Sperm Analysis

Sperm analysis is a fundamental laboratory test used to evaluate male reproductive capacity. The test objectively measures sperm parameters such as count, motility and morphological structure. It is applied as the standard diagnostic method in determining the causes of infertility.

Sperm concentration assessment examines in detail the sperm concentration within the ejaculate. This measurement is a quantitative indicator of production capacity and provides important information in distinguishing pathologies such as hormonal imbalance, infection or duct obstruction.

Sperm motility analysis identifies progressive and non-progressive movement patterns and directly reveals fertilization potential. Motility disorders indicate physiological insufficiencies associated with factors such as varicocele, oxidative stress or exposure to environmental toxins.

Sperm morphology evaluation identifies cellular structural abnormalities and calculates the proportion of normal forms. Morphological deviations constitute critical diagnostic data for assessing the reflections of genetic anomalies, testicular damage or systemic diseases on reproductive function.

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What Is Sperm Analysis?

Sperm analysis is a fundamental laboratory test performed to evaluate a man’s reproductive capacity. Parameters such as sperm count, motility, shape (morphology) and other characteristics in the semen sample are examined. It is one of the first-step tests especially in infertility evaluation. The results provide important information about male reproductive health and help plan further investigations or treatment when necessary.

What should be considered before giving a sperm sample?

The first step to obtaining a reliable sperm analysis result begins even before the sample is collected. Sperm cells are living cells that are extremely sensitive to external conditions. In particular, functional features such as motility and viability are very quickly affected by temperature changes, time and environmental factors. Therefore, strict rules must be followed to ensure the accuracy of the test.

The most important points in this preparation phase are:

• Sexual abstinence (no ejaculation) for 2 to 5 days.
• Emptying the bladder just before giving the sample.
• Washing hands and the genital area thoroughly with soap and rinsing well.
• Ensuring that no foreign substance (water, soap, saliva) comes into contact with the sample collection container.
• Strictly avoiding the use of lubricants (creams, oils, etc.).
• Using the special sterile container provided by the laboratory (non-toxic to sperm).
• Collecting the entire sample, especially the first portion of the ejaculate, into the container.

The period of sexual abstinence is very important for making results comparable. Abstinence of less than 2 days may cause the sperm count and volume to appear low. Abstinence longer than 5 days can result in sperm staying for a long time in the storage area (epididymis), and these “stored” sperm may show artificially reduced motility and viability.

The prohibition of lubricants is vital, as almost all products on the market are spermicidal and can cause motility to drop to zero, rendering the test completely invalid.

Ideally, the sample should be produced by masturbation in a quiet, dedicated, sterile room in the laboratory. If this is not possible and the sample is to be brought from home, two additional critical rules apply.

The sample must reach the laboratory no later than 30 to 60 minutes after ejaculation.

During transport, the sample must be kept at a temperature close to body temperature (between 20°C and 37°C).

Sperm cells are highly sensitive to “cold shock”. If the sample is exposed to cold air, the tail structure of the sperm can be irreversibly damaged, and this may lead to a falsely “immotile” (asthenozoospermia) result in an otherwise healthy individual. The safest way to transport the container is to keep it close to the body, such as in an inside pocket or under the arm.

It must be remembered that sperm analysis is a “snapshot” of that moment. Stress, lack of sleep, a recent infection or even a minor error during collection can affect the result. Therefore, when an abnormal result is obtained, the test is usually repeated after 2–3 months (one full sperm production cycle) before making a definitive diagnosis.

How is the sperm sample initially examined in the laboratory?

When the sample arrives at the laboratory, critical information such as identification details, abstinence period and time of delivery are recorded, and the sample is immediately placed in special 37°C incubators. The initial evaluation is a macroscopic examination performed with the naked eye before looking under the microscope.

The first aspect assessed is liquefaction. Semen, when it first emerges, has a coagulated, gel-like consistency due to proteins from the seminal vesicles. Thanks to enzymes secreted by the prostate (primarily PSA), it gradually dissolves at 37°C and becomes fluid within 15 to 30 minutes. This liquefaction is essential for sperm to move freely. In the laboratory, this process is expected to be complete within 60 minutes. If liquefaction does not occur or is significantly delayed, this may hinder sperm movement.

The second feature assessed is viscosity (consistency). This is distinct from liquefaction. The “flow” characteristics of the liquefied semen are examined. While a normal sample drips from a pipette as separate drops, a highly viscous sample forms a string longer than 2 cm. High viscosity is a condition that makes it difficult for sperm to progress through semen and often indicates a prostate infection.

Next, the volume is measured. This is crucial for calculating the total sperm count. The most accurate measurement is made by weighing (1 gram of semen is considered approximately equal to 1 mL). According to the 2021 WHO 6th Edition criteria, the lower limit for volume is 1.4 mL. Values below this (hypospermia) may indicate incomplete collection, short abstinence period, hormonal problems or obstruction of the ejaculatory ducts.

Finally, the pH value is assessed. Semen should be slightly alkaline (basic). Normal pH is accepted as geq 7.2. If the pH is very low (acidic) and this is accompanied by low volume and zero sperm (azoospermia), a congenital obstruction of the ejaculatory ducts or absence of the seminal vesicles (CBAVD) is suspected. High pH (>8.0) usually indicates infection.

Why is sperm motility so important?

After macroscopic evaluation is completed, a small drop of semen is placed on a special pre-warmed (37°C) glass slide and examined under a microscope. One of the first and most critical parameters assessed here is sperm motility. To reach the egg, sperm must travel a long and challenging path through the female reproductive tract. To complete this journey, it is not enough for sperm to be merely “alive”; they must also move “forward”.

The WHO 6th Edition guideline classifies motility into three main groups:

• Progressive Motility (PR): These are the “champion” sperm. They move actively and in a specific direction (in straight lines or large circles). This is the main group with potential to reach the egg.
• Non-Progressive Motility (NP): These sperm are alive and their tails move, but they do not make forward progress. They merely vibrate in place or move in very small circles.
• Immotility (IM): Sperm that show no tail movement.

Two important percentages are obtained from this evaluation:

• Total Motility (PR + NP): The sum of progressive and non-progressive motility. The lower reference limit is 42%.
• Progressive Motility (PR): The proportion of sperm that move forward. The lower reference limit is 30%.

A low proportion of progressively motile sperm is called asthenozoospermia (reduced sperm motility). This significantly restricts the sperm’s ability to reach the egg and lowers the success rates of treatments such as intrauterine insemination (IUI).

If the proportion of immotile (IM) sperm is very high (for example over 60%), an important question arises: are these immotile sperm dead, or are they alive but unable to move? This distinction is vital for planning IVF (ICSI). To clarify this, a sperm vitality test is performed. In this test, a special stain (Eosin-Nigrosin) is used. Viable sperm have an intact cell membrane and do not take up the dye (they appear white). Dead sperm have a damaged membrane and take up the dye, appearing pink/red. The lower reference limit for vitality is 54%.

If a patient has 80% immotile sperm, but the vitality test shows 70% viability, this indicates that the sperm are not dead but likely have a structural tail problem (e.g. Kartagener’s syndrome). These live but immotile sperm can develop into healthy embryos when injected directly into the egg using ICSI (intracytoplasmic sperm injection).

How is sperm count (concentration) measured?

When sperm analysis is mentioned, “sperm count” is usually the first thing that comes to mind. For this measurement, special counting chambers (haemocytometers) such as Makler or Neubauer slides, which contain millimetric squares, are used. The sample is diluted with a special solution to stop sperm movement and then loaded into these squares. An experienced technician counts the sperm heads within these squares one by one under the microscope.

Two core values are obtained from this counting:

• Concentration: The number of sperm in 1 milliliter (mL) of semen. The WHO 2021 lower reference limit is 16 million sperm (16 times 10^6/mL) per milliliter.
• Total Sperm Count: This is actually more important than concentration. It is calculated as (Concentration times Total Volume). The entire ejaculate is expected to contain at least 39 million sperm (39 times 10^6).

Values below these limits are referred to as oligozoospermia (low sperm count). If no sperm cells are seen in the sample under the microscope, this is called azoospermia (zero sperm) and requires much more advanced urological and genetic investigation.

Why is sperm morphology (shape) important?

Sperm morphology, that is, the examination of the physical structure of sperm (head, midpiece and tail), is the most complex, most expertise-requiring and sometimes most debated part of the analysis. This evaluation provides indirect information about the quality of the “manufacturing” process (spermatogenesis) in the testes, where sperm are produced.

The Kruger “Strict Criteria” are used in the assessment. This is essentially a “beauty contest” with very strict rules. For a sperm to be considered “normal”, all of its parts must be flawless. The head must be smooth and oval in shape, its size must fall within specific micrometric limits, the acrosome (the cap that allows penetration of the egg) must cover 40–70% of the head, the midpiece must be regular, and the tail must be single and unbroken.

Any slight deviation in any of these parts (slightly large head, slightly thick midpiece, double tail, cytoplasmic droplet, etc.) classifies that sperm as “abnormal”.

According to these strict criteria, the lower reference limit for the percentage of normal-shaped sperm is only 4%. This low rate often worries patients. However, it simply means that even in the most fertile and healthy men, the vast majority of sperm (96%) are morphologically “imperfect”. A morphology value below 4% (teratozoospermia) does not by itself constitute a diagnosis of “infertility”. However, it is an important indicator that the ability of sperm to naturally penetrate and fertilize the egg may be reduced. Very severe morphology defects (such as globozoospermia) with values below 1% can lead to fertilization failure in conventional IVF and may necessitate direct ICSI.

Which advanced tests are performed if the basic sperm test is normal but pregnancy does not occur?

Sometimes couples present with complaints of not being able to achieve pregnancy even though all basic sperm test parameters (count, motility, morphology) are within normal ranges. This situation is referred to as “unexplained infertility”. At this point, more advanced tests that examine the “internal structure” and “function” of sperm become relevant.

• Sperm DNA Damage (Fragmentation)

Sperm is a “cargo package” carrying the father’s genetic material (DNA). From the outside, this package may look normal (good morphology) and move quickly (good motility). However, if the “genetic cargo” (DNA) inside the package is damaged, meaning there are breaks in the chain (fragmentation), healthy embryo development may not be possible even if fertilization occurs.

High Sperm DNA Fragmentation (SDF) is strongly associated with low fertilization rates, slow or arrested embryo development, failure to achieve pregnancy and, most importantly, recurrent miscarriages. This damage may stem from problems in sperm production in the testes, smoking, environmental toxins, infections or prolonged storage of sperm in the reproductive tract.

Special tests such as SCD (halo test) or TUNEL are used to measure this damage. These tests provide a “DNA Fragmentation Index” (DFI).

• DFI < 15% (Normal)
• DFI 15–30% (Borderline or moderate damage)
• DFI > 30% (High damage)

When a high DFI is detected, conventional IVF or intrauterine insemination is avoided and direct ICSI is preferred. If the damage is very high, it may even be necessary to obtain sperm directly from the testes using TESE, where DNA damage is generally lower than in ejaculated sperm.

• HBA (Hyaluronan Binding Assay) Test

This is a very important functional test that shows whether a sperm is “mature” or not. In natural fertilization, the egg cell is surrounded by an outer layer rich in a substance called hyaluronan. Only physiologically fully matured sperm can bind to this “lock” (hyaluronan) using special “keys” (receptors) on their heads.

In the HBA test, the semen sample is placed in a special laboratory chamber coated with hyaluronan. Mature and healthy sperm bind to this surface, while immature sperm continue to swim freely. The proportion of bound sperm is counted to obtain an “HBA score”.

If the HBA score is low (usually <65%), this indicates a maturation defect in sperm. In standard ICSI, the embryologist selects the “best-looking” sperm under the microscope, but that sperm may still be immature. When the HBA score is low, a special technique called Physiological ICSI (PICSI) is used. In PICSI, instead of randomly selecting a sperm, the embryologist waits for sperm to bind to the hyaluronan gel. The sperm that prove their ability by binding to this gel are selected and injected into the egg. This method mimics natural selection in the laboratory and may improve embryo quality.

How are sperm prepared for IVF or intrauterine insemination?

The semen sample (ejaculate) obtained from the man can never be used as it is for insemination or IVF. Semen is a mixture of seminal plasma (the fluid component), dead cells, leukocytes, debris and motile sperm. Seminal plasma can cause cramps if introduced into the uterine cavity and contains factors that may impair sperm’s fertilizing capacity:

Therefore, the aim of “sperm preparation” (or sperm washing) is to isolate the most motile, healthiest and most functional sperm population from this mixture and concentrate it into a small volume. There are two main methods most commonly used for this purpose:

• Density Gradient Centrifugation (DGC): This technique is a type of “filtering” method that separates cells according to their density. Mature, healthy and morphologically normal sperm are the most dense cells in the semen. Immature sperm, leukocytes and other debris are less dense. A “track” composed of special solutions is layered in a tube. Using centrifugation (spinning at high speed), only the densest and strongest sperm manage to pass through these layers and settle at the bottom of the tube. All unwanted cells remain in the upper layers. This method is ideal for patients with low sperm count (oligozoospermia), low motility (asthenozoospermia) and abnormal morphology (OAT syndrome), in whom it is necessary to “filter and concentrate” a small number of good sperm out of a large background population.
• Swim-Up Technique: This method is a positive selection technique based on the ability of sperm to “swim the fastest”. The semen sample or washed sperm pellet is placed at the bottom of a tube and a special culture medium is layered on top. The tube is kept at 37°C for 30–60 minutes. During this time, the strongest and fastest sperm “swim upwards” from their original environment into the clean medium above. At the end of the incubation period, only this upper fraction, which contains the most motile sperm, is collected. It is generally considered a more “gentle” method and is thought to select sperm with less DNA damage. However, sperm recovery is lower compared to DGC. Therefore, it is mainly preferred in patients whose initial sperm count and motility are already good (normozoospermia).

Frequently Asked Questions