| Objective: To explore mechanism of female mice gut microbiome affect the development of conception and for the use of intestinal flora adjustment and improvement of infertility.Methods:1.The experimental group: 264 female Swiss(ICR)mice were provided by the experimental animal center of Dalian Medical University(Dalian,China),where they were maintained under specific pathogen-free conditions and were divided into gut microbiota dysbiosis group and control group randomly2.The experimental batches: Including four batches(spring,summer,autumn and winter),66 female Swiss mice one batch.The intestinal flora disorder group and normal control group contained 33 respectively.3.The experimental cycle: 46 days for each batch mice experimental cycle,experimental group intestinal flora disturbance from the first day to 28 days,then these female mice were mated with male mice for 3:1 and were checked for pregnant.We observed female mice pregnancy bolt for 3 days consecutively,appeared pregnancy bolt female mice alive another 16 days and then executed,no pregnancy bolt mice immediately executed.4.Establishment of the intestinal dysbacteriosis model: Female mice were treated with 400 mg/ml ceftriaxone sodium dose of 0.2ml two times a day intra-gastrically,six to eight hours at intervals for four weeks.At the same time thenormal control group intragastric administration with saline water,dose the same with above method.5.Pregnancy bolt examination: We detected the pregnancy bolt in the second morning after mated female and male mice last night and observed the female mice vaginal white solidification residue.Residue was pregnancy bolt as conception.6.Samples collection: Collecting female mice ovary,serum,embryonic mice intestinal tract,intestinal contents and feces for index detection.7.ELISA method was applied to groups of mice serum estrone,estradiol,testosterone,androstenedione level detection.8.The application method of PCR-DGGE to analysis groups of mice intestinal flora,clear difference of strip plastics recycling,purification and sequencing,sequencing results via GenBank to determine the bacterial species.9.Collecting the ovary of female mice HE staining slices,observing ovarian morphological changes.Results:1.The conception number of gut microbiota dysbiosis group was79 conception rate was 81.4%,the conception number of normal control group was 69,conception rate was 71.1%,the difference between the two groups was significantly(P<0.05).Compared with the normal control group,gut flora disorder group conception rate was higher than normal group 10.3%.2.The change of seasons and intestinal dysbacteriosis effects on pregnancy were different,compared with normal control group,among them,the winter flora disorder group with the most obvious change,conception rate was 87.0%;Spring and summer change of intestinal dysbacteriosis group followed,conception rate were 81.80% and76.0% respectively.The autumn intestinal dysbacteriosis group change was not obvious,conception rate 62.9%.3.Compared with the normal control group,the number of stillbirths in the flora disorder group was 32,still-birth rate was 4.44%,significantly difference between thetwo groups(P<0.05),the flora disorder group still-birth rate was higher than the normal control group.4.In female mice fetuses intestinal we found 16 sRNA sequence of intestinal flora.5.Compared with the control group,the pregnant mice in the gut microbiota dysbacteria group Estrone and Estradiol levels were decreased(p<0.05);Androstenedione content was increased(p<0.05),no significant change in testosterone level(p>0.05).6.Compared the ovary with the normal control group,the flora disorder group granular cell layer and theca folliculi layer were thin,the number of ovarian follicles increased,the morphology of ovarian follicles were obviously bigger than the control.Conclusions1.Lack of gut bacteria caused conception rate increased,still-birth rate rised.2.Lack of gut bacteria effected sex hormones.3.Lack of gut bacteria caused morphological changes in the ovary4.In mice fetuses intestinal we found 16 sRNA sequence of intestinal flora.Intestinal5.Dysbacteriosis can replace morphology and PCR-DGGE technique with total nucleic acid concentration. |
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