RU2795567C1 - Method for laboratory diagnosis of male reproductive function based on evaluation of spermatozoa dna fragment dispersion - Google Patents

Abstract

FIELD: medical diagnostics.

SUBSTANCE: perform sampling and processing of biological material; after that, carry out diagnostics and, on the basis of the received data make a conclusion about male reproductive function, use ejaculate as biological material. Processing of the ejaculate is carried out as follows: placed an aliquot of 100 mcL of fresh ejaculate on a dry disposable glass slide; place the glass slide in a thermostat at 37°C for 25-30 minutes until it is completely dry; then take out the slide with dried aliquot and left for 2 mins at room temperature 22°С; place the slide with dried aliquot in a sealed plastic container for storage at -22°C for a period of 28 days for subsequent delayed study. After 28 days of storage, before assessment of spermatozoa DNA fragmentation take out the glass slide with a frozen air-dried aliquot, leave it at a temperature of +22°C; add to the dried aliquot of the ejaculate 50 mcL of phosphate-buffered saline; mix with a pipette tip until a homogeneous suspension is formed; place the resulting suspension in 0.5 ml Eppendorf conical microtube; then coat the glass slide with a positively electrically charged surface to ensure that the cellular material is attached to the surface; mix the ejaculate suspension with inert agarose microgel; cool and expose to denaturant and lysing solution; apply to a glass slide; stain and evaluate spermatozoa DNA fragmentation by counting 300 spermatozoa per sample on a laboratory counter using bright field microscopy under x1000 immersion lens; estimate DFI ejaculate fertility by taking into account spermatozoa DNA fragmentation according to the formula. If DFI is less than or equal to 15%, make a conclusion about normal reproductive function, if DFI is greater than 15%, but less than 25%, make a conclusion about doubtful reproductive function, if DFI is greater than 25%, make a conclusion about abnormality.

EFFECT: invention provides increase in the diagnostic reliability of male reproductive function.

3 cl, 1 dwg, 6 tbl

Description

The field to which the invention relates

The invention relates to medicine and can be used in the diagnosis of disorders of spermatogenesis and infertility in urology, andrology, gynecology, reproduction, dermatovenereology, oncology to identify the etiology and pathogenesis of pathoso-ospermia.

State of the art

Human spermatozoa are easily susceptible to oxidative stress due to the influence of systemic factors (aging, comorbidity - diabetes mellitus, cancer, varicocele) and lifestyle (obesity, smoking, drugs, xenobiotics, ecology, ejaculation frequency). In 25-30% of cases diagnosed as male idiopathic infertility, a high percentage of spermatozoa with DNA fragmentation is found (Agarwal et ah, 2017), which leads to infertility, miscarriages and miscarriages.

DNA packaging in the sperm head is the result of a complex process that requires significant compaction and redistribution of chromatin during the final stages of spermatogenesis. The normal DNA of mature spermatozoa is highly resistant to physical or chemical denaturation.

Today, three mechanisms are known that can explain damage to sperm DNA:

- defective chromatin condensation during spermatogenesis [1];

- apoptosis [2]

- oxidative stress (OS) [3], to which human spermatozoa are easily exposed due to the influence of systemic factors (old age, comorbidity - diabetes mellitus, cancer, varicocele, etc.) and lifestyle (obesity, smoking, drugs, xenobiotics, ecology) , frequency of ejaculation, etc.) [4-7].

Basic ejaculate analysis (spermogram) remains the main test for evaluating male infertility, but the predictive value of common spermogram parameters (sperm count, motility, morphology) is low because of their high intra- and interlaboratory variability and low predictive value [8].

Spermogram cannot assess sperm chromatin damage, while scientific studies and clinical practice indicate that the integrity of sperm DNA affects their functionality, thus emphasizing the importance of FDNKS tests for assessing fertility disorders [9, 10].

In this regard, along with the basic analysis of the ejaculate, the World Health Organization (WHO) recommends additional semen examination using tests to detect OS and FDNKS [11].

Since the birth of assisted reproductive technologies (ART) and up to the present, FDNKS has been considered one of the main causes of male infertility due to impaired fertilization, implantation, production of poor quality embryos, an increase in the frequency of spontaneous abortions and a decrease in the frequency of pregnancy with ART [8, 12-17 ].

Known methods for diagnosing male reproductive function and their shortcomings. To assess the integrity of sperm chromatin, a range of methods and tests with varying degrees of diagnostic and prognostic capabilities are known:

1) tests for assessing DNA damage - structural analysis of sperm chromatin (SCSA / sperm chromatin structure assay); identification of damaged DNA fragments (single or double break) labeled with dUTP using terminal deoxynucleotidyl transferase (TUNEL/nick-end labeling of dUTP using terminal deoxynucleotidyl transferase); evaluation of chromatin dispersion (SCD / sperm chromatin dispersion test) or halo test (halo assay); single-cell gel electrophoresis (SCGE/single-cell gel electrophoresis or Comet assay);

2) tests to assess the quality of chromatin condensation - staining with aniline blue (aniline alue/AB); staining with chromomycin A3 (chromomycin A3/CMA3); acridine orange staying/AO staining [10, 18-19].

All these methods differ in their diagnostic accuracy (sensitivity, specificity) and clinical value due to their inherent advantages and disadvantages associated with different technologies for detecting sperm DNA damage [10,20] (Table 1)

There are also a number of methods for diagnosing male infertility.

Evaluation and calculation of the concentration of neutrophilic granulocytes / peroxidase-positive leukocytes in the ejaculate is carried out using cytochemical staining with ortho-toluidine (see WHO Guidelines for the study and processing of human ejaculate. -Publishing house "CAPITAL PRINT", Moscow, 2012, fifth edition, ISBN 978- 5-905106-09-05 (section 2.18.1.5-1.7; Fig. 2.14; 3.11).

The disadvantages of this method are the following:

1. there is no commercially available kit for staining, reagent: as a rule, 6 reagents are offered, which are difficult to buy due to the fact that they are precursors,

2. it is difficult to prepare for use, due to the fact that modern laboratories use, as a rule, exclusively ready-made sets of reagents and do not have the necessary verified equipment: balances, titration equipment,

3. used ortho-toluidine is a particularly dangerous carcinogen, which causes a risk to the health of personnel,

4. the results are considered in the improved Neubauer hemocytometer, which is not available on the Russian laboratory market of equipment and consumables with the necessary registration certificate,

5. The calculation formula is adapted to the Neubauer hemocytometer, which causes certain difficulties.

A known method for assessing and calculating the concentration of neutrophilic granulocytes (leukocytes)/peroxidase-positive cells (PPC) in the ejaculate. In its implementation, a semi-quantitative assessment of the content of leukocytes in the ejaculate on a test strip is used using an immunochromatographic method based on the enzymatic activity of leukocyte esterase (without cytochemical staining) (see V.V. Dolgov, S.A. Lugovskaya, N.D. and etc. Laboratory diagnosis of male infertility. - M. - Tver: LLC Publishing House "Triada", 2006. - 145 p., 199 ill., 5-94789-144-1. P. 70).

The disadvantages of this method are as follows:

1. The method allows only an approximate determination of the concentration and only of neutrophilic granulocytes / peroxidase-positive leukocytes in the ejaculate,

2. Does not give an accurate idea of the presence or absence of an inflammatory process in a man.

3. The method of determination is non-quantitative (semi-quantitative "on a test strip": > or <1x10 ⁶ / ml), which is contrary to WHO recommendations.

4. Leukocyte esterase appears in the biofluid when leukocytes are destroyed, which can lead to a false positive diagnosis (pyospermia).

There is a method for quantifying the content of round cells (neutrophilic and other leukocytes, macrophages, spermatogenesis cells) (concentration) without cytochemical staining of the PPC in the ejaculate (see E.E. Bragina. Protocol for spermiological research. - Andrology and genital surgery. - 2014, No. 1, p. 20).

The disadvantages of this method are the following.

1. The concentration of only round cells (all in a row) is determined, and not only the AUC in the ejaculate.

2. The method does not give an accurate idea of the presence or absence of an inflammatory process in a man.

3. The method is not suitable for monitoring the content of leukocytes in semen.

4. Identification of leukocytes occurs by subjective assessment ("by eye") by indirect identification signs, which does not comply with WHO recommendations.

There is a known method for determining the concentration of spermatozoa in 1 milliliter (ml) of ejaculate (see WHO Guidelines for the study and processing of human ejaculate, CAPITAL PRINT publishing house, Moscow, 2012, fifth edition, ISBN 978-5-905106-09-05 (section 2.7 , 2.8.1, 2.8.4, pp. 34-47).

The disadvantages of this method are the following.

1. The calculation of the concentration is carried out only for spermatozoa in 1 milliliter (ml) of ejaculate.

2. Counting is carried out in an improved Neubauer hemocytometer, which is not available on the Russian laboratory market for equipment and consumables with a registration certificate.

3. The semen sample is fixed and diluted with a working solution consisting of a complex prescription of chemicals - precursors (paragraph 2.7.5 of the WHO manual), which are not used in a modern clinical diagnostic laboratory. One of these precursors is a particularly dangerous carcinogen - formalin.

4. The estimation of the required dilution for further calculation of sperm concentration is based on complex mathematical calculations and is also adapted to the Neubauer hemocytometer (WHO Manual, pp. 40-41 chapter 2.8.1).

5. All calculation formulas are adapted to the Neubauer hemocytometer.

There is a known method for measuring the concentration of spermatozoa in 1 milliliter (ml) of ejaculate (see E.E. Bragina. Protocol for spermiological research. - Andrology and genital surgery. - 2014, No. 1, p. 18-19).

According to this method, preparation of the preparation, fixation of the ejaculate, preparation of the counting chamber, filling the windows of the hemocytometer with ejaculate, sedimentation of the spermatozoon in the chamber, determination of the number of large squares necessary for counting spermatozoa, measurement of the number of spermatozoa in 1 milliliter (ml) of ejaculate in the preparation in the Goryaev chamber and issuing a conclusion.

The disadvantages of this method are the following.

1. Only the measurement of sperm concentration in 1 milliliter (ml) of ejaculate is carried out.

2. Fixation and dilution of the semen sample is carried out with several working solutions (50 g NaHCO ₃ , 10 ml 36-40% formaldehyde solution (saturated solution)). One of them is a precursor and is a particularly dangerous carcinogen formaldehyde. Fixation with saline solution is long (until the motile cells completely stop), which affects the turn around time of the test in the laboratory / TAT (turn around time) and introduces additional errors.

3. Evaluation of the quality of the required dilution for further calculation of the concentration of spermatozoa is multi-stage and complicated: when preparing the preparation (10 µl, coverslip 22x22 mm), the depth of the preparation is approximately 20 µm. A special field-of-view sperm count is used to determine the optimal dilution. With a microscope field of view diameter (measured with a micrometer lens) of 500 μm, the detection of 4 spermatozoa in the field of view approximately corresponds to 1 million / ml. If the diameter of the field of view is 250 microns - 1 spermatozoon in the field of view corresponds to 1 million / ml. In this case, Table 2 should be used after multiplying the number of detected spermatozoa by 4).

4. The step of determining the number of large squares required for sperm count also complicates the research process, which is reflected in the TAT.

J.I. Martinez-Salamanca, S. Minhas (Vice-chair),

P. Verze; Guidelines Associates: L. Boeri, P. Capogrosso, A. Cocci, K. Dimitropoulos,

G. Hatzichristodoulou, A. Kalkanli, V. Modgil, U. Milenkovic, G. Russo, T. Tharakan, © European Association of Urology 2021; EAU Guidelines on urological Infections. 2022. Accessed January 28, 2022. https://d56bochluxqnz.cloudfront.net/documentd/full-guideline/EAU-JN-Urological-Infections-2022.pdf].For the identification of microorganisms in infertile couples and in patients with suspected male accessory gland infection (MAGI) clinical guidelines (CR) of the European Association of Urology / European Association of Urology (EAU, 2022) along with the SLE and post-massage urine ejaculate is recommended [EAU Guidelines on Sexual and Reproductive Health A. Salonia (Chair), C. Bettocchi, J. Carvalho, G. Corona, T.N. Jones,

JI Martinez-Salamanca, S. Minhas (Vice-chair),

P. Verze; Guidelines Associates: L. Boeri, P. Capogrosso, A. Cocci, K. Dimitropoulos,

G. Hatzichristodoulou, A. Kalkanli, V. Modgil, U. Milenkovic, G. Russo, T. Tharakan, © European Association of Urology 2021; EAU Guidelines on Urological Infections. 2022. Accessed January 28, 2022. https://d56bochluxqnz.cloudfront.net/documentd/full-guideline/EAU-JN-Urological-Infections-2022.pdf].

Regulatory legal acts (NLA) of Russian colleagues recommend using urethral scraping (US) / urethral discharge (UV) or ejaculate to search for infectious agents [Order of the Ministry of Health of Russia dated July 31, 2020 N 803n "On the procedure for using assisted reproductive technologies, contraindications and restrictions to their application"; "Clinical guidelines "Male infertility" (approved by the Ministry of Health of Russia), developer: All-Russian public organization "Russian Society of Urologists", approved by the Scientific and Practical Council of the Ministry of Health of the Russian Federation, 2021; Federal clinical guidelines. Dermatovenereology 2015: Skin diseases. Transmitted infections - 5th ed., revised and supplemented - M.: Delovoy Express, 2016. - 768 pp.; Order of the Ministry of Health of Russia dated December 29, 2012, No. 1673 n "On approval of the standard of primary health care for chronic prostatitis (examination for diagnosis and treatment); Order of the Ministry of Health of Russia No. 1675n dated December 29, 2012 "On approval of the standard for primary health care for nonspecific and other urethritis"; Order of the Ministry of Health of Russia dated November 9, 2012 No. 696n "On approval of the standard specialized medical care for acute prostatitis, orchitis and epididymitis"; Order of the Ministry of Health of the Russian Federation dated October 30, 2012 No. 556n "On approval of the standard of medical care for infertility using assisted reproductive technologies"].

At present, on the way to the search for the etiological factor of MAGI in combination with male infertility, the existing laboratory algorithms involving SV/US and prostate secretion (SPS) seem doubtful due to the low diagnostic value of the declared loci, as well as due to the lack of the possibility of reproduction and standardization in terms of the number and the quality of the taken biomaterial [Sapozhkova Zh.Yu., Shabalova I.P., Kasoyan K.T. The study of ejaculate sediment in the diagnosis of sexually transmitted infections: Textbook. Moscow, 2017; Sapozhkova Zh.Yu., Milovanova G.A., Patsap O.I. Laboratory diagnosis of male infertility. Markers and methods. Part II // Laboratory and Clinical Medicine. Pharmacy. 2021. V. 1, No 2. P.65 - 79. DOI: 10.14489/1 p.2021.02.pp. 065-079].

Thus, the known methods are low informative and unreliable. In this regard, the task is to create timely and correct diagnostics of the quantitative composition of the ejaculate sediment biotope, which, along with well-known diagnostic approaches, will allow avoiding the appointment of additional expensive laboratory and instrumental methods and unreasonable antibiotic therapy, which reflects the concept of health and longevity management based on correct planning of medical and economic efficiency. in a healthcare organization.

A known method for diagnosing a violation of male reproductive function based on microbiological (MBA) (cultural) studies of whole ejaculate and on the basis of molecular (NAAT / PCR-RT) studies of UO / US and life expectancy (Order of the Ministry of Health of Russia dated 29.12. 2012, No. 1673 n “On approval of the standard of primary health care for chronic prostatitis (examination for diagnosis and treatment)), which is the closest to the claimed method and pleasant as the closest analogue (prototype).

The disadvantages of the known method include:

- most of the etiological agents of infectious and inflammatory processes are uncultivated or poorly cultivated due to their biological properties;

- the existing laboratory algorithms involving VR/US and SLE have low efficiency due to the lack of diagnostic value of the listed loci, since the invasive process of obtaining these types of biomaterial is associated with discomfort and, often, pain in the patient, which, as a result, does not allow taking a full-fledged biological sample for research.

Determination of the number of obligate anaerobic opportunistic pathogens (OAUPMO) and their ratio in the biotope, as well as the identification of resistant types of bacterial communities associated with impaired male fertility, is not possible using MBA due to the limited diagnostic capabilities of the method. As a result, there is a risk of false-negative results, which determines the difficulties of etiotropic therapy, the transition of the disease to a chronic form and the recurrent nature of the course.

At present, the ideal method for measuring FDNCS and its optimal thresholds has not been determined, but the four main tests - TUNEL, SC-SA, SCGE / Comet assay and SCD / halo assay / Halo test seem to be reliable for providing information on the quality of spermatozoa, which important for establishing male factor infertility [19, 21].

The essence of the invention

The invention is aimed at solving the problem of increasing the diagnostic reliability of the results of diagnosing male reproductive function based on the assessment of the dispersion of DNA fragments of spermatozoa.

The result achieved by using the presented method is to expand the range of diagnosis of etiological factors of the infectious nature of the violation of male reproductive function, increase the information content and reliability of diagnosis.

The result is achieved due to the fact that in the method of laboratory diagnostics of male reproductive function, including the collection and processing of biological material, the implementation of diagnostics and, based on the data obtained, the formation of a conclusion about male reproductive function, while ejaculate is used as biological material, ejaculate processing is carried out as follows : an aliquot of 100 μl of fresh ejaculate is placed on a dry disposable slide; place the glass slide in a thermostat at 37°C for 25-30 minutes until completely dry; take out a glass slide with a dried aliquot and leave for 2 min at room temperature 22°C; place the slide with the dried aliquot in a sealed plastic container for storage at a temperature of -22°C for a period of 28 days for a subsequent delayed study; after 28 days of storage, before assessing sperm DNA fragmentation, a glass slide with a frozen air-dried aliquot is taken out, left at a temperature of +22°C; add to the dried aliquot of the ejaculate 50 μl of phosphate-buffered saline; mix with a pipette tip until a homogeneous suspension is formed; the resulting suspension is placed in a 0.5 ml Eppendorf conical microtube; a glass slide is coated with a positively electrically charged surface to allow attachment of cellular material to the surface; the ejaculate suspension is mixed with an inert agarose microgel; cooled and exposed to a denaturing agent and a lysing solution; applied to a glass slide; staining and evaluating sperm DNA fragmentation by counting 300 sperm per sample on a laboratory counter using bright field microscopy under an immersion lens x1000; ejaculate fertility is estimated taking into account DNA fragmentation - spermatozoa according to the formula:

Where

DFI - sperm DNA fragmentation index,

S is the number of spermatozoa with a small halo around the spermatozoon,

WH - number of spermatozoa without halo,

WHD - number of destroyed spermatozoa without halo,

N is the number of test spermatozoa,

at DFI<=15% they make a conclusion about the presence of normal reproductive function, at DFI>15%, but <25% they make a conclusion about doubtful reproductive function, and at DFI>25% - about the presence of pathology.

The technical result is enhanced by the fact that fragmentation of sperm DNA is classified as follows: fragmentation is absent in the absence of breaks and/or damage to the nuclear DNA of spermatozoa, while the size of the halo is greater than or equal to the inner diameter of the nucleus; there is partial fragmentation of DNA, there are breaks and / or damage to the nuclear DNA of spermatozoa, while the size of the halo is less than the large halo and greater than the small halo of the spermatozoa; spermatozoa S with a small halo include spermatozoa in which DNA fragmentation is present, with breaks and / or damage to the nuclear DNA of spermatozoa, the size of the halo is less than 1/3 or the same as the inner diameter of the nucleus; WH spermatozoa without a halo include spermatozoa with DNA fragmentation, breaks and / or damage to the nuclear DNA of spermatozoa, without a halo; WHD spermatozoa include those in which DNA fragmentation is present, complete degradation of the nuclear DNA of spermatozoa takes place.

Before diagnostics: high-viscosity ejaculate samples are liquefied with Bromelain phosphate-salt solution and mixed without foaming or air bubbles, the test time is determined to obtain a result between 30-60 minutes after semen collection.

To stain a slide with an aliquot of ejaculate applied, an ejaculate staining kit is used, including a solution for fixing and prestaining, a pink staining solution, a blue staining solution.

The following reagents are used in the Ejaculate Staining Kit:

Description of graphic materials

The essence of the invention and the possibility of achieving a technical result will be more clear from the following description with reference to the figures of graphic materials, which show:

fig. 1 - Microscopic picture of the preparation, control visualization of the five evaluation categories of sperm DNA fragmentation according to Fernandez et al.

The following symbols are used in the figure:

1 - microscopic picture of the preparation: spermatozoa with a large halo (Large / L) (spermatozoa whose halo size is larger or the same as the inner diameter of the nucleus),

2 - Microscopic picture of the preparation: spermatozoa with a medium-sized halo (Medium / M) (sperm with a halo smaller than the large halo and larger than the small halo of the spermatozoa), some breaks / damage to the nuclear DNA of the spermatozoa,

3 - Microscopic picture of the specimen: small halo spermatozoon (Small/S) (spermatozoon whose halo size is less than 1/3 or the same as the inner diameter of the nucleus), significant breaks/damages in the nuclear DNA of spermatozoa,

4 - Microscopic picture of the preparation: spermatozoa without halo (Without HALO /WH), significant breaks/damages in the nuclear DNA of spermatozoa,

5 - Microscopic picture of the preparation: a destroyed spermatozoon without a halo (Without HALO -Degraded / WHD) (a spermatozoon without a halo and with a nucleus that is uneven or weakly colored).

Implementation of the method

For the period July-August 2022, at the clinical base of the organization "International School of Cytology & Medical School of Innovations" (Moscow), a one-stage study was conducted in which the diagnostic method was applied under the same conditions to all the examined male volunteers in the amount of 492 people.

The average age of the examined men was 38.28 years (standard deviation 8.64; median age 38 years, minimum age 23 years, maximum age 59 years).

Criteria for inclusion in the study:

- the presence of complaints in patients of unsuccessful attempts to conceive for 1-5 years;

- observance of sexual abstinence within 3-5 days;

- the absence of active complaints characteristic of MAGI (male accessory gland infections / infections of the male accessory gonads) and / or a history of MAGI, namely specific or nonspecific urethritis, chronic or acute prostatitis, orchitis, epididymitis.

Exclusion Criteria:

- azoospermia,

- cryptozoospermia,

- the use of antibacterial, antiviral drugs in the last 4 weeks before the examination;

- the use of local drugs during the 3 weeks preceding the examination,

- the presence of patients with chronic pelvic pain syndrome,

- presence of lower urinary tract symptoms,

- the presence of chronic scrotal pain syndrome,

- the presence of erectile dysfunction,

- absence of karyotype disorders, microdeletions of the AZF locus of the Y chromosome, mutations of the CFTR gene;

- the presence of sexual partners in patients - women with recurrent disorders of the vaginal biocenosis, with inflammatory diseases of the pelvic organs.

There were no patients who dropped out of the study.

All volunteers signed an informed consent to participate in the study.

A month before the Halo test (halo or halo around the spermatozoon, the shape of the halo depends on the state of the spermatozoon), all the examined men (n=492) received the results of the spermogram, where various scenarios of pathospermia were identified, as well as normospermia.

As part of research and clinical development, to determine the cause of infertility, all volunteers underwent a study of the ejaculate for FDNKS on pre-prepared samples of sperm preparations collected and delivered in accordance with the recommendations of the WHO 6th edition (2021) [11].

Each ejaculate sample was divided into two parts: one part in an aliquot of 100 μl was used directly for preparation of preparations with subsequent storage and further analysis for FDNKS, and the rest was used for spermogram [22].

The ejaculate was processed according to the following steps [22]:

1) an aliquot of 100 µl of fresh ejaculate was placed on a dry disposable slide

glass;

2) put the glass in a thermostat at 37°C for 25-30 minutes until the aliquot is completely dry;

3) a glass slide with a dried aliquot was removed from the thermostat and left for 2 min at room temperature 22°C;

4) the glass preparation was placed in a sealed plastic container for storage at a temperature of -22°C for a period of 28 days for a subsequent delayed study;

5) after 28 days of storage, immediately before the evaluation of FDNKS, the frozen glass preparations dried in air were taken out of the plastic container and left to room temperature 22°C;

6) added to the dried aliquots of ejaculate, 50 μl of recovery liquid (phosphate-buffered saline);

7) mix gently with a pipette tip until a homogeneous suspension is formed;

8) the resulting suspension was placed in a 0.5 ml Eppendorf-type conical microtube;

9) used the prepared ejaculate sample for further analysis using the Kit includes:

1) Eppendrof-type test tube with low-melting agarose (Reagent No. 1) - 10 pcs.;

2) Phosphate-saline buffer solution (Reagent No. 2) 50 ml;

3) Denaturing solution (Reagent No. 3) 100 ml;

4) Lysing solution (Reagent No. 4) 100 ml;

5) Dehydrating solution 1 (Reagent No. 5) 50 ml;

6) Dehydrating solution 2 (Reagent No. 6) 50 ml;

7) Dehydrating solution 3 (Reagent 7) 50 ml;

8) Solution for fixing and prestaining (Reagent No. 8) 50 ml;

9) Staining solution "pink" (Reagent No. 9) 50 ml;

10) Staining solution "blue" (Reagent No. 10) 50 ml.

Prior to being placed on a pre-coated (positively charged surface) glass slide to ensure the best adhesion of the cellular material to the surface, the samples were mixed with an inert agarose microgel, cooled and exposed to a denaturing agent and a lysing solution. The slides were then stained and the FDNCS was assessed by counting 300 spermatozoa per sample on a laboratory counter using bright-field microscopy under a x1000 immersion lens [23, 24].

The principle of operation of the Kit is as follows: intact (intact/unfragmented/whole) DNA molecules expand after denaturation and extraction of nuclear proteins to form nucleoli with large specific halos (halos) of untwisted DNA loops that emerge from the central nucleus. With DNA fragmentation (damage to integrity), dispersion does not develop or is minimal and is accompanied by the formation of nucleoli without a scattering halo, or the halo (halo) is weakly expressed.

Sperm halo in the studied preparations were classified into 5 categories (Fig. 1):

1. No DNA fragmentation - optimal quality of the ejaculate - no breaks/damages in the nuclear DNA of the spermatozoa microscopic picture of the preparation: spermatozoa with a large halo (large/l) (spermatozoa whose halo size is larger or the same as the internal diameter of the nucleus) (1).

2. DNA fragmentation is present, but without a significant effect on embryogenesis - some breaks/damages in the nuclear DNA of spermatozoa Microscopic picture of the preparation: spermatozoa with a medium-sized halo (medium/m) (2).

3. DNA fragmentation is present, suboptimal quality of the ejaculate - significant breaks / damage to the nuclear DNA of spermatozoa Microscopic picture of the preparation: spermatozoon with a small halo (small / s) (spermatozoon whose halo size is less than 1/3 or the same as the inner diameter of the nucleus) ( 3).

4. DNA fragmentation is present, suboptimal quality of the ejaculate - significant breaks/damages in the nuclear DNA of spermatozoa microscopic picture of the preparation: spermatozoon without halo (without halo / wh) (4).

5. DNA fragmentation is present, poor quality ejaculate - complete degradation of sperm nuclear DNA Microscopic picture of the preparation: destroyed sperm without halo (without halo - degraded / whd) (sperm without halo and with a nucleus that is uneven or weakly colored) (5).

The percentage of spermatozoa with fragmented DNA was calculated according to the following formula in accordance with the claimed claims:

Where:

DFI (DNA Fragmentation Index) - sperm DNA fragmentation index,

S is the number of spermatozoa with a small halo,

WH - number of spermatozoa without halo,

WHD - number of destroyed spermatozoa without halo,

N is the number of test spermatozoa.

The results of the analysis on the patient report were recorded according to the following categories:

Number of test spermatozoa (N), %

Number of spermatozoa without DNA fragmentation (L+M), %

Number of spermatozoa without DNA fragmentation (L+M), %

Number of spermatozoa with DNA fragmentation (S+WH), %

Number of destroyed/degraded spermatozoa (WHD), %

Sperm DNA Fragmentation Index (DFI), %

Next, the laboratory analysis results were evaluated according to the following threshold values in accordance with the claims:

- norm: DFI<=15%,

- doubtful result: from DFI>15% to <25%,

- pathology: DFI>25%.

Statistical processing of the results was performed using standard computer programs: GraphPad Prism 8.0.1 and Microsoft® Excel® for Microsoft 365 MSO (version 2201 Build 16.0.14827.20158). To assess the reliability of the results obtained, we used the Pearson coefficient (p) for parametric statistics, the correlation coefficient (r) and methods of descriptive statistics.

Results of experimental studies

54.1% (266/492=54.1%) of men with a mean age of 36.58 years (standard deviation 8.12; median age 34 years, minimum age 23 years, maximum age 59 years) did not have FDNCS. detected (DFI<=15%).

According to the correlation matrix, this category of volunteers showed a weak direct correlation between a low level of FDNCS and asthenozoospermia (r=0.20; ρ 0.0001) and viscosipathia (r=0.13; p 0.0001), as well as a weak inverse correlation with oligoastenoteratozoospermia (r=-0.12; ρ 0.0001). The absence of pyospermia in men without FDNCS was also noted (Table 2).

Table 2 shows the results of calculating the following parameters:

Parameter 1 / Parameter 1 - Normozoospermia / normozoospermia,

Parameter 2 / Parameter 2 - Oligoastenoteratozoospermia / oligoasthenoteratozoopermia,

Parameter 3 / Parameter 3 - Asthenozoospermia / asthenozoospermia,

Parameter 4 / Parameter 4 - Oligostenozoospermia / ologoasthenozoospermia,

Parameter 5 / Parameter 5 - Dyskinesia / dyskinesia,

Parameter 6 / Parameter 6 - Viscosipathia / viscosipathia,

Parameter 7 / Parameter 7 - Leukospermia (Pyospermia) / 1 eusozreggsha,

Parameter 8 / Parameter 8 - Hemospermia / hemospermia,

Parameter 9 / Parameter 9 - Oligospermia / oligospermia,

Parameter 10 / Parameter 10 - Agglutination / agglutination,

Parameter 11/ Parameter 11 - Necrozoospermia/necrozoospermia,

Parameter 12/ Parameter 12 - FDNKS/DFI index.

21.3% (105/492=21.3%) of men with a mean age of 38.71 years (standard deviation 8.33; median age 39 years, minimum age 23 years, maximum age 56 years) had doubtful values FDNKS (DFI in the range of >15% to <25%).

According to the correlation matrix, in this category of volunteers, a weak direct correlation was noted between the questionable level of FDNCS and oligoasthenoteratozoos-permia (r=0.24; ρ 0.0001), oligospermia (r=0.23; p 0.0001) and mean age infertile men aged 39 (r=0.17; ρ 0.0001); weak inverse relationship with asthenozoospermia (r=-0.22; p 0.0001). (Table 3).

Table 3 shows the results of calculating the following parameters based on the results of experimental studies:

Parameter 1 / Parameter 1 - Normozoospermia / normozoospermia,

Parameter 2 / Parameter 2 - Oligoastenoteratozoospermia / oligoasthenoteratozoopermia,

Parameter 3 / Parameter 3 - Asthenozoospermia / asthenozoospermia,

Parameter 4 / Parameter 4 - Oligostenozoospermia / ologoasthenozoospermia,

Parameter 5 / Parameter 5 - Dyskinesia / dyskinesia,

Parameter 6 / Parameter 6 - Viscosipathia / viscosipathia,

Parameter 7 / Parameter 7 - Leukospermia (Pyospermia) / leucospermia,

Parameter 8 /Parameter 8 - Hemospermia/hemospermia

Parameter 9 / Parameter 9 - Oligospermia / oligospermia,

Parameter 10 / Parameter 10 - Agglutination / agglutination,

Parameter 11/ Parameter 11 - Necrozoospermia/necrozoospermia,

Parameter 12/ Parameter 12 - FDNKS/DFI index.

24.6% (121/492=24.6%) of men with a mean age of 41.55 years (standard deviation 9.06; median age 43 years, minimum age 23 years, maximum age 59 years) had FDNCS. (DFI>25%).

According to the correlation matrix, this category of volunteers showed a weak direct correlation between the level of FDNCS and the average age of infertile men 44 years (r=0.28; ρ 0.0001) (Table 4).

Table 3 shows the results of calculating the following parameters based on the results of experimental studies:

Parameter 1 / Parameter 1 - Normozoospermia / normozoospermia,

Parameter 2 / Parameter 2 - Oligoastenoteratozoospermia / oligoasthenoteratozoopermia,

Parameter 3 / Parameter 3 - Asthenozoospermia / asthenozoospermia,

Parameter 4 / Parameter 4 - Oligostenozoospermia / ologoasthenozoospermia,

Parameter 5 / Parameter 5 - Dyskinesia / dyskinesia,

Parameter 6 / Parameter 6 - Viscosipathia / viscosipathia,

Parameter 7 / Parameter 7 - Leukospermia (Pyospermia) Leisosereggta,

Parameter 8 / Parameter 8 - Hemospermia / hemospermia,

Parameter 9 / Parameter 9 - Oligospermia / oligospermia,

Parameter 10 / Parameter 10 - Agglutination / agglutination,

Parameter 11/ Parameter 11 - Necrozoospermia/necrozoospermia,

Parameter 12/ Parameter 12 - FDNKS/DFI index.

According to the analysis of the entire surveyed sample of infertile men (n=492), a weak direct correlation was established between the age of men and the level of FDNCS (r=0.1563, p=0.0005) (Table 5).

A weak direct correlation was established between the FDNKS index and oliasthenoteratoso-ospermia (r=0.19; p<0.0001), pyospermia (r=0.29; p<0.0001); necrozoospermia (r=0.16; p=0.0004) (Table 6).

Conclusion.

The absence of FDNCS was found in 54.1% (266/492=54.1%) of men, whose mean age was 36.58 years (DFI<=15%); a weak direct correlation was noted between the normal level of FDNCS and asthenozoospermia (r=0.20; ρ 0.0001) and viscosipathia (r=0.13; p 0.0001); weak inverse correlation with oligoasthenoteratozoospermia (r=-0.12; ρ 0.0001); the absence of pyospermia was noted.

A questionable result for the presence of FDNCS was found in 21.3% (105/492=21.3%) of men, whose mean age was 38.71 years (DFI in the range of >15% to <25%); there was a weak direct correlation between the questionable level of spermatozoa DNA fragmentation (FDNKS) and oligoasthenoteratozoospermia (r=0.24; ρ 0.0001), oligospermia (r=0.23; ρ 0.0001) and a mean age of 39 years (r=0 .17, p 0.0001); weak inverse relationship with asthenozoospermia (r=-0.22; ρ 0.0001). FDNCS was found in 24.6% (121/492=24.6%) of men, with a mean age of 41.55 (DFI>25%); there was a weak direct correlation between a high level of FDNC and a mean age of 44 years (r=0.28; p 0.0001).

In the entire surveyed sample of infertile men (n=492), a weak direct correlation was established between the level of FDNCS and age (r=0.1563, p=0.0005), oliasthenoteratozoos-permia (r=0.19; p<0.0001) , pyospermia (r=0.29; p<0.0001) and necrozoospermia (r=0.16; p=0.0004).

The results of this study showed that the proposed method for laboratory diagnosis of male reproductive function based on the evaluation of the dispersion of DNA fragments of sperm complements the routine spermogram, providing specific information to the clinician about the quality of the genetic material of the ejaculate, which significantly increases the information content of the diagnosis and improves the effectiveness of treatment.

The validated test protocol contributes to the standardization of the technology for detecting the test for the dispersion of DNA fragments of spermatozoa in Russian clinical diagnostic laboratories and in specialized embryological laboratories in order to screen for violations of male reproductive function.

The results of a fast and accurate Halo test can be useful for clinicians to develop new treatment and diagnostic strategies and tactics in men with impaired fertility, both for the natural process of reproduction and for ART.

Thus, the considered clinical cases of the use of diagnostics by the claimed method clearly show the advantages of using the proposed diagnostic method, which allows you to expand the range of recognizable up to 90%.

The analysis of practicing laboratory medicine doctors and urologists-andrologists of the Russian Federation on working with ejaculate sediment seems to be a valuable professional case for revising the protocols for laboratory examination of male patients in preconception preparation of a married couple, including those recommended for discussion and revision of the existing CR and RLA of the Russian Federation.

The application of the proposed diagnostic method allows avoiding the appointment of additional laboratory methods, instrumental diagnostic procedures and unreasonable antibiotic therapy, which reflects the concept of health and longevity management based on the correct planning of medical and economic efficiency in a healthcare organization.

Thus, the application of the presented method allows to eliminate the disadvantages inherent in known methods.

LITERATURE

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Claims (27)

1. A method for laboratory diagnosis of male reproductive function, including the collection and processing of biological material, the implementation of diagnostics and, based on the data obtained, the formation of a conclusion about male reproductive function, while ejaculate is used as biological material, characterized in that ejaculate processing is carried out as follows: an aliquot of 100 µl of fresh ejaculate is placed on a dry disposable glass slide; place the glass slide in a thermostat at 37°C for 25-30 minutes until completely dry; take out a glass slide with a dried aliquot and leave for 2 min at room temperature 22°C; place the slide with the dried aliquot in a sealed plastic container for storage at a temperature of -22°C for a period of 28 days for a subsequent delayed study; after 28 days of storage, before assessing sperm DNA fragmentation, a glass slide with a frozen air-dried aliquot is taken out, left at a temperature of +22°C; add to the dried aliquot of the ejaculate 50 μl of phosphate-buffered saline; mix with a pipette tip until a homogeneous suspension is formed; the resulting suspension is placed in a 0.5 ml Eppendorf conical microtube; a glass slide is coated with a positively electrically charged surface to allow attachment of cellular material to the surface; the ejaculate suspension is mixed with an inert agarose microgel; cooled and exposed to a denaturing agent and a lysing solution; applied to a glass slide; staining and evaluating sperm DNA fragmentation by counting 300 sperm per sample on a laboratory counter using bright field microscopy under a x1000 immersion lens; ejaculate fertility is estimated taking into account sperm DNA fragmentation according to the formula

where DFI - sperm DNA fragmentation index; S is the number of spermatozoa with a small halo around the spermatozoon; WH - number of spermatozoa without halo; WHD - number of destroyed spermatozoa without halo; N is the number of test spermatozoa; if DFI is less than or equal to 15%, a conclusion is made about the presence of normal reproductive function, if DFI is greater than 15%, but less than 25%, a conclusion is made about doubtful reproductive function, and if DFI is greater than 25%, the presence of pathology. 2. The method according to p. 1, characterized in that before the diagnosis: - samples of ejaculate with increased viscosity are diluted with Bromelain phosphate-saline solution and mixed without the formation of foam and air bubbles, - determine the time to perform the test to obtain a result in the interval between 30-60 minutes after collection of sperm. 3. The method according to claim 1, characterized in that for staining a glass slide with an applied ejaculate aliquot, an ejaculate staining kit is used, including a solution for fixing and prestaining, a “pink” staining solution, a “blue” staining solution.

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