The hypo-osmotic swelling (HOS) test, introduced in 1987 to assess the functional integrity of the human sperm membrane, appears to have withstood the test of time. Many articles have been published on the subject since its inclusion in the third edition of the World Health Organization (WHO) manual in 1992 [1, 2, 3, 4, 5].

Natural fertilization

Immediately following ejaculation, semen forms into a coagulum which effectively traps all sperm within a viscous medium. During coitus, the ejaculated coagulum becomes deposited around the external orifice of the cervix and the posterior fornix of the vagina. Freshly ejaculated, this coagulated semen protects sperm from the otherwise harsh vaginal environment, and facilitates sperm transport through the cervix and into the fallopian tubes.

As the coagulum begins to liquefy, otherwise trapped sperm are released into the surrounding environment. A majority of motile sperm with normal forms will then rapidly penetrate and migrate into the cervical mucus. A tiny fraction of these sperm might eventually reach the oocyte within the fertilization area. Those sperm remaining behind within the vagina are inactivated and summarily destroyed, likely due to the relatively high acidity of the vaginal confines.

The capacitation reaction

Interestingly enough, healthy sperm, although fully formed at the moment of ejaculation, are not yet able, in and of themselves, to fertilize an egg. Prior to making contact with the oocyte, sperm must undergo further physiological maturation possible only within the female reproductive tract, a process called “capacitation.” (Artificial capacitation, performed in vitro, is commonly utilized in the laboratory.)

Such capacitation is inhibited outside the female in order to conserve sperm fertilization capacity. Specifically, decapacitating factor(s) present within the seminal plasma itself prevent sperm from undergoing spontaneous and independent capacitation reactions. Once semen has been ejaculated into the vagina, however, the cervical mucus also prevents these decapacitating factor(s) from ever entering the inner female reproductive tract, facilitating the capacitation processes going on within.

Male fertility issues plague over 40% of couples experiencing procreative difficulties. Problems with the male reproductive system can involve compromised sperm quality, anatomical and physiological irregularities, or hormonal deficiencies. Fertility specialists are now able to combat and treat many of these fertility concerns through a host of clinically proven methods.

Specifically, therapeutic methods for successful male fertility treatment focus on optimization of sperm procurement. Whether sperm are procured directly by the patient, with clinical or even surgical correction, the fertility specialist's goal is acquisition of as many viable spermatozoa as possible. Once an optimal number of sperm have been procured, various laboratory methods can then enhance sperm quality, making it suitable for intrauterine insemination techniques.

In other cases necessitating intervention by the clinician, sperm are aspirated directly from the male reproductive tract. Once a sufficient number of viable spermatozoa have been procured, procedures such as intracytoplasmic sperm injection (ICSI) can then be performed.

In fact, sperm procurement and processing methods have become sophisticated to the point of delegating even azoospermia to the history books. Azoospermic ejaculates can now be pelleted to extract a few sperm using high-g force centrifugation, suitable for ICSI procedures. Fertility specialists can also aspirate sperm directly from the testes. Cases of arrested spermatogenesis no longer result in sterility: spermatids may be harvested ex vivo and further processed in the laboratory for use in assisted reproductive techniques.

These sperm processing procedures can generally be considered common to both intrauterine insemination (IUI) and assisted reproductive technology (ART) procedures.

Sperm washing is the primary method for seminal plasma removal from the sperm. Should difficulties arise, or sperm quality pose a further need for enhancement, then additional sperm treatment methods should be implemented.

In situations where sperm must be preserved for future use, semen cryopreservation is employed to freeze sperm for longterm storage:

Sperm washing procedure

Sperm washing, employing dilution and then centrifugation, is the primary procedure for separating sperm from seminal plasma.

The rationale for removing sperm from the seminal plasma is twofold:

• Removal of decapacitation factor(s) and other detrimental elements; and

• Concentration of all sperm within the ejaculate

Viscosity reduction

Sperm washing for most semen samples begins with an initial four- to fivefold media dilution (see below) of the original seminal plasma, especially for those samples containing high sperm concentration or extremely high viscosity.

If a semen sample contains low sperm concentration in relatively high volume, then a relatively low dilution rate is necessary. Note that significantly high dilution procedures might require protein supplementation.

Media and protein supplementation

Recommendations for media and protein supplementation are somewhat arbitrary and subject to laboratory personnel opinion.

Sexual stimulation, penile erection and subsequent orgasm generally result in ejaculation. When successfully induced in a clinical environment, ejaculation provides a primary procurement source for sperm.

Two vital subcomponents concurrently responsible for ejaculation include semen emission and expulsion. Any difficulties with either or both of these functions will affect sperm quantity and quality, necessitating a change in spermprocurement strategy.

Semen production and release

Spermatozoa produced in the testicles, when mixed with accessory sex glands' fluids, becomes semen (typically referred to as an “ejaculate,” or simply as the “sample”).

The neurophysical mechanism inducing sperm emission and expulsion is the orgasm.Thehumanorgasm is typically dependent upon a feedback mechanism between direct penile stimulation and excitation of various portions of the central nervous system.

As such, orgasm is a vital component of the sperm procurement process. Difficulties with orgasm inducementmust be addressed or circumvented to facilitate any sperm retrieval regimen.

Once orgasm has been achieved, emission and expulsion should autonomously occur.

During emission, bladder neck and external urethral sphincter remain closed to contain any deposited seminal fluid. Physical closure of the bladder neck is essential for prevention of retrograde semen flow back into the bladder.

Introduction

Sperm from various sources and procured through various methods have been successfully utilized for assisted reproductive technology (ART) procedures.

Naturally ejaculated fresh semen and stored, cryopreserved semen have both proven acceptable, effective sources.

• The ART procedures include:

• In vitro fertilization (IVF)

• Gamete intrafallopian transfer (GIFT)

• Intracytoplasmic sperm injection (ICSI)

All viable sperm within any given prepared ejaculate are utilized to maximize success rates for intrauterine insemination (IUI) procedures.

In contrast, a mere 100000 (maximum) motile spermatozoa are sufficient for ART procedures such as IVF and GIFT.

And, since the female reproductive tract naturally induces sperm capacitation, IVF and GIFT procedures, although requiring fewer viable sperm than IUI, none the less require in vitro means of capacitation.

In vitro sperm capacitation induction

Once decapacitation factor(s) present in the seminal plasma have been removed from the sperm, they may be artificially induced to undergo capacitation. Since capacitation incubation time might actually be hastened by using the right type of media with IVF and GIFT, their proper selection is important to the overall outcome.

• Media: Of the many commercially available media products, these three are common:

• Ham's F-10 medium (Irvine Scientific, Santa Ana, CA 92705)

• Human tubal fluid medium and modified human tubal fluid (MHTF) medium (Irvine Scientific, Santa Ana, CA 92705)

• Preimplantation stage one (P-1) medium (Irvine Scientific, Santa Ana, CA 92705)

Recent advances in sperm procurement and processing have made fertility conditions once deemed hopeless a thing of the past. Even cases of sterility are now either treatable or entirely circumvented through various clinical techniques, including surgical correction, hormone treatment, and various sperm enhancement technologies.

This anthology presents these latest sperm procurement and processing methods in chapters written by acknowledged experts in their respective fields. The editor hopes that this collection will provide clinical and laboratory professionals from all areas of fertility science with both a comprehensive overview and a handy reference resource.

The primary biological function of the male reproductive system is procreation facilitation. The system accomplishes this through two fundamental processes: generation of fertility potential within the male, and subsequent male genome delivery directly into the female reproductive tract.

Spermatozoa (or “sperm”), as the exclusive male genome vehicle, is the vital carrier and component of male fertility potential. “Sperm procurement methods” are clinical procedures designed to acquire viable sperm for analysis, analysis interpretation and, if deemed necessary, sperm utilization for various assisted reproductive techniques and artificial insemination procedures.

Any difficulty with sperm procurement, analogous to problems with male fertility as a whole, can involve biological deficiencies with sperm creation, delivery, or both. The various procurement methods described in this text are all designed with one purpose in mind: acquisition of as many viable spermatozoa as possible, given initial patient conditions.

Introduction

Sperm processing procedures that mimic the sperm separation abilities of periovulatory mucus can favorably influence reproductive outcome. These methods analogously and effectively filter progressively motile and morphologically normal sperm from the overall sample population. The resultant sample contains an enriched sperm population with higher fertilizing potential.

Central to this process, as detailed in the previous section, is the separation and elimination of decapacitation factor(s) and contaminants from the seminal plasma. These may be in the form of cellular debris (gelatinous pieces, epithelial cells), bacteria, mycoplasmas, Chlamydia, Trichomonas, and various blood components (leukocytes and erythrocytes).

The goal should be a thorough filtering of motile sperm, and the recovery of most, if not all, viable spermatozoa. Such separation procedures should be reliable, repeatable, and relatively simple to perform.

These separation procedures can be broadly categorized into three major groups:

On occasion, fresh ejaculate is unavailable, making the use of cryopreserved sperm necessary (see Semen cryopreservation procedure, page 119). Prior to use, frozen sperm must be thawed, washed, and processed.

In addition, the deleterious effects of cryopreservation are more pronounced during the thawing and washing procedure, necessitating certain considerations:

Sperm migration method

Procedures based on this concept mimic sperm migration through the pericervical mucus.

Naturally motile sperm will effortlessly and efficiently move from one medium to another, whereas nonmotile or morphologically compromised sperm will have greater difficulty. The sperm migration method inspires procedures which take advantage of healthy sperm's natural tendency for migration.