CN115128277A - Group of biomarkers for evaluating PCOS (Primary biliary cirrhosis) oocyte quality, application and screening method - Google Patents
Group of biomarkers for evaluating PCOS (Primary biliary cirrhosis) oocyte quality, application and screening method Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a group of biomarkers for evaluating PCOS oocyte quality, application and a screening method. The expression levels of the group of biomarkers were significantly different between the PCOS population and the fertile population. The group of biomarkers can provide a detection and evaluation means with high efficiency, rapidness, strong specificity and high sensitivity for oocyte quality evaluation and embryo transplantation guidance of PCOS infertility patients. The screening method screens out differential protein to analyze potential mechanism in the follicular development process by respectively evaluating proteome expression profiles of follicular fluid of PCOS population and pregnant population, and screens out biomarker with application prospect as noninvasive index for clinically evaluating oocyte quality. The method has the characteristics of high efficiency, rapidness, strong specificity and high sensitivity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a group of biomarkers for evaluating PCOS oocyte quality, application and a screening method.
Background
Polycystic ovary syndrome (PCOS) is an endocrine disturbance syndrome with coexistence of reproductive dysfunction and abnormal carbohydrate metabolism common to women in the reproductive age, is a main cause of anovulatory infertility, and affects about 5% -15% of women in the reproductive age. The fertility of a PCOS patient is reduced, and partial PCOS patients who receive in vitro fertilization and embryo transfer (IVF-ET) cycle ovulation promotion treatment have the problems that although the obtained number of eggs is large, the quality of the oocytes is poor, the cycle cancellation rate is high, the fertilization rate is low, and the number of high-quality embryos is small. Therefore, the selection of high quality oocytes is important for technologies such as in vitro fertilization and embryo transfer, and how to evaluate the quality of oocytes is a key problem facing the field of assisted reproduction.
Folliculogenesis is a highly coordinated biological event during oocyte development and release, PCOS results in recruitment of primordial follicles, follicular stunting and ovulation failure due to abnormally elevated androgen and/or insulin levels. However, due to the complex etiology and high heterogeneity of PCOS, the influencing factors in the oocyte development process are not completely clarified, and how to evaluate and improve the quality of the oocytes of PCOS patients in order to obtain high-quality embryos and successfully pregnancy is still a hotspot and difficulty in the field of assisted reproduction.
In the prior art, oocyte quality evaluation methods are generally divided into non-invasive techniques and invasive techniques, and the non-invasive techniques are used for judging by observing the form of oocytes and other methods which do not damage the biological activity of the oocytes, and comprise ovary weight and follicle size evaluation, perivitelline space and first polar body form evaluation, spindle body form observation, osmotic pressure treatment, in-vitro embryo culture and the like. Invasive techniques are based on biochemical changes in oocytes at different developmental stages for evaluation, and mainly include ATP assay, GSH assay, Cyclin B assay, mitochondrial and cytoplasmic compartmentalization assay, and gene expression differential assay. The determination result of the invasive technology has quantifiability, accuracy and objectivity, but the non-invasive technology is established on the basis of biochemical change of cells and can cause irreversible damage to the cells, while the non-invasive technology does not damage the cells and influence further development of the cells, but has the defects of lack of quantifiable indexes, deviation of subjective judgment, difficulty in determining observation time and the like, so that the finding of a quantifiable, accurate, objective and non-invasive method is crucial to the evaluation of the quality of the oocytes.
Disclosure of Invention
The invention aims to provide a group of biomarkers for evaluating PCOS oocyte quality, application and a screening method.
The invention provides a group of biomarkers for evaluating PCOS oocyte quality, wherein the biomarkers are a plurality of follicular fluid difference proteins, and the follicular fluid difference proteins comprise up-regulated proteins and down-regulated proteins;
the ratio of the expression level of the up-regulated protein in the PCOS population to the expression level of the up-regulated protein in the fertile population is greater than or equal to 1.2;
the ratio of the expression level of said down-regulated protein in said PCOS population to the expression level thereof in said pregnant population is less than or equal to 0.83.
Further, the up-regulated protein includes at least one of inhibin beta C chain, pantothenic acid esterase, proteoglycan 4, angiotensinogen, protein 1 containing FERM and PDZ structural domain, lymphatic endothelial hyaluronic acid receptor 1, phosphatidylinositol proteoglycan-3, hydrocephalus inducing protein homolog, cholinesterase, N-acetyl chitosanase, and nerve cell adhesion molecule L1.
Further, the down-regulation protein comprises at least one of monocyte differentiation antigen CD14, alpha-2 glycoprotein rich in leucine, olfactory mediator-like protein 3, laminin subunit alpha-1, intercellular adhesion molecule 2, vascular cell adhesion protein 1, serine glycine, cadherin-1, complement factor H-related protein 2, complement factor H-related protein 4, metalloproteinase inhibitor 2, cystatin-M, pepsin A-4, cadherin-11, multidrug resistance-related protein 9, immunoglobulin heavy chain allosteric 3-15, transforming growth factor beta receptor type 3, and cartilage matrix protein-2.
The invention provides the use of a panel of biomarkers as described above in the assessment of oocyte quality in a PCOS population.
The invention provides a kit for evaluating the quality of PCOS oocytes, which contains the biomarkers for evaluating the quality of PCOS oocytes.
Furthermore, the kit contains 4-5 up-regulated proteins and/or down-regulated proteins.
The invention provides a screening method of the biomarker for evaluating the PCOS oocyte quality, which is characterized by comprising the following steps:
s1, collecting follicular fluid of the PCOS population and follicular fluid of the pregnant population respectively;
s2, respectively carrying out non-labeled proteomics detection and analysis on the collected follicular fluid of the two groups of people;
the non-standard proteomics detection and analysis process comprises the steps of firstly extracting protein in follicular fluid, then carrying out enzymolysis on the obtained protein to obtain a peptide fragment, and then carrying out mass spectrometry on the peptide fragment;
s3, processing the mass spectrometry data obtained in the step S2, and screening out proteins with significant differential expression in follicular fluid of two groups of people to obtain the biomarker for evaluating the PCOS oocyte quality;
wherein the screening standard is that the expression quantity in the PCOS population and the expression quantity in the pregnant population have the difference multiple of more than or equal to 1.2 or less than or equal to 0.83; and assume a value P of less than 0.05.
Further, the pregnant group is a healthy pregnant group, and the infertility factor is a male factor.
Further, the specific process of step S2 is as follows:
s2-1, extracting protein: pretreating the extracted protein by adopting a high-abundance protein removal chromatographic column to obtain a protein sample from which albumin, IgG and other high-abundance proteins are removed;
s2-2, determining the protein concentration of the protein sample by using a BCA method;
s2-3, carrying out reductive alkylation treatment on the protein sample;
s2-4, carrying out FASP enzymolysis on the protein sample subjected to reductive alkylation treatment;
s2-5, desalting the protein sample after FASP enzymolysis;
s2-6, carrying out liquid chromatography-mass spectrometry analysis on the protein sample subjected to desalting treatment.
Further, in step S2, the mass spectrometry includes eluting and separating peptide fragments and mass spectrometry detection; wherein, the elution separation peptide section is subjected to gradient elution by adopting a mobile phase A and a mobile phase B;
the mobile phase A is a formic acid-water solution with the volume ratio of 1:1000, and the mobile phase B is a formic acid-acetonitrile solution with the volume ratio of 1: 1000;
before gradient elution, dissolving the peptide segment after enzymolysis by using a sample dissolving solution, wherein the sample dissolving solution is a formic acid-water solution with the volume ratio of 1: 1000;
the gradient elution process comprises fully oscillating the dissolved peptide fragment solution, and performing LC-MS mass spectrometry on 2ug of the solution; the analysis time is 120min, and the flow rate is 300 nL/min; gradient elution conditions were as follows, wherein the proportions of mobile phase B are all volume percent: 0min-5min, 2% -8% of B; 5-90 min, 8-24% of B; 90-110 min, 24-32% B; 110min-115min, 32% -90% B; 115min-120min, keeping 90% B;
and after the gradient washing is finished, detecting the separated peptide fragment by adopting a mass spectrometer.
The invention has the beneficial effects that:
(1) the group of biomarkers for evaluating the PCOS oocyte quality can provide a detection and evaluation means with high efficiency, rapidness, strong specificity and high sensitivity for evaluating the oocyte quality of a PCOS infertility patient and guiding embryo transplantation.
(2) The screening method of the biomarker of the PCOS oocyte quality screens out differential protein to analyze a potential mechanism in the follicular development process by respectively evaluating the proteome expression profiles of the follicular fluid of a PCOS crowd and a pregnant crowd, and screens out the biomarker with application prospect to be used as a noninvasive index for clinically evaluating the oocyte quality.
(3) The screening method of the biomarker of PCOS oocyte quality of the invention screens the proteins which are differentially expressed in the follicular fluid of PCOS infertility patients and pregnant groups by a Label free quantitative proteomics technology, does not depend on an isotope labeled non-labeled proteome quantitative technology, performs mass spectrum analysis on proteolysis peptide segments by a liquid mass spectrometry technology, does not need to use an expensive stable isotope Label as an internal standard, only needs to analyze mass spectrum data generated during large-scale protein identification, and compares the signal intensity of corresponding peptide segments in different samples, thereby quantifying the protein corresponding to the peptide segments, and has the advantages of no limitation of samples, low detection cost and the like.
(4) The screening method of the PCOS oocyte quality biomarker screens differential protein among different comparison groups according to fold difference (FC) and a P value (P-value), verifies and screens a group of biomarkers for evaluating the PCOS oocyte quality through a low throughput technology, and predicts the PCOS oocyte quality and development potential through the biomarkers.
(5) The kit for evaluating the PCOS oocyte quality can provide a detection and evaluation means with high efficiency, rapidness, strong specificity and high sensitivity for evaluating the oocyte quality of a PCOS infertility patient and guiding embryo transfer.
Drawings
FIG. 1 is a flow chart of the specific screening of biomarkers in example 1 of the present invention;
FIG. 2 is a heatmap of differential protein expression in follicular fluid of PCOS group and pregnant human group (CON) in example 1 of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The group of biomarkers for evaluating the quality of PCOS oocytes has a significant difference in expression level between PCOS population and pregnant population.
By detecting the expression quantity of the biomarker, the difference of the oocytes of PCOS (human chorionic gonadotropin) crowds compared with oocytes of pregnancies can be judged, so that a more objective evaluation result can be provided for the oocytes of the PCOS crowds, and the success rate of subsequent processes such as excretion promotion treatment, in-vitro fertilization and embryo transfer can be effectively improved.
The follicular fluid is composed of secretion of granulosa cells, theca cells and oocytes and plasma protein diffused through the basement membrane of the theca capillary, provides microenvironment for the development of the oocytes, and can be used as a medium for communication between the oocytes and the follicular cells in the follicular development process. During follicular formation, the primordial follicle develops into a primary growing follicle which in turn develops into a secondary growing follicle, also known as the pre-antral follicle. Surrounding the follicle with several layers of granulosa cells, the granulosa cells accumulate follicular fluid, which increases with oocyte size. The composition of follicular fluid is very similar to plasma composition, which reflects the metabolic activity of granulosa cells and membrane cells and changes in follicular permeability, while changes in the protein composition of follicular fluid reflect changes in the secretion process of follicular cells and plasma composition in pathological conditions. The follicular fluid has a complex composition and contains a variety of active ingredients, such as growth factors, interleukins, reactive oxygen species, anti-apoptotic factors, proteins, polysaccharides, and various steroid hormones, which play an important role in oocyte maturation. Therefore, the knowledge of the proteome of follicular fluid can deepen the understanding of the process of follicular development, better evaluate the quality of oocytes and understand the underlying mechanisms of follicular development. Through analyzing the components of the follicular fluid, the proteins related to oocyte development and maturation are found, potential biomarkers influencing the quality of the oocytes are screened, the oocyte quality is favorably predicted, and the success rate of IVF treatment is improved.
The biomarker can provide a detection and evaluation means with high efficiency, rapidness, strong specificity and high sensitivity for oocyte quality evaluation of PCOS infertility patients and embryo transplantation guidance.
Specifically, the biomarkers of the invention are a plurality of follicular fluid differential proteins, including upregulated proteins and downregulated proteins; the ratio of the expression amount of the up-regulated protein in the PCOS group to the expression amount of the up-regulated protein in the pregnant group is greater than or equal to 1.2; the ratio of the expression level of the down-regulated protein in the PCOS population to the expression level of the down-regulated protein in the fertile population is less than or equal to 0.83.
In an embodiment of the present invention, the number of the follicular fluid differential proteins is 29, wherein the number of the upregulated proteins is 11 and the number of the downregulated proteins is 18.
The up-regulated protein is specifically as follows:
u1, accession number P55103, protein annotated as Inhibin beta C chain, chinese name Inhibin beta C chain;
u2, accession No. O95497, protein annotated as Pantetherinase, the Chinese name pantothenase;
u3, accession No. Q92954, protein annotation as Proteoglycan 4, chinese name Proteoglycan 4;
u4, accession number P01019, protein annotation, Angiotensinogen, chinese name;
u5, accession number Q5SYB0, protein annotation FERM and PDZ domain-containing protein 1, Chinese name FERM and PDZ domain-containing protein 1;
u6, accession number Q9Y5Y7, protein annotation is Lymphatic vessel endothial hyaluronic acid receptor 1, Chinese name Lymphatic endothelial hyaluronic acid receptor 1;
u7, accession number P51654, protein annotated as Glypican-3, Chinese name phosphatidylinositol proteoglycan-3;
u8, accession number Q4G0P3, protein annotation is Hydrocephalus-inducing protein homolog, Chinese name Hydrocephalus-inducing protein homolog;
u9, search number P06276, protein annotation for Cholinesterase, Chinese name Cholinesterase;
u10, accession number Q01459, protein annotated as Di-N-acetylchitinase, Chinese name N-acetyl chitosanase;
u11, accession number P32004, protein annotation Neural cell adhesion molecule L1, chinese name Neural cell adhesion molecule L1;
the down-regulation proteins are specifically as follows:
d1, accession number P08571, protein annotation is Monocyte differentiation antigen CD14, chinese name Monocyte differentiation antigen CD 14;
d2, accession number P02750, protein Annotation Leucine-rich alpha-2-glycoprotein, Chinese name Leucine-rich alpha-2 glycoprotein;
d3, accession No. Q9NRN5, protein annotated as Olfactomedin-like protein 3, chinese name olfactory mediator-like protein 3;
d4, accession number P25391, protein Annotation Lamin sunburnt alpha-1, Chinese name Laminin subunit alpha-1;
d5, accession number P13598, protein annotation is Intercellular adhesion molecule 2, chinese name Intercellular adhesion molecule 2;
d6, accession number P19320, protein annotation as Vascular cell attachment protein 1, herein named Vascular cell adhesion protein 1;
d7, accession number P10124, protein annotated as serglcin, chinese name serine glycine;
d8, accession number P12830, protein annotated as Cadherin-1, chinese name Cadherin-1;
d9, accession number P36980, protein Annotation as complete factor H-related protein 2, Chinese name Complement factor H-related protein 2;
d10, accession number Q92496, protein annotation is complete factor H-related protein 4, Chinese name Complement factor H-related protein 4;
d11, accession number P16035, protein annotation is Metalloproteinase inhibitor 2, chinese name Metalloproteinase inhibitor 2;
d12, accession number Q15828, protein annotation for Cystatin-M, Chinese name Cystatin-M;
d13, accession number P0DJD7, protein annotation Pepsin A-4, Chinese name Pepsin A-4;
d14, accession number P55287, protein annotated as Cadherin-11, Chinese name Cadherin-11;
d15, accession number Q96J65, protein annotated as Multidrug resistance-associated protein 9, Chinese name Multidrug resistance-associated protein 9.
D16, accession number A0A0B4J1V0, protein annotated as Immunoglobulin heavy variable 3-15, Chinese name Immunoglobulin heavy chain allosteric 3-15.
D17, accession number Q03167, protein annotated as Transforming growth factor beta receptor type 3, the chinese name Transforming growth factor beta receptor type 3.
D18, accession No. O00339, protein annotation for Matrilin-2, Chinese name cartilage matrix protein-2.
The biomarkers of the invention can be used for evaluating the quality of oocytes of PCOS people.
The kit for evaluating the quality of PCOS oocytes of the present invention contains the above-described biomarker for evaluating the quality of PCOS oocytes.
Preferably, the kit contains 4-5 of the up-regulated proteins and/or down-regulated proteins; weighting each biomarker in a group of PCOS follicular fluid protein markers according to the fold difference and the biological function thereof, calculating and comparing by an algorithm, screening 4-5 most representative differential proteins, and combining to obtain a kit.
The screening method of the biomarker for evaluating the PCOS oocyte quality comprises the following steps:
s1, collecting follicular fluid of a PCOS population and follicular fluid of a pregnant population respectively;
s2, respectively carrying out non-labeled proteomics detection and analysis on the collected follicular fluid of the two groups of people;
the non-standard proteomics detection and analysis process comprises the steps of firstly extracting protein in follicular fluid, then carrying out enzymolysis on the obtained protein to obtain a peptide fragment, and then carrying out mass spectrometry on the peptide fragment;
s3, processing the mass spectrometry data obtained in the step S2, and screening out proteins with obvious differential expression in follicular fluid of two groups of people, namely obtaining a biomarker for evaluating PCOS oocyte quality;
wherein, the screening standard is that the FC value of the difference multiple of the expression quantity in the PCOS population and the expression quantity in the pregnant population is more than or equal to 1.2 or less than or equal to 0.83; and assume a value P of less than 0.05.
The invention screens out differential protein to analyze the potential mechanism in the follicular development process by respectively evaluating the proteome expression profiles of follicular fluid of different PCOS populations, and screens out biomarkers with application prospect to be used as noninvasive indexes for clinically evaluating oocyte quality.
The screening method adopts unlabeled (Label free) quantitative proteomics detection and analysis, is an unlabeled proteomics quantitative technology independent of isotope labeling, performs mass spectrometry on proteolysis peptide fragments through a liquid chromatography-mass spectrometry technology, does not need to use expensive stable isotope labels as internal labels, only needs to analyze mass spectrum data generated during large-scale protein identification, and compares the signal intensity of corresponding peptide fragments in different samples, thereby quantifying the proteins corresponding to the peptide fragments, and has the advantages of no limitation of samples, low detection cost and the like.
The screening method screens differential proteins among different comparison groups according to Fold Change (FC) and a P value (P-value), verifies and screens a group of biomarkers for evaluating the PCOS oocyte quality through a low throughput technology, and predicts the PCOS oocyte quality and development potential through the biomarkers.
The invention is illustrated by the following specific examples:
example 1
This example used the screening method of the present invention for specific biomarker screening
The specific screening steps of this example are shown in FIG. 1.
1. Screening for PCOS patients;
PCOS patients were selected for ovulation-promoting treatment according to the diagnosis standard of polycystic ovarian syndrome (2011 China medical society, "diagnosis of PCOS and consensus of therapists").
Specifically, patients with PCOS in the cohort need to meet the following conditions simultaneously:
1) meets the diagnostic criteria of Rotterdam: clinical or biochemical modification of hyperandrogenism; ② dilute the egg or no ovulation; and thirdly, the ovary is found to be changed like a polycycle by ultrasonic. A diagnosis of PCOS that corresponds to any two of the three items;
2) the patient is 38 years old or less;
3) the ovulation promoting scheme is antagonist + HCG trigger;
4) the number of eggs obtained by IVF cycle excretion promoting treatment is more than 10.
Accordingly, the exclusion criteria is that there is one of any of the following:
1) those who do not meet the selection criteria
2) Greater than or equal to 2 gestational losses;
3) factors affecting pregnancy: premature ovarian failure, ectopic cyst of ovarian uterine follicular fluid, ectopic uterine follicular fluid, diabetes, hyperthyroidism, etc.;
4) hormonal or metabolic drugs are used within the first three months of the IVF cycle.
2. The female group capable of being pregnant has the following discharging standards:
and (3) inclusion standard: 1) the age of a female with the reproductive age in the first egg taking period in the hospital is less than or equal to 38 years old; 2) patients with primary or secondary infertility; 3) can be accompanied by infertility caused by fallopian tube factors and male factors; 4) normal menstrual cycle, 21-35 days of menstrual cycle; 5) peripheral blood basal testosterone is normal; 6) the ovulation promoting scheme is antagonist + HCG trigger; 7) the number of eggs obtained in IVF cycle promoting treatment is more than 10.
Exclusion criteria: 1) those who do not meet the inclusion criteria; 2) greater than or equal to 2 gestational losses; 3) factors affecting pregnancy: premature ovarian failure, ectopic cyst of ovarian uterine follicular fluid, ectopic uterine follicular fluid, polycystic ovarian syndrome, diabetes, hyperthyroidism, etc.; 4) hormonal or metabolic drugs are used during the first three months of the IVF cycle.
Based on the above grouping criteria, 10 PCOS and 10 normal fertile women were recruited in the experiment, and the basic information of the patients is shown in table 1:
TABLE 1 basic information of the patients in this example
3. And (3) carrying out ovulation promotion treatment on the two groups of patients meeting the conditions, and carrying out ovum taking and follicular fluid collection, wherein the ovulation promotion treatment method specifically comprises the following steps:
1) injecting HCG, washing vulva and vagina with 0.1% PVP and normal saline daily, and sterilizing and spreading towel conventionally;
2) the vaginal ultrasonic probe is covered with a sterile rubber sleeve, and a puncture frame is arranged;
3) carrying out ultrasonic observation on the positions of the uterus and the ovary and the number and the size of follicles by using vagina, distinguishing the echoes of the follicles and blood vessels, and selecting a proper puncture route;
4) selecting a proper ovum pickup needle, inserting the ovum pickup needle from the posterior/lateral fornix of the vagina under the guidance of vaginal ultrasound, puncturing the follicle, sucking follicular fluid, keeping the negative pressure at 14kPa, and feeding the collected follicular fluid and ovum into an embryo culture room to pick up the ovum;
5) after the ovum is picked up in the embryo culture room, the residual follicular fluid specimen is placed in the ovum pick-up tube;
6) centrifuging the collected follicular fluid at 800Xg for 10 minutes, and taking the supernatant fluid as a follicular fluid specimen;
7) the oocyte fates of the PCOS patient such as oocyte MII ovum, fertilization rate, D3 high-quality embryo rate and the like are tracked, and the PCOS patient is divided into a high-quality embryo rate group and a low-quality embryo rate group.
Wherein the high-quality embryo rate group is D3 high-quality embryo rate more than 70%; the low-quality embryo rate group is D3 high-quality embryo rate less than 30%. The high-quality embryo rate of D3 is (number of high-quality embryos of D3/number of normal fertilized eggs). times.100%.
The D3 high-quality embryo refers to embryo which is derived from normal fertilized egg, has 7-9 embryonic cells at 3 days after fertilization, has a cell size according with the development stage, has a fragmentation degree of less than 10%, and has no multinucleated phenomenon.
4. Follicular fluid of two groups of people is taken as samples respectively, and is subjected to Label free proteomics detection and analysis respectively, and the method comprises the following specific steps:
1) pretreatment of proteome samples: equilibrating the high-abundance protein-removed chromatographic column (Thermofeisher Cat. A36370) to room temperature for more than 30 minutes in advance; thawing the sample on ice, centrifuging at 4 ℃ and 10000rpm for 10 minutes, and sucking 10 mu L of the middle layer; removing the screw cap of the chromatographic column, and directly adding no more than 10 microliter of sample into the resin slurry in the chromatographic column; covering the chromatographic column and turning over the chromatographic column for several times until the resin is completely uniform in the solution; slowly rotate the reverse through the mixer at room temperature, incubate for 20 minutes, ensure sample and resin mix during incubation, or gently vortex once every few minutes; after incubation, the substrate cap was broken off above the centrifuge tube mouth and the upper cap was unscrewed to allow communication up and down. Placing the micro-column into a 2ml collection tube, and centrifuging for 2 minutes at 1000 Xg; removing the column containing the resin; the filtrate contains a sample depleted of albumin, IgG and other high abundance proteins for further processing or storage at-20 ℃ for future use; adding 6 times volume of 100% acetone, and precipitating at-20 deg.C overnight; the next day, the pellet was centrifuged, 500 μ L of pre-cooled ethanol: acetone: the precipitate was washed 2 times with a 50:50:0.1 mixture, centrifuged at 13,000rpm for 15 minutes at 4 ℃, re-precipitated with 6M guanidine hydrochloride +300mM TEAB and stored in a refrigerator at 4 ℃.
2) Protein concentration determination by BCA method: taking a part of the extracted protein sample to carry out protein concentration determination, and the specific operation method comprises the following steps: preparing a standard substance and a working solution, namely firstly preparing a BSA standard substance system, wherein the standard substance diluent is 1 x PBS. The BSA standard system was then formulated according to the scale of standard 2.
TABLE 2 BSA standard systems
Preparing a BCA working solution, and calculating the volume of the required total BCA working solution.
The total BCA working solution volume is (standard + sample to be tested) x number of replicates × BCA working solution required for each sample.
The specific steps for preparing the BCA working solution are as follows: to 50 volumes of BCA reagent a, 1 volume of BCA reagent B (a: B50: 1) was added and mixed well. Assay with 96-well plate (sample: BCA working solution ═ 1: 8): diluting a sample to be detected by 10 times by using 1 XPBS; adding 25 mu L of standard substance and sample to be detected into a 96-well plate; add 200. mu.l BCA working solution to each well and mix well by shaking for 30 s. Covering a 96-well plate, and incubating for 15min at 37 ℃; cooling to room temperature, and detecting absorbance at a wavelength of 570nm on an enzyme-labeling instrument; a standard curve was drawn based on the absorbance of the BSA standard. And calculating the protein concentration of the sample according to the standard curve and the dilution times of the sample.
3) Reductive alkylation: taking a 26 mu g protein solution sample, and using 25mM ammonium bicarbonate to fix the volume to 100 mu L; adding 1M DTT to the protein solution; 2 μ L of 1M DTT/100 μ L protein was added at a final concentration of 20 mM. Mix well and incubate at 57 ℃ for 1 hour. 10. mu.L of 1M iodoacetimide/100. mu.L of the solution at a final concentration of 90mM was added thereto, and the mixture was mixed well and left at room temperature for 40 minutes in the dark. Iodoacetimide is now available.
4) FASP enzymolysis: adding the protein subjected to reductive alkylation into a 10K ultrafiltration tube, centrifuging at 12,000rpm, discarding the solution at the bottom of the collection tube, washing with ammonium bicarbonate solution for 4 times, adding Typsin prepared from ammonium bicarbonate, and performing enzymolysis at 37 ℃ overnight; the next day, the peptide fragments after enzymolysis and digestion are collected by centrifugation, concentrated and dried.
5) Desalting: desalting the polypeptide after centrifugal concentration and drying by using a Monospin desalting column, wherein the desalting method is to dissolve the dried mixed peptide section by using a 0.1% trifluoroacetic acid solution; activating the desalting column with 100% acetonitrile; equilibrating the desalting column with 0.1% trifluoroacetic acid solution; adding the re-dissolved sample into a desalting column, and centrifuging; then adding 0.1% trifluoroacetic acid solution to clean the desalting column; adding 50% acetonitrile solution, centrifuging, eluting the peptide segment, and collecting the elution solution by using a new centrifugal tube; the eluted solution is centrifugally concentrated and dried to remove the acetonitrile.
6) Liquid mass spectrometry analysis: the mobile phase A is formic acid, water and 1:1000 and V/V, and the mobile phase B is formic acid, acetonitrile and 1:1000 and V/V; the peptide fragment is dissolved by a sample dissolving solution, wherein the dissolving solution is formic acid, water and 1: 1000V/V. After sufficient oscillation, 2ug of each sample was subjected to LC MS-MS; the analysis time of each sample is 120min, and the flow rate is 300 nL/min; gradient elution conditions were as follows, where the proportions of mobile phase B are all volume percent: 0min-5min, 2% -8% B; 5min-90min, 8% -24% B; 90-110 min, 24-32% B; 110min-115min, 32% -90% B; 115min-120min, keeping 90% B; the separated peptide fragment enters a mass spectrometer Obritrap Fusion Lumos for detection.
5. Screening for PCOS follicular fluid protein markers:
1) and performing database retrieval on the obtained mass spectrum detection data: specifically, a protome discover 2.4 is used for searching a library, after a series of optimization processing is carried out on mass spectrum data, similarity comparison scoring is carried out on the mass spectrum data and a database, and protein identification is carried out. The library search parameters are shown in table 3.
TABLE 3 database search parameters
2) Statistics of protein identification results
A. Protein relative molecular weight distribution: all identified proteins were counted according to their relative molecular weight distribution.
B. Number distribution of Unique peptide fragments: all identified proteins were counted according to the Unique peptide number distribution.
C. Peptide fragment sequence length distribution: all identified proteins were counted according to the peptide fragment sequence length distribution.
D. Protein coverage distribution: all identified proteins were counted according to protein coverage distribution.
3) Protein quantitative analysis: the peak Intensity of the peptide fragment associated with the primary mass spectrum (Precusos Intensity) was used as a quantitative parameter for the Label free. When a plurality of peptide fragments are detected by one protein in the library searching result, the peak intensity of all the peptide fragments detected by the protein is weighted and calculated, and the peak intensity of the protein expression can be obtained (Abundance). Further, the peak intensities of the respective proteins in each sample were normalized to obtain abundance (normalized), and the respective proteins in all samples were quantitatively analyzed by means of the abundance (normalized). When two groups are compared pairwise, calculating the mean value of all the normalized signals of the samples in each group, calculating the ratio Fold change between the groups, and calculating two groups of p values (p-values) by using Mann-Whitney U test.
4) Screening for differential proteins: the screening standard is that the ratio of the expression quantity in the PCOS group to the expression quantity in the pregnant group is more than or equal to 1.2 or less than or equal to 0.83; and assume a value P of less than 0.05.
After screening, the biomarkers of the invention are obtained.
The heatmap of the follicular fluid differential expression proteins of the group with high embryo production rate (HP) and the group with low embryo production rate (LP) selected in this example is shown in FIG. 2.
Example 2
This example demonstrates the biomarker obtained in example 1.
The follicular fluid differential proteins of each of the PCOS population and the fertile population screened in example 1 were subjected to an ELISA test with an expanded sample size to verify the protein expression level of these differential proteins in follicular fluid.
The specific steps of ELISA experiment of each follicular fluid differential protein are as follows:
1) preparing enzyme and related reagents required by an ELISA experiment, a standard substance of follicular fluid differential protein, follicular fluid of PCOS people and follicular fluid of pregnant people;
2) adding a proper standard substance or sample into a microplate according to the requirements of an ELISA experimental kit, and simultaneously adding a responsive antibody mixture in the kit;
3) the microplate was sealed and incubated for 1 hour at room temperature on a flat shaker set at 400 rpm;
4) wash with corresponding volume of 1 × wash buffer for at least 10 seconds; after the last washing, inverting the tray, slightly beating clean paper towels, and removing redundant liquid;
5) an appropriate volume of TMB developer was added to each well and incubated for 10 minutes in the dark on a plate shaker flask set at 400 rpm. The optimal incubation time may vary in view of the changing environmental conditions of the laboratory (note: addition of stop solution will change the color from blue to yellow and increase the signal intensity by about 3-fold to avoid saturation of the signal, proceed to the next step before the high concentration standard solution reaches a blue color where o.d.600 equals 1.0.)
6) To each well, 100. mu.L of stop solution was added to terminate the color reaction. Mix by shaking on a shaker for one minute. The absorbance at 450nm wavelength was recorded. And finally, calculating the protein concentrations of the follicular fluid of the PCOS population and the follicular fluid of the pregnant population according to the standard curve, and verifying the difference of the expression levels of the PCOS population and the pregnant population.
Through verification, the expression amounts of the 27 differential proteins in the invention in two groups of people have obvious difference, which shows that the biomarkers in the invention can be used for evaluating the quality of oocytes of PCOS people.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A set of biomarkers for assessing PCOS oocyte quality, wherein the biomarkers are a plurality of follicular fluid differential proteins, including upregulating proteins and downregulating proteins;
the ratio of the expression amount of the up-regulated protein in the PCOS population to the expression amount of the up-regulated protein in the pregnant population is greater than or equal to 1.2;
the ratio of the expression level of said down-regulated protein in said PCOS population to the expression level thereof in said pregnant population is less than or equal to 0.83.
2. The panel of claim 1, wherein the upregulating proteins comprise at least one of inhibin β C chain, pantothenase, proteoglycan 4, angiotensinogen, FERM-and PDZ-domain containing protein 1, lymphatic endothelial hyaluronic acid receptor 1, phosphatidylinositol proteoglycan-3, hydrocephalus-inducing protein homolog, cholinesterase, N-acetyl chitosanase, neuronal adhesion molecule L1.
3. The set of biomarkers for assessing PCOS oocyte quality according to claim 1, wherein the downregulating proteins include at least one of monocyte differentiation antigen CD14, leucine rich alpha-2 glycoprotein, olfactory mediator-like protein 3, laminin subunit alpha-1, intercellular adhesion molecule 2, vascular cell adhesion protein 1, serine glycine, cadherin-1, complement factor H related protein 2, complement factor H related protein 4, metalloproteinase inhibitor 2, cystatin-M, pepsin a-4, cadherin-11, multidrug resistance related protein 9, immunoglobulin heavy chain allosteric 3-15, transforming growth factor beta receptor type 3, cartilage matrix protein-2.
4. Use of a panel of biomarkers according to any one of claims 1 to 3 for assessing oocyte quality in a PCOS population.
5. A kit for evaluating the quality of PCOS oocytes, wherein the kit contains the biomarker for evaluating the quality of PCOS oocytes according to any one of claims 1 to 3.
6. The kit for evaluating the quality of a PCOS oocyte according to claim 5, wherein the kit contains 4 to 5 of the up-regulated protein and/or the down-regulated protein.
7. A screening method for biomarkers for assessing PCOS oocyte quality according to any one of claims 1 to 3, comprising the steps of:
s1, collecting follicular fluid of the PCOS population and follicular fluid of the pregnant population respectively;
s2, respectively carrying out non-labeled proteomics detection and analysis on the collected follicular fluid of the two groups of people;
the non-standard proteomics detection and analysis process comprises the steps of firstly extracting protein in follicular fluid, then carrying out enzymolysis on the obtained protein to obtain a peptide fragment, and then carrying out mass spectrometry on the peptide fragment;
s3, processing the mass spectrometry data obtained in the step S2, and screening out proteins with significant differential expression in follicular fluid of two groups of people to obtain the biomarker for evaluating the PCOS oocyte quality;
wherein the screening is carried out according to the standard that the expression level of the protein in the PCOS population and the expression level of the protein in the pregnant population have a difference multiple of more than or equal to 1.2 or a difference multiple of less than or equal to 0.83.
8. The screening method of biomarkers for evaluating PCOS oocyte quality according to claim 7,
the pregnant people are healthy pregnant people, and the infertility factor is a male factor.
9. The screening method of biomarkers for evaluating PCOS oocyte quality according to claim 7,
the specific process of step S2 is as follows:
s2-1, extracting protein: pretreating the extracted protein by adopting a high-abundance protein removal chromatographic column to obtain a protein sample from which albumin, IgG and other high-abundance proteins are removed;
s2-2, determining the protein concentration of the protein sample by using a BCA method;
s2-3, carrying out reductive alkylation treatment on the protein sample;
s2-4, performing FASP enzymolysis on the protein sample subjected to reductive alkylation treatment;
s2-5, desalting the protein sample after FASP enzymolysis;
s2-6, carrying out liquid chromatography-mass spectrometry analysis on the protein sample after desalting treatment.
10. The screening method for biomarkers for evaluating PCOS oocyte quality according to claim 7, wherein in said step S2, the mass spectrometry comprises eluting and separating peptide fragments and mass spectrometry detection; wherein, the elution separation peptide section is subjected to gradient elution by adopting a mobile phase A and a mobile phase B;
the mobile phase A is a formic acid-water solution with the volume ratio of 1:1000, and the mobile phase B is a formic acid-acetonitrile solution with the volume ratio of 1: 1000;
before gradient elution, dissolving the peptide segment after enzymolysis by using a sample dissolving solution, wherein the sample dissolving solution is a formic acid-water solution with the volume ratio of 1: 1000;
the gradient elution process comprises fully oscillating the dissolved peptide fragment solution, and performing LC-MS mass spectrometry on 2ug of the solution; the analysis time is 120min, and the flow rate is 300 nL/min; gradient elution conditions were as follows, wherein the proportions of mobile phase B are all volume percent: 0min-5min, 2% -8% B; 5min-90min, 8% -24% B; 90-110 min, 24-32% B; 110min-115min, 32% -90% B; 115min-120min, keeping 90% B;
and after the gradient washing is finished, detecting the separated peptide fragment by adopting a mass spectrometer.
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