Metabolomic and microbiome analysis of cervicovaginal mucus in in vitro fertilization-embryo transfer: Toward predicting pregnancy success.

Purpose: In the context of in vitro fertilization-embryo transfer (IVF-ET), factors other than egg quality may be key determinants of treatment success, in particular, maternal factors related to uterine endometrial receptivity and unidentified factors. We therefore aimed to analyze the metabolome and microbiome in IVF-ET patients who did and did not achieve pregnancy. Methods: Cervicovaginal mucus was collected from patients undergoing IVF-ET. Metabolite analysis was conducted by liquid chromatography-mass spectrometry and the microbiota were determined by the polymerase chain reaction using universal 16S-rRNA gene bacterial primers by MiSeq sequencing. Patients were classified as pregnant (N = 10) or nonpregnant (N = 13). Metabolic pathways were examined by MetaboAnalyst. Results: Three metabolic pathways, including alanine-aspartate-glutamate metabolism, arginine biosynthesis, and cysteine-methionine metabolism, were commonly decreased at the time of embryo transfer irrespective pregnant outcomes. Notably, pyruvate was decreased in the pregnant group. Amino acid metabolites showed inverse correlations with the presence of anaerobic microbiota in the nonpregnant group. Conclusions: Metabolism decreased during embryo transplantation, with a notable decrease in pyruvate metabolism, particularly in patients who became pregnant. The behavior of metabolites in the pregnant and nonpregnant groups suggests that metabolome analysis in the cervicovaginal mucus may be a diagnostic marker for predicting pregnancy.

Changes to the cervicovaginal microbiota and cervical cytokine profile following surgery for cervical intraepithelial neoplasia.

Persistent HPV infection associated with immune modulation may result in high-grade squamous intraepithelial lesions (CIN)2/3. Currently, there is little information on the cervicovaginal microbiome, local cytokine levels and HPV infection related to CIN. Follow-up of patients after local surgery provides an opportunity to monitor changes in the cervicovaginal environment. Accordingly, we undertook this longitudinal retrospective study to determine associations between HPV genotypes, cervicovaginal microbiome and local cytokine profiles in 41 Japanese patients with CIN. Cervicovaginal microbiota were identified using universal 16S rRNA gene (rDNA) bacterial primers for the V3/4 region by PCR of genomic DNA, followed by MiSeq sequencing. We found that Atopobium vaginae was significantly decreased (p < 0.047), whereas A. ureaplasma (p < 0.022) increased after surgery. Cytokine levels in cervical mucus were measured by multiplexed bead-based immunoassays, revealing that IL-1ß (p < 0.006), TNF-α (p < 0.004), MIP-1α (p < 0.045) and eotaxin (p < 0.003) were significantly decreased after surgery. Notably, the level of eotaxin decreased in parallel with HPV clearance after surgery (p < 0.028). Thus, local surgery affected the cervicovaginal microbiome, status of HPV infection and immune response. Changes to the cervicovaginal microbiota and cervical cytokine profile following surgery for cervical intraepithelial neoplasia may be important for understanding the pathogenesis of CIN in future.

MicroRNA­126­3p suppresses HeLa cell proliferation, migration and invasion, and increases apoptosis via the PI3K/PDK1/AKT pathway.

We previously reported that relative to normal cervical mucus, microRNA 126­3p (miR­126­3p) is present in significantly greater amounts in the cervical mucus of patients with overt cervical cancer or precursor lesions. Here, we investigated the effects of enforced miR­126­3p expression in the cervical cancer cell line, HeLa, on proliferation, migration, invasion, apoptosis and protein expression. We transfected HeLa cells with miR­126­3p miRNA and found that proliferation, migration and invasion by cell counting, wound healing, cell migration and invasion assay were significantly reduced in these cells relative to those transfected with a negative control mimic. The levels of phosphoinositide 3 kinase (PI3K), phosphorylated 3­phosphoinositide­dependent protein kinase­1 (p­PDK1) and p­AKT proteins were lower in the miR­126­3p­transfected cells. Phosphorylated 70S6K (p­p70S6K), phosphorylated glycogen synthase kinase 3ß (p­GSK3ß), phosphorylated S6K (p­S6K), cyclin D1, phosphorylated p21­activated kinase 1 (p­PAK1), Rho associated coiled­coil containing protein kinase 1 (ROCK1), myotonic dystrophy­related CDC42­binding kinases α (MRCKα) and phospholipase C γ1 (p­PLCγ1) were also downregulated. This suggests that downstream effectors of the PI3K/PDK1/AKT pathway are targets for inhibition by miR­126­3p. In contrast, apoptotic­related proteins including the BCL­2­associated agonist of cell death (Bad), B­cell lymphoma­extra­large (Bcl­xL) and BCL­2­associated X (Bax), were all upregulated by miR­126­3p, resulting in increased caspase 3/7 activity and apoptosis. Thus, enforced expression of miR­126­3p inhibited cell migration and invasion and also induced apoptosis by regulating the PI3K/PDK1/AKT pathway in HeLa cells. Hence, high levels of miR­126­3p may inhibit cervical carcinogenesis, and targeting the PI3K/PDK1/AKT pathway via miR­126­3p could represent a new approach for treating patients with cervical cancer.