Umbilical cord prostaglandins in term and preterm parturition

Abstract

Objective: Prostaglandins (PGs) are considered the universal mediators of parturition. Amniotic fluid PGE₂ and PGF_2α concentrations increase before the onset of spontaneous labor at term, as well as during labor. This study was conducted to determine if the concentrations of umbilical cord PGE₂ and PGF₂α change with advancing gestational age, spontaneous labor at term, and preterm labor (with and without funisitis).

Methods: Umbilical cord (UC) tissue samples were obtained from women (N = 158) with singleton pregnancies in the following groups: (1) term deliveries without labor (TNL; n = 20); (2) term deliveries with labor (TIL; n = 20); (3) spontaneous preterm deliveries (sPTD) with (n = 20) and without acute funisitis (n = 20); and (4) preeclampsia without labor (n = 78). The concentrations of PGs were determined in different locations of the UC. PGE₂ and PGF_2α were measured by specific immunoassays. Non-parametric statistics were used for analysis.

Results: (1) In spontaneous preterm deliveries, the median UC PGE₂ concentration was higher in cases with funisitis than in those without funisitis (233.7 pg/µg versus 87.4 pg/µg of total protein, p = 0.001); (2) the median UC PGE₂ concentration in sPTD with funisitis was also higher than that obtained from samples who had undergone labor at term (233.7 pg/µg versus 116.1 pg/µg of total protein, p = 0.03); (3) the UC PGE₂ and PGF_2α concentration increased as a function of advancing gestational age before 36 weeks (PGE₂: ρ = 0.59, p < 0.001; PGF_2α: ρ = 0.39, p = 0.01), but not after 36 weeks (PGE₂: ρ = −0.1, p = 0.5; PGF_2α: ρ = −0.2, p = 0.2); (4) the median UC concentrations of PGE₂ and PGF_2α at term was similar in samples obtained from women with and without labor (PGE₂: TNL 133.7 pg/µg versus TIL 116.1 pg/µg of total protein, p = 0.9; PGF_2α: TNL 8.4 pg/µg versus TIL 8.1 pg/µg of total protein, p = 0.7); and (5) there was no correlation between UC PG concentration and gestational age at term pregnancy (PGE₂: ρ = 0.01, p = 0.9; PGF_2α: ρ = 0.07, p = 0.7).

Conclusions: (1) PGE₂ concentrations in the UC are higher in the presence of acute funisitis than in the absence of this lesion; (2) spontaneous labor at term was not associated with a change in the UC concentration of PGE₂ and PGF_2α; and (3) the UC concentrations of PGE₂ and PGF_2α increased as a function of gestational age. We propose that UC PGs act as inflammatory mediators generated in the context of fetal systemic inflammation.

Keywords:

• Chorioamnionitis
• eicosanoids
• funisitis
• intra-amniotic inflammation
• microbial invasion of the amniotic cavity
• PGE₂
• PGF_2α
• preeclampsia
• pregnancy

Introduction

Prostaglandins (PGs) are considered key mediators of human parturition [1–19], and can induce myometrial contractility [20–34], cervical remodeling [35–58], and participate in extracellular matrix degradation leading to ruptured membranes [7,59–70]. The main PGs found in amniotic fluid are prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) [16,71–73], and concentrations of these eicosanoids increase prior to the onset of term labor [11,16,17], during labor [8,72] or rupture of membranes at term [7], and in spontaneous preterm labor [particularly in the setting of microbial invasion of the amniotic cavity (MIAC)] [4–6,12,65,73–82].

The increased concentrations of PGs in amniotic fluid have been considered important in both normal labor at term [7,8,11,12,16,71,72,82–84], and preterm labor [4–6,16,65,73–78,81]. The amnion is an important source of PGs in the amniotic fluid [85–94]. This membrane can be anatomically divided into three distinct regions: the reflected, placental, and umbilical amnion [95–97]. Gene expression profile studies indicate that there are differences between placental and reflected amnion [97–100]. Indeed, we previously reported that the placental amnion is responsible for the tonic production of PGs with advancing gestational age, while the reflected amnion increases the production of PGs during labor [66]. Such difference in PG production suggests that the placental amnion and the reflected amnion play different roles in the regulation of eicosanoids during pregnancy and labor [66].

The umbilical cord (UC) is a source of PGs [95,101–103]. PGE₂ and PGF_2α have been localized in the amniotic cells covering the UC and in the endothelium of the umbilical veins [103]. Furthermore, McCoshen et al. claimed that the UC is the major source of PGE₂ found in the amniotic fluid of women in labor at term [95]. However, incubation of a segment of the UC, as well as the placental and reflected amnion in a perfusion chamber, allowed demonstration of the PGE₂ output from the UC which did not change before or after labor, and the placental and reflected amnion PGE₂ output were two-fold greater in tissues collected after labor [95]. This observation suggests that the pattern of PG production by the UC is different from that of the reflected and placental amnion [95]. The current study is conducted to assess the concentrations of PGE₂ and PGF_2α in UC segments from women who had undergone labor at term, women delivered by cesarean section without labor, and a group who had preterm labor and delivery (with and without funisitis).

Materials and methods

Tissue samples were retrieved from the Bank of Biological Specimens at the Perinatology Research Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), U.S. Department of Health and Human Services (DHHS), Detroit, Michigan. Samples were taken from the following clinical groups, all of whom had singleton gestations: (1) women at term not in labor (TNL; n = 20); (2) women at term in labor (TIL; n = 20); (3) women with spontaneous preterm labor and delivery (sPTD; n = 20) without acute funisitis; (4) women with sPTD with acute funisitis (n = 20); and (5) women with preeclampsia who underwent cesarean delivery without labor over a range of gestational ages 25–39 weeks (n = 78). To compare the PG concentrations at different locations in a given cord, three samples from each UC (1 cm apart from UC insertion, 1 cm apart from the cord clamping, and mid-segment) were obtained from additional term delivery (n = 10) cases. Samples of UC tissue were flash-frozen using liquid nitrogen, and stored at −80 °C until use. All women provided written informed consent prior to the collection of samples, and the Institutional Review Boards of the NICHD and Wayne State University approved the collection and use of materials and clinical data for research purposes.

Clinical definition

Gestational age was determined by the last menstrual period and confirmed by ultrasound examination, or by ultrasound examination alone if the sonographic determination of gestational age was not consistent with menstrual dating. Preterm labor was diagnosed by the presence of at least two regular uterine contractions every 10 min in association with cervical changes in patients with a gestational age between 20 and 36 6/7 weeks which led to preterm delivery (defined as birth prior to the 37th week of gestation). Spontaneous term labor was defined as the presence of regular uterine contractions with a frequency of at least one every 10 min associated with cervical changes after 37 weeks of gestation. Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton’s jelly, according to criteria previously published [104–109]. Preeclampsia was defined as new onset hypertension during pregnancy (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg on at least two occasions, 4 h to 1 week apart) and proteinuria (≥300 mg in a 24-h urine collection or one dipstick measurement ≥2+) [110].

Prostaglandins assays

Tissue samples were pulverized in liquid nitrogen using a mortar and pestle for protein isolation. Total protein lysates were obtained using T-PER® Tissue Protein Extraction Reagent with a protease inhibitor cocktail (Thermo Scientific, Rockford, IL). To conduct the immunoassays for PGE₂ and PGF_2α content, 6.25 µg of total protein in 5 µl were added to 95 µl of assay buffer, and meclofenamic acid (10 µg/ml; Sigma-Aldrich Corporation, St. Louis, MO) was added to all samples. The assays were performed using the Prostaglandin E₂ and Prostaglandin F_2α enzyme immunoassay kits (Assay Designs, Inc., Ann Arbor, MI). The inter-assay coefficient of variation was 8.1%, and the intra-assay coefficient of variation was 6.7%.

Statistical analyses

Pearson’s chi-square test and Fisher’s exact test were used to compare proportions for categorical variables, and the Mann–Whitney U test was used to compare medians for continuous variables. Spearman’s correlation was used to examine the relationship between continuous variables such as gestational age at delivery and PG concentration. A repeated-measures analysis of variance test was performed to compare PG concentrations in different locations within each UC. A piecewise linear regression was performed to detect a change point in PG concentrations in preeclampsia using the SiZer package in the R program (www.r-project.org). Other statistical analyses were conducted using SPSS version 18.0 software (SPSS, Inc., Chicago, IL).

Results

Prostaglandin concentration in the umbilical cord in term and preterm labor/delivery

The clinical characteristics of the study groups as well as umbilical concentrations of PGE₂ and PGF_2α are displayed in Table 1. The median UC concentrations of PGE₂ and PGF_2α were not different among women at term, according to whether the samples had been obtained in women who had undergone labor (PGE₂: TNL 133.7 pg/μg versus TIL 116.1 pg/µg of total protein, p = 0.9; PGF₂α: TNL 8.4 pg/μg versus TIL 8.1 pg/µg of total protein, p = 0.7). The UC concentrations of PGE₂ and PGF_2α showed no correlation with gestational age at delivery in term pregnancies (PGE₂: ρ = 0.01, p = 0.9; PGF_2α: ρ = 0.07, p = 0.7).

Table 1. Clinical characteristics and concentrations of prostaglandins (PGE₂ and PGF_2α) in the umbilical cord for each study group.

	Term delivery without labor (n = 20)	Term delivery with labor (n = 20)	p-value*	sPTD without funisitis (n = 20)	sPTD with funisitis (n = 20)	p-value†	p-value‡
Maternal age (years)	25.5 (20–35)	26.0 (17–35)	0.8	25.5 (20–36)	23.0 (16–39)	0.7	0.4
Nulliparity	10% (2/20)	20% (4/20)	0.7	10% (2/20)	30% (6/20)	0.2	0.5
GA at delivery (weeks)	39.0 (37.1–40.1)	39.1 (37.1–40.4)	0.9	31.8 (27–34)	32.3 (28.6–34)	0.9	<0.001
Birth weight (g)	3323 (2545–4485)	3258 (2550–3695)	0.5	1818 (881–2690)	1665 (1010–2335)	0.6	<0.001
Male gender	70% (14/20)	55% (11/20)	0.3	45% (9/20)	50% (10/20)	0.8	0.8
PGE2	133.7 (44.8–252.4)	116.1 (33.5–354.9)	0.9	87.4 (6.1–258.6)	233.7 (61.3–1000)	0.001	0.03
PGF2α	8.4 (4.1–15.2)	8.1 (4.2–14.8)	0.7	7.1 (0.6–13.1)	9.6 (4.3–43.8)	0.051	0.3

Data are presented as median (interquartile range) and percentage with (n).

sPTD, spontaneous preterm delivery; PGE2 and PGF_2α concentrations were expressed by pg/µg of total protein; GA: gestational age.

*Comparison between term delivery without labor and term delivery with labor.

†Comparison between sPTD without funisitis and sPTD with funisitis.

‡Comparison between TIL and sPTD with funisitis.

The median PGE₂ concentration in the UC of patients with spontaneous preterm delivery was significantly higher among patients with acute funisitis than in those without funisitis (233.7 pg/μg versus 87.4 pg/µg of total protein, p = 0.001). The median PGF_2α UC concentration was also higher in patients with funisitis than in those without funisitis (9.6 pg/μg versus 7.1 pg/µg of total protein, p = 0.051).

The UC concentration of PGE₂ (but not that of PGF_2α) in cases of spontaneous preterm labor/delivery with funisitis was higher than that measured in cases who had undergone labor at term (PGE₂: 233.7 pg/µg versus 116.1 pg/µg of total protein, p = 0.03; PGF_2α: 9.6 pg/µg versus 8.1 pg/µg of total protein, p = 0.3).

The concentration of prostaglandins in the umbilical cord during gestation

The evaluation of UC PG concentration is not possible in women with uncomplicated pregnancies. Therefore, we assembled a study group comprised of women with preeclampsia who underwent indicated cesarean delivery. Table 2 shows the clinical characteristics of patients with preeclampsia. All women with preeclampsia were delivered by cesarean in the absence of labor (n = 78). None of the patients had acute histologic chorioamnionitis or funisitis after pathologic evaluation of the placenta. We found a positive correlation between PG concentrations in the UC and gestational age at delivery (PGE₂: ρ = 0.60, p < 0.001; PGF_2α: ρ = 0.35, p = 0.002).

Table 2. Clinical characteristics and the umbilical cord concentrations of PGE₂ and PGF_2α for term and preterm preeclampsia cases.

	Preeclampsia (n = 78)
Maternal age (years)	26.4 (± 6.1)
Nulliparity	45% (35/78)
Gestational age at delivery (weeks)	35.7 (25.3–39.0)
Birth weight (g)	2051 (± 859)
Male gender	58% (45/78)
PGE2 (pg/µg of total protein)*	95.1 (11.5–714.1)
PGF2α (pg/µg of total protein)*	5.5 (1.2–19.9)

Data are presented as mean (± SD) and percentage with (n).

*[median (range)].

Since our findings showed that PG concentrations in the UC did not change with gestational age at term, we attempted to establish if there was a point of change in UC PG concentration in preeclampsia. A piecewise linear regression revealed that the slopes before 36 weeks provided an estimate of 20.1 pg/µg of total protein/week and 0.63 pg/µg of total protein/week – this difference was statistically significant for both PGE₂ and PGF_2α. In both cases, the slopes after 36 weeks were estimated as −20.0 pg/µg of total protein/week and −0.53 pg/µg of total protein/week, but they were not significantly different from zero at a 5% significance level (Figure 1). These results indicate that the correlation between PG concentrations and gestational age changed significantly at about 36 weeks of gestation. Among patients with preeclampsia who were not in labor, there was an increase of PGE₂ and PGF_2α concentrations in the UC as a function of gestational age before 36 weeks (PGE₂: ρ = 0.59, p < 0.001; PGF_2α: ρ = 0.39, p = 0.01) but not after 36 weeks (PGE₂: ρ = −0.1, p = 0.5; PGF_2α: ρ = −0.2, p = 0.2).

Figure 1. PGE₂ and PGF_2α concentrations in the umbilical cord as a function of gestational age including preterm and term gestations with preeclampsia. Umbilical cord PGE₂ and PGF_2α concentrations increased with advancing gestational age until 36 weeks (PGE₂: ρ = 0.59, p < 0.001; PGF_2α: ρ = 0.39, p = 0.01), but not after 36 weeks (PGE₂: ρ = −0.1, p = 0.5; PGF_2α: ρ = −0.2, p = 0.2).

A comparison of the concentrations of prostaglandins at different locations in the same umbilical cord

The concentrations of PGs at three different locations in the same UC were determined. Figure 2 illustrates concentrations of PGE₂ and PGF_2α at each UC sampling site. There were no significant differences in the concentrations of PGE₂ and PGF_2α among the three sampling sites (PGE₂ and PGF_2α: p = 0.7 and p = 0.7, respectively).

Figure 2. PGE₂ and PGF_2α umbilical cord concentrations in different sites of the umbilical cord. There was no significant difference in the umbilical cord concentrations of PGE₂ and PGF_2α among the three different sampling sites (PGE₂; p = 0.7; and PGF_2α; p = 0.7). (I: 1 cm apart from umbilical cord insertion. C: 1 cm apart from the cord clamping. M: mid-segment). Connected lines referred to prostaglandin concentration in a particular segment of the umbilical and in another segment in each patient.

Discussion

Principal findings of the study

(1) The UC concentrations of PGF_2α and PGE₂ after spontaneous preterm labor with funisitis were significantly greater than in preterm labor without this lesion; (2) spontaneous labor at term was not associated with a change in the UC concentrations of PGF_2α and PGE₂; and (3) among women who underwent a cesarean delivery without labor (because of preeclampsia), the UC concentrations of PGF_2α and PGE₂ increased as a function of advancing gestational age in the second and third trimesters.

Prostaglandins, different regions of the amnion, and the umbilical cord

Amniotic fluid PGs increase prior to the onset of labor [11,16,17] and further increase during labor [8,72]. The amnion is considered to be the main source of PGs (in particular, PGE₂) in the amniotic fluid [85,87–94]. The human amnion can be divided into three areas: (1) placental amnion; (2) reflected amnion; and (3) amnion covering the UC. The term “placental amnion” refers to that covering the chorionic plate of the placenta, while the reflected amnion is part of the extraplacental membranes. A small portion of amnion covers the surface of the UC [95–97]. McCoshen et al. proposed that each component of amnion contributes to the production of PGE_2, and that the UC is the major source of prostaglandin in the amniotic fluid in patients in labor at term [95]. This work was based on in vitro experiments in which the production of PGs by different types of amnion was studied, and calculations about the relative contribution of each type were made. The investigators reported that before labor, the UC accounts for the greatest tissue mass (76%), compared to the placental amnion (14%) and reflected amnion (10%) [95]. They further claimed that the UC accounts for 66% of the total PGE₂ output before labor, and 44% after labor, proposing that the UC was the primary source of PGE₂ in the amniotic fluid [95].

Previously, we compared the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), a key enzyme involved in prostaglandin production by amnion [111–114], in placenta versus reflected amnion [66]. We reported that: 1) before the onset of labor, PTGS2 expression was greater in placental amnion, rather than reflected amnion; 2) after labor, there was a significant increase in PTGS2 expression in reflected, but not in placental amnion; and 3) there was little expression of PTGS2 in preterm labor in the absence of acute inflammatory lesions [66].

Changes in umbilical cord prostaglandins with advancing gestational age, labor, and acute inflammatory lesions of the placenta

The results of the current study indicate that the UC concentrations of PGE₂ and PGF_2α increase with advancing gestational age. Such findings were based on studies of UC derived from patients with the diagnosis of preeclampsia who underwent cesarean delivery for obstetrical indications. It is noteworthy that the prostaglandin concentration increased until approximately 36 weeks of gestation, and there was no detectable increase thereafter. Labor at term was not associated with an increase in the prostaglandin concentration in the UC. However, in the presence of funisitis (acute inflammation of the UC) the prostaglandin concentration increased significantly in cases with spontaneous preterm labor.

Amniotic fluid prostaglandins with microbial invasion of the amniotic cavity

Amniotic fluid concentrations of eicosanoids and arachidonate lipoxygenase products are increased in the amniotic fluid of women with preterm labor and MIAC [4–6,59,74,75,82]. MIAC is frequently associated with acute histologic chorioamnionitis and funisitis [82,107,108,115–148]. Amniotic fluid PGs in cases of MIAC and/or intra-amniotic inflammation are thought to derive from amnion cells and local cells in the amniotic cavity, as well as from the human fetus. Microorganisms and their products (e.g. lipopolysaccharide endotoxin) could stimulate resident macrophages in the amniotic fluid, as well as fetal cells suspended in amniotic fluid or contained within fetal mucosa or skin to produce PGs which could be found in amniotic fluid. In a similar way, bacterial products could stimulate the amniotic epithelial cells surrounding the UC to produce PGs, which could find their way into the amniotic fluid. In the context of a fetal systemic inflammatory response syndrome (FIRS) [104,131,149–156], prostaglandin production could be increased, as is the case for the adult systemic inflammatory response syndrome [157–162]. PGs contained within the UC may exert a local effect, such as modifying vascular reactivity of the umbilical arteries and vein [102,163–168], altering vessel permeability [169] or changing extracellular matrix metabolism in Wharton’s jelly. McCoshen et al. proposed that PGs generated in the UC could diffuse into the amniotic cavity [95]. Experimental evidence is required to prove this concept.

Conclusion

Prostaglandin concentrations in the UC are increased in the context of funisitis. We propose that PGE₂ and PGF_2α act as inflammatory mediators in the fetal inflammatory response syndrome, whose pathologic hallmark is funisitis.

Declaration of interest

This work was supported in part by the Perinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, MD, and Detroit, MI, USA. The authors declare no conflicts of interest.

Acknowledgements

The authors are grateful to the patients who agreed to provide the materials of our studies; to the nurses, laboratory staff, and clinicians who made this work possible; and to Maureen McGerty (Wayne State University) for her critical reading of the manuscript. J.-S. H., R. R., N. G. T., L. Y., P. C., C. J. K., Y. M. K. worked on the design of the study, data analysis, manuscript preparation; D. C. L., S. A. performed experiments, data analysis; and J. S. K. contributed in histopathological analysis and manuscript preparation.