At the time of oocyte retrieval or intrauterine insemination (IUI), IVF laboratory should already be familiar with the male partner’s semen profile and can refer to features that might influence the choice of sperm preparation method used. Semen is a non-sterile body fluid that can transmit infection, and viral screening tests should be confirmed as negative before the sample is handled for preparation in the IVF laboratory. Aseptic technique should be maintained throughout.
The choice of sperm preparation method or combination of methods depends upon the assessment of:
Ratio between motile/immotile counts,
Presence of antibodies, agglutination, pus cells or debris.
Ejaculated semen is a viscous liquid composed of a mixture of testicular and epididymal secretions containing spermatozoa, mixed with prostatic secretions produced at the time of ejaculation. This seminal plasma contains substances that inhibit capacitation and prevent fertilization. The purpose of sperm preparation is to concentrate the motile spermatozoa in a fraction that is free of seminal plasma and debris.
Early IVF practice involved preparing sperm by simple washing and centrifugation, but this method also concentrates cells, debris and immotile sperm, which can jeopardize fertilization. Researchers have demonstrated that leukocytes and dead sperm in semen can generate reactive oxygen species (ROS), and these can initiate lipid peroxidation in human sperm membranes. Peroxidation of sperm membrane unsaturated fatty acids leads to a loss of membrane fluidity, which inhibits sperm fusion events during the process of fertilization.
When preparing sperm for assisted conception, it is advantageous to separate motile sperm from leukocytes and dead sperm as effectively and efficiently as possible. However, if ICSI is the treatment of choice, sperm fusion events are, of course, bypassed, and direct high-speed centrifugation of these suboptimal sperm samples does not appear to jeopardize fertilization by ICSI.
Sperm samples that show moderate to high counts (>35 × 106 motile sperm/mL) with good forward progression and motility can be prepared using a basic overlay and swim-up technique. Discontinuous buoyant density gradient centrifugation is the method of choice for samples that show
Poor forward progression,
Large amounts of debris and (or) high numbers of cells,
Chemical Enhancement of Sperm Motility Prior to Insemination
Pentoxifylline is a methylxanthine-derived phosphodiesterase inhibitor which is known to elevate spermatozoa intracellular levels of cAMP in-vitro. It has been postulated that the resulting increase in intracellular adenosine triphosphate (ATP) enhances sperm motility in samples that are assessed as having poor progressive motility, with an increase in fertilization and pregnancy rates for suboptimal semen samples. 2-De-oxyadenosine has also been used to achieve a similar effect. The protocol involves a 30-minute pre-incubation of prepared sperm with the stimulant. The resulting sperm suspension is then washed to remove the stimulant, and the preparation is used immediately for insemination.
Determination of Reactive Oxygen Species (ROS) in Seminal Fluid
Important, free radicals are atoms or molecules with one or more unpaired electrons, which makes them extremely reactive. Having an incomplete outer valence shell, they attempt to interact with other molecules in order to gain electrons. Once a molecule loses an electron to a free radical, it then itself becomes a free radical, initiating a chain reaction that can potentially be disastrous for cells. ROS are radical and non-radical derivatives of oxygen. The most common ROS in spermatozoa is the superoxide anion radical that eventually forms hydrogen peroxide. Seminal plasma contains two types of antioxidants to combat free radical-induced damage: enzymatic antioxidants, such as superoxide dis-mutase, catalase and peroxidase, and non-enzymatic antioxidants, such as vitamins, glutathione and pyruvate.
Although excessive generation of ROS is involved in the pathogenesis of a wide range of diseases, such as neurodegenerative disease, cancer and infertility, they are also essential in a variety of physiological functions such as cellular signaling. Homeostasis is the key, as in all cell functions, and an excessive use of antioxidants to treat male infertility may be detrimental, triggering, amongst other problems, a decrease in spermatogenesis and fertility.
ROS can be detected in seminal fluid via both direct and indirect methods:
Direct methods: Cytochrome c reduction, electron spin resonance, nitroblue tetrazolium techniques and flow cytometry.
Indirect methods: Redox potential, measuring the level of lipid peroxidation products or oxidative DNA damage and measuring reactive nitrogen species by fluorescence spectroscopy.
The chemiluminescence assay is the most commonly used technique in andrology laboratories. This relies on the measurement of light after reagents are added to human sperm to cause a reaction:
Luminol measures both intracellular and extracellular ROS, such as hydrogen peroxide, superoxide anions and hydroxyl radicals.
Lucigenin measures only extracellular ROS, in particular the superoxide anion.
ROS can be measured in ejaculated semen, or in processed sperm following swim-up or density gradient techniques. A variety of luminometers are commercially available, varying in design, features and price.
Sperm Preparation for ICSI
A combination of sperm preparation methods can be used. Extremely oligospermic/asthenozoospermic samples cannot be prepared by buoyant density centrifugation or swim-up techniques.
Surgical Sperm Retrieval
Until the mid-1990s, virtually all patients with obstructive or non-obstructive azoospermia had untreatable male sterility. This situation was completely reversed by the ability to combine ICSI with surgical techniques to recover samples from the epididymis and directly from the testis.
Epididymal sperm can be obtained by open microscopic surgery (MESA) or by percutaneous puncture (PESA), using a 21-gauge “butterfly” or equivalent needle to aspirate fluid. If large numbers of sperm are found, they can be processed by buoyant density gradient centrifugation. Samples with less sperm can be washed and centrifuged with IVF medium a number of times, and the concentrated sample is then added to micro-droplets in the injection dish. Motile spermatozoa “swim out” to the periphery of the droplets, where they can be collected and transferred to clean drops of medium for injection later.
Testicular sperm is obtained by open biopsy (testicular sperm extraction, TESE) or by percutaneous needle biopsy (testicular fine needle aspiration, TEFNA), and samples can be processed in a variety of ways:
Crush the biopsy sample between two microscope slides, and expose sperm by shredding the tissue either with glass slides, by needle dissection, by dissection using micro-scissors, or by maceration using a micro-grinder. Concentrate the debris by centrifugation and examine under high-power microscopy to look for spermatozoa. Large quantities of debris are invariably present, and it may be difficult to find sperm (especially with cases of focal spermatogenesis). Further processing steps will depend upon the quality of the sample: it may be loaded onto a small single-step buoyant density gradient, or sperm simply harvested “by hand” under the microscope. Use a large needle, assisted hatching pipette or biopsy pipette to collect and pool live sperm in a clean drop of medium.
Tubules in the biopsy sample can be carefully unraveled under the dissecting microscope, using fine watchmakers’ forceps. Cut the tubules into small lengths of 1–2 cm and “milk” the contents by squeezing from the middle to an open end (analogous to squeezing a tube of toothpaste). The cells can be picked out of the dish and examined under the ICSI microscope, or placed into a centrifuge tube of clean medium for further preparation. An alternative approach is to slit the segments of tubules rather than “milking” to release the cells. Fresh testicular sperm is often immotile and combined with Sertoli cells, but free-swimming sperm is usually seen after further incubation. In cases of obstructive azoospermia, pregnancies have been achieved from testicular sperm incubated for up to 3 days after the biopsy procedure, but the proportion of motile sperm in a testicular biopsy sample is usually highest after 24 hours’ incubation. Incubation at 32° C instead of 37°C may also be of benefit. If fresh sperm is to be used, the biopsy procedure should be carried out the day before the planned oocyte retrieval. In cryopreserved samples, a higher proportion of frozen testicular sperm has been found to retain their motility on thawing if they have been incubated for 24–48 hours before freezing. However, in cases of non-obstructive azoospermia, incubation for longer than 24 hours is not recommended. When biopsied sperm are processed in advance, any motile sperm found using an injection needle can be stored in an empty zone before freezing. This has the advantage that the sperm is then readily available at the time of ICSI, which can save a considerable time.
Pathology of Azoospermia
The Mean Johnsen Score (MJS) score is an assessment of the degree of spermatogenesis found in a biopsy: a number of tubules are assessed, and each one is given a score for the most advanced stage of spermatogenesis seen:
1 – no cells present in the tubule,
2 – Sertoli cells,
3 – Spermatogonia,
4–5 – Spermatocytes,
6–7 – Spermatids,
8–10 – Spermatozoa.
MJS = average of all the tubules assessed, i.e.:
MJS – 2 is the Sertoli cell only syndrome,
MJS – 8–10 is normal spermatogenesis,
MJS between two and eight represents varying degrees of subnormal spermatogenesis, but a qualitative description is required.
There is a correlation between testicular size and MJS.