Altered Cytokine Gene Expression in Peripheral Blood Monocytes across the Menstrual Cycle in Primary Dysmenorrhea: A Case-Control Study

<div><p>Primary dysmenorrhea is one of the most common gynecological complaints in young women, but potential peripheral immunologic features underlying this condition remain undefined. In this paper, we compared 84 common cytokine gene expression profiles of peripheral blood mononuclear cells (PBMCs) from six primary dysmenorrheic young women and three unaffected controls on the seventh day before (secretory phase), and the first (menstrual phase) and the fifth (regenerative phase) days of menstruation, using a real-time PCR array assay combined with pattern recognition and gene function annotation methods. Comparisons between dysmenorrhea and normal control groups identified 11 (nine increased and two decreased), 14 (five increased and nine decreased), and 15 (seven increased and eight decreased) genes with ≥2-fold difference in expression (<em>P</em><0.05) in the three phases of menstruation, respectively. In the menstrual phase, genes encoding pro-inflammatory cytokines (IL1B, TNF, IL6, and IL8) were up-regulated, and genes encoding TGF-β superfamily members (BMP4, BMP6, GDF5, GDF11, LEFTY2, NODAL, and MSTN) were down-regulated. Functional annotation revealed an excessive inflammatory response and insufficient TGF-β superfamily member signals with anti-inflammatory consequences, which may directly contribute to menstrual pain. In the secretory and regenerative phases, increased expression of pro-inflammatory cytokines and decreased expression of growth factors were also observed. These factors may be involved in the regulation of decidualization, endometrium breakdown and repair, and indirectly exacerbate primary dysmenorrhea. This first study of cytokine gene expression profiles in PBMCs from young primary dysmenorrheic women demonstrates a shift in the balance between expression patterns of pro-inflammatory cytokines and TGF-β superfamily members across the whole menstrual cycle, underlying the peripheral immunologic features of primary dysmenorrhea.</p> </div

Multivariate analysis of cytokine gene expression profiles from controls (NM1-NM3) and dysmenorrheic (DM1–DM6) samples on the first day of menstruation.

<p>A) Heat map showing hierarchical clustering of individual arrays by gene expression. B) 3D PCA score plot showing separate clustering of expression profiles corresponding to DM vs. NM. C) OPLS-DA model results for DM vs NM group. D) S-plot of OPLS-DA model for DM vs. NM group.</p

DAVID analysis of differentially expressed genes in women with primary dysmenorrhea on the first day of menstruation.

<p>DAVID analysis of differentially expressed genes in women with primary dysmenorrhea on the first day of menstruation.</p

DAVID analysis of differentially expressed genes in women with primary dysmenorrhea on the fifth day of menstruation.

<p>DAVID analysis of differentially expressed genes in women with primary dysmenorrhea on the fifth day of menstruation.</p

DAVID analysis of genes differentially expressed in women with primary dysmenorrhea on the seventh day before menstruation.

<p>DAVID analysis of genes differentially expressed in women with primary dysmenorrhea on the seventh day before menstruation.</p

PLS-DA score plot (3D) of PBMC cytokine gene expression between controls and primary dysmenorrhea groups.

<p>DS, the secretory phase in the primary dysmenorrhea group (seventh day before menstruation); DM, the menstrual phase in the primary dysmenorrhea group (first day of menstruation); DP, the proliferative phase in the primary dysmenorrhea group (fifth day of menstruation); NS, NM, NP, the secretory, menstrual and proliferative phase, respectively, in unaffected controls.</p

Quantitative RT-PCR array analysis of differentially expressed genes in women with primary dysmenorrhea on the fifth day of menstruation.

<p>Quantitative RT-PCR array analysis of differentially expressed genes in women with primary dysmenorrhea on the fifth day of menstruation.</p

A simplified representation of biological cross-talk between multiple TNFα/IL-1-induced actions and the TGF-β superfamily member signaling pathway.

<p>TGF-β family members may interfere with the multiple roles of TNF-α/IL-1via HO-1, p300, PPARα, and PPARγ.</p

Functions and possible roles of genes differentially expressed in women with primary dysmenorrhea during the menstrual cycle.

<p>Functions and possible roles of genes differentially expressed in women with primary dysmenorrhea during the menstrual cycle.</p

Genes revealed by quantitative RT-PCR analysis to be differentially expressed in women with primary dysmenorrhea on the seventh day before menstruation.

<p>Normalized ΔCt = Ct (GOI) – avg. (Ct (HKG)), where GOI is each gene of interest, and HKG are the housekeeping genes. Fold change (FC) = 2^{−ΔΔCt =}2^{−ΔCt (Dys)} ÷ 2^{−ΔCt (Nor)}.</p