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 Are there phase-specific changes in the early secretory (ES) phase human tubal lavage proteome that can inform and potentially optimize IVF culture media? SUMMARY ANSWER The 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. WHAT IS KNOWN ALREADY The 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. STUDY DESIGN, SIZE, DURATION Proteome-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 = 482) were collected from superovulated wild type C57BL/6 female mice and cultured in microdrops over 5 days for the assessment of blastocyst development. PARTICIPANTS/MATERIALS, SETTING, METHODS Human 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. MAIN RESULTS AND THE ROLE OF CHANCE Comparison 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. LIMITATIONS, REASONS FOR CAUTION Although 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. WIDER IMPLICATIONS OF THE FINDINGS This 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. STUDY FUNDING/COMPETING INTEREST(S) This 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. TRIAL REGISTRATION NUMBER N/A

Region-Specific Decellularization of Porcine Uterine Tube Extracellular Matrix: A New Approach for Reproductive Tissue-Engineering Applications

“…On the other hand, the isthmus has a more rigid and less flexible structure, being the region that connects the uterine tube to the uterus [53]. The ampulla has an intermediate structural configuration compared to the other two, allowing a greater interaction between the embryo and the tubal tissue [54]. Due to these particularities, the development of decellularization protocols is delicate, being necessary the standardization of each step to preserve the ECM without compromising the efficiency of cells removal processes.…”

Region-Specific Decellularization of Porcine Uterine Tube Extracellular Matrix: A New Approach for Reproductive Tissue-Engineering Applications

“…On the other hand, the isthmus has a more rigid and less flexible structure, being the region that connects the uterine tube to the uterus [18]. The ampulla has an intermediate structural configuration compared to the other two, allowing a greater interaction between the embryo and the tubal tissue [52]. Due to these particularities, the development of decellularization protocols is delicate, the standardization of each step being necessary to preserve the ECM without compromising the efficiency of the cell removal processes.…”

Integrative Proteomics and Transcriptomics Profiles of the Oviduct Reveal the Prolificacy-Related Candidate Biomarkers of Goats (Capra hircus) in Estrous Periods

“…Hydrogen sulfide (H 2 S) is a potent vasodilator and angiogenic factor with potent angiogenic activity, and cystathionine β-synthase (CBS) upregulation increases endogenous H 2 S production, which plays a role in uterine vasodilation and is involved in pregnancy [ 64 , 65 ]. The trophic role of the oviduct is widely supported during early mitotic active embryonic development [ 66 ], where the vasculature acts as a nutrient delivery system and therefore provides nutrients to the embryo and gametes in the oviduct. In mammalian cells, PES1 is associated with more than 50% of telomerase activity, and data suggest that PES1 is a key cofactor in telomerase assembly and can regulate telomerase activity and positively regulate cellular senescence and accretion [ 67 ].…”