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Serum Hyaluronic Acid Levels During Pregnancy and Labor

From the Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu; and the Diagnostics Division, Chugai Pharmaceutical Co., Ltd., Tokyo, Japan.

Address reprint requests to: Hiroshi Kobayashi, MD, PhD, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-3192, Japan

	Abstract

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Objective: To study the changes in concentrations of serum hyaluronic acid in uncomplicated human pregnancies.

Methods: We determined the concentrations of serum hyaluronic acid, using a specific enzyme-linked immunosorbent assay, in 70 nonpregnant women, 250 women during their pregnancies, and 68 women at the time of parturition. Results were analyzed for statistical significance with Scheffé test for multiple comparisons.

Results: During pregnancy, mean (± standard deviation) serum hyaluronic acid levels were 11.4 ± 4.5, 13.6 ± 2.8, 20.6 ± 1.5, and 46.9 ± 7.9 ng/mL at 5–14 (n = 47), 15–26 (n = 46), 27–37 (n = 58), and 38–40 (n = 99) weeks’ gestation, respectively. Pregnant women in labor (n = 68) had significantly higher levels (100.4 ± 11.3 ng/mL) than did women at term but not in labor (P < .01).

Conclusion: Maternal serum hyaluronic acid concentrations increase as pregnancy progresses and serum levels increase significantly at term. Hyaluronic acid may be associated with cervical ripening during parturition.

Hyaluronic acid is a high molecular weight, nonsulfated linear glycosaminoglycan composed of repeating units of (ß, 14)-D-glucuronic acid-(ß, 13)-N-acetyl-D-glucosamine.¹ It is a key molecule of the extracellular matrix, found whenever there is rapid tissue proliferation, regeneration, and repair.² The extracellular matrix hyaluronic acid is produced in chorionic villi, fetal lung, and connective tissue fibroblasts located in the uterine cervix during parturition.^3–5 The lower uterine segment and the amniotic fluid are also sources of hyaluronic acid.⁶

The uterine cervix is a fibrous organ composed largely of collagen, proteoglycan, and hyaluronic acid.⁷ The principal change observed in these constituents as pregnancy progresses is an increase in hyaluronic acid.⁷ Cervical ripening during parturition is characterized by changes of the uterine cervix, with softening and water retention.⁸ These changes reflect the ripening processes that precede the more rapid and dramatic events of parturition.

Circulating hyaluronic acid levels have been reported to increase significantly at parturition in guinea pigs.⁹ These authors⁹ also reported that the increase in hyaluronic acid probably results from an increase in production or mobilization from the extracellular matrix of the uterine cervix and the lower uterine segment into the circulation by lymphatic drainage at parturition.

Hyaluronic acid–binding protein being made available,¹⁰ we developed a specific enzyme-linked immunosorbent assay (ELISA) to measure hyaluronic acid directly in maternal serum. The purpose of this article is to demonstrate the changes in levels of circulating hyaluronic acid through gestation in pregnant women and at parturition.

	Materials and Methods

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A cross-sectional study was conducted involving pregnant women. Sera were obtained from 70 nonpregnant healthy women, 25–41 years old, and from 250 pregnant women, 20–37 years old, each with a singleton gestation, who had been followed at the obstetric department of the Hamamatsu University School of Medicine and its related hospitals between January 1994 and September 1997. The study population was divided into six groups. The first group included 70 nonpregnant healthy women. None of the women were taking any medication, and all underwent routine laboratory screening. The second group included 47 women undergoing serum sampling at 5–14 weeks’ gestation. The third group included 46 women from whom sera were collected at 15–26 weeks’ gestation. The fourth group included 58 women undergoing serum sampling at 27–37 weeks’ gestation. The fifth group included 99 women who were not in labor or who had an elective cesarean delivery at term (38–40 weeks’ gestation). The indication for cesarean delivery was a previous cesarean delivery or a breech presentation in an otherwise uncomplicated pregnancy. The 68 women in the sixth group were in spontaneous active labor at term (38–40 weeks’ gestation) and delivered vaginally. All 250 pregnant women had uncomplicated pregnancies, delivered term neonates, and did not have any clinical signs of infection or preeclampsia. Samples obtained from women with pregnancies complicated by multiple gestations and fetal anomalies were excluded from analysis. Patients who received prostaglandins or oxytocin for induction or augmentation of labor also were excluded from this study. Treatment decisions were made by the patients’ physicians and were not affected by participation in this study. The gestational age was confirmed by a reliable menstrual history and an ultrasound examination at 10 and/or 20 weeks’ gestation. Obstetric histories, weight, blood pressure, urinary protein levels, glucose levels, and any complications were recorded at each visit. Demographic variables for each group included age, gravidity, parity, reproductive history, race, marital status, birth weight, sex, 5-minute Apgar scores, Bishop scores, and birth hematocrit level.

In a parallel study, three women were followed longitudinally throughout gestation and underwent clinical examination and blood sampling during the three trimesters. Samples were taken sequentially from 8 weeks’ gestation until delivery. All women gave their informed consent to participate in these studies, which were approved by the Hamamatsu University Hospital Ethical Committee.

Peripheral blood samples were drawn from the anti-cubital vein with a polypropylene syringe and transferred to chilled tubes. The tubes were centrifuged immediately at 4C (2000 x g for 15 minutes). All serum samples were kept at -20C until assay.

Hyaluronic acid–binding protein was prepared after trypsin digestion of bovine nasal cartilage proteoglycans. The bovine nasal cartilage hyaluronic acid–binding protein was purified by affinity chromatography on hyaluronic acid covalently coupled to Sepharose 4B (Pharmacia Fine Chemicals AB, Uppsala, Sweden).¹¹ A purified preparation of hyaluronic acid–binding protein was supplied by Chugai Pharmaceutical Co., Ltd., and Seikagaku Kogyo Inc., Tokyo, Japan. A purified preparation of hyaluronic acid–binding protein was biotinylated according to the method of Guesdon,¹² using N-hydroxysucciminidyl biotinamidocaproate (Sigma, St. Louis, MO) in accordance with the manufacturer’s suggested procedures.

Immobilization of purified hyaluronic acid–binding protein (2 µg/mL, 100 µL per well) to 96-well microtiter plates was carried out in 50 mM sodium carbonate buffer (pH 9.5) containing 100 mM NaCl, overnight at 4C. All subsequent additions in ELISA were done at 23C in Tris-buffered saline containing 2% (w/v) bovine serum albumin. Ten-microliter aliquots of the samples and 90 µL of Tris-buffered saline were added to each well coated with purified hyaluronic acid–binding protein and were incubated for 2 hours at 23C. After seven washes with Tris-buffered saline, 100 µL of biotinylated hyaluronic acid–binding protein (2 µg/mL) was added to each well and incubated for another hour at 23C. Detection of bound biotinylated hyaluronic acid–binding protein was with an avidin peroxidase (1:1000; Dako, Copenhagen, Denmark). Finally, the wells were washed seven times and 100 µL of 0.05% tetramethyl-benzidine containing 0.03% H₂O₂ was added to each well. After 10 minutes of incubation at 23C, the enzyme reaction was terminated by adding 50 µL of 2 M H₂SO₄ to each well, and the A450 of each well was measured with a microplate reader (Model 2550; Bio-Rad, Tokyo, Japan). In these experimental conditions, the lowest detectable level of hyaluronic acid was 1.0 ng/mL. The purified hyaluronic acid was used for the assay standard.

Solid-phase competition assay was performed as follows: Microtiter plate wells were coated with 100 µL of a solution containing the purified hyaluronic acid (100 µg/mL) and the solution was incubated for 16 hours at 4C. Wells were washed three times and blocked in Tris-buffered saline containing 2% bovine serum albumin for 1 hour at 23C. Biotinylated hyaluronic acid–binding protein (2 µg/mL, 50 µL) was added to wells in the presence of various concentrations of competitors (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin; 0-100 µg/mL, 50 µL per well) and incubated for 2 hours at 23C. After seven more washes with Tris-buffered saline, avidin peroxidase was added to wells, and this was followed by color reaction. All samples were assayed in triplicate.

All statistical analysis was performed using StatView for Macintosh (Hulinks Co., Tokyo, Japan). Continuous variables were compared with the independent two-tailed t test. Pearson ² was used to analyze noncontinuous variables. One-way analysis of variance was performed with post hoc analysis by using Scheffé F procedure for statistical interpretation. P < .05 was considered significant.

	Results

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The demographic and clinical variables were compared in each group to evaluate similarity in characteristics between groups after sample selection. No statistically significant differences were found in each group with respect to maternal age, gravidity, parity, reproductive history, race, marital status, birth weight, sex, 5-minute Apgar scores, Bishop scores, and birth hematocrit level.

The ELISA was validated for measuring hyaluronic acid in serum. A standard curve was produced that characteristically yielded a broad range of detection and a high degree of sensitivity (Figure 1). Data at the lower scale on the graph are displayed in the inset in Figure 1. The data represent the mean determination (± standard deviation) for each concentration of hyaluronic acid from three separate assays. Hyaluronic acid concentrations are plotted. Optical density 450 was plotted against hyaluronic acid concentration, and a standard curve was constructed. Absorbance values increased from 0.09 at baseline to 2.0 at the highest dose of hyaluronic acid (1000 ng/mL). The concentration of the unknown samples was read from the standard curve. Dilution curves parallel to the hyaluronic acid standard curve were observed for some samples, when the standard or serum was diluted with assay buffer. The fact that human serum dilution curves were parallel to the standard curve demonstrated that the assay was not affected by unknown serum factors and could be used for direct detection of hyaluronic acid in serum. The useful range for measuring hyaluronic acid levels was from 1.0 to 1000.0 ng/mL. In the present experimental conditions, the lowest detectable level of hyaluronic acid was 1.0 ng/mL. No deterioration of hyaluronic acid immunoreactivity was observed after three repeated freezings and thawings of serum samples. The interassay and intra-assay coefficients of variation were 6.8% (n = 10) and 8.5% (n = 10), respectively.

View larger version (14K): [in this window] [in a new window]   Figure 1. Standard curve of the hyaluronic acid enzyme-linked immunosorbent assay.

View larger version (18K): [in this window] [in a new window]   Figure 2. Specificity of binding of hyaluronic acid–binding protein to immobilized hyaluronic acid. Values are mean ± standard deviation (bar) from three experiments. HA = hyaluronic acid; CS = chondroitin sulfate; DS = dermatan sulfate; HS = heparan sulfate; Hep = heparin.

View larger version (14K): [in this window] [in a new window]   Figure 3. Hyaluronic acid levels in maternal serum samples. Values are mean ± standard deviation. a versus b, P < .05; a versus c, a versus d, b versus c, b versus d, and c versus d, P < .01.

View larger version (16K): [in this window] [in a new window]   Figure 4. Serum hyaluronic acid profiles for three sequentially sampled pregnant women. Arrows indicate delivery.

	Discussion

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Our observation is partly in accordance with the results of a study by Rajabi et al,⁹ who reported that circulating hyaluronic acid levels increase significantly at parturition in guinea pigs. The levels observed by Rajabi et al⁹ were ten-fold higher than those in human samples. This may be due to species difference. In addition, these investigators found no correlation between elevated levels and gestational age. They found that elevated levels were present only at delivery. This is in contrast to the elevated serum levels in our human subjects. The previous animal study involved a cross-sectional sampling of the population and was not a longitudinal study of levels in the same subjects. Our study involved sequential samples from the same women throughout pregnancy. The maternal hyaluronic acid profiles for three typical pregnancies demonstrated that a significant and progressive increase in serum hyaluronic acid levels occurred; the highest values were found at term. Pregnant woman in labor had higher levels than did women at term who were not in labor.

Dilation of the uterine cervix at parturition is reported to be associated with an increase in cervical hyaluronic acid content.^7,13 The observed softening and swelling of the uterine cervix may be caused by an accumulation of hyaluronic acid in the extracellular matrix, because hyaluronic acid possesses unique viscoelastic properties and high avidity for water.¹⁴ Ripening of the cervix signals the onset of labor and facilitates the subsequent delivery. The large increase in serum hyaluronic acid concentrations at term is likely caused by the release of this substance from the lower uterine segment and uterine cervix as well as from the amniotic fluid into the maternal circulation.^3–5 An increased leakage of hyaluronic acid into the maternal serum, probably through the lymph vessels, therefore seems the best explanation for the high concentrations of hyaluronic acid at parturition. It is possible that the increasing levels in maternal circulation could result from increasing leakage rather than from an increased amount produced by the ripening of the cervix. The fact that laboring patients have higher serum levels could be due to leaking hyaluronic acid into serum once the cervix dilates.

It is apparent that an increase in hyaluronic acid levels occurs close to term at the time of cervical ripening and that hyaluronic acid could be associated with cervical ripening during parturition. We cannot say, however, whether the elevated hyaluronic acid levels are cause for or result of or secondary to the onset of labor and whether the higher levels are directly responsible for cervical ripening. Nevertheless, the increase in hyaluronic acid levels toward term may allow locally produced hyaluronic acid to be involved in the sequence of events culminating in parturition.

	Footnotes

 
We are grateful to Drs. T. Kobayashi, N. Kanayama, T. Nishiguchi, and N. Tokunaga for supplying plasma samples and to Dr. K. Sumimoto for statistical assistance. A purified preparation of hyaluronic acid–binding protein was supplied by Chugai Pharmaceutical Co., Ltd., and Seikagaku Kogyo Inc., Tokyo, Japan.

PII S0029-7844(98)00526-2

Received May 19, 1998. Received in revised form October 4, 1998. Accepted October 15, 1998.

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