Maternal intestinal L. vaginalis facilitates embryo implantation and survival through enhancing uterine receptivity in sows

Abstract

Background: The embryo implantation quality during early pregnancy is the predominant factor for embryo survival and litter performance in sows. Gut microbiota is demonstrated to show a correlation to pregnancy outcomes by participating in regulating maternal metabolism. However, the specific functional microbiota and its mechanical effects on regulating embryo implantation and survival remain unclear. The objective of this study was to clarify whether embryo implantation and litter performance were affected by maternal intestinal microbiota, and to identify specific microbial communities and its mechanism in regulating embryo implantation.

Results: In this study, we first conducted 16S rRNA sequencing and metabolomic analysis revealing the intestinal microbiota and metabolism of 42 sows with different litter size to select the potential functional microbiota that may contribute to embryo survival. Then, we explored the effects of that microbiota on embryo implantation and litter performance through microbiota transplantation in mice and sows. We found that maternal intestinal L. vaginalis exhibits enrichment in sows with higher litter size, which could facilitate embryo implantation and survival and ultimately increases litter size in mice. We further employed transcriptomic analysis to determine the characteristics of uterus, which found an enhanced uterine receptivity after L. vaginalis gavage. The plasma untargeted metabolomic analysis after L. vaginalis gavage in mice and targeted metabolomics analysis of in vitro cultured medium of L. vaginalis were used to evaluate the metabolic regulation of L. vaginalis and to reveal the underlying functional metabolites. Next, an increasing adhesion rate of endometrial-embryonic cells and an obvious increasing formation of pinopodes in cell surface of porcine endometrial epithelial cells were observed after treatments of L. vaginalis metabolites, especially galangin and daidzein. Also, the gene expression levels related to uterine receptivity were increased after treatments of L. vaginalis metabolites in porcine endometrial epithelial cells. Finally, we found that L. vaginalis or its metabolites supplementation during early gestation significantly increased the litter performance in sows.

Conclusions: Overall, intestinal microbial-host interactions can occur during early pregnancy and may be contribute to maternal metabolic changes and influence pregnancy outcomes in mammals. Our study provides insights of maternal intestinal L. vaginalis to enhance uterine receptivity and to benefit embryo/fetal survival through a gut-uterus axis, contributing to advanced concept and novel strategy to manipulate gut microbiota during early pregnancy, and in turn to improve embryo implantation and reduce embryo loss in sows. Video Abstract.

Keywords: L. vaginalis; Embryo implantation; Litter performance; Sows; Uterine receptivity.

Fig. 1

A distinct intestinal microbiota composition in sows with different reproductive performances. A Overview of study design for unveiling relationship of maternal intestinal microbiota and reproductive performance in sows with higher or lower litter size. B–C Number of litter size and liver litter size in sows. D–F Levels of estradiol (E₂), progesterone (P₄), and short chain fatty acids (SCFAs) in feces of sows at day 28 of pregnancy. G Intestinal different bacteria showed in genus and species level between sows with higher and lower litter size. H–I The abundance of intestinal C. butyricum and L. vaginalis in sows detected by qRT-PCR. J–M The scatter plots of correlation coefficient for abundance of C. butyricum and L. vaginalis correlated with litter size and live litter size. Statistical analyses were performed using unpaired Students’ t-test (B–F) or Mann–Whitney U test (G). ns, not significant; *p < 0.05; **p < 0.01. J–M Correlation coefficients were analyzed using Pearson correlation analysis, and least square linear regression lines (red line) with 95% confidence interval (pink shading) are provided for visual representation of the non-parametric testing

Fig. 2

Untargeted metabolomics analysis reveals intestinal metabolism changes in sows with different reproductive performances. A–B PLS-DA analysis and volcano plot (High L × Y group vs Low L × Y group) of fecal metabolome of sows at day 28 of pregnancy. C Pathway enrichment analysis of different metabolites. The color gradients and circles’ size respectively correspond to the fluctuation of -log10 (p value) and impacts. D Correlation between intestinal different metabolites and bacteria and litter size. Correlation coefficients were analyzed using Spearman correlation analysis. n = 21

Fig. 3

Microbiota transplantation of L. vaginalis improves embryo implantation and increases litter sizes in mice. A Experimental scheme for the generation and analysis of microbiota transplantation on embryo implantation efficacy at early pregnancy and offspring survival at delivery. B–H The total litter size, live litter size, total litter weight, average litter weight, coefficient of variation of litter pups’ weight, and the ratios of male or female of litter pups at delivery after microbiota transplantation in mice. I–K Number of embryo implantation sites, embryo survival ratio, and number of corpus luteum at day 6 of pregnancy. L Experimental scheme for the generation and analysis of bacterial cells or metabolites of L. vaginalis supply on embryo implantation efficacy. M–O Number of embryo implantation sites, embryo survival ratio, and number of corpus luteum at day 6 of pregnancy in mice after bacterial cells or metabolites of L. vaginalis supply. In A and L, the red arrow points to the date of gavage. Statistical analyses were performed using unpaired Students’ t-test. ns, not significant; *p < 0.05; **p < 0.01

Fig. 4

Targeted metabolomics analysis exhibits ex vivo cultured metabolites of L. vaginalis. A Growth curve of L. vaginalis measured by turbidimetric method. B PCA analysis of targeted metabolome of L. vaginalis ex vivo cultured for 0, 4, 12, and 30 h. C Heatmap of metabolites showing products and substrates of L. vaginalis. D Fold changes and levels of metabolites after L. vaginalis cultured for 30 h. E Pathway enrichment analysis of different metabolites (30 h vs 0 h) of L. vaginalis. n = 6

Fig. 5

Effects of L. vaginalis transplantation on histomorphology of uterus and colon, colonic microbiota composition, and steroid hormones synthesis in mice. A–B Representative H&E-stained sections of uterine implantation site and colonic tissue of mice at day 6 of pregnancy. The gestational sac was marked with blue arrows. C α-diversities of colonic bacteria of mice at day 6 of pregnancy. D Colonic microbial communities clustered using PCoA of weighted Unifrac matrix. E–F The abundance of L. vaginalis detected by qRT-PCR in colon of mice at day 4 and day 6 of pregnancy. G–H Levels of estradiol and progesterone in plasma of mice at day 4 and day 6 of pregnancy. I The relative mRNA level of genes related to steroid hormones synthesis in ovaries of mice at day 4 of pregnancy. Statistical analyses were performed using Mann–Whitney U test (C) or unpaired Students’ t-test (F–H). ns, not significant; *p < 0.05; **p < 0.01

Fig. 6

Microbiota transplantation of L. vaginalis enhances uterine receptivity. A Volcano plot showing different expressed genes (DEGs) in uterus of mice at day 4 of pregnancy. B Network plot showing the subset of enriched GO terms in the top 20 clusters. C GSEA analysis showing enriched KEGG pathways related to cell–cell adhesion. D Circos plot visualizing putative relationship between DEGs and GO terms. E Bar plot showing mRNA level of genes related to embryonic development, cell–cell adhesion, and angiogenesis. Statistical analyses were performed using unpaired Students’ t-test. *p < 0.05; **p < 0.01. n = 4

Fig. 7

Microbiota transplantation of L. vaginalis modulates maternal plasma metabolism. A PLS-DA analysis of plasma metabolome in mice at day 4 of pregnancy. n = 5. B The differential abundance score (L. vaginalis vs PBS) of top 20 enriched KEGG pathways in plasma metabolome analysis of mice at day 4 of pregnancy. C Correlation analysis showing the top 20 enriched KEGG pathways for plasma metabolome (blue bar) and uterine transcriptome (red bar) of mice at day 4 of pregnancy. D Expression profile and VIP of different metabolites. E ROC analysis of different metabolites including daidzein, norethindrone oxime, taurocholic acid, galangin, and caproic acid. Statistical analyses were performed using unpaired Students’ t-test. *p < 0.05; **p < 0.01

Fig. 8

L. vaginalis metabolites enhance endometrial epithelial cells receptivity and improves litter performances in sows. A Schematic representation of the microfluidic chip used for simulating endometrial-embryo adhesion. B Physical photo of the microfluidic chip. C Fluorescence microscopy viewing the attachment of PTCs to endometrial microfluidic chip. D Scanning electron microscopy viewing the microvilli on the cell surface of PEECs. E Experimental scheme for the generation and analysis of bacterial cells or metabolites of L. vaginalis supply during early gestation on offspring survival in sows. F–J Litter performance including total litter size, live litter size, litter size of litter pup’s weight higher than 0.95 kg, average litter pup’s weight, and total litter weight of sows. K The abundance of intestinal L. vaginalis in sows detected by qRT-PCR. Statistical analyses were performed using unpaired Students’ t-test. ns, not significant; *p < 0.05; **p < 0.01