1. Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm 
Alessandra A. Vireque, Ph.D., Alessandra Tata, Ph.D., Oswaldo F.L.L.O. Silva, M.D., Edson G. LoTurco, Ph.D., Augusto Azzolini, M.Sc., Christina R. Ferreira, Ph.D., Marilda H.Y. Dantas, B.S., Rui A. Ferriani, M.D., Ph.D., Rosana M. Reis, M.D., Ph.D. 
Fertility and Sterility 
Volume 106, Issue 2, Pages e6 (August 2016)
DOI: /j.fertnstert
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Significant ions between TEST and TEST-PC samples. (A) Ions of significance for group discrimination observed by principal component analysis (PCA) can be effectively indicated by means of volcano plots. The volcano plot is a combination of fold change and t tests. Note that for unpaired samples the x-axis is log (FC). For paired analysis, the x-axis is the number of significant counts. For the y-axis, −log10 (P value) for both cases. Threshold for fold change was set to 5 and P=.05. (B) Seven ions were indicated as significantly distinct between the two experimental groups. (C) Changes indicated by the volcano plot can be observed also visually in the heat map. Due to the high number of ions (n = 185) displayed in the heat map, regions where the significant differences occur were cut, and individual ions are highlighted in yellow.
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3. Figure 2
Significant ions between TEST-Y and TEST-Y-PC samples. (A) Volcano plot indicated just one ion (of m/z ) of significance when the threshold for fold change was set to 5 and P=.05. (B) Heat map of the TEST-Y and TEST-Y PC samples, showing the m/z , highlighted in yellow.
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4. Figure 3
Detail at greater magnification of fresh and cryopreserved human spermatozoa treated with purified Lα-phosphatidylcholine and/or egg yolk cryoprotectants. (A) Fresh untreated sperm. (B) Postthaw sperm recovered from TEST, (C) TEST-Y, (D) TEST-PC, (E) TEST-Y-PC, and (F) cryoprotectant-free medium (FMB). SEM: scanning electron microscopy 20,000×. A high-resolution version of panels A–F of this image is available as virtual microscope eSlide VM02545, eSlide VM02546, eSlide VM02547, eSlide VM02548, eSlide VM02549, and eSlide VM02550.
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5. Supplemental Figure 1
Three-dimensional principal component analysis (PCA) of the five experimental groups. A clear tendency can be observed discriminating samples exposed to soy-PC (TEST-PC and TEST-Y-PC) from those not supplemented with soy-PC (TEST, TEST-Y, and control) before freezing.
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6. Supplemental Figure 2
Principal component analysis (PCA) for the TEST and TEST-PC groups. (A) Data normalization by autoscaling. (B) PCA scores and loadings plot showing a clear tendency of discrimination of TEST from TEST-PC samples on PC2, which explains 16.3% of the data variability.
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7. Supplemental Figure 3
(A) Data normalization by autoscaling. (B) Principal component analysis (PCA) scores and loadings plot showing a slight tendency of discrimination of TEST-Y from TEST-Y PC samples on PC2, which explains 13% of the data variability.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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8. Supplemental Figure 4
Univariate receiver operating characteristic (ROC) curves obtained with the test set (n = 20) for the ions of m/z (AUC: 0.943); (AUC: 0.852); and (AUC: 0.828). The classification of the validation set by using only the ROC curve of the m/z was 82.5% correct; when using these three ions (m/z , , and ), the correct classification was 77.5%.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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9. Supplemental Figure 5
Fresh untreated human sperm and cryopreserved human spermatozoa exposed to purified Lα-phosphatidylcholine and/or egg yolk cryoprotectants. (A) Fresh untreated sperm. (B) Postthaw sperm recovered from TEST, (C) TEST-Y, (D) TEST-PC, (E) TEST-Y-PC, and (F) cryoprotectant-free medium (FMB). SEM = scanning electron microscopy 2,500×. A high-resolution version of the panels A-F of this image are available as virtual microscope eSlide VM02551, eSlide VM02552, eSlide VM02553, eSlide VM02554, eSlide VM02555, and eSlide VM02556.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

10. Supplemental Figure 6
Morphologically abnormal human spermatozoa after freezing in the absence of cryoprotectants (FMB). Note alterations in acrosome (acrosomal swelling), head-neck junction, and tail membranes (arrows). SEM = scanning electron microscopy 9,500×.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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