A Metabolomics Study Of Follicular Fluid With The Theory Of “Obesity Causes Infertility” From Fu Qingzhu’s Female Discipline

Objective:We aimed to discuss the clinical outcomes of infertility patients with spleen deficiency phlegm dampness type simple overweight/obesity based on the theory of“Obesity causes infertility”from Fu Qingzhu’s female discipline.With the purpose of evaluating the clinical efficacy of Xiaonang Yusi Decoction in treating infertility patients with spleen deficiency phlegm dampness type simple overweight/obesity by in vitro fertilization and embryo transfer（IVF-ET）,and to elucidate the scientific meaning of the theory of“Obesity causes infertility”with the metabolomics and explore the targets and mechanisms of traditional Chinese medicine.Methods:A prospective randomized controlled trial was conducted to recruit102 infertility patients undergoing IVF-ET assisted pregnancy due to tubal or male side factors alone,of whom 68 were simple overweight/obesity patients,and they were randomly divided into two groups:Thirty-four overweight/obesity patients were treated with traditional Chinese medicine group（BMI＿TCM group）and 34 overweight/obesity patients with normal control（BMI＿OB group）.The other 34 patients were the blank control group（BMI-CON group）who received IVF due to the female fallopian tube factors or the male factor.All patients in the three groups were superovulated using a gonadotropin releasing hormone antagonist（Gn RH-ant）protocol.Patients in BMI＿TCM group received oral traditional Chinese medicine（TCM）Xiaonang Yusi Decoction beginning on the 5th day of the menstrual cycle before controlled ovarian hyperstimulation（COH）,at one payment per day,water decoction,divided into doses in the morning and night,until the patient was discontinued on the h CG day.The IVF treatment status（number of oocytes retrieved,total number of normal fertilized oocytes,normal fertilization rate,total number of available embryos,number of good quality embryos,number of remaining embryos,fresh embryo transfer pregnancy rate,fresh embryo transfer miscarriage rate,cumulative pregnancy rate,miscarriage rate）and the change in score of traditional Chinese medicine syndrome of spleen deficiency phlegm dampness type between the evaluation groups were observed.In the experimental section,metabolites were measured in follicular fluid samples obtained at the time of oocyte retrieval by transvaginal puncture from patients in three groups using an ultra performance liquid chromatography triple quadrupole mass spectrometer（UHPLCQTRAP MS）.The experimental procedures included sample preparation,QC preparation,sample LC-MS/MS mass spectrometry and data analysis.Carry out identification quantity statistics and chemical classification attribution statistics for the measured metabolites.Reliable data models were built by multidimensional statistical analysis and differentially expressed metabolites among the three groups were sought by differential analysis between groups.In order to understand the mutual regulatory relationship and related pathways between metabolites,bioinformatics analysis was carried out on the selected significantly different metabolites（P<0.05）,including cluster analysis,correlation analysis,pathway analysis,etc.To elucidate the theory of“Obesity causes infertility”from the metabolomics level and explore the related mechanisms of Xiaonang Yusi Decoction interventions for overweight/obesity.Results:（1）The clinical trial was finally completed by 95 patients,including 34 in the overweight/obesity weight intervention group,30 in the overweight/obesity normal control group,and 31 in the blank control group.The clinical trial results showed that there was no significant difference in the baseline characteristics of the three groups of patients（P>0.05）.The score of spleen deficiency phlegm dampness syndrome was significantly（P<0.05）reduced after TCM intervention compared with that before treatment.The symptoms of simple overweight/obesity patients with spleen-deficiency and phlegm-dampness type,such as excessive weight loss,mental fatigue,limb fatigue,abdominal distension,loose stool,tongue coating,etc.,were significantly improved.There were no significant differences in the number of oocytes retrieved,the levels of serum estradiol（E₂）and progesterone（P）trigger day or the number of remaining embryos among the three groups（P>0.05）.But the level of luteinizing hormone（LH）on the trigger day of patients in BMI＿OB group was significantly higher than that in BMI＿CON group（P<0.05）.Compared with patients in BMI＿CON group,patients in BMI＿TCM group and patients in BMI＿OB group had a prolonged Gn days and an increased in the total number of Gn,but decreased in the total number of normally fertilized oocytes and the normal fertilization rate.Besides the number of available embryos on the day 3 after oocyte pick-up（OPU）and the number of top-quality embryos（TQEs）in BMI＿OB group were significantly lower than those in BMI＿CON group.Interestingly,after the intervention of traditional Chinese medicine,the total number of normal fertilized oocytes and high-quality embryos in BMI＿TCM group were significantly higher than those in BMI＿OB group（P all<0.05）.There was a significant difference among the three groups in terms of miscarriage rate,which was significantly higher in BMI＿OB group patients compared with BMI＿CON group patients by pairwise comparison（50%vs.16%,P=0.029）.However,when focusing on the number of fresh embryo transfer cycles,fresh embryo transfer pregnancy rate,fresh embryo transfer abortion rate,and cumulative pregnancy rate of embryos formed by oocyte retrieval in this cycle,there was no significant difference among the three groups（P all>0.05）.（2）The results of high-throughput targeted metabolomics analysis showed that233 different metabolites were identified in the follicular fluid of the three groups of patients.By comparing BMI＿CON groups and BMI＿OB group,18differential metabolites were found,of which 11 metabolites were down regulated and 7 metabolites were up regulated,involving 15 metabolic and signal transduction pathways.Comparison between BMI＿TCM group and BMI＿OB group found 28 different metabolites,of which 24 were down regulated and 4were up regulated,involving 30 metabolic and signal transduction pathways.Conclusion:（1）In simple overweight/obesity infertility patients with hyposplenic sputum dampness type,the duration of Gn use was prolonged,the total amount of Gn and the miscarriage rate were increased,but the total number of normal fertilized oocytes,the normal fertilization rate,the number of available embryos,the number of TQE were decreased.（2）Xiaonang Yusi Decoction can improve the related TCM symptoms of simple overweight/obesity infertility patients with spleen deficiency and phlegm dampness.In addition,Xiaonang Yusi Decoction can also increase the total number of normal fertilized oocytes and high-quality embryos in patients with simple overweight/obesity infertility of spleen deficiency and phlegm dampness type,and reduce the miscarriage rate.（3）The composition and contents of metabolites in the follicular fluid of simple overweight/obesity infertility patients with hyposplenic sputum dampness type can be affected by the decoction of Xiaonang Yusi Decoction,and these substances may be targets for TCM intervention.