Rakesh Sharma and Ashok Agarwal

647© Springer Nature Switzerland AG 2020S. J. Parekattil et al. (eds.), Male Infertility, https://doi.org/10.1007/978-3-030-32300-4_52

Sperm Processing and Selection

Rakesh Sharma and Ashok Agarwal

52.1 Introduction

Assisted reproductive technologies (ART) such as intrauter-ine inseminations (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) are effective treat-ment options that allow infertile couples the opportunity to have their families. However only a third of the ART cycles result in live births, and it is unclear why so many attempts result in non-fertilization [1]. Male infertility is a factor in 50–60% of infertility cases [2, 3]. Infertile men tend to have abnormal sperm parameters, such as low sperm concentra-tion, poor motility, abnormal morphology, and elevated lev-els of sperm DNA damage [4, 5]. Additionally, about 40–88% of sperm samples from infertile men have high levels of reac-tive oxygen species (ROS) [6–8]. Low concentrations of ROS are required for physiological sperm functions such as

capacitation, acrosome reaction, and hyperactivation, and the overproduction of ROS is usually due to the inability of antioxidants to neutralize ROS [9–12]. A high level of ROS and decreased levels of antioxidants can cause oxidative stress, which decreases sperm motility, DNA integrity, and viability and increases midpiece defects [5, 12–14]. Poor DNA integrity is correlated with lower in vitro fertilization pregnancy rates, irregular pre-implantation development, early loss of pregnancy, and increased disease rates in off-spring conceived through ART [15–18].

In natural conception, only a small fraction out of the mil-lions of sperm that are deposited in the vagina very near to the cervix reach the oocyte. This indicates the presence of a strict and efficient sperm selection process that naturally occurs in the female genital tract. Subsequently sperm are drawn through the cervix, uterus, uterotubal junction, and oviductal isthmus to reach the egg in the oviductal ampulla. The sperm are presented with different structural, fluidic, ionic, and molecular environments resulting in a complex process of sperm migration [19]. In in vitro fertilization, an oocyte is incubated with about 50,000 sperm from an initial sample containing about 100 million sperm. In intracytoplas-mic sperm injection (ICSI), a single sperm is selected and directly microinjected into the oocyte. Therefore the funda-mental challenge of sperm selection is dictated by the sperm biology, sample volume, sperm concentration, and lifetime in vitro. The ideal time for sperm selection process is about 10 min for 1 mL of sample containing 100 million/mL sperm. This indicates an extremely high biological sorting rate of ~100 kHZ which is extremely higher than the current cell sorting technologies [20]. The two most common sperm selection methods for sperm preparation for ART are density gradient centrifugation and swim-up based on sedimentation and migration, respectively. The density gradient technique separates about 36% of sperm from 0.5 mL raw semen in about 30 min, whereas swim-up selects about ~12% of the sperm population from 1 mL of the sperm population in about 1 h. This results in about 18–19% and 5% sperm improvement in sperm motility [21].

Key Points• Sperm selection is important in assisted reproduc-

tive technology to obtain good-quality sperm with high DNA integrity.

• Centrifugation-based techniques can produce reac-tive oxygen species, result in oxidative stress, and decrease DNA integrity.

• Non-centrifugation-based techniques reduce oxida-tive stress and select sperm with high DNA integrity.

• Advances tests such as magnetically activated cell sorting, hyaluronidase binding, and microfluidics are promising and warrant further investigation in ART outcomes.

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R. Sharma · A. Agarwal (*) American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USAe-mail: [email protected]

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https://doi.org/10.1007/978-3-030-32300-4_52

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The current sperm processing techniques suffer from a number of limitations:

• The methods do not resemble the natural process in vivo.• Selected sperm population is contaminated with poor

motile cells.• Presence of leukocyte contamination.• Iatrogenic injury due to prolonged exposure and oxidative

stress due to centrifugation [22].• Further visual inspection which is related to the embry-

ologist specific step which can influence the success rate between the clinics [23].

Therefore, there is a need to develop and implement improved sperm sorting techniques and protocols in order to select sperm containing normal DNA and lesser ROS, improve ART success rates, and decrease birth defects. New sperm selection methods should closely mimic the natural selectivity of the female genital tract to ensure that only the healthy sperm are selected. In this paper, we review current sperm selection technologies and their respective effects on sperm morphology and function as well as ART outcomes.

52.2 Reduction of Semen Viscosity

Human semen normally liquefies within 5–20 min after ejac-ulation [24]. However, some ejaculates fail to liquefy, and some are viscous by nature. Semen viscosity is a problem since it can reduce sperm motility. To reduce viscosity, the semen can be mixed with a sperm wash medium. However liquefaction achieved by this method might not be adequate for highly viscous samples. In such cases, the viscosity can be reduced by forcing the viscous semen through a needle with a narrow gauge which is another option [24]. The com-mon method to reduce viscosity is by treating the sample with 5 mg of trypsin. If the sample fails to liquefy after 20 min, trypsin powder is directly added to the sample, and after swirling, the sample is incubated for an additional 10 min. This results in complete liquefaction of the sample.

52.3 Conventional Sperm Selection Methods

A variety of selection techniques have been introduced that are based on centrifugation, filtration, or sperm migration. Among the centrifugation techniques, density gradient cen-trifugation has been proposed as the gold standard for sperm preparation. The latest developments in sperm selection are focused on the sperm surface combined with or without a standard preparation protocol as illustrated in advanced sperm selection section.

52.3.1 Simple Sperm Wash

Both one-step and two-step sperm washing methods involve resuspension of sperm in the culture medium after complete liquefaction. The one-step wash technique does remove or reduce any cellular component such as the number of leuko-cytes, immature spermatozoa, or other cellular debris. It only removes the seminal plasma. Furthermore, centrifugation causes additional harm by formation of reactive oxygen spe-cies (ROS) by abnormal spermatozoa and leukocytes [25]. Increased levels of ROS result in DNA damage in spermato-zoa, decreased sperm motility, increased numbers of apop-totic spermatozoa, and decreased sperm plasma membrane integrity [26].

52.3.2 Swim-Up

It is one of the most commonly used migration techniques for sperm preparation. Sperm culture medium containing antioxidants provides the nutritional support. In the con-ventional swim-up technique, a prewashed pellet obtained after a soft spin is placed at the bottom of an overlying medium. In addition, after complete liquefaction, semen sample (0.5 mL) can be carefully layered at the bottom of a round bottom tube containing about 2 mL of the sperm wash medium. The tube is placed at an angle of 45° and incubated for 60 min. Depending on the original sample, multiple tubes can be prepared. At the end of incubation, using sterile technique, clear supernatant is aspirated into a separate tube (Fig. 52.1). The sample can be centrifuged at 1600 rpm for 7 min and the pellet resuspended in 0.5 mL of sperm wash medium. The swim-up procedure uses the active motion of the spermatozoa. All motile spermatozoa move out of the pellet into the clear supernatant. Highly motile, morphologically normal intact spermatozoa are enriched in the absence of other cells, proteins, and debris within the supernatant. A modified swim-up method called the direct swim-up is used for oligospermic samples [27]. In this method, sperm swim out directly from the semen rather than from the cell pellet. Round bottom tubes are used to maximize the surface area between the semen and the medium [25]. Swim-up method is inexpensive, and highly motile sperm can be obtained. The disadvantages are that the sperm recovery is relatively low. Only 5 to 10% of sperm cells are retrieved. When a pellet is used, sperm are trapped in the pellet and may not move into the clear medium. In addition, centrifugation results in the generation of ROS [28]. Furthermore if the sample is contaminated with leukocytes, the close cell-to-cell contact may further result in production of reactive oxygen species (ROS).

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52.3.3 Density Gradient Centrifugation

Density gradient centrifugation is considered the gold stan-dard technique for sperm preparation. It separates cells based on the density, motility, and centrifugation speed. Morphologically normal and abnormal spermatozoa have different densities. Mature morphologically normal sperm are denser (1.10 g/mL) compared to immature and morpho-logically abnormal sperm (1.06–1.09 g/mL) [29]. Components of the density gradient sperm separation proce-dure include a colloidal suspension of silica particles stabi-

lized with covalently bonded hydrophilic silane supplied in HEPES. There are two gradients: a lower phase (80%) and an upper phase (40%). Sperm washing medium (modified HTF with 5.0 mg/mL human albumin) is used to wash and resuspend the final pellet (Fig. 52.3).

Percoll™, a colloidal suspension of silica particles coated with polyvinylpyrrolidone, was widely used by ART labora-tories until it was withdrawn from the market for clinical use. Media containing silane-coated silica particles are com-monly used. Isolate™ (Irvine Scientific, Santa Ana, CA), IxaPrep™, Sperm Preparation Medium™ and SupraSperm™ (Origio, MediCult, Copenhagen, Denmark), SpermGrad™ (Vitrolife, San Diego, CA), SilSelect™ (FertiPro NV, Beernem, Belgium), and PureSperm™ (NidaCon Laboratories AB, Gothenburg, Sweden) are commonly used [24]. This method allows for the enrichment of mature and motile sperm, and recovery rates of 30–80% can be achieved depending on the initial semen sample and the technical skill of the individual doing the procedure.

A gradient is prepared by carefully layering 2 mL of lower phase at the bottom of the 15 mL graduated centrifuge tube, and a 2 mL of upper layer is layered on top without mixing the two gradients [30]. Up to 2 mL of a completely liquefied semen sample is layered on top and centrifuged for 20 min (Figs. 52.2 and 52.3). During this procedure, highly motile spermatozoa move actively in the direction of the sedimentation gradient and therefore can reach lower areas quicker than poorly motile or immotile cells. The resulting interphases between seminal plasma and 40%, 40%% and 80% containing the leukocytes, cell debris, and morphologi-cally abnormal sperm with poor motility are discarded. The highly purified motile sperm cells are enriched in the soft pellet at the bottom. The pellet is resuspended in 2 mL of the medium and centrifuged again at 1600 rpm for 7 min. The clear pellet is finally resuspended in 0.5 mL of sperm wash-ing medium before use in intrauterine insemination. Centrifugal force and time should be kept at the lowest pos-sible values (<300 g) in order to minimize the production of ROS by leukocytes and non-viable sperm cells [31]. Also, non-viable sperm cells and debris should be separated from viable sperm cells as soon as possible to minimize oxidative damage. Double density gradients comprise the commonly used sperm preparation protocol for ART [25].

Compared to swim-up, density gradient takes only 30 min. It is relativity easy to perform under sterile conditions. Spermatozoa from oligozoospermic patients can also be sep-arated by this method. Density gradient eliminated the major-ity of the leukocytes in the ejaculates. The disadvantages are that the interphases between the layers may take some time; there are reports that sperm prepared by density gradient still have some degree of DNA fragmentation compared to sper-matozoa prepared by swim-up [32].

Fig. 52.1 Sperm selection by swim-up technique (Reprinted with per-mission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)

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52.4 Preparation of Assisted Ejaculation Samples

Patients with spinal cord injury often have ejaculates with a high sperm concentration and low sperm motility [33]. These ejaculates are also contaminated with red blood

cells and white blood cells. In these patients, ejaculates can be obtained by electroejaculation using direct penile vibratory stimulation or indirect rectal stimulation. These ejaculates can be effectively prepared with density gradient centrifugation [33].

52.5 Preparation of Retrograde Ejaculation Samples

Retrograde ejaculation occurs when the semen is directed into the urinary bladder during ejaculation. If there are an inadequate number of spermatozoa in the ejaculate, sperm cells in the urine need to be retrieved. The patient is first asked to urinate without entirely emptying his bladder. Then, he is asked to ejaculate and urinate again into another speci-men cup containing 9 mL of warm sperm wash medium to alkalinize the urine. The urine sample volume is noted and analyzed after centrifugation. Both the concentrated retro-grade specimen and the antegrade specimen are prepared with density gradient centrifugation technique to obtain motile sperm for use in ART [34].

52.6 Sperm Preparation Techniques for Cryopreserved Semen

There are many medical conditions where sperm banking is indicated to preserve fertility. Cancer patients both young adolescents and adults are the most common group of men

Fig. 52.3 HTF resuspended sample centrifuged to produce viable sperm pellet (Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)

Fig. 52.2 Double density gradient wash procedure; separation of sem-inal plasma, abnormal nonmotile sperm, and viable motile sperm (Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)


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who are referred for sperm cryopreservation. In addition, patients with medical conditions such as lupus, multiple sclerosis, varicocele, testicular torsion, spinal cord injury, and ejaculatory dysfunction, gender reassignment, and trav-elling husbands can also benefit from sperm cryopreserva-tion. Protocols for sperm freezing include conventional slow freezing [35] and vitrification [36, 37]. In the slow freezing technique, after liquefaction, a TEST-yolk buffer (TYB) aliquot equal to 25% of the original volume of specimen is added to the semen sample using sterile technique. The spec-imen and TYB combination are placed on a test tube rocker for 5 min to ensure that the sample is gently mixed. The pro-cess of adding a 25% aliquot of TYB followed by gentle mixing should be repeated three more times so that the total volume of TYB added over the four different aliquots equals that of the original semen sample. The samples are aliquoted into cryovials and stored in liquid nitrogen (LN2) [35].

Vitrification is a technique based on the ultra-rapid freez-ing of cells by directly immersing in LN2. In this technique, there is no formation of ice crystals. Vitrification of sperma-tozoa is challenging due to the unique properties of the sper-matozoa. A superior preservation of motility and viability is seen in sperm preserved by vitrification when compared to standard slow freezing. Spermatozoa are osmotically fragile, and use of high concentration of permeable cryoprotectants is toxic and also potentially mutagenic [36]. Samples can be processed by swim-up or other method and vitrified samples loaded onto straws or cryoloops and immersed in LN2. Cryoprotectant-free vitrification can be accomplished by using high cooling rates by directly plunging samples into LN2, ~720,000 K/min, and increasing the surface area for heat exchange using extremely small sample volume [37]. Sperm can be stored during vitrification using cryoloop, droplet, open straws, and open pulled straws.

52.7 Preparation of Epididymal and Testicular Sperm

Sperm can be obtained from the epididymis or the testicular tissue in case of epididymal obstruction or complete azo-ospermia. A large number of sperm can be collected from the epididymis which are not contaminated with other non-germ cells such as red blood cells [38]. Sperm wash technique can be used if the number of cells obtained is low and if sufficient numbers are collected; density gradient centrifugation can be used to prepare the spermatozoa for ART. Spermatozoa obtained from the testes by open biopsy or by percutaneous needle biopsy contain large numbers of non-germ cells such as red blood cells. Spermatozoa have to be separated from non-germ cells. Sperm motility in spermatozoa is generally

low. Hypoosmotic solution or pentoxifylline is occasionally used to increase the motility of epididymal and testicular spermatozoa before ICSI [24].

52.8 Advanced Sperm Preparation Methods

Advanced sperm preparation techniques allow the spermato-zoa to be selected on their surface charge and morphology and overcome the limitations of classical sperm selection procedures. New insights into the molecular biology of the spermatozoa have allowed the molecular selection strategies including hyaluronic acid-mediated sperm selection, annexin V magnetic-activated cell sorting (MACS), and the latest technology of selecting sperm by microfluidics.

52.8.1 Zeta Potential and Sperm Birefringence

The electrical potential between the sperm membrane that is negatively charged and its surrounding is called zeta poten-tial. Negative charge is due to the presence of the epididymal proteins that are present on the sperm membrane surface [39]. Zeta potential is lower in sperm with DNA damage, and this property can be used to select sperm with intact DNA [40]. The washed sperm (~100 μL) are suspended in a 15 mL of serum-free HEPES-HTF medium. The tube is rapidly pulled after rotating it a couple of times in a latex glove. This allows the negatively charged sperm to stick to the walls of a positively charged plastic tube (Fig. 52.4). Immature- abnormal sperm in the suspension are discarded. The tube is maintained at room temperature without agitation for about 1 min and centrifuged at x300g for 5 min. The sperm retain-ing the negative zeta potential are attached to the walls of the tube. These can be recovered in a 0.2 mL of serum- supplemented HEPES-HTF medium, thereby neutralizing the charge on the wall of the test tube [40]. Markers of apop-tosis were significantly reduced in zeta selected sample [41]. Zeta selection results in a significant reduction in progressive motility and is not very helpful when used in cryopreserved sperm [41]. High-quality spermatozoa can be separated from the poor-quality sperm using a positively charged centrifuge tube. In ART, sperm sample containing superior motility, normal morphology, and intact DNA can be separated by this technique [42]. Protamine-deficient sperm are eliminated, and sperm with DNA integrity are retained resulting in high fertilization rate. Negative zeta potential sperm in IVF had a higher fertilization rate (65.79%) compared with sperm iso-lated with double density gradient centrifugation. Embryo cleavage and pregnancy rates were high in sperm used in


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ICSI selected by MACS compared with DGC in oligo-, astheno-, and teratozoospermic men [43, 44].

The subacrosomal protein filaments are longitudinally arranged in mature sperm. Therefore mature sperm nucleus exhibits higher birefringence that can be examined by polar-ized light microscopy. This allows the evaluation of birefringence and selection of mature sperm [45]. Acrosome intact sperm with high DNA integrity can be selected from the acrosome-reacted spermatozoa with DNA fragmentation using this technique [45, 46].

52.8.2 Ultrastructural Sperm Selection

Subtle defects in the sperm morphology in the acrosome, nucleus, mitochondria, post-acrosomal lamina, and neck can be observed using real-time inverted light microscope equipped with Nomarski optics enhanced by digital imaging. It achieves an ultra-high magnification microscopy (6300x) called motile sperm organelle morphological examination (MSOME) [47, 48]. The ultrastructural morphology of the sperm head components correlates with sperm fertilizing capacity in vitro [49]. MSOME was also shown to be posi-tively associated with both fertilization rate and pregnancy outcome [47]. Sperm selected by strictly defined morpho-logically normal nuclei, especially when sperm with vacu-oles were avoided, significantly improved the incidence of pregnancy in couples with previous ICSI failures [48]. A cor-relation was also reported between higher fraction of sperm with high DNA fragmentation and presence of large nuclear vacuoles. These results support the use of MSOME for rou-tine selection of sperm for ICSI [50]. The procedure however is time-consuming, and the selected sperm may still be

exposed to oxidative stress. In a retrospective study by Bradley et al. [51] examining the efficacy of interventions such physiological intracytoplasmic sperm injection (PICSI), MSOME or intracytoplasmic morphologically selected sperm injection (IMSI), testicular sperm, or other interven-tion such as frequent ejaculation alone or in combination with PICSI or IMSI, or testicular sperm in combination with IMSI with high sperm DNA fragmentation (SDF) on preg-nancy rate, birth rate, miscarriage rate. High SDF patients who underwent IMSI intervention had the poorest outcomes of all intervention groups and were very similar to those of the no intervention group. Furthermore a recent meta- analysis included 9 randomized controlled trials and 2014 couples (IMSI = 1002; ICSI = 1012) compared regular ICSI for assisted reproduction. The results from this study show lack of evidence that IMSI improves clinical pregnancy rates compared to ICSI [52].

52.8.3 Hyaluronic Acid-Mediated Sperm Selection

This is a novel selection technique comparable to the earlier sperm-zona pellucida binding. Hyaluronic acid receptors are present on the plasma membrane on acrosome-intact sperm and are indicative of sperm maturity [53]. It is also the main component of the extracellular matrix of the cumu-lus oophorus. Mature sperm bind to the hyaluronic acid and therefore have a better chance of reaching the oocytes for fertilization. Sperm can be selected by physiological intra-cytoplasmic sperm injection (PICSI) which is a plastic dish containing spots of HA attached to its base. Sperm are attached to HA by the head, and the sperm can be easily

Fig. 52.4 Selection of spermatozoa using zeta potential principle. The negatively charged mature sperm sticks to the walls of the positively charged centrifuge tube, while non-mature sperm in the suspension are discarded (Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)


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selected for microinjection. The frequency of sperm with chromosomal disomy is significantly reduced when com-pared with ejaculated sperm. Hyaluronic acid binding also excludes immature sperm with cytoplasmic extrusion, pres-ence of sperm with histones, and DNA fragmentation indi-cating selection of sperm with reduced oxidative stress [53]. Pregnancy has been reported after the use of PICSI [54]. A viscous medium containing HA called SpermSlow slows the active sperm and allows the selection of the appropriate sperm.

52.8.4 Electrophoretic Sperm Selection

Sperm can be separated based on their size and charge on their surface by electrophoretic separation [55]. It consists of two outer chambers separated by two inner chambers by polyacrylamide restriction membranes of 15 kDa pore size. Semen is placed in the electrophoretic device, and a current is applied. The pore size allows the competent spermatozoa to move in the applied electric field [56]. Normally differen-tiated sperm are rapidly separated and collected at the adja-cent chamber. Normally differentiated sperm are charged negatively. The resulting population shows a low incidence of DNA damage. It compares favorably with the density gra-dient separation technique in purity, absence of ROS, and superior viability and morphology of the isolated spermato-zoa. Motility has been reported to be affected by electropho-resis [55]. Ainsworth et al. effectively used the sperm selected with high DNA integrity, to establish pregnancy from the semen sample with high DNA fragmentation by ICSI [56].

52.8.4.1 Microflow CellA microflow cell consists of an outer chambers connected with a platinum-coated titanium electrodes, and the inner chamber is divided into two compartments – the inoculation (loading) chamber and the collection chamber. A polycar-bonate membrane 5 μm thick separates the two compart-ments (Fig. 52.5). The membrane filters out the good-quality sperm from the contaminating cells such as the leukocytes and germ cells. A 400 μL semen sample is loaded in the inoc-ulation chamber and the buffer in both loading and collecting chambers, and sample is equilibrated for 5 min at 23° C. A constant current of 75 mAmps is applied with a variable volt-age of 18–21 mV [55]. The highly motile good quality sper-matozoa are sorted out and are ready to be used in ART.

Microelectrophoresis technique can be used to select the negatively charged spermatozoa from the seminal ejaculate as well as after density gradient selection [57]. The micro-electrophoresis chamber consists of the egg inoculation and the bubble restriction chambers. Microelectrophoresis is car-ried out under ICSI stage. A 10–15 μL semen sample is elec-trophoresed in the buffer, and increasing current (6–14 mA)

and variable voltage (3–100 V) are applied. The sperm are monitored under the inverted microscope under 200x, and good-quality sperm are selected for ICSI [57]. This tech-nique allows the selection of viable, morphologically nor-mally, motile sperm with high DNA integrity. Selected sperm are negatively charged and free from oxidative stress and exhibit normal zona pellucida binding [58, 59]. Selected sperm used in ICSI have resulted in pregnancy [56].

52.8.5 Annexin V and MACS Separation

Phosphatidylserine is a phospholipid that is present on the inner leaflet of the plasma membrane. It moves to the outer surface when the membrane is damaged. Thus externaliza-tion of the phosphatidylserine residue is a marker of apopto-sis [60]. Reactive oxygen species (ROS) not only affects nuclear and mitochondrial DNA but also is involved in the activation of apoptosis signaling cascade parts [61, 62]. Annexin V is a phospholipid-binding protein. It has a strong affinity for phosphatidylserine residue. It cannot penetrate the sperm membrane, and its binding to the sperm membrane signifies that the sperm integrity is compromised and the sperm phosphatidylserine has been externalized. Therefore annexin V is used to label sperm that have a compromised membrane integrity and are less able to fertilize the egg [63].

Magnetically activated cell sorting (MACS) uses a col-loidal super-paramagnetic microbeads conjugated with annexin V antibodies. A strong magnetic field is employed, and the sperm that are non-apoptotic pass through the mag-netic field, whereas those that are apoptotic are tagged and retained in the magnetic field [64–66]. This allows the rapid separation and selection of non-apoptotic sperm from apop-totic sperm [64] (Fig. 52.6a–c). Selection of non-apoptotic spermatozoa for use in ART is based on the ability of phos-phatidylserine residues on the external surface of the sper-matozoa in early stages of apoptosis.

Higher-quality sperm have been obtained using sperm selected by density gradient and MACS than by density gra-dient alone [67]. While density gradient removes immature sperm cells, debris, and leukocytes, the annexin V MACS removes already damaged sperm with altered membranes, activated apoptosis signaling, and DNA fragmentation [68]. All these indirectly result in a significant reduction in oxida-tive stress. Similarly sperm selected by MACS before cryo-preservation had a larger number of sperm with intact mitochondrial membranes, a reflection of mitochondrial sur-vival after cryopreservation, than sperm prepared by cryo-preservation alone (36.1% ± 18.9%) [67]. Hence sperm selection by MACS before cryopreservation can be used to improve motility and cryosurvival rate. The clinical signifi-cance of using annexin V MACS technique is that it allows the selection of sperm with improved motility, viability, and


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morphology and significantly improved fertilization rates and oocyte penetration [68, 69]. Improved pregnancy rates have been reported compared to sperm prepared by density gradient alone [43].

52.8.6 Microfluidic Separation of Sperm

This is the latest in sperm selection technologies. Microfluidic devices use microchannels made from polydimethylsiloxane (PDMS) silicon polymers that are nontoxic and transparent [70]. Lab-on-chip approaches have been used to select sperm based on motility [71–73], chemotaxis [73–75], optical forces [76, 77], and electrophoresis [78]. Sperm can be selected using (1) passively driven microfluidic device [71, 79], (2) chemoattractant microfluidic device [80], (3) chemo-taxis device [74], (4) microfluidic fertilization device [81],

(5) macro-microfluidic sperm sorter [82], (6) Zech selector [83], (7) circular microfluidic device [20], (8) microgroove and channel device [84], and (9) boundary-following behavior- based passive microfluidic device [85].

The popular passive microfluidic device selects spermato-zoa based on the boundary-following behavior (Fig. 52.7). This device consists of radial network of channels (52 μ width) which separates sperm into left, right, and straight swimmers. Using a plastic syringe, an aliquot of raw semen (200 μL) is loaded into the inner ring and kept undisturbed for 15 min at 37° C. Motile sperm move and flow through the microchannel in the medium that mimics the viscosity of the reproductive tract fluid. Dead or immotile sperm are retained in the inlet, and motile sperm are collected from the micro-channel outlet [85].

Sperm are selected according to the normal motility, mor-phology, and high DNA integrity [20, 71, 82, 83, 86, 87].

Fig. 52.5 Microflow cell separation of spermatozoa from leukocytes using polycarbonate separation membranes and sorting based on the movement in the applied electric field (Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)


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a

c

b

Fig. 52.6 Sperm selection by (a) magnetic-activated cell sorting and collection device. The MACS columns are placed on the stand surrounded by magnetic field. (b) Loading the MACS columns with liquefied semen (apoptotic and non-apoptotic sperm) labeled with annexin V-coated micromagnetic beads. (c) Activated magnetic field retains the apoptotic sperm bound to micromagnetic beads coated with annexin V in the column and allows the non-apoptotic healthy sperm cells to flow through the selection column (Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)

Fig. 52.7 Microfluidic device used for sorting sperm based on their swimming patterns: left-hand side (left swimmers), right-hand side (right swimmers), or straight (straight swimmers). Live sperm navigate from the inlet toward the outlet, while dead sperm and debris remain in the inlet (Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2011–2019. All Rights Reserved)


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Selection of sperm by microfluidic technology has great potential in IVF and ICSI setting. A simple clinically appli-cable lab-on-a-chip method was reported for sperm selection based on progressive motility in 500 parallel microchannels [20]. In this one-step procedure, 1 mL of semen could be processed under 20 min resulting in over 80% improvement in selected sperm DNA integrity.

The major advantage of microfluidic devices over con-ventional selection techniques is the ability to work with small sperm sample volume, the short processing times, and the ability to manipulate single cells in a noninvasive manner [88–90]. The yield of the selected sperm by microfluidics is about 41% and comparable to recovery rates of currently used methods [70]. Another advantage is the one-step pro-cess that eliminates centrifugation and the exposure to reac-tive oxygen species and thereby preserves the DNA integrity [20, 82]. Fertilization of ova with preselected spermatozoa of superior quality by microfluidic technique was accomplished using a robotic-assisted platform and IVF on a chip [91].

DNA fragmentation is significantly decreased in sperm separated with the microfluidic sperm sorting system [88, 92] compared the swim-up method with a microfluidic device, resulting in a significantly lower rate of DNA dam-age (16.4% swim-up vs. 8.4% microfluidic). Using radial array of microchannels to select the most motile sperm, an 80% improvement in sperm DNA integrity after sorting was reported [20].

The use of a microfluidic device shortened the time in the ICSI treatment of porcine sperm and increased the number of viable embryos without reducing the in vitro production effi-ciency. An application in human ART is suggested [88, 93]. The technique requires only a low concentration of sperm in a murine IVF model [94]. A robotic-assisted reproduction platform was developed to carry out IVF on a chip by fertil-izing the preloaded ova with superior-quality spermatozoa selected by microfluidic technique [91]. Thus, the microflu-idic sperm sorting proved to have a great potential in clinical IVF and ICSI for achieving early embryo development.

52.9 Specific Indications of Sperm Selection Techniques: Clinical Implications

A variety of techniques can be used to select sperm for use in ART both intrauterine insemination and IVF and ICSI. Conventional methods such as sperm preparation by swim-up and density gradient are very popular. In addition, the introduction of newer techniques such as preparation by MACS alone or in combination with density gradient separa-tion is also being used. The goal is to use a method that selects a highly motile sperm population with intact DNA from the raw semen. In this context, sperm selection utilizing the microfluidic techniques is important and is gaining popu-

larity. It allows the rapid selection of sperm with intact DNA in a one-step process which replaces the previous multi-stage processes involving centrifugation steps associated with oxi-dative stress and iatrogenic risks.

52.10 Future Directions

Sperm separation from seminal fluid removes also the natural protective antioxidants contained in the seminal fluid. To pre-vent excessive oxidative stress to the sperm, antioxidants like human serum albumin must be added to sperm preparation and incubation media for assisted reproduction. Standard sperm selection techniques like density gradient centrifuga-tion are able to reduce oxidative stress by depletion of imma-ture sperm and leukocytes. Prolonging the selection methods, sperm selection with enhanced motility may be achieved but at the expense of DNA damage due to oxidative stress. Advanced sperm separation techniques focus rather on the depletion of already damaged sperm. In ART, procedures such as IVF and especially ICSI require fewer spermatozoa, so lower sperm recovery is not an adverse factor, and therefore microfluidic techniques are more versatile. Microfluidics offers new oppor-tunities to better understand human sperm migration and to use this understanding to prepare sperm for intrauterine insemination, IVF, and ICSI. New sperm sorting technologies have been shown to improve DNA integrity, morphology, and motility, but whether these improvements are significant over the conventional centrifuged-based techniques is unclear [46], and these devices need thorough evaluation.

52.11 Conclusion

In summary, we have described a number of sperm prepara-tion methods that are available to process sperm for use in ART. Each infertile couple must be carefully examined to determine the best sperm preparation method. Future research should seek to improve the efficacy and the safety of the sperm preparation techniques. Advanced sperm selec-tion strategies include selection according to surface charge, sperm birefringence. sperm morphology under ultra-high magnification, ability to bind to hyaluronic acid, sperm apoptosis, and microfluidic separation. These techniques may improve the chances of selecting structurally intact and mature sperm with high DNA integrity for and help improve fertilization and pregnancy rates.

52.12 Review Criteria

An extensive search of studies examining the relationship between sperm selection techniques and improvement in sperm quality and ART outcome was performed using search


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engines such as Google Scholar and PubMed. The start and end dates for these searches were September 1992 and September 2018, respectively. The overall strategy for study identification and data extraction was based on the following keywords: “sperm selection techniques,” “centrifugation,” “reactive oxygen species,” “oxidative stress,” “DNA frag-mentation,” “density gradient techniques,” “electrophoretic cell separation,” “hyaluronic acid binding,” “magnetically activated cell sorting,” “microfluidics,” and “ART and preg-nancy rate.” Articles published in languages other than English were excluded. Data published in conference or meeting proceedings, websites, or books was also excluded.

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Rakesh Sharma and Ashok Agarwal