Sperm Preparation for AI: Sperm Washing, Swim Up, Density Gradient Centrifugation

The article focuses on representing the basic overview and comparing three main Sperm Preparation Techniques for Artificial Insemination. At present, the issues of researching the essence of male’s infertility: its causes, testing and Treatment have become an interdisciplinary research mainstream focus for both paradigms of modern andrology and male Reproductive Endocrinology. The purpose of this study was to determine the main Sperm Preparation Techniques for Artificial Insemination. Having observed the numerous scientific articles and analytical overviews concerning this question, we found out three main Sperm Preparation Techniques for Artificial Insemination, which can be categorized in the following way: 

1. Sperm Washing; 
2. Swim up; 
3. And density gradient centrifugation methods. 

Introduction

The spermatozoon is a highly regionalized cell with localized membrane domains that have specific functions. The domains have heterogeneous fluidity, shape, diffusion coefficients and composition of phospholipids, glycolipids and steroids. There are five specialized regions: the acrosomal, equatorial, postacrosomal, midpiece and tail region. The spermatozoon membrane is a dynamic system undergoing many changes, often domain specific, as the spermatozoon passes through the reproductive tract. These maturational changes are thought to be essential for eventual fertilization of the oocyte.

An improved understanding of sperm morphology and physiology with special emphasis on the role of integrity of the male gamete in both fertilization and embryogenesis, has led experts in Reproductive Medicine to an increased demand on Sperm Separation Techniques: a good Sperm Preparation Technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte.

An ideal Sperm Processing Technique should be gentle and one that recovers a highly functional sperm population [36]. Serial centrifugation of the semen is known to induce sperm dysfunction mediated through production of reactive oxygen species by spermatozoa and leucocytes [2; 4; 35; 38; 76]. Therefore, more gentle sperm selection techniques such as double density gradient centrifugation and swim–up procedures have evolved and are widely applied in clinical practice

When couples have difficulty conceiving, sometimes nature just needs a little nurturing. Sperm Washing is one process that has proven particularly helpful. Being one of the Assisted Reproductive Techniques, the Artificial Insemination, needs a selection of the ejaculated spermatozoa before the performance of the treatment. In fact, some components of the seminal fluid may become an obstacle to the fertilization when the In Vitro Fertilization or the Intrauterine Insemination are performed [13]. Spermatozoa and leukocytes produce many oxygen radicals that alter the possibility of the sperm–oocyte fusion after repeated centrifugations. So, the selection of the sperms from the other components with methods like the swim up technique or the gradient density centrifugation must be preferred [2].

There has been a surge by researchers to develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are incapable of fertilizing oocytes [1]. Intrauterine Insemination (lUI) and Ovarian Stimulation are still extensively applied Treatment modalities for male subfertility.  Some different techniques are used to prepare the spermatozoa for the Artificial Insemination, but the choice strongly depend on the quality of the semen, that is on the concentration, motility and morphology, in order to obtain the higher number of good high–quality spermatozoa, even from the poorest semens. The principle techniques of sperm preparation consist of migration, density gradient centrifugation and filtration techniques. While for the migration the method is based on movement of the spermatozoa, for density gradient centrifugation and filtration techniques the method is based on a combination of the motility and the retention at phase borders and adherence to filtration matrices, respectively [36].

Having compared the efficacy of various methods of processing semen samples, there have been established several andrological indications for preparation of high–quality spermatozoa: viability, motility and morphology were used to present qualitative parameters [concentration of viable and motile spermatozoa with normal morphology increases the chances of successful fertilization.

1. Sperm Preparation procedures [pre–wash procedures]

1.1. What is semen?

Semen is the fluid that a man ejaculates. This fluid is produced at several different sites in the body. The semen consists of a suspension of spermatozoa stored in the epididymis that, at the moment of the ejaculation, is mixed with the secretions of the accessory glands. These glands are mainly the prostate and the seminal vesicles, while the bulbourethral glands and the epididymis represent only the minor contribution of the ejaculate. There are two main fractions present in the seminal fluid: the first one is prostatic, rich in spermatozoa. The last fraction of the semen consists of vesicular fraction, less rich in spermatozoa [12]. The sperm within the semen are the cells that actually fertilize the egg and are therefore the most important to assess. However, the sperm account for only 1% to 2% of the semen volume. (In fact, only 5% of the total fluid comes from the testes, which is why men with no sperm in the ejaculate, including after a vasectomy, cannot tell the difference either in the feeling they have when they ejaculate, or in the appearance of the ejaculate itself). Problems with the surrounding fluid may also interfere with the movement and function of the sperm so both the sperm and the fluid must be tested.

1.2. Collection of semen for diagnostic purposes or research purposes

The sample should be collected in a private room near the laboratory, in order to limit the exposure of the semen to fluctuations in temperature and to control the time between collection and analysis.

The specimen container is placed on the bench or in an incubator (37 °C) while the semen liquefies. The specimen container should be kept at ambient temperature, between 20°C and 37°C, to avoid large changes in temperature that may affect the spermatozoa after they are ejaculated into it. It must be labelled with the man’s name and identification number, and the date and time of collection.

2. Sperm Preparation procedures [Pre–wash procedures]: basic semen testing, standard semen fluid tests and specialized semen tests for diagnostic purposes or research purposes

The most important first step in any man’s evaluation is the Semen Analysis. The Semen Analysis allows experts to identify problems to be addressed in order to maximize the quality of the man’s semen. This may reduce the need for more complicated interventions for the female partner. It will also allow us to rule out significant medical problems that may contribute to poor analysis results.

Basic Semen Testing for diagnostic purposes or research purposres

Semen is the fluid that a man ejaculates. It is produced at several different sites in the body. The sperm within the semen are the cells that actually fertilize the egg. While it is most important to assess the sperm, they account for only 1% to 2% of the semen volume. Problems with the surrounding fluid may also interfere with the movement and function of the sperm. Therefore, both the sperm and the fluid must be tested. Semen Analysis is the laboratory testing of freshly ejaculated semen that usually has been produced by masturbation. Under a microscope, the number, shape and movement of sperm are measured. A Semen Analysis is a vital part of diagnosing male infertility. Testing should be done at a specialized laboratory that uses methods approved by the World Health Organization (WHO); special equipment and expertise are needed to do an accurate Semen Analysis. There is no specific, magic number of sperm in the Semen Analysis of men whose partners will get pregnant. The partners of some men with a very poor Semen Analysis may conceive easily. The partners of some men with an excellent Semen Analysis may experience difficulty. However, men with good Semen Analysis results will, as a group, conceive at significantly higher rates than those with poor Semen Analysis results. The semen analysis will help determine whether there is a male factor involved in the couple’s sub–fertility. In those cases, we will recommend an evaluation. There are certain findings of the Semen Analysis which suggest specific potential problems. For example, an increased white blood cell count may indicate infection or inflammation. However, other abnormalities in many of the main parameters are non–specific. For example, there are a number of different causes for a decreased sperm count or diminished sperm movement. Some of these causes have other serious medical implications, others don’t. A thorough evaluation helps determine the cause of an abnormal Semen Analysis.

2.1. Initial macroscopic semen examination

Semen Analysis begins with a simple inspection soon after liquefaction, preferably at 30 minutes, but no longer than 1 hour after ejaculation, to prevent dehydration or changes in temperature from affecting semen quality.

2.1.1. Semen liquefaction

Immediately after ejaculation into the collection vessel, semen is typically a semisolid coagulated mass. Within a few minutes at room temperature, the semen usually begins to liquefy (become thinner), at which time a heterogeneous mixture of lumps will be seen in the fluid. As liquefaction continues, the semen becomes more homogeneous and quite watery, and in the final stages only small areas of coagulation remain. The complete sample usually liquefies within 15 minutes at room temperature, although rarely it may take up to 60 minutes or more. If complete liquefaction does not occur within 60 minutes, this should be recorded. Normal liquefied semen samples may contain jelly–like granules (gelatinous bodies) which do not liquefy; these do not appear to have any clinical significance. The presence of mucus strands, however, may interfere with Semen Analysis.

2.1.2. Semen viscosity

After liquefaction, the viscosity of the sample can be estimated by gently aspirating it into a wide–bore (approximately 1.5 mm diameter) plastic disposable pipette, allowing the semen to drop by gravity and observing the length of any thread. A normal sample leaves the pipette in small discrete drops. If viscosity is abnormal, the drop will form a thread more than 2 cm long. Alternatively, the viscosity can be evaluated by introducing a glass rod into the sample and observing the length of the thread that forms upon withdrawal of the rod. The viscosity should be recorded as abnormal when the thread exceeds 2 cm. In contrast to a partially unliquefied sample, a viscous semen specimen exhibits homogeneous stickiness and its consistency will not change with time. High viscosity can be recognized by the elastic properties of the sample, which adheres strongly to itself when attempts are made to pipette it. High viscosity can interfere with determination of sperm motility, sperm concentration, detection of antibody–coated spermatozoa and measurement of biochemical markers.

2.1.3. Appearance of the liquefied semen [ejaculate]

A normal liquefied semen sample has a homogeneous, grey–opalescent appearance. It may appear less opaque if the sperm concentration is very low; the colour may also be different, i.e. red–brown when red blood cells are present (haemospermia), or yellow in a man with jaundice or taking certain vitamins or drugs.

2.1.4. Semen volume

The volume of the liquefied semen [ejaculate] is contributed mainly by the seminal vesicles and prostate gland, with a small amount from the bulbourethral glands and epididymides. Precise measurement of volume is essential in any evaluation of semen, because it allows the total number of spermatozoa and non-sperm cells in the ejaculate to be calculated.

Low semen volume is characteristic of obstruction of the ejaculatory duct or congenital bilateral absence of the vas deferens, a condition in which the seminal vesicles are also poorly developed. Low semen volume can also be the result of collection problems (loss of a fraction of the ejaculate), partial retrograde ejaculation or androgen deficiency.

High semen volume may reflect active exudation in cases of active inflammation of the accessory organs.

2.1.5. Semen pH

The pH of semen reflects the balance between the pH values of the different accessory gland secretions, mainly the alkaline seminal vesicular secretion and the acidic prostatic secretion. The pH should be measured after liquefaction at a uniform time, preferably after 30 minutes, but, in any case within 1 hour of ejaculation since it is influenced by the loss of CO2 that occurs after production.

If the pH is less than 7.0 in a semen sample with low volume and low sperm numbers, there may be ejaculatory duct obstruction or congenital bilateral absence of the vas deferens, a condition in which seminal vesicles are also poorly developed.

2.2. Initial microscopic semen examination

An initial microscopic examination of the sample involves scanning the preparation. This provides an overview of the sample, to reveal: 

1. Mucus strand formation; 
2. Sperm aggregation or agglutination; 
3. The presence of cells other than spermatozoa, e.g. epithelial cells, “round cells” (leukocytes and immature germ cells) and isolated sperm heads or tails; 
4. Assessment of sperm motility; 
5. Determination of the dilution required for accurate assessment of sperm number.

2.2.1. Thorough mixing and representative sampling of semen

The nature of the liquefied ejaculate makes taking a representative sample of semen for analysis problematical. If the sample is not well mixed, analysis of two separate aliquots may show marked differences in sperm motility, vitality, concentration and morphology.

2.2.2. Aggregation of spermatozoa

The adherence either of immotile spermatozoa to each other or of motile spermatozoa to mucus strands, non–sperm cells or debris is considered to be nonspecific aggregation and should be recorded as such.

2.2.3. Agglutination of spermatozoa

Agglutination specifically refers to motile spermatozoa sticking to each other, 

1. Head–to–head, 
2. Tail–to–tail [heads are seen to be free and move clear of agglutinates]; 
3. Tail–tip–to–tail–tip, 
4. Tn a mixed way [clear head–to–head and tail–to–tail agglutinations]; 
5. Tangle [heads and tails enmeshed. 

Heads are not clear of agglutinates as they are in tail–to–tail agglutination]. The motility is often vigorous with a frantic shaking motion, but sometimes the spermatozoa are so agglutinated that their motion is limited. Any motile spermatozoa that stick to each other by their heads, tails or midpieces should be noted.

The major type of agglutination (reflecting the degree (grades 1–4) and the site of attachment should be recorded [71]:

1. Grade 1: isolated <10 spermatozoa per agglutinate, many free spermatozoa;
2. Grade 2: moderate 10–50 spermatozoa per agglutinate, free spermatozoa;
3. Grade 3: large agglutinates of >50 spermatozoa, some spermatozoa still free;
4. Grade 4: gross all spermatozoa agglutinated and agglutinates interconnected.

The presence of agglutination is not sufficient evidence to deduce an immunological cause of infertility, but is suggestive of the presence of anti–sperm antibodies; further testing is required. Severe agglutination can affect the assessment of sperm motility and concentration.

2.2.4. Cellular elements other than spermatozoa

The ejaculate contains cells other than spermatozoa, some of which may be clinically relevant. These include epithelial cells from the genitourinary tract, as well as leukocytes and immature germ cells, the latter two collectively referred to as “round cells” [40]. 

2.2.5. Sperm motility (sometimes referred to as “mobility”): 

This describes the percentage of sperm that are moving. In even the best specimens, many sperm are not moving. The (immotile) sperm may either be dead or just not moving. This actually is a significant difference, especially in specimens with a 0% motility. Motility is measured as a percentage. If all the sperm are moving, motility is 100 or 100%. If no sperm are moving, motility is 0%. Most specimens are in between. The sperm are placed on an etched slide, and numerous boxes in the grids are counted. Counts are kept of the moving sperm and the immotile (non–moving) sperm. The percentage of moving sperm (the total moving divided by the total number of sperm) is calculated. Since specimens are not the same throughout (i.e. they are not homogeneous) sometimes multiple areas of the slide must be examined to come up with an average. The measurement is tricky because the moving sperm are of course moving in and out of the boxes. Normal motility is fifty percent or more of the moving sperm. Low sperm motility can be caused by issues with production, as well as issues occurring within the ducts that store and transport the sperm. For example, infections may decrease the motility of the sperm in the ejaculate, even if they were originally produced with a good motility.

2.2.5.1. Categories of sperm motility [sperm movement]

A simple system for grading motility is recommended that distinguishes spermatozoa with progressive or non–progressive motility from those that are immotile. The motility of each spermatozoon is graded as follows:

1. Progressive Motility (PR): spermatozoa moving actively, either linearly or in a large circle, regardless of speed;

2. Non–progressive motility (NP): all other patterns of motility with an absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed;

3. Immotility (IM): no movement.

Forward Progression: this parameter looks at the quality of the movement itself, when looking at the sperm that are moving. In other words, are they “fast swimmers” or “slow swimmers?”. Forward Progression (FP) is measured on a scale. The World Health Organization (WHO) uses a scale of 1–4. The table below reviews what each number is meant to represent. Usually it is reported as a single number, which is the average FP.

0      No Movement

1      Movement, None Forward

1+    Occasional Movement of a Few Sperm

2      Slow, Undirected

2+    Slow, Directly Forward Movement

3–    Fast, Undirected Movement

3      Fast, Directed Forward Movement

3+    Very Fast Forward Movement

4      Extremely Fast Forward Movement

What are the implications of forward progression?

It is crucial that this number be reported. It is important not only to measure what percentage of the sperm are moving (motility) but also how well they are moving (forward progression) to get a sense of the sperm’s overall movement. If a man has a high motility, but a low forward progression (i.e. a lot of sperm just wiggling back and forth in place) the sperm will not be able to make their way through their partner’s reproductive tract and fertilize an egg, because they can’t move forward. A man with a lower motility, but a great forward progression, would have fewer sperm moving, but they would be moving forward quickly, which would give them a better chance of fertilizing an egg.

Total Motile Count (TMC) is the number of moving sperm in the entire ejaculate. It is calculated by multiplying the volume (cc) by the concentration (million sperm/cc) by the motility (percent moving). There should be more than 40 million motile sperm in the ejaculate. Total motile count is measured simply as a number, reported in millions. For example, a TMC of 53 means that in the man’s entire specimen, the whole ejaculate, he is calculated to have 53 million moving sperm. Some men have a very high volume, and still produce good numbers of moving sperm. However, their concentrations may be low, because the sperm are found in a lot of fluid, i.e. the fluid is diluting out the count. Some men may have excellent concentrations, but low motilities, so be very few moving sperm in the ejaculate. TMC accounts for the all three of these parameters, and gives a value that is useful in making clinical decisions. 40 million moving sperm in the ejaculate is considered the low end of normal. TMC is a very valuable parameter. It helps specialists make clinical decisions. If the TMC is over 10 million, there is a reasonable chance that taking the processed sperm and placing them directly into the uterus (IUI or Intrauterine Insemination) will be successful. Most specialists feel that if the TMC, after maximizing the man’s sperm production, is less than 10 million, that In Vitro Fertilization (IVF) is indicated.

2.2.6. Sperm Vitality:

Sperm Vitality, as estimated by assessing the membrane integrity of the cells, may be determined routinely on all samples, but is especially important for samples with less than about 40% progressively motile spermatozoa. This test can provide a check on the motility evaluation, since the percentage of dead cells should not exceed (within sampling error) the percentage of immotile spermatozoa. The percentage of viable cells normally exceeds that of motile cells. The percentage of live spermatozoa is assessed by identifying those with an intact cell membrane, from dye exclusion or by hypotonic swelling. The dye exclusion method is based on the principle that damaged plasma membranes, such as those found in non–vital (dead) cells, allow entry of membrane–impermeant stains. The hypo–osmotic swelling test presumes that only cells with intact membranes (live cells) will swell in hypotonic solutions.

Sperm Vitality should be assessed as soon as possible after liquefaction of the semen sample, preferably at 30 minutes, but, in any case within 1 hour of ejaculation, to prevent observation of deleterious effects of dehydration or of changes in temperature on vitality.

2.2.7. Sperm numbers

The number of spermatozoa in the ejaculate is calculated from the concentration of spermatozoa, which is measured during semen evaluation. For normal ejaculates, when the male tract is unobstructed and the abstinence time short, the total number of spermatozoa in the ejaculate is correlated with testicular volume [7; 11] and thus is a measure of the capability of the testes to produce spermatozoa [63] and the patency of the male tract. The concentration of spermatozoa in the semen, while related to fertilization and pregnancy rates, is influenced by the volume of the secretions from the seminal vesicles and prostate and is not a specific measure of testicular function.

The terms “total sperm number” and “sperm concentration” are not synonymous. Sperm concentration refers to the number of spermatozoa per unit volume of semen and is a function of the number of spermatozoa emitted and the volume of fluid diluting them. Total sperm number refers to the total number of spermatozoa in the entire ejaculate and is obtained by multiplying the sperm concentration by the semen volume.

Concentration (sometimes referred to as “the count”): is a measurement of how many million sperm there are in each milliliter of fluid. There are various techniques for obtaining this number. Some prove to be more accurate than others. The most accurate method uses one–time–only use slides. These slides have a built–in cover that allows only an exact amount of semen into the chamber. Thus, the depth is consistent. Reusable chambers change in depth as they are cleaned and reused. Measuring concentration takes surprising expertise, both in preparing the slides and reading them. Average sperm concentration is more than 60 million per milliliter (>60 million/cc). Counts of less than 20 million per milliliter (<20 million/cc) are considered sub–fertile. Concentration depends on how many sperm are made and ejaculated, and how much fluid is made and ejaculated. Men with low sperm production will of course have low concentrations no matter what the volume. Some men make high volumes of semen. This dilutes out the sperm, and causes the man’s concentration to be lower. Two men who ejaculate the same number of sperm, will have different concentrations if they ejaculate a different volume. Thus, an expert will look at the volume as well as the concentration to assess sperm production.

The generalization that total sperm number reflects testicular sperm productivity may not hold for electro–ejaculates from men with spinal cord injury, those with androgen deficiency, or for samples collected after prolonged abstinence or partial retrograde ejaculation.

The term “sperm density” (mass per unit volume) should not be used when sperm concentration (number per unit volume) is meant.

Volume: is a measurement of the quantity of the ejaculate. How much fluid is there? The Semen Analysis is measured in milliliters (ml) which is the same as cubic centimeters (cc). For reference, a teaspoon equals 5cc. Normal volume is two cc or more. The volume may be low if a man is anxious when producing a specimen, if the entire specimen is not caught in the collection container, if there are hormonal abnormalities, or if there are ductal blockages.

2.2.7. Normal sperm morphology

Morphology describes the shape of the sperm. The sperm are examined under a microscope and must meet specific sets of criteria for several sperm characteristics in order to be considered normal. Most commercial laboratories will report WHO morphology (use World Health Organization criterion). A more sophisticated, and more useful way of assessing shape, called Strict, Kruger, or Tygerberg Morphology is also used by speciality labs. Strict is much more time consuming to perform, and takes a lot of time and experience to master, so it is usually not used by commercial or hospital labs.

The concept of morphologically normal spermatozoa

The variable morphology of man’s spermatozoa makes assessment difficult, but observations on spermatozoa recovered from the female reproductive tract, especially in postcoital endocervical mucus [31; 65] and also from the surface of the Zona Pellucida [49; 66], have defined the appearance of potentially fertilizing (morphologically normal) spermatozoa. By the strict application of certain criteria of sperm morphology, relationships between the percentage of normal forms and various fertility endpoints (Time–To–Pregnancy (TTP), pregnancy rates in vivo and in vitro) have been established [19; 24; 25; 34; 59; 68; 73; 75], which may be useful for the prognosis of fertility. The underlying philosophy of the classification system described here is to limit what is identified as normal to the potentially fertilizing subpopulation of spermatozoa prevalent in endocervical mucus. Using these guidelines, the range of percentage normal values for both fertile and infertile men is likely to be 0–30%, with few samples exceeding 25% normal spermatozoa [68]. This low value will inevitably produce low thresholds; indeed, reference limits and thresholds of 3–5% normal forms have been found in studies of In–Vitro Fertilization [19], Intrauterine Insemination [75] and In–Vivo Fertility [74].

The woman’s oocyte Zona Pellucida also selects a subpopulation of morphologically similar spermatozoa, but such “zona–preferred” spermatozoa display a wider range of forms [33; 48; 52; 53; 54; 55]. The percentage of motile spermatozoa in semen from fathers displaying “zona–preferred” morphology is also low: 8–25% [59].

Spermatozoa consist of a head, neck, middle piece (midpiece), principal piece and endpiece. As the endpiece is difficult to see with a light microscope, the cell can be considered to comprise a head (and neck) and tail (midpiece and principal piece). For a spermatozoon to be considered normal, both its head and tail must be normal. All borderline forms should be considered abnormal.

The head should be smooth, regularly contoured and generally oval in shape. There should be a well–defined acrosomal region comprising 40–70% of the head area [68]. The acrosomal region should contain no large vacuoles, and not more than two small vacuoles, which should not occupy more than 20% of the sperm head. The post–acrosomal region should not contain any vacuoles.

The midpiece should be slender, regular and about the same length as the sperm head. The major axis of the midpiece should be aligned with the major axis of the sperm head. Residual cytoplasm is considered an anomaly only when in excess, i.e. when it exceeds one third of the sperm head size [69].

The principal piece should have a uniform caliber along its length, be thinner than the midpiece, and be approximately 45 mm long (about 10 times the head length). It may be looped back on itself, provided there is no sharp angle indicative of a flagellar break.

2.2.8. Classification of abnormal sperm morphology

Man’s semen samples contain spermatozoa with different kinds of malformations. Defective spermatogenesis and some epididymal pathologies are commonly associated with an increased percentage of spermatozoa with abnormal shapes. The morphological defects are usually mixed. Abnormal spermatozoa generally have a lower fertilizing potential, depending on the types of anomalies, and may also have abnormal DNA. Morphological defects have been associated with increased DNA fragmentation [32], an increased incidence of structural chromosomal aberrations [46], immature chromatin [22] and aneuploidy [23; 64]. Emphasis is therefore given to the form of the head, although the sperm tail (midpiece and principal piece) is also considered.

The following categories of defects should be noted:

Head defects: large or small, tapered, pyriform, round, amorphous, vacuolated (more than two vacuoles or >20% of the head area occupied by unstained vacuolar areas), vacuoles in the post-acrosomal region, small or large acrosomal areas (<40% or >70% of the head area), double heads, or any combination of these.

Neck and midpiece defects: asymmetrical insertion of the midpiece into the head, thick or irregular, sharply bent, abnormally thin, or any combination of these.

Principal piece defects: short, multiple, broken, smooth hairpin bends, sharply angulated bends, of irregular width, coiled, or any combination of these.

Excess Residual Cytoplasm (ERC): this is associated with abnormal spermatozoa produced from a defective spermatogenic process. Spermatozoa characterized by large amounts of irregular stained cytoplasm, one third or more of the sperm head size, often associated with defective midpieces [68; 69] are abnormal. This abnormal excess cytoplasm should not be called a cytoplasmic droplet [21].

Cytoplasmic droplets (membrane–bound vesicles on the midpiece at the head–neck junction) are normal components of physiologically functional man’s spermatozoa. If swollen, they may extend along the length of the midpiece, as observed by phase–contrast, differential–interference–contrast and X–ray microscopy of living cells in semen, cervical mucus and medium [1; 30]. Cytoplasmic droplets are osmotically sensitive and are not well preserved by routine air–drying procedures [18]. They are not obvious in stained preparations, where they may appear as small distensions of the midpiece. Cytoplasmic droplets are less than one third the size of the sperm head in fixed and stained preparations [69] and are not considered abnormal.

2.2.9. Indices of multiple sperm defects

Morphologically abnormal spermatozoa often have multiple defects (of the head, midpiece or principal piece, or combinations of these defects). A detailed assessment of the incidence of morphological abnormalities may be more useful than a simple evaluation of the percentage of morphologically normal spermatozoa. Recording the morphologically normal spermatozoa, as well as those with abnormalities of the head, midpiece and principal piece, in a multiple–entry system gives the mean number of abnormalities per spermatozoon assessed.

Three indices can be derived from records of the detailed abnormalities of the head, midpiece and principal piece in a multiple–entry system:

1. The multiple anomalies index (MAI) [42] 
2. The teratozoospermia index (TZI) [67];
3. The sperm deformity index (SDI) [8; 9].

2.2.10.  Assessment of specific sperm defects

Occasionally, many spermatozoa will have a specific structural defect. For example, the acrosome may fail to develop, giving rise to the “small round–head defect” or “globozoospermia”. If the basal plate fails to attach to the nucleus at the opposite pole to the acrosome at spermiation, the heads are absorbed and only tails are found in semen (the pinhead defect).

Pinheads (free tails) are not counted as head defects, since they possess no chromatin or head structure anterior to the basal plate. Because free tails (pinheads) and free heads are not counted as spermatozoa (defined as having a head and tail), they are not considered to be sperm abnormalities.

Men whose spermatozoa all display one of these defects are usually infertile. Such cases are rare, but it is critical that they are identified and correctly reported. Thus, report the presence of specific sperm defects, e.g. free sperm heads, pinheads (free tails), heads lacking acrosomes.

2.2.11. Testing for antibody coating of spermatozoa

Anti–sperm antibodies: some men may produce antibodies to their own sperm. These antibodies may decrease fertility rates in a number of ways. They may impede the movement of sperm through a woman’s cervical mucous, inhibit the binding of a sperm to the egg, and/or inhibit its penetration into the egg. Men who are most at risk for developing antibodies are those with previous testicular and epididymal infection, trauma, surgery, or large varicoceles. The presence of these antibodies is often not predictable from other semen parameters or from the man’s history.

If spermatozoa demonstrate agglutination (i.e. motile spermatozoa stick to each other head–to–head, tail–to–tail or in a mixed way), the presence of sperm antibodies may be the cause. Sperm antibodies can be present without sperm agglutination; equally, agglutination can be caused by factors others than sperm antibodies. The mere presence of sperm antibodies is insufficient for a diagnosis of sperm autoimmunity. It is necessary to demonstrate that the antibodies interfere severely with sperm function; this is usually done by a sperm–mucus penetration test. Antibodies can also interfere with zona binding and the acrosome reaction.

Anti–Sperm Antibodies (ASAs) in semen belong almost exclusively to two immunoglobulin classes: IgA and IgG. IgM antibodies, because of their larger size, are rarely found in semen. IgA antibodies may have greater clinical importance than IgG antibodies [39; 44].

If spermatozoa have antibodies on their surface, the latex beads will adhere to them. The motile spermatozoa will initially be seen moving around with a few or even a group of particles attached. Eventually the agglutinates become so massive that the movement of the spermatozoa is severely restricted. Sperm that do not have coating antibodies will be seen swimming freely between the particles.

Standard semen fluid tests for diagnostic purposes or research purposes

Many parameters can be measured but the most important are: 

1. Round Cell Concentration, 
2. White Blood Cell Concentration, and 
3. Viscosity.

Round Cell Concentration

Besides the sperm, there are often other cells in the semen, seen under the microscope. Round cells can be immature sperm, debris (junk), or White Blood Cells (WBC’s). This number is usually reported as a concentration of round cells, for example, how many round cells there are, in millions, per cc. of the ejaculate. Round cell concentrations of greater than one million per cc, may be significant. The presence of non–sperm cells in semen may be indicative of testicular damage (immature germ cells), pathology of the efferent ducts (ciliary tufts) or inflammation of the accessory glands (leukocytes). The number of non-sperm cells in semen (epithelial cells, “round cells” (germ cells and leukocytes) or isolated sperm heads and tails) can be estimated in fixed wet preparations by the use of a haemocytometer in the same way as for spermatozoa. However, semen that has been diluted adequately for counting spermatozoa will normally be too dilute for accurate estimation of non–sperm cells, unless high concentrations are present. The prevalence of round cells relative to spermatozoa can be assed from slides. Alternatively, their concentration can be assessed during the estimation of peroxidase–positive cells.

White Blood Cell Concentration

Of the round cells found in the semen, it is felt that only if increased numbers of WBC’s is considered significant.

White blood cells are considered significant if more than one million are found in each milliliter of the ejaculate. However, white blood cells cannot be differentiated from the other round cells normally found in the semen (debris and immature sperm) without special staining. Thus, if more than one million round cells are found in the ejaculate, a portion of the ejaculate should be specially stained to look for an increased number of white blood cells. A high white blood cells concentration may be an indication of either infection or inflammation, which can have a negative impact on the sperm. If the white blood cell count is elevated, semen cultures should be performed on a subsequent specimen. Unfortunately, the semen culture cannot be performed on the original specimen as it must be the first step performed on the specimen in order to keep it sterile.

Assessment of leukocytes in semen

Leukocytes, predominantly Polymorphonuclear Leukocytes (PMN, neutrophils), are present in most human ejaculates [40; 72]. They can sometimes be differentiated from spermatids and spermatocytes in a semen smear. Differentiation is based on differences in staining coloration, and on nuclear size and shape [40]. Polymorphonuclear leukocytes can easily be confused morphologically with multinucleated spermatids, but stain a bluish colour, in contrast to the more pinkish colour of spermatids [40]. Nuclear size may also help identification: monocyte nuclei exhibit a wide variation in size, from approximately 7 mm for lymphocytes to over 15 mm for macrophages. These sizes are only guidelines, since degeneration and division affect the size of the nucleus.

Excessive numbers of leukocytes in the ejaculate (leukocytospermia, pyospermia) may be associated with infection and poor sperm quality.

Leukocyte–dependent damage to spermatozoa depends on the total leukocyte number in the ejaculate and the number of leukocytes relative to the number of spermatozoa.

Leukocytes can impair sperm motility and DNA integrity through an oxidative attack. However, whether the level of leukocytic infiltration observed is damaging depends on factors that are impossible to infer from a semen sample, such as the reason for, timing and anatomical location of the infiltration, as well as the nature of the leukocytes involved and whether they are in an activated state [5; 10; 72].

Viscosity

When a man ejaculates, the fluid is clumpy and viscous (thick). However, when it is exposed to body temperature enzymes that are present in it, break down certain proteins. This makes the fluid runny and non–viscous (thin). Some men have thick semen even after the semen has been at body temperature for greater than an hour. This is called increased viscosity. Increased viscosity can make it more difficult for the sperm to break through the semen fluid, and make their way through their partner’s reproductive tract, and ultimately fertilize an egg. Increased viscosity may indicate an infection, and cultures should be performed. If a man has persistently increased viscosity, it may be worthwhile to wash the sperm, to separate the sperm from the fluid. Then it is put directly into his partner’s vagina (Intrauterine Insemination or IUI).

Specialized semen tests for diagnostic purposes or research purposes

In certain situations, specialized tests are needed. These depend on the findings at the time of the analysis and can often be performed on that specimen. Only a specialized laboratory will be able to perform these tests appropriately and accurately.

Spun Specimen

Even if no sperm are seen on the test slide, the sperm count may still not be zero (as there may be very low numbers of sperm in the ejaculate). This has very important implications as it may determine if the couple can conceive using Advanced Reproductive Techniques. This must be assessed by spinning down the specimen so that any sperm that might be in the semen are concentrated in a pellet on the bottom of the tube. This pellet is then meticulously and exhaustively examined to see if any sperm are found.  

If sperm are found, it is important to consider freezing (cryopreserving) them for later use. For men with very low counts, often there are no sperm in a particular specimen.  If a couple is doing an In Vitro Fertilization, requiring one live sperm per egg, having frozen sperm as a backup is crucial, in case there are no sperm in the man’s ejaculation on the day they retrieve his partner’s eggs.

Viability

Sperm may be alive, but not moving. A specialized staining technique is used to determine what percentage of the sperm are alive and is indicated when the motility (percent moving) is less that thirty percent.

Some men have no moving sperm in their ejaculate. However, if some are found to be alive, they can actually be injected into his partner’s egg.

The initial viability testing done on a particular specimen kills the sperm on the slide. Specialized testing can be done on the specimen the day sperm are needed to inject into eggs, which do not kill the sperm, but just differentiate which are alive. (Only alive sperm can lead to fertilization when injected into an egg.)

The initial viability testing is done on a Semen Analysis, in order to help predict how many living, even if not moving sperm, a man can be expected to have in his ejaculations.

Fructose

In men with no sperm or very low numbers of sperm in the ejaculate, it is important to determine whether the sperm are not being produced at all, or whether they are being produced but are blocked from “getting into” the semen. A fructose test can help differentiate between these two problems. Fructose is only made in certain glands, the seminal vesicles, which may be absent in men with missing or incomplete ducts.

Post–Ejaculatory Urinalysis (PEU)

Some men ejaculate all or part of the sperm backward into the bladder. This can be detected by having a man ejaculate and immediately afterward urinate into a separate cup. The post-ejaculatory urine is then centrifuged to see if any sperm are present.

Additional sperm–oocyte interaction tests

The binding of spermatozoa to the Zona Pellucida initiates the acrosome reaction, releases free and exposes bound lytic acrosomal components, and allows the spermatozoa to penetrate through the zona matrix, driven by the increased flagellar thrusting of hyperactivated motility. To evaluate the binding events, non–viable, non–fertilizable human oocytes from autopsy, surgically removed ovaries or failed In–Vitro Fertilization may be used. These tests can be performed using oocytes that have been stored in salt, but are usually limited by the lack of availability of human oocytes [43; 45; 50; 60].

Zona Pellucida binding tests

One Zona Pellucida binding assay, the hemizona assay [16], involves microdissection of the Zona Pellucida into equal halves and the exposure of each matching half to the same concentration of test or control spermatozoa. Another sperm–zona binding assay [33; 47; 51; 57; 58; 61; 62] involves labelling the relative contributions made by leukocyte and sperm subpopulations to the reactive–oxygen–generating capacity of the cell suspension:

(a) in the presence of leukocyte contamination, a burst of ROS generation is observed on addition of the leukocyte–specific probe FMLP. The subsequent addition of PMA generates a sustained, intense chemiluminescent signal from both the spermatozoa and leukocyte populations;

(b) in the absence of leukocyte contamination, the FMLP response is lost, while PMA elicits a pronounced chemiluminescent signal from the spermatozoa.

Assessment of the acrosome reaction

The physiological acrosome reaction occurs at the Zona Pellucida after sperm binding. The Zona Pellucida–induced acrosome reaction can be assessed on spermatozoa removed from the surface of the Zona Pellucida or exposed to disaggregated human Zona Pellucida proteins [6; 26; 27; 29; 52; 54; 55].

In cases of teratozoospermia and oligozoospermia, some men may have otherwise normal semen analyses, but spermatozoa that display disordered Zona Pellucida–induced acrosome reactions. Others may have spermatozoa that exhibit normal Zona Pellucida binding but have a poor Zona Pellucida–induced acrosome reaction [60]. These tests are limited by the restricted availability of human Zonae Pellucidae. Other stimuli, such as calcium ionophores, will induce the acrosome reaction but the results are not related to those obtained from the Zona Pellucida–induced acrosome reaction [55; 56]. Acrosomal status after induction of the acrosome reaction can be assessed by microscopy or flow cytometry [20; 28; 37] with fluorescently labelled lectins, such as Pisum sativum (pea agglutinin) or Arachis hypogaea (peanut lectin), or monoclonal antibodies against the acrosome antigen CD46.

The acrosome reaction is an exocytotic process that occurs after spermatozoa bind to the Zona Pellucida and must take place before the spermatozoon can penetrate the oocyte vestments and fuse with the oocyte. Calcium influx is believed to be an initiating event in the normal acrosome reaction. Inducing calcium influx by using a calcium ionophore is one way of testing the competence of capacitated spermatozoa to undergo the acrosome reaction [3]. This is the basis of this assay, also called the Acrosome Reaction after Ionophore Challenge (ARIC) test. However, further evaluation is needed before testing of acrosome status can be considered a routine clinical assay.

3. Sperm Washing as the preparational process for performing an Intrauterine Insemination (IUI)

Spermatozoa may need to be separated from seminal plasma for a variety of purposes, such as diagnostic tests of function and therapeutic recovery for insemination and assisted reproductive technologies (ART). If tests of sperm function are to be performed, it is critical that the spermatozoa are separated from the seminal plasma within 1 hour of ejaculation, to limit any damage from products of non–sperm cells.

3.1. When spermatozoa may need to be separated from seminal plasma

Although seminal plasma helps spermatozoa penetrate cervical mucus, some of its components (e.g. prostaglandins, zinc) are obstacles to the achievement of pregnancy when natural barriers are bypassed in ART, such as Intrauterine Insemination (IUI) or In–Vitro Fertilization (IVF). The separation of human spermatozoa from seminal plasma to yield a final preparation containing a high percentage of morphologically normal and motile cells, free from debris, non–germ cells and dead spermatozoa, is important for clinical practice. Diluting semen with culture media and centrifuging is still used for preparing normozoospermic specimens for IUI [15]. However, density–gradient centrifugation and direct swim–up are generally preferred for specimens with one or more abnormalities in semen parameters. Glass–wool columns are reported to be as effective as density gradients for the separation of spermatozoa from semen with suboptimal characteristics [41; 70].

3.2. The choice of Sperm Preparation Technique

The choice of Sperm Preparation Technique is dictated by the nature of the semen sample [17]. For example, the direct swim–up technique is often used when the semen samples are considered to be largely normal, whereas in cases of severe oligozoospermia, teratozoospermia or asthenozoospermia, density gradients are usually preferred because of the greater total number of motile spermatozoa recovered. Density gradients can also be altered to optimize handling of specific properties of individual samples: the total volume of gradient material can be reduced, limiting the distance that the spermatozoa migrate and maximizing total motile sperm recovery, or the centrifugation time can be increased for specimens with high viscosity. Each laboratory should determine the centrifugal force and centrifugation time necessary to form a manageable sperm pellet. When sperm numbers are extremely low, it may be necessary to modify the centrifugal force or the time, in order to increase the chances of recovering the maximum number of spermatozoa.

3.3. Efficiency of sperm separation from seminal plasma and infectious organisms

The efficiency of a Sperm Selection Technique is usually expressed as the absolute sperm number, the total number of motile spermatozoa, or the recovery of morphologically normal motile spermatozoa. Swim–up generally produces a lower recovery of motile spermatozoa (<20%) than does density–gradient centrifugation (>20%). Swim–up and density–gradient centrifugation also produce different levels of contamination with seminal components in the final Sperm Preparation. Using the prostatic secretion zinc as a marker of soluble seminal components, demonstrated time-dependent diffusion of zinc from semen into the overlaying swim–up medium. The final zinc concentration in swim-up preparations was greater than that after density–gradient preparation. Semen samples may contain harmful infectious agents, and technicians should handle them as a biohazard with extreme care. Sperm Preparation Techniques cannot be considered 100% effective in removing infectious agents from semen.

3.4. General principles of Sperm Preparation Techniques

Three simple Sperm Preparation Techniques are described in the following sections. For all of them, the culture medium suggested is a balanced salt solution supplemented with protein and containing a buffer appropriate for the environmental conditions in which the spermatozoa will be processed. For Assisted Reproduction Procedures, such as Intracytoplasmic Sperm Injection (ICSI), In–Vitro Fertilization (IVF), Artificial Insemination (AI) or Gamete Intrafallopian Transfer (GIFT), it is imperative that the human serum albumin is highly purified and free from viral, bacterial and prion contamination. Albumins specifically designed for such procedures are commercially available. If the incubator contains only atmospheric air and the temperature is 37 °C, the medium should be buffered with Hepes or a similar buffer, and the caps of the tubes should be tightly closed. If the incubator atmosphere is 5% (v/v) CO2 in air and the temperature is 37 °C, then the medium is best buffered with sodium bicarbonate or a similar buffer, and the caps of the test–tubes should be loose to allow gas exchange. Adherence to this will ensure that the culture pH is compatible with sperm survival. The final disposition of the processed spermatozoa will determine which buffered medium is appropriate.

What is the essence of Sperm Washing process?

Sperm Washing, also known as Sperm Processing or Sperm Preparation, is the name given to the laboratory technique that separates sperm from seminal fluid for use in Infertility Treatments. Sperm Washing prepares a semen sample for an Intrauterine Insemination (IUI), which involves placing sperm inside a woman’s uterus to facilitate fertilization. The most common reasons for choosing an Intrauterine Insemination (IUI) are low sperm count, decreased sperm mobility or an ejaculation dysfunction. But the procedure may also be used when the cause of infertility is unknown, or when there is a hostile cervical condition or cervical scar tissue.

For an Intrauterine Insemination (IUI) to be performed, the semen sample must be washed free of debris, white blood cells, and prostaglandins, which can cause the uterus to contract. The processing also removes dead sperm and concentrates the sperm into a small volume which can easily be handled by the uterus. There are three main methods of Sperm Washing; the swim-up, density gradient wash, and simple (centrifugation) wash.

The type of Sperm Wash used depends on the individual characteristics of each semen specimen, which will be determined beforehand by a thorough Semen Analysis. To optimize man’s chances of success, experts analyze both the fresh (original) and washed specimen for sperm count, motility, viscosity and other important variables.

What are the main techniques of Sperm Washing for Intrauterine Insemination (IUI)?

There are three main techniques of Sperm Washing, used for the Artificial Insemination: the simple (centrifugation) wash, swim–up and density gradient wash. The type of wash used depends on the individual characteristics of each semen specimen.

3.1. Simple (centrifuge) [Sperm Washing Technique]

For the best quality samples (number and motility of sperms) the Sperm Washing is often performed [14] for the Artificial Insemination. The centrifuge wash (or simple wash) should be performed on a semen sample that has a decreased concentration (concentration depends on how many sperm are made and ejaculated, and how much fluid is made and ejaculated) and/or motility (motility describes the percentage of sperm that are moving. In even the best specimens, many sperm are not moving. The (immotile) sperm may either be dead or just not moving. This actually is a significant difference, especially in specimens with a 0% motility. Additionally, it is essential to emphasize the viability criteria: viability means that sperm may be alive, but not moving. The initial viability testing done on a particular specimen kills the sperm on the slide. Specialized testing can be done on the specimen the day sperm are needed to inject into eggs, which do not kill the sperm, but just differentiate which are alive, because only alive sperm can lead to fertilization when injected into an egg. The initial viability testing is done on a Semen Analysis, in order to help predict how many living, even if not moving sperm, a man can be expected to have in his ejaculations). A sample containing round cells and debris should not be prepared by this method. 

Semen is diluted in a test tube with a special solution of antibiotics and protein supplements. After that it is placed in a centrifuge, a machine that spins around at extremely high speeds. As the sperm mixture is spun, sperm cells fall to the bottom of the test tube, producing a mass of dense, highly active sperm. The pellet is recovered, resuspended and again centrifuged. The final pellet is resuspended in approximately 0.5 mls of media. These sperm can then be removed from the test tube and used in Intrauterine Insemination (IUI). A simple Sperm Wash takes about 20 to 40 minutes. The repeated centrifugations without the separation of the good sperms from leukocytes and dead sperms can produce many oxidative species and the damage of the sperms function [2].

3.2. Swim–up [Sperm Washing Technique]

Spermatozoa may be selected by their ability to swim out of seminal plasma and into culture medium. This is known as the “swim–up” technique. The swim up is the most common technique used in IVF laboratories performed on patients and is preferred if the semen sample has a normal number of good sperms (normozoospermia), and is not recommended for samples of high viscosity, with high numbers of round cells, or with a high content of debris. In case of normozoospermia this technique is the most successful one. By this technique, the sperms are selected on their motility and the capability to swim out of the seminal plasma.

In this procedure, the washing media is gently placed over the semen in a conical tube. The specimen is then placed in an incubator for approximately one hour. During this time, the sperm are allowed to swim up into the media, with the purpose of collecting the most motile, normal sperm which are free of debris. The supernatant is collected and centrifuged twice with Sperm Washing media. The final pellet is then resuspended in approximately 0.5 mls of media. Modified Sperm Washing media must be used to process the sample. It is available from Irvine Scientific.

3.3. Discontinuous (density) gradient [Sperm Washing technique]

This is the preferred technique to select the greater number of motile spermatozoa in cases of severe oligozoospermia, teratozoospermia or asthenozoospermia. In this method, good quality sperms can be separated from dead sperms, leukocytes and the other components of the seminal plasma by a density discontinuous gradient. Cells with different density and motility can be selected during the centrifugation by the colloidal silica coated with silane of the gradient; the sperms with high motility and good morphology are at the bottom of the tube, finally free from dead spermatozoa, leukocytes, bacteria and debris.

The discontinuous (density) gradient method should be used on samples containing round cells, debris, or those with increased viscosity, but with a relatively normal concentration and motility. The gradient is achieved by layering media of two different densities in a conical tube. The semen is then placed on top of the gradient and the tube is then spun to allow the specimen to proceed through the gradient. The resulting pellet should contain the motile, normal sperm, while the dead sperm and debris are caught up in the gradient media. The pellet is then resuspended in washing media and centrifuged twice. The final pellet is resuspended in a final volume of approximately 0.5 mls of media. There are several commercially available kits. Conception Technologies carries the Enhance–S Plus kits and Irvine Scientific carries the Isolate Sperm Separation Medium.

In each of the represented–above cases the laboratory performs an analysis on both the fresh specimen and the washed specimen. This will include an assessment of the count, motility, volume and viscosity.

Conclusion

Prolonged exposure of sperm to seminal plasma results in a marked decline in both motility and viability. Sperm incubated in synthetic culture medium free of seminal plasma contamination show no such declines. It is essential, therefore, that spermatozoa for clinical procedures such as In Vitro Fertilization (IVF)/Intracytoplasmic Sperm Injedion (ICSI) be separated from the seminal environment as soon as possible after ejaculation.

The most common methods for Sperm Processing are the swim–up technique and the separation through a discontinuous colloidal density gradient. Alternatively, simple washing can be employed in cases of severely oligozoospermic ejaculates. The advantage of swim–up and density gradient are that they selected the sperm population exhibiting better motility, in contrast to the non-selective concentration of spermatozoa obtained through a simple wash procedure.

Sperm Processing by swim–up or density gradient eliminate immotile and dead spermatozoa, along with exfoliated cells, cellular debris, and amorphous material. Sperm Preparation by swim–up removes seminal plasma and concentrates the most motile spermatozoa in a very small volume of sperm culture media. However, the sperm yield is low in cases of oligozoospermic ejaculates with low motility. For this reason, swim–up is preferred for normozoospermic specimens. Conversely, density gradient centrifugation is usually preferred to process ejaculates with low sperm number, motility, or morphology, as it allows the elimination of leukocytes and other microorganisms which are trapped in the gradient interphases. Density gradients can be altered to optimize sperm recovery by decreasing the gradient volume which limit the distance that the spermatozoa migrate, or by increasing the centrifugation time in cases of hyperviscous ejaculates.

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