Several commercially available products are opti- mized for purification of human sperm for assisted reproductive technology. All batches are strictly tested to assure high levels of sterility, low endotoxin levels
IUI SpermAssistTM
CRYO SpermCryoProtect
IVF NidOilTM
ICSI SpermCatchTM
NidOilTM
Single-layer preparation PureSperm® SpeedKit
Swim-up preparation ReadySwimTM Density gradient preparation
Ready-to-use gradient PureSperm® 40/80
Do-it-yourself gradient
PureSperm® Buffer PureSperm® 100
PureSperm® Wash
Figure 15.5 PureSperm® product range that uses integrated and optimized sperm preparation for assisted reproductive technology (Nidacon International AB, Goteborg, Sweden). IVF,in vitrofertilization; ICSI, intracytoplasmic sperm injection; CRYO, cryoprotectant; IUI, intrauterine insemination
SPERM PROCESSING
199
G
A × 2 B × 2 C × 2 D × 2 E × 2 F × 2
H I J K
Sterile pipette
New
New sterile
pipette
New sterile pipette New sterile
pipette New tube
sterile Pasteur pipette
New sterile Pasteur pipette
Another new sterile Pasteur pipette
PureSperm 80%
2 ml
Carefully layer 2 ml PureSperm 40%
on top of the PureSperm 80%
Use a sterile Pasteur pipette to layer carefully liquefied semen (up to 1.5 ml) onto the top layer taking care not to disrupt the layers
Centrifuge at 300g for 20 min.
Do not use the brake
Aspirate seminal plasma and gradient material, layer by layer.
Do not dip pipette more than 1−2 mm into any layer.
Leave 2 mm of PureSperm 80% and pellet in the tube
Carefully pass Pasteur pipette to bottom of tube
Aspirate pellet Pellet
Combine sperm pellets from both tubes
Resuspend pellet in 5 ml PureSperm Wash
Centrifuge at 500 g for 10 min, using a second conical tube containing an equal volume of water as a balance.
Do not use the brake
Aspirate PureSperm Wash supernatant leaving no more than 1 mm depth of liquid above the pellet
Resuspend pellet in SpermAssist, 0.5 ml
Determine sperm concentration and motility. Dilute with sperm medium required for application
Figure 15.6 Preparation of PureSperm®density gradients for human sperm. It is recommended to prepare two gradients for each ejaculate in steps A–F (Nidacon International AB, Goteborg, Sweden)
and optimal performance. Ready-to-use products, such as 40% and 80% dilutions (PureSperm®40 and PureSperm® 80, respectively), save preparation time in the laboratory. PureSperm 40 and PureSperm 80 are formulated to minimize sudden pH and osmo- lality changes during the transition of sperm from the semen sample to the fertilization medium.
This helps to avoid premature hyperactivation and improves the fertilization potential. They maintain a pH between 7.4 and 7.8 at room temperature and at 37ºC, thus providing suitable conditions for good sperm motility, survival time and fertilization poten- tial. The preparation contains glucose as a usable energy substrate for normal cell metabolism and sperm function.
Nicotine (systematic name, 5-(N-2,3-dihydrox- ypropylacetamido)-2,4,6-tri-iodo-N ,N′bis(2,3- dihydroxypropyl)isophthalamide) is a non-ionic iodi- nated gradient medium which readily dissolves in water to produce non-toxic autoclavable solutions. To separate viable cells in a morphologically intact state by isopycnic centrifugation, it is necessary to use a gra- dient medium that provides gradients of an appropri- ate density/tonicity. It is important to maintain isotonic conditions throughout the gradient; other- wise, the cells will change in volume and, therefore, density, and large changes in tonicity across the gradi- ent can damage the cells irreversibly. The properties of iodinated gradient media make them suitable for sep- arating cells and sperm (Ford and Rockwood, 1982).
Calculus
Ejaculatory ducts
Figure 15.7 Testicular sperm processing methods. (a) Cystoscopic view after transurethral resection of the ejaculatory ducts, showing obstructing ductal calculi within lumens, bilaterally. (b) Appearance of the same vasal anastomosis after inner-layer 10-0 nylon sutures have been tied and cut. The inner layer has been completed, and the anastomosis is watertight. (c) View of the epididymis after epididymal tunic aperture creation. Note pearly white and dilated tubule appearance. (d) Scrotum longitudinally or transversely incised.Tunica vaginalis longitudinally deepened. Tunica albuginea is exposed surface. From Brannigan et al., 2003, with permission
a b
c d
‘Accudenz’ facilitates a higher rate of recovery of sperm and motile sperm. Sperm motility is lower in Accudenz compared to Percoll pellets; however, the long-term retention of sperm motility is substantially improved in Accudenz at 24 h. Sperm activation status monitored by chlortetracycline fluorescence indicated that after 4 h of incubation the incidence of fully acrosome-reacted spermatozoa in the Accu- denz versus Percoll pellets was 6.2±0.3% versus 13.1±1.0%, a 100% increase in Percoll.
Accudenz yields a higher concentration of motile sperm with improved retention of motility, velocity and acrosomal integrity and without an increase of sperm with diminished cellular maturity.
TEST yolk treatment
TEST (TES and Tris buffer) yolk treatment enhances sperm quality by increasing the ability of sperm to penetrate zona-free hamster oocytes. Such treat- ments also enhance the ability of sperm to bind to the human zona pellucida, further improving IVF outcome. These sperm enhancement functions have been substantiated (and with no apparent deleterious effects on sperm). To enhance fertiliza- tion potential further, the treated sperm sample can be additionally processed using column adherence methods.