Dynamic changes in cervical glycosaminoglycan composition during normal pregnancy and preterm birth

Abstract

Glycosaminoglycans (GAG) have diverse functions that regulate macromolecular assembly in the extracellular matrix. During pregnancy, the rigid cervix transforms to a pliable structure to allow birth. Quantitative assessment of cervical GAG is a prerequisite to identify GAG functions in term and preterm birth. In the current study, total GAG levels increased at term, yet the abundance, chain length, and sulfation levels of sulfated GAG remained constant. The increase in total GAG resulted exclusively from an increase in hyaluronan (HA). HA can form large structures that promote increased viscosity, hydration, and matrix disorganization as well as small structures that have roles in inflammation. HA levels increased from 19% of total GAG in early pregnancy to 71% at term. Activity of the HA-metabolizing enzyme, hyaluronidase, increased in labor, resulting in metabolism of large to small HA. Similar to mice, HA transitions from high to low molecular weight in term human cervix. Mouse preterm models were also characterized by an increase in HA resulting from differential expression of the HA synthase (Has) genes, with increased Has1 in preterm in contrast to Has2 induction at term. The Has2 gene but not Has1 is regulated in part by estrogen. These studies identify a shift in sulfated GAG dominance in the early pregnant cervix to HA dominance in term and preterm ripening. Increased HA synthesis along with hyaluronidase-induced changes in HA size in mice and women suggest diverse contributions of HA to macromolecular changes in the extracellular matrix, resulting in loss of tensile strength during parturition.

Fig. 1.

Quantification of GAG content and chain length in nonpregnant and pregnant mice. Cervices were collected from cycled nonpregnant (DE, diestrus; PE, proestrus; E, estrus; and ME, metestrus), pregnancy d 6–d 18 and postpartum 1 d (pp) and analyzed by FACE. A, Total GAG expression (sum of HA, CS/ DS, and HS). B, Total sGAG (CS/DS and HS). C, Total HA. D, Estimation of chain length. CS/DS chain length ranges from 40–75 disaccharides. *, P < 0.05 when compared with pregnant d 6 cervix (n = 4–6 samples per time point).

Fig. 2.

Dynamic changes in mouse cervical GAG composition in nonpregnant estrus, gestation d 6, gestation d 18, and postpartum samples. Most notable is the predominance of CS/DS GAG in early pregnancy (67%) as opposed to HA predominance (71%) in late pregnancy. Four to six samples were evaluated for each time point. NP, Not pregnant.

Fig. 3.

Hyaluronidase activity is increased IL and early postpartum in mouse cervix (panel A, n = 4 per time point; *, P < 0.05 when compared with the d 15 group). Consistent with the timing of increased hyaluronidase activity, lower MW HA is increased postpartum as shown in panel B. Cervical tissues were collected from pregnancy d 16–d 18 and postpartum 4–24 h (pp). Representative gel from one of three experiments.

Fig. 4.

Human cervical tissue HYAL2 mRNA expression is increased at term. *, P < 0.05 when compared with the NP (n = 4–5 samples). NP, NIL, and IL represent nonpregnant, term not in labor, and term in labor (panel A). Assessment of HA size distribution in cervical mucus from women at gestation wk 35–39. Lanes 1 and 2 contain HA MW standards. Lanes 3–8 evaluate samples from women at 35–39 wk gestation whose cervix is not dilated, and lanes 9 and 10 contain samples from women at 40–42 wk gestation with dilation more than or equal to 2 cm (panel B).

Fig. 5.

HA, but not sGAG, is elevated in premature cervical ripening. Cervices were collected on the 15th day of pregnancy from untreated (d 15), RU486 vehicle (−), RU486 (0.5 mg) (+), and LPS sham surgery (sh) or LPS-treated mice (+) and from gestation d 18 (d 18). Samples were analyzed by FACE. *, P < 0.05 when compared with d 15 untreated cervix; n = 3–4 (panel A). Assessment of HA size distribution in mouse cervical tissue of preterm models. HA Marker, HA MW standards, d 15, gestation d 15; RU-d 15, RU486-treated; and LPS-d 15, LPS-treated. Gel represents one of three experiments performed (panel B).

Fig. 6.

Cellular localization of cervical HA in preterm models is confined to the stroma in contrast to term pregnancy (d 18) where HA is localized to both epithelial and stromal ECM. Green, HA; blue, nucleus; S, stroma; E, epithelia.

Fig. 7.

Gene expression of HA synthases (Has1, Has2, and Has3) and hyaluronidases (Hyal1 and Hyal2). The study groups are d 15, d 15RU486 control or treatment (−, +), d 15 LPS sham surgery or treatment (sh, +), and d 18. Gene expression is normalized to d 15 untreated of each group. *, P < 0.05 when compared with d 15 untreated cervix (n = 5–6/group). RU486 and LPS groups were tested separately with d 15 untreated group using one-way ANOVA.

Fig. 8.

Cervical Has2 expression is regulated in part by estrogen. A, Gene expression of HA synthases (Has1, Has2, Has3) and hyaluronidases (Hyal1 and Hyal2) in cervical tissues (d 18) from mice treated with (+) or without (−) tamoxifen. The estrogen-regulated gene trefoil factor 1 (Tff1) was used to assess responsiveness of tissue to tamoxifen treatment. Data represent mean ± sem of four animals per group. B, Cervical HA content in gestation d 18 mice treated with (+) or without (−) tamoxifen; n = 3–4 (P < 0.05). C, Gene expression of Has1, Has2, Has3, Hyal1, Hyal2, and Tff1 in cervical tissues from ovariectomized (ovx) mice treated with sham pellets (Sham), E2, E2+P4, or E2+P4+RU486. D, Cervical HA content in ovariectomized (ovx) mice treated with E2 (0.05 mg pellet per mouse) or E2 and P4 (0.05 mg pellet per mouse) for 10 d. RU486 (500 μg/mouse) was injected 12 h before tissue collection. Data represent mean ± sem of four animals per group. *, P < 0.05 compared with control cervix or ovx sham in tamoxifen and ovx study, respectively. Tmx, Tamoxifen.