#METABOLOMICS WORKBENCH yli_20221117_075202 DATATRACK_ID:3584 STUDY_ID:ST002368 ANALYSIS_ID:AN003863 VERSION 1 CREATED_ON 12-12-2022 #PROJECT PR:PROJECT_TITLE Sperm Environmental Epigenetics and Development Study (SEEDS) PR:PROJECT_TYPE C18 Reversed-Phase Broad Spectrum Metabolomics PR:PROJECT_SUMMARY Infertility is one of the most common reproductive health disorders affecting PR:PROJECT_SUMMARY 16% of couples in the U.S. Most concerning are the new meta-analysis data PR:PROJECT_SUMMARY showing that sperm counts among men in developed countries have declined over PR:PROJECT_SUMMARY 50% in the past four decades. With no sign of reversing this downward PR:PROJECT_SUMMARY trajectory, we may not only be facing a fertility crisis, but low sperm count PR:PROJECT_SUMMARY also has wider public health implications, including increased risks in PR:PROJECT_SUMMARY morbidity and mortality. Given this dramatic decrease in sperm quality over a PR:PROJECT_SUMMARY short period, genetic influences are likely not attributable, but rather, PR:PROJECT_SUMMARY environmental factors encountered over the life-course. The objective of this PR:PROJECT_SUMMARY pilot project is to determine the feasibility of generating metabolomic data PR:PROJECT_SUMMARY from human seminal plasma collected as part of the ongoing SEEDS cohort. PR:INSTITUTE NC HHEAR Hub PR:DEPARTMENT Untargeted Analysis PR:LABORATORY Sumner Lab PR:LAST_NAME Li PR:FIRST_NAME Yuanyuan PR:ADDRESS 500 Laureate Way, Kannapolis, NC 28081 PR:EMAIL yuanyli4@unc.edu PR:PHONE 9843770693 PR:DOI http://dx.doi.org/10.21228/M8ZH8S #STUDY ST:STUDY_TITLE Sperm Environmental Epigenetics and Development Study (SEEDS) ST:STUDY_SUMMARY Infertility is one of the most common reproductive health disorders affecting ST:STUDY_SUMMARY 16% of couples in the U.S. Most concerning are the new meta-analysis data ST:STUDY_SUMMARY showing that sperm counts among men in developed countries have declined over ST:STUDY_SUMMARY 50% in the past four decades. With no sign of reversing this downward ST:STUDY_SUMMARY trajectory, we may not only be facing a fertility crisis, but low sperm count ST:STUDY_SUMMARY also has wider public health implications, including increased risks in ST:STUDY_SUMMARY morbidity and mortality. Given this dramatic decrease in sperm quality over a ST:STUDY_SUMMARY short period, genetic influences are likely not attributable, but rather, ST:STUDY_SUMMARY environmental factors encountered over the life-course. The objective of this ST:STUDY_SUMMARY pilot project is to determine the feasibility of generating metabolomic data ST:STUDY_SUMMARY from human seminal plasma collected as part of the ongoing SEEDS cohort. ST:INSTITUTE Wayne State University ST:LAST_NAME Pilsner ST:FIRST_NAME Rick ST:ADDRESS 275 E. Hancock Street, Detroit, MI, USA ST:EMAIL rpilsner@wayne.edu ST:PHONE 917-557-2499 ST:SUBMIT_DATE 2022-11-17 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS S087 S_3 Seminal quality:LSQ | Live Birth:Live birth RAW_FILE_NAME=S_3 SUBJECT_SAMPLE_FACTORS S075 S_4 Seminal quality:LSQ | Live Birth:Live birth RAW_FILE_NAME=S_4 SUBJECT_SAMPLE_FACTORS S048 S_1 Seminal quality:LSQ | Live Birth:Not-live birth RAW_FILE_NAME=S_1 SUBJECT_SAMPLE_FACTORS S069 S_2 Seminal quality:LSQ | Live Birth:Not-live birth RAW_FILE_NAME=S_2 SUBJECT_SAMPLE_FACTORS S099 S_5 Seminal quality:LSQ | Live Birth:Not-live birth RAW_FILE_NAME=S_5 SUBJECT_SAMPLE_FACTORS N/A NIST_1_1 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=NIST_1_1 SUBJECT_SAMPLE_FACTORS N/A NIST_1_2 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=NIST_1_2 SUBJECT_SAMPLE_FACTORS N/A NIST_2_1 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=NIST_2_1 SUBJECT_SAMPLE_FACTORS N/A NIST_2_2 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=NIST_2_2 SUBJECT_SAMPLE_FACTORS N/A SP_1_1 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=SP_1_1 SUBJECT_SAMPLE_FACTORS N/A SP_1_2 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=SP_1_2 SUBJECT_SAMPLE_FACTORS N/A SP_2_1 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=SP_2_1 SUBJECT_SAMPLE_FACTORS N/A SP_2_2 Seminal quality:N/A | Live Birth:N/A RAW_FILE_NAME=SP_2_2 SUBJECT_SAMPLE_FACTORS S092 S_7 Seminal quality:NSQ | Live Birth:Live birth RAW_FILE_NAME=S_7 SUBJECT_SAMPLE_FACTORS S131 S_9 Seminal quality:NSQ | Live Birth:Live birth RAW_FILE_NAME=S_9 SUBJECT_SAMPLE_FACTORS S133 S_10 Seminal quality:NSQ | Live Birth:Not-live birth RAW_FILE_NAME=S_10 SUBJECT_SAMPLE_FACTORS S038 S_6 Seminal quality:NSQ | Live Birth:Not-live birth RAW_FILE_NAME=S_6 SUBJECT_SAMPLE_FACTORS S113 S_8 Seminal quality:NSQ | Live Birth:Not-live birth RAW_FILE_NAME=S_8 #COLLECTION CO:COLLECTION_SUMMARY Semen samples were collected in a sterile plastic specimen cup after a 2–3-day CO:COLLECTION_SUMMARY abstinence period. To separate motile sperm from the seminal plasma, semen CO:COLLECTION_SUMMARY samples were processed using a two-step (80 and 40%) gradient fractionation. The CO:COLLECTION_SUMMARY seminal plasma was removed from the top of the gradient and placed in a sterile CO:COLLECTION_SUMMARY tube and frozen at -80oC until analyses. CO:SAMPLE_TYPE Seminal plasma CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Seminal plasma samples (500 µL each) were shipped from Dr. Pilsner’s lab at SP:SAMPLEPREP_SUMMARY the University of Massachusetts Amherst to the NC HHEAR Hub with dry ice and SP:SAMPLEPREP_SUMMARY stored at -80 °C until analysis. NIST blood plasma aliquots (SRM 1950, 50 µL) SP:SAMPLEPREP_SUMMARY were provided by the NC HHEAR hub and used external reference materials. All SP:SAMPLEPREP_SUMMARY samples (including study samples and NIST reference materials) were thawed at 4 SP:SAMPLEPREP_SUMMARY °C overnight. The seminal plasma sample (50 µL) was transferred from the SP:SAMPLEPREP_SUMMARY shipped original tubes to a new set of pre-labeled Lo-Bind Eppendorf tubes for SP:SAMPLEPREP_SUMMARY sample preparation. A quality control total pool was made by pooling an SP:SAMPLEPREP_SUMMARY additional 7-µL seminal plasma from each of the original samples into a new SP:SAMPLEPREP_SUMMARY Lo-Bind Eppendorf tube and then distributed into multiple aliquots within 50 µL SP:SAMPLEPREP_SUMMARY each to use as quality control study pools (QCSPs) ran throughout the whole SP:SAMPLEPREP_SUMMARY study. LC-MS grade water aliquots (50 µL) were used as blanks. All samples, SP:SAMPLEPREP_SUMMARY including study samples, QCSPs, NIST reference material samples, and blanks, SP:SAMPLEPREP_SUMMARY were mixed with 400 µL methanol containing 500 ng/mL tryptophan-d5 (internal SP:SAMPLEPREP_SUMMARY standard) and vortexed by the multiple tube vortex mixer for 2 min at 5000 rpm SP:SAMPLEPREP_SUMMARY at room temperature. The supernatants (350 µL) were transferred into SP:SAMPLEPREP_SUMMARY pre-labeled 2.0 mL Lo-bind Eppendorf tubes and then dried by a SpeedVac SP:SAMPLEPREP_SUMMARY overnight. For immediate analysis, 100 µL of water-methanol solution (95:5, SP:SAMPLEPREP_SUMMARY v/v) was used to reconstitute the dried extracts, and the samples were SP:SAMPLEPREP_SUMMARY thoroughly mixed on the multiple tube vortex mixer for 10 min at 5000 rpm at SP:SAMPLEPREP_SUMMARY room temperature and then centrifuged at 4°C for 10 min at 16,000 rcf. The SP:SAMPLEPREP_SUMMARY supernatant was transferred to pre-labeled autosampler vials for data SP:SAMPLEPREP_SUMMARY acquisition by the instrument. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reverse phase CH:INSTRUMENT_NAME Thermo Scientific™ Vanquish™ UPHPLC CH:COLUMN_NAME Acquity UPLC HSS T3 C18 (100 x 2.1mm, 1.8um) CH:COLUMN_PRESSURE 6000-10000 CH:COLUMN_TEMPERATURE 50 CH:FLOW_GRADIENT Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3. CH:FLOW_GRADIENT 16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5 6. 22.00 CH:FLOW_GRADIENT 0.4 99.0 1.0 5 CH:FLOW_RATE 0.4 ml/min CH:INJECTION_TEMPERATURE 8 CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% methanol; 0.1% formic acid CH:RANDOMIZATION_ORDER Randomized CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:LABORATORY_NAME UNC-NRI Sumner/Li lab AN:ANALYSIS_TYPE MS AN:SOFTWARE_VERSION Xcalibur 4.1.31.9 AN:OPERATOR_NAME Madison Schroder AN:DETECTOR_TYPE Orbitrap AN:DATA_FORMAT Profile #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Instrument: Thermo Q Exactive HFx Software: Xcalibur 4.1.31.9 for data MS:MS_COMMENTS acquisition; Progenesis QI 2.4 for data preprocessing MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 320 °C MS:CAPILLARY_VOLTAGE 3.5 KV MS:COLLISION_ENERGY 20-45, ramp MS:COLLISION_GAS N2 MS:DRY_GAS_FLOW 55 MS:DRY_GAS_TEMP 400°C MS:FRAGMENTATION_METHOD CID MS:DESOLVATION_GAS_FLOW 55 MS:MS_RESULTS_FILE ST002368_AN003863_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END