To Investigate The Mechanism Of Follicular Fluid Metabolomics In Early Embryo Loss After In Vitro Fertilization-Embryo Transfer Based On The Theory Of "Kidney Dominant Reproductive"

Objective: Through the collection of clinical syndromes of early spontaneous abortion,summarize the distribution rule of TCM syndromes,in order to expect to reveal the change rule of early spontaneous abortion syndromes and provide guidance for clinical diagnosis and treatment.At the same time,targeted metabolomics technology was used to screen and identify follicular fluid markers associated with early spontaneous abortion,providing preliminary ideas for predicting early pregnancy outcomes after IVF-ET.Methods: The clinical symptoms of patients with early spontaneous abortion who visited the Reproductive and Genetic Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2021 to August 2022 were investigated.The characteristics of TCM syndromes of early spontaneous abortion were summarized by systematic cluster analysis.The patients were divided into groups.Descriptive analysis,chi-square test and other statistical methods were used to summarize the general conditions and syndrome characteristics of patients in each syndrome group,and the correlation between each index and TCM syndromes was analyzed.From April 2019 to August 2021,57 patients with clinical pregnancy after in vitro fertilization-embryo transfer(IVF-ET)were selected from the Reproductive Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine for the first time,including 35 patients in the early spontaneous abortion group(divided into 15 patients in the conventional abortion group and 20 patients in the aneuploid abortion group)and 22 patients in the continuous pregnancy group.Follicular fluid composition(FF)was analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry(HPLC-MS/MS)in the two groups,and the data were processed using univariate statistical analysis and orthogonal partial least squares discriminant analysis(OPLS-DA)to screen FF differential metabolites in the abortion and ongoing pregnancy groups.Twenty-six patients with clinical pregnancy after in vitro fertilization-embryo transfer(IVF-ET)were selected from October 2021 to June 2022 in the Reproductive Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine,including 14 patients with early spontaneous abortion(divided into routine abortion group(n = 4)and aneuploid abortion group(n = 10))and 12 patients with persistent pregnancy.The differential metabolites screened by non-target metabolism in the early stage were qualitatively and quantitatively analyzed by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry,and the relevant metabolic pathways were analyzed by KEGG Pathway to target and verify the differential metabolites screened by non-target metabolism in the early stage and find follicular fluid markers associated with early spontaneous abortion.Results:(1)Among 183 cases of early spontaneous abortion,126 cases(68.9%)were kidney deficiency syndrome,which was the main syndrome of early spontaneous abortion.Among them,there were 34 cases of kidney qi deficiency syndrome,accounting for 18.6%;26 cases of kidney yin deficiency syndrome,accounting for 14.2%;23 cases of kidney yang deficiency syndrome,accounting for 12.6%;17 cases of kidney yin and yang deficiency syndrome,accounting for 9.3%;9 cases of kidney deficiency and blood stasis syndrome,accounting for 4.9%;8 cases of kidney deficiency and blood-heat syndrome,accounting for 4.4%;6 cases of kidney deficiency and blood deficiency syndrome,accounting for 3.3%;and 3 cases of kidney deficiency and damp-heat syndrome,accounting for1.6%.In addition,there were 28 cases of liver depression syndrome,accounting for 15.3%;12 cases of blood stasis syndrome,accounting for6.6%;7 cases of qi and blood deficiency syndrome,accounting for 3.8%;5cases of blood-heat syndrome,accounting for 2.7%;3 cases of liver depression and spleen deficiency syndrome,accounting for 1.6%;and 2 cases of spleen and kidney yang deficiency syndrome,accounting for 1.1%.(2)The most common symptoms in 183 patients with early spontaneous abortion were soreness and weakness of waist and knee in 126 cases(68.9%),followed by dizziness and tinnitus in 109 cases(59.6%),fatigue in 98 cases(53.6%),abdominal pain in 95 cases(51.9%),oligomenorrhea in 87 cases(47.5%),low back cold pain in 83 cases(45.4%),hot flashes and night sweats in 77 cases(42.1%),and irritability in 74 cases(40.4%).(3)differential metabolites between patients with early spontaneous abortion and those with persistent pregnancy were mainly concentrated in amino acid metabolism,fatty acid metabolism,steroid metabolism,purine metabolism,and B vitamin metabolism.Subgroup analysis revealed that meso-tartaric acid,β-hyodeoxycholic acid,2-hydroxy-3-methylbutyric acid,ketoleucine,7-methylguanosine,docosatetraenoic acid(DTA),docosapentaenoic acid(DPA),eicosatrienoic acid(ETA),docosahexaenoic acid(DHA),eicosenoic acid(EA),methionine,corticosterone,pantothenic acid,and creatinine were up-regulated in FF of patients in the conventional abortion group,and cysteine and tryptophan were up-regulated in FF of patients in the persistent pregnancy group;while glycochenodeoxycholic acid,glycoursodeoxycholic acid,2-hydroxy-3-methylbutyric acid,indoleacetic acid,sphingomyelin,meso-tartaric acid,7-methylguanosine,and ketoleucine were downregulated in the aneuploid abortion group compared with the conventional abortion group;However,glycolic acid 3-sulfate and phenylacetic acid were up-regulated in the aneuploid abortion group compared with patients in the persistent pregnancy group.Conclusion: Clinical epidemiological investigation showed that the TCM syndrome types of patients with early spontaneous abortion were mainly kidney deficiency syndrome,and the clinical symptoms were mainly soreness and weakness of the waist and knees,which were consistent with the concept of " kidney dominant reproductive ".Combined analysis of non-target and targeted metabolism in follicular fluid showed that metabolites such as amino acids,fatty acids,steroids,purines,and B vitamins were associated with the occurrence of early spontaneous abortion.Targeted identification of these differential metabolites in follicular fluid may provide guidance for routine embryo transfer as a non-invasive screening tool and provide the possibility to reduce the occurrence of early spontaneous abortion in infertile patients.