CN110361534B - Chemical markers for evaluating embryo and predicting success rate of in vitro fertilization and application thereof - Google Patents
Chemical markers for evaluating embryo and predicting success rate of in vitro fertilization and application thereof Download PDFInfo
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Classifications
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
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Abstract
The application provides chemical markers for evaluating embryos and predicting success rate of in vitro fertilization and applications thereof. In particular, the application provides a method of evaluating an embryo for in vitro fertilization comprising measuring the amount of leukemia inhibitory factor in a culture broth of in vitro culture of said embryo in a subject. The present application also provides a method for predicting success rate of in vitro fertilization-embryo transfer, comprising in vitro culturing embryos for transfer and measuring the amount of leukemia inhibitory factor in the culture broth. The application also provides kits and devices for use in the methods. The application also provides the use of leukemia inhibitory factor in the preparation of kits and devices for assessing embryo for in vitro fertilization or predicting success rate of in vitro fertilization-embryo transfer.
Description
Technical Field
The application relates to the technical fields of molecular biology and assisted reproduction. In particular, the application relates to methods for assessing embryo quality and devices and kits for use in the methods. The application also relates to a method for predicting success rate of in vitro fertilization and to a device and kit for use in the method.
Background
In vitro fertilization-Embryo Transfer (In Vitro Fertilization Pre-Embryo Transfer, IVF-ET), the selection of the Embryo with the most developmental potential is critical to success. The use of morphological methods to evaluate selection of embryos with the most developmental potential has become a common approach [1,2]. Although this approach relatively increases pregnancy rates, it has limitations, with over 70% of IVF embryos failing to successfully gestate [3]. Detection of morphological features of embryos has been further perfected over the past decade and a real-time monitoring system has been employed [4,5]. However, the clinical success rate of pregnancy for ART has not increased in recent years [6,7].
The pregnancy process is a very complex physiological event, depending on the development of the embryo, the synchronous coordination of the endometrium, etc. Leukemia inhibitory factor (leukemia inhibitory factor, LIF) is a member of the interleukin IL-6 family, a secreted glycoprotein. Leukemia inhibitory factor can inhibit the growth of myeloid leukemia cell line M1 and promote differentiation [13]. LIF is a cytokine with a wide range of biological functions. Studies have shown that LIF affects reproductive activity in various ways. In recent years, extensive studies have shown that LIF may affect reproductive activities including follicular growth, embryo growth, differentiation, etc. [8]. LIF burst in preovulatory follicular fluid increases, and LIF levels in follicular fluid are positively correlated with egg cell mass (egg cell mass). However, there has been no study of the secretion of LIF from embryos and its effects.
Recent advances in omics technology (genomics, transcriptomics, proteomics and metabolomics), including improvements in platform sensitivity, have allowed the study of new molecular approaches for embryo selection. Defining and characterizing embryo secretions will also expand the knowledge of early embryogenesis and facilitate understanding of the role of embryos in the implantation process. Research and development into the group of embryo secretory proteins (secretome) has expanded understanding of early embryogenesis and has facilitated understanding of the role of embryos in the transfer process, thereby better understanding of embryo selection
There is also a need in the art to develop new methods for predicting the quality of embryos, i.e., their transfer potential, for in vitro fertilization-embryo transfer in order to balance the maintenance of embryo viability from the detection procedures, while obtaining the detailed information needed to assess embryo quality.
Disclosure of Invention
The present application is based on the study and discovery of leukemia inhibitory factor (leukemia inhibitory factor, LIF) levels in the medium of individual embryos for in vitro fertilization-embryo transfer, and the correlation between them and successful pregnancy, and the discovery that LIF is a chemical marker for assessing embryos for in vitro fertilization-embryo transfer and predicting success rate of in vitro fertilization-embryo transfer. The present application thus provides methods of assessing embryos for in vitro fertilization and predicting success rate of in vitro fertilization-embryo transfer by measuring the amount of leukemia inhibitory factor in the culture broth of in vitro culture of said embryos in a subject, and devices and kits therefor.
In particular, the application provides a method of evaluating an embryo for in vitro fertilization comprising measuring the amount of leukemia inhibitory factor in a culture broth of in vitro culture of said embryo in a subject. In one aspect of the application, the amount of leukemia inhibitory factor refers to the concentration of leukemia inhibitory factor in the culture broth. The concentration of leukemia inhibitory factor can be measured by methods known in the art, including ELISA methods and the like. In one aspect of the application, the method comprises culturing in vitro the embryo to be transplanted of the subject and measuring the concentration of leukemia inhibitory factor in the culture broth. The method of the application may further comprise the step of comparing leukemia inhibitory factor in the in vitro culture broth of the embryo to be transferred in the subject with a normal control (e.g. a normal average value for a certain population, e.g. a population divided according to race, age group or physical condition) or other embryo to be transferred in the subject. In the method of the application, embryos with higher leukemia inhibitory factor concentrations in the embryo in vitro culture broth are of higher quality, i.e. with higher transfer potential.
In yet another aspect of the application, the subject in the method is a infertility patient with tubal occlusion.
In yet another aspect of the application, in assessing embryos for in vitro fertilization, the age of the subject is assessed in addition to measuring and assessing leukemia inhibitory factor concentration in embryo in vitro culture broth. In the method of the application, the lower the age of the subject, the higher the embryo quality it uses for transfer, and the higher the success rate of pregnancy.
The application also provides a method for predicting success rate of in vitro fertilization-embryo transfer comprising measuring the amount of leukemia inhibitory factor in a culture broth of in vitro culture of said embryo in a subject. In one aspect of the application, the method comprises culturing in vitro the embryo to be transplanted of the subject and measuring the concentration of leukemia inhibitory factor in the culture broth. The method of the application may further comprise the step of comparing the leukemia inhibitory factor in the in vitro culture broth of the embryo to be transferred in the subject with a normal control or other embryo to be transferred in the subject. In the method of the application, embryo with higher concentration of leukemia inhibitory factor in embryo in vitro culture solution is used for in vitro fertilization-embryo transplantation, and then the success rate is predicted to be higher. In yet another aspect of the application, the subject in the method is a infertility patient with tubal occlusion. In yet another aspect of the application, in assessing in vitro fertilization-embryo transfer, the age of the subject is assessed in addition to measuring and assessing leukemia inhibitory factor concentration in the embryo in vitro culture broth. In the method of the present application, the lower the age of the subject, the higher the success rate of in vitro fertilization-embryo transfer is predicted.
The application also provides a kit or device for use in the above-described method of assessing embryos for in vitro fertilization-embryo transfer. The present application also provides a kit or device for use in the above-described method for predicting success rate of in vitro fertilization-embryo transfer.
The application also provides the use of leukemia inhibitory factor in the preparation of a kit or device for evaluating embryos for in vitro fertilization. The application also provides the use of leukemia inhibitory factor in the preparation of a kit or device for predicting success rate of in vitro fertilization-embryo transfer.
The kit or device of the present application comprises reagents for detecting the amount of leukemia inhibitory factor in the culture broth of in vitro culture of the embryo of a subject. LIF levels can be measured using methods common in the art. In one aspect of the application, LIF levels can be determined by measuring protein amounts using immunological methods, for example by ELISA with antibodies that specifically recognize LIF. The kit or device of the application may be a kit containing reagents for detecting LIF levels in a single or multiple samples of solution (e.g., embryo culture fluid or supernatant thereof). The kit or device of the application may also be a device (e.g. a dipstick or a small protein detector) for measuring LIF levels in single or multiple samples of a solution (e.g. embryo culture fluid or supernatant thereof), or may be a large throughput protein detector that can handle a large number of samples. In yet another aspect of the application, the kit or device is for detecting the amount of leukemia inhibitory factor in culture broth of in vitro culture of embryos of an infertility patient with oviduct obstruction. In yet another aspect of the application, the kit or device further comprises means for assessing the age of the subject, e.g. a card or information input for the subject to fill in the age, etc.
In the present application, "in vitro fertilization-embryo transfer" (sometimes abbreviated as "in vitro fertilization") refers to a technique in which sperm and eggs of a mammal, particularly a human, are subjected to fertilization in an in vitro artificial controlled environment, and then the in vitro fertilized embryo is transferred to the mother.
In the present application, "evaluation of an embryo" refers to evaluating the quality of an embryo for in vitro fertilization-embryo transfer, in particular its transfer potential, i.e. the ability to successfully gestate after embryo transfer. Embryos assessed as good are embryos with high pregnancy rates after in vitro fertilization and embryo transfer. Assessment of embryos can help predict success rate of in vitro fertilization, i.e., the rate of pregnancy (including biochemical pregnancy rate or clinical pregnancy rate) in subjects receiving in vitro fertilization and embryo transfer.
In the present application, "predicting in vitro fertilization" or "predicting success rate of in vitro fertilization-embryo transfer" refers to predicting pregnancy rate of a subject receiving in vitro fertilization-embryo transfer, i.e., pregnancy outcome of in vitro fertilization-embryo transfer. The embryo with higher pregnancy rate can be obtained. The embryo with lower pregnancy rate is not good.
Drawings
FIG. 1 shows the amount of LIF in an in vitro culture broth of embryos. A is the amount of LIF in the embryo culture medium of the good and bad embryos of the gestational group. B is the amount of LIF of the embryo culture medium of the fine embryo and the bad embryo of the non-pregnant group.
FIG. 2 shows the LIF amount and ROC curve in vitro culture of good embryos. A is the amount of LIF in the medium of pregnant and non-pregnant embryos in the elite embryo. B is an ROC curve evaluating the value of embryo culture fluid LIF in predicting clinical pregnancy.
Detailed Description
The spirit and advantages of the present application will be further illustrated by the following examples, which are only intended to illustrate the application and not to limit it.
Example 1 subject and sample
The subjects included 52 women receiving assisted reproductive therapy from day 7, 2016, 2 to day 9, 12. All subjects had normal uterine cavities and endometrium, at least two belonging to high quality blastula according to Gardner scoring criteria (Gardner grading standard). All subjects provided written informed consent according to the institutional review board's guidelines for protecting human subjects.
EXAMPLE 2 ovarian stimulation
The ovaries of the subjects were stimulated by three methods, including a long regimen, a short regimen, and an antagonist regimen, respectively. Ovulation is triggered with HCG when the diameter of at least two follicles reaches 18mm and the remaining half is greater than 15 mm. After about 34.5 hours, the ova were retrieved transvaginally under ultrasound guidance.
EXAMPLE 3 embryo culture
All subjects obtained were fertilized with metaphase II (MII) oocytes. At 37 ℃,5% CO 2 ，5％O 2 ，90％N 2 Embryos were cultured individually in 50 μLP1 medium (Irvine Scientific, santa Ana, calif.) in ambient. All embryos were cultured to blastocyst stage (blastcys phase) and graded under the microscope for in vitro transplantation according to Gardner scoring criteria. All eggs are transplanted by taking out the eggsAfter 120 hours, the treatment was performed under direct ultrasound guidance using Wallace catheter (Wallace catheter). LIF expression was then tested in the supernatant of the original culture broth used for embryo culture using a specific ELISA assay.
EXAMPLE 4 luteal support and pregnancy diagnosis
After implantation, progesterone was administered by intramuscular injection, 60mg daily, for 14 days for luteal support. If pregnancy is confirmed, the use of progestin is not ended until week 10 of pregnancy. The subject was diagnosed as a biochemical pregnancy based on HCG >10U in the blood on day 14 post-transplantation. The subject was diagnosed with clinical pregnancy by ultrasound B-mode examination of gestational sac and fetal heart beat at about day 64 post-implantation.
Example 5LIF detection
LIF levels were determined using a specific ELISA kit (RD, human LIF Quantikine ELISA Kit).
When embryos are transferred between the culture media, a small amount of medium is taken away. To achieve testing of equal volumes of embryo culture supernatant, 100 μl of dilution buffer was added to microplate test wells. 40. Mu.L of supernatant from each embryo culture broth was manually added to the wells for measurement. The LIF standard provided by the kit is diluted to obtain a calibration curve within the expected expression range of embryo LIF, and the calibration curve is adjusted according to the dilution of the sample. LIF concentrations were determined from absorbance at 450nm using EL800Universal Microplate Reader (Bio-Tek Instruments Inc., winioski, VT).
EXAMPLE 6 statistical analysis
Differences between groups were assessed by Student's t test or Mann-Whitney rank sum test, depending on whether the data was normally distributed. The LIF concentration cut-off and sensitivity/specificity were analyzed with the subject's working characteristics curve (ROC). P values <0.05 were significant.
Example 7 subject parameters and LIF levels
The cause of infertility in all subjects is tubal obstruction. Following the method of examples 1-3, subjects were fertilized in vitro, embryos obtained, cultured, and the embryo culture supernatants were tested for LIF levels. Subjects were divided into two groups according to LIF levels, high LIF (. Gtoreq. 40,26) and low LIF (. Gtoreq.40 pg/mL, 26), respectively. The mean age of the high LIF group was 31.73+ -5.01 years and the low LIF group was 33.92 + -4.69 years. Basal FSH levels, LH levels, E2 levels and endometrial thickness were not significantly different between the high LIF group and the low LIF group. Ovulation stimulation methods used in the high LIF group included long regimens (11 cases), short regimens (7 cases) and antagonist regimens (8 cases). In the low LIF group, long regimens (21 cases), short regimens (4 cases), and antagonist regimens (1 case) are included. The high LIF group had 7 cases of primary infertility and 19 cases of secondary infertility, and the low LIF group had 5 cases of primary infertility and 21 cases of secondary infertility, respectively. Pregnancy rates were significantly higher in the high LIF group (69.23%) than in the low LIF group (34.62%) (table 1).
TABLE 1
EXAMPLE 8 comparison of LIF levels in culture of good and bad embryos
A total of 208 parts of embryo culture fluid were collected from pregnant and non-pregnant subjects. The medium was divided into medium of good embryos (n=110) and medium of bad embryos (n=98) according to Gardner blastula scoring criteria. Embryos with scoring criteria above and including 4BC are referred to as good embryos, and 4BC is referred to below as bad embryos. The LIF level of the embryo culture medium of the gestational group of good embryos was 46.91pg/mL, which is 40.74+ -23.55 pg/mL higher than that of the embryo culture medium of the bad embryos (FIG. 1A), but there was no statistically significant difference. LIF levels of 39.06+ -23.49 pg/mL in embryo culture media of non-pregnant good embryos were higher than 35.85 + -24.17 pg/mL in poor embryo culture media (FIG. 1B), with no statistically significant differences.
Example 9 comparison of culture LIF levels of embryos from gestated and non-gestated groups
Embryos judged to be good according to Gardner blastula scoring criteria were used for transfer. After 10 weeks of embryo transfer, the subjects were examined with B-ultrasound. Patients were classified into gestational groups and non-gestational groups according to the presence or absence of gestational cysts as a result of the B-ultrasonic examination. LIF levels were measured in embryo culture media of both groups of patients and found to be 52.41 + -31.76 pg/mL in embryo culture media of pregnant groups, which is significantly higher than 39.06+ -23.49 pg/mL (P < 0.05) in non-pregnant groups (FIG. 2A).
To assess the value of embryo culture fluid LIF in predicting clinical pregnancy, a subject work characteristic curve (ROC) curve was drawn (fig. 2B). Area Under Curve (AUC) values were determined with the highest specificity and sensitivity as the best predicted point. The best predicted point, sensitivity, specificity and AUC values were 36.79ng/mL,82.6%,72.4% and 0.788 (95% CI: 0.665-0.911), respectively (FIG. 2B). As demonstrated in fig. 2B, LIF expression levels of embryo culture media can be used as predictors of IVF-ET results.
Example 10 univariate analysis and multivariate analysis of pregnancy Rate influencing factors
Using univariate analysis and multivariate analysis, no statistically significant correlation was found between basal FSH levels, LH levels, endometrial thickness, E2 and pregnancy rates (table 2).
Age (p=0.041) and embryo culture fluid LIF levels (p=0.02) were found to be important predictors of clinical pregnancy (table 3).
TABLE 2 univariate analysis of pregnancy rate influencing factors
TABLE 3 multivariate analysis of pregnancy rate influencing factors
In order to select embryos suitable for transfer, morphological screening of embryos is widely used. However, morphological indexes still have limitations, and the high quality of morphology cannot represent an absolutely better developmental potential embryo. It is desirable to find other complementary or supplemental assessment methods that reflect embryo function or physiological status. Researchers have explored a number of effective methods to select the best potential embryo to improve the clinical pregnancy rate of in vitro fertilization-embryo transfer, i.e., IVF-ET. For example, using morphological feature monitoring, many morphological features related to embryo quality have been reported, such as cell cycle time and cell shape, etc. [9,10]. Analysis of the embryo secretory proteome (the protein produced and secreted by the developing embryo) can provide a non-invasive method for embryo assessment [11]. Identification of chemical markers in human embryo culture media that are related to embryo quality and transfer potential is an important tool in combination with morphological parameters in the selection of embryos suitable for transfer [12].
The present application has been developed by observing and studying the expression of Leukemia Inhibitory Factor (LIF) in individual embryos in their embryo culture medium in a large number of subjects undergoing in vitro fertilization. The present application unexpectedly found that LIF concentration in embryo culture fluid has a significant correlation with clinical pregnancy rate: the embryo of the subject who is successfully pregnant has more LIF in the medium than the embryo of the subject who is not pregnant. As can be seen, LIF is a chemical marker that can be used to identify human embryo culture media that are related to embryo quality and transfer potential.
Thus, the present application provides the use of LIF as a method for identifying and predicting embryo quality and transfer potential in embryo culture media, and thus provides a kit useful for rapidly and efficiently judging the quality of embryos for in vitro fertilization and predicting success rate of pregnancy. The method and the kit provided by the application can help to improve pregnancy potential, simultaneously reduce the risk of multiple pregnancy to the maximum extent, benefit the technical field of artificial fertilization, further improve pregnancy rate and realize conventional single embryo transfer.
References mentioned in this patent specification are included in the following references and are disclosed in the present application by reference in their entirety.
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3.Holm P,Booth PJ,CallesenH.Kinetics of early in vitro development of bovine in vivo-and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media.Reproduction.2002；123(4):553-565.
4.Pribenszky C,Losonczi E,Molnár M,et al.Prediction of in-vitro developmental competence of early cleavage-stage mouse embryos with compact time-lapse equipment.Reprod Biomed Online.2010；20(3):371-379.
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The foregoing is illustrative of the present application and is not to be construed as limiting thereof. The practice of the application will employ, unless otherwise indicated, conventional techniques of organic chemistry, polymer chemistry, biotechnology, and the like, it being apparent that the application may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the application will be apparent to those skilled in the art to which the application pertains. Many modifications and variations are possible in light of the teachings of the application and, thus, are within the scope of the application.
Unless otherwise indicated, the unit "degree" of temperature as presented herein refers to degrees celsius, i.e., degrees celsius.
Claims (2)
1. Use of a leukemia inhibitory factor in the manufacture of a kit for assessing the success rate of in vitro fertilization-embryo transfer of embryos of infertility patients with salpingemphraxis or for predicting the success rate of in vitro fertilization-embryo transfer of embryos of infertility patients with salpingemphraxis, wherein the kit further comprises a device for assessing the age of a subject, wherein the use further comprises in vitro culturing the embryos for transfer and measuring the amount of leukemia inhibitory factor in the culture broth.
2. The use of claim 1, wherein the kit comprises reagents for detecting the amount of leukemia inhibitory factor in a culture broth of in vitro culture of embryos of a subject.
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