Menstrual cycle rhythmicity: metabolic patterns in healthy women

Abstract

The menstrual cycle is an essential life rhythm governed by interacting levels of progesterone, estradiol, follicular stimulating, and luteinizing hormones. To study metabolic changes, biofluids were collected at four timepoints in the menstrual cycle from 34 healthy, premenopausal women. Serum hormones, urinary luteinizing hormone and self-reported menstrual cycle timing were used for a 5-phase cycle classification. Plasma and urine were analyzed using LC-MS and GC-MS for metabolomics and lipidomics; serum for clinical chemistries; and plasma for B vitamins using HPLC-FLD. Of 397 metabolites and micronutrients tested, 208 were significantly (p < 0.05) changed and 71 reached the FDR 0.20 threshold showing rhythmicity in neurotransmitter precursors, glutathione metabolism, the urea cycle, 4-pyridoxic acid, and 25-OH vitamin D. In total, 39 amino acids and derivatives and 18 lipid species decreased (FDR < 0.20) in the luteal phase, possibly indicative of an anabolic state during the progesterone peak and recovery during menstruation and the follicular phase. The reduced metabolite levels observed may represent a time of vulnerability to hormone related health issues such as PMS and PMDD, in the setting of a healthy, rhythmic state. These results provide a foundation for further research on cyclic differences in nutrient-related metabolites and may form the basis of novel nutrition strategies for women.

Figure 1

Hormone levels according to menstrual cycle phase. Changing concentrations of female sex hormones (progesterone, luteinizing hormone, follicular stimulating hormone, estradiol) that characterize the 5 phases (menstrual, follicular, periovulatory, luteal and pre-menstrual) of the menstrual cycle (adapted with permission). Follicular stimulating hormone concentration changes overlayed.

Figure 2

Study schema. Thirty-four women (BMI 22.9 +/− 3.5 kg/m², age 26.6 +/− 5.9 yrs) provided 4 blood and urine samples that each uniquely fit into 1 of 5 phase timepoints based on 4 sex hormone measurements (LH, FS, estradiol, and progesterone) and self-reported menstrual cycle timing. A total of 401 metabolites were measured which included 263 plasma, 114 urine, and 19 clinical and vitamin analyses. Metabolite profiling was conducted and statistically significant rhythmicity is depicted for the amino acid, lipid and organic acid panels. Biochemical pathway interconnectivity was identified between the urea cycle, 1 carbon metabolism, glutathione metabolism and the citric acid cycle. M-menstrual, F-follicular, O-Periovulatory, L-luteal, P-premenstrual phases.

Figure 3

Metabolites vary across menstrual cycle phase. This heatmap with color gradients indicates rhythmicity across the menstrual cycle. Lower amino acid and lipid metabolite concentrations are visualized in the luteal phase. Phase means of logarithmically transformed metabolite data are row standardized in the heatmap to obtain Z scores. Two cells that are close in color represent similar Z scores, ranging from blue (Z equals minus 2) to red (Z equals plus 2). Amino acid, lipid, organic acid and sex hormone variables are ordered according to main biochemical pathways or classes and depicted at q < 0.20 after contrast analyses. Menstrual (M), Follicular (F), Periovulatory (O), Luteal (L), Premenstrual (P) phases are depicted. LPC- Lysophosphatidylcholine, LPE- Lysophosphatidylethanolamine, PC-phosphatidylcholine.

Figure 4

Amino acid variability by cycle phase. Mean log intensity is depicted along with individual variability for threonine, ornithine, arginine, alanine, glycine, serine, methionine, asparagine, proline, glutamine, tyrosine, gamma-glutamyl-alanine, citrulline, o-acetyl-serine, alpha-aminobutyric acid, and gamma-glutamylglutamine at one time point for each of the 5 menstrual phases (M = menstrual, F = follicular, O = periovular, L = luteal, p = premenstrual). Each colored line represents an individual. Amino acids are depicted which have 2 or more contrast comparisons meeting the multiple testing threshold of q < 0.20. Statistically significant luteal phase reductions can be observed.

Figure 5

Rhythmic metabolites in the urea cycle, neurotransmitter metabolism connect with 1 carbon, glutathione metabolism and the citric acid cycle. The metabolites with FDR controlled rhythmicity participate in inter-related, biochemical pathways including nitrogen metabolism (the urea cycle), neurotransmitter metabolism, methylation (1 carbon metabolism), oxidative stress (glutathione metabolism) and energy metabolism (citric acid cycle). NOS = Nitric oxide synthase; BH4 = Tetrahydrobiopterin; BH2 = Bihydrobiopterin; MTHFR = Methylenetetrahydrofolate reductase; THF = Tetrahydrofolate; MTR = Methionine synthase; DMG = Dimethylglycine; TMG = Trimethylglycine; B6 = Vitamin B6. Compounds boxed with dotted lines (NOS, BH4, BH2, MTHFR, THF, MTR, 5-methyl THF, DMG, TMG, homocysteine) were not evaluated or not significant (dopamine). All metabolites without dotted lines met the multiple testing threshold q < 0.20. *Cystathionine was statistically significant with p value < 0.05, but did not meet the multiple testing threshold.