JP5092149B2 - Test method for sperm function - Google Patents

Description

  The present invention relates to a sperm function test method. Specifically, the present invention relates to a sperm function test method using sperm nuclear DNA fragmentation as an index, a sperm function test method using sperm head vacuoles as an index, and a sperm function test method combining these test methods. The present invention also relates to a sperm nuclear DNA fragmentation detection method and a sperm head vacuole measurement method. Furthermore, the present invention relates to a method for diagnosing male infertility, characterized in that sperm function is evaluated by the sperm function test method. The present invention also relates to a sperm sorting method characterized by evaluating a sperm function by a sperm function test method using a sperm head vacuole as an index and selecting a sperm having a good function.

  In advanced countries such as Japan and Europe, about 10% of all couples have infertility problems, and the ratio of infertility cases is increasing due to recent problems such as stress society and environmental hormones. Since infertility patients have a variety of causes, the cause is usually identified through repeated examinations, and a treatment method suitable for the cause is received.

  About half of infertility cases are thought to be due to male factors. The majority of male infertility is due to insufficient spermatogenesis, sperm loss and sperm dysfunction.

  The sperm has a total length of about 50 μm, and its structure can be roughly divided into three parts: the head, the middle piece of the tail (flagella), and the main part of the tail (flagella). The head is covered with a plasma membrane, most of which consists of a nucleus containing chromatin, and there is a precursor that produces and secretes the enzymes required for fertilization at the tip. The middle part of the tail consists of mitochondria and is the source of kinetic energy. The main part of the tail is composed of a skeletal component related to movement and its related protein, and the movement of sperm occurs due to the contraction and extension. Mammalian finished sperm nuclei are highly agglomerated, especially rodent sperm nuclei are completely agglomerated and no vacuoles are observed in the nucleus, whereas the degree of aggregation of human sperm nuclei is uneven. There are multiple large and small nuclear vacuoles (clinically called head vacuoles). Within the sperm nucleus, chromatin exists in a highly condensed state. As a basic structure for achieving such an aggregation state, a structure called a toroid containing a double-stranded DNA of about 50-60 kb is recognized, where double-stranded DNA is condensed by the nuclear protein protamine. Each toroid is tethered to the matrix attachment region of the nuclear matrix by a toroid linker.

The basis of the test for male infertility is a semen test including sperm, which measures semen volume, sperm density, motile sperm concentration, sperm motility rate, sperm malformation rate, and the like. The standard values for normal semen are, according to the World Health Organization (WHO) standard established in 1999, the amount of semen is 2.0 ml or more per ejaculation, the pH is 7.2 or more, and the sperm concentration is 20 ×. 10 ⁶ / ml or more, total sperm count 40 × 10 ⁶ or more, and sperm motility rate within 60 minutes after ejaculation 50% or more of fast moving sperm or 25% or more of fast moving sperm, normal sperm rate is Kruger test 15% or more and sperm survival rate is 75% or more. Male infertility is diagnosed and classified based on semen findings from such tests.

  On the other hand, even in the case of sperm that is judged to be normal in quantity, form, and motility, there are cases in which there is an abnormality in the nucleus of the sperm, which causes infertility. As a method for detecting abnormalities in the sperm nucleus, such as sperm chromatin defects and DNA damage, a sperm chromatin diffusion test (SPDT) is known (Non-patent Documents 1 to 7). In this test, double-stranded DNA in the toroid is dissociated into single-stranded DNA, then the toroid linker is cleaved with sodium dodecyl sulfate (SDS) or a lysate to release the toroid from the nuclear matrix, and the DNA in the toroid is further removed. And the DNA extending out of the nucleus is detected by dye staining. If the chromatin and DNA are normal, the detected DNA is long, and thus a ring-shaped stained image called halo is detected around the sperm head. On the other hand, such stained images are small or not recognized in sperm having a defect in chromatin or DNA damage. That is, in this test, abnormalities in the sperm nucleus are detected based on the size of the DNA stained image. However, in this test, since the sperm head and tail are separated by the use of SDS or a lysis solution, the separated head is measured, but the measured head is separated by chemical treatment. In addition to the severed head, there is a problem that the head of an already dead one is included. Accordingly, there is a demand for providing a more accurate method for detecting abnormalities in the sperm nucleus. In addition to this test, sperm chromatin structure test (SCSA) is known as a method for detecting abnormalities in the sperm nucleus (Non-patent Document 8). This test is a method of detecting the proportion of abnormal DNA by staining normal DNA and abnormal DNA with two types of fluorescent dyes that bind to each DNA. Such a fluorescence method requires a quick operation in terms of fluorescence intensity, fluorescence time, and the like, and also requires a device for measuring fluorescence, which causes problems such as complicated operation and high costs. Have. Therefore, there is a demand for providing a method for detecting abnormalities in the sperm nucleus in which these problems are eliminated.

  In recent years, with the spread of treatment by reproductive support techniques, for example, in vitro fertilization (IVF) such as intracytoplasmic sperm microinjection (ICSI) and intrauterine insemination (IUI). In the selection of sperm for use in such treatment, evaluation of sperm function has been carried out. Even sperm without motility survive, and there are sperm that can be fertilized by ICSI. A sperm swelling test is known as a method for confirming such sperm. Also, seemingly normal sperm may have abnormal fertility. As a test method for insemination ability of sperm, for example, a hamster test or the like is known. In addition, methods for evaluating cervical mucus permeability and acrosome reaction induction have been reported.

  In addition, intraspermologically selected perm microinjection (IMSI) based on morphological selection has recently been used in clinical practice, and in addition to the morphology of each part of sperm, the morphology of the inner vacuole Properties (number, size, contents, etc.) are attracting attention. It is known that when excessive nuclear membranes or cytoplasmic components remain in the sperm head, active oxygen is likely to be produced, and as a result, oxidative stress is likely to cause fragmentation of sperm nuclear DNA (non- Patent Documents 7 and 9), the nature of the nuclear vacuole may be an indicator of the maturity of the sperm nucleus. In ICSI using sperm that shows normal morphology but has large vacuoles, it has been reported that the fertility rate is reduced (Non-patent Document 10), but the criteria and determination method in this report have not been established. In addition, the relationship between the morphological characteristics of vacuoles and fertility is still unclear. For this reason, establishment of criteria and determination methods for selecting sperm based on the morphological characteristics of vacuoles is required.

Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG.The sperm chromatin dispersion test: A simple method for the determination of sperm DNA fragmentation.J Androl 24 (1): 59-66 (2003). Muriel L, Segrelles E, Goyanes V, Gosalvez J, Fernandez JL. Structure of human sperm DNA and background damage, analyzed by in situ clinical and digital image analysis.Mole Human Reprod 10 (3): 1-7 (2004). Fernandez JL, Muriel L, Goyanes G, Segrelles E, Gosalvez J, Enciso M, Lafromboise M, De Jonge C. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertili Steril 84 (4): 833-842 (2005). Enciso M, Muriel L, Fernandez JL, Goyanes V, Segrelles E, Marcos M, Montejo JM, Ardoy M, Pacheco A, GosalvezJ. Infertile men with Varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test. J Androl 27 (1): 106-111 (2006). Perez-Llano B, Enciso M, Garcia-Casado P, Sala R, Gosalvez J. Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders. Theriogenology 66: 2137-2143 (2006). Muriel L, Garrido N, Fernandez JL, Remohi J, Pellicer A, De Los Santos MJ, Meseguer M. Value of the sperm deoxyribonucleic acid fragmentation level, as measureed by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertili Steril 85 (2): 371-383 (2006). Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT.Comparsion of chromatin assays for DNA fragmantation evaluation in human sperm.J Androl 27 (1): 53-59 (2006). Evenson DP, Larson KL, Jost LK.The sperm chromatin structure assay (SCSA (R)): clinical use for detecting sperm DNA fragmentation related to male infertility and comparisons with other techniques.Andrology Lab Corner.J Androl 23 (1): 25 -43 (2002). Aitken RJ, De Iuliis GN. Value of DNA integrity assays for fertility evaluation. Soc Reprod Fertil Suppl. 65: 81-92 (2007). Berkovitz A, Eltes F, Ellenbogen A, Peer S. Feldberg D, Bartoov B. Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Human Reprod 21 (7): 1787-1790 (2006).

  There are various causes of sperm function decline in male infertility, and many tests are required for the determination, but many of the conventional tests have problems such as accuracy, operability and economy. It was. In recent years, in the treatment of infertility, with the widespread use of assisted reproduction techniques, such as microinsemination such as ICSI and IMSI, in vitro insemination, and in utero insemination, it is important to select sperm for such treatment. Occupies a position. An object of the present invention is to provide an inspection method that is useful for determination of sperm function and in which the above problems are solved.

  The present inventor has conducted intensive research to achieve the above object, found a method for detecting sperm nuclear DNA fragmentation with high accuracy, and a data classification method and a sperm function test method useful for simply analyzing sperm function. Established. Furthermore, the present inventor has established a method for evaluating sperm function by the sperm head vacuole and a data analysis method for the evaluation. The present invention has been achieved based on these findings.

That is, the present invention is a method for detecting sperm nuclear DNA fragmentation,
(1) dissociating double-stranded DNA in the toroid in the sperm nucleus into single-stranded DNA;
(2) cleaving the toroid linker to release the toroid from the nuclear matrix;
(3) extending the DNA in the toroid;
(4) including a step of detecting extended DNA, wherein the steps (2) and (3) are performed using Triton X-100 (Triton X-100, where “Triton” is a trade name). It relates to a method characterized in that it is performed.

  Further, the present invention provides the sperm nuclear DNA fragmentation detection wherein the step of detecting the elongated DNA is a step of measuring the size of a circular stained image formed on the sperm head after dyeing the elongated DNA. Regarding the method.

Furthermore, the present invention compares the width (A) of the stained image with the head width (B) of the untreated sperm and classifies the stained images according to the value of A / B as follows:
(I) A / B value is 3.0 or greater,
(Ii) the value of A / B is less than 3.0 and 1.5 or greater,
(Iii) the value of A / B is less than 1.5 and 1.0 or greater;
(Iv) A / B value is less than 1.0 and intense staining;
(V) A / B value is less than 1.0 and weakly or unstained,
The sperm showing the stained image classified as (i) or (ii) above is classified into any one of the above (iii), (iv) and (v) The sperm showing the stained image relates to the sperm nuclear DNA fragmentation detection method, wherein the sperm nuclear DNA is evaluated to be fragmented.

  Furthermore, the present invention relates to a sperm function test method for detecting sperm nuclear DNA fragmentation using any one of the above-described sperm nuclear DNA fragmentation detection methods and evaluating sperm function using sperm nuclear DNA fragmentation as an index.

  Further, the present invention evaluates sperm function using sperm nuclear DNA fragmentation as an index, and sperm function using DNA fragmentation index (DFI), which is the ratio of the number of nuclear DNA fragmented sperm to the total number of measured sperm, as an index. In this case, the sperm function test method determines that the sperm function is defective when the DFI is 10% or more.

Furthermore, the present invention is a method for measuring sperm head vacuole,
(A) measuring the number and width of vacuoles in the sperm head;
(B) Comparing the vacuole width (C) with the sperm head width (D) and classifying the vacuoles according to the value of C / D;
(I) A vacuole (vacuum L) having a C / D value greater than 1/2 is score 3,
(II) A vacuole (vacuum M) having a C / D value of 1/3 or larger and 1/2 or smaller is score 2,
(III) A vacuole (vacuum S) having a C / D value smaller than 1/3 is score 1,
(IV) score 0 if no vacuole,
Classifying and scoring according to
(C) Formula 1 below;
A step of calculating a sperm head vacuolar area score by
The present invention relates to a sperm head vacuole measurement method including

  Furthermore, the present invention provides a sperm head vacuole measurement using the sperm head vacuole measurement method, and the sperm function using the number of sperm head vacuoles and / or the sperm head vacuole area score as an index. The present invention relates to a sperm function test method characterized by

  The present invention also provides a sperm function test method for evaluating sperm function using the sperm nuclear DNA fragmentation as an index, and the number of sperm head vacuoles and / or the sperm head vacuole area score as an index. The present invention relates to a sperm function test method comprising a combination of sperm function test methods for evaluating functions.

  Furthermore, the present invention provides a method for determining whether or not a semen sample is a semen sample derived from a male infertility patient, wherein the function of sperm contained in the semen sample is evaluated by any one of the sperm function testing methods. In addition, the present invention relates to a method for determining that a semen sample evaluated as having a poor sperm function is a semen sample derived from a male infertility patient.

  Furthermore, the present invention evaluates sperm function using the sperm function test method, wherein the sperm function is evaluated using the number of sperm head vacuoles and / or the sperm head vacuole area score as an index. The present invention relates to a sperm selection method characterized by selecting a sperm having a good function.

  According to the present invention, a sperm function test method using sperm nuclear DNA fragmentation as an index, a sperm function test method using sperm head vacuoles as an index, and a sperm function test method combining these test methods can be provided. The present invention can also provide a method for detecting sperm nuclear DNA fragmentation and a method for measuring sperm head vacuole.

  The sperm function testing method according to the present invention using sperm nuclear DNA fragmentation as an index is highly accurate, does not require complicated operations, and does not require special equipment, and thus can be performed at low cost. Furthermore, since the test can be performed with a small amount of specimen, the burden on the patient is small. Moreover, the data analysis of the sperm function test method can be performed quickly and easily by the classification method established in the present invention. As described above, the present sperm function test method has an extremely superior advantage compared to the sperm function test method that uses sperm nuclear DNA fragmentation as an index.

  The sperm function test method using the sperm head vacuole as an index does not require complicated operations, and the data analysis method established in the present invention enables quick and simple data analysis of the sperm function test method. Conventionally, since a sperm function test method using the sperm head vacuole as an index has not been carried out, the provision of this sperm function test method is useful in pursuing and diagnosing various causes of male infertility. Moreover, since this sperm function test | inspection method can be implemented in the state in which a sperm is alive, it is useful for selection of the sperm with the favorable function used for in vitro fertilization.

  The present invention thus exhibits excellent effects in the fields of diagnosis and treatment of male infertility and research on the cause thereof.

  The present invention provides a method for examining sperm function. This test method of sperm function is useful for diagnosis and treatment of male infertility. “Sperm function” means the insemination ability of sperm. Here, “insemination ability of sperm” means the ability of a sperm to perform a series of processes that reach the egg and fuse and then form a fertilized egg to reach embryogenesis. “Embryonic development” refers to the process from the fertilized egg through adult cell division to adulthood. When saying “good sperm function”, it means that the sperm has fertility. On the other hand, when saying “poor sperm function”, it means that the sperm does not have fertility or has low fertility and therefore does not eventually lead to embryo development. “Male infertility” refers to infertility that is thought to be caused by male factors such as insufficient spermatogenesis, sperm loss and sperm dysfunction. “Infertility” refers to a condition in which there is normal sexual activity and no pregnancy for two years.

  More specifically, the sperm function test method according to the present invention is a sperm function test method using sperm nuclear DNA fragmentation as an index, a sperm function test method using sperm head vacuoles as an index, and a combination of these test methods. It relates to the sperm function test method.

  The present invention also provides a sperm nuclear DNA fragmentation detection method and a sperm head vacuole measurement method.

This sperm nuclear DNA fragmentation detection method comprises the following steps:
(1) dissociating double-stranded DNA in the toroid in the sperm nucleus into single-stranded DNA;
(2) cleaving the toroid linker to release the toroid from the nuclear matrix;
(3) extending the DNA in the toroid;
(4) a step of detecting the elongated DNA;
(See FIG. 1).

  “Sperm nuclear DNA fragmentation” refers to a state in which DNA stored in the nucleus existing in the sperm head is shorter than normal. “Toroid” refers to a donut-shaped structure containing a sperm-type linear nuclear DNA strand, which includes a double-stranded DNA of about 50-60 kb. In the toroid, double-stranded DNA is condensed by the nucleoprotein protamine. “Toroid linker” refers to DNA that anchors toroids to the matrix attachment region of the nuclear matrix. The “nuclear matrix” refers to a skeleton structure in the nucleus composed of nucleic acids and proteins. “Extending DNA” refers to stretching condensed DNA.

  In this sperm nuclear DNA fragmentation detection method, the step (2) of cleaving the toroid linker to release the toroid from the nuclear matrix and the step (3) of extending the DNA in the toroid are carried out using Triton X-100. It is characterized by that. A conventional sperm chromatin diffusion test reported as a sperm nuclear DNA fragmentation detection method includes the same steps, but the steps (2) and (3) are performed using SDS or a lysis solution. Since the head and tail of the sperm are separated by the use of SDS or a lysate, the separated head is measured by the conventional method, but the measured head is separated by the chemical treatment. In addition to the head, a head that has already died is included, resulting in a problem that the accuracy of the test decreases. Since the head and tail are not cut in Triton X-100, this sperm nuclear DNA fragmentation detection method is more accurate than the conventional method. The concentration of Triton X-100 used is preferably 0.1% to 2%, and more preferably 1%, for example. The concentration of Triton X-100 is not limited to the exemplified concentration, and the above steps (2) and (3) can be carried out, and the sperm protein is not denatured and the sperm head and tail are not separated. Any concentration may be used.

  Further, in this step, a neutral polymer surfactant or the like capable of dissociating a binding protein or the like with a large molecule, which is not strong in protein denaturation like SDS, can be used. Preferably, the above Triton X-100 is used. For example, other Triton series (Triton WR-1339, Triton X-305, Triton X-405, Triton X-114, Triton CF-10, etc.) and NP-40 (Nonidet P40, Nonidet are trademarks) Furthermore, Tween 20 (Tween 20, Tween is a trade name) or the like can be used.

  The step (1) of dissociating the double-stranded DNA in the toroid in the sperm nucleus into single-stranded DNA can be performed, for example, by acid treatment, but any means that can dissociate the double-stranded DNA into single-stranded DNA. There is no particular limitation to acid treatment, and any means can be used. As the acid treatment, hydrochloric acid treatment is preferably used. When hydrochloric acid is used, the concentration is preferably in the range of 0.01N to 1N, and more preferably 0.08N, for example.

  The step (4) of detecting the elongated DNA can be carried out by measuring the size of a circular stained image called halo formed on the sperm head after dyeing the elongated DNA. Any dye can be used as long as it is a dye generally used for staining DNA or nuclei. Preferably, a light staining solution is used. For dyeing DNA and nuclei, fluorescent dyes such as propidium iodide (PI), DAPI (4 ′, 6-diamidino-2-phenylindole), Hoechst 33258 can be used. In order to maintain high quantitative accuracy in relation to time, conditions such as rapid measurement are required. In addition, an apparatus for measuring fluorescence is required, and there are problems such as operational complexity and high costs. On the other hand, light staining can be stored semi-permanently after the staining, so that repeated measurement is possible and the quantitativeness is high. In light staining, the staining solution is inexpensive, the stained image can be observed with an optical microscope, and the operation process is simple and easy. Wright dyeing can be performed with reference to a commonly used method.

  The size of the stained image can be measured by a commonly used measuring method corresponding to the dye used for staining. When light staining is used as the pigment, the stained image can be observed with an optical microscope. When a fluorescent dye is used as the dye, the stained image can be observed with a fluorescence microscope. The size of the stained image can be measured visually under a microscope. In consideration of quantitativeness, it is preferable to connect a computer to a microscope, capture the image data of the stained image into the computer, and measure the size of the stained image on the computer screen.

  “Circle” refers to a round disk shape and includes not only a perfect circle but also an ellipse.

  If chromatin or DNA is normal, the detected DNA is long, and therefore a large circular stained image is detected. On the other hand, in a sperm having a chromatin defect or DNA damage, since the DNA is fragmented, such a stained image is small or not recognized. That is, DNA fragmentation can be evaluated based on the size of the stained image, thereby detecting abnormalities in the sperm nucleus.

  Stained images based on the size of the circular stained image (halo) formed on the sperm head are classified into five stages based on the width of the head of untreated sperm (see FIG. 2 and Table 1).

  Based on the classification of the stained images, sperm classified as type 1 and type 2 are those in which DNA is not fragmented, and sperm classified as one of type 3, type 4 and type 5 has no DNA. It can be evaluated that it is fragmented. That is, it can be determined that the sperm classified into type 1 and type 2 has a good function, and the sperm classified into any of type 3, type 4 and type 5 has a poor function.

  In addition, a DNA fragmentation index (DFI) is calculated from the number of sperm classified into type 3, type 4 and type 5 based on the classification of the stained image and the total number of sperm counted according to the following formula 2. Can do.

  In fact, when this sperm nuclear DNA fragmentation detection method was used to detect and compare DNA fragmentation of sperm collected from normal volunteers and male infertility patients, sperm in which sperm nuclear DNA fragmentation was observed in male infertility patients was found. Remarkably many were observed. Specifically, in normal volunteers, 98.4% of sperm are classified into type 1 and type 2 with respect to the total number of sperm counted, and 0.5% of sperm are classified into any of types 3 to 5. On the other hand, in male infertility patients, 48.4% of sperm were classified into type 1 and type 2 with respect to the total number of sperm counted, and sperm classified into any of types 3 to 5 44.0% (see Example 1). That is, the DFI of sperm of this normal volunteer and this male infertility patient was 0.5% and 44.0%, respectively. From this result, it is clear that sperm function can be evaluated using DFI of sperm as an index.

  In this sperm function testing method, the sperm nuclear DNA fragmentation detection method is performed on a specimen containing sperm, and sperm function can be evaluated using sperm nuclear DNA fragmentation as an index. The evaluation can be performed by, for example, the above DFI. Specifically, when the DFI is 10% or more, more preferably 20% or more, still more preferably 30% or more, and even more preferably 40% or more, it can be determined that the sperm function is poor.

The method for measuring the sperm head vacuole is:
(A) measuring the number and width of vacuoles in the sperm head;
(B) classifying and scoring vacuoles based on the ratio of vacuole width to sperm head width;
(C) calculating a sperm head vacuole area score from the number and score of vacuoles in each class;
including.

  Measurement of morphological properties such as the number and width of vacuoles can be performed with an electron microscope. Alternatively, the measurement can be performed using a differential interference microscope, and the measurement can be performed with high accuracy by performing computer image analysis. Therefore, these measurements can be performed without using an electron microscope.

  The measurement of the morphological properties of the vacuole is preferably carried out on a sperm having a tail and a flat orientation (a sperm having a flat surface with respect to the observation surface). Malformed spermatozoa are excluded from this measurement.

  The vacuoles are classified based on the ratio of the vacuole width to the maximum width of the sperm head (about 5 μm), and classified into three stages and scored (see FIG. 6 and Table 2). The score when no vacuole is observed is 0.

  The sperm head vacuole area score is calculated by the following formula 1 from the score of the above classification, the number of vacuoles classified into the classification, and the total number of sperm under measurement.

  The number of sperm head vacuoles and / or the sperm head vacuole area score are highly correlated with the DNA fragmentation index, so the number of sperm head vacuoles and / or the sperm head vacuole area score is used as an index Thus, sperm function can be evaluated. The number of sperm head vacuoles and / or the sperm head vacuole area score can be calculated for each sperm and used to evaluate the function of each sperm. In addition, the number of sperm head vacuoles and / or sperm head vacuole area scores are measured for a plurality of sperm and calculated as the average number of vacuoles per sperm and / or the average sperm head vacuole area score. Can be used to evaluate sperm function. A sperm having a small number of sperm head vacuoles and / or a sperm head vacuole area score, or an average number of vacuoles and / or an average sperm head vacuole area score, can be determined to have good sperm function.

  This sperm function test method carries out this sperm head vacuole measurement method for specimens containing sperm, and evaluates sperm function using the number of sperm head vacuoles and / or sperm head vacuole area score as an index. To do.

  Furthermore, this sperm function test method is carried out for a specimen containing sperm by combining both the sperm function test method using sperm nuclear DNA fragmentation as an index and the sperm function test method using sperm head vacuole as an index. Can be a sperm function testing method. The correlation between both methods is quite high, and the accuracy of inspection can be improved by combining both methods. Moreover, the combination of both methods increases the diversity of diagnosis and is useful for diagnosing infertility by cause. For example, the correlation between the two methods is quite high, but on the other hand, it is judged to be normal by the evaluation using sperm nuclear DNA fragmentation as an index, but is judged to be abnormal by the evaluation using sperm head vacuole as an index. And, on the contrary, cases that are judged as normal in the evaluation using the sperm head vacuole as an index, but that are judged as abnormal in the evaluation using sperm nuclear DNA fragmentation as an index may be detected. . Such a decrease in sperm function in cases can be fully attributed to different causes.

  The present invention also provides a method for determining whether or not a semen specimen is a semen specimen derived from a male infertility patient using the present sperm function test method. A semen sample evaluated as having poor sperm function by this sperm function test method can be determined to be a semen sample derived from a male infertility patient. When this determination is performed, it is preferable to evaluate the functions of a plurality of sperm in the sperm function test method. That is, in the sperm function testing method using sperm nuclear DNA fragmentation as an index, sperm nuclear DNA fragmentation is measured for a plurality of sperm, DFI is calculated from the measurement results, and sperm function is evaluated using DFI as an index. Is preferred. Further, in the sperm function test method using the number of sperm head vacuoles and / or the sperm head vacuole area score as an index, a plurality of sperm are measured and the average number of vacuoles per sperm and / or the average sperm are measured. It is preferable to calculate a head vacuole area score and evaluate sperm function using the average number of vacuoles and / or the average sperm head vacuole area score as an index.

  The present invention also provides a method of selecting sperm with good sperm function using a sperm function test method using the number of sperm head vacuoles and / or sperm head vacuole area score as an index. Sperm with good sperm function can be used for in vitro insemination, such as ICSI and IMSI. When selecting a sperm with a favorable sperm function, it is preferable to evaluate each function about several sperm. That is, in the sperm function testing method using the number of sperm head vacuoles and / or the sperm head vacuole area score as an index, a plurality of sperm is measured, and the number of sperm vacuoles and / or sperm head empty is measured. The vesicle area score is calculated and the vacuole number and / or sperm head vacuole area score is compared between sperm. Those having a small number of vacuoles and / or sperm head vacuole area score can be determined to have good sperm function, and thus such sperm is selected.

  Examples of specimens containing sperm include collected semen, sperm contained in the semen, and sperm collected from the semen. The collection of semen can be carried out in the same manner as the collection method used in general male infertility testing methods. Specifically, it is collected in a wide-mouthed container that can be sealed by a method (masturbation). The collected semen is preferably left for about 30 minutes to be liquefied before testing. Recovery of sperm from semen can be carried out by a method known per se, such as a swim-up method or a density gradient centrifugation method. Although it is possible to test using sperm collected after simple washing without doing anything, in order to increase accuracy, pretreatment by a swim-up method, a density gradient centrifugation method or the like is preferable. The collected sperm may be used in the sperm function testing method immediately after the collection, or may be used after freezing at -20 ° C or -80 ° C. Any solution for storing and washing sperm can be used as long as it is a physiological salt solution having a buffering capacity such as various culture solutions usually used in clinical practice or phosphate buffered saline.

  Sperm can be used physically immobilized. “Physical immobilization” means fixing only the position of a cell, for example, a sperm in a living state, and immobilizing it by denaturing the chemical components of the cell like chemical fixation. Different. Physical immobilization can be carried out by a method known per se. For example, sperm or a specimen containing sperm can be suspended by suspending the sperm in a solution that can suppress the movement of the sperm and dropping it onto the solid phase. A low melting point agarose gel can be preferably exemplified as a solution that can contain sperm and suppress its movement. The solid phase for immobilizing sperm may be any solid phase that can observe sperm under a microscope, and examples thereof include a slide glass. Specifically, the sperm is physically fixed by suspending a sperm or a specimen containing sperm in a low melting point agarose gel and dropping it on a slide glass, and then solidifying the agarose gel under low temperature such as on ice. it can. In this operation, it is preferable for observing sperm because the surface can be kept flat by placing the cover glass on the dropped solution and solidifying and removing the solution.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

Evaluation of human sperm nuclear DNA fragmentation [1] Materials and methods Human semen was provided after obtaining informed consent from normal volunteers and patients treated for infertility. The sperm was collected from the semen by the swim-up method.

After washing the sperm, it was counted and adjusted to a concentration of 2-3 × 10 ⁷ / ml (solution A). Thereafter, a process of physically fixing the solution containing sperm on the cover glass (sample preparation step 1), an acid treatment for dissociating the double-stranded DNA in the toroid in the sperm nucleus into single-stranded DNA (sample preparation) Step 2), a treatment for cleaving the toroid linker to release the toroid from the nuclear matrix (sample preparation step 3), a treatment for extending DNA (sample preparation step 4), and dye staining. Specifically, sample preparation 1 was performed as follows. First, Solution A and low melting point agarose were mixed at a volume ratio of 1: 2 (Solution B) and allowed to stand on a hot plate at 23 ° C. 5 μl of the solution B was dropped onto a slide glass, and the cover glass was put on, then the slide glass was transferred onto ice to harden the agarose gel, and the cover glass was removed. Next, sample preparation 2 was performed as shown below. HCL adjusted to 0.08N with distilled water was dropped onto the surface of 300-400 μl slide glass, and the slide glass was stored for 5 minutes in the dark. Sample preparation 3 was performed as shown below. The hydrochloric acid solution was removed by tapping the slide glass, and the solution for neutralization / dissolution 1 (NLS1; 0.4 M Tris buffer, pH 7.5, 0.4 M dithiothreitol (DTT), 1% Triton X-100 50 mM ethylenediaminetetraacetic acid (EDTA) was poured and the slides were stored in the dark for 10 minutes. And sample preparation 4 was performed as shown below. NLS1 was removed from the slide glass, NLS2 (0.4M Tris buffer, pH 7.5, 1% Triton X-100, 2M NaCl) was poured, and the slide glass was stored in the dark for 5 minutes. Finally, dye staining was performed as shown below. The glass slide was washed with a trisboric acid solution (TBE) containing EDTA and dehydrated with 50%, 70%, 90%, and 98% ethanol. Thereafter, it was air-dried for 5 minutes, and light staining was performed. The light staining solution was a commercially available solution, mixed with distilled water so that the volume ratio was 1: 2, and poured onto a slide glass. The slide glass was stored in a dark place for 10 minutes, washed with running water for 3 minutes, soaked in distilled water, and dried by air drying for 10 to 15 minutes to prepare a specimen. The stained image was observed with an optical microscope equipped with a CCD camera, and the image data was taken into a computer for data analysis.

[2] Evaluation Method and Evaluation Criteria FIG. 1 shows a stained image obtained for normal sperm. FIG. 1 shows a stained image of sperm in each sample preparation step. In each of the samples obtained in the sample preparation steps 1 and 2, the toroids are tethered to the nuclear matrix, and thus the DNA is aggregated. Therefore, small dot-like stained images were observed (Panel A and Panel in FIG. 1). B). In the sample obtained in the sample preparation step 3, since the toroid is liberated, a circular stained image was observed (panel C in FIG. 1). In the sample obtained in the sample preparation step 4, since the DNA was elongated, a large circular stained image was observed (panel D in FIG. 1).

  Thus, if the chromatin and DNA are normal, the detected DNA is long, and therefore a large circular stained image called a halo is detected around the sperm head. On the other hand, in a sperm having a chromatin defect or DNA damage, since the DNA is fragmented, such a stained image is small or not recognized. That is, DNA fragmentation can be evaluated by the size of the halo, thereby detecting abnormalities in the sperm nucleus.

  FIG. 2 shows the classification and definition of stained images according to the size of the halo detected around the sperm head. Here, the size of the halo was classified into five levels based on the maximum width (about 5 μm) of the unprocessed sperm head (see Table 1 above).

  Moreover, the definition of sperm nuclear DNA fragmentation was set according to the type of stained image. According to this definition, it can be evaluated that type 1 and type 2 are sperm in which nuclear DNA is not fragmented, and type 3, type 4 and type 5 are sperm in which nuclear DNA is fragmented. Furthermore, a DNA fragmentation index (DFI) can be calculated by the above formula 2.

  The width of the halo was measured by computer analysis of the image data. Specifically, the width of the halo was measured by applying a scale to each stained image on a computer screen. FIG. 3 shows an example of image data analysis results when sperm nuclear DNA fragmentation evaluation was performed on human normal semen. A total of 22 spermatozoa were found in the image, all of which were type 1 spermatozoa. Subdividing Type 1 into two types, Type 1-1 (T1-1) and Type 1-2 (T1-2), based on scale (based on size), type 1-1 sperm 19 and type 1-2 sperm. By finely classifying T1, there is a possibility that the object of analysis is expanded and the accuracy is further improved.

  Images of sperm with no DNA fragmentation and sperm with DNA fragmentation are shown in FIG. 4 panel A and panel B, respectively.

[3] Results FIG. 5 and Table 3 show the results of evaluating sperm nuclear DNA fragmentation for normal human semen and infertility treatment target patient semen.

  In normal human semen, type 1 and type 2 spermatozoa, in which DNA was evaluated as not fragmented, accounted for 98.4%. On the other hand, in semen subject to treatment of infertility, 44.0% accounted for 44.0% of type 3 and type 5 sperm, and 48.4% accounted for type 1 and type 2 sperm. there were. Thus, the analysis results of this sperm nuclear DNA fragmentation evaluation method were consistent with infertility cases.

  According to this sperm nuclear DNA fragmentation evaluation method, the size of the stained image of sperm nuclear DNA is measured, and by applying the classification shown in FIG. Can be detected. Moreover, in the said sperm nuclear DNA fragmentation evaluation method, since the sperm Triton X-100 is used for dissociation from a double stranded DNA to a single strand, and the cutting | disconnection of a toroid linker, the tail part of a sperm does not cut | disconnect from a head. Therefore, it is possible to evaluate only raw sperm by selecting and analyzing only sperm having a tail on the analysis image, and as a result, the accuracy of analysis is high.

Evaluation of human sperm head vacuole [1] Materials and methods Human semen was provided after obtaining informed consent from volunteers. The sperm was collected from the semen by the swim-up method.

  2 μl of the collected sperm was dropped on the surface of the slide glass, covered with a cover glass, and observed. The sperm concentration may be arbitrarily determined according to the amount to be collected, but the smaller the sperm overlap, the easier and faster the measurement. It is preferable to adjust the density to such a level.

  Under a bright field of an optical microscope, the sperm is observed using a differential interference contrast (DIC, also called Nomarski) method, and is a sperm having a tail and having a flat orientation (flat relative to the observation surface). The number of vacuoles existing in the head and the size of the vacuoles were measured. Malformed sperm was excluded from the measurement.

  The average number of sperm head vacuoles per sperm (average number of vacuoles / sperm; v / s) was calculated for each semen specimen, and the correlation with the sperm nuclear DNA fragmentation index was examined. In addition, the area of the sperm head vacuole (sperm vacuum score (area); SVS) was scored, and the correlation with the sperm nuclear DNA fragmentation index was examined.

[2] Evaluation Method and Evaluation Criteria FIG. 6 shows the classification of sperm head vacuole size. Here, the size of the vacuole was classified into three stages based on the maximum sperm head width (about 5 μm) (Table 2 above). And the magnitude | size of each vacuole was represented by the score. That is, the score of vacuoles classified into L, M, and S was set to 3, 2, and 1, respectively. The score when no vacuoles were observed was 0.

  The area of the sperm head vacuole was scored according to Equation 1 above using the score of the vacuole size.

[3] Results FIG. 7 shows an analysis image of the size of the sperm head vacuole using an optical microscope DIC (Nomarski) image. This sperm sample is one analysis image of a sperm sample created in plural from one semen sample. A total of 3 spermatozoa were observed in the images, and the total number of vacuoles present in these was 2, 1 and 2 respectively for L, M and S. As a result of measuring 37 sperm specimens prepared from this sperm sample, the total number of vacuoles present in the counted 214 spermatozoa was 20, 47 and 176 for L, M and S, respectively. The average number of sperm head vacuoles present in the sperm sample per sperm was calculated as (20 + 47 + 176) /214=1.14. Further, the sperm head vacuole area score of this sperm sample was calculated as (20 × 3 + 47 × 2 + 176 × 1) /214=1.54 from the above equation 2.

  The results of examining the correlation between the average number of sperm head vacuoles (s / v) and the sperm head vacuole area score (SVS) and DFI for 20 semen samples in this manner were shown in FIGS. 8 and 9, respectively. Shown in The correlation coefficient is shown in Table 4.

  The correlation coefficient between SVS and DFI was 0.642, and the correlation coefficient between s / v and DFI was 0.556, both of which were determined to have a fairly high correlation. Sperm with a high DFI can be evaluated as having poor function and causing infertility. Therefore, a sperm with high SVS and s / v can be evaluated as having poor function and causing infertility.

  According to this sperm head vacuole evaluation method, the number and size of vacuoles are measured, and SVS and s / v are calculated using the classifications and scores shown in FIG. The function can be easily evaluated.

It is a figure which shows the state of the DNA toroid and DNA in each process process in a sperm nucleus DNA fragmentation evaluation method, and the light staining image of the human normal sperm sample in this process. Panel A is a sample (sample A) in which sperm is physically immobilized on a slide glass, panel B is a sample obtained by acid-treating sample A (sample B), and panel C is a sample B that is Triton X-100 and DTT. Panel D shows a sample (Sample D) obtained by treating Sample C with Triton X-100 and NaCl. It is a schematic diagram showing the classification and definition of sperm nuclear DNA fragmentation according to the size of the halo detected around the sperm head in the sperm nuclear DNA fragmentation evaluation method. It is a figure which shows the image data analysis result when performing sperm nucleus DNA fragmentation evaluation about human normal semen. A total of 22 spermatozoa were found in the image, of which 19 were type 1-1 and 3 were type 1-2. It is a figure which shows the image of the sperm which the DNA is not fragmented (panel A), and the sperm panel B where the DNA is fragmented. It is a figure which shows the result of having evaluated sperm nuclear DNA fragmentation about normal human semen and the semen of a patient subject to infertility treatment. It is a figure which shows the classification | category of the magnitude | size of a sperm head vacuole. It is a figure which shows the analysis image of the size of a sperm head vacuole. A total of 3 spermatozoa were found on the screen, and the total number of vacuoles present in them was 2, 1 and 2 respectively for L, M and S. It is a figure which shows the correlation with the average number of sperm head vacuoles, and DFI. The vertical axis shows the average number of sperm head vacuoles (average number of vacuoles / sperm), and the horizontal axis shows DFI. It is a figure which shows the correlation with a sperm head vacuole area score and DFI. The vertical axis represents the sperm head vacuole area score (sperm vacuum core (area)), and the horizontal axis represents DFI.

Claims (4)

A method for detecting sperm nuclear DNA fragmentation for sperm function testing ,
(1) dissociating double-stranded DNA in the toroid in the sperm nucleus into single-stranded DNA;
(2) cleaving the toroid linker to release the toroid from the nuclear matrix;
(3) extending the DNA in the toroid;
(4) a step of detecting the extended DNA by measuring the size of a circular stained image formed on the sperm head after dyeing the extended DNA, wherein (2) and (3) ) Is performed using Triton X-100 (registered trademark),
The width (A) of the stained image and the head width (B) of the untreated sperm are compared, and the stained image is classified according to the value of A / B as follows:
(I) A / B value is 3.0 or greater,
(Ii) the value of A / B is less than 3.0 and 1.5 or greater,
(Iii) the value of A / B is less than 1.5 and 1.0 or greater;
(Iv) A / B value is less than 1.0 and intense staining;
(V) A / B value is less than 1.0 and weakly or unstained,
The sperm showing the stained image classified as (i) or (ii) above is classified into any one of the above (iii), (iv) and (v) A sperm nuclear DNA fragmentation detection method , wherein the sperm showing the stained image is evaluated that the sperm nuclear DNA is fragmented . Sperm nuclear DNA fragmentation is detected using the method for detecting sperm nuclear DNA fragmentation according to claim 1, and the sperm showing the stained image classified as (i) or (ii) has a good sperm function. The sperm function test | inspection method which evaluates that the sperm which shows the dyeing | staining image classified into any of said (iii), (iv), and (v) has a bad sperm function. The sperm function is evaluated using the DNA fragmentation index (DFI), which is the ratio of the number of nuclear DNA fragmented sperm to the total number of sperm measured, as an index, wherein the number of nuclear DNA fragmented sperm is the above (iii), The sperm function testing method according to claim 2, wherein the sperm function test method determines that the sperm function is poor when the number of spermatozoa indicates the stained image classified into (iv) and (v) and the DFI is 10% or more. The function of the sperm contained in the semen sample is evaluated using the sperm function test method according to claim 2 or claim 3, and the semen sample evaluated as having a poor sperm function is derived from a male infertility patient A method for testing male infertility, comprising determining that the sample is a semen sample of the male.