13.2.1 Swim-Up

The swim-up technique selects highly motile and morphologically normal spermatozoa based on their ability to swim out of the seminal plasma into the upper layer containing medium. This technique can be performed using a washed sperm pellet (conventional swim-up) or whole liquefied semen specimen (direct swim-up) [7]. The conventional swim-up method consists of several centrifugation steps, which results in the generation of reactive oxygen species (ROS), leading to decreased sperm motility and plasma membrane integrity, increased apoptosis, and DNA fragmentation [8]. The direct swim-up technique was specially developed for oligozoospermic semen samples to overcome the negative impact of centrifugation steps on spermatozoa [8,9]. The migration–sedimentation method was developed for spermatozoa with reduced motility, and the swim-down technique allows selection of highly motile sperm in a downward gradient [9].

The direct swim-up method is cost-effective and the easiest technique for sperm separation by migration. The liquefied semen specimen is placed into round-bottom tubes, followed by stratification with sperm wash medium. The tubes with the samples are placed at a 45° angle in the incubator for 1 h at 37 °C, followed by aspiration of viable sperm from the upper layer [9,10]. The viable sperm swims up through the medium, while the nonmotile sperm remains at the bottom of the tube (Figure 13.1). Finally, sperm analysis is done to determine whether the sperm parameters such as count and motility are sufficient for IUI [11]. Ideally, a total motile sperm count of 5 × 10⁶ is utilized for IUI [12]. Youglai et al. reported that there was no difference between the conventional and direct swim-up methods with respect to sperm DNA damage and ability of recovered spermatozoa to fertilize the oocyte [13].

Figure 13.1 Swim-up procedure. The upper layer contains sperm wash medium and the bottom one the liquefied semen sample. After incubation, the motile spermatozoa will swim up into the upper layer, and the nonviable sperm, leukocytes, and cell debris will remain at the bottom of the tube.

The swim-up technique is fast and cost-effective. The selected sperm are highly motile, with improved motion characteristics compared to unselected ones [14]. Furthermore, sperm selected with swim-up before freezing (cryopreservation) have higher post-thaw motility, acrosome integrity, and ability to undergo acrosome reaction [14]. However, one of the major limitations of this technique is the low rate of sperm recovery (5–10 percent) [9].

13.2.2 Density Gradient Centrifugation

The density gradient centrifugation technique is considered to be the gold standard approach for semen selection [15]. This method separates motile from nonmotile sperm cells and cellular debris according on their density. For this, the spermatozoa cross a gradient made with colloidal silicon and stop at a specific density layer according to their isopycnic point [8]. Live and motile spermatozoa have different density compared to immature or dead spermatozoa. Live and motile spermatozoa have slightly higher density of at least 1.10 g/ml, and settle in the bottom layer (80 percent). Abnormal nonmotile sperm have a slightly lower density of 1.06–1.09 g/ml, and they are retained in the middle layer (40 percent) [16]. The cell debris and leukocytes are collected at the interphase between the seminal plasma and the 40 percent upper phase [16]. There are several approaches to this technique, which include continuous and discontinuous density gradient methods [9].

The two-layer discontinuous density gradient technique is easy to perform and consists of adding the lower phase (80 percent), layering the upper phase (40 percent), followed by the liquefied semen specimen in a graduated conical centrifuge tube. The tube is centrifuged for 20 minutes at 1600 rpm (Figure 13.2) [9]. After centrifugation, the supernatant consisting of seminal plasma, abnormal nonmotile sperm, and both interphases are completely removed and the sperm pellet at the bottom of the tube is resuspended with 2 ml of sperm wash medium. This is further centrifuged for 7 min at 1600 rpm. Again, the supernatant is discarded and the washed sperm pellet is resuspended in a final volume of 0.5 ml of sperm wash medium. The density gradient centrifugation is an easy and fast method. In contrast with the swim-up method, it has a higher rate of sperm recovery, and is the preferred method for semen specimens with low sperm concentration or abnormal morphology or motility. However, there is a higher risk of obtaining spermatozoa with low DNA integrity and contaminated with endotoxins [9].

Figure 13.2 Density gradient centrifugation procedure. The lower phase is followed by the upper phase and semen sample. After centrifugation, the viable mature spermatozoa will be present in the bottom layer. The abnormal nonmotile and seminal plasma will be in the upper and seminal plasma layers, respectively.

13.3.1 Magnetic-Activated Cell Sorting

The magnetic-activated cell sorting (MACS) technique uses annexin V-conjugated paramagnetic microbeads to differentiate and eliminate apoptotic cells from a sperm suspension. In apoptotic spermatozoa, the phospholipid phosphatidylserine is translocated from the inner leaflet to the outer leaflet of the plasma membrane. Consequently, the phosphatidylserine is considered an apoptotic marker. This technique is based on the fact that annexin V protein has high affinity for phosphatidylserine expressed at the outside plasma membrane of apoptotic sperm [5,9].

The liquefied semen specimen is incubated with annexin V-conjugated paramagnetic beads for 15 min at room temperature. During the incubation step, the apoptotic spermatozoa attach to the annexin V-coated microbeads as a result of interaction between phosphatidylserine and annexin V protein. Then, the sperm suspension is passed through the column, which is placed on a stand surrounded by a magnetic field. While the non-apoptotic spermatozoa (annexin V-negative) passes through the magnetic field along with the solution, drop by drop, in the collection tube, the apoptotic (annexin V-positive) spermatozoa are retained in the magnetic field (Figure 13.3). The viable, non-apoptotic (annexin V-negative) sperm collected in the solution can be used for ART [17].

Figure 13.3 (a) MACS stand. (b) After incubation, sperm suspension is placed into a column which is placed on a stand surrounded by a magnetic field. (c) While the non-apoptotic spermatozoa pass through the column into the solution, the apoptotic spermatozoa, attached to the magnetic microbeads, are retained in the magnetic field.

This technique is simple, reliable, and noninvasive. Sperm sorted with MACS have higher progressive motility, normal morphology, high DNA integrity, and non-disturbed mitochondrial membrane potential [5]. This technique alone allows obtaining sperm with lower DNA fragmentation by 30 percent. When MACS is used in combination with density gradient centrifugation, a further relative reduction in sperm DNA fragmentation of 40 percent is obtained [6]. The microbeads used in the MACS technique are considered harmless for sperm cells as they are composed of microspheres (50 nm in diameter) made up of iron [18]. However, care should be taken that no microbeads are injected into the oocyte along with the spermatozoa during ICSI. As the MACS technique does not eliminate leukocytes and cell debris, it should be performed before or after other semen sample processing techniques such as swim-up and density gradient centrifugation [6].

13.3.2 Microfluidics

Microfluidics is a relatively new technique introduced in the field of sperm selection. It uses a nontoxic and transparent microchannel made of polydimethylsiloxane (PDMS) silicon polymers that separates sperm with high motility and normal morphology [17]. There are several approaches within this technique. However, the passively microfluidic device had demonstrated better results in selecting motile sperm without the need for centrifugation steps [19].

These devices mimic the female reproductive tract with respect to fluid viscosity and temperature. The dead and nonmotile sperm, leukocytes, and cell debris in the liquefied semen are unable to move and are retained in the inlet. While sperm with DNA breaks swim straight, sperm with high DNA integrity swim to the left and right outlets (Figure 13.4) [17]. This allows the selection of sperm with normal morphology, high chromatin condensation, and high DNA integrity from a low-volume semen sample (1 ml) [20]. The spermatozoa are collected in a 100 μl final volume of the buffer.

Figure 13.4 Microfluidic device able to select sperm according to their motility capacity. Immotile and immature sperm, leukocytes, and cell debris remain in the inlet compartment. Motile sperm with high levels of DNA damage swim straight. Motile spermatozoa with high DNA integrity swim to the right and left compartments.

13.3.3 Motile Sperm Organelle Morphology Examination

Motile sperm organelle morphology examination (MSOME) was developed as a sperm selection technique based on morphologic characteristics at the subcellular level (head components, mid-piece, tail region, mitochondria, nucleus chromatin content, presence and size of vacuoles). The spermatozoa are assessed in real time at a high magnification (6300×) using an inverted light microscope equipped with Nomarski optics [21].

This technique allows the visualization of sperm at a higher magnification compared with the one used for routine semen analysis. It makes possible the selection of spermatozoa without subcellular morphological abnormalities [6]. The MSOME protocol involves mixing 1 μl of sperm suspension with 5 μl of human tubal fluid (HTF) (containing 7 percent polyvinylpyrrolidone solution). The mixture is placed in a glass Petri dish and observed under an inverted light microscope at a high magnification. Then, the images are captured and the morphologically normal sperm can be selected for ICSI. The combination of MSOME and ICSI has been named intracytoplasmic morphologically selected sperm injection (IMSI) [21]. This is a costly, time-consuming, labor-intensive process and requires a skilled technologist to select the best sperm [22,23].

13.3.4 Flow Cytometry

The flow cytometric cell sorting technique is used to differentiate specific cell populations from a sample. This method allows assessing important sperm parameters such as sperm count, viability, acrosomal reaction, mitochondrial membrane potential, DNA integrity, and capacitation status [24]. Specific subpopulations of sperm cells, such as cells with high DNA integrity, high mitochondrial membrane potential, and non-apoptotic cells can be analyzed and sorted according to their physical, chemical, or fluorescent markers [17,24]. Moreover, flow cytometry can differentiate between X- and Y-sperm carrying chromosomes and can be used as a selection technique to reduce the risk of having a child with X- or Y-linked disorders [24].

The sperm cells are washed, diluted, and suspended in appropriate medium, such as modified Tyrode’s albumin–lactate–pyruvate (TALP) buffer or binding buffer [17,25]. Next, they are either directly (primary antibodies – fluorescent-conjugated monoclonal antibodies) or indirectly (using secondary conjugated antibodies) labeled with fluorescent probes. The primary antibody recognizes and binds directly to a target antigen, whereas the secondary antibody binds to a primary antibody that is attached to a target antigen. Then, the sperm are passed thorough the flow channel and excited by the laser to generate fluorescence signals. The photomultiplier detects the light scatter and fluorescent signals. The optic system records and amplifies the signals obtained by the photomultiplier tubes. The signals recorded by the machine are converted into data files that can be analyzed using software.

This process allows sorting the sperm based on their emitted fluorescence. Finally, different sperm subpopulations are collected in separate wash media [17,24]. The sorted spermatozoa can be used either for IUI, IVF, or ICSI.

It is considered that sperm sorting using flow cytometry efficiently recovers non-apoptotic spermatozoa from a sperm suspension [17]. However, a recent study by De Geyter et al. reported that removal of DNA-fragmented spermatozoa using flow cytometry did not significantly improve the ICSI outcome compared to the swim-up technique [26]. Also, it should be taken into consideration that the sperm cells pass through the flow system very quickly, and they reach a speed of about 90 km/h when they exit the nozzle at the routine sorting pressure of 50 psi (standard pressure). This can damage the plasma membrane and affects viability and motility of the spermatozoa. Suh et al. demonstrated that lowering the standard pressure from 50 psi to 40 psi can improve the parameters of sorted sperm cells, such as concentration and motility [25].

13.3.5 Electrophoretic Sperm Selection

Plasma membrane of matured spermatozoa with normal morphology contains a high concentration of sialic acid, which induces negative charge to the sperm. The electrophoretic approach allows differentiating mature sperm from immature and dysfunctional sperm, as well as from leukocytes based on the size and charge [17]. Microcell flow and microelectrophoresis are two methods used for electrophoretic sperm selection.

The microcell flow consists of two outer and two inner chambers, separated by a 5-μm pore polycarbonate membrane. The inner chamber consists of an inoculation chamber and a collection compartment. The outer chamber is connected with platinum-coated titanium electrodes [6]. The semen specimen (400 μl) is loaded into the inoculation chamber and allowed to migrate from the negative (cathode) to positive pole (anode) for 5 min at 23 °C, with a constant current of 75 mA and variable voltage of 18–21 V, followed by collection of mature sperm from the collection compartment [27]. The spermatozoa with high negative charge are able to migrate from the cathode to the anode within 5 min because the plasma membrane contains a high concentration of sialic acid. The immature sperm cannot migrate within this time because they contain lower concentrations of sialic acid in the plasma membrane, so carry less negative charge. Larger cells such as leukocytes and immature germ cells are filtered/trapped at the polycarbonate membrane level (Figure 13.5) [17]. This technique allows recovery of sperm of good quality (motility, normal morphology, and DNA integrity), able to capacitate and bind to the zona pellucida. Furthermore, the selected sperm are free from leukocytes and other contaminants [5,27].

Figure 13.5 Microcell flow device consisting of a polycarbonate membrane able to let highly negative-charged mature spermatozoa migrate from cathode to anode. The leukocytes, immature spermatozoa, and cell debris stop at the polycarbonate membrane level.

The microelectrophoresis approach differentiates sperm with low levels of DNA damage from abnormal spermatozoa based on electrical charge. The microelectrophoresis consists of an electrophoresis chamber, an egg injection chamber, two bubble restriction chambers, one conductive bridge, and a power pack [28]. After density gradient centrifugation and adjusting sperm concentration at 20 × 10⁶/ml, 10–15 μl of selected sperm is loaded into the electrophoretic buffer at increasing current from 6 mA to 14 mA and variable voltage (30–100 V). The ability of sperm to migrate from cathode to anode is monitored with an inverted microscope, and one spermatozoa is picked up for ICSI [28].

13.3.6 Zeta Potential

The electrical potential at the slipping plane of the sperm cell in suspension, away from the interface, is called sperm zeta potential or electrokinetic potential [5]. The plasma membrane of mature sperm is negatively charged, and it has a zeta potential ranging from –16 mV to –20 mV [5,9]. This technique is based on the fact that mature spermatozoa have a more negative charge than does cellular debris. In addition, mature spermatozoa with zeta potential adhere to surfaces positively charged in a medium without proteins.

Briefly, 100 μl of washed sperm is loaded into a glass tube and mixed with 5 ml of serum-free HEPES-HTF medium. Glass tubes are preferred as they are positively charged (2–4 mV) [29]. The glass tube is placed into a latex glove, rotated two or three times, and withdrawn from the glove. The tube is incubated for 1 min at room temperature. During the incubation, mature spermatozoa with negative zeta potential stick to the walls of the tube (Figure 13.6). Then the tube is centrifuged at 300 g for 5 min, which does not change the electric charge in the tube. The supernatant is discarded and 200 μl serum-supplemented HEPES-HTF medium is added to wash the mature sperm adhering to the tube’s walls. The addition of serum neutralizes the surface charge by binding to anions and cations in the solution [29]. During all the steps, the tube should be held vertically.

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Related posts:

1. Chapter 2 – Evaluation of Candidates for Sperm Retrieval
2. Chapter 5 – Epididymal Sperm Retrieval
3. Chapter 7 – Surgical and Nonsurgical Sperm Retrieval Techniques in Patients with Ejaculatory Dysfunctions
4. Chapter 12 – Sperm Cryopreservation

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