Diagnostic technique for abnormal chromatin packaging in male infertility

FIELD: medicine.

SUBSTANCE: diagnostic technique for abnormal chromatin packing in male sterility is based on a quantitative estimation of chromatin hydrolysis depth in sperm nuclei immobilised on slides. Hydrolysis by micrococcal nuclease is followed by preparation washing from soluble chromatin. Chromatin DNA without hydrolysis by nuclease and after hydrolysis is stained by fluorochrome DAPI and examined for the distinctions in a degree of fluorescence of the initial and nuclease-processed samples. A check value is a nature of chromatin hydrolyzate of fertile donor sperm cells characterised by the spermogram values. The prepared images are analysed by comparing brightness of the sperm cell images, and the abnormal chromatin packaging is shown by DNA amount extracted by nuclease relatively to total DNA.

EFFECT: use of the declared technique allows simplifying diagnosing of male sterility within the therapies in extracorporeal fertilisation and homologous insemination programs.

6 cl, 1 ex, 2 tbl

 

The invention relates to the field of medicine and veterinary medicine, namely to the field of assisted reproductive technologies, in particular to increase the pregnancy rate, and can be used in the treatment of infertility in programs of in vitro fertilization and homologous insemination by sperm sorting of samples containing cells with abnormal chromatin packaging, and diagnostics of the need for a specific therapy or restorative treatments with unfavorable prognosis regarding the effectiveness of assisted reproductive technologies.

Now who accepted methods of diagnosing abnormalities of spermatozoa, leading to male infertility, based on the estimate of their quantity, motility and detection of DNA breaks. At the same time it is shown that one common cause of male infertility is abnormal or not fully completed compaction of the DNA genome in semen.

Known (EN, application 2006130740) the method of determining the integrity of chromatin/DNA in the sperm cells of animals, including the stage of processing of a sample containing sperm cells, denaturing the DNA solution, one stage of processing lytic solution for extraction of nuclear proteins, the stage of determining the integrity of chromatin/DNA of sperm, characterizes the Yuexiu fact, that lyse solution does not contain denaturing proteins detergent and does not destroy much of the tails of sperm.

The disadvantage of this method should recognize that assesses the integrity of DNA in the presence or absence of tears, and this figure does not correlate with the pathology associated with incomplete or abnormal chromatin packaging.

Also known (RU, patent No. 2262107) method of determining maturity of sperm by assessing the degree of condensation of the chromatin of spermatozoa in the study of ejaculate. In the process of defining comparing the fluorescence intensities of sperm DNA before and after violent decondense chromatin heparin by means of static processing method Kolmogorov-Smirnov with the definition of the similarity index curves of the fluorescence intensity of n, a value that is less than 48,5 judge immature sperm, and when the value n is greater than 48,5 judge ripeness of sperm.

The disadvantage of this method should recognize that the property of chromatin to deconcentrate under the action of heparin only indirectly reflects anomalies in its packaging.

Also known (RU, patent No. 2118822) a method for the diagnosis of male infertility by flow-cytofluorimetric analysis of sperm chromatin, characterized in that state of chromatin OC is Nivat on the fluorescence intensity of nuclei of cells, painted bromide by ethidium before and after treatment with heparin, and in the presence of semen, in which the intensity of fluorescence increased source more than 30% of the cells, or it increases more than 2 times in more than 50% of the cells after treatment with heparin, diagnose male infertility.

The disadvantage of this method should be recognized that the method is not strictly quantitative, and how and when decondensation heparin, only indirectly reflect anomalies in the packaging of chromatin. In addition, this method requires complex and expensive equipment and cannot be used for widespread screening.

Known (Fernandez J.L. J.Androl. 2003, v.24(1), h/h/59-66) a method for diagnosing anomalies packaging of the chromatin of spermatozoa in male infertility, with the implementation of the method carried out a study of the state of chromatin using a fluorescent microscope and DAPI.

Significant disadvantages of the method should recognize the complexity and long time (about 2 weeks) sample preparation. It is fundamentally important that the method does not allow to quantify accurately the proportion of spermatozoa with damaged DNA compaction and objectively describe the depth of this pathology.

The technical problem to be solved by the developed method consists in establishing the diagnosis and what omali packaging of the chromatin of spermatozoa in male infertility.

The technical result obtained by the implementation of the developed method consists in the simplification and cheapening of the method while increasing the informativeness of the study.

To achieve the technical result of the proposed use of the developed method for the diagnosis of abnormalities of the packaging of the chromatin of spermatozoa in male infertility. According to the developed method to determine the number of spermatozoa in 1 ml, sperm concentration adjusted to 10-50 million/ml with PBS buffer, the cover glass is covered with a layer of polylysine, conduct immobilization of spermatozoa on the prepared cover glass, fix and paint the kernel part of immobilized spermatozoa DNA-binding fluorochrome DAPI obtaining control samples, the second part of immobilized spermatozoa treated Micrococcus a nuclease, spend fixation and staining of the nuclei of spermatozoa treated Micrococcus a nuclease, DNA-binding fluorochrome DAPI stained preparations of control sperm and sperm treated Micrococcus a nuclease, photographed using a fluorescent microscope and analyze the image by comparing fluorescence intensity images of sperm. The decrease in the fluorescence intensity of the poison is R spermatozoa after treatment Micrococcus the nuclease reflects the release of soluble (available for hydrolysis Micrococcus by nuclease) chromatin of the nuclei. Quantitatively, the proportion of soluble chromatin corresponds to the difference between the fluorescence intensity of nuclei in control and after treatment Micrococcus the nuclease and expressed as a percentage of control. The increase in the share of soluble chromatin shows the presence of anomalies in the packaging of chromatin, which are one of the causes of male infertility.

During the implementation of the developed method, you must perform the following procedures.

1. To determine the number of spermatozoa in 1 ml solution. To do this, use a standard camera Goryaeva, which in Hematology used for counting blood corpuscles. Determine the number of sperm in the sample. The sperm concentration was adjusted to 10-50 million/ml with PBS buffer. This procedure is necessary to calculate the concentration micrococcal nucleases. The resulting suspension cannot be stored in a frozen state!

2. To prepare the glass. To do this, cover a total area of 24×24 mm2cut by the cutter into fragments with an area of about 3×5 mm2. To reduce the size of the glasses should be for use in subsequent procedures standard tubes "Eppendorf" with a volume of 1.7 ml Next glass tear solution polylysine (concentration 25 µg/ml) and left for 30 min at room temperature, covered from dust. The volume of solution should be such that pokr is th most of the glass (10-50 μl, depending on the area). After 30 minutes the pipette away the excess solution polylysine with glass back in the tube for later use. The solution polylysine must be stored at a temperature of 20°C. Further, the glass loosely in a closed Petri dish is placed in a thermostat at 37°C until complete dryness polylysine. Glass is recommended to be prepared immediately prior to diagnosis, as during long-term storage, they lose their adhesive properties.

3. To carry out the immobilization of spermatozoa on the prepared glasses. This covered polylysine glass is placed in a plastic Petri dish with a diameter of 4 cm, in a Cup pour 2 ml of sperm suspension, diluted if necessary, PBS. The sperm is then precipitated in a centrifuge at acceleration 670g, 10 minutes At this stage, the drugs should not be dry.

4. To hold the fixation and staining of the nuclei of the sperm DNA-binding fluorochrome DAPI in control. For this glass with immobilized spermatozoa treated with 0.5% solution of Triton X-100 in PBS for 10 min at room temperature and washed in three changes of PBS for 5 min For staining DNA glass is placed in a solution of DAPI (0.1 mg/ml in PBS) for 10 min, washed off the dye two changes of PBS for 5 min After staining glass with spermatozoa conclude on slides in a drop of Moviol with DABCO (volume drops about µl). Promised so drugs are controlled. Prisoners in Moviol drugs can be stored in the refrigerator for 1-2 months.

5. To handle the sperm Micrococcus the nuclease. For this glass with immobilized sperm transfer in tubes with PBS with 0.5 mM PMSF, leave on for 5 minutes and carefully selected solution. The procedure is repeated 2-3 times. The subsequent stage is carried out in ice. In tubes with glass add PBS with 0.5 mM PMSF and 0.5% Triton X100 and incubated for 10 min in ice. After incubation, the glass is washed 2 times with PBS 0.5 mM PMSF, carefully selecting the liquid. Then hold incubation of the slides in the reaction buffer: 10 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 2 mM MgCl2and add micrococcal nuclease 10 IU/mg, 20 IU/mg, 30 IU/mg, 40 u/mg, 80 IU/mg. Incubation carried out for 10 min at room temperature. To stop reaction for samples add 100 mM EDTA to a final concentration of 10 mM and incubated for 30 min in ice. Glass 2-3 times washed with reaction buffer without EDTA.

6. To hold the fixation and staining of the nuclei of spermatozoa treated Micrococcus a nuclease, DNA-binding fluorochrome DAPI. Drugs placed in fixative (3% paraformaldehyde in PBS) for 15 min at room temperature, washed from release two changes of PBS for 5 min For staining DNA glass is placed in a solution of DAPI (0.1 μg/ml in PBS) for 10 min, washed off the dye is two changes of PBS for 5 minutes After staining glass with spermatozoa conclude on slides in a drop of Moviol with DABCO (volume drops about 5 µl).

7. Registration and image processing. Stained preparations of control sperm and sperm treated Micrococcus a nuclease, photographed on a microscope Zeiss Axiovert 200M equipped with a camera Hamamatsu Orca II-ERG2 and connected to the computer. To register and view images using the Zeiss Axio Vision v4.5. A survey carried out on the lens X20 (you can use X40). The obtained images are exported in .tif and analyzed using the software ImageJ. For image analysis, conduct the following:

7.1. The program ImageJ, you must call the Analyze menu->Set measurements and put the marker opposite the Integrated density, removing all other tokens.

Open the image and enlarge it to a convenient scale (~h).

7.2. Using the menu you can make the dull parts of the sperm is clearly visible;

7.3. To allocate sperm (the free select tool, similar to the Lasso tool in Photoshop), to measure the total brightness. Then, without resetting the selection to move it in the blank area (black background) and re-measure the brightness. The total brightness of the sperm and the background will be automatically added to the table. To select spermatozoa with high accuracy is not required

7.4. To copy the received data into the spreadsheet Excel.

7.5. Subtract pairs of the background brightness from the brightness of the object for each measurement.

7.6. The obtained data are expressed graphically.

In the future, the essence of the developed method will be discussed in detail.

The essence of the proposed method in identifying anomalies packaging of the chromatin of sperm on his availability for hydrolysis Micrococcus the nuclease.

The method consists in quantifying the depth of hydrolysis of DNA chromatin Micrococcus the nuclease. After hydrolysis of chromatin produce staining of the nuclei of sperm fluorochrome DAPI. Control is the nature of the hydrolysis of the chromatin of sperm donors and sperm parameters corresponding norm.

The method is as follows.

Fresh sperm cells taken from healthy donors and infertile patients without treatment from related somatic and not differentiated cells, immobilized on the glass pre-coated with polylysine. For immobilization of spermatozoa, you can use a commercial glass coated with a special adhesive for cell structures. Using centrifugation at 650 g treated with 0.5% solution of Triton X-100 in PBS for 10 min at room temperature and washed in three changes of PBS for 5 minutes in each shift. At this point you can about the tis single washing, and instead of PBS, you can use another buffer with the appropriate pH and ionic strength.

Then the glass is incubated in a solution of 20 mm Tris-Hcl (pH 8), 2 mm CaCl2, 2 mm MgCl2. You can use 10 mm Tris-HCl, however, in this case, the concentration of CaCl2and MgCl2should be 1 mm. To the medium was added micrococcal nuclease in the concentration range from 10 IU/mg to 80 u/mg Sample is incubated for 3 minutes at a temperature of not lower than 10°C and above 40°C. the hydrolysis Reaction is stopped by adding EDTA to 10 mm in ice.

Received the drugs are divided into 2 groups: glass with the sperm cells, not treated with nuclease (control), and glass with spermatozoa treated with nuclease. Drugs placed in fixative (3% paraformaldehyde in PBS) for 15 min at room temperature, washed from release two changes of PBS for 5 minutes Instead of paraformaldehyde you can use any release, stabilizing chromatin in the nuclei of the sperm. For staining DNA glass is placed in a solution of DAPI (0.1 μg/ml in PBS) for 10 min, washed off the dye two changes of PBS for 5 minutes Instead of fluorochrome DAPI you can use any DNA-binding fluorochrome. After staining glass with spermatozoa conclude on slides in a drop of Moviol with DABCO (volume drops about 5 µl). You can use other environment conclusions, not about lagausie own fluorescence. The relative amount of DNA in the control and the treated spermatozoa determined by the fluorescence intensity of fluorochrome. For this purpose, the samples are photographed in fluorescent microscope Zeiss Axiovert 200M equipped with a camera Hamamatsu Orca II-ERG2 and connected to the computer. You can use any fluorescence microscope equipped with a camera and connected to the computer. To register and view images using the Zeiss Axio Vision v4.5. A survey carried out on the lens X20 (you can use X40). The obtained images are exported in .tif and analyzed using the software ImageJ. Method tests the differences between the amount of DNA in the sperm cells, not treated Micrococcus the nuclease and DNA content in spermatozoa treated Micrococcus the nuclease. Differences in the content of insoluble nuclease chromatin are expressed in percentage of the control.

The method was tested on samples of healthy sperm donors and sperm samples infertile patients provided by the family planning Center. The method was implemented as described above. Below are the results.

4.1. Examples of healthy donors:

93,9
No. of donorsInsoluble chromatin (%)
1613
21088,68
200888,73

4.2. Examples infertile patients

No. of patientsInsoluble chromatin (%)
165,36
262,42
377,33

Therefore, for semen collected from healthy donors, the amount of DNA extracted by micronucleate, from the total amount of DNA does not exceed 3%, and sperm taken from infertile patients, the content of extracted DNA from total DNA, usually increased 10-20 times and in some cases reaches 60% and above.

Received data corraleros with electronmicroscopical studies of the same donors and patients and represent an accurate assessment of the content in the ejaculate infertile patients of abnormal sperm.

1. The way to diagnose anomalies packaging of the chromatin of spermatozoa in male infertility, including the study of the state of chromatin using a fluorescent microscope and a colorant, characterized in that stoopidest the number of spermatozoa in 1 ml of solution, the sperm concentration was adjusted to 10-50 million/ml with PBS buffer, the cover glass is covered with a layer of polylysine, conduct immobilization of spermatozoa on the prepared cover glass with an area of about 3×5 mm2fix and paint the kernel part of immobilized spermatozoa DNA-binding fluorochrome DAPI obtaining control samples, the second part of immobilized spermatozoa treated Micrococcus a nuclease, spend fixation and staining of the nuclei of spermatozoa treated Micrococcus a nuclease, DNA-binding fluorochrome DAPI stained preparations of control sperm and sperm treated Micrococcus a nuclease, photographed using a microscope and analyze the image by comparing the brightness of the images of sperm, and an anomaly packaging of the chromatin of sperm judged by the amount of DNA extracted by the nuclease, relative to the total amount of DNA in the control.

2. The method according to claim 1, characterized in that the determination of the number of spermatozoa is performed using the standard camera Goryaeva, which in Hematology used for counting blood corpuscles.

3. The method according to claim 1, characterized in that the glass cover with a solution of polylysine with a concentration of 25 μg/ml and left for 30 min at room the first temperature, covering of dust.

4. The method according to claim 1, characterized in that the glass is prepared immediately before diagnosis.

5. The method according to claim 1, characterized in that, during the fixation of the nuclei of the sperm DNA-binding fluorochrome DAPI glass with immobilized spermatozoa treated with 0.5%solution of Triton X-100 in PBS for 10 min at room temperature and washed in three changes of PBS for 5 minutes

6. The method according to claim 1, characterized in that for staining DNA glass is placed in a solution of DAPI concentration of 0.1 μg/ml in PBS for 10 min, washed off the dye two changes of PBS for 5 minutes



 

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FIELD: medicine.

SUBSTANCE: method involves application of a diagnostic multiplex panel (DMP) designed for simultaneous identification of a set of possible microorganisms which can be found in a biological sample, with applying primer extension reaction to high conserved nucleotide sequences in sampled microorganisms. The biological sample is immobilised either on, or in a solid substratum in a first position, then transferred to the other position and extracted on the solid substratum so that to extract DNA of any microorganism which is found in the sample. It is followed by amplification of nucleic acid on the extracted DNA of microorganisms, then target sequences are mixed with primers with using the DMP. It results in genotyping of any microorganisms found and identifying of the required microorganisms. For implementation of the method, the diagnostic multiplex panel (DMP) applicable for genotyping of pathogenic microorganisms, and a testing kit for microorganisms is used.

EFFECT: use of the invention allows reliable selection of the biological samples and their testing, providing simultaneous testing of a set of microorganisms.

22 cl, 2 dwg, 3 tbl, 1 ex

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