The proteome of human Fallopian tube lavages during the phase of embryo transit reveals candidate proteins for the optimization of preimplantation embryo culture.

Abstract

STUDY QUESTION\nAre there phase-specific changes in the early secretory (ES) phase human tubal lavage proteome that can inform and potentially optimize IVF culture media?\n\n\nSUMMARY ANSWER\nThe human tubal lavage proteome during the ES phase relative to the menstrual phase reveals substantial differential protein abundance in pathways such as glycolysis, redox homeostasis and activation of 14-3-3 zeta-mediated signaling.\n\n\nWHAT IS KNOWN ALREADY\nThe Fallopian tube is uniquely suited to the development of the preimplantation embryo as it transits the tube during the ES phase of the menstrual cycle. Euploid cleavage-stage embryo arrest may reflect incomplete recapitulation of in-vivo conditions by current media formulations.\n\n\nSTUDY DESIGN, SIZE, DURATION\nProteome-wide analysis of distal tubal lavage specimens collected from 26 healthy women undergoing open microtubal anastomosis surgery from January 2013 to January 2018 was performed. Specimens were grouped by menstrual cycle phase in order to analyze phase-specific differences in protein abundance. For the murine embryo assay, single-cell embryos (N\u2009=\u2009482) were collected from superovulated wild type C57BL/6 female mice and cultured in microdrops over 5\u2009days for the assessment of blastocyst development.\n\n\nPARTICIPANTS/MATERIALS, SETTING, METHODS\nHuman tubal lavage specimens were processed for label-free mass spectrometry. Reported menstrual cycle day was confirmed by measuring serum hormones. Key protein targets in the ES phase were validated via immunoblot. The ES phase-specific increase in 14-3-3 zeta protein was confirmed via ELISA of conditioned media obtained from primary human Fallopian tube epithelial cell culture. A murine embryo assay was performed to investigate the impact of graduated concentrations of 14-3-3 zeta on the blastocyst development rate.\n\n\nMAIN RESULTS AND THE ROLE OF CHANCE\nComparison of the ES and menstrual phase human tubal lavage proteomes revealed 74 differentially expressed proteins with enrichment of pathways and biological processes involved in the regulation of carbohydrate metabolism, oxidative stress and cell survival. The adapter-regulator protein 14-3-3 zeta was among the most significantly increased in the ES phase. Supplementation of embryo culture media with 14-3-3 zeta at concentrations tested did not significantly improve the murine blastocyst development.\n\n\nLIMITATIONS, REASONS FOR CAUTION\nAlthough select associations were recapitulated in the conditioned media from sex steroid exposed primary human tubal epithelial cells, cell culture represents an in-vitro approximation. Changes to embryo culture media, such as protein supplementation, must undergo rigorous preclinical safety testing prior to adoption for human use.\n\n\nWIDER IMPLICATIONS OF THE FINDINGS\nThis study represents the first description of the human Fallopian tube lavage proteome across the menstrual cycle, revealing a unique proteomic signature during the ES phase. Although supplementation of culture media with 14-3-3 zeta at appropriate concentrations showed no significant impact on the murine blastocyst development rate, other biologically plausible candidate proteins for individual or high throughput testing strategies are identified.\n\n\nSTUDY FUNDING/COMPETING INTEREST(S)\nThis work was funded in part by an Army Medical Department Advanced Medical Technology Initiative grant from the United States Army Medical Research and Materiel Command s Telemedicine and Advanced Technology Research Center. There are no competing interests.\n\n\nTRIAL REGISTRATION NUMBER\nN/A.