Male infertility: diagnosis

Diagnosis of male infertility include anamnesis of life and history of the disease, physical examination, detailed examination of the reproductive system, while assessing the nature of infertility (primary or secondary), its duration, the previous examination and treatment.

[1], [2], [3], [4]

Male infertility: clinical examination

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

When assessing somatic status, attention is paid to timely constitutional and sexual development, determining the type of physique, body weight / height. Secondary sexual characteristics and gynecomastia are classified by stages, body weight and height are estimated from nomograms.

Evaluation of the urogenital status includes examination 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 with the help of Prader's orchidometer.

A rectal finger examination of the prostate and seminal vesicles is performed to reveal the state of the adnexal sexual glands.

Male infertility: clinical examination

• primary interrogation (anamnesis collection);
• general medical examination;
• examination of the genito-urinary area;
• counseling therapist, genetics, sex therapist (according to indications);
• medical genetic research.

Laboratory diagnosis of male infertility

The most important method in assessing the functional state of sexual glands and the fertility of men is the study of sperm.

Relatively high stability of spermatogenesis parameters for each individual allows one to limit one semen analysis under the condition of normozoospermia. With pathoscopy, the analysis should be performed twice at intervals of 7-21 days with sexual abstinence 3-7 days. If the results of the two studies differ dramatically, a third analysis is performed. Collection of sperm is carried out by masturbation in a sterile plastic container. Previously tested by the manufacturer for toxicity to spermatozoa. Or in a special condom. The use of interrupted sexual intercourse or a conventional latex condom to produce ejaculate is unacceptable. The sample collected not completely is not analyzed. All manipulations with storage and transportation of sperm are carried out at a temperature of at least 20 C and not more than 36 C. Of the two spermograms, choose the best result. At the same time, it is taken into account that the highest discriminating index of sperm fertility is sperm motility.

The following WHO normative indicators for sperm evaluation have now been adopted.

Normative indicators of sperm fertility

Characteristics of spermatozoa
Concentration	> 20x10 6 / ml
Mobility	> 25% of the category "a" or> 50% of the category "a" + "in the"
Morphology	> 30% of normal forms
Viability	> 50% of live sperm
Agglutination	Absent
MAR-test	<50% of mobile spermatozoa coated with antipodes
Scope	> 2.0ml
PH	7.2-7.8
Type and viscosity	Normal
Dilution	<60 min
Leukocytes	<1, .0х10 6 / ml
Flora	Absent or <10 3 cfu / ml

The motility of spermatozoa is estimated in four categories:

• a - rapid linear progressive motion;
• c - slow linear and nonlinear progressive motion;
• with - there is no progressive movement or movement on the spot;
• d - the spermatozoa are immovable.

Terms used in the evaluation of semen analysis

Normozoospermia	Normal sperm counts
Oligozoospermia	The concentration of spermatozoa <20.0 × 10 6 / ml
Teratozoospermia	Normal forms of spermatozoa <30% with normal numbers and moving forms
Asthenozoospermia	Sperm motility <25% of the category "a" or <50% of the category "a" + "in"; at normal indicators of quantity and morphological forms
Oligoasthenoteratozoospermia	Combinations of three variants of pathozoospermia
Azoospermia	There are no spermatozoa in the sperm
Aspermia	Ejaculation is absent

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

Biochemical examination of sperm is performed in order to study the physiological properties of seminal fluid, which is important in assessing the violation of spermatogenesis. Practical value was determined in the sperm of citric acid, acid phosphatase. Zinc ions, fructose. The secretory function of the prostate is assessed by the content of citric acid, acid phosphatase and zinc. There is a clear correlation between these parameters, while 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 for azoospermia, when low levels of fructose, pH and high levels of citric acid indicate a congenital absence of seminal vesicles. Normative indices 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 these survey parameters, other available methods can also be included, for example, the determination of ACE activity. The testicular isoform of the enzyme has been little studied. At the same time, it was found that the ACE activity in the ejaculate at the liquidators of the consequences of the Chernobyl nuclear power plant accident was 10 times higher than that of the sperm donors and was 3 times higher than in patients with chronic prostatitis.

In the diagnosis of functional disorders of the reproductive system of different etiology, the developed methods for determining proteins possessing various functions are used. In the ejaculate there are specific and nonspecific proteins: transferrin, haptoglobin, lactoferrin, microglobulin fertility, saliva-spermal alpha-globulin, complement components C3 and C4 and a number of other proteins. It has been established that any disturbance of spermatogenesis or diseases of the reproductive organs leads 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 the infectious etiology of the process, a cytological analysis of the discharge urethra, secretion of the prostate and seminal vesicles, bacteriological analysis of sperm and secretion of the prostate is performed. PCR-diagnosis of chlamydia, mycoplasmas, ureaplasma, cytomegalovirus, herpes simplex virus. Indirect signs indicating an infection - a change in the normal volume of sperm, increased viscosity of the ejaculate, impaired mobility and agglomeration of spermatozoa, deviations in biochemical indicators of sperm and secretion of sexual glands.

Diagnosis of immunological infertility is carried out in patients in all cases of pathozoospermia and the detection of agglutinates of spermatozoa or in infertility of an unknown genesis that does not show signs of reproductive harm. To do this, perform immunodiagnosis with the detection of anti-spermatozoa antibodies class G, A, M in the sperm and in the blood serum by spermoagglutination and spermimobilization. However, these methods have a number of significant drawbacks and are very time consuming.

The MRA test (biased agglutination test) is currently the most promising diagnostic method, including the use of latex beads coated with human IgG and a monospecific antiserum against the human IgG Fc fragment.

One drop (5 μl) of latex suspension is applied to the slide. Test sample and antiserum. First, mix a drop of latex with sperm, and then with antiserum. Sperm counts are performed using a phase contrast microscope at a 400-fold magnification. The test is considered positive if 50% or more of the mobile spermatozoa are covered with latex beads.

AR. In 5-10% of cases, the cause of infertility of an unclear genesis is a violation of spontaneous and / or induced AP. In a normally occurring process, the binding of spermatozoa to the egg results in the release of a complex of enzymes from the sperm head, among which acrolein plays the main role. Providing destruction of the egg shell and penetration of the sperm into it. The following normal AP parameters were taken: spontaneous (<20U units), induced (> 30U units), inducibility (> 20 and <30 conventional units).

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

Determining the level of sex hormones that regulate spermatogenesis is an important circumstance in assessing fertility.

The level of sex hormones in healthy men

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

Regulation of spermatogenesis is carried out by the hypothalamic-pituitary system by the synthesis of LHRH and gonadotropins. Which through the receptors of target cells in the gonads regulate the formation of sex hormones. The production of these hormones is provided by specific testicular cells: Leydig cells and Sertoli cells.

The function of Sertoli cells is aimed at ensuring normal spermatogenesis. They synthesize androgen-binding proteins, which 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 by the feedback type.

Spermatogenesis represents several stages of transformation of primary germ cells into spermatozoa. Among the mitotically active cells (spermatogonia), two populations A and B are isolated. Subpopulation A passes through all stages of development and differentiation to the spermatozoon, and subpopulation B remains in reserve. Spermatogonia divide up to first-order spermatocytes, which enter the meiosis stage, forming second-order spermatocytes with a haploid set of chromosomes. From these cells mature spermatids. At this stage, morphological intracellular structures are formed, which create the final result of differentiation - spermatozoa. However, these spermatozoa are not able to fertilize the egg. This property they acquire when passing through the appendage of the testicle within 14 days. It was found that the spermatozoa obtained from the head of the epididymis do not have the mobility necessary for the penetration of the egg cell membrane. Spermatozoa from the appendage of the appendage are a mature gamete with sufficient mobility and fertility. Mature spermatozoa have an energy reserve that allows them to move along the female reproductive tract at a rate of 0.2-31 μm / s, while retaining the ability to move in the female reproductive system from several hours to several days.

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

Any damage to the membrane of the spermatozoon is accompanied by inhibition of its mobility and impaired fertility.

Male infertility: medical genetic research

Medico-genetic research includes the study of karyotype of somatic cells, which allows to determine the numerical and structural anomalies of mitotic chromosomes from peripheral blood lymphocytes and germ cells in the ejaculate and / or testicular biopsy. 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 conducting a sterile couple and allows reducing the risk of having children with hereditary diseases. Infertile men, chromosomal abnormalities occur an order of magnitude more often than in fertile anomalies. Structural chromosomal abnormalities disrupt the course of normal spermatogenesis, leading to a partial block of spermatogenesis at different stages. It was noted that numerical chromosomal abnormalities predominate in azoospermia, and structural disorders are accompanied by oligozoospermia.

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 - is widely discussed. Herpes simplex virus, hepatitis and human immunodeficiency virus, in violation of the fertilizing ability of spermatozoa. Despite the many works on the existence of these pathogens in the sex of men and women, there are conflicting conclusions about the role they play in the infertility. First of all, this is due to the fact that these infections are detected in both fertile and infertile couples.

The impact of the immunological effects of STIs on fertility is a separate area of modern research. The secrets of the accessory genital glands contain antigenic substances that can stimulate the formation of antibodies. In this case, the antibodies are formed locally in these glands or come 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 known at the present time are specific tissue substrates of the prostate and seminal vesicles.

Laboratory diagnosis of male infertility:

• sperm analysis (spermogram);
• definition of antisperm antibodies;
• assessment of the acrosomal response (AP);
• determination of the level of generation of free radicals:
• cytological examination of the secretion of the prostate and seminal vesicles;
• research on 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 thyroid peroxidase and thyroglobulin).

Instrumental diagnosis of male infertility

Instrumental diagnostics includes thermography and echographic research. Thermographic analysis of the scrotum organs, which allows to identify subclinical stages of varicocele and to monitor the effectiveness of surgical treatment. It is carried out using a special thermographic plate or a remote thermal imager. In patients with varicocele, thermo-asymmetry of the right and left half of the scrotum is noted during thermography in the range from 0.5 ° C to 3.0 ° C on the side of varicose veins. This method also allows you to set the temperature ratios for hydrocele, inguinal hernia, inflammatory diseases of the scrotum. Ultrasound is performed to assess the anatomical and functional state of the prostate and seminal vesicles, preferably with a transrectal sensor. Instruments with 3D echography (3D) should be used. Doppler and color Doppler mapping can be used both as an independent method and as an additional tool for more accurate diagnosis.

Ultrasound of the thyroid gland and the determination of its function (for the content of hormones triiodothyronine, thyroxine, thyroid-stimulating hormone in the blood) are performed for patients with suspected nodal toxic goiter or for its diffuse increase, as well as for other diseases.

X-ray examination. To exclude primary disturbances in the hypothalamus and / or in the pituitary gland with hyperprolactinaemia or hypothalamic-pituitary insufficiency, X-rays are performed: radiography of the skull. MRI or CT scan.

CT becomes increasingly important in the diagnosis of pathological changes in the hypothalamic-pituitary region and becomes a method of choice in the examination of patients in view of the obvious advantage over conventional radiography.

A testicle biopsy is the final method, it is performed with idiopathic azoospermia, when there is a normal volume of testicles and a normal concentration of FSH in the blood plasma. Apply closed (puncture, percutaneous) and open biopsy. Open biopsy is considered more informative due to obtaining more material, it is performed more often. Histological data are classified as follows:

• normospermatogenesis - the presence of a complete set of cells of spermatogenesis 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, for a definitive decision on the choice of treatment tactics or the use of the method of intracytoplasmic sperm injection, testicular biopsy is performed with inadequate values of blood hormone concentration and hypogonadism.

Instrumental diagnosis of male infertility:

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

Immunological male infertility

At present, it is known that the incidence of immunological infertility is 5-10% in different populations, 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.

The change in immunological regulation in the body due to auto-, iso-and alloimmunization leads to the formation of antisperm antibodies (class G, A and M immunoglobulins). Antisperm antibodies can be present in one of the sexual partners, or both in the serum, in various secrets of the reproductive system (cervical mucus, ejaculate, etc.). Among antisperm antibodies, spermimmobilization is distinguished. Spermoagglutinating and spermolyzing antibodies. There are several reasons for the appearance of antisperm antibodies in men and women. In the male reproductive system, spermatozoa appear after the formation of immune tolerance to the body's own tissues. Therefore, the testicles have a hematotestick barrier formed at the level of the basal membrane of the convoluted tubule and Sertoli cells and interfering with the interaction of spermatozoa with immunocompetent cells. Various factors that damage this barrier lead to the appearance of immune reactions. These include inflammatory diseases of the testis and adnexa of the sexual glands (orchitis, epididymitis, prostatitis, vesiculitis), trauma and surgical interventions (hernia repair, orchopexy, vasectomy), impaired blood circulation in the genitals (varicocele), exposure to scrotum organs of high and low temperatures, anatomical changes (obstruction of the vas deferens, agenesis v. Deferens, inguinal hernia). It should be noted that it is impossible to exclude the possibility of pregnancy even if one or both spouses have antisperm antibodies.

There are following methods of immunodiagnosis of male infertility:

Investigation of general immunity

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

Investigation of local immunity

• Laboratory and diagnostic methods.
  • Microsperoagglutination.
  • Macrospermoagglutination.
  • Spermimmobilization.
  • Indirect fluorescence.
  • Flow cytofluorometry: evaluation of antisperm antibodies and evaluation of acrosomal response.
• Biological methods. Samples for compatibility and penetration ability of spermatozoa.
  • The Shuvarsky-Guner test (postcoital test). Determine the mobility of spermatozoa in the cervical mucus under study.
  • Test of Kremer. Measurement of the penetrating ability of spermatozoa in capillary tubes.
  • The Kurzrok-Miller test. The permeability of spermatozoa to cervical mucus is assessed.
  • A test of Buvo and Palmer. Cross-penetration test using donor sperm and ceric mucus.
  • MAr-test.
  • Test of venation of the transparent zone of the ovum of the golden hamster with spermatozoon. It is believed that the ability of the spermatozoon to connect to membranes of the lacking shell of hamster oocytes characterizes the acrosomal reaction and the ability to penetrate.
  • Hamzona analysis is one of the methods for assessing the acrosomal response.
  • Fertilization of oocytes in vitro. Cross-fertilization tests using donated spermatozoa and mature ova.
• The study of the biochemistry of vaginal mucus depending on the phase of the menstrual cycle (determination of pH, glucose content, various ions, etc.)