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Obstetrics & Gynecology 2001;97:229-234
© 2001 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Granulocyte Colony-Stimulating Factor in Preterm and Term Pregnancy, Parturition, and Intra-amniotic Infection

DARLENE A. CALHOUN, DO, NASSER CHEGINI, PhD, BRUNO M. POLLIOTTI, PhD, DSc, JASON A. GERSTING, MS, RICHARD K. MILLER, PhD and ROBERT D. CHRISTENSEN, MD

From the Division of Neonatology, Department of Pediatrics, Gynecology and Obstetrics, University of Florida College of Medicine, Gainesville, Florida; and theDepartment of Gynecology and Obstetrics, University of Rochester School of Medicine and Dentistry, Rochester, New York.

Address reprint requests to: Darlene A. Calhoun, DO, Department of Pediatrics, University of Florida College of Medicine, P.O. Box 100296 JHMHC, Gainesville, FL 32610-0296, E-mail: calhoda{at}peds.ufl.edu


   Abstract
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Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To determine the sources of granulocyte colony-stimulating factor (G-CSF) in amniotic fluid and to examine its relation to labor and clinically diagnosed intra-amniotic infection.

Methods: We assessed G-CSF and G-CSF receptor expression in placentas (n = 50) from 5–40 weeks’ gestation, and G-CSF concentrations were measured in amniotic fluid (n = 146), bronchoalveolar lavage fluid (n = 8), and paired maternal serum, cord blood, neonatal serum, and neonatal urine samples (n = 16).

Results: Immunohistochemical staining and messenger RNA analysis showed placental expression of G-CSF and G-CSF receptor throughout gestation. The number of decidual stromal cells expressing G-CSF receptor was significantly higher in women with intra-amniotic infection compared with women without infection (27 ± 2 versus 18 ± 3 cells per high power field, P = .02). Amniotic fluid concentrations of G-CSF were not significantly different in noninfected preterm compared with term samples (1708 ± 1673 versus 1612 ± 2100 pg/mL, P = .9). Labor was not associated with a significant increase in amniotic fluid G-CSF concentrations (1864 ± 3151 versus 1612 ± 2100 pg/mL, P = .77, term labor versus no labor; 3335 ± 5364 versus 1708 ± 1673 pg/mL, P = .09, preterm). Concentrations of G-CSF in maternal serum, amniotic fluid, bronchoalveolar lavage fluid, and neonatal urine were increased during intra-amniotic infection (all P < .05).

Conclusion: Amniotic fluid G-CSF concentrations were similar in preterm and term pregnancies and were not significantly influenced by labor. Intra-amniotic infection was associated with an increased number of placental cells expressing the G-CSF receptor and higher concentrations of G-CSF in amniotic fluid, maternal serum, neonatal urine, and neonatal bronchoalveolar lavage samples.

Granulocyte colony-stimulating factor (G-CSF) is recognized for its effects on proliferation, differentiation, and survival of neutrophil progenitors.1 These actions occur after binding to its specific receptor.2 It has been learned recently that G-CSF and its receptor are expressed in placenta3 and a variety of fetal tissues.4 Preterm labor is a major clinical problem, and one of its causes is intra-amniotic infection.5,6 Recently, amniotic fluid G-CSF concentrations were shown to indicate intra-amniotic infection.7 However, the source(s) and physiologic role(s) of G-CSF in amniotic fluid are not known. We previously reported that the placenta produces large quantities of G-CSF,8 but whether this is the primary source of amniotic fluid G-CSF is not known. The objectives of this study were to determine the sources of G-CSF in amniotic fluid and to examine the effect of gestational age, labor, and clinically diagnosed intra-amniotic infection on G-CSF and its receptor in the maternal-fetal unit.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
All studies were approved by the institutional review boards at the University of Florida and the University of Rochester. Placentas (n = 40) were selected after elective termination of normal pregnancies (5–24 weeks, n = 15) and vaginal or cesarean delivery in pregnancies of 24–40 weeks’ gestation (n = 15 preterm and ten term) without clinical evidence of infection. For comparison, placentas were obtained from ten pregnancies 24–34 weeks’ gestation affected with clinically diagnosed intra-amniotic infection9 and from pregnancies matched for gestational age (± 1 week) without evidence of infection. Clinical intra-amniotic infection was defined as maternal fever (at least 38C) during labor, accompanied by uterine tenderness, maternal tachycardia, fetal tachycardia, or foul-smelling amniotic fluid.9 A portion of placenta was excised and washed before fixation in Bouin’s solution (Sigma, St. Louis, MO) for immunohistochemistry, 4% paraformaldehyde for in situ polymerase chain reaction (PCR) or frozen at -80C for PCR analysis. Total cellular RNA was extracted from 30 representative placentas using the guanidinium thiocyanate method and converted to cDNA using Maloney murine leukemia virus reverse transcriptase (Gibco-BRL, Rockville, MD).4 Granulocyte colony-stimulating factor receptor messenger RNA was also examined by in situ PCR4 in a separate section from five placentas.

Representative placentas (n = 20) were immuno-stained with an enzyme-labeled biotin-streptavidin technique (Ventana, Tucson, AZ).4 Granulocyte colony-stimulating factor (1:20, Oncogene Research Products, Cambridge, MA) and G-CSF receptor antibodies (1:10, Santa Cruz Biotechnology Inc., Santa Cruz, CA) were applied, and counter-staining was done. Some sections were prepared for computerized image analysis (Imaging Research Inc., St. Catharines, Ontario, Canada)10 of the G-CSF receptor. A minimum of 50 cells per slide was examined in each placenta (n = 20; same placentas as used for immunohistochemistry).

Amniotic fluid samples (n = 146) were collected by transabdominal amniocenteses performed for medical indications, including amnioreduction, Rh sensitization, and assessment of fetal lung maturity and intra-amniotic infection. Information relevant to the pregnancy at the time of the amniocentesis was collected. Patients with (n = 16) and without (n = 130 [69 term and 61 preterm]) clinically diagnosed intra-amniotic infection were included. Labor was defined as the presence of regular uterine contractions occurring with a minimum frequency of two every 10 minutes accompanied by changes in cervical effacement, dilatation, or both.11 All fluids were centrifuged at 1500 g for 10 minutes and frozen at -80C.

In a separate study, matched maternal serum, cord blood, neonatal serum, and neonatal urine were obtained from pregnancies with (n = 8) and without (n = 8) clinically diagnosed intra-amniotic infection. Postdelivery maternal serum samples were obtained within 24 hours after delivery. Bronchoalveolar lavage samples (n = 8) were collected on the first day of life from neonates (30.5 ± 6 weeks) requiring intubation for respiratory distress secondary to prematurity, hyaline membrane disease, or sepsis. Sterile saline (1.5 mL/kg) was passed through a sterile catheter and retrieved into a sterile trap. Neonatal gastric secretions (n = 5) were collected as part of routine suctioning on the first day of life. From these samples, G-CSF was quantified by enzyme-linked immunosorbent assay (Quantikine Human G-CSF Immunoassay, R & D Systems, Minneapolis, MN).7,8,12–14

Adult and neonatal serum, amniotic fluid, neonatal gastric secretions, and neonatal urine (n = 5 for each specimen) in which the G-CSF concentration was at least 100 pg/mL were used for western analysis. Each specimen was extracted, lysed on ice, spun, and stored at -80C. Protein concentrations were determined by the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Protein was denatured by heating at 98C for 5 minutes. The electrophoretically separated proteins were transferred to a nitrocellulose filter and incubated at 4C overnight before probing with anti-human G-CSF antibody. The ABC-Alkaline Phosphatase and Alkaline Phosphatase Substrate Kits (Vector Laboratories, Inc., Burlingame, CA) were used to detect G-CSF. Molecular weight markers (Sigma) were used to determine protein size. Recombinant human G-CSF (Amgen, Thousand Oaks, CA) and native G-CSF (ATCC 5636) were used as positive controls.

Descriptive data are given as mean ± standard deviation (SD). Measures of relative optical density of G-CSF-receptor protein were analyzed using a generalized least-squares analysis of variance blocking on sample and allowing for unequal variances per group. Graphical displays are given as box plots, which are a five-number summary including the minimum, lower quartile, median, upper quartile, and maximum values. Granulocyte colony-stimulating factor concentrations in amniotic fluid were analyzed by analysis of covariance with the factors of gestational age, labor, and intra-amniotic infection. The results were displayed as a two-way analysis of variance. Specific contrasts were then used to examine within-group comparisons. All two-group comparisons in the maternal serum study were analyzed using exact Wilcoxon rank-sum tests.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
Polymerase chain reaction products for G-CSF (143 bp) and G-CSF receptor (340 bp) were identified in all placentas. Granulocyte colony-stimulating factor receptor transcripts were also identified by in situ PCR and localized to the syncytiotrophoblast layer of the placental villi in a site corresponding to that demonstrated for the protein. Representative photomicrographs are shown (Figure 1Go, A–C).



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  		Figure 1. In situ polymerase chain reaction of granulocyte colony-stimulating factor receptor, which was localized to the apical surface of the villi (A, arrows). The positive control (B) shows intense staining of all cells. The negative control is shown (C) (original magnification, x 400).

 
Granulocyte colony-stimulating factor (Figure 2Go, A–F) and G-CSF receptor (Figure 2Go, G–L) were also demonstrated using immunohistochemistry. No difference in relative optical density of G-CSF receptor was noted in placentas at different gestational ages or during intra-amniotic infection (Figure 3Go). However, the total number of decidual macrophages expressing G-CSF receptor was higher in placentas from women with clinically diagnosed intra-amniotic infection than from those without infection (27 ± 2 versus 18 ± 3 cells [high-power field] per placenta, P = .02).



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  		Figure 2. Immunohistochemical staining for granulocyte colony-stimulating factor (G-CSF) and G-CSF receptor. Representative placental sections at 10–14, 25–27, and 38–40 weeks’ gestation are shown. (A–C) noninfected placentas; D–F, placentas from women with intra-amniotic infection stained with G-CSF antibody. (G–I) noninfected placentas; J–L, placentas from women with intra-amniotic infection stained with G-CSF receptor antibody (original magnification, x 400).

 


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  		Figure 3. Relative optical density (ROD). Placentas from women with intra-amniotic infection (IAI) and without intra-amniotic infection are plotted by trimester using a box plot with crossbars given by the minimum, median, and maximum values as well as the lower and upper quartiles.

 
Amniotic fluid from women who were not in labor but were preterm (n = 41; 33.9 ± 2.1 weeks) had G-CSF concentrations within the same range as women not in labor at term (n = 42; 37.6 ± 0.5 weeks; 1708 ± 1673 versus 1612 ± 2100 pg/mL, P = .9). Similarly, no difference was detected in amniotic fluid G-CSF concentration from women who were preterm and in labor (n = 20; 34.3 ± 1.5 weeks) versus term and in labor (n = 27; 3335 ± 5364 versus 1864 ± 3151 pg/mL, P = .16). Granulocyte colony-stimulating factor concentrations in amniotic fluid of women with intra-amniotic infection who were preterm (n = 16) were not significantly different than those without infection (n = 61; 5520 ± 6594 pg/mL versus 2241 ± 3402 pg/mL, P < .07).

Before delivery, maternal serum G-CSF concentrations were significantly higher in women with intra-amniotic infection (n = 8) than without (n = 8) infection (Table 1Go). Within 24 hours after delivery, maternal G-CSF concentrations did not differ between the groups. Granulocyte colony-stimulating factor concentrations in urine (n = 5) and bronchoalveolar lavage samples (n = 4) of infants after intra-amniotic infection were significantly higher than in those without infection. The molecular weight of G-CSF in serum, amniotic fluid, neonatal gastric secretions, and neonatal urine were identical to native G-CSF (18,800 daltons). The molecular weight of recombinant G-CSF was 18,000 daltons.


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  		Table 1. Granulocyte Colony-Stimulating Factor Concentrations in Maternal Serum, Cord Blood, Neonatal Serum, Urine, and Bronchoalveolar Lavage Fluid
 

   Discussion
Top
Abstract
 Materials and Methods
 Results
 Discussion
References
 
High concentrations of G-CSF in amniotic fluid have been considered an indicator of intra-amniotic infection,7 but the sources of G-CSF in amniotic fluid are unknown. We detected G-CSF in all amniotic fluid specimens examined and in all its component sources. In amniotic fluid from noninfected pregnancies, G-CSF concentrations did not change between 21–41 weeks’ gestation, nor did it change with labor. However, during clinically diagnosed intra-amniotic infection G-CSF concentrations increased and were higher in the serum of women with intra-amniotic infection before delivery and in neonatal urine and bronchoalveolar lavage samples. Although the presence of G-CSF has been reported in amniotic fluid during clinically diagnosed intra-amniotic infection,7,12–14 its relationship to labor and gestational age was not previously reported.

In contrast to previous reports,12,13 we detected no increase in G-CSF concentrations during labor. Several potential explanations for this discrepancy exist. One might be differences in routes of retrieval of amniotic fluid. Whereas the samples in our study were obtained transabdominally, others12,13 used primarily transvaginal approaches. Second, in the absence of bacterial infection, some cytokines such as interleukin-1 (IL-1),15 IL-6,16 and IL-817 increase during labor whereas others, such as IL-16,18 do not. Alternatively, subclinical infection in the control group could have obscured differences.

McCracken et al3 proposed that G-CSF has a central role in placental development. Although we did not demonstrate an increase in G-CSF receptor expression in individual cells during intra-amniotic infection, we did observe an increase in the number of placental cells expressing the receptor. This is in agreement with Stallmach et al14 and might indicate a role for G-CSF during intra-amniotic infection. Other proteins, including integrin subunit {alpha}6, transforming growth factor beta and its receptor, and granulocyte-macrophage colony-stimulating factor receptor, are expressed temporally by the placenta.19–21 However, the functional significance of the regulation of specific placental genes during gestation remains unknown.

In pregnancies without intra-amniotic infection, G-CSF concentrations in maternal serum, cord blood, neonatal serum, and neonatal urine were generally less than 100 pg/mL. However, in the presence of intra-amniotic infection there were variable increases in G-CSF concentrations. The variability might reflect differences in the severity of infection but could indicate differences in the transplacental transfer of G-CSF.22,23 Because the pregnancies without intra-amniotic infection were not normal pregnancies, their use introduces the potential for variability related to the specific condition associated with the pregnancy (ie, preeclampsia).

Stallmach and Karolyi24 proposed that cytokines generated at the fetomaternal interface increased fetal granulocytopoiesis during intra-amniotic infection. The sensitivity of fetal hematopoiesis to G-CSF administered to the mother was shown by Medlock et al.25 We also demonstrated that the administration of recombinant G-CSF to pregnant women with an imminent preterm delivery resulted in transplacental transfer of a measurable quantity of G-CSF and a significantly greater neonatal bone marrow proliferative pool.22

During the later stages of pregnancy, fetal urine and tracheobronchial secretions contribute significantly to amniotic fluid volume.26 In urine and tracheobronchial secretions of neonates delivered of women with intra-amniotic infection, we found that G-CSF concentrations were significantly higher than those of neonates delivered of women without intra-amniotic infection. Stallmach et al14 reported that G-CSF immunoreactivity during intra-amniotic infection was prominent in the alveolar epithelium and in fibroblasts of the lung and kidney. Our finding of increased G-CSF during intra-amniotic infection in bronchoalveolar lavage fluid and urine of neonates further substantiates that these cells could be responsible for G-CSF production. Although the precise origin of G-CSF in amniotic fluid cannot be determined from this analysis, it is clear that its molecular size is similar in the different compartments of the maternal-fetal unit.

Amniotic fluid G-CSF is produced in various placental, maternal, and fetal sites and increases during intra-amniotic infection. Because granulocytopoiesis is increased in fetuses during intra-amniotic infection,24 and neonates generate G-CSF during bacterial sepsis,27 we speculate that increases in amniotic fluid G-CSF might be a means by which the fetus is protected from overwhelming sepsis in utero.


   Footnotes
 
Supported by grants HD-01180, HL-44951, and RR-00083 from the National Institutes of Health and a grant from the Children’s Miracle Network Telethon.

The authors thank Douglas W. Theriaque, MS, and Alan D. Hutson, PhD, for their assistance with the statistical analyses.

PII S0029-7844(00)01120-0

Received June 28, 2000. Received in revised form September 18, 2000. Accepted October 12, 2000.


   References
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 Materials and Methods
 Results
 Discussion
 References
 
1. Avalos B, Gasson J, Hedvat C, Quan SG, Baldwin GC, Weisbart RH, et al. Human granulocyte colony-stimulating factor: Biologic activities and receptor characterization on hematopoietic cells and small cell lung cancer cell lines. Blood 1990;75:851–7.[Abstract/Free Full Text]

2. Avalos B, Parker J, Ware D, Hunter M, Sibert K, Druker B. Dissociation of the Jak kinase pathway from G-CSF-receptor signaling in neutrophils. Exp Hematol 1997;25:160–8.[Medline]

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7. Hoskins I, Zandieh P, Schatz F, Lee C. Amniotic fluid granulocyte colony-stimulating factor levels: A rapid marker for diagnosing chorioamnionitis. Am J Reprod Immunol 1997;38:286–8.

8. Li Y, Calhoun D, Polliotti B, Sola M, Al-Mulla Z, Christensen R. Production of granulocyte colony-stimulating factor by the human placenta at various stages of development. Placenta 1996;8:611–8.

9. Gilstrap L, Leveno K, Cox S, Burris J, Mashburn M, Rosenfeld C. Intrapartum treatment of acute chorioamnionitis: Impact upon neonatal sepsis. Am J Obstet Gynecol 1988;159:579–83.[Medline]

10. Matthews S, Challis J. Regulation of corticotropin-releasing hormone (CRH) and vasopression (AVP) messenger ribonucleic acid (mRNA) expression in the developing ovine hypothalamus: Effects of stress and glucocorticoids. Am J Physiol 1995;268:E1096–107.

11. Maymon E, Ghezzi F, Edwin S, Mazor M, Yoon BH, Gomez R, et al. The tumor necrosis factor alpha and its soluble receptor profile in term and preterm parturition. Am J Obstet Gynecol 1999;181: 1142–8.[Medline]

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14. Stallmach T, Hebisch G, Joller-Jemelka H, Orban P, Schwaller J, Engelmann M. Cytokine production and visualized effects in the fetomaternal unit: Quantitative and topographic data on cytokines during intrauterine disease. Lab Invest 1995;73:384–92.[Medline]

15. Romero R, Brody D, Oyarzum E, Mazor M, Wu YK, Hobbins SC, et al. Infection and labor III. Interleukin-1: A signal for the initiation of parturition. Am J Obstet Gynecol 1989;160:1117–23.[Medline]

16. Romero R, Avila C, Santhanam U, Sehgal P. Amniotic fluid interleukin-6 in preterm labor. J Clin Invest 1990;85:1392–1400.

17. Romero R, Ceska M, Avila C, Mazor M, Behnke E, Lindly T. Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition. Am J Obstet Gynecol 1991;165:813–20.[Medline]

18. Athayde N, Romero R, Maymon E, Gomez R, Pacora P, Yoon BH, et al. Interleukin 16 in pregnancy, parturition, rupture of fetal membranes, and microbial invasion of the amniotic cavity. Am J Obstet Gynecol 2000;182:135–41.[Medline]

19. Roelen BA, Lin H, Knezevic V, Freund E, Mummery C. Expression of TGF-betas and their receptors during implantation and organogenesis of the mouse embryo. Dev Biol 1994;166:716–28.[Medline]

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21. Jokhi P, King A, Jubinsky P, Loke Y. Demonstration of the low affinity alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R alpha) on human trophoblast and uterine cells. J Reprod Immunol 1994;26:147–64.[Medline]

22. Calhoun D, Christensen R. A randomized, double-blind, placebo controlled pilot trial of recombinant granulocyte colony-stimulating factor administration in women prior to preterm delivery. Am J Obstet Gynecol 1998;179:766–71.[Medline]

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25. Medlock E, Kaplan D, Cecchini M, Ulich T, del Castillo J, Andresen J. Granulocyte colony-stimulating factor crosses the placenta and stimulates fetal rate granulopoiesis. Blood 1993;81:916–22.[Abstract/Free Full Text]

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27. Calhoun D, Lunøe M, Du Y, Hutson A, Veerman M, Christensen R. Granulocyte colony-stimulating factor (G-CSF) serum and urine concentrations in neutropenic neonates before and following the intravenous administration of recombinant G-CSF. Pediatrics 1999; 105:392–7.




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