INTRODUCTION:

This article explains in depth the general semen analysis and addresses the different semen preparation methods currently being used in assisted reproductive laboratories (ART) today. Male infertility factor contributes around one-third of all aided reproductive referrals¹.

Therefore, it is important to conduct an adequate study of their semen when examining couples applying for fertility therapy. In 2010, the latest version of the WHO laboratory manual for human semen analysis and processing (WHO Manual) was released. This is the gold norm for both semen research and the techniques of planning and explain them all in depth.

Several ART laboratories still use the 1999 reference manual, although improvements resulting from the 2010 upgrade are now starting to trickle in. Both sets of reference criteria will be referred to in this section, with the newer principles specifically shown in italics^2,3.

Recent revisions to the WHO manual were necessary, as earlier versions did not take into account the time to become pregnant (fecundity) when determining a fertile man's usual semen characteristics⁴.

Semen analysis:

There are four main stages of semen analysis:

1. Patient history

2. Sample production

3. Semen analysis

4. Interpretation of results

Patient history:

When evaluating semen, it is important to evaluate specific patient background, as often very clear variables may have major impacts on outcomes such as fever disorder, prescription, previous medical and surgical records and occupational and recreational habits.

Sample production:

Ideally, sample processing rooms should be located as near as possible to the laboratory, so that samples can be examined as quickly as possible after liquefaction. The semen sample should be converted into a clean single-use container specifically identified with the full name and initial identity of the recipient. Washed containers should never be used, since they may contain soap or contamination from their previous content.

Masturbation should generate the sample, and note the time taken. It should also be noticed whether the entire sample was extracted or not on an accompanying slip. If the first portion of the ejaculate is absent, it will impair the examination. This latter part is less abundant in sperm³. The average production time for this is 10-15 minutes⁵. If the patient takes significantly longer than this, then clinical decisions should be considered regarding the cryopreservation of the samples for use on the day of treatment. This will both alleviate the patient's stress of producing a sample on the day, and ensure that when required, a useable sample is available. Samples should only be produced after 2-7 days of abstinence in order to accurately evaluate and reproduce results of the semen analysis. Shorter abstinence cycles will result in both semen volume and sperm concentration declining. In comparison, improved abstinence cycles may result in a higher percentage of non-moving sperm. If additional tests are needed then abstinence periods should be sufficient to allow comparable results⁶.

Semen analysis:

In order to achieve an accurate estimation of the particular semen characteristics of the recipient, it is important that at least two samples be analyzed. Specific parameters tend to be unpredictable and it is possible to collate the outcomes of more than one study to determine the features of a typical sample.

Basic study of semen is composed of four components: length, sperm motility, count and morphology. As soon as the sample is made, the liquefaction should be accelerated by putting it in a warmed non-CO2 incubator. Liquefaction refers to the normal transition in consistency of the semen over time from a gel-like vesicular secretion to a liquid^3,6.

Samples cannot be reliably examined since sperm cannot travel freely. Typical liquefaction time is 20 minutes but almost immediately after liquefaction Ejaculation: Ejaculation. Delayed semen liquefaction, more than 1 hour after ejaculation, can suggest an accessory gland function disorder, or an infection. In order to keep the sample from evaporating and degrading inside the seminal plasma, semen processing can take place as soon as liquefaction has arisen, preferably 30 minutes after development and definitely before 60 minutes lapsed. The appearance of mucus streaks, which can also be seen during initial examination, can interfere with the parameters of motility³. Standard semen is a homogenous grey-opalescent color, and illness can be shown by differences in this color. When a sample is oligospermic, it becomes less visible. A red/brown color suggests red blood cells are present. This can be suggestive of illness, which should therefore be reviewed with the patient and clinician to ensure that no significant symptoms are apparent. Semen can also look yellow due to jaundice, or other vitamin supplements³.

Viscosity and thickness:

The first step of the testing semen is to assess volume and viscosity. Volume is measured by using a pipette graduated from 5ml to 10ml. Average semen amount is between 2 and 5mL (1.5ml). Low semen volume can be characteristic of ejaculatory duct obstruction or congenital bilateral absence of the vas deferens together with an azoospermic sample, i.e. a sample that contains no sperm. This may indicate increased abstinence or inflammation of the accessory glands, particularly if the volume is too high Where the pH is low (3). It is customary to measure the sample pH using pH paper with a scale 6.0–10.0 during volume calculation. PH should also be evaluated within 1 hour of development, as pH is impaired by CO2 loss that happens after ejaculation and hence increases over time. The viscosity is then assessed by allowing the sample to gravitate and analyze the stream into its bottle. The viscosity is divided into 4 categories:

1. Graph looks watery, reduced

2. Standard, leaves the pipette in discreet, tiny drops

3. Slightly increased-it is easy to load into a pipette tip

4. Substantially increased – does not load easily into a pipette tip (in this case, cutting the end of the tip would encourage the sample) abnormally increased viscosity is described as if the gravitating semen thread is more than 2cm long³.

Increased viscosity does not seem to have any therapeutic significance; however, it decreases initial parameters of mobility and thus limits sperm's ability to infiltrate in vivo cervical mucus. After processing, and elimination of seminal useful sperm It returns plasma, normal parameters of movement⁷.

Motility of the Sperm:

Two separate aliquots of about 10μl should be put on a clean glass slide and examined under a microscope in phase contrast for the existence in mucus stains, agglutination, existence of inflammation due to elevated numbers of circular cells and, most notably, the presence of sperm. The magnification needed depends on what is being observed, but a minimum of 400 ranges is recommended to detect the presence of bacteria³.

This slide can be used to measure sperm motility at a magnification power of 200 ranges or 400 ranges depending on the concentration. Using a larger force would promote focused sample analysis. The measurement of ejaculated sperm motility is an extremely significant function which has a strong correlation with fertility⁸. It can be measured using computer-aided sperm analysis (CASA), but the calculation of motility manually using a cell counter and a phase contrast microscope is considered much more reliable in the clinical setting. Sperm was divided into four groups for measuring motility:

a) Progressive motility > 20μm/s at 20°C

b) Slow or sluggish

c) Non-progressive motility < 5μm/s

d) Immotile

The sperm count should be spread over at least five viewing fields. Then these values are expressed in percentage form. This is to be repeated on a second aliquot and compared with the results. If the difference between the highest categories on the two aliquots is less than 10 percent, then this is deemed acceptable; if not, a third slide should be prepared and analyzed, and the results averaged.

WHO 2010 simplified motility calculation by classifying sperm into three categories. (A) Progressively mobile, these sperm move actively, either in a straight line or in a circle at any speed, i.e. preceding a and b. (B) non-progressive motility, this includes all other types of mobile sperm, i.e. shaking, twitching or moving in very small circles, and (c) immobile sperm, with no movement whatsoever; It should be remembered the sperm velocity Is temperature-dependent and can double as compared to room temperature when measured at body temperature⁹.

Concentration:

Because of its connection with fertilization rates and time to conception, determining an accurate concentration is important⁶. A count is deemed normal if the sperm concentration reaches 20M/ml (15M/ml. Because of the viscous nature of the sperm, use a positive displacement pipette is necessary so about eliminating dilution mistakes. A number of dilutions may be used, but if 1 in 20 is used then the sperm count in five large boxes of a Neubauer hemocytometer is the equivalent of the sperm count per ml. It is therefore important not to over count the sperm count; thus, only entire sperm (those with heads and tails) can be counted.

To stop measuring the sperm twice, only sperm can be counted with the bulk of its head in the square and even then, only if it is on one of the two boundary lines assigned to count. This will discourage overstatement. There are other ways of sperm counting, including the Makler chamber and the Howell Fertility Counting chamber, all of which have a grid of ten by ten counting and use undiluted semen. The benefit of this process, i.e. ease of use and speed, is counteracted by a large rate of error, as precaution must be taken not to overfill these chambers. The chambers do not allow the formation of a single layer of sperm when overfilled, and thus add a source of error. Although these methods can be very popular for a busy, non-specialized laboratory, they should not take the place of the approach suggested for accurate analysis by the WHO manual¹⁰.

Morphology of the Sperm:

In determining a semen sample sperm morphology is important; however, there is no definite distinction between fertility and natural sperm infertility. If there are highly defective morphological abnormalities so the ability to fertilize is reduced considerably.

A natural, human sperm has a smooth, regular oval shape, with a well-defined acrosome region that occupies around 40–70% of the head area. The head should be whole and does not include vacuoles. Standard sperm is about 4.1 microns long and has a median width of 2.8 microns³. The midpiece should be 0.6 microns in width, standard and should be about 4 microns in length. The tail, or main portion, should be lengthwise uniform and shorter than the midpiece.

It should be unbroken and have no kinks or coils, and should be around 10 times the head height, i.e. 45 microns long. There are numerous head, collar, midpiece and tail anomalies. Complete and succinct explanations can be found in WHO Handbook³. Analysis requires processes which are both stained and unstained. There are also sperm stain kits available for commercial use. Easy morphological testing on raw semen may be done. Although such procedure for diagnostic laboratories may not be effective, however, it could be appropriate in a clinical setting. If highly irregular sperm rises than ICSI (intracytoplasmic sperm injection) can be recommended. This is particularly important for globozoospermic samples where there is a lack of acrosome and traditional fertilization would not usually be feasible.

Other aspects:

Besides concentration, count and morphology, there are further studies that can be carried out to create a full semen content study. Antibodies of anti-sperm (ASABs) Mature sperm is produced after puberty, and is often recognized by the immune system as foreign proteins. When the sperm is within the testis, the Sertoli cells are covered by close junctions. An immune response can occur when there is a breach of this 'blood-testis' barrier. Vasectomy, testicular biopsy, and testicular damage are the most common causes for this, e.g. torsion and infection¹¹. Antibodies are then formed and secreted into the seminal and prostatic fluids. These antibodies may cause sperm to agglutinate, prevent progression forward and thereby decrease their ability to fertilize, or become cytotoxic and destroy sperm. Such antibodies inhibit contact with the sperm-cervical mucus or block zonal binding.

If there is an improvement in ASABs, so the use of ICSI to minimize the likelihood of missed fertilization that may be linked with impaired sperm motility or binding is of therapeutic advantage (12). The Mixed Agglutination Reaction (MAR) assay is one of the ways an antisperm antibodies can be tested. This procedure is achieved by combining a small semen aliquot with latex beads, which are filled with immunoglobins. If there are antibodies then the motile sperm form clumps with the beads; if not, the sperm will begin to float freely across the slide¹².

Most labs still use the Immunobead test, though, since this will establish the antibodies (IgA, IgG, and IgM) are present. In this procedure, the semen is mixed and incubated with latex beads filled with IgA or IgG. If there are antibodies so the beads bind themselves directly to the blood. This test offers more detail than other studies, because the beads will bind to the head, collar, midpiece or tail and thereby decide the exact position of the ASABs¹³.

Vitality of the Sperm:

Sperm potency is usually tested by measuring the plasma membrane's ability to resist such dyes or stains from being applied. In andrology laboratories, sperm vitality is not routinely analyzed. Such methods, however, may be useful in identifying rare cases of necrospermia, as opposed to total sperm motility, such as. Syndrome Kartagener⁴.

A further means of identifying sperm vitality is the Hypo-Osmotic Swelling Test (HOST). This test is based on live sperm being able to withstand moderate hypotonic conditions by measuring sperm plasma membrane's ability to transport water. Dead sperm, the membranes of which are no longer intact, show no swelling and therefore cannot function during the fertilization process. Live sperm will show controlled swelling, and will curl their tails. The tails should recover when the sperm is placed back in normal media, and they can be used for ICSI¹⁴.

Reactive oxygen species:

Generating Reactive Oxygen Species (ROS), or free radicals, is normally associated with leucocyte infiltration that occurs in semen. ROS was associated with capacitational roles and pathways for cell signaling when present at low levels¹⁵.

However, where such samples are prepared on gradients of density, ROS may also be created in abundance. Spermatozoa within these samples has the capacity to produce additional ROS. This is a symptom of incomplete sperm maturation or irregular morphology, which was associated with infertility of the male component. These sperm maintain disproportionate amounts of residual cytoplasm of spermatid in the midpiece and are often associated with decreased ability to fertilize¹⁶.

ROS are oxygen metabolites that contain hydrogen peroxide, nitric oxide, superoxide anion that hydroxyl radical. This is important for the andrology lab, as they can cause pathological cellular damage when ROS is present at high levels. There can be oxidizing damage to cellular lipids, proteins and DNA. While most cells have antioxidant defense mechanisms, sperm function can be compromised when this fail.

DNA fragmentation:

Human sperm chromatin is highly concentrated, but breaks between single and double helix can occur over time. This are usually restored by essential mechanisms; but this damage becomes non-repairable at some point and can be transferred to any subsequent embryos¹⁷. A high degree of heterogeneity of DNA inside the human spermatozoa can be a cause of infertility of the male component ignored by standard semen study.

Sperm fertilizing ability with increased DNA fragmentation is not necessarily affected; however, increased genetic defects to blastocyst stage can have major implications for embryo development. Owing to a linked rise in abortion rates, there is also a bad prognosis for a successful birth¹⁷.

Sperm preparation:

Seminal fluid includes a wide variety of substances other than sperm that need to be extracted to maximize the sperm count required for fertilization. In time, strength and motility are dwindling. This is because the seminal plasma has decapacitation causes. Therefore, it is important that samples be processed after processing as soon as possible in order to remove sperm from the seminal plasma and its harmful effects.

The presence of even a small amount of seminal plasma inside the preparation will prevent capacitation, so it is necessary to create a clean preparation to concentrate the appropriate fertilizing sperm¹⁸. During semen storage, sperm damage must be reduced by preventing significant temperature variations and excessive centrifugation. The centrifugal forces used as mobile sperm is removed from seminal

Preparation methods:

There are four main preparation methods:

1. Dilution and washing

2. Sperm migration

3. Density gradient

4. Commercially available products

Whatever process is used, prior to first use, all plastic ware, glass and media should be tested for sperm and embryo toxicity, with a sperm survival test. Generally speaking, the choice of preparation method can differ between laboratories but ultimately depends on the sample itself. Many materials are formulated with a gradient of two densities. However, it is better to prepare an extreme oligoasthenospermic sample on a gradient of 40 percent as this will condense the sample and typically the viable sperm will swim out in long drops.

A long drop is an elongated drop in the media under oil that puts the preparation in. The drops are allowed to settle and then Sperm is analyzed, if present, may then be picked up individually using the injection pipette if appropriate for ART.

Dilution and washing:

Fresh semen is filtered and differentiated by a wide amount of cultivated paper. The drawback of this approach is that the seminal plasma is easily wiped out by a simple process. The downside is that all sperm, including dead sperm, are pelleted, thereby reducing the fertilizing capacity by impairing the usable motility of the healthy sperm. It also does not minimize ROS and can only be used for ICSI⁴.

Sperm migration (swim up):

Here, it distinguishes functional sperm by movement. Small liquefied semen aliquots are put under a sheet of culture media and left for swimming out. Typically, this is achieved by deliberately using a semen-filled syringe and a needle. It is necessary to ensure that the syringe does not produce air bubbles, and that the semen is slowly removed.

If the allotted quantity is put under publicity, the needle is drawn rapidly upwards, preventing any semen drawing up. By layering culture media over the semen, it is possible to create the swim up; however, care must be taken not to dislodge the lower layer. It can use multiple tubes in Order maximizing the yield of samples. To increase semen/media interface the tubes should be left at an angle. The samples are left to move the mobile portion of the sperm into the media after 30-45 minutes, depending on the quality of the sample. Sample, the supernatant is collected and centrifuged for 5 minutes at 500g (0.5 rcf) for concentration of the sample.

In order to calculate the number of motile sperms present, a motility and concentration analysis should then be carried out. The entire process should be carried out at a temperature of 37°C (10). A swim up has many advantages, the biggest being that it creates a highly motile preparation. This method is useful for samples with an increased amount of cellular debris or round cells, as these do not obstruct the gradients. It is also an appropriate technique to use for samples that do not respond very well to the method of centrifugation and remains cost-effective as it does not require any other expensive gradients or chemicals than the culture media. The draw backs of this method however are that it is not suitable for every sample. Very viscous samples are poorly responsive to this technique, as the semen tends to pull up into the media and thus 'contaminate' the sperm/media solution. Prolonged exposure of sperm to the seminal plasma means that samples with significant levels of ASABs are not suitable to be used with this technique, for, ideally, the sperm should be removed as quickly as possible from the semen¹⁸.

Density gradient:

In the ART laboratory the most common method of preparing sperm is selective washing. This uses centrifugation by density gradient to fractionate sperm subpopulations. During centrifugation the sperm reaches a point in the gradient of density which corresponds to its own density. Motile sperm are isolated by high-speed centrifugation from immotile sperm and seminal plasma by numerous gradients of commercially obtained silane-coated silica particles, e.g. Pure sperm (Nidacon Universal AB, Sweden, Gothenburg).

During centrifugation typical sperm with more diluted DNA flows deeper down the tube. Round cells and rare cytoplasmic droplet shapes cannot make it into the pellet. However, if adequate precaution is not taken, over-centrifugation will cause accumulation of ROS. Also, caution should be taken to avoid overloading the gradient, as this can 'bar' the interface layer and keep usable sperm from passing^6,10.

There are many benefits of density gradients; they are ideal for all samples including viscous samples and are much easier to conduct than a swim up. Typically, there is an increase in viable sperm that can be collected and therefore a strong overall yield and the gradients usually prevent ROS production¹⁹. For example, silicon particles are expensive and not all samples react well to the centrifugation process, but there are drawbacks. In these cases, a gradient followed by swimming up may be done in order to increase the amount of motile sperm available for use.

In the following way the density gradient is equipped. Typically, the lower, or more compact, layer is positioned in the bottom of a conical sterile tube. This is normally 80% of silica particles in 1-3ml, but may be 90% or 95%. Then the tube is swirled to facilitate reduction of friction between the tube and the top meniscus.

The upper layer is deliberately applied by rubbing the solution gently over the interface layer and allowing it to slip down the tube walls providing a smooth interface. The heavier layer may be underlaid, but caution must be taken not to pop bubbles that would weaken the interface. These men is placed on top, the tube is closed, and centrifuged for 20 minutes at 300g (0.3 rcf). The upper layer and interface should be properly stripped after centrifugation, and then discarded.

The pellet can be collected with a soft brush, and then cleaned twice to eliminate any residual fragments of silica. We use 2/5 min washes at 500g. The sample is resuspended at an acceptable volume after final wash based on the procedure to be used. A concentration of 5-10M/ml and 1-2M/ml is appropriate for IVF, and for ICSI. 40% Centrifugation: this is a variant of the above-mentioned double density procedure which is used to extract seminal plasma from extreme oligoasthenoteratozoospermic (OATS) or blood/tissue from surgically extracted sperm. This procedure concentrates samples to facilitate sperm regeneration, which should be used for ICSI only.

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Received on 22.09.2020 Modified on 20.10.2020

Research J. Pharm. and Tech. 2021; 14(9):4941-4946.

DOI: 10.52711/0974-360X.2021.00859