Metabolic profiling of follicular fluid and plasma from natural cycle in vitro fertilization patients—a pilot study  Cassey McRae, M.Chem., N. Ellissa Baskind, M.B.Ch.B., Nicolas M. Orsi, Ph.D., Vinay Sharma, F.R.C.O.G., Julie Fisher, Ph.D.  Fertility and Sterility  Volume 98, Issue 6, Pages 1449-1457.e6 (December 2012) DOI: 10.1016/j.fertnstert.2012.07.1131 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Principal components analysis (PCA) scores (A) and loadings (B) plots and partial-least-squares discriminant analysis (PLS-DA) scores (C) and X-weights (D) for the follicular fluid (FF) spectral data. For the PCA, the first two principal components (PCs) gave cumulative R2 and Q2 values of 0.58 and 0.31, respectively, and for the PLS-DA PC1 had R2X(cum) of 0.40, R2Y(cum) of 0.67, and Q2(cum) of 0.54. Both models show a deviation of midfollicular (black squares) and periovulatory (red triangles) FF. The FF aspirated at the time of the LH-surge (blue squares) is clustered with the periovulatory FF rather than the midfollicular FF. The loadings plots show that this separation occurs on the basis of FF lactate, pyruvate, and glucose concentrations. The dotted ellipses have no statistical meaning; the large solid black ellipse represents the 95% confidence region. Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 PCA scores (A) and loadings (B) plots and PLS-DA scores (C) and X-weights (D) for the blood plasma spectral data. For the PCA, PCs 1 and 3 had R2X values of 0.30 and 0.13 and Q2 values of 0.19 and 0.09, respectively. For the PLS-DA PCs 1 and 2 had R2X(cum) of 0.42, R2Y(cum) of 0.88, and Q2(cum) of 0.47. Both models show a deviation of midfollicular (black squares) and periovulatory (red triangles) plasma. The LH-surge plasma (blue squares) is not clustered with the periovulatory samples as in the case of the FF. The loadings plots show that this separation occurs on the basis of glucose, acetate, trimethylamine, glycine, and glycoprotein concentrations. The dotted ellipses have no statistical meaning; the large solid black ellipse represents the 95% confidence region. Abbreviations as in Figure 1. Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 (A) Follicular fluid (FF) spectrum contaminated with flushing medium. (B) The same FF spectrum after subtraction of an identical spectrum of the flushing medium. (C) Spectrum of an FF sample that is not contaminated with flushing medium. An NMR spectrum of authentic flushing medium was obtained under the exact same conditions as the FF samples, and this was subtracted from the spectra of the FF samples that were contaminated by flush. The HEPES signal at 2.98 ppm was used as a concentration reference for the amount of flush fluid contamination because no other molecules in the natural FF give rise to signals at this point, as indicated in (C); therefore, the area under this estimated signal (and thus the concentration) is purely for a component of the flush. The flushing medium spectrum was multiplied by a factor that meant that subtraction gave zero intensity at 2.98 ppm. In doing this, all the other components of the flush fluid were subtracted at the appropriate level, as shown in (B). Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental figure 2 Validation plot after permutation testing (20 permutations) of the partial-least-squares discriminant analysis (PLS-DA) model of the follicular fluid (FF) spectral data. The validation plot shows the values of R2Y/Q2Y for the original and permuted models on the y-axis and the correlation coefficients between the original model and the permuted models on the x-axis. Thus, the original model’s R2Y and Q2Y values are displayed on the far right of the plot (with a correlation of 1.0) and the permuted models’ values on the left side of the plot, if the original model should have higher values of R2Y and Q2Y than the permuted models. Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental figure 3 Principal component (PC) 3 versus PC1 scores plot for the blood plasma spectral data with the bins containing the glucose signals excluded. The cumulative R2 and Q2 values of the two PCs were 0.46 and 0.18, respectively. The same separation between the midfollicular (black squares) and periovulatory (red triangles) plasma is observed, confirming that this grouping is not based on diet only. Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental figure 4 Validation plot after permutation testing (20 permutations) of the principal components analysis model of the plasma spectral data. Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental figure 5 PCA scores and loadings plots of the FF and plasma spectral data shown in Figures 1 and 2, indicating the deviation of patients who had a live birth (circled in green) and a miscarriage (circled in purple) following their second IVF cycles. (A) PC2 versus PC1 scores plot of the FF spectral data. The midfollicular FF sample of the patient who went on to have a live birth following her second IVF cycle is outlying from the other patients along PC2, but her periovualtory sample (the one corresponding to the oocyte that resulted in a live birth) was not. It may be the case that markers of good oocyte quality are present only in the early FF. However, because no embryo was collected in the first cycle, it is not possible to know whether this cycle would have led to a live birth or not. Both the midfollicular and periovulatory FF samples of the patient who had a miscarriage following her second IVF cycle are outlying, to a lesser extent, along PC1. (B) PC3 versus PC1 scores plot of the plasma spectral data. The live birth patient’s periovulatory plasma deviates from the other patients’ periovulatory samples, and the midfollicular plasma of the miscarriage patient is outlying along PC3. (C) PC2 versus PC1 loadings plot of the FF spectral data, showing that the midfollicular FF of the live birth patient had higher levels of alanine and glutamine in her FF than the other patients, whereas the miscarriage patient had higher FF levels of 3-hydroxybutyrate and acetoacetate. (D) PC3 versus PC1 loadings plot of the plasma spectral data, showing that the live birth patient had higher plasma levels of lactate and the miscarriage patient, again, had higher levels of ketones in her plasma than the other patients. It is interesting that the levels of ketones were high in the FF surrounding the oocyte that led to a miscarriage, because ketones have been linked to oxidative stress (OS) (45–47) and the role of OS in miscarriage has been well documented (48–55). It is also interesting that these ketones could be detected in the patient’s FF and blood during the miscarriage patient’s first cycle as well. Abbreviations as in Supplemental Figures 1 and 3. Fertility and Sterility 2012 98, 1449-1457.e6DOI: (10.1016/j.fertnstert.2012.07.1131) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions