**1. Introduction**

Spontaneous preterm labor (PTL) is one of the major causes of preterm birth (PTB), which accounts for 56% of the total PTB [1] and it possesses deteriorating effects, both long and short term, on the health of the mother and the offspring [2]. Moreover, statistics showed that 30% of pregnant women who experienced spontaneous PTL are often spontaneous with no known signs of PTL (herein termed as spontaneous unexplained PTL with intact membrane (sPTL-IM)) [3].

pathophysiological complications. For this, we have applied an existing proteomic approach (two dimensional-gel electrophoresis (2D-GE)-coupled matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS)) to identify differentially expressed protein during sPTL-IM by comparing the fetal and maternal placenta proteome between the sPTL-IM and spontaneous term labor with intact membrane (sTL-IM)

Spontaneous Unexplained Preterm Labor with Intact Membrane: Finding Protein Biomarkers…

http://dx.doi.org/10.5772/intechopen.74925

3

The study was approved by the Medical Research and Ethics Committee (NMRR-13-1660- 18742) and written informed consent was obtained from all subjects prior to placenta collection. Placenta was taken from five asymptomatic pregnant women who experienced sPTL-IM (22 and 36 + 6 weeks of gestation). The control group comprised pregnant women who underwent spontaneous labor with ≥37 completed weeks of gestation (*n* = 10) and had at least one previous history of spontaneous PTB. Gestational age was determined from the last menstrual period and by ultrasound measurements up to the second trimester. Another ultrasound measurement was performed during the second trimester to rule out congenital malformations. All subjects were patients who sought for prenatal care and delivered in Hospital Serdang, Malaysia, during 2014–2015 and represented the Malay population in Peninsula Malaysia who carried a singleton pregnancy. Risk factor was at least one prior spontaneous PTB of a singleton infant due to spontaneous PTL-IM (>20 to <37 weeks of gestation). Pregnant women with known obstetrical complications such as multiple gestations, chronic infective diseases, hypertension, preeclampsia, pregestational diabetes, severe anemia, placenta previa, known malignancy, known or suspected congenital malformation of the fetus, symptoms/ signs of PTL, fetal loss in second trimester, intrauterine growth restriction (IUGR), had an immunodeficiency virus, cervical incompetence, planned cervical cerclage, uterine malformation, antepartum hemorrhage, and previous cervical surgery were excluded from the investigation. Subjects with uncertain gestation were also excluded from this study. Moreover, obstetric complications needing iatrogenic and preterm prerupture of membranes (PPROM) deliveries were also not recruited in the study. It is believed that PPROM did not share the same condition as spontaneous PTL [12]. Each patient was given an anonymized ID number. Demographic and clinical characteristics of these pregnant women from whom preterm and

term samples utilized in this study were collected and are presented in **Table 1**.

The chemicals used in this study include phosphate buffer saline (PBS, Sigma-Aldrich, USA, P4417), protease inhibitor mix (GE Healthcare, USA, 80-6501-23), β-mercaptoethanol (Invitrogen, US, 11528926), urea (GE Healthcare, USA, 17-1319-01), thiourea (GE Healthcare, USA, RPN6301), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, GE

placentas, respectively, prior to the study of their functions.

**2. Materials and methods**

**2.1. Sample population**

**2.2. Reagents**

Current diagnosis for spontaneous PTL-IM was based on several predictors which include risk factors assessment, cervical measurement, and/or biochemical markers specifically fetal fibronectin and phosphorylated insulin-like growth factor-binding protein 1 [4]. These protein biomarkers possess a major limitation where they can only be detected 14 days prior to onset, providing a short time frame to plan for preventive care and treatment [5]. On the other hand, positive-predictive values of other newly found biomarkers including thioredoxin or interleukin 1 receptor antagonist had shown to be relatively poor [4, 5].

It has become evident that existing and newly found protein biomarkers often lack specificity and sensitivity to provide an effective prediction measure against sPTL-IM among pregnant women [6, 7]. Specific and sensitive predictive tool is important to allow the effective use of treatment or care for the risked mothers and avoid the use of unnecessary medical interventions, which further reduces medical costs. Furthermore, throughout every stages of pregnancy from conception to parturition, it is believed that levels of certain protein biomarkers fluctuate. Hence, the identification of these proteins that are associated with sPTL-IM is a sensational method to establish reliable novel biomarkers in assisting sPTL-IM prediction among pregnant women, as well as unwinding its underlying complex pathophysiologies [6, 7].

Since results from easily accessible biological fluids do not always reflect accurate localized information on the state of a pregnancy and impending labor, the use of gestational tissues can be advantageous. The placenta is an organ connecting developing fetus with the mother that plays roles during labor [8]. Interestingly, the study reported that protein expression in the fetally origin tissues (chorion) is different from the maternally origin tissues (decidua basalis) [9]. Thus, both the fetal and the maternal side of the placenta are a potential source of preliminary discovery of biomarker associated to the processes of sPTL-IM. Unfortunately, studies that applied proteomic approaches to discover protein alterations in the placenta mainly focused on pregnancies complicated with gestational diabetes mellitus and preeclampsia [10, 11] while little is known about the proteome from placenta in relation to sPTL-IM. Therefore, investigating protein levels in delivered placentas offers researchers not only to identify potential biomarker panels but also to study the delivery pathophysiology of sPTL-IM.

Given the clinical significance of sPTL-IM and the presence of proteomics technology, placenta has become a good source for proteome study associated with sPTL-IM. The purpose of this research was to discover protein biomarkers that could potentially be used to identify the risk of sPTL-IM among pregnant women. In addition, this will also allow us to understand any functional classes of proteins that are associated with sPTL-IM and disclose its pathophysiological complications. For this, we have applied an existing proteomic approach (two dimensional-gel electrophoresis (2D-GE)-coupled matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS)) to identify differentially expressed protein during sPTL-IM by comparing the fetal and maternal placenta proteome between the sPTL-IM and spontaneous term labor with intact membrane (sTL-IM) placentas, respectively, prior to the study of their functions.
