Bovine lactoferrin in preventing preterm delivery ...

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Bovine lactoferrin in preventing preterm delivery associated with sterile inflammation1

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Rosalba Paesano, Miriam Pietropaoli, Francesca Berlutti, and Piera Valenti

Abstract: Preterm delivery (PTD) occurs before the 37th week of gestation. Iron deficiency anemia and inflammatory processes either related to infection or sterile inflammatory response represent risk factors for PTD. Bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, can represent a new therapeutic approach for PTD treatment. Here an open-label cohort and subcohort study is reported. The cohort was designed to assess the effect of bLf oral administration on iron and inflammatory homeostasis in anemic pregnant women. The subcohort including women of the cohort with PTD threat was additionally treated with bLf intravaginal administration. A significant improvement of hematological parameters was observed in the women’s cohort together with a consistent decrease of serum interleukin-6 (IL-6) levels. Combined administration of oral and intravaginal bLf to the women’s subcohort with PTD threat decreased IL-6 in both serum and cervicovaginal fluids, cervicovaginal prostaglandin F2a, and suppressed uterine contractility. BLf administration blocked further shortening of cervical length and the increase of fetal fibronectin thus prolonging the length of pregnancy. The deliveries occurred between the 37th and 38th week of gestation. These results provide strong evidence for a role of bLf in PTD treatment, thus extending the therapeutic potential of this multifunctional natural protein. Key words: lactoferrin, preterm delivery, inflammation, anemia, IL-6. Résumé : L’accouchement est qualifié de prématuré s’il survient avant la 37ième semaine de gestation. L’anémie ferriprive et les processus inflammatoires reliés soit à l’infection ou à une réponse inflammatoire stérile représentent des facteurs de risque d’accouchement prématuré. La lactoferrine bovine (bLf), un régulateur émergeant important de l’homéostasie du fer et inflammatoire, peut constituer une nouvelle approche thérapeutique de traitement de l’accouchement prématuré. Une cohorte a été constituée afin d’évaluer l’effet de l’administration orale de bLf sur l’homéostasie du fer et inflammatoire chez des femmes enceintes souffrant d’anémie. Un sous-groupe comprenant des femmes de la cohorte présentant un risque élevé d’accouchement prématuré a été également traité par une administration intravaginale de bLf. Une amélioration significative des paramètres hématologiques a été observée chez les femmes de la cohorte, parallèlement à une diminution constante des niveaux d’interleukine-6 (IL-6) sérique. L’administration combinée de bLf orale et intravaginale au sous-groupe de femmes présentant un risque élevé d’accouchement prématuré a diminué les niveaux d’IL-6 du sérum et du liquide cervico-vaginal, de prostaglandine F2a cervico-vaginale, et a supprimé la contractilité utérine. L’administration de bLf bloquait de plus le raccourcissement du col utérin et augmentait la fibronectine fétale, prolongeant ainsi la durée de la grossesse. Les accouchements se sont produits entre la 37ième et la 38ième semaine de gestation. Ces résultats apportent la preuve que la bLf peut jouer un rôle dans le traitement de l’accouchement prématuré, ajoutant ainsi au potentiel thérapeutique de cette protéine naturelle multifonctionnelle. Mots‐clés : lactoferrine, accouchement prématuré, inflammation, anémie, IL-6. [Traduit par la Rédaction]

Introduction Preterm delivery (PTD) that occurs of gestation complicates ~10% of all berg et al. 2008). Iron deficiency (ID), iron deficiency flammatory processes either related to

before the 37th week pregnancies (Goldenanemia (IDA), and ininfection or sterile in-

flammatory response represent risk factors for PTD (School 2005; Christiaens et al. 2008; Estrada-Gutierrez et al. 2010; Genc and Ford 2010). Cervical and vaginal infections (Leitich et al. 2003; Estrada-Gutierrez et al. 2010) as well as cervicovaginal sterile inflammations play a key role in PTD (Houben et al. 2009). Conflicting data are reported on the association

Received 15 July 2011. Revision received 23 September 2011. Accepted 28 September 2011. Published at www.nrcresearchpress.com/bcb on 31 January 2012. R. Paesano. Department of Woman Health and Territorial Medicine, Sapienza University of Rome, Via di Grottarossa, 1035-1039, 00189 Rome, Italy; Clinica Fabia Mater, Via Olevano Romano 25, 00171 Rome, Italy. M. Pietropaoli. Microbo Srl, Biotechnology Company, P. zza S. Apollonia 3, 00153 Rome, Italy. F. Berlutti and P. Valenti. Department of Public Health and Infectious Disease, Sapienza University of Rome, p.le A. Moro, 5, 00185 Rome, Italy. Corresponding author: Piera Valenti (e-mail: [email protected]). 1This

article is part of Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Biochem. Cell Biol. 90: 468–475 (2012)

doi:10.1139/O11-060

Published by NRC Research Press


Paesano et al.

between maternal serum interleukin-6 (IL-6) concentration and PTD (Murtha et al. 1998; Goepfert et al. 2001; Sorokin et al. 2010; Wei et al. 2010). Conversely, IL-6 levels in amniotic and cervicovaginal fluids appear strongly associated with PTD thus indicating that the inflammation at the maternalfetal interface, rather than systemic inflammation, plays a major role in PTDs (Wei et al. 2010). IL-6 stimulates PTD through classic and transsignaling pathways (Lee et al. 2011). In the classic pathway, IL-6 binds to a transmembrane cognate receptor, IL-6R, activating Janus kinases that in turn phosphorylate STAT3, a signal transducer and activator of transcription (Heinrich et al. 2003). STAT3 activation and translocation to the nucleus results in a concerted transcriptional increase of genes involved in iron (Wrighting and Andrews 2006) and inflammatory homeostasis (McLoughlin et al. 2005; Wessling-Resnick 2010). IL-6 through the classical signaling pathway stimulates the synthesis of prostaglandin F2a (PGF2a), an important inducer of uterine contractions and premature rupture of membranes (Mitchell et al. 1991). In the transsignaling pathway, IL-6 complexes to a soluble form of IL-6R (sIL-6R), activating dimerization of gp130, a signal-transducing glycoprotein. Soluble gp130 (sgp130), a natural inhibitor of IL-6 transsignaling, prevents the agonistic IL-6–sIL-6R complex from interacting with membrane-bound gp130 (Heinrich et al. 2003). The decreased sgp130 in amniotic fluid is associated with PTD and premature rupture of membranes (Lee et al. 2011). This has important implications as the coordinated interplay of IL-6 and PGF2a regulates delivery both at preterm and at term (Lyon et al. 2010). Prostaglandin synthase inhibitors as well as the anti-inflammatory molecules suppress uterine activity thus prolonging the length of pregnancy (Gotsch et al. 2009). Lactoferrin (Lf), an iron-binding glycoprotein (Baker and Baker 2005) synthesized by exocrine glands and neutrophils, is emerging as an important regulator of iron and inflammatory homeostasis. Oral administration of bovine Lf (bLf) is safe and effective in both decreasing serum IL-6 levels and increasing hematological parameters in pregnant women suffering from ID and IDA (Paesano et al. 2009, 2010a, 2010b). The bLf in vivo anti-inflammatoty efficacy encourages new therapeutical approaches to prevent PTD threat related to inflammatory processes. Here an open-label cohort and subcohort study is reported. The cohort included anemic pregnant women without PTD threat treated with bLf oral administration. The subcohort included women of the cohort with PTD threat additionally treated with intravaginal bLf administration. The bLf combined therapy prevented PTDs. Maternal and fetal side effects were not observed.

Materials and methods Study design We conducted an open-label study in accordance with the ethical principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Clinica Fabia Mater, Via Olevano Romano, 25 Rome, Italy (FM MOD 26/02/2010). All pregnant women gave written informed consent. This open-label study included cohort and subcohort samplings.

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Pregnant women from 20 to 40 years with singleton physiological pregnancies, ID and IDA, and normal uterine cavity, intact membranes were enrolled in the cohort regardless of trimester, and women of the cohort with ascertained PTD threat not related to infections were included in the subcohort. Women suffering from severe anemia (hemoglobin < 7 g/dL) were excluded from the cohort. Women were excluded from the cohort and subcohort if their fetus had identified congenital anomalies and they had recurrent miscarriages, prior preterm birth, previous caesarean section, complicated pregnancies, multiple pregnancy, cervical cerclage, premature rupture of membranes, fetal complications such as intrauterine growth restriction, serious disease such as preeclampsia or hypertension, vaginal bleeding, previous or current cervical and vaginal infections, previous PTD threats, allergies to milk proteins, or were taking topic or systemic antibiotic therapy within 4 weeks prior to the enrollment or other concomitant iron supplementations, or recent blood transfusion(s). Cohort and subcohort samplings Cohort Eligible and consenting women were enrolled regardless of trimester when at least 1 of 4 hematological parameters (red blood cells, hemoglobin, total serum iron, and serum ferritin) indicated ID and IDA and were treated with bLf oral administration. ID and IDA were defined by red blood cells 50 ng/mL), and fetal fibronectin (fFN) (250 ng/mL indicated premature rupture of

Biochem. Cell Biol. Vol. 90, 2012

membranes as well as premature labor risk (Mitchell et al. 1991). fFN concentrations were determined by ELISA (Adeza Biomedical Company, Sunnyvale, Calif.). Within 22 and 36 weeks of gestational age, fFN values 50 ng/mL indicated rupture of membranes (Goepfert et al. 2001). Vaginal infections Vaginal infection was defined by the Gram stain according to the criteria of the Nugent score (Nugent et al. 1991). Vaginal samples were analyzed to detect pH values, the whiff, and the presence of Lactobacillus spp. A vaginal pH corresponding to 4.5, the absence of the whiff, and the predominance of Lactobacillus spp. through microscopic examination indicating a normal flora was expressed as a Nugent score of 0 to 3. Vaginal pH values >4.5, the whiff, and a replacement of lactobacilli with predominantly anaerobic bacteria such as Gardnerella vaginalis, Prevotella spp., Peptostreptoccus spp., and Mobiluncus spp. indicating bacterial vaginosis was expressed as a Nugent score of 7 to 10. Cervical infections Cervical samples were analyzed to screen for Mycoplasma hominis, Chlamydia trachomatis, and Ureaplasma urealyticum. Maternal side effects The safety and compliance detected by monitoring maternal vital sign assessments, adverse events, and clinical laboratory evaluations during scheduled visits were registered in the patient file. The side effects of bLf oral administration were monitored through the following parameters: gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation. In addition, the following clinical laboratory parameters were evaluated every 30 days: hematocrit, glycemia, uricemia, bilirubin, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, cholesterol, triglyceride acid, and electrolytes. The side effects of bLf intravaginal administration were monitored through vaginal irritation, itching, and burning. Fetal and newborn side effects Fetal vital sign assessments were monitored by ultrasonographic measurements of intrauterine growth and through the amount of amniotic fluid, expressed as the amniotic fluid index (AFI). AFI, an index of the fetal well-being, can be influenced by uterine contractions. An AFI of ≤5 cm was considered oligohydramnios, 5–8 cm borderline, and m>8 to 24 cm normal (Petrozella et al. 2011). Newborn weight and Apgar score were registered. Apgar score is a practical method of evaluating the physical condition of a newborn shortly after delivery (Apgar 1953). An Apgar score of 0–3 at 5–10 min of age is predictive of high morbidity and mortality, while an Apgar score of 9–10 means an infant is in the best possible condition. Statistical analysis Calculations for statistical differences on the assayed parameters before and after therapy were carried out using ANOVA. P-values ≤0.0001 were considered significant. Published by NRC Research Press

Paesano et al.


Results Effect of bLf oral administration on pregnant women suffering from ID and IDA Over a study period of 12 months, pregnant women were enrolled at their first visit at Clinica Fabia Mater, regardless of trimester of gestation. When at least 1 of 4 hematological parameters (red blood cells, hemoglobin, total serum iron, and serum ferritin) indicated ID and IDA, the women were included in the cohort and treated with one capsule containing 100 mg of bLf twice a day for at least 4 weeks until delivery. Among 163 enrolled pregnant women, 70 and 93 were at their second and third trimester of gestation, respectively. Two pregnant women at second trimester of pregnancy were lost at scheduled visits. All enrolled women received bLf oral administration for at least 4 weeks. The number of red blood cells and the concentration of hemoglobin, total serum iron, and serum ferritin as well as serum IL-6 levels of 161 pregnant women before the treatment and at the delivery are reported as mean values, regardless of the total weeks of bLf oral administration (Fig. 1). The mean values shown in Fig. 1 also include the hematological values of the pregnant women at PTD risk related to infections (excluded from the subcohort) and not related to infections (included in the subcohort). As shown (Fig. 1), a significant improvement of hematological parameters including red blood cell number, hemoglobin, total serum iron, and serum ferritin concentrations was observed in the women’s cohort orally receiving bLf, together with a consistent decrease in serum IL-6 levels (P = 0.0001). Effect of bLf oral and intravaginal administration on women with PTD threat Among the 161 pregnant women in the cohort receiving bLf oral administration, 18 perceived uterine contractions in the preterm period. As the maternal perception of uterine contractions is scanty evidence about the predictive PTD, the actual risk of PTD was detected through ultrasonographic cervical length and by IL-6, PGF2a, and fFN concentrations in cervicovaginal fluids, the best methods for predicting PTD (Krupa et al. 2006). Cervical and vaginal samples were also analyzed to screen for putative infections. Among 18 women with the perception of PTD threat, 4 were objectively found to not be at risk of PTD as detected by a cervical length >30 mm, by cervicovaginal fluid analysis of fFN 1000 pg/mL, confirmed an infective process as well as the mean values of PGF2a >250 ng/mL were predictive of labor and delivery in very short time (Mitchell et al. 1991). These women were excluded from the subcohort.

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The remaining 11 women showing a cervical length 120 pg/mL, PGF2a >50 ng/mL, and fFN