研究目的
Investigating the biochemical alterations in granulosa cells from patients with unilateral ovarian endometriosis using infrared and Raman microspectroscopy to understand the impact on fertility.
研究成果
The study concludes that unilateral ovarian endometriosis causes similar biochemical impairments in granulosa cells from both the affected and contralateral ovaries, including protein alteration, oxidative stress, and metabolic dysregulation, suggesting systemic effects that impair overall ovarian function and fertility.
研究不足
The study is limited by the sample size (20 patients) and the focus on specific biochemical markers; it may not capture all aspects of endometriosis impact, and results are based on in vitro analysis which might not fully represent in vivo conditions.
1：Experimental Design and Method Selection:
The study used Fourier Transform Infrared Microspectroscopy (FTIRM) and Raman MicroSpectroscopy (RMS) to analyze luteinized granulosa cells (GCs) from patients with unilateral ovarian endometriosis and controls. Samples were collected, prepared, and analyzed using specific protocols for each technique, with multivariate analysis (PCA) for data interpretation.
2：Sample Selection and Data Sources:
GCs were collected from 20 patients (10 with unilateral ovarian endometriosis and 10 controls with tubal, idiopathic, or male infertility) during the luteinization phase of the ovarian cycle. Inclusion criteria included age 35±
3：8, non-smokers, regular cycles, and FSH<10 IU/I. List of Experimental Equipment and Materials:
Equipment included a Hyperion 3000 Vis-IR microscope with MCT_A detector and Vertex 70 interferometer for FTIRM, and a Horiba Jobin-Yvon LabRAM HR800 spectrometer with 532-nm diode laser for RMS. Materials included CaF2 optical windows, paraformaldehyde, silica particles suspension (PureSperm), and various media and solutions.
4：Experimental Procedures and Operational Workflow:
For FTIRM, GCs were deposited on CaF2 windows, air-dried, and spectra collected in transmission mode. For RMS, cells were fixed in paraformaldehyde, and point spectra were acquired. Data were processed using OPUS
5：1 software and analyzed with PCA and statistical tests. Data Analysis Methods:
Spectral data were vector normalized, subjected to PCA, and curve-fitted for specific band ratios. Statistical analysis used one-way ANOVA and Tukey's test with Prism6 software.
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Hyperion 3000 Vis-IR microscope
3000
Bruker Optics
Used for FTIR microspectroscopy measurements to analyze biochemical composition of cells.
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Vertex 70 interferometer
70
Bruker Optics
Interferometer coupled with the microscope for FTIR measurements.
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OPUS software
7.1
Bruker Optics
Software used for spectral data processing, including averaging, atmospheric compensation, and integration.
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LabRAM HR800 spectrometer
HR800
Horiba Jobin-Yvon
Used for Raman microspectroscopy measurements to analyze biochemical composition of cells.
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CaF2 optical windows
1-mm thick, 13-mm diameter
Substrate for depositing GC samples for FTIR measurements to avoid water contributions.
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PureSperm
40 and 80
Nidacon
Silica particles suspension used in gradient centrifugation to separate GCs from red blood cells.
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R Studio
RStudio, Inc.
Software environment used for developing in-house algorithms for multivariate analysis (PCA).
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GRAMS/AI software
7.02
Galactic Industries, Inc.
Software used for curve fitting of IR spectra with Gaussian algorithm.
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Prism6 software
6
Graphpad Software, Inc.
Statistical software used for ANOVA and Tukey's multiple comparisons test.
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