Abstract
Infertility is a difficult and stressful condition that impacts about 15 percent of couples attempting to conceive for the first time [1]. In about half of these cases a male factor is causative and, in general, constitutes a major health issue. While the cornerstone of the evaluation of male infertility remains the basic semen analysis, the sperm penetration assay (SPA) is a useful laboratory test for predicting the capacity of an individual male’s spermatozoa to fertilize a female oocyte. This assay supplements standard semen parameters and aids clinicians in identifying couples who will have a high chance of success with in vitro fertilization. The test was first developed in the 1970s and gained momentum when, in 1976, Yanagimachi and colleagues noted that enzymatic removal of the zona pellucida of hamster ova allowed penetration by human spermatozoa [2]. The goal of the SPA is to measure the spermatozoa’s ability to undergo capacitation, acrosome reaction, fusion and penetration through the oolemma (egg plasma membrane), and decondensation within the cytoplasm of hamster oocytes resulting in the formation of the male pronucleus [3].
13.1 Introduction
Infertility is a difficult and stressful condition that impacts about 15 percent of couples attempting to conceive for the first time [1]. In about half of these cases a male factor is causative and, in general, constitutes a major health issue. While the cornerstone of the evaluation of male infertility remains the basic semen analysis, the sperm penetration assay (SPA) is a useful laboratory test for predicting the capacity of an individual male’s spermatozoa to fertilize a female oocyte. This assay supplements standard semen parameters and aids clinicians in identifying couples who will have a high chance of success with in vitro fertilization. The test was first developed in the 1970s and gained momentum when, in 1976, Yanagimachi and colleagues noted that enzymatic removal of the zona pellucida of hamster ova allowed penetration by human spermatozoa [2]. The goal of the SPA is to measure the spermatozoa’s ability to undergo capacitation, acrosome reaction, fusion and penetration through the oolemma (egg plasma membrane), and decondensation within the cytoplasm of hamster oocytes resulting in the formation of the male pronucleus [3].
13.2 Principle/Mechanism
Hamster oocytes have become widely used in this assay because of one unique feature – their promiscuity. First described in 1956 by Braden et al., hamster oocytes do not possess the same barriers to fertilization by sperm of other mammalian species and have thus become the oocytes of choice for testing fertilization potential [4]. During traditional fertilization, the male spermatozoa bind to the zona pellucida, a prohibitive glycoprotein layer surrounding the mammalian oocyte plasma membrane and ensure that fertilization remains species-specific. In traditional fertilization, the acrosome reaction that occurs with spermatozoa binding to the zona pellucida involves the release of acrosin (a serine protease) and N-acetylglucoaminindase. These enzymes aid in breakdown of the zona pellucida to allow human spermatozoa fusion with the hamster oolemma – ultimately allowing fertilization and a measurable outcome. The SPA methodology requires analogous enzymes to remove the zona pellucida of hamster oocytes [5].
Currently, no standardized SPA protocol exists. However, the World Health Organization (WHO) has described a protocol for the SPA that involves five detailed steps – 1) sperm preparation, 2) ova preparation, 3) sperm and ova incubation, 4) mounting ova and 5) scoring the final slides [3].
13.2.1 Protocol
The protocol described below is the protocol described in the WHO Laboratory Manual for the Examination and Processing of Human Sperm [6].
All equipment and reagents should be stored at room temperature (unless otherwise indicated). All procedures should be carried out at room temperature (unless otherwise indicated).
I. BWW stock solution
1. Hyaluronidase (300–500 IU/mg)
2. Trypsin Type I (10,000 BAEE U/mg)
3. Wax (melting point 48–66°C)
4. Petroleum jelly
6. Zona-free hamster oocytes: these can be purchased commercially or obtained by superovulation of hamsters
7. Dimethyl sulfoxide (DMSO).
1. Mix the semen sample well.
2. Prepare the semen samples by density-gradient centrifugation or swim-up.
3. Remove most of the supernatant from the pellet.
4. Dislodge the pellet by gentle pipetting and establish the concentration of spermatozoa in the pellet.
5. Dilute the pellet to approximately 10 × 106 spermatozoa per mL in approximately 0.5 mL of medium.
6. Incline the tube at an angle of 45 degrees to the horizontal to increase the surface area.
7. Incubate the sperm suspensions for 18–24 hours at 37°C in an atmosphere of 5 percent (v/v) CO2 in air to induce capacitation (loosen the cap of the tube to allow gas exchange). If a CO2 incubator is not available, use a Hepes-buffered medium, cap the tubes tightly and incubate at 37°C.
8. Return the tubes to the vertical position for 20 minutes to allow settling of any immotile cells after capacitation.
9. Aspirate motile spermatozoa from the top third of the supernatant, being careful not to disturb the dead spermatozoa at the interface, and transfer them to a new tube.
10. Adjust the concentration to 3.5 × 106 motile spermatozoa per mL of medium.
11. With a positive-displacement pipette, aspirate known volumes (50–150 μL) of a sperm suspension and slowly dispense them into a small Petri dish. With a plastic disposable pipette, cover the droplet with prewarmed mineral oil equilibrated in CO2, being careful not to disturb the sperm suspension. Add enough oil to surround and just cover each droplet of spermatozoa.
1. Recover the oocytes within 18 hours after the injection of hCG by sacrificing the animals according to methods approved by the relevant animal care and use committee.
2. Place the hamsters on their back and dampen the abdominal fur with 95 percent (v/v) ethanol.
3. Grasp the skin with toothed forceps and cut through the skin and muscle with scissors to expose the uterus and ovaries.
4. Wipe the forceps and scissors free of fur with 95 percent (v/v) ethanol.
5. Push the intestines out of the abdominal cavity to expose the uterine horns.
6. Grasp one uterine horn with the forceps and lift it out of the abdominal cavity to expose the oviduct, ovary and ovarian ligament.
7. The oviducts are excised and placed in the first of three ice-cold saline washes after which the oviducts are singularly transferred to a 35 × 10 mm culture dish containing iced saline on a dissecting microscope.
IV. Collecting the cumulus masses
1. Examine the ovaries by transillumination in a dissecting microscope to locate the cumulus cells containing the oocytes in the swollen portion of the oviduct.
2. Hold the oviduct with the forceps and puncture the swollen area with a 21-gauge needle. The cumulus mass will pour out of the puncture hole.
3. Tease out the cumulus mass with the needle. Squeeze the oviduct with the forceps to remove all of the cumulus mass.
V. Recovering and treating the oocytes
1. Gather the cumulus cells with the needle and forceps and place the cells in a watch glass dish, spot plate or other shallow container containing 0.1 percent (1g/L) hyaluronidase (300–500 IU/mL) in warm, CO2-equilibrated BWW.
2. Incubate the container, covered with aluminum foil to protect the cells from light, for 10 minutes at room temperature. Observe the separation of the cumulus cells in a dissecting microscope.
3. Use a flame-drawn glass pipette to transfer freed oocytes from the hyaluronidase to the warm equilibrated BWW.
4. Rinse the recovered oocytes twice in BWW by transferring them into fresh drops of warm, equilibrated BWW. This can be done in a glass multi-well dish or spot plate. Rinse the pipette with BWW between each oocyte transfer.
5. Treat the oocytes with 0.1 percent (1g/L) trypsin (10,000 IU/mL) for approximately one minute at room temperature to remove the zonae pellucidae. Observe the digestion of the zona in a dissecting microscope and remove the oocytes as soon as the zona has dissolved.
6. Wash the oocytes three more times with BWW.
7. Warm the isolated oocytes to 37°C and introduce them into the sperm suspensions. Alternatively, they may be stored at 4°C for up to 24 hours before use.
1. Dispense the zona-free hamster oocytes into several droplets, with about five oocytes per drop (i.e. for 20 oocytes per semen sample prepare four aliquots of five oocytes per drop).
2. Load groups of about five oocytes into the glass pipette with little medium so as not to dilute the sperm suspensions too much.
3. Insert the pipette tip directly into the center of one droplet of sperm suspension and slowly dispense the oocytes. Maintain positive pressure to prevent the mineral oil from entering the pipette and take care not to introduce air bubbles into the sperm suspension.
4. Wipe any excess oil from the pipette tip after removal from the sperm suspension.
5. Repeat step 3 until all oocytes have been transferred to the sperm suspensions.
6. Rinse the pipette thoroughly in BWW after each egg transfer to prevent cross contamination of spermatozoa.
7. Incubate the gametes for three hours at 37°C in an atmosphere of 5 percent (v/v) CO2 in air.
8. Recover the oocytes from the oil droplets. Take care to wipe any oil from the tip of the pipette before transferring the oocytes to BWW.
9. Wash the oocytes free of loosely adherent spermatozoa with the flame-drawn Pasteur pipette, by rinsing in BWW.
1. Place four pillars of wax-petroleum jelly mixture in a rectangular pattern to support the coverslip (22 mm × 22 mm, thickness number 1.5, 0.17 mm) at its corners.
2. Place a small droplet of oocyte-containing BWW in the center of the four pillars.
3. Lower the coverslip over the wax pillars and gently press it down, to begin to flatten the oocytes. A well-flattened oocyte is required for optimal observation of decondensed sperm heads.
4. If necessary, add more BWW to flood the slide to prevent squashing of the oocytes.
5. Examine the preparation by phase-contrast microscopy at 200× magnification.
6. Count the number of decondensed sperm heads with an attached or closely associated tail.
7. Record the percentage of eggs penetrated by at least one spermatozoon and the number of spermatozoa per penetrated egg.
8. Record the presence of any spermatozoa that remain bound to the surface of the oocytes after the initial washing procedure, since this may give some indication of the proportion of the sperm population that has undergone the acrosome reaction.
For additional information or further details of the protocol, please see the WHO Laboratory Manual for the Examination and Processing of Human Sperm.
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