Uterine Lavage Fluid 16S rRNA Gene Sequencing in Rats with Embryo Implantation Dysfunction
This study aimed to determine the alterations in the microbiota of uterine lavage fluid in rats with embryo implantation dysfunction (EID) following treatment with the modified Shoutai Wan (JWSTW) herbal formula. JWSTW is composed of seven medicinal herbs: Tu Si Zi, E Jiao, Sang Ji Sheng, Xu Duan, Dan Shen, Dang Gui, and Xiang Fu. Seven-week-old Sprague-Dawley (SD) rats were used in the experiment and housed in the specific pathogen-free (SPF) animal facility. A rat model of EID was established via subcutaneous injection of a mifepristone oil solution into the neck region. After one week of adaptive feeding, female rats with regular estrous cycles were selected. At 5:00 PM daily, female and male rats were housed together at a ratio of 2:1. The following morning at 7:00 AM, vaginal smears were collected from the female rats. The presence of sperm in the smear was designated as gestational day 1. The mated female rats were randomly divided into different groups: (1) the control group; (2) the model group; (3) the JWSTW group, in which rats were orally administered JWSTW of 8.4 g/kg. Both the control group and the model group were administered an equal volume of sterile water by gavage. With the exception of the control group, all female rats were administered mifepristone for modeling on the morning of gestational day 4. The control group was administered an equivalent volume of sesame oil at the same time point. Female rats were subjected to oral gavage daily at 9:00 AM from day 1 to day 6 of gestation. One hour after oral administration, rats were anesthetized using an intraperitoneal injection of pentobarbital, uterine lavage fluid was collected from Control (n=6), Model (n=6), and JWSTW (n=6) rats under sterile conditions. Each uterine hornwas gently flushed with sterile PBS (volume: 2 ml), and the recovered fluid was immediately frozen. Microbial DNA was extracted using the Mag-Bind SoilDNA Kit (Omega). The 16S rRNA gene V3-V4 was amplified using specific primerswith a Barcode and a high-fidelity DNA polymerase and sequenced on an Illumina Novaseq 6000. Sequence processing and ASV inference were performed using QIIME2/DADA2, taxonomy was assigned against the Silva database Version 138, and beta-diversity ordination (PCoA/NMDS) and LEfSe analyses were conducted (parameters:LDA>2).