RU2804586C1 - Method for predicting the production of excellent and good quality embryos in assisted reproductive technology programs for asthenozoospermia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: clinical and laboratory diagnostics, reproductology and embryology, concerns the prediction of obtaining good quality embryos. The method includes the use of the Androflor test in the ejaculate, the parameters of which correspond to the criteria for asthenozoospermia, used for fertilization, followed by calculation of the prognostic index. In this case, based on microbiological analysis, the prognostic index ECHO-Pro-A is calculated using a formula that takes into account age, total bacterial mass, content of: Staphylococcus spp., Streptococcus spp., Corynebacterium spp., Gardnerella vaginalis, Megasphaera spp./Veillonella spp./Dialister spp., Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, Anaerococcus spp., Eubacterium spp., Haemophilus spp., Pseudomonas aeruginosa/Ralstonia spp./Burkholderia spp., Enterobacteriaceae spp./Enterococcus spp. If the ECHO-Pro-A value is greater than 0.2, the probability of obtaining more than 40% of embryos of good and excellent quality is low; if ECHO-Pro-A is less than 0 - high; with values of 0-0.2 - the forecast is uncertain.

EFFECT: predicting the effectiveness of the embryological stage of ART in couples planning an IVF procedure, with ejaculate parameters that meet the criteria for asthenozoospermia.

1 cl, 3 ex, 2 tbl, 6 dwg

Description

The invention relates to medicine, namely to the field of clinical and laboratory diagnostics, reproductology and embryology, the purpose of which is to predict the level of blastulation of the resulting embryos in assisted reproductive technology (ART) programs and to optimize the treatment of infertility in patients using in vitro fertilization (IVF).

Currently, the problem of infertility is acquiring not only medical, but also socio-demographic significance. The need for ART is constantly growing, but the pregnancy rate during treatment with in vitro fertilization does not exceed 38.5% per embryo transfer [1].

In this regard, the search for methods to increase the effectiveness of ART is an urgent task of modern reproductive medicine, and the effectiveness of IVF programs depends not only on the receptivity of the endometrium, but also on the number of good quality embryos obtained[2]. Despite the observance of aseptic conditions when working with human germ cells and embryos at the embryological stage, in some cases bacterial contamination of culture media is possible, which in turn can lead to the arrest of embryo development [12]. One of the main causes of bacterial contamination is ejaculate. When preparing men for ART, it is mandatory to examine a scraping of the urethra (or ejaculate) for STIs, which, of course, does not allow for a full assessment of other microorganisms that are likely the cause of erased forms of the inflammatory process of the urogenital tract (UGT) and unsuccessful ART protocols. The ability to predict the number of good-quality embryos obtained based on an assessment of the microbial composition of the ejaculate will allow optimizing the preparation of patients for ART. It has previously been shown that bacteriospermia is associated with the production of fewer good quality embryos and a decrease in clinical pregnancies, and the presence of bacteria in sperm in quantities greater than 10 ³ CFU/ml is associated with a decrease in sperm motility (asthenozoospermia)[3].

Currently, the search and testing of markers of the implantation potential of embryos are underway, which make it possible to analyze the metabolomic, proteomic, transcriptomic status of the follicular fluid and culture media in which embryos are cultured [4, 5, 6, 7]. The influence of the male factor on the effectiveness of ART and in particular the influence of ejaculate microorganisms is an insufficiently studied issue.

The search for additional markers that can improve the effectiveness of treatment in a particular couple remains the most important task in reproductology.

From the point of view of the stages of preparation of a couple for ART, it seems promising to search for a relationship between the microbiological parameters of the ejaculate and the effectiveness of the embryological stage.

Analogues of predicting the production of embryos of excellent and good quality, taking into account the microbial composition of the ejaculate, were:

1. A method for predicting the effectiveness of IVF programs for tubal-peritoneal infertility associated with chronic endometritis (RF patent No. RU2 677 467 C1, 2017) [8]. When the concentration of lactoferrin in the blood serum is up to 1.8 μg/ml and IL-8 up to 10 pkg/ml or an isolated increase in the concentration of lactoferrin up to 2.5 μg/ml with the level of IL-8 up to 10 pkg/ml, a high probability of a positive IVF result is predicted and transfer a fresh embryo into the uterine cavity.

Disadvantages of the method. When using the method, the man’s anamnestic data is not taken into account, and the microbial composition of the ejaculate is not assessed, with the exception of examination for STIs. When using the method, sensitivity and specificity are not indicated.

2. A method for predicting the outcome of an IVF program and embryo transfer (RF patent No. RU2581027, 2015) [9]. When the concentration of anti-Mullerian hormone in venous blood is less than 1.14 ng/ml and a high level (more than 45) of Spielberg-Hann anxiety, an unfavorable outcome of the IVF program for a given woman is predicted.

Disadvantages of the method. When using the method When using the method, the man’s anamnestic data and data on the microbial composition of the ejaculate are not taken into account. Data on calculating the specificity and sensitivity of the method for determining the level of anxiety are not provided; the method for determining the level of anxiety itself does not exclude a certain amount of subjectivity.

3. A method for predicting the result of in vitro fertilization and embryo transfer (IVF and ET) (RF patent No. RU 2476142С1, 2011)[10]. At the first stage of preconception preparation, a woman’s anamnestic data is collected, at the second stage a gynecological ultrasound examination is performed, at the third stage the carriage of the methylenetetrahydrofolate reductase (MTFHR) mutation at position C677T is identified. The obtained data is entered into the formula and the P index is calculated; as a result, P index values of more than 0.11 predict an unfavorable outcome of IVF and PE; with an index value of less than -0.21, a favorable outcome of IVF and PE is judged.

Disadvantages of the method. When describing the method, data on the microbiological study of the ejaculate used for fertilization are not taken into account. The method is inconvenient for clinical use, because has several stages of research.

4. A method for predicting the results of in vitro fertilization (IVF) in patients with autoimmune diseases (RF patent No. RU2345715C2, 2006)[11]. A woman’s history of adnexitis is determined, the presence of HLA antigens B18, Cw3, Cw4 in the phenotype, the number of platelets in the coagulogram, and the prognostic index F is calculated. If F is more than 0, a successful outcome of IVF is predicted; if F is less than 0, it is judged that it is inappropriate IVF at this stage due to the expected lack of a positive IVF result.

Disadvantages of the method. The method can only be used in patients with autoimmune diseases. The method involves determining a large number of variable indicators: B18, Cw3, Cw4, platelet count, which is financially expensive for patients. The sensitivity and specificity of the method are not specified.

There is no prototype of the proposed method in the literature.

Thus, the authors were tasked with developing an effective method for predicting the production of good and excellent quality embryos in assisted reproductive technology programs for asthenozoospermia.

This goal is achieved through a comprehensive study of the ejaculate at the stage of pre-gravid preparation, including assessment of spermogram parameters according to the WHO standard, and determination of the microbial composition of the ejaculate using quantitative polymerase chain reaction (PCR) with real-time detection of results (RT-PCR) using a test system Androflor (DNA-Technology TS LLC, Moscow).

The material for the study (0.5 ml of native ejaculate) was collected on the day of egg fertilization in an Eppendorf tube containing 0.5 ml of physiological solution. DNA was isolated using the PROBA-GS reagent kit (DNA-Technology TS LLC, Moscow). RT-PCR was performed according to the manufacturer's instructions. (https://www.dna-technology.ru/equipmentpr/nabory-reagentov-dlya-pcr-nabory-reagentov-dlya-vydeleniya-nukleinovyh-kislot-i-3).

24 indicators were determined: the total bacterial mass (TBM) of the sample and 23 groups of microorganisms, of which 4 were determined qualitatively, 19 - quantitatively. The quantities of detected microorganisms in the sample were expressed in genome equivalents per ml (GE/ml). A sample conclusion is shown in Fig. 1.

To process and analyze the obtained data, the computer program StatPlus:mac 8.0.3 (AnalystSoft, USA) was used.

In the proposed method for predicting the receipt of embryos of good and excellent quality, for the first time in reproductology, a unique formula is proposed for determining the probability of obtaining at least 40% of embryos of good and excellent quality before the start of the ART program. The presence of at least 40% of day 5 embryos (blastocysts) of good and excellent quality according to the Istanbul consensus workshop on embryo assessment classification is one of the indicators of the success of the embryological stage of ART [13]. Thus, the proposed method meets the “inventive step” criterion.

For the study, men undergoing ART treatment collected ejaculate in a sterile container in accordance with WHO recommendations for the collection of ejaculate for microbiological studies (WHO Guideline 2.2.4)[14]. Analysis of sperm concentration and motility was carried out using a Biola AFS-500 analyzer (NPF Biola, Moscow). The morphology of spermatozoa was assessed in stained preparations at 100x microscope magnification using an immersion lens. The Leukoskrin test was used to count peroxidase-positive leukocytes.

The method allows you to calculate the prognosis for cases of asthenzoospermia. The criteria for asthenozoospermia corresponded to samples with a decrease in total (a+b+c) sperm motility (less than 40%) or progressive (a+b) motility (less than 32%). The remaining parameters of the spermogram corresponded to the norm: ejaculate volume - no less than 1.5 ml, sperm concentration - no less than 15 million/ml, number of sperm with normal morphology - no less than 4%, pH 7.2, leukocyte count - less than 1 million/ml .

The study included 80 patients. Depending on the number of formed blastocysts of good and excellent quality at the embryological stage of cultivation, patients were divided into 2 groups. The first group included 34 patients from whom at least 40% of blastocysts were obtained of good and excellent quality. The second group included 46 patients in whom less than 40% of blastocysts of good and excellent quality were formed.

For the convenience of estimating the probability of obtaining at least 40% of embryos of good and excellent quality for couples in which the spermogram parameters of the man met the criteria for asthenozoospermia, we have developed a forecasting method by calculating the prognostic index ECHO-Pro-A (Embryos of Good and Excellent Quality Forecast Asthenospermia) To develop a prognostic index in the resulting matrices of clinical and laboratory indicators, a discriminant analysis of variables in the study groups was carried out.

To determine the relative contribution of each individual microorganism to the formation of the probability of obtaining more than 40% blastocysts of good and excellent quality and to develop a prognostic index, a ranking of features was carried out using the calculation of canonical discriminant function coefficients (CDCF).

The most significant microorganisms necessary to calculate the probability of obtaining embryos of excellent and good quality were the following groups of microorganisms: Streptococcus spp., Staphylococcus spp., Corynebacterium spp., Haemophilus spp., Pseudomonas aeruginosa/Ralstonia spp./ Burkholderia spp., Enterobacteriaceae/ Enterococcus spp., Gardnerella vaginalis. Eubacterium spp., Megasphaera spp./Veillonella spp./Dialister spp., Anaerococcus spp., Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum,. The total bacterial mass (TBM) was also taken into account. Data were defined in the format Lg GE/ml.

The ECHO-Pro-A index is calculated using the formula:

ECHO-Pro-A=0.05*X1-0.14*X2+0.4*X3-0.48*X4+0.36*X5-0.17*X6+0.3*X7-0, 51*Х8+0.7*Х9+0.18*Х10-0.58*Х11+0.3*Х12-.17*Х13+0.98*Х14+0.11*Х15-0.41, where

X1 - age, years

X2 - MBP, Lg GE/ml

X3 - Staphylococcus spp., Lg GE/ml

X4 - Streptococcus spp., Lg GE/ml

X5 - Corynebacterium spp., Lg GE/ml

X6 - Gardnerella vaginalis, Lg GE/ml

X7 - Megasphaera spp./Veillonella spp./Dialister spp., Lg GE/ml

X8 - Ureaplasma urealyticum, Lg GE/ml

X9 - Ureaplasma parvum, Lg GE/ml

X10 - Mycoplasma hominis, Lg GE/ml

X11 - Anaerococcus spp., Lg GE/ml

X12 - Eubacterium spp., Lg GE/ml

X13 - Haemophilus spp., Lg GE/ml

X14 - Pseudomonas aeruginosa/Ralstonia spp./Burkholderia spp., Lg GE/ml

X15 - Enterobacteriaceae spp./Enterococcus spp., Lg GE/ml

If the Echo-Pro-A value is greater than 0.2, it is predicted that the probability of obtaining more than 40% of blastocysts of good and excellent quality is low; if the Echo-Pro-A value is less than 0, it is high; with values of 0-0.2, the prognosis is uncertain.

The average Echo-Pro-A value in groups 1 and 2 was 0.26 (-0.43 - 0.23) and 0.37 (0 - 1.63), respectively. The differences are statistically significant (p<0.001).

In FIG. Figure 2 graphically presents the values of the ECHO-Pro-N index in groups 1 and 2.

To evaluate the effectiveness of the presented forecasting method, ROC analysis was carried out. The ROC curve for the Echo-Pro-A index is depicted in FIG. 3. The area under the curve (AUC) was 0.76 (95% CI 0.65-0.87), which corresponds to the good quality of the forecasting model.

An examination sample was used to determine the sensitivity and specificity of the presented prediction method. The sensitivity and specificity indicators were 76.47% and 67.39%, respectively, and the effectiveness of the method was 71.25%. Sensitivity and specificity for various values of the ECHO-Pro-A index are presented in the table and in Figure 3.

The novelty of the proposed method lies in the fact that for the first time, based on indicators of the microbial composition of the ejaculate, spermogram parameters and the age of the man, a highly effective prediction of obtaining a sufficient number of embryos on the 5th day of development of excellent and good quality is carried out.

Distinctive features of the method: using the Androflor test, data on the microbial composition of the ejaculate is obtained from patients with asthenozoospermia and, based on the data obtained, the probability of obtaining at least 40% blastocysts of excellent and good quality is calculated.

Examples of practical use of the proposed method.

Example 1. Married couple K. applied for an IVF program for secondary infertility for 7 years, the cause of infertility was the lack of ovulation. Patient K., 41 years old, patient K., 40 years old, from the anamnesis: urgent surgical delivery 20 years ago; 8 years ago, regressing pregnancy at 7 weeks. Past gynecological diseases: cervical ectopia. Previous gynecological procedures: 10 years ago, GSG - the fallopian tubes are patent, suspicion of adenomatous endometrial hyperplasia; Histology results: small fibrous polyps in the endometrium, atrophic glands. Previous attempts at ART: 4 years ago, 2 attempts were made at intrauterine artificial insemination with my husband’s sperm, without effect. 3 years ago the first attempt at IVF was made, but pregnancy did not occur.

This is the second IVF attempt. A sample of native ejaculate was taken on the day of oocyte aspiration; the spermogram parameters correspond to asthenozoospermia. Conclusion based on the results of the Androflor test: DNA of pathogenic microorganisms was not detected. Candida spp. - below the threshold level. The structure of the bacterial microbiome is changed: opportunistic microorganisms are present. The results are presented in Fig. 4.

The ECHO-Pro-A index was calculated,

ECHO-Pro-A=0.05*X1-0.14*X2+0.4*X3-0.48*X4+0.36*X5-0.17*X6+0.3*X7-0, 51*Х8+0.7*Х9+0.18*Х10-0.58*Х11+0.3*Х12-0.17*Х13+0.98*Х14+0.11*Х15 -0.41,

where X2=4.5 Lg GE/ml, X3=3.4 Lg GE/ml, X5=3.8 Lg GE/ml, X7=3.3 Lg GE/ml, X11=3.8 Lg GE/ml , X12=4.3 Lg GE/ml, X15=3.2 Lg GE/ml, ECHO-Pro-N=2.116 index. The prognosis for obtaining embryos of excellent and good quality is poor. As a result of follicle puncture, 9 oocytes were obtained, 7 of them were mature. As a result of fertilization using the classical IVF method, 6 normally fertilized zygotes were obtained; on the 5th day, 1 blastocyst of poor quality grew from them. The number of blastocysts of excellent and good quality was 16.7%.

Example 2. A married couple M. applied for an IVF program for secondary infertility for 7 years, the cause of infertility is idiopathic. Patient M., 42 years old, patient M., 37 years old, history of medical abortion 12 years ago, term birth 10 years ago, without complications. Previous gynecological diseases: ectopia of the cervix, 6 years ago a ureaplasma infection was treated in a couple. Previous gynecological procedures: 4 years ago, GHA - the fallopian tubes are patent, suspicion of endometrial hyperplasia. This is the first attempt at IVF.

On the day of oocyte aspiration, a sample of native ejaculate was taken; the spermogram parameters correspond to asthenozoospermia. Conclusion based on the results of the Androflor test: The structure of the bacterial microbiome corresponds to the norm. No pathogenic microorganism DNA was detected. Candida spp. below threshold level. The results are presented in Fig. 5.

The ECHO-Pro-A index was calculated,

ECHO-Pro-A=0.05*X1-0.14*X2+0.4*X3-0.48*X4+0.36*X5-0.17*X6+0.3*X7-0, 51*Х8+0.7*Х9+0.18*Х10-0.58*Х11+0.3*Х12-0.17*Х13+0.98*Х14+0.11*Х15-0.41, where X2=3.3 Lg GE/ml, X4=3.1 Lg GE/ml.

ECHO-Pro-A index = -2.36 or less than 0. The prognosis for obtaining embryos of excellent and good quality is good. As a result of follicle puncture, 40 oocytes were obtained, 20 of which were mature. As a result of fertilization using the classical IVF method, 17 normal zygotes were obtained, of which 17 blastocysts grew on the 5th day of cultivation, 16 of which were of good and excellent quality. The number of blastocysts of excellent and good quality was 80%.

Example 3. A married couple P. applied for an IVF program for primary idiopathic infertility for 5 years. Patient P., 37 years old, patient P., 35 years old; From the anamnesis: hysterosalpingography (HSG) was performed 5 years ago - the fallopian tubes are passable. Over the past 4 years, menstrual irregularities and acyclic bleeding have been identified; this year, ultrasound data revealed signs of endometrial hyperplasia. This is the first attempt at IVF.

On the day of follicle puncture, a sample of native ejaculate was taken to study the microbial composition; the spermogram parameters corresponded to asthenozoospermia. Conclusion based on the results of the Androflor test: The structure of the bacterial microbiome corresponds to the norm. No pathogenic microorganism DNA was detected. Candida spp. below threshold level. The results are presented in Fig. 6.

The ECHO-Pro-A index was calculated.

ECHO-Pro-A=0.05*X1-0.14*X2+0.4*X3-0.48*X4+0.36*X5-0.17*X6+0.3*X7-0, 51*Х8+0.7*Х9+0.18*Х10-0.58*Х11+0.3*Х12-0.17*Х 13+0.98*Х14+0.11*Х15-0.41 , where X2 is equal to 3.9 Lg GE/ml, X13 is equal to 3.4 Lg GE/ml, other values are equal to 0.

ECHO-Pro-A index = 0.166. The prognosis for obtaining embryos of excellent and good quality is uncertain. On the day of follicle puncture, 5 oocytes were aspirated, 4 of them were mature. As a result of fertilization using the IVF method, 4 normal zygotes were obtained, on the 5th day 3 blastocysts grew from them, one of which was of good quality. The number of blastocysts of excellent and good quality was 25%.

Thus, the proposed method makes it possible to predict the effectiveness of the embryological stage of ART in couples planning an IVF procedure, with ejaculate parameters that meet the criteria for asthenozoospermia.

This method can be used at the stage of preliminary examination of men planning ART. If deviations in the microbial composition of the ejaculate are detected according to the results of the Androflor test, the andrologist can carry out corrective therapy before the start of ART treatment, as well as select tactics for managing the embryological stage of the ART program. If the prognosis for obtaining a sufficient number of embryos is low, fertilization by intracytoplasmic sperm injection (ICSI) may be recommended, as well as the use of a more thorough stage of “washing” the sperm used for fertilization.

The method is easy to use, as it does not require additional invasive interventions, is easily accessible, provides quick acquisition of the necessary information, and is applicable before starting infertility treatment using ART. Evaluation of the ejaculate microbiota using RT-PCR can be performed simultaneously with a spermogram from one sample.

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Claims (18)

A method for predicting the production of embryos of excellent and good quality, including the “Androflor” test in the ejaculate, the parameters of which correspond to the criteria for asthenozoospermia used for fertilization, with subsequent calculation of the prognostic index, characterized in that the ECHO-Pro-A prognostic index is calculated based on microbiological analysis, which is calculated by the formula ECHO-Pro-A=0.05*X1-0.14*X2+0.4*X3-0.48*X4+0.36*X5-0.17*X6+0.3*X7-0, 51*Х8+0.7*Х9+0.18*Х10-0.58*Х11+0.3*Х12-0.17*Х13+0.98*Х14+0.11*Х15-0.41, where X1 is age, years, X2 - MBP, Lg GE/ml, X3 - Staphylococcus spp., Lg GE/ml, X4 - Streptococcus spp., Lg GE/ml, X5 - Corynebacterium spp., Lg GE/ml, X6 - Gardnerella vaginalis, Lg GE/ml, X7 - Megasphaera spp./Veillonella spp./Dialister spp, Lg GE/ml, X8 - Ureaplasma urealyticum, Lg GE/ml, X9 - Ureaplasma parvum, Lg GE/ml, X10 - Mycoplasma hominis, Lg GE/ml, X11 - Anaerococcus spp., Lg GE/ml, X12 - Eubacterium spp., Lg GE/ml, X13 - Haemophilus spp., Lg GE/ml, X14 - Pseudomonas aeruginosa/Ralstonia spp./Burkholderia spp., Lg GE/ml, X15 - Enterobacteriaceae spp./Enterococcus spp., Lg GE/ml, if the Echo-Pro-A value is more than 0.2, the probability of obtaining more than 40% of embryos of good and excellent quality is low; if the Echo-Pro-A value is less than 0, it is high; with values of 0-0.2, the prognosis is uncertain.