Summary of Study ST003266

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002028. The data can be accessed directly via it's Project DOI: 10.21228/M8GC01 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003266
Study TitleMetabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia (MS data)
Study SummaryBackground: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia, or low sperm motility, is a common cause of male infertility with complex etiology, involving genetic and metabolic alterations, inflammation, and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility. Methods: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and asthenozoospermia (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate. Results: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa’s metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered in asthenozoospermia. Conclusions: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy, and lipid metabolism in asthenozoospermia. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the etiology of decreased motility in asthenozoospermia.
Institute
University of Aveiro
DepartmentDepartment of Chemistry
Last NameGuerra-Carvalho
First NameBárbara
AddressCampus Universitário de Santiago, 3810-193 Aveiro, Portugal
Emailbarbaraggcarvalho@gmail.com
Phone234 370 360
Submit Date2024-06-15
Num Groups2
Total Subjects57
Num Males57
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-07-25
Release Version1
Bárbara Guerra-Carvalho Bárbara Guerra-Carvalho
https://dx.doi.org/10.21228/M8GC01
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002028
Project DOI:doi: 10.21228/M8GC01
Project Title:Metabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia
Project Summary:Background: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia, or low sperm motility, is a common cause of male infertility with complex etiology, involving genetic and metabolic alterations, inflammation, and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility. Methods: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and asthenozoospermia (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate. Results: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa’s metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered in asthenozoospermia. Conclusions: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy, and lipid metabolism in asthenozoospermia. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the etiology of decreased motility in asthenozoospermia.
Institute:University of Aveiro
Department:Department of Chemistry
Last Name:Guerra-Carvalho
First Name:Bárbara
Address:Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
Email:barbaraggcarvalho@gmail.com
Phone:234 370 360
Project Comments:Study part 1 of 2

Subject:

Subject ID:SU003386
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:20 to 49 years old
Gender:Male
Human Inclusion Criteria:Participants enrolled in this study were randomly recruited among men who attended fertility consultations at the Centre for Reproductive Genetics Professor Alberto Barros (Porto, Portugal) and the Centro Hospitalar Universitário de Santo António (Porto, Portugal) from March to June 2021. Adult men (age ≥ 18 years) diagnosed with asthenozoospermia (AS, sperm total motility < 42%) were recruited for AS group and men with normal sperm parameters (sperm concentration ≥ 15 million cells/mL, sperm count ≥ 39 million cells and sperm total motility ≥ 42%) were recruited for the control group (normozoospermia (NZ) group).
Human Exclusion Criteria:Men with a history of abusing drugs or alcoholism, under treatment or exposed to drugs known to interfere with fertility, diagnosed with acute genital inflammatory disease or with systemic disease known to affect fertility before sperm collection, survivors of cancer treatment, or men diagnosed with leukocytospermia were excluded from this study. Smoking was not included as an exclusion criterion for this study.

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Group Sample source
SA354284Not applicable2Asthenozoospermia Pooled spermatozoa
SA354285Not applicable1Asthenozoospermia Pooled spermatozoa
SA354286N49Asthenozoospermia Spermatozoa
SA354287N15Asthenozoospermia Spermatozoa
SA354288N64Asthenozoospermia Spermatozoa
SA354289N58Asthenozoospermia Spermatozoa
SA354290N56Asthenozoospermia Spermatozoa
SA354291N54Asthenozoospermia Spermatozoa
SA354292N53Asthenozoospermia Spermatozoa
SA354293N14Asthenozoospermia Spermatozoa
SA354294N44Asthenozoospermia Spermatozoa
SA354295N28Asthenozoospermia Spermatozoa
SA354296N43Asthenozoospermia Spermatozoa
SA354297N26Asthenozoospermia Spermatozoa
SA354298N17Asthenozoospermia Spermatozoa
SA354299N29Asthenozoospermia Spermatozoa
SA354300N3Asthenozoospermia Spermatozoa
SA354301N34Asthenozoospermia Spermatozoa
SA354302N4Asthenozoospermia Spermatozoa
SA354303N27Asthenozoospermia Spermatozoa
SA354304Not applicable4Normozoospermia Pooled spermatozoa
SA354305Not applicable3Normozoospermia Pooled spermatozoa
SA354306N60Normozoospermia Spermatozoa
SA354307N51Normozoospermia Spermatozoa
SA354308N52Normozoospermia Spermatozoa
SA354309N57Normozoospermia Spermatozoa
SA354310N59Normozoospermia Spermatozoa
SA354311N6Normozoospermia Spermatozoa
SA354312N7Normozoospermia Spermatozoa
SA354313N61Normozoospermia Spermatozoa
SA354314N62Normozoospermia Spermatozoa
SA354315N68Normozoospermia Spermatozoa
SA354316N69Normozoospermia Spermatozoa
SA354317N5Normozoospermia Spermatozoa
SA354318N70Normozoospermia Spermatozoa
SA354319N8Normozoospermia Spermatozoa
SA354320N9Normozoospermia Spermatozoa
SA354321N50Normozoospermia Spermatozoa
SA354322N42Normozoospermia Spermatozoa
SA354323N48Normozoospermia Spermatozoa
SA354324N20Normozoospermia Spermatozoa
SA354325N10Normozoospermia Spermatozoa
SA354326N11Normozoospermia Spermatozoa
SA354327N12Normozoospermia Spermatozoa
SA354328N13Normozoospermia Spermatozoa
SA354329N16Normozoospermia Spermatozoa
SA354330N18Normozoospermia Spermatozoa
SA354331N19Normozoospermia Spermatozoa
SA354332N2Normozoospermia Spermatozoa
SA354333N21Normozoospermia Spermatozoa
SA354334N47Normozoospermia Spermatozoa
SA354335N23Normozoospermia Spermatozoa
SA354336N24Normozoospermia Spermatozoa
SA354337N25Normozoospermia Spermatozoa
SA354338N35Normozoospermia Spermatozoa
SA354339N37Normozoospermia Spermatozoa
SA354340N38Normozoospermia Spermatozoa
SA354341N41Normozoospermia Spermatozoa
SA354342N45Normozoospermia Spermatozoa
SA354343N46Normozoospermia Spermatozoa
SA354344N30Normozoospermia Spermatozoa
Showing results 1 to 61 of 61

Collection:

Collection ID:CO003379
Collection Summary:Human seminal samples were obtained through masturbation into a specific sterile container after 2 to 4 days of sexual abstinence. After collection, samples were left at 37°C until complete liquefaction and sperm parameters (semen pH, ejaculate volume, sperm concentration, sperm count and sperm motility) were analyzed according to the WHO guidelines 5. After routine semen analysis, spermatozoa were separated from the seminal fluid through centrifugation at 500.g for 5 min. Pelleted spermatozoa were washed two times with phosphate buffer saline (PBS) before being stored at -80°C.
Sample Type:Spermatozoa
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003395
Treatment Summary:Participants enrolled in this study were randomly recruited among men who attended fertility consultations at the Centre for Reproductive Genetics Professor Alberto Barros (Porto, Portugal) and the Centro Hospitalar Universitário de Santo António (Porto, Portugal) from March to June 2021. Adult men (age ≥ 18 years) diagnosed with asthenozoospermia (AS, sperm total motility < 42%) were recruited for AS group and men with normal sperm parameters (sperm concentration ≥ 15 million cells/mL, sperm count ≥ 39 million cells and sperm total motility ≥ 42%) were recruited for the control group (normozoospermia (NZ) group).

Sample Preparation:

Sampleprep ID:SP003393
Sampleprep Summary:Pellets containing 20 million spermatozoa were resuspended in 500 µL of ice-cold methanol/water (4:1, v/v) solution and subjected to ultrasonic homogenization in a Vibra Cell sonicator (Sonics & Materials, Inc.) for 2.5 min at 4°C, with 5 s ON - 5 s OFF intervals with 20% amplitude using a ¼” probe. Samples were then incubated at -20°C for 30 min to allow protein precipitation, and centrifuged at 14000.g for 30 min at 4°C. After centrifugation, supernatants were collected into a new 1.5 mL centrifuge tube, dried in SpeedVac Plus SC 210A (Thermo Savant, NY, USA), and stored at -20°C before LC-MS analysis.

Combined analysis:

Analysis ID AN005350 AN005351
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column Ascentis Si HPLC Pore column (15 cm x 1 mm, 3µm), Sigma-Aldrich Ascentis Si HPLC Pore column (15 cm x 1 mm, 3µm), Sigma-Aldrich
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE
Units normalized areas normalized areas

Chromatography:

Chromatography ID:CH004052
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Ascentis Si HPLC Pore column (15 cm x 1 mm, 3µm), Sigma-Aldrich
Column Temperature:40°C
Flow Gradient:100% of B was held in isocratic mode for 1 min (t = 0-1 min); then it was linearly decreased to 50% of B within 14 min (t = 15 min); 50% of B was held in isocratic mode for 5 min (t = 20 min); finally, returned to 100% of B in t = 20.01 which was held in isocratic mode for 10 min (t = 30 min).
Flow Rate:200µL/min
Internal Standard:LeuTyr, 0.05 µg
Solvent A:2.5% acetonitrile/2.5% methanol/95% water; 5 mM ammonium formate; 0.1% formic acid
Solvent B:90% acetonitrile/10% water; 5 mM ammonium formate; 0.1% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS005080
Analysis ID:AN005350
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Spermatozoa metabolite content was analyzed by HPLC-ESI-MS and HPLC-ESI-MS/MS using a hydrophilic interaction liquid chromatography (HILIC) column, on an UltiMate 3000 UHPLC system (Thermo Scientific, Germering, Germany) coupled to a Q-Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher, Scientific, Bremen, Germany). The metabolite extracts were resuspended in 47.5 µL of ice-cold methanol/water (4:1, v/v) solution and 2.5 µL of internal standard (LeuTyr, 0.05 µg). Three quality control samples were prepared: NZ quality control, containing 5 µL of each NZ sample (n = 39); AS quality control, containing 5 µL of each AS sample (n = 18); and Total quality control, containing 160 µL of the NZ quality control and 45 µL of the AS quality control. Five µL of each sample were injected into the HPLC column (Ascentis Si HPLC Pore column, 15 cm x 1 mm, 3µm, Sigma-Aldrich), with a flow rate of 200µL/min, at 40°C. One injection per biological sample was performed for the full scan MS run, and quality control samples were used for the MS/MS experiments. The mass spectrometer was simultaneously operated in positive (electrospray voltage of 3.2 kV) and negative (electrospray voltage of 2.9 kV) ionization modes, with a capillary temperature of 320 °C, sheath gas (nitrogen) flow of 35 units and auxiliary gas flow of 3 units. Data acquisition was performed in full scan mode with a high resolution of 70 000, automatic gain control (AGC) target of 1 x 106 and scan range of m/z 65 – 900. Data-dependent MS/MS was acquired for the 10 most abundant species with a resolution of 17 500, AGC target of 1 x 105, dynamic exclusion of 30 s and intensity threshold of 1 x 104. Cycles consisted of one full scan MS followed by 10 data-dependent MS/MS scans. Collision energies of 20, 30 and 40 were used in both positive and negative modes. Data acquisition and processing were performed using the Xcalibur data system (V 3.06, Thermo Fisher Scientific, Waltham, MA, USA). Metabolites were identified using the Mass Spectrometry-Data Independent Analysis Software V 4.60 (MS-DIAL) 25 and integrated using the MZmine V 2.53 software 26. LC-MS lipidomics data were normalized by dividing the area of the ion corresponding to the molecular species by the sum of the areas of all quantified species, and metabolomics data were normalized by dividing the area of the internal standard.LC-MS metabolomics and lipidomics data were log-transformed and EigenMS was used to normalize data.
Ion Mode:POSITIVE
Capillary Temperature:320 °C
Capillary Voltage:3.2 kV
Collision Energy:20, 30 and 40
  
MS ID:MS005081
Analysis ID:AN005351
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Spermatozoa metabolite content was analyzed by HPLC-ESI-MS and HPLC-ESI-MS/MS using a hydrophilic interaction liquid chromatography (HILIC) column, on an UltiMate 3000 UHPLC system (Thermo Scientific, Germering, Germany) coupled to a Q-Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher, Scientific, Bremen, Germany). The metabolite extracts were resuspended in 47.5 µL of ice-cold methanol/water (4:1, v/v) solution and 2.5 µL of internal standard (LeuTyr, 0.05 µg). Three quality control samples were prepared: NZ quality control, containing 5 µL of each NZ sample (n = 39); AS quality control, containing 5 µL of each AS sample (n = 18); and Total quality control, containing 160 µL of the NZ quality control and 45 µL of the AS quality control. Five µL of each sample were injected into the HPLC column (Ascentis Si HPLC Pore column, 15 cm x 1 mm, 3µm, Sigma-Aldrich), with a flow rate of 200µL/min, at 40°C. One injection per biological sample was performed for the full scan MS run, and quality control samples were used for the MS/MS experiments. The mass spectrometer was simultaneously operated in positive (electrospray voltage of 3.2 kV) and negative (electrospray voltage of 2.9 kV) ionization modes, with a capillary temperature of 320 °C, sheath gas (nitrogen) flow of 35 units and auxiliary gas flow of 3 units. Data acquisition was performed in full scan mode with a high resolution of 70 000, automatic gain control (AGC) target of 1 x 106 and scan range of m/z 65 – 900. Data-dependent MS/MS was acquired for the 10 most abundant species with a resolution of 17 500, AGC target of 1 x 105, dynamic exclusion of 30 s and intensity threshold of 1 x 104. Cycles consisted of one full scan MS followed by 10 data-dependent MS/MS scans. Collision energies of 20, 30 and 40 were used in both positive and negative modes. Data acquisition and processing were performed using the Xcalibur data system (V 3.06, Thermo Fisher Scientific, Waltham, MA, USA). Metabolites were identified using the Mass Spectrometry-Data Independent Analysis Software V 4.60 (MS-DIAL) 25 and integrated using the MZmine V 2.53 software 26. LC-MS lipidomics data were normalized by dividing the area of the ion corresponding to the molecular species by the sum of the areas of all quantified species, and metabolomics data were normalized by dividing the area of the internal standard.LC-MS metabolomics and lipidomics data were log-transformed and EigenMS was used to normalize data.
Ion Mode:NEGATIVE
Capillary Temperature:320 °C
Capillary Voltage:2.9 kV
Collision Energy:20, 30 and 40
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