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α1-Antitrypsin insufficiency is a possible contributor to preterm premature rupture of membranes

Joel Baron1, Eyal Sheiner1, Avishag Abecassis2, Efrat Ashkenazi2, Galit Shahaf2, Shimrit Yaniv Salem1, Tamar Madar1, Guy Twina1, Arnon Wiznitzer1, Gershon Holcberg1 & Eli C. Lewis2

1Department of Obstetrics and Gynecology and 2Department of Clinical Biochemistry, Faculty of Health Sciences, Soroka University Medical Center, Ben Gurion University of the Negev, Beer-Sheva, Israel

The Journal of Maternal-Fetal and Neonatal Medicine

2012

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7

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937

© 2012 Informa UK, Ltd.

10.3109/14767058.2011.600369

1476-7058

1476-4954

The Journal of Maternal-Fetal and Neonatal Medicine, 2012; 25(7): 934–937© 2012 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2011.600369

Correspondence: Joel Baron, Soroka University Medical Center, Beer-Sheva, Israel. Tel: 972-50-460205 Fax: 972-8-6400111 E-mail: baronj@bgu.ac.il

Objective: α1-Antitrypsin (AAT) is protective of tissue damage induced by enzymes of inflammatory cell source. Inflammatory cells are involved in preterm labor, preterm premature rupture of membrane (PPROM) and term premature rupture of membrane (PROM). The purpose of this research was to examine whether plasma concentration and activity of AAT differ between these manifestations. Methods: In a prospective case control study, blood samples were assayed for AAT concentration and activity in 71 individuals. AAT concentration and activity were measured by standard methods. Results: No significant differences were found between AAT levels (p = 0.497) and activity (p = 0.879) in preterm and term labor. AAT levels and activity in PPROM and PROM were not significantly different as well (p = 0.748 and p = 0.880, respectively). While 69 out of 71 patients displayed normal circulating levels of AAT, 2 PPROM patients out of 15 had abnormally low, previously undiagnosed,AAT concentra-tions, and had subsequently developed complications that were absent in the other groups. Conclusions: No statistically signifi-cant differences were demonstrated in the levels of AAT between patients with preterm and term labor, nor between preterm and term PROM. Yet, unexpectedly, patients that had marked AAT deficiency belonged exclusively to the PPROM group.

Keywords: α1-antitrypsin, premature rupture of membranes, preterm labor, inflammation, pregnancy

Introductionα1-Antitrypsin (AAT) in an anti-inflammatory circulating 52-kDa glycoprotein that is primarily produced by the liver, and also produced locally by human amniotic epithelial cells [1,2]. Circulating AAT levels increase throughout pregnancy [3]. Mean AAT concentrations in pregnant and non-pregnant women varies in previous studies. In one study, non-pregnant women had AAT plasma concentration of 3.04 mg/ml, while pregnant individuals had 3.74 mg/ml in the first trimester, 4.06 mg/ml in the second trimester and 4.27 mg/ml in the third trimester [3]. The activity of AAT appears to rise during early pregnancy compared to non-pregnant women [3]. In another study, AAT concentrations varied between 0.93–3.54 mg/ml [4]. Kanayama et al. reported that cases of preterm premature rupture of membranes (PPROM) had a decrease concentration of AAT in their amniotic fluid [5]. The authors suggested that low concentrations of AAT in amni-otic fluid might cause PPROM [5]. In a later study O’Brien et al.

compared amniotic fluid AAT concentration from patients with PPROM to pregnancies with preterm labor (PTL) and found no difference in amniotic fluid AAT concentrations [6]. Decreased AAT activity in human amnion cells during premature rupture of membranes (PROM) and PPROM compared to term labor was also documented [7].

PPROM and PTL are commonly encountered in obstetrics. PROM occurs in approximately 8% of pregnancies [8]. PPROM complicates 3% of all pregnancies and is associated with approxi-mately 30% of preterm deliveries [9,10]. The mechanism of membrane rupture is not fully understood. It has been suggested that activation of catabolic enzymes such as collagenase, matrix metalloproteinases, cathepsin G and elastase contributes to the pathology. These enzymes are elevated at sites of inflammation, such that are predominated by tumor necrosis (TNF)-α, and digest connective tissue in the amniotic membrane; combined with the mechanical forces in the uterus, this process might result in rupture of the membranes [11]. Preterm PROM occurs probably due to the same mechanisms as term PROM, with the distinction of premature activation of these pathways and more advanced inflammation [12].

Preterm delivery is a common obstetric problem. Despite advances in understanding the related mechanisms and risk factors for PTL, it is still an increasing phenomena throughout the world [12]. Uncontrolled inflammation is one of the major contributors to PTL, particularly to early PTL [13,14].

The purpose of this study was to examine whether an associa-tion can be identified between reduced AAT plasma concentra-tions and activity, in PTL, PPROM and PROM.

MethodsA single-center prospective case control study was conducted in a sample of 71 individuals. PROM was defined as rupture of membranes at least one hour prior to the onset of labor at or beyond 37 weeks of gestation. PPROM was defined as PROM prior to 37 weeks of gestation. Term labor was defined as labor after completed 37 weeks gestation. PTL was defined as labor before 37 weeks gestation. Inclusion criteria consisted of preg-nant women within 24–41 weeks of gestation with either normal pregnancy, PROM, PPROM or preterm delivery. To differentiate PROM and PPROM from patients that had a simultaneous labor, only patients that had a cervical dilation of less than 2 cm on admission were included in the PROM and PPROM groups. Labor was defined as cervical dilation of 3 cm or more with regular

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contractions. Rupture of membrane was defined by history of broken water, proven by vaginal pooling and positive nitrazine testing. Ultrasound examination was a routine part of the assess-ment including amniotic fluid index. Exclusion criteria consisted of multiple gestations, as well as admittance with hypertensive disorders, diabetes mellitus or fever >38°C. Blood samples were collected from four groups of patients; 16 with term labor, 19 with PTL, 15 with PPROM and 21 patients with PROM. Samples were drawn at the delivery room upon admission, allowed to clot and then centrifuged for 10 min at 5000 RPM at 4°C. Two milliliters of serum were then transferred in aliquots to 0.5 ml tubes and frozen at −80°C until assayed.

AAT levels and activity were both determined. Several instances exhibit an inactive modification of circulating AAT, as in gestational diabetes and juvenile diabetes [15,16]. Such variations are present as normal circulating levels, but func-tionally inactive AAT. AAT levels were determined by standard human AAT ELISA according to the manufacturer’s instructions (Immunology Consultants Laboratories, OR, USA). Elastase inhibition was determined by kinetic assay according to manufac-turer recommendations (Sigma-Aldrich, Rehovot, Israel). Assays were performed in duplicates and repeated twice for each sample in separate sessions. Statistical analysis was performed using the Mann–Whitney U-test to evaluate the differences between groups in AAT concentration and activity. Results are presented as mean±SD.

ResultsGeneral obstetric characteristics, including maternal age, parity and gestational age in the 4 groups are presented in Table I. The mean gestational age in term and PTL was 39.5 ± 1.35 and 33 ± 3.2 weeks, respectively. The mean gestational age in term and preterm PROM was 39.2 ± 1.1and 33.5 ± 2.6 weeks, respectively.

As shown in Figure 1, no significant difference was found in circulating AAT concentrations between preterm and term labor (mean 1.02 ± 0.21 mg/ml vs. 1.059 ± 0.44 mg/ml; p = 0.497). Accordingly, as shown in Figure 2, there was no significant difference in AAT activity between preterm and term labor (0.118 ± 0.036 vs. 0.116 ± 0.04 relative anti-elastase activity; p = 0.879). Comparing preterm and term PROM (Figure 3), no significant differences in AAT concentrations were observed (1.048 ± 0.38 mg/ml vs. 1.054 ± 0.32 mg/ml; p = 0.748). AAT activity (Figure 4) was comparable between preterm and term PROM (0.1156 ± 0.02 vs. 0.1506 ± 0.123 relative anti-elastase activity; p = 0.880).

Two of the 15 PPROM patients had abnormally low AAT levels and activity, while the entire PROM group, as well as the two remaining groups, had normal readouts. The low circulating concentrations of AAT in these 2 women correlated with reduced AAT activity (Figure 5, points a and b). Interestingly, patient a had pregnancy with an intra-uterine contraceptive device, and patient b developed chorioamnionitis with neonatal sepsis.

DiscussionSince inflammation is involved in PTL and PROM, we hypoth-esized that the naturally-occurring anti-inflammatory circulating protein, AAT, could negatively correlate with these parturients. Because of the possibility that alterations in AAT may be reflected not only in its concentrations but also in its activity, changes in AAT anti-elastase function were evaluated as well.

Nevertheless, this study did not demonstrate significant differences in AAT concentration and activity between patients with preterm and term labor, nor between preterm and term PROM.

Interestingly, our results have shown that AAT concentrations were lower than the levels reported previously, albeit within the normal range for adults [3]. The differences might reflect different populations or, conversely, different laboratory measurement techniques. Furthermore, the reason for the elevated plasma AAT concentration that were previously documented [3] could be due to the fact that it was not measured during actual labor or PROM; both processes might involve inflammatory processes that may lead towards a reduction in detectable free-form AAT. In addi-tion, AAT concentrations in pregnancy have a large variation [3,4]. To the best of our knowledge, there are no current reports that directly address plasma AAT levels in PPROM and PTL.

The study has several weaknesses, primarily due to its relatively small sample size. Also, we could not measure intra-amniotic AAT because amniocentesis was not performed in study patients. The strengths of this prospective study include the extension of parameters to include AAT activity at term and PTL, as well as in PROM and preterm PROM. Also, the 2 patients with low AAT concentrations and activity in the PPROM group deserve further attention since lower levels and activity of AAT may reflect greater protease activity and an elevated risk of inflammation in the amnion fluid, facilitating preterm PROM or chorioamnionitis [7]. Our findings suggest that further studies should investigate a possible causative relationship between AAT deficiency and the occurrence of PPROM. If such an association were to be found, it could lead to future prevention of PPROM in selected patients by screening for AAT levels, and by subsequent AAT augmentation therapy as readily available for genetic AAT deficiency [17].

In conclusion, although we could not demonstrate a signifi-cant difference between patients with preterm and term labor, or between preterm and term PROM as far as circulating AAT levels and activity are concerned, it is most notable that two individuals that had AAT deficiency were detected in our study and belonged to the 15-patient PPROM group. Considering that the frequency of AAT deficiency in the population is less than 1:5000 [18] and that AAT deficiency is currently largely under-diagnosed, these findings warrant thorough investigation as to whether AAT defi-ciency is undetected in pregnant women and holds risk for related complications. Thus, further large scale prospective studies should focus on the association between AAT and PPROM. Such an association might lead to future prevention of PPROM in selected patients by administration of AAT accordingly.

Table I. General obstetric characteristics of the study population. Term labor Preterm labor PROM PPROMMaternal age (years) (mean ± SD) 24.4 ± 4.9 26.7 ± 5.8 28.2 ± 5.3 28.4 ± 3.8Parity 1 8 9 10 82–5 6 8 9 66+ 2 2 2 1Gestational age (weeks) (mean ± SD) 39.5 ± 1.35 33 ± 3.2 39.2 ± 1.1 33.5 ± 2.6PROM, premature rupture of membrane; PPROM, preterm premature rupture of membrane; SD, standard deviation.

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Figure 1. A1AT concentration in term and preterm labor.Figure 2. A1AT activity in term and preterm labor.

Figure 3. A1AT concentration in term and preterm rupture of membranes.

Figure 5. A1AT concentration and activity in term labor, preterm labor, PROM and PPROM. Point a represents a patient that has had pregnancy with an intra-uterine contraceptive device. Point b represents a patient that has developed chorioamnionitis with neonatal sepsis.

Figure 4. A1AT activity in term and preterm rupture of membranes.

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Declaration of Interest: The authors report no conflicts of interest.

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