What is semen analysis and how is it interpreted?

Semen analysis is the most important examination in the evaluation of male fertility and provides essential information about the health of the male reproductive system, reflecting the functionality of the testicles, the patency of the system and its secretory activity.

The parameters analyzed in semen analysis have clinical value for the possibility of natural conception, aid in identifying reversible medical conditions that may affect fertility, and are essential for the proper management and success of assisted reproduction and IVF.

The evaluation of sperm parameters is currently based on the standards set by the World Health Organization (WHO) and according to the most recent laboratory manual (6th Edition, 2021).

According to WHO the semen analysis is a fundamental step for:

1. Diagnosis of fertility and infertility
2. Evaluation of reproductive health and function
3. Guidance in the selection of assisted reproduction procedure
4. Monitoring response to treatment
5. Measuring the effectiveness of male contraception

Sample collection process

The guidelines recommend abstinence from sexual intercourse for 2-7 days prior to sample collection. Scientific evidence shows that a shorter period of abstinence is associated with improved sperm motility, morphology and DNA integrity, while longer abstinence is associated with a larger sample volume and higher sperm count.

The semen is collected in a sterile container and kept at a temperature of 37°C. If the sample is collected at home, it must be transported to the laboratory according to the instructions and within one (1) hour avoiding temperature fluctuations as these can affect sperm quality. Potential loss of sample during collection should be reported to ensure a representative result for the patient. Semen evaluation begins immediately after liquefaction of the sample that occurs within 15-60 minutes of ejaculation.

The properties of seminal fluid

Semen is a heterogeneous fluid that is released at the time of ejaculation. It consists of cellular and non-cellular parts. The cellular fraction includes mature and immature spermatozoa, epithelial cells, leukocytes and in some cases erythrocytes, while the non-cellular fraction is the seminal fluid.

Seminal fluid consists of 50-65% of seminal vesicle secretions and 20%-30% of prostate secretions. The testes and epididymis yield about 5% of the total volume (~0.15 ml), while the bulbourethral glands contribute <5% of the total ejaculate, producing a clear, protein-rich secretion.

The epididymal fraction is rich in neutral α-glucosidase which is also the main indicator of epididymal function and L-carnitine. The seminal vesicle secretion contains fructose which contributes to sperm energy, metabolism and motility, while prostaglandins enhance sperm motility and inhibit the female immune response against sperm. Prostatic secretions are rich in citric acid which binds Ca2+ and regulates ejaculate liquefaction and coagulation in synergy with proteolytic enzymes.

Physical and chemical characteristics of the semen sample

The liquefaction timing, appearance and colour, volume, viscosity and pH are initially evaluated in the sample.

• Time of liquefaction reflects prostate gland function, and prolonged time (>60 minutes) may indicate possible prostate dysfunction.
• The appearance and color of the sample provides information regarding the function of the reproductive system and variations from the normal shade and texture may indicate the presence of other pathologies and inflammations or be affected by medication intake.
• Semen volume reflects the overall functionality of the reproductive system including the accessory glands. A larger volume (>6ml) can be observed in normal cases, but also in cases of infection or inflammation of the reproductive system, as well as in prolonged abstinence from ejaculation. Reduced volume (<1.4ml) may be associated with inflammations and infections of the reproductive system, agenesis or obstruction of the reproductive ducts, retrograde ejaculation and incomplete ejaculation due to stress.
• Viscosity reflects the flow resistance of the seminal fluid and when increased it can considerably affect sperm motility. High viscosity may indicate dysfunction of the prostate gland and seminal vesicles during ejaculation or may even be a result of inflammation or infection of the accessory glands by pathogenic microorganisms.
• Semen pH is an indicator of balance between the acidic secretion of the prostate and the alkaline secretion of the seminal vesicles. A more acidic pH (pH < 7) is notable in cases of congenital agenesis of vas deferens or obstruction of the ejaculatory ducts, while inflammation of the accessory glands and especially the prostate and seminal vesicles may produce a more alkaline pH.

Microscopic examination of the specimen

Microscopic assessment in semen analysis involves the evaluation of the concentration and total number of spermatozoa in the sample, sperm motility and presence of typical morphological forms.

• The concentration in the sample is the number of sperm per unit volume (ml) of semen. This value is calculated by loading a sample from the ejaculate into a special cytometric chamber with a fixed mesh with gridlines and counting at least 200 spermatozoa under phase contrast microscopy. Concentration is mathematically calculated following repeated measurements to confirm the result.
• The total sperm count expresses the total number of sperm in the ejaculate and it is a measure of testicular productivity since it is proportional to the volume of the total ejaculate. This number is mathematically through sperm concentration and semen volume. Total sperm count and progressive motility are useful parameters in fertility assessment as they indicate the total number of potentially functional sperm available.

A reduction in sperm production (oligozoospermia) beyond reference values or complete absence of spermatozoa from the ejaculation (azoospermia) is observed in various pathological conditions which can be the result of genetic factors, disorders of the physiology and function of the reproductive system, endocrine disorders but also acquired factors such as infections and environmental influences, as also some medical treatments may affect the observed sperm count.

• Sperm motility is evaluated immediately after liquefaction of the sample and specifically spermatozoa movement is differentiated between rapid, slow progressive and non-progressive movement and immotile. To assess motility, an aliquot of the sample is loaded into the measurement chamber and examined under a phase contrast microscope by evaluating at least 200 spermatozoa in at least 5 different fields.

Decreased motility (asthenozoospermia) may result from high viscosity, infection or inflammation of the testes and accessory glands, from pathological or anatomical variations (eg, varicocele, cryptorchidism, testicular cancer) or may be due to lifestyle and environmental factors including exposure to pollutants, alcohol and drug use, smoking and psychological stress.

• Sperm morphology is one of the most demanding qualitative parameters of semen analysis in both terms of evaluation and interpretation, as there is considerable variation in the individual morphological elements of the spermatozoa contained in the semen that requires thorough training and methodological consistency to differentiate. The evaluation of morphologically normal spermatozoa is carried out by differential staining of the various parts of their structure (head and acrosome, mid-piece, tail and cytoplasmic residues) and assessment of morphologically normal spermatozoa is based on strict morphological criteria proposed by the WHO. During morphological evaluation, the abnormalities are simultaneously categorized, as specific abnormalities are an indication of specific dysfunctions during spermatogenesis, while they provide important information for the correct management of male infertility. Abnormal morphology is attributed to reproductive dysfunctions related to the production or maturation of sperm, while specific morphological defects such as e.g. globozoospermia and multiple tail abnormalities are indicative of genetic defects.

Additional parameters in semen analysis

Semen analysis may also include other important factors for evaluating male fertility, such as sperm vitality, formation of aggregations or agglutinations, the presence of round cells, such as white and red blood cells, epithelial cells, microorganisms and gelatinous forms.

• Sperm viability assessment helps distinguish between immotile but alive spermatozoa and immotile apoptotic or dead spermatozoa and is recommended for all specimens with a progressive motility lower than 40%. Sperm vitality is determined by assessing sperm with cell membrane integrity as a property of functionality.
• The adhesion of motile spermatozoa to each other forming clumps is termed agglutination. When other cellular or non-cellular elements are involved in the formed clumps, then this is characterized as an aggregation and may contain both motile and immotile spermatozoa. Sperm agglutination is an indication of an immunological reaction in the environment of spermatogenesis and may affect motility in the sample. Agglutinates are assessed according to the number of spermatozoa involved and the point of agglutination (head, mid-piece, tail or mixed). The presence of agglutinations may be indicative of the presence of antisperm antibodies and further testing is required to confirm this as an immunological cause of infertility. The presence of aggregates is a non-specific observation but it is reported in semen analysis, since in extensive forms it may affect the quality of the sample.
• Apart from spermatozoa, other cells are commonly present in semen and these may include white and red blood cells, immature spermatozoa, and epithelial cells. In order to distinguish white blood cells from immature germ cells, a special staining technique is applied to a sperm sample to accurately identify and quantify leukocyte population. When the concentration of white blood cells exceeds a concentration of one million cells/ml, then it is an indication of an inflammatory response of the genital system that may be linked to an infection. The activation of leukocytes in the male reproductive system drives the production of reactive oxygen species (ROS) up to 1,000 times more than normal, thus contributing to the generation of oxidative stress that affects the genetic integrity of spermatozoa.

Sperm chart reference limits

WHO in its recent update of their laboratory manual for the examination and processing of human semen (2021) includes reference limits which derive from scientific studies and involve a total of 3,587 men who had a successful natural conception and pregnancy in a maximum time of 12 months of trying. The meta-analysis of the data included various geographic regions around the globe that  represented most male populations, and the distributions of values ​​were determined according to the standard methods used in medicine to define reference ranges.

Semen analysis has a significant clinical importance and offers many indications of fertility and reproductive function, but it does not constitute a distinct boundary between fertile and infertile men. The interpretation of the test results is carried out by the medical specialist who, in combination with the medical history and clinical examination, determines whether further investigations and/or individualized treatment and/or medical management through assisted reproduction is needed. Reproduction as a multifactorial biological process surrounds the fertility of the couple and therefore a single test is not predictive of fertility.

Possible factors of price differentiation

A number of factors can cause male infertility, including genetic factors (eg, Kallmann syndrome, Klinefelter syndrome, Y chromosome microdeletions), endocrine disruptions (eg, hypothalamic-pituitary disorders and hypogonadism), disorders of the physiology and function of the reproductive system (e.g. cryptorchidism, varicocele, obstruction or agenesis of the ejaculatory ducts of the sperm, inflammations and infections of the reproductive system), systemic diseases, malignancies, but also medical treatments related to genotoxicity or/and inhibition of spermatogenesis.

Important and common factors that may individually affect sperm parameters and male fertility are lifestyle related, such as smoking, alcohol and substance abuse, poor diet, obesity, scrotal heat induction and psychological stress. Additionally, recent scientific evidence suggests that oxidative stress plays an important role in male infertility and has been recognized as a causative factor in 30%-80% of infertile men. Infertility affects about one in eight couples of reproductive age, with the male factor being solely responsible in 20% and a contributing factor in a further 30% of cases. Nevertheless, the modern methods of investigating and diagnosing male infertility, and the evolution of available medical treatments have significantly helped in the effective treatment of most manifestations of male infertility.

References:

1. Baskaran S, Finelli R, Agarwal A, Henkel R. Diagnostic value of routine semen analysis in clinical andrology. Andrologia2021 Mar;53(2):e13614.
2. Boitrelle F, Shah R, Saleh R, Henkel R, Kandil H, Chung E, Vogiatzi P, Zini A, Arafa M, Agarwal A. The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis. Life (Basel) 2021;11(12):1368.
3. WHO laboratory manual for the examination and processing of human semen, 6^th Edition 2021 https://www.who.int/publications/i/item/9789240030787