RU2498314C1 - Method for prediction of risk of disturbed fertility in males suffering obesity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a method for prediction of a risk of disturbed fertility in males suffering obesity, involving venous blood sampling followed by the allele-specific polymerase chain reaction using a fluorescent labelled oligonucleotide probe and melting line analysis. Having found the allele A of CYP19A aromatase gene polymorphisms rs2414096 and rs749292 enables predicting the risk of disturbed fertility in males suffering obesity.

EFFECT: new prognostic indices for the estimated risk of disturbed fertility in males with obesity.

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Description

The invention relates to medicine, namely to urology, and can be used to predict fertility disorders in obese men.

A known method for determining the fertility of men (RU 2052202 C1, Posiseeva et al., 10.01.1996). The content of thermostable alpha-1-glycoprotein is examined in sperm fluid and, at a level of the latter above 16 μg / ml, reduced sperm fertility is diagnosed. A method for diagnosing early reproductive dysfunction in men is also described (RU 2413949 C1, Tarasova et al., 10.03.2011). In the blood serum by the method of enzyme immunoassay, the concentrations of the cytokines IL-8, IL-10 and IL-1RA are determined, then the diagnostic index DI is calculated. When DI is greater than 0, early reproductive dysfunctions in men are diagnosed, and with DI less than 0, a conclusion is made that there are no laboratory signs of disturbances in male reproductive function. However, these methods do not involve identifying a risk group for infertility in men with obesity.

The invention (RU 2342660 C2, Semenov, 12/27/2008) describes a method for predicting fertility disorders in men. By flow cytometry of sperm, the ratio of lymphocytes with the phenotype CD25 + and CD95 + is examined. With its values less than 0.71 and more than 1.20, the prognosis of fertility is considered unfavorable. However, this method does not allow to identify the risk group for infertility among men with obesity.

Closest to patentable is a method for predicting impaired sperm fertility in men in the form of asthenozoospermia, in which peripheral blood is examined by gene analysis (RU 2314530 C1, Fetisova et al., 10.01.2008 - prototype). Fragments of the glutathione-S-transferase M1 and P1 genes are amplified on the DNA extracted from lymphocytes. If one of the genotypes GSTM1 0/0, GSTP1 B / V, GSTP1 B / C and GSTP1 C / C is identified, the risk of asthenozoospermia is predicted. However, this method makes it possible to determine only a violation of sperm motility and does not apply to morphology and concentration.

However, it is known that infertility does not develop in all men with obesity. One of the possible factors that can explain the hormonal changes in obese men, as well as the differences in the effect of obesity on a particular individual, is the aromatase gene polymorphism, which is responsible for the conversion of testosterone to estradiol in various body tissues, including adipose tissue (see a review of the literature in St. Hamidov S.I. et al. in. Problems of Reproduction, 2010, N 6, pp. 79-88). The existence of polymorphisms of the aromatase gene CYP19A1, which is localized on chromosome 15q21.2, has been shown. It was proved that in men with a higher frequency of TTTA repeats, the concentration of estradiol in the blood serum was significantly higher and depended on weight. In addition, it was shown that only men with a higher frequency of TTTA repeats in the CYP19A1 aromatase gene have a chance of lowering estradiol levels after losing weight (Hammoud A et al. An aromatase polymorphism modulates the relationship between weight and estradiol levels in obese men. Fertil steril; 2009; 10: 37).

M.R. Sowers et al. (Sowers MR et al. Aromatase gene (CYP 19) polymorphisms and endogenous androgene concentrations in a multiracial / multiethnic, multisite study of women at midlife. Am J Med. 2006 Sep; 119 (9 Suppl 1): S 23-30)) studied the relationship of aromatase gene polymorphism with androgen concentration in middle-aged women. Five single nucleotide polymorphisms of the aromatase gene CYP19A1 (rs2414096, rs936306, rs2446405, rs1008805, rs749292) were analyzed. Three out of five polymorphisms were associated with changes in androgen concentration. However, a direct correlation between the relationship of the polymorphisms of this gene with fertility in men with obesity has not been described in the literature.

The present invention is to search for criteria for predicting the risk of fertility disorders in obese men, which is the technical result of the invention.

A patented method for predicting the risk of fertility disorders in obese men includes venous blood sampling, an allele-specific polymerase chain reaction using a fluorescently-labeled oligonucleotide probe and analysis of the melting curve, determination of rs2414096 and rs749292 polymorphism genotypes of the aromatase CYP19s24 and rs749292 polymorphisms of the CYP19A1 aromatase gene polymorphisms r24140, and identification rs749292, in the presence of which predict the risk of impaired fertility in men with obesity.

To determine the role of CYP19A1 aromatase gene polymorphisms in the development of infertility in obese men, 85 obese men were examined who underwent a genetic study. Description of aromatase CYP19A1 gene polymorphisms is given in Table 1.

Table 1 Aromatase gene polymorphisms CYP19A1 Polymorphism Replacement Chromosome position Gene localization rs2414096 A> G 15q21.1: 51529779 Intron rs749292 A> G 15q21.1: 51558731 Intron rs936306 C> T 15q21.1: 51579598 3'UTR

The obese men examined were divided into two groups. The main group (group 1) included 50 men with a BMI of more than 30 kg / m ² and idiopathic pathozoospermia; the control group (group 2) included 35 fertile patients with a BMI of more than 30 kg / m ² .

A comparative analysis of patients of the two groups did not reveal statistically significant differences in anthropometric indicators, the presence or absence of bad habits.

The data presented in Table 2 substantiate the relationship of infertility in men with obesity and the presence of the A allele of polymorphism rs2414096. In men with obesity and infertility, almost 2 times more often than in fertile men, carriage of a homozygous genotype AA is detected. The incidence of allele A in infertile men was 60% compared with 42.9% in the control group (p = 0.027). The risk of developing infertility (OR) in obese men with the presence of the A allele in the hetero- or homozygous form was 2.0 (95% CI: 1.1-3.7).

table 2 The frequency of polymorphism rs2414096 aromatase gene CYP19A1 Genotype Fertile Infertile quantity % quantity % AA 7 20.00 19 38.00 AG 16 45.70 22 44.00 Gg 12 34.30 9 18.00 Allele frequency BUT thirty 42.90 60 60.00 * G 40 57.10 40 40.00 Note: * - significance of the difference p = 0,027.

A similar association was also found for rs749292 polymorphism. In infertile obese men, the AA homozygous genotype was 1.7 times more likely than in men in the control group (38.0% and 22.9%, respectively). The risk of infertility in the presence of the allele A of the rs749292 polymorphism was 1.92 (95% CI: 1.04-3.55). Data on the frequency of rs749292 polymorphism in groups are presented in Table 3.

Table 3 The frequency of polymorphism rs749292 aromatase gene CYP19A1 Genotype Fertile Infertile quantity % quantity % AA 8 22.9 19 38 AG fourteen 40 21 42 Gg 13 31.1 10 twenty Allele frequency BUT thirty 42.9 59 59.0 * G 40 57.1 41 41.0 Note: * - significance of differences p = 0,038.

The frequency of the C and T alleles of rs936306 polymorphism in infertile and fertile men with obesity was almost the same: 81.4 and 18.6% in fertile patients and 83.0 and 17.3% in patients with idiopathic infertility. Data on the frequency of rs936306 polymorphism in groups are presented in Table 4.

Table 4 The frequency of polymorphism rs936306 in groups Genotype Fertile Infertile quantity % quantity % SS 23 65.7 35 70 ST eleven 31,4 13 26 TT one 2.9 2 four Allele frequency FROM 57 81.4 83 83.0 T 13 18.6 17 17.0

After determining the frequency of occurrence of aromatase gene polymorphisms and their relationship with the development of infertility in obese men, we compared the spermogram and hormone levels in patients of the main group depending on the genotype of the rs2414096 and rs749292 aromatase gene polymorphisms, i.e. those polymorphisms for which a relationship with the development of infertility in men with obesity has been identified.

During the examination, patients with the rs749292 polymorphism of the aromatase gene CYP19A1 were divided by us depending on the genotype: genotype AA (19 men), genotype AG (21 men) and genotype GG (10 men).

In the mathematical processing of data in patients with the AA genotype, the levels of LH, FSH and testosterone were significantly lower than the similar hormone levels in patients with the AG and GG genotypes. The average estradiol level was higher in patients with AA genotype (p <0.05). The above data are presented in table 5.

Table 5 Hormone levels depending on the genotype of rs749292 polymorphism of the CYP19A1 aromatase gene Hormone Genotype AA (N = 19) Genotype AG (N = 21) Genotype GG (N = 10) LH, honey / ml 2.8 ± 0.2 * 4.0 ± 0.3 4.9 ± 0.5 FSH, honey / ml 3.1 ± 0.3 * 6.1 ± 0.5 6.0 ± 0.7 TTG, honey / l 2.4 ± 0.2 2.0 ± 0.2 1.9 ± 0.3 Thyroxine, nmol / L 96.1 ± 4.8 103.8 ± 4.3 93.8 ± 6.8 Estradiol, pmol / L 134.9 ± 3.7 * 97.2 ± 4.4 86.8 ± 5.2 Progesterone, nmol / L 1.4 ± 0.1 1.3 ± 0.1 1.2 ± 0.2 Prolactin, honey / l 253.4 ± 25.9 244.7 ± 21.9 222.6 ± 26.5 Testosterone, nmol / L 11.1 ± 0.7 * 17.2 ± 0.9 20.1 ± 1.0 Cortisol, nmol / L 305.6 ± 28.2 352.0 ± 30.0 388.5 ± 37.3 Leptin, ng / ml 13.6 ± 1.3 12.6 ± 1.0 10.5 ± 1.2 Note: * - p <0.05 when comparing hormone indices in patients, depending on the genotype of rs749292 polymorphism of the CYP19A1 aromatase gene.

When analyzing the characteristics of spermogram indices in men with obesity and idiopathic pathozoospermia, depending on the genotype of the rs749292 polymorphism of the CYP19A1 aromatase gene, the following features were found. All three groups were comparable in terms of ejaculate volume and sperm motility (p> 0.05). At the same time, sperm concentration in men with AA genotype was significantly lower than in men with GG genotype, and sperm concentration in men with AG genotype was intermediate. A similar situation was noted in the analysis of the number of normal forms of sperm - 14.1 ± 1.3% and 20.8 ± 2.5%, respectively (p <0.05) (see Table 6).

Table 6 Spermogram indices depending on the genotype of aromatase rs749292 polymorphism Spermogram indicators Genotype AA (N = 19) Genotype AG (N = 21) Genotype GG (N = 10) The volume of ejaculate, ml 3.2 ± 0.2 2.9 ± 0.2 2.6 ± 0.2 The concentration of sperm, million / ml 34.8 ± 7.9 42.3 ± 8.3 46.8 ± 9.1 * Mobility category A,% 11.2 ± 1.1 12.7 ± 1.2 13.5 ± 1.5 Mobility category A + B,% 26.7 ± 2.0 30.1 ± 2.1 31.5 ± 3.2 Morphology,% 14.1 ± 1.3 18.3 ± 2.1 20.8 ± 2.5 * Note: * - p <0.05 when comparing the genotypes of AA and GG polymorphism of rs749292 aromatase gene CYP19A1.

When evaluating the hormonal profile in men with idiopathic pathozoospermia and obesity, depending on the genotype of rs2414096 polymorphism of the aromatase CYP19A1 gene, the results are obtained, which are presented in Table 7.

Table 7 Hormone levels depending on the genotype of the rs2414096 polymorphism of the aromatase CYP19A1 gene Hormone Genotype AA (N = 19) Genotype AG (N = 22) Genotype GG (N = 9) LH, honey / ml 2.5 ± 0.2 * 4.3 ± 0.2 4.7 ± 0.6 FSH, honey / ml 2.5 ± 0.2 * 6.4 ± 0.4 6.1 ± 0.8 TTG, honey / l 2.3 ± 0.2 2.2 ± 0.2 1.8 ± 0.3 Thyroxine, nmol / L 98.9 ± 4.8 99.6 ± 4.4 96.9 ± 7.0 Estradiol, pmol / L 137.9 ± 2.8 * 96.9 ± 3.7 84.8 ± 6.4 Progesterone, nmol / L 1.4 ± 0.1 1.4 ± 0.1 1.2 ± 0.2 Prolactin, honey / l 255.4 ± 27.5 242.4 ± 20.5 223.3 ± 27.3 Testosterone, nmol / L 11.2 ± 0.8 * 17.0 ± 0.9 20.1 ± 1.3 Cortisol, nmol / L 318.5 ± 30.0 342.8 ± 27.3 386.7 ± 42.9 Leptin, ng / ml 14.5 ± 1.3 11.4 ± 0.9 11.7 ± 1.3 Note: * - p <0.05 when comparing AA genotypes with AG and GG genotypes of rs2414096 polymorphism of CYP19A1 aromatase gene

In the mathematical processing of data in patients with the AA genotype, the levels of LH, FSH and testosterone were significantly lower than the similar hormone levels in patients with the AG and GG genotypes. The average estradiol level was higher in patients with AA genotype (p <0.05).

The data of a spermological study when analyzing the influence of the genotype of polymorphism rs2414096 of the aromatase gene CYP19A1 are presented in table 8.

Table 8 Spermogram indices depending on the genotype of rs2414096 polymorphism of the aromatase CYP19A1 gene Spermogram indicators Genotype AA (N = 19) Genotype AG (N = 22) Genotype GG (N = 9) The volume of ejaculate, ml 3.2 ± 0.2 2.8 ± 0.2 2.8 ± 0.3 The concentration of sperm, million / ml 38.3 ± 6.7 42.9 ± 8.4 52.5 ± 12.6 * Sperm motility (category A),% 11.0 ± 1.0 12.1 ± 1.5 14.3 ± 1.1 Sperm motility (category A + B),% 25.0 ± 1.9 27.1 ± 2.3 33.3 ± 2.2 * Morphology,% 13.0 ± 1.2 18.0 ± 1.2 23.2 ± 4.2 ** Note: * - p <0.05 when comparing the genotypes of AA and GG polymorphism rs2414096 aromatase gene CYP19A1; ** - p <0.05 when comparing AA genotype with AG and GG genotypes of rs2414096 polymorphism of CYP19A1 aromatase gene.

In patients with the AA genotype of these polymorphisms, the levels of LH, FSH and testosterone are significantly higher than in men with the AG and GG genotypes. At the same time, men with AA genotype have higher estradiol values.

When evaluating spermogram indices in patients with a homozygous form of rs2414096 and rs749292 aromatase gene polymorphisms, a decrease in the concentration of spermatozoa and the number of spermatozoa with active motility (category A and A + B) in the ejaculate is noted. In addition, in patients with the AA genotype of the rs2414096 aromatase gene polymorphism, the content of sperm with a normal structure in the ejaculate is significantly lower (morphology).

In the presence of the AA genotype of rs2414096 and rs749292 polymorphisms of the CYP19A1 aromatase gene, more active aromatization of testosterone to estradiol occurs, which leads to a relative decrease in testosterone concentration and a corresponding increase in estradiol levels, which in turn leads to a decrease in FSH and LH, thus closing the vicious circle, so how a decrease in LH concentration decreases testosterone production by Leydig cells. These hormonal changes lead to a decrease in spermatogenesis, the features of the changes of which are described above.

Thus, the experimental data presented demonstrate the achievement of a technical result: a relationship between infertility in men with obesity and the presence of the A allele A of the polymorphisms rs749292 and rs2414096, which can be used as a prognostic risk criterion for fertility disorders in obese men, is revealed.

Claims (1)

A method for predicting the risk of fertility disorders in obese men, including venous blood sampling, carrying out an allele-specific polymerase chain reaction using a fluorescently-labeled oligonucleotide probe and analyzing the melting curve, determining the genotypes of rs2414096 and rs749292 polymorphisms of the CYP19A aromatase gene r24 and rs749292 of the rs4096 aromatase polymorphism of r24140 and rs1406 aromatase polymorphisms of almase rs749292, in the presence of which predict the risk of impaired fertility in obese men.