following the use of HA-selected sperm versus DGC-prepared sperm for IVF and ICSI do not seem to be significantly different.
Sperm Ultramorphology
Normal sperm morphology, evaluated after fixation and staining at a magnification of ×1000, is considered a major determinant of successful fertilization both in vivo and in vitro. However, sperm is typically selected for ICSI under light microscopy at a magnification of ×400 from unstained and unfixed samples, which are conditions that are inadequate for the evaluation of subtle structural malformations, DNA damage, and chromosomal aberrations. As a result, sperm selected for ART, especially ICSI, based on light microscopy alone can be suboptimal in quality.
The motile sperm organelle morphology examination (MSOME) is a recently developed alternative that allows the real-time assessment of sperm ultrastructure at a magnification of ×6300. A microdroplet of motile sperm, prepared using conventional techniques, is examined under oil inversion, with an inverted light microscope fitted with Nomarski optics and digital enhancement. The MSOME assesses 6 sperm organelles (acrosome, postacrosomal lamina, neck, tail, mitochondria, and nucleus), which are classified as being either normal or abnormal. Of the 6 organelles, the nucleus seems to be most important in influencing ART outcome.
An increasing number of ART centers now include MSOME in ICSI protocols; the combination of MSOME and ICSI is termed intracytoplasmic morphologically selected sperm injection (IMSI). Because MSOME identifies structural details that are undetectable by light microscopy, such as nuclear vacuoles that indicate abnormal chromatin packaging, this method is much more stringent than the evaluation of sperm morphology according to strict criteria. Indeed, IMSI has been shown to increase fertilization, implantation, and pregnancy rates, while lowering aneuploidy and miscarriage rates, compared with standard ICSI. However, IMSI is expensive, requires special technical training, involves additional sperm manipulation, and takes considerably longer to perform than ICSI. Extended exposure of sperm to nonphysiologic conditions may pose additional risks to the male germ cell.
Sperm Surface Electric Charge
During epididymal transit and maturation, human sperm acquire a net negative surface electric charge, because of the acquisition of sialylated and negatively charged CD52 membrane glycopolypeptides. The expression of CD52 is positively correlated with normal sperm morphology and capacitation, and serves as the basis of sperm separation by electrophoresis.
A sperm sample is loaded into a microflow cell fitted with a polycarbonate membrane containing 5-μm pores, large enough to permit the passage of spermatozoa, yet small enough to exclude larger cells like leukocytes and immature germ cells. The application of an electrical current promotes the active transit of mature sperm across the field into a collection chamber from where they can be retrieved. Electrophoretic sperm selection is fast and minimizes the generation of ROS because it excludes leukocytes and involves no centrifugation steps. Separated sperm capacitate and bind to the zona pellucida (ZP) normally. The efficiency of this technique has been proven with oligospermic samples, testicular sperm, and frozen spermatozoa. Some investigators have expressed concern that the use of electrophoresis may negatively impact fertility, but definitive evidence for this is lacking. Unfortunately, the apparatus required for this technique is expensive, making it inaccessible to many andrology laboratories.
A second technique based on the sperm electrokinetic potential (ie, the difference in electrical charges between the membrane of mature sperm and its surroundings, typically −16 to −20 mV) is called the zeta potential . Because of their negative charge, spermatozoa adhere to glass surfaces when a culture medium is not supplemented with serum or albumin. The zeta method isolates mature sperm by allowing them to adhere to the inner surface of a positively charged test tube. Because it does not involve the application of electricity, the zeta method is considerably cheaper and likely safer than electrophoresis, but sperm recovery is too low to accommodate oligospermic samples. Additionally, the zeta method has not been tested in a humid environment, which has the potential to neutralize electrical charges. X-chromosome–bearing spermatozoa exhibit a higher net negative charge than Y-bearing spermatozoa, but early concerns that electrophoresis or the zeta method may skew the ratio of X- and Y-chromosome–bearing sperm are not supported by recent investigations.
Separation of Apoptotic and Nonapoptotic Sperm
In viable cells, annexin V, a 35-kDa protein, is bound to the negatively charged phosphatidylserine (PS) on the inner leaflet of the plasma membrane. Early in apoptosis, PS is externalized to the outer leaflet of the plasma membrane, a change that is positively correlated with nuclear DNA damage and has implications for fertilization and pregnancy failure following ART. Given this association, methods to identify and isolate spermatozoa that do not show PS on their surface have been developed.
In magnetic activated cell sorting (MACS), a sperm sample is incubated with annexin V-conjugated microbeads, which bind specifically to apoptotic sperm with externalized PS. The bead-sperm mixture then runs through a magnetic column, which retains the labeled cells and allows the unlabeled cells to flow freely. Because MACS is unable to separate leukocytes or other cells from viable sperm, this technique is usually used in combination with DGC, which adds to processing time and involves an additional centrifugation step. The cost of specialized equipment is also a consideration. Nevertheless, available data demonstrate that MACS is fast, highly specific, and safe. A recent meta-analysis of patients undergoing IVF or ICSI showed that MACS-selected sperm were associated with a significantly higher pregnancy rate than sperm selected with DGC and the swim-up procedure alone. No significant difference in miscarriage rates was identified.
Two other sperm selection techniques based on the same principle have been described in the literature, namely, annexin V-activated glass wool filtration and flow cytometric cell sorting of fluorescently labeled annexin V-PS positive spermatozoa. Both are currently in experimental stages, with limited clinical data.
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