DOI: 10.5061/dryad.sbcc2fr7t
title: Uterine lumen fluid is metabolically semi-autonomous

description: Uterine lumen fluid (ULF) is central to successful pregnancy establishment
 and maintenance, and impacts offspring wellbeing into adulthood. The
 current dogma is that ULF composition is primarily governed by endometrial
 glandular epithelial cell secretions and influenced by progesterone. To
 investigate the hypothesis that ULF is metabolically semi-autonomous, ULF
 was obtained from cyclic heifers, and aliquots incubated for various
 durations prior to analysis by untargeted semi-quantitative metabolomic
 profiling. Metabolite flux was observed in these ULF isolates, supporting
 the idea that the biochemical makeup of ULF is semi-autonomously dynamic
 due to enzyme activities. Subsequent integrative analyses of these, and
 existing, data predict the specific reactions underpinning this
 phenomenon. These findings enhance our understanding of the mechanisms
 leading to pregnancy establishment, with implications for improving
 fertility and pregnancy outcomes in domestic animals as well as women.
description: <b>Animals</b> All animal procedures were conducted in accordance with the Guide for the Care and Use of Agriculture Animals in Research and Teaching and approved by the Institutional Animal Care and Use Committee at the University of Missouri. Fifteen (15) Holstein heifers were used averaging 15 months of age with a mean (± SD) body condition score (scale of 1-5) of 3.04 ± 0.44 and 766.6 ± 62.8 kg weight. On a random day of their estrous cycle, heifers were synchronized to estrus using an established protocol. Heifers initially received a dose of gonadotropin releasing hormone (GnRH), coupled to the insertion of a controlled internal drug release (CIDR) intravaginal P4 insert (1.9g, CIDR, Zoetis, NJ, USA). CIDR removal occurred 6 days thereafter, at which time, a PGF<sub>2</sub><sub>a</sub> dose (25 mg Dinoprost, Lutalyse, Zoetis, NJ, USA) was also administered, and heat detection patches (Estrotect, TN, USA) applied. A second PGF<sub>2</sub><sub>a</sub> dose was administered 24 h later and heifers were visually observed for standing estrus at 12 h intervals for 72 h. Estrus was designated by heat detection patch activation. Thereafter, heifers were distributed in two experimental groups for ULF retrieval on Days 12 and 16 post-estrus. On the day of ULF retrieval, the presence of an ovarian <i>corpus luteum</i> was confirmed by transrectal ultrasonography prior to uterine lavage. In brief, 10 ml PBS was gently expelled into the uterine body using a silicone catheter coupled to a syringe. The uterine body was gently massaged transrectally, and ULF was recovered by generating a mild negative pressure using the syringe. Upon retrieval, ULF was immediately processed as described below. All flushes were performed by the same technician. Of the 15 heifers initially enrolled, two were excluded on account of not exhibiting signs of estrus. The remaining 4 were removed, as ULF flushes were visibly contaminated with blood based on color. <b>Uterine lavage collection and processing</b> Uterine lavages were immediately equally apportioned into 7 aliquots. All but two aliquots from each lavage were incubated at 38°C with shaking (Genie Temp Shaker 100; Scientific Industries; Bohemia, NY, USA) for either 2, 5, 10, 15, or 20 min. Following incubation, these aliquots were immediately placed at 4°C to effectively quench enzymatic activity and then clarified by centrifugation at 1,000 <i>x g</i> for 15 min at 4°C (5424R; Eppendorf, Hamburg, Germany). One aliquot (Time 0) was maintained at 4°C for 20 min prior to centrifugation as described. The supernatants were submerged in N<sub>2</sub>(<i>l</i>) and stored at -80 °C until shipment for analysis on dry ice, as described below. A ‘control’ aliquot was not processed and immediately flash frozen in N<sub>2</sub>(<i>l</i>) and stored at -80°C until total protein was quantified using a Qubit 3.0 Fluorometer (ThermoFisher, Waltham, MA, USA) as per manufacturer instructions. <b>Metabolomic profiling</b> Metabolomic analyses were performed by ultrahigh performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) by Metabolon Inc. (Durham, NC, USA). Briefly, protein was precipitated and extracted using the automated MicroLab STAR system (Hamilton Company) with methanol under vigorous centrifugation at 680 <i>x g</i> for 2 min (Geno/Grinder 2000, Glen Mills) prior to methanol removal by TurboVap (Zymark) and overnight incubation in N<sub>2</sub>. Each sample was subsequently divided into 4 fractions – two for analysis by reverse phase (RP) UPLC-MS/MS with positive ion mode electrospray ionization (ESI), one for analysis by RP UPLC-MS/MS with negative four-ion mode electrospray ionization (ESI), and one for analysis by hydrophilic interaction liquid chromatography (HILIC) UPLC-MS/MS with negative ion mode ESI. Sample extracts were then dried and reconstituted in solvents as outlined below. The first fraction, analyzed under positive ionization, was subject to gradient elution (Waters UPLC BEH 1.7 μm C18 column 2.1 x 100 mm) in water and methanol with 0.05% perfluoropentanoic acid and 0.1% formic acid. The second fraction, run under positive ESI, was identically eluted, using the same column, but with an elution buffer additionally comprising acetonitrile. The third fraction, analyzed under negative ionization, was also eluted by gradient buffer comprising methanol, water, and 6.5 mM ammonium bicarbonate (pH 10.8). The last fraction, ran under negative ESI, was eluted using a HILIC (Waters UPLC BEH Amide 1.7 μm column 2.1 x 150 mm) with a water and acetonitrile plus 10 mM ammonium formate (pH 10.8) gradient. Samples were analyzed using a Waters Acquity UPLC coupled to a Thermo Scientific Q-Exactive high resolution MS interfaced with heated electrospray ionization (HES-II) source and Orbitrap mass analyzer operating at 35,000 mass resolution and with a scan range between 70-1000 <i>m</i>/<i>z</i>. Metabolites were quantified against known internal and recovery standards, run in parallel at random intervals. Identification was based on retention time and a <i>m</i>/<i>z</i> within ± 10 ppm. The technical (instrument) median relative standard deviation was 5% with a total process variability of 10%. Three controls were analyzed in parallel with the experimental samples: (<i>i</i>) a pooled aliquot of all experimental samples, serving as a technical replicate control; (<i>ii</i>) ultra-pure water samples served as process blanks, also run in between the experimental samples at defined intervals; and (<i>iii</i>) a cocktail of quality control metabolites, absent from endogenous compound measurements, were spiked into each sample. The latter internal standard enabled instrument performance monitoring and chromatographic alignment.<b><i> </i></b>
description: These raw data in .csv format have been uploaded in accompaniment
 to a publication entitled “Uterine Fluid is Metabolically Semi-Autonomous”
 by Constantine A. Simintiras, Jessica N. Drum, Hongyu Liu, M. Sofia
 Ortega, and Thomas E. Spencer in Communications Biology.
 Specifically, bovine uterine lumen fluid (ULF) was flushed on
 Days 12 (n=5) and 16 (n=4) post-estrous and aliquoted. Each aliquot was
 incubated at 38°C for either 0 min (control), 2 min, 5 min, 10 min, 15
 min, or 20 min. Following minor sample processing, as delineated in the
 Methods, each aliquot was subjected to ultrahigh performance liquid
 chromatography tandem mass spectroscopy (UPLC-MS/MS). This experiment was
 designed to test the hypothesis that ULF is metabolically semi-autonomous
 — i.e. that select biochemical reactions are active within ULF
 independently of external influences.  The raw
 chromatographic data obtained are provided herein. For example, the cell
 corresponding to “Day 12 Heifer 1 Time 5 min” and “1-methylhistidine” is
 the raw intensity data for the chromatographic peak observed for
 1-methylhistidine in the aliquot of ULF from the first heifer flushed on
 Day 12 and incubated for 5 minutes. Unidentified metabolites are depicted
 as ND (not detected).