JP2007530030A - Pregnancy possibility diagnosis method and diagnostic kit - Google Patents

Abstract

The present invention relates to a method and a diagnostic kit for diagnosing fertility. In the present invention, follicular fluid was collected from a woman undergoing a test tube fertilization program and the activity of MMP-9 was examined. According to the present invention, pregnancy was not possible at all from the group having no or low MMP-9 activity, and the pregnancy rate was about 50-60% from the group having MMP-9 activity of a certain level or higher. Therefore, by using the diagnostic method and diagnostic kit of the present invention, the degree of expression of MMP-9 is measured, the possibility of pregnancy in the assisted reproduction method is predicted, and the efficiency of the assisted reproduction method is improved. Can do.
[Selection] Figure 1

Description

  The present invention relates to a method and a diagnostic kit capable of early diagnosis of fertility. More specifically, the present invention relates to a method for diagnosing fertility by measuring MMP activity in follicular fluid.

  Since the first test tube baby was born in 1979, various assisted reproduction technology (ART) for treating infertile patients has been developed and applied. Examples of ART include in vitro fertilization-embryo transfer (IVF-ET), intracytoplasmic sperm injection (ICSI), testicular sperm extraction method (testicular TE) And sperm cell injection (ROSI) and embryo freezing. At present, assisted reproduction is important in the treatment of infertility due to various causes, and it has become common, but problems such as high cost, complicated process and low success rate remain.

  ART tends to increase, and the pregnancy rate especially in IVF is increasing, but it does not exceed expectations, and the actual birth rate is about 15-30%. According to US statistics, the pregnancy rate by IVF in 1985 was 23.8% per cycle and the evening rate was 19.3% per cycle. According to 1997 statistics, the pregnancy rate was as high as 29.3% per cycle and the evening rate of 24.0%. However, there was no difference between 1996 and 1997 statistics.

  In the assisted reproduction (ART) cycle, successful human pregnancy depends on follicular development, the number of oocytes collected, fertilization, fertilized egg development and implantation (Sakkas D, et al., 2001). . Among them, blastocyst implantation in the endometrium is a complex process that depends on structural changes from the development of the endometrium and fertilized eggs. Tissue remodeling requires uterine preparation, endometrial fertilized egg rupture and the appearance of a continuous endometrial matrix. Persuasive evidence supports the hypothesis that MMPs are essential for the destruction of ECM components in this process. From this point of view, MMPs are presumed to be related to cell meiosis, penetration and tissue reproducibility during development. That is, MMP is known to be involved in the remodeling of intimal tissue associated with the physiological cycle by inducing changes in ECM (extracellular matrix), and MMP molecules and the like have various expression modes associated with the physiological cycle. Involved in the ovulation and implantation process.

  Among MMPs, MMP-9 (matrix metalloproteinase-9), known as type IV Collagenase / gelatinase B (92 kDa type IV collagenase / gelatinase B), has many types of collagen (IV, V and XI, elasto). Proteoglycans and gelatin are degraded (Hibbs et al., 1985: Murphy et al., 1991). In addition, previous studies have shown that human cervical fibroblasts (Sato, H. and Seiki, M., 1993), trophoblasts (Simonovitz, S. at al., 1996) and the uterus. It has been demonstrated that MMP-9 secreted from endometrial stromal cells (Huang, HA, et al., 1998) is stimulated by hormones and cytokines. MMP-9 has been proposed to mediate different stages of cyclical changes in female reproduction, such as menstrual cycle, ovulation, implantation, parting, and regression of the mammary gland after lactation (Jeziorska, M., et al. Librach, C. L. et al., 1991; Vadillo-Ortega, F, et al., 1995). Ovulation is a process that occurs when LH rises from the pituitary gland before ovulation, and as a result, mature eggs are discharged from the ovarian follicle. Such a process requires a lot of tissue formation, and MMP-9 is important for inducing proteolytic activity necessary at the time of ovulation, but the relationship between human follicular fluid and MMP-9 is important. It has not been revealed.

  An object of the present invention is to provide a method and a diagnostic kit capable of diagnosing fertility in a method of assisted reproduction such as test tube fertilization.

  To achieve this goal, follicular fluid and MMP activity collected from women participating in the test tube fertilization program were measured. As a result, it was shown that there was no pregnancy at all from the group with low MMP-9 activity, and the pregnancy rate was 50-60% in the group with high MMP-9 activity. Therefore, by measuring the expression level of MMP-9 in the follicular fluid using the method and kit of the present invention, the possibility of pregnancy in ART can be predicted, and the efficiency of ART can be improved. The inventors of the present invention tested whether the expression of MMP-9 from the follicular fluid is related to pregnancy in the in vitro fertilization cycle. The results of the present invention showed that MMP-9 expression from ovulation fluid is related to high implantation rate and high pregnancy rate.

  That is, the present invention provides a method for diagnosing fertility by measuring the activity of MMP-9 contained in the follicular fluid of follicles having mature oocytes.

  Specifically, the present invention is characterized by being a method for diagnosing fertility by measuring the above-mentioned MMP-9 activity by the zymographic method.

  More specifically, the present invention is characterized by a method for diagnosing fertility by selecting a follicle having a particle size of 17 mm or more and measuring MMP-9 activity.

  The present invention also provides a fertility diagnostic kit capable of measuring MMP-9 activity in follicular fluid using a polyacrylamide gel containing a protein substrate that is degraded by MMP-9. To do.

  More specifically, the present invention is characterized in that in the diagnostic kit for diagnosing fertility, the protein substrate is selected from collagen IV, collagen V, collagen XI, elastin, proteoglycan and gelatin.

  Changes in female endocrine status during the reproductive cycle and gestation are the result of many tissue reformations in the uterus (Wewer et al., 1986; Aplin et al., 1988). For example, various basement membrane components such as type IV collagen, laminin, fibronectin and proteoglycan in the uterus become altered throughout the menstrual cycle and pregnancy (Alin et al., 1988). . Similarly, murine intrauterine matrix cells undergo remodeling of extracellular fluid matrix components (Wewer et al., 1986) during this process, and MMPs and TIMPs are ovulated, implanted and It is thought to be a core mediator that degrades the matrix into the decidual process (Behrendsen et al., 1992: Cross et al., 1994; Leftebre et al., 1995); Harvey et al., 1995; Leco et al., 1996; Alexander et al., 1996).

  In the present invention, MMP-9 activity present in human follicular fluid during ovulation is proved by gelatin substrate zymography technology, and MMP-9 activity is higher than that in non-pregnant group. Significantly increased in pregnant groups (P <0.01). However, MMP-2 activity, another enzyme known to be involved in cell remodeling, was highly expressed in all follicular fluids of pregnant and non-pregnant groups and there was no difference. The MMP-9 activity measured from each group was quantified and compared using a density measuring instrument, and as a result, the average MMP-9 activity of the pregnancy group was 61,759 units. These results clearly suggest that MMP-9 activity is a prerequisite for pregnancy.

  Many studies suggest that the role of MMP-9 is important in pathological conditions such as tissue formation and many biological tissue treatments, ovulation, inflammation, arthritis, wound treatment, tumor penetration, morphogenesis (Behrendsen et al., 1992; Cross et al., 1994; Leftebre et al., 1995; Harvey et al., 1995; Leco et al., 1996; Alexander et al., 1996). However, little is known about physiologically required MMPs and protein substrates generated in the body. In follicular fluids with substrate specificity, including the membrane component collagen IV, the presence of MMP-9 is thought to require tissue reproducibility during follicular growth and development. Ovulation occurs as a result of regulated enzymatic degradation and loss of collagenases at the follicular wall (Espey, 1994; Luck and Zhao, 1995).

  Collagenase is thought to be important in ovulation of other species that are not human, and a fibrous collagenase, such as MMP-1, that disrupts the formation of fibrous collagen that imparts an outer coat structure to the ovarian matrix. including. Expression of MMP-9 was detected by zymography, immunohistochemistry and RCR from mouse and human pre-implication embryo, EC cells and bleach products. In the presence of fibroblast growth factor-4, blastocyst culture increased secretion of MMP-P. MMP-9 has been shown to progressively increase in mouse blastocysts and trophoblast cell layers during fertilized egg implantation. In the present invention, the cleavage rate between the MMP-9 expression group and the non-expression group from fertilization to the 8-cell stage, the expression of MMP-9 is not significantly different due to cell division. During the bed period, MMP-9 has been shown to be necessary.

  On the other hand, successful pregnancy relies on the penetration of trophoblasts in the decidua at the time of implantation, resulting in further penetration of extracellular trophoblasts into the uterine wall (Fisher et al. , 1985; Librach et al., 1985; Librach et al., 1991; Cross et al., 1994). These events require destruction of the extracellular fluid matrix and cell migration. MMPs are an important family of zinc-dependent enzymes with broad substrate specificity that allow for destruction of the extracellular matrix (Hulboy et al., 1997). In rat and human fetal membranes, the total amount of MMP-9 increased with labor (Bryant-Greenwood and Yamamoto, 1995; Draper et al., 1995; Lei et al., 1995).

  This study demonstrated that deficiency of MMP-9 expression in follicular fluid results in infertility. That is, it is suggested that MMP-9 expression is essential in the implantation and pregnancy states, and that this expression is closely related to MMP-9 expression in the ovulation phase. In conclusion, MMP-9 plays an important role in regulating follicle growth and development of extracellular matrix destruction, follicle migration, embryonic egg and other cellular actions.

  The present invention will be described below through examples. The examples are for illustrative purposes and are not intended to limit the specific form of the invention.

<Materials and methods>
Study Subject According to the Hospital Female Insemination (IVF) program of Dongguk University, 54 patients with various infertility problems were mobilized in this study. There are fallopian tube factors (n = 15), endometriosis (n = 11), ovulation arrest (n = 9), and infertility with unknown causes (n = 19). The patient's years were 21 to 33 years old (average 31.3 years). On the other hand, male infertility factors such as azoospermia and rare spermosis were excluded from this study.

<Reproductive assisted treatment process>
1. Oocyte preparation Ovulation induction was performed with a gonadotropin releasing hormonal agonist (GnRH-a). From the middle luteal phase, 900 puserelin (Hoechst, Germany) was administered by nasal spray to suppress the pituitary gland, and from the 3rd to 5th days of the follicular phase, human menopausal gonadotropin (hMG, Pergon) Serono or IVF-M, LG. Korea). When the dose of hMG is adjusted according to the follicular reaction using asphyxiation ultrasound and the follicle particle size is 2 or more, 10,000 IU human chorionic gonadotropin (human choriogonadotropin) ) (HCG, IVF-C, LG, Korea) was administered to induce embryos. 36 hours after hCG injection, the follicle was inhaled using choking ultrasound and oocytes were collected. The collected oocytes were analyzed with the characteristics of the oocyte using a dissecting microscope (SMZ-10, Nikon, Japan) in an operating machine (IVF chamber, USA) adjusted to 37 ° C. and 5% CO ₂ conditions. The degree of maturation of the oocyte was confirmed based on the presence or absence of GV in the cytoplasm. Thereafter, the cells were transferred to a culture dish (3037, Falcon, USA) containing P1 medium supplemented with 10% SSS and cultured in a CO ₂ incubator until fertilization time. The number of oocytes used for fertilization was adjusted to 5 or less per culture dish.

2. In vitro fertilization After collecting oocytes, semen was collected by masturbation in a sample container (Baxter, USA). Sperm concentration and motility were measured according to WHO criteria. The semen was left at room temperature for 10-20 minutes to induce liquefaction. The liquefied semen was transferred to a 15 ml conical tube (2099, Falcon, USA) and then twice at 1,500 rpm (5 minutes) on Ham's F-10 with 10% SSS attached. 3 minutes) was centrifuged. After centrifugation, the upper layer solution excluding the pellet was removed, and Ham's F-10 to which 1 ml of 10% SSS was added was carefully added so that the pellet would not scatter. Sperm floated inside. The suspended sperm was used for fertilization while being stored in a 5 ml tube (2003, falcon, USA). Sperm used for fertilization was injected into a culture solution containing oocytes at the time of fertilization so as to be 1 × 10 ⁵ / ml. The next morning, were oocytes removed and fertilized using a syringe needle (320310, BD, USA) under a dissecting microscope in an operating machine adjusted to 37 ° C, 5% CO ₂ conditions? I checked. A female pronucleus and a male pronucleus were formed, and the presence of two polar bodies was confirmed as fertilization.

3. Embryo transplantation Only embryos that were successfully fertilized were selected and cultured in P1 culture for 48 hours, and then 2-5 embryos of 8-cell stage or higher were selected and transferred. In most cases, a Tomcat catalyzer (8890-793021, Sherwood, USA) was used for transplantation.

4). Progesterone Administration and Confirmation of Pregnancy 100 mg progesterone in oil, Progest, Samil, Korea was injected intramuscularly into patients daily after embryo transfer. Around 10 days after the transplantation, a biochemical pregnancy was defined as having a blood β-hCG concentration of 10 mIU / ml or more. It was also defined as clinical pregnancy by confirming fetal heart activity by suffocation ultrasonography at least during pregnancy.

<Preparation of follicular fluid>
The follicular fluid was collected by an ultrasonic-guided aspiration method from a follicle having a particle size of 17 mm or more in a woman whose cycle was elevated by IVF in vitro fertilization. The collected oocytes and follicular fluid were subjected to ultrasonic inhalation, and only the follicular fluid that was hardly mixed with blood was collected and used. The collected follicular fluid was centrifuged (3,500 rpm) for 30 minutes to remove blood cells and granulocytes, and then only the upper layer fluid was collected. The upper layer liquid was sterilized with a 0.2 μm filter and stored in a −20 ° C. refrigerator. The follicular fluid was dissolved upon use.

<Measurement of MMP-9 activity using zymography method>
Expression of MMP-2 and MMP-9 in the follicular fluid was detected using a zymograph that was modified from the description of Riley et al. (1999) in the description of Rawdanowicz et al. (1994). The follicular fluid was separated using SDS-PAGE (7.5% (w / v gels; Minigel apparatus; Bio-Red, Hemel Hemphead) containing 1 mg / ml gelatin. The gel was 2.5% ( v / v) Washed twice with Triton X-100 and digestion buffer (200 mM NaCl, 50 mM Tris-HCl, 2.5 mM CaCl ₂ , 1 mM ZnCl ₂ , pH 7.5; all chemicals from Sigma Chemical Chem) , St Louis, Mo.) and stored for 18 hours at 37 ° C. The gel was stained with 0.5% (w / v) Coomassie blue R250 (Bio Red, Richmond, Calif.) In 30% (v / v ) Methanol 10% (v v) were stained at room temperature in _{glacial acetic acid in H 2 O)} .

<Numericalization of MMP-9 activity after zymography>
Gelatinase activity was quantified using Gel Documentation semi-automated image analysis (Core-Bio System, Seoul, Korea), which measures the strength and surface of the band layer after scanning the gel.

<Statistics>
Results were performed by averaging at least 3 replicates. Significance for the mean was evaluated by Students t-test. When P <0.05, it was determined to be statistically significant.

<Result>
In follicular fluid, the gelatinase activity appearing at 92 kDa was consistent with MMP-9 activity as demonstrated by zymography (FIG. 1). The total amount of MMP-9 activity in the pregnant group was significantly higher than in the non-pregnant group (P <0.01).

  Also, however, the gelatinase activity appearing at 72 kDa size is consistent with the MMP-2 activity that is abundant in all female follicular fluids. However, there was no difference in MMP-2 activity between pregnant and non-pregnant groups (FIG. 2).

  The average age of women in this study was 31 years (± 3.3). Oocytes were successfully recovered after one treatment cycle from all test groups (n = 54). Oocyte collection was performed 36-37 hours after hCG injection. The average fertilization rate in the pregnancy group was 71.82% (± 11.7). The non-pregnant group that could not get pregnant was 69.1% (± 13.1). As a result, all women transplanted 2-7 fertilized eggs. Sixteen were confirmed with chemical pregnancy, of which one became miscarriage and 15 continued and died. There was no difference between pregnant and non-pregnant groups by age. The average years were 29 (± 3.3) and 31 (± 3.2), respectively (Table 1).

  As a result of measuring the MMP-9 activity present in the follicular fluid, the mean MMP-9 activity of the pregnancy group was 61,759 densitometric units. A total of 54 women were included in this study group, but looking at the classification of each group, tube factor (n = 15), endometriosis (n = 11), cause unknown (N = 19) and ovulation arrest (n = 9), and the average degree of MMP-9 activity of each group was compared (FIG. 3). That is, MMP-9 activity in the anovulatory group was significantly lower than the other three groups (p <0.001).

  On the other hand, the final total implantation rate and the whole pregnancy rate were 18.6% and 29.6%, respectively. In Table 2, based on the degree of MMP-9 activity, the pregnancy rate and the implantation rate were divided into four groups arbitrarily. The MMP-P activity of the first group is 30,000 to 40,000 units, the second group is 40,000 to 50,000 units, the third group is 50,000 to 60,000 units, and the fourth group is 60, Classified into more than 000 units.

  Interestingly, when the MMP-9 activity level was below 50,000 units, the implantation rate and pregnancy rate were all 0%. In contrast, when the MMP-P activity level was 50,000 units or more, the implantation rate and pregnancy rate were 32.15 and 47.3%, respectively, and only when the MMP-9 activity was high. Pregnancy was successful. Implantation and pregnancy were not performed at MMP-9 activity below 50,000 units. Therefore, the inventors presented the fact that implantation and pregnancy would not occur without MMP-9 activity (Table 2).

  According to the present invention, in the assisted reproduction method, it is possible to solve the problem that the probability of connection to actual childbirth is low, and to diagnose the possibility of pregnancy at an early stage.

  Furthermore, according to the present invention, it is possible to classify a group in which MMP-9 is not expressed and find a new treatment method that can increase the pregnancy rate in advance of the conventional assisted reproduction method.

References

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FIG. 1 is a photograph of the activity of MMP-9 and MMP-2 in human follicular fluid measured by zymographic analysis. Samples were washed by electromigration on a 7.5% SDS-PAGE gel containing 0.1% gelatin, so that the gelatin was digested overnight from the zymographic incubation buffer. did. FIG. 1A is a zymograph for MMP-9 activity, and FIG. 1B is a zymograph for MMP-2 activity. Lane C is the MMP-9 control group, lanes 1-5 are the pregnancy group, and lanes 6-10 are the non-pregnancy group. FIG. 2 is a graph showing the relationship between the expression of proMMP-9 and the pregnancy rate from the follicular fluid of test tube fertilization. FIG. 3 is a graph showing the relationship between the cause of infertility and the expression of MMP-9 in patients studied in this study.

Claims (5)

  A method for diagnosing fertility, comprising diagnosing fertility by measuring the activity of MMP-9 contained in the follicular fluid of a follicle having a mature oocyte.   The method of diagnosing fertility according to claim 1, wherein the activity of MMP-9 is measured by a zymography method.   The method for diagnosing fertility according to claim 1, wherein the follicle is selected to have a particle diameter of 17 mm or more.   A fertility diagnostic kit characterized by measuring the activity of MMP-9 contained in a follicular fluid using a polyacrylamide gel containing a protein substrate that is degraded by MMP-9.   The fertility diagnostic kit according to claim 4, wherein the protein substrate is selected from collagen IV, collagen V, collagen XI, elastin, proteoglycan and gelatin.

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