Changes in concentrations of cervicovaginal immune mediators across the menstrual cycle: a systematic review and meta-analysis of individual patient data

Abstract

Background: Hormonal changes during the menstrual cycle play a key role in shaping immunity in the cervicovaginal tract. Cervicovaginal fluid contains cytokines, chemokines, immunoglobulins, and other immune mediators. Many studies have shown that the concentrations of these immune mediators change throughout the menstrual cycle, but the studies have often shown inconsistent results. Our understanding of immunological correlates of the menstrual cycle remains limited and could be improved by meta-analysis of the available evidence.

Methods: We performed a systematic review and meta-analysis of cervicovaginal immune mediator concentrations throughout the menstrual cycle using individual participant data. Study eligibility included strict definitions of the cycle phase (by progesterone or days since the last menstrual period) and no use of hormonal contraception or intrauterine devices. We performed random-effects meta-analyses using inverse-variance pooling to estimate concentration differences between the follicular and luteal phases. In addition, we performed a new laboratory study, measuring select immune mediators in cervicovaginal lavage samples.

Results: We screened 1570 abstracts and identified 71 eligible studies. We analyzed data from 31 studies, encompassing 39,589 concentration measurements of 77 immune mediators made on 2112 samples from 871 participants. Meta-analyses were performed on 53 immune mediators. Antibodies, CC-type chemokines, MMPs, IL-6, IL-16, IL-1RA, G-CSF, GNLY, and ICAM1 were lower in the luteal phase than the follicular phase. Only IL-1α, HBD-2, and HBD-3 were elevated in the luteal phase. There was minimal change between the phases for CXCL8, 9, and 10, interferons, TNF, SLPI, elafin, lysozyme, lactoferrin, and interleukins 1β, 2, 10, 12, 13, and 17A. The GRADE strength of evidence was moderate to high for all immune mediators listed here.

Conclusions: Despite the variability of cervicovaginal immune mediator measurements, our meta-analyses show clear and consistent changes during the menstrual cycle. Many immune mediators were lower in the luteal phase, including chemokines, antibodies, matrix metalloproteinases, and several interleukins. Only interleukin-1α and beta-defensins were higher in the luteal phase. These cyclical differences may have consequences for immunity, susceptibility to infection, and fertility. Our study emphasizes the need to control for the effect of the menstrual cycle on immune mediators in future studies.

Keywords: Cervix; Chemokine; Cytokine; Female genital tract; Menstrual cycle; Meta-analysis; Systematic review; vagina.

Fig. 1

PRISMA-IPD flow diagram. Identification and selection of included studies

Fig. 2

Concentrations of immune mediators. Concentration ranges for all immune mediators measured in at least 2 studies. The box-and-whisker plots show concentrations including all studies and sample types. The middle bar shows the median, with the edges of the box ranging from the 25th to the 75th percentiles and the whiskers ranging from the 5th to the 95th percentiles. The percentages shown at the right reflect the percent of samples detected above the lower limit of detection

Fig. 3

Primary meta-analyses. A The log2 difference between phases (log2-pg/mL of the luteal phase minus log2-pg/mL of the follicular phase). B The logistic difference between phases (log-odds of proportion detectable in luteal vs. follicular phase). Each row represents a different immune mediator, with the symbols showing the mean and the lines showing the 95% confidence intervals. Gray symbols indicate individual studies and blue the meta-estimates as determined by inverse-variance pooling random effects models. Filled symbols indicate p < 0.05 while open symbols indicate p > 0.05. Positive numbers indicate higher during the luteal phase (relative to the follicular phase), while negative numbers indicate lower during the luteal phase (relative to the follicular phase). Symbol shape and shade of blue indicate the GRADE strength of evidence

Fig. 4

Validation and exploratory experiments. A Concentrations of validation cytokines. Each symbol shows the concentration in a single sample. Lines connect samples from the same participant. Pale grey symbols are below the lower limit of detection. B Differences in concentrations between phases of the menstrual cycle for the validation cytokines. Black shows the new data generated in this study, gray shows all other studies. Error bars for several CCL2 and IL-1α studies extend off-scale. Black-filled symbols indicate p<0.05, open symbols indicate p>0.05. C Concentrations of exploratory cytokines, as in A. D Differences in concentrations between phases of the menstrual cycle for the exploratory cytokines, as in B. Error bars for several IgA and IgM studies extend off-scale

Fig. 5

Subgroup analysis: Does the effect of menstrual cycle differ by sample type? Meta-analyses comparing all studies (black circles) to studies grouped by sample type (menstrual cup: red diamonds; sponge: blue inverted triangles; CVL: purple squares; swab: green triangles)

Fig. 6

Sensitivity analyses. A Correlation of effect sizes (log2-pg/mL of the luteal phase minus log2-pg/mL of the follicular phase) of meta-estimates derived from one- and two-stage meta-analysis. Each symbol indicates an immune mediator. B Correlation of effect sizes (log2-pg/mL of the luteal phase minus log2-pg/mL of the follicular phase) of meta-estimates derived from underlying univariate models or multivariate models adjusted for relevant covariates. Each symbol indicates an immune mediator. C Percentage of samples with red blood cells detected using the indicated detection methods. Dark red indicates positive, light red indicates trace detection, and grey indicates negative

Fig. 7

Secondary outcomes: Sample type and assay method comparison. A Comparison of concentrations recovered from CVLs to concentrations recovered from other sample types. Each symbol represents one immune mediator. The circles show the mean log2 difference between the indicated sample types and CVLs. B CXCL8 concentrations recovered by sample type. Each box plot shows a single study, colored by sample type in that study, with menstrual cup shown in red, sponge shown in blue, swab shown in green, and CVL shown in white. The studies are sorted by median concentration. C Comparison of concentrations recovered from 10 mL CVLs to concentrations recovered from 5 mL CVLs. Each symbol represents one immune mediator. The circles show the mean log2 difference between 5 and 10 mL CVLs. D CXCL8 concentrations recovered by CVL volume. Each box plot shows a single study, colored by CVL volume in that study, with 5 mL shown in grey and 10 mL shown in white. The studies are sorted by median concentration. E Comparison of concentrations detected by ELISA to concentrations detected by other assays. Each symbol represents one immune mediator. The circles show the mean log2 difference between the indicated assays and ELISAs. F CXCL8 concentrations measured by assay type. Each box plot shows a single study, colored by assay type in that study, with Luminex shown in green, MSD shown in orange, and ELISA shown in white. The studies are sorted by median concentration

Fig. 8

Secondary outcomes: Method of determining menstrual phase. A Progesterone concentrations (log10 ng/mL) and days since last menstrual period. Each symbol is a single sample. Samples are categorized into follicular (blue) or luteal (green) phases based on days since LMP (top) or serum progesterone (bottom). The same samples are shown in both plots. Gray symbols have undefined phase. B The number of samples categorized as follicular phase, luteal phase, or undefined by serum progesterone and by days since LMP. Squares are colored based on whether the methods categorized those samples as the same phase (green), opposite phases (orange), or one method was undefined (gray)