Microfluidics in andrology

INTRODUCTION

About 30–50% of clinical infertility cases occur because of male factor with sperm defects.^[1] Sperm parameters including extremely low sperm concentration, poor sperm motility, and abnormal morphology, hormonal disturbances, and physical and psychological problems may contribute to natural fertility problems. Earlier, these abnormalities were not completely overcome by contemporary infertility treatments such as intra uterine insemination and conventional in vitro fertilization (IVF), but the advent of intracytoplasmic sperm injection (ICSI) in early 1990s has allowed patients with oligozoospermia or azoospermia to father children.^[2] ICSI enables the fertilization of an egg with one sperm selected from ejaculated spermatozoa. Therefore, sperm quality parameters, including motility, morphology, viability, DNA integrity, apoptosis, and maturity, are the critical determinants of successful assisted reproductive techniques (ART) outcomes.^[3] Several methods such as double-density centrifugation and conventional swim-up procedure are performed for the isolation and the collection of the high-motility spermatozoa from semen samples and removing any impurities that interfere with fertilization.^[4] However, several studies found that single or multiple centrifugation steps induce sperm DNA damage and the generation of reactive oxygen species (ROS)^[5,6] producing adverse consequences during the postimplantation development of the embryo rather than before it.^[7] Therefore, there is a need to develop sperm separation technique that facilitates the retrieval of spermatozoa with normal DNA integrity from ejaculated semen.

SPERM PREPARATION TECHNIQUES

Different sperm preparation techniques are used to prepare spermatozoa for artificial insemination but the choice is strongly dependent on the quality of the semen. So, a prewash analysis of the semen is suggested to quantify the concentration, motility, and morphology of spermatozoa to obtain the higher number of motile spermatozoa even from the poor semen. The most common techniques of sperm preparation are swim-up technique and density gradient technique. Recent advances such as Microfluidics sperm sorting is also one of the sperm preparation techniques used to isolate highly motile spermatozoa with good integrity and will be described below:

1. Swim-up technique

   • Swim-up is one of the most commonly used techniques for sperm preparation in IVF laboratories and is preferred if the semen sample has a normal number of good sperms (normozoospermia). Using this technique, sperms can be selected on the basis of their motility and the capability to swim out of the seminal plasma.^[8] Swim-up can be performed using a cell pellet or a liquefied semen sample. In conventional swim-up, a liquefied semen sample is washed twice with sperm wash media and the pellet obtained by a soft spin is placed in an overlaying culture medium in a conical tube. The common steps of this technique are as follows:

     1. Allow specimen to liquefy completely for 15–30 min in an incubator at 37°C before processing.

     2. Perform a prewash analysis, that is, volume, sperm count, and motility.

     3. Gently mix the specimen with 4 ml of sperm wash media (human tubular fluid, HTF) using a sterile pipette.

     4. Centrifuge the tubes at 1500 rpm for 10 min.

     5. Carefully aspirate the supernatant without disturbing the pellet and resuspend the pellet in 2 ml of fresh HTF. Transfer the resuspended sample into a sterile round bottom tubes using a sterile pipette.

     6. Centrifuge the tubes at 1500 rpm for 5 min.

     7. Again carefully aspirate the entire supernatant from the round bottom tube using a pipette, with the tip placed just above the pellet surface.

     8. Resuspend the pellet in a 0.5 ml HTF using a 1 ml sterile pipette and incubate the tubes at a 45° angle for 20 min for swim-up in vertical rack in a 37°C incubator.

     9. Record the final volume and motility of the sperms.

2. Density gradient centrifugation (DGC) technique

   • DGC separates sperm cells based on their density. It is preferred in the case of oligozoospermia, teratozoospermia, and asthenozoospermia.^[8] Thus, at the end of centrifugation, each spermatozoon is located at the gradient level that matches its density. This method uses two gradients: a lower phase (90%) and an upper phase (45%), and sperm washing medium (modified HTF with 5.0 mg/ml human albumin) is used to wash and resuspend the final pellet. Below are some of the main steps of the process:

     1. Place all components of the 45 and 90% gradient and semen samples in an incubator at 37°C for 20 min.

     2. Transfer 1 ml of the lower phase into a sterile conical centrifuge tube.

     3. Layer 1 ml of the 45% gradient on top of the 90% gradient using a transfer pipette. Slowly dispense the upper phase lifting the pipette up the side of the tube as the level of the 45% gradient rises. A distinct line separating the two layers will be observed. This two-layer gradient is stable for up to 2 h.

     4. Measure semen volume to be loaded using a sterile 3 ml pipette. Remove a drop of semen for a prewash analysis.

     5. Gently place 1 ml of liquefied semen over the upper phase.

     6. Centrifuge for 20 min at 2000 rpm.

     7. Remove supernatant using a transfer pipette without disturbing the pellet, add 2 ml of HTF, and resuspend pellet. Mix gently with pipette until sperm pellet is in suspension.

     8. Transfer this suspension into a sterile round bottom tube and centrifuge for 10 min at 1500 rpm.

     9. Again, remove supernatant from the centrifuge tube using a transfer pipette down to the pellet.

     10. Resuspend the final pellet in a volume of 0.5 ml using a 1 ml sterile pipette with HTF and incubate the tubes for 20 min for swim-up. Record the final volume.

3. Microfluidic sperm sorter (MFSS)

   • Microfluidic sperm sorters are used to isolate morphologically normal human spermatozoa with high motility and with no debris based on fluid dynamics in a microenvironment or nanoenvironment, relying on variables such as fluid density, viscosity, velocity, and size/geometry of the environment.^[9] Microfluidics have been widely applied to biomedical fields, because the control of fluid transport is useful for cell analysis systems, drug delivery systems, assisted reproductive technologies, and in numerous biological applications specifically for the miniaturization and simplification of laboratory techniques.^[10] Sperm selection in this method is based on the two gravity-driven laminar flows within the central microfluidic channel. The semen and the medium dispensed into inlet A and B, respectively, flow parallel to each other and exit through their respective outlets (A/D and B/C). Spermatozoa are sorted depending on their ability to swim across these two streams. Only motile spermatozoa swim toward outlet C whereas immotile spermatozoa keep flowing to outlet D.^[11] Hence, this method does not allow spermatozoa to undergo added physical stress from sources such as a centrifuge and thus minimize DNA damage.

   • Among the sperm sorting via microfluidic devices, Horsman et al.^[12] sorted motile spermatozoa based on their ability to move out of their initial streamlines in the laminar fluid flow using a microfabricated device. Cho et al.^[13] put forward a horizontally set up gravity-driven pumping system to simplify the sorting step without centrifugation step to separate 100% motile sperms. Nowadays, there are some innovative sperm separation devices that have been developed. For example, Lin et al.^[14] suggested a microfluidic device with diffuser chamber, which separates the sperm by the velocity gradient. Ainsworth et al.^[15] proposed a rapid and safe method to isolate the motile human spermatozoa with less DNA damage by size and electric field using an electrophoretic system. In particular, the channels and reservoirs were coated with 1% bovine serum albumin to improve the surface hydrophilicity.^[16] By varying the channel dimensions, numerical simulations were applied to confirm the separation efficiency of sperm sorter. The device isolates motile sperm from immotile sperm and other cellular debris.^[17]

     1. Media flow in ART microfluidic devices

        • Early devices used for ART applications have employed gravity-driven passive flow, with hydrostatic pressure in media reservoirs as an important variable to drive media flow through microchannels.^[16] Many have utilized manually applied pressure via externally attached syringes to input/output ports to cause media flow through microfluidic devices. Later on, the use of Hamilton syringes attached to a programmable infusion pump has been adapted.^[18] In addition, one study showed the use of a tilting culture system in conjunction with microfluidic channels using gravity, which offered more consistent control over flow. Finally, a Braille pumping system using tiny electric piezo-actuators has been used successfully to peristaltically move media along microchannels during embryo culture, while the embryos remain largely undisturbed.^[19]

     2. Fabrication of microfluidic device

        • Microfluidic channels are fabricated by a standard soft lithography technique using polydimethylsiloxane (PDMS)^[16] silicone, borosilicate glass, Pyrex, quartz, or the combinations of these materials. Construction materials for microfluidic channels may vary from materials selected for cell substrate. Microchannels are manufactured by techniques involving molding, photolithography, and chemically or mechanically etching channels into suitable materials. PDMS polymer is the most commonly used material for microchannel fabrication, which is selected due to its inherent use and fabrication advantages, such as flexibility, ease of soft-lithography patterning, and low autofluorescence for use with microscopy.^[17]

     3. Procedure

        • Microfluidic sperm sorter commonly used in our laboratory is Sperm Sorter QUALIS which is a cyclo-olefin polymer-based MFSS chip manufactured by Menicon Co. Ltd. It has four chambers named chamber A, B, C, and D and microchannels being connected to each other [Figure 1]. The common steps of this method are as follows:

          1. Before sperm separation, semen samples were filtered using a 20 mm pore size filter to remove foreign bodies (optional).

          2. Each ejaculated sample was carefully diluted at 1:1 ratio with sperm sorting medium (G-MOPS™ PLUS supplemented with HAS, manufactured by Vitrolife Sweden AB V. Frölunda, Sweden (Ref.: 10130)) and maintained at approximately 37°C before using the MFSS.

          3. Then the MFSS was fixed in a 60-mm dish.

          4. After fixing of the MFSS, 100 μl of the sperm-sorting medium was loaded in chambers A, B, C, and D to create streamlines in the microfluidic channels and then the medium was pulled out from all chambers.

          5. Twenty microliters of the medium was loaded in chambers C and D, 100 μl of the medium was loaded in chamber B, and 65 μl of sperm suspension was loaded in chamber A.

          6. The amount of medium in chamber B was adjusted such that the width of the laminar flow from chamber A was approximately 40% of the overall width of the central microchannel under the microscope [Figure 2].

             

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             Figure 1

             Microfluidic sperm sorter having four chambers, named chamber A, B, C, and D, and microchannels being connected to each other

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             Figure 2

             The amount of medium in chamber B was adjusted such that the width of the laminar flow from chamber A was approximately 40% of the overall width of the central microchannel

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          7. After 20 min, 20 μl of completely isolated motile spermatozoa were extracted from chamber C and used only for ICSI.

Review of literature

This paper has presented a systematic review of the effects of microfluidics on sperm motility, morphology, and integrity over the past 20 years since the idea of microfluidics was developed two decades back. To start with, we searched for the articles related to keywords such as microfluidics, sperm DNA fragmentation (SDF), and DGC on the PubMed site of National Center of Biotechnology Information and selected five articles whose detailed description is provided in Table 1.

Table 1 Various studies showing the effect of microfluidics on sperm integrity

Schulte et al.^[20] in their article reported that semen samples prepared by microfluidics have reduced the percentage of fragmented DNA and increased the motility when compared to unprocessed semen, serial centrifugation, swim-up, and DGC. Shirota et al.^[21] in 2016 reported that low SDF was detected in the case of MFSS preparation than centrifugation and swim-up procedures. Another paper by Wang et al.^[22] in 2011 reported that sperm motility, sperm morphology, SDF, and tail hypo-osmotic swelling rate were significantly improved in microfluidic sorted sperms. Furthermore, Kishi et al.^[7] reported that the rate of SDF was lower in samples sorted by MFSS than the swim-up preparations. Moreover, Schuster et al.^[23] analyzed the increased sperm motility and morphology in MFSS preparations.