Male Infertility - Diagnosis

Diagnosis of male infertility includes a medical history and medical history, physical examination, detailed examination of the reproductive system, while assessing the nature of infertility (primary or secondary), its duration, previous examination and treatment.

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Male Infertility: Clinical Examination

Sexual and ejaculatory functions are assessed as follows. The average frequency of vaginal intercourse should be at least 2-3 times a week. Erection is considered adequate if it was sufficient for vaginal intercourse. Ejaculation is characterized as adequate if it occurred intravaginally. Anejaculation, premature ejaculation (before intromission), and extravaginal ejaculation are considered inadequate.

When assessing the somatic status, attention is paid to the timely constitutional and sexual development, determination of the body type, and the body weight/height ratio. Secondary sexual characteristics and gynecomastia are classified by stages, body weight and height are assessed using nomograms.

Assessment of the urogenital status includes inspection and palpation of the scrotum organs, indicating the position, consistency and size of the testicles, appendages and vas deferens. It is taken into account that the normal size of the testicle corresponds to 15 cm3 and more, they are determined using the Prader orchidometer.

To determine the condition of the accessory sex glands, a digital rectal examination of the prostate and seminal vesicles is performed.

Male Infertility: Clinical Examination

• initial survey (anamnesis collection);
• general medical examination;
• examination of the genitourinary system;
• consultations with a therapist, geneticist, sexologist (as indicated);
• medical genetic research.

Laboratory diagnostics of male infertility

The most important method in assessing the functional state of the sex glands and male fertility is sperm analysis.

The relatively high stability of spermatogenesis parameters for each individual allows one semen analysis to be performed, provided that there is normozoospermia. In case of pathozoospermia, the analysis should be performed twice, with an interval of 7-21 days, with sexual abstinence of 3-7 days. If the results of the two studies differ sharply from each other, a third analysis should be performed. Sperm is collected by masturbation into a sterile plastic container. previously tested by the manufacturer for toxicity to sperm. or into a special condom. The use of interrupted intercourse or a regular latex condom to obtain ejaculate is unacceptable. An incompletely collected sample is not analyzed. All manipulations with sperm storage and transportation are performed at a temperature of no lower than 20 C and no higher than 36 C. The best result is selected from the two spermograms. It is taken into account that the highest discriminatory indicator of sperm fertility is sperm motility.

The following WHO standard values are currently accepted for sperm evaluation.

Normal sperm fertility rates

Characteristics of spermatozoa
Concentration	>20x10 6 /ml
Mobility	>25% category "a" or >50% category "a"+"b"
Morphology	>30% normal forms
Viability	>50% live sperm
Agglutination	Absent
MAR test	<50% motile spermatozoa coated with antigens
Volume	>2.0ml
RN	7.2-7.8
Type and viscosity	Normal
Liquefaction	< 60 min
Leukocytes	<1.0x10 6 /ml
Flora	Absent or <10 3 CFU/ml

Sperm motility is assessed in four categories:

• a - fast linear progressive movement;
• in - slow linear and nonlinear progressive movement;
• c - no progressive movement or movement in place;
• d - spermatozoa are immobile.

Terms Used in Evaluating Semen Analysis

Normozoospermia	Normal sperm counts
Oligozoospermia	Sperm concentration <20.0x10 6 /ml
Teratozoospermia	Normal sperm forms <30% with normal count and motile forms
Asthenozoospermia	Sperm motility <25% category "a" or <50% category "a"+"b"; with normal indicators of quantity and morphological forms
Oligoasthenoteratozoospermia	Combinations of three variants of pathozoospermia
Azoospermia	There are no sperm in the semen
Aspermia	No ejaculation

In the absence of sperm and the presence of orgasm, a study of the sediment of post-orgasmic urine is performed after centrifugation (for 15 minutes at a speed of 1000 revolutions per 1 minute) to detect spermatozoa in it. Their presence indicates retrograde ejaculation.

Biochemical examination of sperm is performed to study the physiological properties of seminal fluid, which is important in assessing spermatogenesis disorders. Determination of citric acid, acid phosphatase, zinc ions, and fructose in sperm has gained practical significance. The secretory function of the prostate is assessed by the content of citric acid, acid phosphatase, and zinc. A clear correlation has been noted between these parameters, and only two indicators can be determined: citric acid and zinc. The function of the seminal vesicles is assessed by the content of fructose. This study is especially important to perform in azoospermia, when low levels of fructose, pH, and high citric acid indicate a congenital absence of the seminal vesicles. Standard indicators determined in the ejaculate:

• Zinc (total) - more than 2.4 mmol/l;
• Citric acid - more than 10.0 mmol/l;
• Fructose - more than 13.0 mmol/l.

In addition to the listed examination parameters, other available methods can be included, for example, determination of ACE activity. The testicular isoform of the enzyme has been little studied. At the same time, it was found that ACE activity in the ejaculate of liquidators of the Chernobyl nuclear power plant accident was 10 times higher than that of sperm donors and 3 times higher than that of patients with chronic prostatitis.

In diagnostics of functional disorders of the reproductive system of various etiologies, developed methods for determining proteins with various functions are used. Specific and non-specific proteins are present in the ejaculate: transferrin, haptoglobin, lactoferrin, fertility microglobulin, salivary-spermal alpha-globulin, complement components C3 and C4 and a number of other proteins. It has been established that any disorder of spermatogenesis or diseases of the reproductive organs lead to a change in the concentration of proteins. The level of fluctuations reflects the characteristics of a particular stage of the pathological process.

To exclude infectious etiology of the process, cytological analysis of the discharge of the urethra, prostate and seminal vesicles secretion, bacteriological analysis of sperm and prostate secretion are performed. PCR diagnostics of chlamydia, mycoplasma, ureaplasma, cytomegalovirus, herpes simplex virus. Indirect signs indicating infection are a change in the normal volume of sperm, increased viscosity of the ejaculate, impaired motility and agglomeration of spermatozoa, deviations in biochemical parameters of sperm and secretion of the gonads.

Diagnostics of immunological infertility is performed in patients in all cases of pathozoospermia and detection of sperm agglutinates or infertility of unclear genesis, which has no signs of reproductive dysfunction. For this purpose, immunodiagnostics are performed with detection of antisperm antibodies of class G, A, M in sperm and in blood serum by methods of sperm agglutination and sperm immobilization. However, these methods have a number of significant drawbacks and are very labor-intensive.

The MAR test (displaced agglutination reaction) is currently the most promising diagnostic method, which includes the use of latex beads coated with human IgG and monospecific antiserum to the Fc fragment of human IgG.

One drop (5 μl) of the latex suspension of the test sample and antiserum is applied to a glass slide. The latex drop is first mixed with sperm and then with antiserum. Sperm counting is performed using a phase contrast microscope at 400x magnification. The test is considered positive if 50% or more of the motile sperm are covered with latex beads.

AR. In 5-10% of cases, the cause of infertility of unknown genesis is a violation of spontaneous and/or induced AR. In a normally occurring process, the binding of spermatozoa to the egg leads to the release of a complex of enzymes from the head of the spermatozoon, among which acrosin plays the main role, ensuring the destruction of the egg membrane and the penetration of the spermatozoon into it. The following normal AR values are accepted: spontaneous (<20 conventional units), induced (>30 conventional units), inducibility (>20 and <30 conventional units).

Evaluation of the level of free radicals generation in the ejaculate (FR test). The FR test is one of the important indicators that allows us to characterize sperm fertility. Free radicals are chemical elements that carry unpaired electrons that interact with other free radicals and molecules, participating in oxidation-reduction reactions. Excessive FR formation can lead to activation of lipid peroxidation of the plasma membrane of spermatozoa and cell damage. The source of FR in the genital tract can be spermatozoa and seminal fluid. It is known that in men with pathozoospermia and even normozoospermia, a high level of free radicals can be detected. Indications for the FR test are infertility against the background of normo- and pathozoospermia, normal sexual development in the absence of systemic and hormonal diseases, infections of the reproductive system. Normal FR test values correspond to <0.2 mV.

Determination of the level of sex hormones that regulate spermatogenesis is an important factor in assessing fertility.

Sex hormone levels in healthy men

Hormone	Concentration
FSH	1-7 IU/L
LG	1-8 IU/L
Testosterone	10-40 nmol/l
Prolactin	60-380 mIU/L
Estradiol	0-250 pmol/l

Spermatogenesis is regulated by the hypothalamic-pituitary system through the synthesis of LHRH and gonadotropins, which regulate the formation of sex hormones through receptors of target cells in the gonads. The production of these hormones is provided by specific cells of the testicles: Leydig cells and Sertoli cells.

The function of Sertoli cells is to ensure normal spermatogenesis. They synthesize androgen-binding proteins that transport testosterone from the testicles to the epididymis. Leydig cells produce the bulk of testosterone (up to 95%) and a small amount of estrogens. The production of these hormones is controlled by LH in a feedback manner.

Spermatogenesis is a series of stages in the transformation of primary germ cells into spermatozoa. Among the mitotically active cells (spermatogonia), there are two populations, A and B. Subpopulation A goes through all stages of development and differentiation to a spermatozoa, while subpopulation B remains in reserve. Spermatogonia divide into first-order spermatocytes, which enter the meiosis stage, forming second-order spermatocytes with a haploid set of chromosomes. Spermatids mature from these cells. At this stage, morphological intracellular structures are formed, which create the final result of differentiation - spermatozoa. However, these spermatozoa are not capable of fertilizing an egg. They acquire this property when passing through the epididymis for 14 days. It was found that spermatozoa obtained from the head of the epididymis do not have the mobility necessary to penetrate the egg membrane. Spermatozoa from the tail of the epididymis are mature gametes with sufficient mobility and the ability to fertilize. Mature spermatozoa have an energy reserve that allows them to move along the female genital tract at a speed of 0.2-31 μm/s, maintaining the ability to move in the female reproductive system from several hours to several days.

Spermatozoa are sensitive to various oxidants, as they contain little cytoplasm and, therefore, a low concentration of antioxidants.

Any damage to the sperm membrane is accompanied by inhibition of its motility and disruption of fertile properties.

Male Infertility: Medical Genetic Research

Medical genetic testing includes the study of the karyotype of somatic cells, which allows determining the numerical and structural abnormalities of mitotic chromosomes in peripheral blood lymphocytes and germ cells in the ejaculate and/or testicular biopsy. The high information content of quantitative narcological and cytological analysis of germ cells, as a rule, reveals abnormalities at all stages of spermatogenesis, which largely determines the tactics of managing an infertile couple and reduces the risk of having children with hereditary diseases. In infertile men, chromosomal abnormalities are an order of magnitude more common than in fertile men. Structural chromosomal abnormalities disrupt the course of normal spermatogenesis, leading to a partial block of spermatogenesis at different stages. It is noted that numerical chromosomal abnormalities prevail in azoospermia, and oligozoospermia is accompanied by structural abnormalities.

Male Infertility: Diagnosis of Sexually Transmitted Infections

Currently, the role of infection caused by such pathogens as chlamydia, mycoplasma, ureaplasma and a number of viruses - cytomegalovirus, herpes simplex virus, hepatitis and human immunodeficiency virus, in the disruption of the fertilizing ability of spermatozoa is widely discussed. Despite the many studies on the presence of these pathogens in the genital tract of men and women, there are contradictory conclusions regarding the role they play in the occurrence of infertility. First of all, this is explained by the fact that these infections are detected in both fertile and infertile couples.

The impact of the immunological consequences of STIs on fertility is a separate area of modern research. Secretions of the accessory sex glands contain antigenic substances capable of stimulating the formation of antibodies. In this case, antibodies are formed locally in these glands or enter through the blood, appearing in the secretion of the prostate or seminal vesicles. Within the genital tract, antibodies can affect the motility of spermatozoa and their functional state. Most of the antigens currently known are specific tissue substrates of the prostate and seminal vesicles.

Laboratory diagnostics of male infertility:

• sperm analysis (spermogram);
• determination of antisperm antibodies;
• assessment of acrosome reaction (AR);
• determination of the level of free radical generation:
• cytological examination of the secretion of the prostate gland and seminal vesicles;
• testing for chlamydia, ureaplasmosis, mycoplasmosis, cytomegalovirus, herpes simplex virus;
• bacteriological analysis of sperm;
• hormonal examination (FSH, LH, testosterone, prolactin, estradiol, thyroid stimulating hormone, triiodothyronine, thyroxine, antibodies to thyrocyte peroxidase and thyroglobulin).

Instrumental diagnostics of male infertility

Instrumental diagnostics include thermography and echography. Thermographic analysis of the scrotum organs allows to detect subclinical stages of varicocele and control the effectiveness of surgical treatment. It is performed using a special thermographic plate or a remote thermal imager. In patients with varicocele, thermography reveals thermal asymmetry of the right and left halves of the scrotum within 0.5 °C to 3.0 °C on the side of varicose veins. This method also allows to establish temperature ratios in hydrocele, inguinal hernia, inflammatory diseases of the scrotum organs. Ultrasound is performed to assess the anatomical and functional state of the prostate and seminal vesicles, preferably using a transrectal sensor. Devices with three-dimensional echography (3D) should be used. Dopplerometry and color Doppler mapping can be used both as an independent method and as an additional one for more accurate diagnostics.

Ultrasound of the thyroid gland and determination of its function (based on the content of hormones triiodothyronine, thyroxine, thyroid-stimulating hormone in the blood) is performed on patients with suspected nodular toxic goiter or its diffuse enlargement, as well as other diseases.

X-ray examination. To exclude primary disorders in the hypothalamus and/or pituitary gland in hyperprolactinemia or hypothalamic-pituitary insufficiency, X-ray examinations are performed: skull X-ray, MRI or CT.

CT is becoming increasingly important in the diagnosis of pathological changes in the hypothalamic-pituitary region and is becoming the method of choice when examining patients due to its clear advantage over conventional radiography.

Testicular biopsy is the final method, it is performed in idiopathic azoospermia, when there is a normal testicular volume and normal FSH concentration in the blood plasma. Closed (puncture, transcutaneous) and open biopsy are used. Open biopsy is considered more informative due to obtaining a larger amount of material, it is performed more often. Histological data are classified as follows:

• normospermatogenesis - the presence of a complete set of spermatogenesis cells in the seminiferous tubules;
• hypospermatogenesis - the presence of an incomplete set of germ cells in the seminiferous tubules;
• aspermatogenesis - the absence of germ cells in the seminiferous tubules.

It should be noted that in some cases, in order to make a final decision on the choice of treatment tactics or the use of the intracytoplasmic sperm injection method, a testicular biopsy is performed even with inadequate blood hormone concentrations and hypogonadism.

Instrumental diagnostics of male infertility:

• Ultrasound of the pelvic organs;
• Thyroid ultrasound;
• thermography of the scrotum organs (remote or contact);
• X-ray methods (skull examination, renal phlebography, CT);
• testicular biopsy.

Immunological male infertility

It is currently known that the frequency of immunological infertility in various populations is 5-10%, and immunological disorders of the process of sperm fertilization and early embryogenesis are associated with the presence of specific antibodies to gametes, in particular, to spermatozoa.

Changes in the immunological regulation in the body due to auto-, iso- and alloimmunization lead to the formation of antisperm antibodies (immunoglobulins of class G, A and M). Antisperm antibodies may be present in one of the sexual partners, or in both in the blood serum, various secretions of the reproductive system (cervical mucus, ejaculate, etc.). Among the antisperm antibodies, sperm-immobilizing, spermagglutinating and spermolyzing antibodies are distinguished. There are several reasons for the appearance of antisperm antibodies in men and women. In the male reproductive system, spermatozoa appear after immune tolerance to the body's own tissues has formed. Therefore, there is a hematotesticular barrier in the testicles, formed at the level of the basal membrane of the convoluted tubule and Sertoli cells and preventing the interaction of spermatozoa with immunocompetent cells. Various factors that damage this barrier lead to immune reactions. These include inflammatory diseases of the testicle and accessory sex glands (orchitis, epididymitis, prostatitis, vesiculitis), trauma and surgical interventions (herniotomy, orchiopexy, vasectomy), impaired blood circulation in the genitals (varicocele), exposure of the scrotum organs to high and low temperatures, anatomical changes (obstruction of the vas deferens, agenesis v. deferens, inguinal hernia). It should be noted that the possibility of pregnancy cannot be ruled out even if one or both spouses have antisperm antibodies.

The following methods of immunodiagnostics of male infertility exist:

Study of general immunity

• Laboratory diagnostic methods.
  • Determination of immune status.
  • Determination of antisperm antibodies in the blood serum of men and women

Study of local immunity

• Laboratory diagnostic methods.
  • Microsperoagglutination.
  • Macrospermoagglutination.
  • Sperm immobilization.
  • Indirect fluorescence.
  • Flow cytometry: assessment of antisperm antibodies and assessment of acrosome reaction.
• Biological methods. Tests for compatibility and penetration ability of spermatozoa.
  • Shuvarsky-Guner test (postcoital test). Determines the motility of spermatozoa in the examined cervical mucus.
  • Kremer's test. Measurement of sperm penetration ability in capillary tubes.
  • Kurzrok-Miller test. Evaluates the penetrating ability of spermatozoa into cervical mucus.
  • Buvo and Palmer test. Cross penetration test using donor sperm and cervical mucus.
  • MAR test.
  • Test of ventricular penetration of the zona pellucida of the golden hamster egg by spermatozoa. It is believed that the ability of spermatozoa to bind to membranes of membrane-less hamster oocytes is characterized by the acrosome reaction and the ability to penetrate.
  • Hamzona analysis is one of the methods for assessing the acrosome reaction.
  • In vitro fertilization of oocytes. Cross-fertilization tests using donor sperm and mature eggs.
• Study of the biochemistry of vaginal mucus depending on the phase of the menstrual cycle (determination of pH, glucose content, various ions, etc.)