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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST003266 |
| Study Title | Metabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia (MS data) |
| Study 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 |
| barbaraggcarvalho@gmail.com | |
| Phone | 234 370 360 |
| Submit Date | 2024-06-15 |
| Num Groups | 2 |
| Total Subjects | 57 |
| Num Males | 57 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-07-25 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
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 |
