RU2759147C1 - Method for assessing the reproductive function in men with idiopathic infertility - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, medicine, namely, clinical laboratory diagnostics, and can be proposed as a method for diagnosing male infertility of an unidentified genesis. The method consists in the fact that in the cells of the mobile ejaculate fraction of patients with idiopathic infertility and asthenozoospermia, the double nucleotide polymorphism of the antimicrobial peptide DEFB126 (rs11468374) gene and the level of expression of the gene thereof are determined using a polymerase chain reaction in real time.

EFFECT: detection of a mutation in the homozygous state and reduction in the expression by at least 3.4 times compared with the level of expression established in healthy men with normozoospermia provides a possibility of specifying the cause of male reproductive dysfunction, increasing the accuracy of diagnostics of idiopathic infertility.

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Description

The invention relates to medicine, namely urology, reproductive immunology (reproductology) and clinical laboratory diagnostics, and can be proposed as a method for diagnosing male infertility of unknown origin.

Currently, infertility has acquired the status of a global socio-demographic problem that 15% of couples around the world face in family planning (Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe; 2 Reprod Biol Endocrinol 2015; 13 (1): 37.doi: 10.1186 / s12958-015-0032-1.). The rate of occurrence of the "male" factor, that is, quantitative and qualitative disorders of spermatogenesis, when examining a couple for infertility is close to 50% and is critical for population reproductology (Lebedev G.S., Golubev N.A., Shaderkin I.A., Shaderkina V.A., Apolikhin O.I., Sivkov A.V., Komarova V.A.Men infertility in the Russian Federation: statistical data for 2000-2018.Experimental and clinical urology 2019; (4): 4-12 ). The structural and biochemical foundations of male reproductive function disorders remain not fully understood, which is reflected in the extremely high rate of the number of men diagnosed with idiopathic infertility: according to various sources, this figure ranges from 31 to 75% (Galimov Sh.N. et al. Idiopathic infertility in men: problems and prospects // Materials of the 12th Russian Scientific and Educational Forum “Men's Health and Longevity.” - Moscow, February 19-20, 2014, p. 7).

It is known that in men with diagnosed idiopathic infertility, the examination does not reveal anatomical or functional pathologies of the reproductive system, however, there are deviations from the norm in the parameters of the spermogram, namely, oligoasthenoteratozoospermia, or OAT syndrome (Dohle GR, Colpi GM, Hargreave TV, Papp GK , Jungwirth A, Weidner W. EAU guidelines on male infertility. Eur Urol 2015; 48 (5): 703-711). The indicator of sperm motility depends on a variety of factors, and the most important are the morphology of the cell and the structure of its glycocalyx. Spermogram is a generally recognized integral and optimal way to assess the reproductive function of a man and the functional and morphological state of his spermatozoa, but this method does not allow to establish the cause of infertility of unknown origin due to the visuality and subjectivity of this study, and also does not take into account the biochemical characteristics of the cells under study (WHO laboratory manual for the examination and processing of human semen. - 5th ed. - WHO (Geneva), 2010. - Vol. 270; WHO Guidelines for the study and processing of human ejaculate. F. Kurilo. 5th ed. - M .: Publishing house "Capital print", 2012).

The treatment of idiopathic infertility is an extremely difficult task due to the unknown etiology of the disease, and although the use of assisted reproductive technologies reduces the number of infertile families, this problem does not lose its relevance throughout the world.

In recent years, the contribution of the components of the innate immune system has been actively studied not only in the course of pregnancy and its outcomes, but also in the processes of migration of germ cells, capacitation and fertilization.

Among the numerous components of the sperm glycocalyx, the protein DEFB126, which belongs to the innate immune system, is distinguished. It belongs to the family of β-defensins, which are the first line of defense of the body, since they have direct antimicrobial activity, have an immunomodulatory effect, act as mediators of inflammation and enhance chemotaxis in the inflammation zone (Kovalchuk L.V., Gankovskaya L.V., Meshkova R Ya. Clinical immunology and allergology with the basics of general immunology. Textbook, GEOTAR-Media, 2014).

It has been shown that the DEFB126 protein is specifically expressed by the cells of the male reproductive tract and is part of the structure of the glycocalyx of mammalian spermatozoa. In addition to the immune defense against pathogens and the implementation of the inflammatory reaction, this β-defensin ensures the effective advancement of the sperm inside the female genital tract, successful fertilization of the egg and prevents sperm recognition by the components of the female immune system (Yudin, A.I., Tgeese, S.A., Tollner, T. L., Overstreet, JW & Cherr, GN The carbohydrate structure of DEFB126, the major component of the cynomolgus Macaque sperm plasma membrane glycocalyx. J. Membr. Biol. 207, 119-129, 2005). In the presence of a double-nucleotide deletion in the second exon (rs11468374) in the structure of the gene encoding the DEFB126 molecule, a defective mRNA devoid of a stop codon is formed, which is rapidly degraded by the intracellular mechanism of quality control of the resulting mRNA, as a result of which the structure of the glycocalyx of β-defensin molecules is devoid of ...

A known method for predicting the risk of fertility disorders in obese men (RU 2498314 C1, Hamidov, 10.11.2013). The allele A of polymorphic variants rs2414096 and rs749292 of the aromatase gene CYP19A, which converts testosterone to estradiol in various tissues of the body, was identified by allele-specific polymerase chain reaction using a fluorescently-labeled oligonucleotide probe. We also determined the levels of the sex hormones LH, FSH and TSH and the spermogram indices of the examined obese men, depending on the genotypes of the aromatase gene polymorphisms. In the presence of the AA genotype of the rs2414096 and rs749292 polymorphisms, a more active conversion of testosterone into estradiol occurs, which leads to a decrease in the concentration of testosterone and other sex hormones and impaired spermatogenesis. The disadvantage of this method is the limitation of its use in relation to men without metabolic disorders.

In another known method for diagnosing sperm fertility disorders in men with asthenozoospermia (RU 2314530 C1, Fetisova, 2008-01-10), it is proposed to analyze allelic polymorphism of genes of the glutathione-S-transferase family M1 and P1 and calculate the risk of sperm fertility disorders in the form of asthenospermia in identification of genotypes GSTM1 0/0, GSTP1 B / B, GSTP1 B / C, GSTP1 C / C. The proposed technique is based on the proven role of exogenous toxic substances in the pathogenesis of male infertility and on the fact that functionally defective alleles of the genes of the glutathione-S-transferase family (GSTM1, GSTP1) involved in the second phase of biotransformation can cause increased sensitivity of spermatocytes to toxic effects, which is negative affects their mobility. The disadvantage of this method is the focus on the negative impact of environmental factors leading to biochemical changes in the body, and not taking into account the endogenous causes of reduced motility and fertility of spermatozoa.

The work of Iranian scientists "The role of DEFB126 variation in male infertility and medically assisted reproduction technique outcome" (Boroujeni, P.V., Ebrahimian, S., Abedini, M., Chayjan, MR, Hassani, M. , Gourabi, H. Meybodi, AM (2019). Reproductive BioMedicine Online. Doi: 10.1016 / j.rbmo.2019.05.012). A study was conducted of the association between the presence of a mutation in the DEFB126 gene with the outcomes of assisted reproductive technologies in a population of Iranian men with reproductive dysfunction. Scientists have demonstrated that a double-nucleotide deletion in the DEFB126 gene occurs significantly more often in the group of infertile patients compared to the control group.

The advantage of the proposed method in comparison with the prototype is the study of the prevalence of the mutant allele of the DEFB126 gene in the population of Russian men and determination of its association with gene expression and sperm motility.

We set the task to assess the level of expression of the gene for the antimicrobial peptide DEFB126 and the prevalence of its mutant allele (del) in the population of Russian men with reproductive disorders of unknown origin, and the association of this allele with the indicator of sperm motility.

The technical result achieved by the implementation of the present invention is to improve the diagnosis of male infertility of unknown origin.

The use of the invention makes it possible to diagnose the cause of a decrease in male sperm fertility associated with the presence of a mutation in the gene of the antimicrobial peptide DEFB126, which is a structural component of the glycocalyx of spermatozoa and affecting their motility, and a decrease in its expression, and thus, allows to increase the accuracy of diagnosis of male infertility of unknown origin and expand spectrum of prognostic markers of male infertility.

The aim of the invention is to develop a method for diagnosing male infertility based on determining the level of gene expression of the sperm glycocalyx protein molecule and the presence of a genetic mutation in it, leading to a significant reduction in the mobility of male germ cells in the female reproductive tract.

The essence of the invention is as follows.

To increase the accuracy of the diagnosis of male infertility of unknown origin, the presence of a double-nucleotide deletion (rs11468374) of the gene of the antimicrobial peptide DEFB126 and the level of its expression in the cells of the mobile fraction of the ejaculate are determined by the real-time polymerase chain reaction (RT-PCR) method. To assess the indicator of sperm motility, a semen analysis is performed. Next, the association of cell motility and the presence or absence of the del mutant allele of the DEFB126 gene are assessed. With the simultaneous detection of the del / del genotype of the DEFB126 gene, a decrease in its expression by at least 3.4 times compared to its level detected in healthy fertile donors, and a reduced mobility indicator (with a normal indicator of 50%), it is proposed to diagnose asthenozoospermia, mediated by mutation of the β-defensin gene DEFB126.

Our proposed method is carried out as follows.

To assess sperm motility, the following technique is used, which includes several successive stages: sterile collection of ejaculate for diagnostic or research purposes, its dilution, assessment of the morphofunctional properties of the ejaculate (sperm analysis). To study the expression of the DEFB126 gene after sterile assembly of the ejaculate and its liquefaction, the fraction of motile spermatozoa is separated from the seminal fluid and other cellular components of the ejaculate and infectious agents, nucleic acids are isolated from the resulting cells, a reverse transcription reaction is carried out to synthesize a copy of cDNA on the RNA template, and RT-PCR to assess the expression of the gene under study.

Collection of ejaculate for diagnostic and research purposes is carried out in accordance with the WHO recommendations (WHO Guidelines for the study and processing of human ejaculate. 5th ed. M .: Capital-Print, 2012; p. 292). After observing all the preparatory recommendations, the ejaculate is collected by masturbation into a vessel with a wide throat made of non-toxic plastic for spermatozoa in a special room near the laboratory in order to avoid temperature fluctuations. Further, to liquefy the ejaculate, the container is placed in a thermostat +37 C ° for 25-30 minutes, where it is stirred every 8-10 minutes with oscillatory movements. After 25-30 minutes, you need to make sure that liquefaction has occurred by rotating the container for 20 s.

Sperm motility is assessed immediately after sample liquefaction by phase contrast microscopy. To do this, take two aliquots of semen and prepare two wet preparations, and then study them at a magnification of x200 or x400. For each aliquot, approximately 200 sperm are counted in no more than 5 randomly selected fields of view. First, it is necessary to evaluate the selected area for category A cells - sperm with fast forward movement, then the count of category B cells - sperm with slow forward movement, and then category C cells - immobile spermatozoa or spermatozoa with non-translational movement. Next, using a laboratory counter, the cells of each category are counted and the average percentage and differences between the two percentages for the most common category in each aliquot are calculated.

Further, in accordance with the recommendations described in the "WHO Guidelines for the Study and Processing of Human Ejaculate", using the commercial set "SPERMGRAD ™" (Vitrolife Sweden AB) in strict accordance with the manufacturer's protocol, the fraction of motile spermatozoa is separated from the round ones by centrifuging the ejaculate in a density gradient. cells, leukocytes, degenerating germ cells and cellular debris. This method allows obtaining a fraction of the most motile spermatozoa with normal morphology.

To isolate nucleic acids from the obtained sperm fraction, use a set of reagents for DNA / RNA isolation by affinity sorption on silica gel particles "AmpliPRAIM Ribo-sorb" (InterLabService, RF) strictly in accordance with the protocol. 150 μl of spermatozoa fraction is added to 1.5 ml tubes and 450 μl of lysis buffer heated to 60 ° C is added. The contents are vortexed and centrifuged at 5000 rpm for 5 seconds. Then 25 μl of the resuspended sorbent is added to the test tubes, mixed on a vortex mixer and centrifuged at 10,000 rpm for 30 seconds, after which the supernatant is decanted. Next, 400 μl of washing solution 1 is added to the test tubes, mixed, centrifuged at 10,000 rpm for 30 seconds, and the supernatant is removed. Then add 500 μl of a solution for washing 3, mix and centrifuge at 10,000 rpm for 30 seconds, remove the supernatant. Repeat washing with solution 3. Then add 400 μl of washing solution 4 to the test tube, mix, centrifuge at 10000 rpm for 30 seconds, remove the supernatant. The sorbent deposited on the bottom of the test tubes is dried in a thermostat at a temperature of 60 ° C for 15 minutes with open lids. After that, 50 μl of RNA buffer is added to the tubes, mixed on a vortex mixer and placed in a thermostat for 3 minutes at a temperature of 60 ° C with the lids closed, mixed again and centrifuged at 13000 rpm for 60 seconds. The samples obtained are stored at -70 ° C.

To study the expression of the DEFB126 gene, a reverse transcription reaction (RT) is staged in a volume of 25 μl using the "Kit for carrying out the reverse transcription reaction" (Synthol, RF) for the synthesis of cDNA copies on the RNA template of the gene under study. Deionized water (ddH2O) included in the kit was added to the test tube as a negative control.

The OT reaction is carried out in two successive stages with the preparation of mixture No. 1 and mixture No. 2. Mix No. 1 contains 1 μl of primer Oligo (dT) [15 OU / ml] and 14 μl of ddH2O. The prepared mixture No. 1 is dispensed into tubes of 15 µl per sample and 4 µl of RNA is added, 4 µl of ddH2O is added to a test tube with negative control. Annealing of Random hexamers and Oligo (dT) primer on the mRNA template is carried out at a temperature of 75 ° C for 3 minutes. After incubation, the tubes are cooled to 4 ° C and 11 μl of mixture No. 2 containing 10 μl of 2.5x Reaction mixture, 1 μl of MMLV-RT reverse transcriptase with a concentration of 50 units / μl are added and incubated for 40 minutes at 37 ° C. Further, the inactivation of revertase is carried out at 95 ° C for 5 minutes. The cDNA obtained after the reaction is stored at -70 ° C.

As a control of the progress of the RT reaction and for standardization of the method, a PCR system is used to determine the expression of the β-actin gene.

Real-time PCR is performed using kits for RT-PCR in the presence of SYBR Green I (Synthol, RF) and specific primers. 4 µl of cDNA obtained by RT reaction is added to the finished mixture. The primers used in the reaction were selected in the Vector NTI Designer software and synthesized by Syntol (RF). A sample with a reaction mixture that does not contain mRNA serves as a negative control. Real-time PCR is carried out on a DT-96 device (DNA-Technology, RF) according to the following scheme:

• denaturation 95 ° С - 5 minutes,

• amplification (annealing at 94 ° С - 20 sec, elongation at 64 ° С - 30 sec) - 35 cycles,

• final extension 72 ° С 5 minutes.

The program for determining the polymorphism of the DEFB126 rs11468374 gene is presented below:

• denaturation 94 ° С - 3 minutes,

• amplification (annealing at 94 ° С - 60 seconds, elongation at 60 ° С - 20 seconds, extension 72 ° С - 1 min) 35 cycles,

• final extension 72 ° С 7 minutes.

The expression level of the DEFB126 gene was assessed using the 2 ^-ΔΔCt method in relative units relative to the activity of the housekeeping gene, β-actin.

The results were processed using Microsoft Excel 2016 and STATISTIC A 10.0 software. The data were checked for normal distribution, their variances were determined. The study groups were compared using analysis of variance (ANOVA) with Tukey's post hoc test. The confidence level was taken as 0.05.

Table 1 shows the distribution of indicators of sperm motility in patients and healthy donors in accordance with their genotypes. The genotype DEFB126 del / del was not identified among donors without impairment of fertility. The sperm motility of the donors of the healthy group is significantly higher than the motility of the spermatozoa of the patients with infertility (p <0.0001). It has been shown that in patients with the DEFB126 del / del genotype, sperm motility is reduced by 5.71 times in comparison with the mobility of patients with the DEFB126 wt / wt genotype and by 4.67 times in comparison with patients with the DEFB126 wt / del genotype (p <0 , 0001). A statistically significant difference was also shown in the indicators of sperm motility of patients with the DEFB126 wt / del genotype and donors from the healthy group with the DEFB126 wt / del genotype (p <0.05).

Table 2 shows the distribution of patients' genotypes and levels of DEFB126 gene expression among patients with idiopathic infertility and healthy donors.

A significant decrease in the DEFB126 gene expression among infertile patients by 12.4 times compared with the group of healthy donors was shown (p = 0.00012 according to Tukey's test). A significant difference was shown between the expression rates of sick patients with the DEFB126 wt / wt genotype in comparison with healthy donors with the DEFB126 wt / wt and DEFB126 wt / del genotypes (p <0.001 and p <0.005, respectively); the difference between the indicators of patients with the DEFB126 wt / del genotype and the expression indicators of healthy donors with the DEFB126 wt / wt and DEFB126 wt / del genotypes (p <0.0001); and the difference between the expression rates of patients with the DEFB126 del / del genotype and those of the healthy group donors with the DEFB126 wt / wt and DEFB126 wt / del genotypes (p <0.001 and p <0.01, respectively).

Thus, the following results were obtained:

1. In patients with idiopathic infertility, the expression of the DEFB126 gene is reduced by 12.4 times in comparison with the group of healthy donors (p = 0.00012 according to Tukey's test);

2. In patients with the DEFB126 del / del genotype, sperm motility is reduced by 5.71 times in comparison with the mobility of patients with the DEFB126 wt / wt genotype and 4.67 times in comparison with patients with the DEFB126 wt / del genotype (p = 0, 00013).

To prove the possibility of realizing the declared purpose and achieving the specified technical result, we also present the following data.

Example 1.

Spouses T. went to the clinic in connection with primary infertility for 2 years. During the examination, the spouse revealed a tubal-peritoneal factor of infertility. In a standard laboratory study of the spouse's ejaculate, no abnormalities were found: the motility rate was 50% (category A - 20%, category B - 30%), the rate of sperm with normal morphology was 30%, the concentration was 95 million / ml. Conclusion: normozoospermia. When carrying out molecular genetic analysis to determine the polymorphism of the DEFB126 gene, the mutant allele was not identified, the genotype DEFB126 wt / wt. The relative expression level of the DEFB126 gene in this patient was 1.96 relative units, which corresponds to the values of this indicator in healthy donors. The female factor of infertility was diagnosed.

Example 2.

Patient X. with his wife B. was examined for an infertile marriage. Examination of the spouse revealed no pathological disorders of the reproductive system. During the laboratory study of the ejaculate, pronounced oligoasthenoteratozoospermia was revealed and the male factor of infertility was diagnosed: in the field of view, single spermatozoa with normal morphology of category B, the concentration was 1 million / ml. As a result of the molecular genetic examination, the DEFB126 del / del genotype was determined, which indicates the presence of a mutation in both alleles of the DEFB126 gene. The expression level of the DEFB126 gene was 0.199 relative units, which is 24.5 times lower in comparison with the expression indicators of the control group. The result of the performed genetic analysis makes it possible to establish the cause of the impaired motility of the patient's spermatozoa and impaired fertility associated with a mutation in the homozygous position of the DEFB126 gene and impaired expression.

Example 3.

Spouses A. applied to the clinic for assisted reproductive technologies for an infertile marriage. The examination revealed a combination of male and female factors of infertility: tubal-peritoneal factor in the spouse and asthenoteratozoospermia with spermogram indices: sperm of group A - 10%, group B - 31%, percentage of sperm with normal morphology 20%, concentration was 72 million / ml ... Molecular genetic analysis revealed the DEFB126 wt / del genotype. The expression level of the DEFB126 gene in patient B. was 0.074 relative units, which is 3.4 times lower than in healthy men. It can be concluded that the presence of a mutant allele may be associated with a decrease in the rate of sperm motility in the absence of pathological disorders of the male reproductive tract.

Thus, the above examples demonstrate that the proposed method allows us to identify a genetic disorder associated with a decrease in sperm motility, which makes it possible to increase the efficiency of diagnosing male infertility in the absence of pathology of the reproductive system.

Thus, the above examples demonstrate that the proposed method allows us to identify a genetic disorder associated with a decrease in sperm motility, which makes it possible to increase the efficiency of diagnosing male infertility in the absence of pathology of the reproductive system.

The advantages of the proposed method:

1.Can be used as a screening method for predicting impaired fertility;

2. allows to improve the accuracy and efficiency of diagnosis of idiopathic infertility;

3. is non-traumatic and requires a minimum amount of ejaculate (150 μl of cell fraction);

4. is a fast-paced method.

Claims (1)

A method for assessing the reproductive function of men with idiopathic infertility and asthenozoospermia, characterized in that the expression level and the mutant allele of the gene of the antimicrobial peptide DEFB126 are determined in the cells of the mobile fraction of the ejaculate using the polymerase chain reaction in real time and with a decrease in the expression level of at least 3, Asthenozoospermia caused by a violation of the protein structure of the sperm glycocalyx is diagnosed 4 times in comparison with the expression of a group of healthy donors and the detection of the del / del DEFB126 mutant genotype.