Andrology, Andrological laboratory test

The higher the fragmentation of sperm DNA, the lower their fertility. Furthermore, a high degree of sperm DNA fragmentation is also associated with the risk that the foetus that develops from the egg they fertilise will die in the first trimester of pregnancy.

The main cause of the damage to the sperm DNA is the increased presence of free radicals in the semen. Free radicals can be caused by white blood cells present in the semen when the urinary organs become inflamed, by environmental factors, smoking, certain anticancer drugs and/or radiation.

Highly fragmented DNA in the sperm population can cause recurrent miscarriages. This observation applies to both natural and assisted reproductive pregnancies. Therefore, the characterisation of DNA fragmentation is very important either in the case of non-fertilisation or in the case of serial miscarriages. The fragmentation of the DNA stock should be monitored regularly during the treatment of the patient.

Treatment should focus primarily on eliminating the suspected cause. For example, reducing inflammation, avoiding smoking, or avoiding the use of radiotherapy. If the treatments do not help and the DNA stock is still highly fragmented, a gradient centrifugal purification of the semen sample after freeze-thawing may be attempted. In such cases, this method may be successful and good quality spermatozoa suitable for assisted reproduction may be obtained.

During the examination, we first evaluate the macroscopic data of the ejaculate (semen volume, pH, viscosity, colour, drainage time), and then determine the sperm count, motility (movement) and morphology (shape) by microscopic examination.

This basic test is essential for assessing fertility, but is often not enough even if the results are normal, as in addition to these physical characteristics, the biological and biochemical properties of sperm play a significant role in fertilisation.

The egg (oocyte) is surrounded by cells embedded in loose connective tissue, called the “cumulus”. For the sperm to reach the egg, it must first attach to the jelly-like connective tissue layer and then pass through it. The sperm cannot do this mechanically, just by moving around. The binding of spermatozoa to the cumulus cell layer involves proteins attached to the cell membrane at the front of the sperm head. These proteins are known as hyaluronan binding proteins (HBPs). HBPs anchor the spermatozoa to the outer surface of the cumulus cell layer. After binding, the sperm release enzymes that locally dissolve the jelly layer surrounding the oocyte, opening the way for sperm to enter the oocyte. If the sperm, due to developmental problems, is unable to attach to the tissue surrounding the egg, it will not be able to reach the egg and consequently cannot fertilise it. There is a loss of fertility.

In a male fertilisation test, it is very important to check that the patient’s sperm can bind to the hyaluronan-rich jelly and that the lack of this jelly is not responsible for the failure to conceive.

To measure the ability of sperm to bind as described above, a test called the Hyaluronan Binding Assay (HBA) is used. A defined amount of the patient’s semen sample is dropped onto a hyaluronan-coated glass slide and the number of hyaluronan-bound spermatozoa is counted within a certain time. More than 80% of the spermatozoa are expected to bind to the coated surface. If less than this percentage of sperm are bound, it is suspected that this defect is partly or wholly responsible for the failure to conceive. If the suspicion is confirmed and other reasons do not rule it out, the couple still has hope of having a baby. To do this, however, the sperm must be injected directly into the egg and the embryo (blastocyst), which is at an early stage of development, will be implanted in the uterus. This in vitro fertilisation procedure is called ICSI (Intra Cytoplasmic Sperm Injection).

About 8% of men produce antibodies against their own sperm. The antibodies produced are called “antisperm antibodies”. The immune system of patients with this autoimmune disease recognises their own sperm as foreign and produces immunoglobulin molecules against them that lead to sperm destruction (cytotoxic). Clinically/pathologically relevant antisperm immunoglobulins belong to the IgA and IgG classes. Patients who carry either IgA or IgA and IgG antibodies together are usually unable to naturally inseminate their partners. Since antibodies against sperm are produced by the accessory gonads, they are found in seminal fluid and on the surface of spermatozoa. They are absent in blood plasma and can therefore only be detected by analysis of a semen sample.

The MAR (Mixed Antiglobulin Reaction) test is the most commonly used test for the detection of antibodies against sperm bound to spermatozoa. Suspensions of sperm are mixed with micro-sized rubber (latex) beads to which IgG and/or IgA antibodies are attached. If the spermatozoa contain anti-sperm immunoglobulins, the beads will bind to the spermatozoa and form smaller to larger sperm/bead agglomerates.

If 10-39% of spermatozoa are attached to the beads, it is suspected that the lack of fertility is due to the presence of anti-sperm antibodies. If more than 40% of the sperm are attached to the beads, the lack of fertility is very likely to be due to this cause.

Note: The test can only be performed if the sample contains motile sperm. Samples with very low sperm concentration or low motility may give a “false” negative result!

During metabolic processes, enzymatic reactions in the body produce reactive molecules with short lifetimes and charges. These transient molecules are necessary for the normal (physiological) functioning of the body, but their excessive production can lead to pathological conditions (disease). The part of reactive oxygen species (ROS) that is produced from oxygen is called reactive oxygen species (ROS). The amount of ROS in the body can also increase in response to environmental factors (smoke, radiation, etc.). ROS are also involved in the normal development of spermatozoa (spermatogenesis). However, excessive ROS production damages the hereditary material of spermatozoa. This damage can be manifested, for example, by fragmentation of the sperm DNA. Therefore, when investigating the cause of an infertility problem, it is very important to determine the ROS content and percentage of spermatozoa.

A ROS test based on quantitative flow cytometry determines the total free radicals in spermatozoa. In contrast, the commonly used rapid laboratory tests detect ROS in semen plasma in a semi-quantitative manner (low, medium, high). ROS are expressed in the spermatozoa. Thus, it is more important to know what percentage of spermatozoa are ROS positive and the level of ROS content than to know whether the semen plasma contains ROS.

In order for a sperm to transfer the hereditary material stored in the head part of the sperm to the egg, the acrosome part undergoes a transformation. This transformation is called the acrosomal reaction. The acrosome reaction must occur at the right place and time. The right place is the immediate environment of the egg cell. The right time: when the sperm passes through the layers surrounding the egg cell. It also follows that if, for some reason, the acrosomal reaction takes place elsewhere, earlier or not at all, fertilisation does not occur.

In the test, sperm are isolated from a fresh semen sample and then artificially created to create the conditions that the sperm will encounter as they migrate through the female reproductive tract. This is the so-called capacitation phase, which precedes the acrosome reaction. During capacitation, the sperm prepare for the acrosome reaction. After the capacitation phase, the samples are split in two. One half of the samples is not treated with any substance (unstimulated), the other half is treated (stimulated) with a substance that artificially induces the acrosome reaction in the sperm.

If the proportion of spermatozoa with a premature acrosome reaction is high, or if the induction results in a low number of sperm with an acrosome reaction, then insemination and in vitro fertilisation (IVF) will not be successful as assisted reproductive techniques. Unfortunately, to date, no treatment has been described that can be used to specifically restore the susceptibility to an insufficient acrosomal response in the male reproductive tract. When the cause of infertility is proven to be an inadequate acrosomal response, in vitro assisted reproduction methods should be used instead of treatment. Of these, the methods involving intracytoplasmic injection are also the most successful (ICSI and PICSI).