Metabolomics analysis of follicular fluid in women with ovarian endometriosis undergoing in vitro fertilization

Figures & data

Table 1. Clinical characteristics of the study participants.

Variables	Endometrioma	Controls	p
	n = 12	n = 12
Age (years)	33.3 ± 5.28	31.0 ± 5.21	0.29
BMI^a (kg/m^2)	25.1 ± 2.62	24.6 ± 3.53	0.73
Previous pregnancies	1/12 (8.3%)	1/12 (%8.3)	1.00
Duration of infertility (years)	5.3 ± 3.63	5.7 ± 4.7	0.81
Basal FSH^b (IU/mL)	8.7 ± 4.3	6.9 ± 1.7	0.21
Total gonadotropin dose (IU)	2387 ± 490	1932 ± 662	0.07
Number of oocytes retrieved	8.00 ± 5.3	8.18 ± 3.0	0.92
Number of mature oocytes^c	5.50 ± 3.9	6.81 ± 2.9	0.37
Number of fertilized oocytes	3.83 ± 2.7	4.54 ± 2.2	0.49
Fertilization rate^d(%)	73.7 ± 17.6	68.8 ± 17.7	0.51
Number of cleaved embryos	3.3 ± 2.3	3.6 ± 1.6	0.72
Cleavage rate^e(%)	86.9 ± 13.8	82.9 ± 15.7	0.52
Number of embryos produced	4.2 ± 1.92	3.12 ± 0.83	0.19
Good quality embryo rate (grade I and II)	82.9 ± 25.5	67.8 ± 25.4	0.17
Clinical pregnancy rate^f	3/12 (25%)	2/12 (16.6%)	1.00

Data are reported as mean ± standard deviation.

a: BMI: body mass index; b: FSH: Follicle- stimulating hormone; c: Mature oocytes: Oocytes with the first polar body visible; d: Fertilization rate: number of fertilized oocytes/number of injected oocytes; e: Cleavage rate: number of cleaved embryo/number of fertilized oocytes; f: Clinical pregnancy rate: presence of embryonic heartbeat visualized by ultrasound at 6 weeks after embryo transfer.

Figure 1. ¹H CPMG (Carr-Purcell-Meiboom-Gill) NMR spectrums (δ 0 to 8.5 ppm) recorded at 600 MHz and 298 K of follicular fluid samples from the study subjects. A) ¹H NMR spectrum of control sample. B) ¹H NMR spectrum of patient sample. C) ¹H NMR spectrum of follicular fluid contaminated with flushing medium [HEPES [4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid].

Figure 2. Multivariate statistical analysis results of the data including both the HEPES contaminated samples and samples without any contamination. Principal component analysis (PCA) scores (A) and loadings (B) plots and partial least squares discriminant analysis (PLS-DA) scores (C) and variable importance in projection (VIP) values (D) for the follicular fluid (FF) spectral data including both the contaminated samples and samples without any contamination.For the PCA, the first component accounts for 39.1407% of overall variability and the second component accounts for 19.7594% of overall variability which corresponds to a cumulative R² value of 0.5890. The PLS-DA model has R²X value of 0.5586, R²Y value of 0.8788 and Q² value of 0.8240. PLS-DA model shows a better separation for the endometriosis and control FF spectral data when compared to the PCA model. In the PCA loadings plot (B) and PLS-DA VIP plot (D), lactate (1.25 ppm, 4.03 ppm), β-glucose (4.55 ppm, 3.15–3.84 ppm), valine (0.95 ppm, 0.89 ppm), pyruvate (2.29 ppm) and HEPES peaks (2.55–3.06 ppm) are highlighted as important peaks.

Figure 3. Multivariate statistical analysis results of the data including only samples without any contamination. Principal component analysis (PCA) scores (A) and loadings (B) plots and partial least squares discriminant analysis (PLS-DA) scores (C) and variable importance in projection (VIP) values (D) for the follicular fluid (FF) spectral data including samples without any contamination.For the PCA, the first component accounts for 43.41% of overall variability and the second component accounts for 21.70% of overall variability which corresponds to a cumulative R² value of 0.6511. The PLS-DA model has R²X value of 0.5381, R²Y value of 0.9723 and Q² value of 0.9239. Both models show a good separation for the endometriosis and control FF spectral data. The PCA loadings plot (B) and PLS-DA VIP plot (D) show that this separation occurs mainly because of lactate (1.25 ppm, 4.03 ppm), β-glucose (4.55 ppm, 3.15–3.84 ppm), valine (0.95 ppm, 0.89 ppm) and pyruvate (2.29 ppm) peaks.

Table 2. Resonance assignments and concentration ranges of metabolites in ovarian endometrioma and control follicular fluid samples.

	Concentrations (mM)
				Endometrioma	Controls
Metabolite	ppm (shift)	Multiplicity	Assignment	n = 7	n = 6	p
Lactate	1.25	d	βH	2.75 ± 1.1	1.6 ± 0.5	0.05*
4.03	q	αH
α-glucose	5.15	d	H1	1.72 ± 0.5	1.25 ± 0.2	0.10
3.15–3.84	m	H2-H6
β-glucose	4.55	d	H1	1.25 ± 0.2	0.83 ± 0.3	0.03*
3.15–3.84	m	H2-H6
Alanine	1.39	d	βH	0.38 ± 0.11	0.27 ± 0.09	0.07
Pyruvate	2.29	s	βH	0.19 ± 0.04	0.14 ± 0.03	0.03*
Valine	0.94	d	δH	0.17 ± 0.03	0.12 ± 0.04	0.02*
	0.89	d	δH

* p < 0.05.