Basic techniques for the diagnosis of male infertility

Basic techniques for the diagnosis of male infertility

Infertility is considered to be an inability to achieve pregnancy in the period of 1 year of regular intercourse (3-4 times a week) of non-contracepting partners. The assessment of infertility should be started simultaneously in both the male and female partner. Unfortunately, most often it is the woman alone who visits the doctor as the first, and some men do not feel the need to consult specialists at all [2, 11]. Polish studies (Sanocka, Kurpisz, 2003) prove that in 40-60% of infertile couples the cause is the male factor [2].

A distinction is made between total male infertility which may be the result of congenital or acquired absence of testicles, and relative male infertility resulting from temporary causes which lead to infertility but can be cured. By the duration of infertility, we can distinguish primary infertility (when the man has never made his female partner conceive) and secondary infertility (when the man made his partner conceive in the past but now he is not able to do it). Infertility can also be divided into the congenital and the acquired one [2].

Since 1980, we have been observing the increase in the number of fathers in the age group of 30 to 49 years and decrease in the age group of 25 to 29 years [9]. It is worth noting that men are most fertile in the age of 25 [2]. With age, spermatogenesis changes occur, semen volume decreases gradually as well as the motility and percentage of sperm with proper morphology, but not the sperm concentration [3, 9, 11]. In some countries, the age of sperm donors has been limited to 40-45 [9]. It has also been proved that the pregnancy rate decreases with age: in the case of men aged over 35, the probability that their partner gets pregnant within 12 months is 50% lower than in case of men under 25 years of age [11]. It is well-known that sexual activity decreases with age because of increasing sexual dysfunction [9] which can be the cause or the result of infertility (psychological aspect) [2]. Men maintain reproductivity even in the period of aging; the age impact is much less visible than in women [11].

It is worth mentioning the environmental factors which contribute to the decrease in male fertility. A man’s professional activity can have impact on semen parameters, genetic defects and genetic diseases in his children [3, 9]. The important factors having impact on gonads are: long-lasting exposure to heat at the temperature exceeding 38°C, exposure to ionizing radiation, vibrations, contact with heavy metals, organic solvents, pesticides [2, 3, 11]. In theory, heat sources in environment, wearing tight-fitting underwear, holding a laptop in one’s lap, carrying a telephone in one’s pocket, frequent hot baths may decrease fertility; this, however, has not been confirmed in clinical studies [11]. Currently, thermal effect of mobile phones is disregarded, while the focus is on the intensity and frequency of electromagnetic waves and their influence on the decrease in fertility in men [6]. A work-focused lifestyle is not without significance – shift work, frequent trips have influence on circadian rhythm disorders, not infrequently joining with daily stress and exhaustion, which in combination with low physical activity, overweight may lead to the decrease in testosterone level and potency [2, 3, 11]. It seems, however, that also excessive physical exercise contribute to the decrease in fertility; this is associated with decreased testosterone level and changes in the levels of LH and GnRH in men who work out intensely. Physical effort may lead to higher temperature and microdamages of testicles [10].

It is worth stressing the negative impact of smoking, alcohol and narcotic drugs on male fertility as they decrease the semen quality and testosterone level [2, 11]. Smoking contributes to the decreased motility and count of normal sperm cells in semen, which in addition show lower survival rates [3, 8]. Heroin and morphine negatively affect semen quality and lead to sexual disorders [3]. Also, it should be kept in mind that certain medications may have toxic effect on gonads, e.g. taking anabolic steroids may be lead to long-lasting and irreversible effects [11].

Male fertility can be temporarily impaired by febrile diseases which cause significantly lower concentration and motility of sperm cells. Systemic diseases, e.g. diabetes, cystic fibrosis, have long-lasting impact on fertility [11]. An important role can also be played by infections of lesser pelvis organs, e.g. orchitis in the course of mumps affects semen parameters as it directly interrupts spermatogenesis [9, 11], or infections of additional glands (prostate, seminal vesicles) [7, 9]. Surgeries in the area of the lesser pelvis can lead to the damage of vasa deferentia, disrupt neural pathways causing ejaculation disorders [11].

It is recommended that semen analysis precede the first visit. The first visit starts with the thorough interview. The physician should obtain the information about the patient’s age, duration of infertility and previous fertility, frequency of sexual intercourses as well as disorders of sexual functions, childhood diseases as well as the course of development and adolescence, systemic diseases, allergies, undergone surgeries, sexually transmitted infections, exposure to harmful environmental factors, use of addictive substances (smoking, alcohol, narcotic drugs). If the diagnostic testing for infertility has been carried out for some time already, it is recommended that the patient submits current full medical documentation (results of hormonal tests, semen analysis, descriptions of hospital treatments). It is considered that the physical examination (including penis examination with the evaluation of the location of urethral orifice, palpation of testicles with the measurement of their size, evaluation of the presence and consistency of vasa deferentia and epididymides, presence of varicocele, evaluation of secondary sex characteristics) may be deferred till the semen analysis result is obtained, provided that in the interview the patient has not reported any abnormalities in the structure and function of sexual organs [11].

Every patient subjected to the analysis should be given detailed instructions regarding the conditions of semen sample collection for analysis. It is recommended that semen be collected in a room close to the laboratory, in settings securing the patient’s privacy and at the same time basic hygiene conditions. Semen should be collected by masturbation directly into a sterile container (preferably, it should be provided by the laboratory). It is important that semen analysis be carried out after a period of sexual abstinence (time from the last ejaculation) of 2 to 5 days. Shorter time causes the decreased sperm volume and concentration; longer time influences the increase in the sperm volume and concentration but also increases the percentage of sperm cells with abnormal morphology. Each semen sample has to be marked with the date and hour of sample collection, sample completeness (as the initial part of semen contains the highest sperm concentration), any potential problems with sample collection and the time between the sample collection and its analysis (semen analysis should be carried out within an hour). It is acceptable that a semen sample be collected at home; however, it is very important to ensure that the semen temperature during transportation remains at 20°C to 37°C and that the sample be delivered to the laboratory within 60 minutes. If a semen sample is collected at home during a sexual intercourse, it should be collected to a special condom without spermicides [11, 12].

Evaluation of male infertility factors should include at least two semen analyses performed at least three weeks apart. Spermatogenesis lasts for approximately 70 days, sperm transport to the ejaculatory duct last for another 12 to 21 days, therefore the result of semen analysis is affected by factors from before even several weeks. In the case of the next semen analysis, the patient should observe the same sexual abstinence period as at the first analysis [11, 12].

In 2010, WHO published the new reference values of semen parameters as well as the principles of analysis. Semen samples were collected from men in the overall population (from various countries, continents) who achieved pregnancy with partners within less than 12 months, as just these men were considered fertile and forming the appropriate reference group [4]. On this basis, recommended semen parameter values for fertile men were determined.

Semen analysis includes macroscopic and microscopic examination. In macroscopic examination, semen liquefaction, viscosity, volume, color and pH is evaluated. Immediately after ejaculation, semen is a semiliquid, heterogeneous mass which typically within 15 minutes liquefies and becomes more homogeneous. At present, according to WHO 2010 reference values, the liquefaction time should be under 60 minutes. Semen viscosity is evaluated by letting the semen drops fall freely by gravitation from a disposable plastic pipette with a diameter of ca 1.5 mm while observing whether the semen start to pour in strands instead of drops. If the semen sample is normal, the falling drops will be small; drops which form strands more than 2 cm long indicate abnormal viscosity [12]. High sperm viscosity may interfere with sperm motility, and concentration, antibody detection, biochemical markers. It may also suggest the dysfunction of additional glands (prostate, seminal vesicles), but in practice this parameter has relatively low significance [11]. A properly liquefied semen sample is homogeneous, gray-opalescent [12]. Any deviations from this color may indicate the ongoing pathological process, e.g. red-brown sample may contain blood cells, yellow one may be the result of jaundice, taken vitamins or medicines. The next parameter is semen volume >1.5 ml (WHO 2010 reference values). The major part of the semen volume is the secretion from seminal vesicles and prostate gland [12]. Low semen volume may be caused by the obstruction of ejaculatory duct or congenital bilateral absence of the vas deferens. When the semen volume is under 1 mL, it is worth doing post-ejaculate urinalysis for the purpose of detecting potential retrograde ejaculation (having excluded the problems with sample collection, short abstinence period, congenital bilateral absence of the vas deferens and hypogonadism). Sour secretion of seminal vesicles and prostate gland decreases the semen pH to still alkaline > 7.2. Lower semen pH (below 7.0), low volume and low sperm count may suggest the obstruction of ejaculatory duct or congenital bilateral absence of the vas deferens [11].

Microscopic examination includes the evaluation of agglutination, sperm count, motility, vitality and morphology as well as the concentration of round cells and peroxidase-positive leukocytes [12].

Sperm agglutination is a phenomenon which consists in the mutual adherence of motile spermatozoa which may prevent them from moving in selected direction. There are four grades of agglutination: 1. 50 spermatozoa, the preparation contains unbound sperm; 4. all spermatozoa bound in large agglutination groups and these groups joining one another. Sperm agglutination was also classified by the parts attached in agglutination groups: A. head-to-head agglutination, B. tail-to-tail agglutination, C. tail-tip-to-tail-tip agglutination, D. mixed agglutination (heads join heads and tails join tails) E. tangle agglutination (heads and tails are enmeshed, heads are not clearly visible as they are obscured by joined tails). Strong agglutination may affect sperm motility and concentration. The presence of sperm agglutination is not sufficient to draw conclusions about the immunological causes of infertility but it suggests the presence of antisperm antibodies [11, 12].

It has to be mentioned that the risk of male infertility rises together with the number of the following abnormal semen parameters: concentration, motility, morphology. The probability increases 2 to 3 times when one of these parameters is abnormal, 5 to 7 times when two are abnormal and is 16 times higher when all three of them are abnormal [11]. Sperm motility is the percentage of the total number of spermatozoa displaying various types of movement: progressive (PR) (sperm moving actively along a linear or large circular path, regardless of its speed), non-progressive (NP) (movement in place), immotility (IM). Sperm total motility is the sum of (PR) and (NP). The lower reference limit for sperm total motility according to WHO 2010 is 40%, and for progressive motility (PR) is 32% [12]. Low sperm motility (asthenozoospermia) typically results from testicular dysfunction (presence of antisperm antibodies, infections of reproductive organs, partial obstruction of ejaculatory ducts or the site of reanastomosis after vasectomy, varicocele or extended period of sexual abstinence). High number of live immotile sperm may indicate the diagnosis of rare immotile cilia syndrome (structural anomalies of sperm prevent its movement) [11]. WHO 2010 guidelines ceased to make a distinction between sperm with fast and slow progressive motility; it seems, however, that such distinction has significant clinical and predictive value [1].

Currently, WHO recommends 3 methods of semen vitality assessment: eosin-nigrosin staining, eosin staining and hypo-osmotic swelling test. If no motile sperm is found, it is vitality tests that are used to differentiate live sperm from the dead ones. WHO 2010 reference value for vitality is ≥ 58 % [12].

An important semen parameter is the sperm count. A distinction is made between sperm concentration (a number of sperm per one milliliter of semen, ≥ 15 million/mL according to WHO 2010), and the total sperm count (a number of sperm cells in ejaculate which is sperm concentration multiplied with semen volume, ≥ 39 million according to WHO 2010) [12]. Azoospermia means the absence of sperm in a standard microscopic examination (which should be confirmed in at least two separate analyses). Azoospermia can be obstructive (caused by a blockage at each level of ejaculatory tract, with preserved normal sperm function) or non-obstructive (spermatogenesis disorders, limited or absent spermatogenesis). Oligozoospermia is a condition where the number of sperm cells in 1 milliliter of semen is lower than 15 million (this can be related to varicocele, hypogonadism or specific microdeletions in the segments of the Y chromosome) [11].

Spermatozoa with normal morphology were selected on the basis of studies in which it was examined what sperm could be found in female reproductive tract, in particular in intracervical mucus after sexual intercourse, and what spermatozoa were present on the surface of the cellular zona pellucida [12]. It is considered that sperm morphology is now the best predictive factor of the sperm’s ability to fertilize mature ovum which can be examined in vivo [11]. Anomalies in sperm morphology are called teratozoospermia (it can be related to varicocele as well as primary and secondary testicular failure). Features indicating the anomalies in the sperm head morphology: too small or too large head, unclear outline, shape other than oval (e.g. round or elongated head), more than two vacuoles in the acrosomal region, vacuole surface area exceeding 20% of the sperm head surface, any vacuoles in post-acrosomal region, acrosome too large or too small (covering less than 40% or more than 70% of the sperm head surface), double head. Midpiece anomalies are observed when the midpiece attached to the head is not in its axis, midpiece is too thick, too slender or irregular, bent at sharp angle, it contains cytoplasmic droplets with surface area exceeding 1/3 of the head surface. Tail anomalies include for example: too short tail, double tail, sharp bending at any segment, varying thickness [12].

Concentration of peroxidase-positive leukocytes in ejaculate (WHO 2010 reference value: <1 million/mL) may indicate the acuteness of inflammation. Their significant number (leukocytospermia) may be related to infection and poor semen quality and is the indication for semen culture (Mycoplasma hominis, Ureaplasma urealyticum, Chlamydia). When the number of round cells exceeds the WHO 2010 reference value of <5 million/mL, further test are required in order to differentiate leukocytes form immature spermatozoa and to identify men with true leukocytospermia [11, 12].

Values outside of the normal range confirmed in the result of semen analysis may indicate male factor of infertility and require further endocrinological, microbiological and genetic diagnosis [11].

Technological progress allowed to develop computer-assisted sperm analysis (CASA) which ensures the measurement of sperm concentration and motility characteristics and sometime also the assessment of sperm morphology (CASMA). Semen sample collection is carried out in the same way as for the routine semen analysis. CASA detects and counts sperm heads, but it does not say whether the sperm morphology is flawless (e.g. the head attached directly to the tail). In order to complement this test, computer-aided sperm morphometric assessment (CASMA) was developed with the task to carry out the quantitative analysis of sperm morphology. CASMA assesses the head, the midpiece and potentially the tail of individual spermatozoa and classifies them as normal or abnormal. The computer-assisted techniques of semen assessment, CASA, were created in order to increase the precision of tests, as well as the repeatability, objectification and automatization of semen assessment. However, it should be kept in mind that the CASA results are affected by many factors (e.g. the manner in which a semen sample is prepared, reading frame, sperm concentration), therefore the applicable procedures have to be observed rigorously [11, 12].

Male infertility may be caused by genetic disorders which interfere with the sperm production or transportation; for these reasons, genetic assessment of semen is useful. At present, it is stressed that the presence of following factors is of high importance: chromosomal abnormalities which contribute to testicular function disorders (Klinefelter’s syndrome), mutations of the Cystic Fibrosis gene (CFTR) coding the transmembrane protein which acts as a chloride channel, and Y chromosome microdeletion which is associated with deficient spermatogenesis. The clinical symptoms include azoospermia and severe oligozoospermia, but the clinical consequences may reach even to the next generation [11].

Chromosomal abnormalities are the most common genetic disorder in infertile men (it is estimated that they affect 2.1% to 15.5%) [5], while the Klinefelter’s syndrome (47, XXY; 46XY/47XXY) represents approx. 2/3 of all chromosomal anomalies [11]. The presence of two X chromosomes leads to the dysgenesis of vasa deferentia in approx. 1/3 of men with Klinefelter’s syndrome. Also, azoospermia or significant decrease in spermatogenesis is observed. Moreover, the incidence of sex chromosome aneuploidies in the sperm of men with the Klinefelter’s syndrome (47, XXY) is 2% to 45%, while in men with a mosaic karyotype its amounts to 1.5%-7% [5]. Therefore, it is worth proposing the preimplantation diagnosis with the use of the FISH technique (Fluorescence In Situ Hybridization) to men with the history of non-obstructive azoospermia or severe oligozoospermia in the IVF/ICSI procedure [5, 11].

There is a very strong relationship observed between the congenital bilateral absence of the vas deference and mutations in the cystic fibrosis gene which codes transmembrane protein and acts as a chloride channel (Cystic Fibrosis Transmembrane Conductance Regulator; CFTR) [11]. The mutation in the CFTR gene is responsible for 95% cases of the congenital bilateral absence of the vas deference, which means that nearly all men with cystic fibrosis lack both vasa deferentia [5, 11]. This happens because CFTR codes a protein which takes part in the production of the seminal vesicles and ejaculatory ducts [11]. Men with congenital bilateral absence of the vas deference or a milder form of aplasia and their partners are advised to undergo the test for CFTR mutation before trying to achieve pregnancy with the ART methods, because there is a possibility of passing on to their children both cystic fibrosis (both daughters and sons are at risk) and vas deferens aplasia of varying severity (sons) [5, 11].

The Y chromosome microdeletion occurs in men with azoospermia and severe oligozoospermia. In diagnostics, PCR (Polymerase Chain Reaction) technique is used [5]. Most microdeletions are found in regions located on the long arm of the Y chromosome (Yq11), marked with the acronym AZS (azoospermic factor) and letters a, b, c, etc. which contain genes (e.g. USP9Y, DBY, RBMY, PRY, DAZ) necessary for the normal spermatogenesis [5, 11]. In men with microdeletion in the AZFc region, sperm can be retrieved by means of a testicular biopsy; in those with AFZa or AFZb microdeletion, the prognosis is definitely poorer. Screening for the Y chromosome microdeletions is proposed to men with non-obstructive azoospermia or severe oligozoospermia, who are the candidates for the IVF/ICSI procedure [11].

Authors of article:: Anna Stachowicz, prof. dr hab. Krzysztof Łukaszuk, Kierownik Klinik Leczenia Niepłodności INVICTA

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