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Human Myometrial Genes Are Differentially Expressed in Labor: A Suppression Subtractive Hybridization Study

Mothers and Babies Research Centre (E.C.C., S.Y., R.S.), Endocrine Unit, John Hunter Hospital, Hunter Region Mail Centre, Newcastle, New South Wales 2310, Australia; Victoria University of Technology (S.F., R.J.F.), Werribee, Victoria 3030, Australia; and Singapore KK Women’s and Children’s Hospital (G.Y., K.K.), Singapore 229899

Address all correspondence and requests for reprints to: E.-Cheng Chan, Mothers and Babies Research Center, Endocrine Unit, John Hunter Hospital, Hunter Region Mail Centre, Locked Bag 1, New South Wales 2310, Australia. E-mail: . Cheng.Chan{at}newcastle.edu.au

Abstract

Human parturition is effected by a cascade of factors, of which many are unknown. We aim to identify the genes that are changed by labor in the human myometrium by suppression subtractive hybridization . We also seek to ascertain whether these genes are differentially expressed in the myometrium at the upper or fundal and lower segments of the uterus. Term myometrial tissues were obtained from laboring and nonlaboring women undergoing cesarean section after obtaining informed consent. Total RNA was used in suppression subtractive hybridization (CLONTECH PCR Select) to produce two subtracted cDNA libraries enriched for genes expressed during or before labor, labor and not-in-labor libraries, respectively. Dot blot screening of 400 positive clones, constituting 20% of the two subtracted libraries, revealed 30 differentially expressed clones, 14 of which were up-regulated by labor. Among the 10 known genes that were up-regulated in labor, 6 had apparent immune regulatory and inflammatory roles. Three are well-known inflammatory mediators and modulators that were previously linked with parturition: IL-8, manganese superoxide dismutase (MnSOD), and metalloproteinase-9. Three others, interferon-inducible 1-8d gene, elongation factor 1, and nucleophosmin, have not been previously linked with labor. Constitutively expressed genes, including cyclophilin and -actin, were found to be altered by labor. Quantitative real-time RT-PCR using Taqman probes further confirmed the up-regulation of some of these genes. The amounts of the specific genes assayed were standardized to 18S ribosomal RNA and are expressed as mean ± SEM. Quantitative real-time RT-PCR showed that IL-8 mRNA rose from 0.003 ± 0.002 in nonlaboring samples (n = 38) to 0.24 ± 0.11 (n = 20) in gestational-age-matched spontaneously laboring women (P = 0.035). Similarly, MnSOD rose from 0.11 ± 0.02 (n = 24) to 1.23 ± 0.56 (n = 24) in gestational-age-matched women (P = 0.047). Additionally, cyclophilin, often used as a constitutive or housekeeping gene marker, increased from 0.0008 ± 0.0002 (n = 6) to 0.002 ± 0.0004 (n = 6; P = 0.008) during labor. Notably, MnSOD mRNA was differentially distributed between the upper (0.63 ± 0.18) and lower (0.15 ± 0.05; n = 15; P = 0.022) segments of the uterus, but IL-8 was not (n = 17; P = 0.97). Induced labor further showed significantly higher levels of IL-8 (0.63 ± 0.21; n = 14) than spontaneous labor (0.22 ± 0.11; n = 20; P = 0.046), but not MnSOD (P = 0.1). This work identifies novel as well as known genes that were not previously associated with parturition. It extends previous data indicating that there is differential expression of some, but not all genes within the gravid human uterus. Inflammatory genes constitute a major proportion of the known genes found to be up-regulated in labor, lending support to the hypothesis of an inflammatory mechanism for human parturition. This work further indicates that many factors associated with human labor and their complex interactions remain to be elucidated.

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