Sperm Function Tests

The comprehensive assessment of the male infertility factor requires the performance of a thorough laboratory examination of the male sperm. Apart from the conventional semen parameters, included in the basic semen analysis (spermiogram), modern scientific research provides a series of laboratory tests, which focus on the assessment of the functionality of spermatozoa and their potential insemination capability.

Such selected laboratory tests enable a more thorough assessment of the functional condition of the male sperm and contribute to:

  • A more comprehensive investigation of the possible causes of male infertility – particularly in cases where the basic semen analysis does not indicate any disorder
  • The selection of the most suitable therapeutic strategies depending on the diagnosed disorder
  • The evaluation of effectiveness of therapeutic regimes
  • The provision of prognostic information regarding the success rates of the reproductive attempt (natural or assisted)

The sperm function tests, regardless of the conventional spermatological profile, are an additional diagnostic tool enabling clinical doctors to provide better guidance to sub-fertile couples.

There is no single laboratory test that may define, with full certainty, a sperm as fertile or infertile. It is the combination of laboratory analyzes that indicates the fertility potential of the examined sperm.

In the effort to investigate the causes of a couple’s failure to have children, each case is unique and must be approached with detailed analysis by the attending doctor, who is exclusively competent for evaluating both laboratory and clinical data, and determining the suitable therapeutic strategy.

The scientifically reliable performance of properly selected tests based on the specifications of a strict quality control system, meets the diagnostic requirements of laboratory examination of infertile men.

The G. Lyberopoulos Spermatology Laboratory applies the principles of quality control provided by the NATIONAL ACCREDITATION SYSTEM (15189:2012) and reputed international entities, such as the World Health Organisation and the European Society for Human Reproduction and Embryology (ESHRE). It participates in an External Quality Control System under the auspices of the Andrological Laboratory of the University of Karolinska, Sweden, and the Andrological Laboratory of Kruger and Franken, in Tygerberg, South Africa.

As part of the diagnostic services provided, the Laboratory performs the following sperm function tests, which provide information on the spermatozoa potential fertilization ability.

Sperm Function Tests

Sperm Longevity Test::

Longevity describes the maintenance of spermatozoa motility over time. Sexual intercourse and ovulation rarely coincide, thus high sperm longevity ensures the survival of spermatozoa for a sufficient period of time in the female reproductive system, increasing the possibility to achieve fertilization. Therefore, in vitro analysis of sperm longevity may provide information on the potential fertilization ability of the sperm.

Sperm “resilience” testing::

The assessment of the spermatozoa endurance under intense temperature stress, contributes to the study of the sperm survival ability through time and has been correlated with the outcome prediction of the classical in vitro fertilization (IVF) attempt. Relevant studies have shown that non resilient spermatozoa are only likely to fertilize through the ICSI method.

The integrity and normal function of spermatozoa membrane provides a useful functional assessment of sperm quality and is performed through the Hypo-osmotic Swelling Test – HOS test.

The value of the Hypo-osmotic Swelling Index is high in the sperm of fertile men or men with normal sperm quality, whereas the same value is low in sperm samples manifesting a disorder in terms of number or mobility. It has been positively correlated with parameters such as progressive motility and vitality, and is an indirect indication of oxidative stress.

Regarding the outcome of the reproductive effort, high values of the index constitute positive prognostic information. On the contrary, in cases of low levels of the index, the probability of in vivo conception decreases significantly. Even when performing classical IVF, although fertility rates do not seem to be affected, the pregnancy rate remains low. In such cases, assisted attempt is more effective when using the ICSI method.

One of the important functional properties of sperm, is cervical mucus penetration, which directly depends on the spermatozoa motility and/or morphology, as well as certain kinematic parameters. The results of penetration tests provide:

  • A direct indication of the sperm ability to penetrate the cervical cavity, the first step towards fertilization, and consequently an assessment of the probability to achieve normal conception/li>
  • An indirect indication of the sperm’s fertility potential, based on the significant correlation of cervical mucus penetration with the egg fertilization

Cervical mucus penetration failure a cause of subfertility that may result from:

  1. disorders related to spermatozoa motility and/or morphology
  2. the presence of an immunological agent, i.e. anti-sperm antibodies
  3. the poor quality of cervical mucus

The relevant laboratory tests performed at the Spermatology Laboratory include:

  • Quality assessment of cervical mucus, according to WHO guidelines
  • In vivo assessment of the cervical mucus penetration by spermatozoa after intercourse (Sims-Huhner test)
  • In vitro assessment of the cervical mucus penetration by spermatozoa
    • Microscope slide method (Kurzrok-Miller test)
    • Sperm – cervical mucus contact test
  • Capillary tube method (Kremer test)

The ad hoc suitable laboratory tests regarding the interaction of sperm and cervical mucus are indicated in cooperation with the attending doctor.

The acrosome reaction performed by spermatozoa when approaching the egg is an essential requirement for fertilization, due to the released enzymes that facilitate the egg penetration by the spermatozoon. This process is the final stage of spermatozoa maturity, known as capacitation, and is induced by the increased concentration of intracellular calcium.
The artificial induction of the process followed by microscopic observation with immunofluorescence, defines the percentage of live spermatozoa, in which acrosome loss has occurred. Low spermatozoa response to this laboratory process has been associated with low rates of in vivo and in vitro fertilization.

The assessment focuses on investigating the causes of fertility failure or long-term infertility, regardless of the findings of a conventional semen analysis.

The presence of anti-sperm antibodies is related to infertility in 9-13% of couples unable to conceive. Anti-sperm antibodies may be present in either one or both partners. A complete immunological examination of infertile couples includes screening for the detection of the two most important anti-sperm immunoglobulins, IgG and IgA, in spermatozoa or sperm fluid and blood serum of men, as well as in cervical mucus or blood serum of women.

The degree the anti-sperm antibodies affect the fertilization process depends on:

• The antibodies class
• The binding site of the antibodies on the surface of the sperm cells (type of agglutination)
• The antibodies titer
• The existence of spermatoxic antibodies

The tests applied in the Spermatology Laboratory include:
• IgG – MAR test
• Direct immunobead test for detecting anti-sperm antibodies on spermatozoa
• Indirect immunobead test for detecting anti-sperm antibodies in semen
• Detection of anti-sperm antibodies in cervical mucus
• Titration of anti-sperm antibodies that cause agglutinations (Friberg test)
• Detection of spermatoxic antibodies

The impact of the male DNA damage on the reproductive effort has been correlated with the difficulty of fertilization and/or the creation of poor quality embryos. The combined assessment:

A. of the chromatin integrity and
B. of the fragmentation of sperm DNA
is a necessary tool in the investigation of long-term unexplained infertility.

A. Chromatin Integrity Test
Chromatin is the structural complex of DNA and proteins that constitute the genetic material of spermatozoon in the form of chromosomes. The presence of granular (immature) or hyperoxidised (hyperstabilised) chromatin indicates a disorder during the packaging of the genetic material in the nucleus of spermatozoa, which has been associated with negative effects on the fertilization potential.

B. DNA Fragmentation Test:

The DNA molecule is in the form of a coiled double helix that is structurally stabilized by lateral bonds. If the bonds brake, the helix becomes unstable. The structural distortion in the sperm DNA, known as ‘Fragmentation’, is a measurable feature of its functionality that is not reflected in any of the conventional spermatological parameters (motility, concentration, morphology etc.). Men with spermatological parameters within the reference values, may still have a high DNA Fragmentation Index.

According to extensive scientific research, the integrity of the sperm DNA molecule has been associated with the quality and/or viability of embryos, mainly during the early stages of division (4- to 8-cell and more). DNA Fragmentation Index of high value is correlated to the negative outcome of reproductive effort, regardless of the applied fertilization method (normal, IUI, IVF, ICSI), through:

  • The reduction of sperm fertility
  • The fetal growth inhibition
  • Their implantation failure (miscarriage).

The main factors responsible for the high levels of sperm DNA fragmentation are:

  • Exposure to chemicals, toxic substances, radiation or high temperature
  • Air pollution
  • The use of medicines
  • Varicocele, testicular cancer, inflammation or high fever
  • Increased testicular temperature
  • Smoking
  • Poor nutrition
  • Advanced age of man
  • Any process that causes oxidative stress.

The assessment of the sperm DNA Fragmentation Index is mainly recommended in cases of:

  • Long-term unexplained infertility
  • Low fertilization rates or poor quality embryos in IVF cycles
  • Failure of implantation following IVF
  • Recurrent miscarriage
  • Long-term exposure to agents that are toxic to fertility
  • Oligoasthenoteratozoospermia
  • Advanced age of man

In addition, it provides a complete evaluation of the male fertilization potential during infertility investigation. The test can be particularly useful:

  • while monitoring the effectiveness of anti-oxidant schemes in sperm enhancement
  • post-operational rehabilitation of varicocele
  • while assessing sperm quality of men who have received strong cytotoxic treatment.

Sperm Oxidative Stress Test
The redox system regulates the balance of the effect of oxidizing and reducing agents based on the body’s normal needs. In men, the oxidizing agents (e.g. ROS) play an important role in the spermatogenesis, in sperm motility and spermatozoa “activation”, as well as in other functions. Oxidative stress is caused when the production of oxidants exceeds the normal needs of the body and the quantity of antioxidants is not sufficient to maintain the balance. Oxidative stress is related to reduced sperm count, low spermatozoa motility and can be used as a male sperm quality index. Τhe evaluation of static oxidation-reduction potential (sORP) indicates the necessity of providing antioxidant treatment to patients if needed.

Inflammation in the male genital area is one of the causes of infertility, without always having clinical symptoms, mainly when the concentration of leucocytes in the sperm is less than 1×106/ml. In such cases, the assessment of polymorphonuclear cells elastase levels may be a strong indication of asymptomatic inflammation of the male genital system.
Increased elastase concentration causes:

  • reduced sperm vitality
  • increased cell necrosis
  • negative effect on the reproductive effort, related to low blastocysts growth rates, increased fetal growth inhibition rates and high rate of poor quality embryos, that are unsuitable for embryo transfer or cryopreservation.

It is a sensitive and accurate diagnostic tool for evaluating the possibility of sperm quality improvement following antibiotic treatment. Even in the case of a negative sperm culture test post treatment, high elastase levels indicate low possibility of semen parameters improvement.

When combined with other diagnostic tests, such as conventional semen analysis, microbiological examination and sperm oxidative potential tests, it provides detailed information on assessment and therapeutic management of subclinical infection/inflammation of the male genital system.

The success of an assisted reproductive attempt strongly depends on the selection of the best spermatozoa in terms of quality. This step plays an important role, mainly when applying the ICSI method, where individual spermatozoa are selected and injected directly to the eggs.

According to a variety of modern studies, mature and functional spermatozoa that are capable of being attached to the zona pellucida of the egg, also have the ability to bind to special hyaluronic acid polymers. Therefore, the sperm-hyaluronan binding test provides clinical doctors with the necessary information for the selection of the most suitable assisted reproduction method (IUI, IVF, ICSI) in view of optimizing the success rate.
It is a laboratory test that identifies the percentage of the best quality spermatozoa in the semen sample, which are then isolated and used in assisted reproduction methods, with:

  • significantly lower frequency of disomies and diploidies
  • low DNA fragmentation levels
  • fewer cytoplasmatic residues
  • fewer histones and apoptotic markers
  • high levels of cellular maturation markers
  • better motility and morphology as per Tygerberg.