Proteomic determinants of uterine receptivity for pregnancy in early and mid-postpartum dairy cows

Abstract

Dairy cow subfertility is a worldwide issue arising from multiple factors. It manifests in >30% early pregnancy losses in seasonal pasture-grazed herds, especially when cows are inseminated in the early post-partum period. Most losses occur before implantation, when embryo growth depends on factors present in maternal tract fluids. Here we examined the proteomic composition of early and mid-postpartum uterine luminal fluid in crossbred lactating dairy cows to identify molecular determinants of fertility. We also explored changes in uterine luminal fluid from first to third estrus cycles postpartum in individual cows, linking those changes with divergent embryo development. For this, we flushed uteri of 87 cows at day 7 of pregnancy at first and third estrus postpartum, recovering and grading their embryos. Out of 1563 proteins detected, 472 had not been previously reported in this fluid, and 408 were predicted to be actively secreted by bioinformatic analysis. The abundance of 18 proteins with roles in immune regulation and metabolic function (e.g. cystatin B, pyruvate kinase M2) was associated with contrasting embryo quality. Matched-paired pathway analysis indicated that, from first to third estrus postpartum, upregulation of metabolic (e.g. creatine and carbohydrate) and immune (e.g. complement regulation, antiviral defense) processes were related to poorer quality embryos in the third estrus cycle postpartum. Conversely, upregulated signal transduction and protein trafficking appeared related to improved embryo quality in third estrus. These results advance the characterization of the molecular environment of bovine uterine luminal fluid and may aid understanding fertility issues in other mammals, including humans.

Metabolomic evolution of the postpartum dairy cow uterus

“…Then, metabolomic changes in ULF between timepoints are reported and analyzed regarding their potential effect on embryo quality. This was done both by univariate analysis and pathway enrichment analysis, considering only metabolites or in conjunction with previously generated proteomics data on the same samples (Aranciaga et al, 2021)…”

“…Details about animals enrolled as well as the proportion of embryos of different stages and qualities, and protein abundance in ULF, have been reported elsewhere (Aranciaga et al, 2021). Briefly, 87 animals were enrolled, inseminated, and sampled by flushing at their first estrus after calving.…”

“…Previous work by this group has recently shown a clear increase in the proportion of viable embryos with increased time and number of estrus events after calving (Aranciaga et al, 2021; Berg et al, 2022). Several proteins were suggested as potential biomarkers of uterine suitability for pregnancy, as well as time‐related changes in immune (e.g., myeloid leukocyte activation) and metabolic (e.g., amino acid metabolism) processes (Aranciaga et al, 2021).…”

“…The animal study has been previously described (Aranciaga et al, 2021) and is presented in the Supporting Information: . In brief, healthy crossbred dairy cows ( N = 87) were enrolled during their naturally expressed first and third estrus after calving.…”

“…Embryos recovered (excluding those from ULF discarded due to blood contamination) were assigned a numerical value as a proxy for their quality or likelihood to develop into a healthy calf as in our previous work (Aranciaga et al, 2021). This consisted of five classes corresponding to distinct embryo phenotypes: 1 cell oocytes/embryos (i.e., potentially due to fertilization failure, score = 5), 4‐ to 16‐cell (arrested development; in this study, no 2‐cell embryos were recovered, score = 4), Grade 3 (fair quality, score = 3), Grade 2 (good quality, score = 2), and Grade 1 (excellent quality, score=1), the latter three at either morula or blastocyst stage.…”