**2.1. Sample population**

**1. Introduction**

2 Electrophoresis - Life Sciences Practical Applications

(sPTL-IM)) [3].

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

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

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

interleukin 1 receptor antagonist had shown to be relatively poor [4, 5].

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**.

### **2.2. Reagents**

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 with attached fetal membranes and umbilical cords were collected within 10 min of delivery and thoroughly washed in ice-cold PBS to remove the blood. Placenta tissues were collected from six different locations from the side facing the basal plate (maternal side) as well as the side facing the chorionic plate (fetal side). Relative consistency was maintained during collection by selecting six closest locations between all collected placentas; a grid indicated with six locations was overlaid as a guide. Moreover, the site with calcium deposits, visible connective tissue, and blood clots were avoided. The chorion with adherent decidua was separated from the amnion using sterile forceps. All collected tissues were snapped frozen in

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

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

5

Based on the histology evidence by a trained histologist, no evidence of histological infection

For each individual sample, a total of 500 mg placenta tissues were randomly chosen and pulverized in a mortar with pestle containing liquid nitrogen. Powdered placenta tissues were subjected to protein extraction using DNase/lithium chloride-dense sucrose, and then cleaned up with dichloromethane-methanol precipitation as described previously by Tan et al. [13].

The total placentas protein concentration of the purified placentas tissue lysates from both case and control (maternal and fetal sides) were quantified by 2D Quant kit (GE Healthcare) according to the manufacturer's instruction. Bovine serum albumin was used as a standard

The 2D-placenta proteome of the fetal and maternal sides of sPTL-IM was compared to their term controls, respectively. The purified tissue lysates from both case and control placentas (maternal and fetal sides) were diluted in a rehydration buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% biolyte 3–10, 20 mM DTT, 40 mM Tris, 0.002% (w/v) bromophenol blue to a final concentration of 266 μg, and a total volume of 250 μL. The protein samples were passively rehydrated at room temperature onto the 13 cm, pH 3–10 nonlinear IPG strips (GE Healthcare) for 16 h. Strips were overlaid with a dry strip cover fluid and focused using IPGphor II IEF system (Ettan IPGphor 3 IEF, GE Healthcare, USA) at 20°C and 50 μA/strip of maximum current limit using the following program: step and hold at 500 V for 500 Vh, gradient at 1000 V

for 750 Vh, gradient at 8000 V for 11,250 Vh, and step and hold at 8000 V for 7333 Vh.

After completion of the first dimension IEF, IPG strips were equilibrated for 15 min at room temperature in a buffer containing 6 M urea, 50 mM Tris/HCl pH 8.8, 40% (v/v) glycerol, 2% (w/v) SDS, 0.02% bromophenol blue, and 65 mM DTT, followed by a 15-min incubation at

**2.4. Protein extraction by DNase/lithium chloride-dense sucrose homogenization-**

These purified protein samples were stored at −80°C until 2D-GE was performed.

liquid nitrogen and stored at −80°C until protein extraction.

in placenta parenchyma was reported in all samples.

**coupled dichloromethane-methanol precipitation**

throughout all the independent experiments.

**2.5. Protein quantification**

**2.6. 2D gel electrophoresis**

NS, not significant; data are presented as means ± SEM, except parity data are presented as median (range).\* *P* < 0.05 when compared to the other group.

**Table 1.** Demographic and clinical details of women participated in this study.

Healthcare, USA, 17-1314-01), Tris (Bio-Rad, USA, 1610716), biolyte 3/10 ampholyte (Bio-Rad, USA, 1632094), dithiothreitol (DTT, GE Healthcare, USA, 17-1318-02), hydrochloric acid solution (HCl, Fisher Chemical, UK, SA48-500), ethylenediaminetetraacetic acid (EDTA, Sigma, USA, E9884), potassium chloride (KCl, Sigma-Aldrich, USA, P9541), sucrose (Fisher Reagent, USA, S25590), methanol (Fisher Reagent, USA, A4521), dichloromethane (DCM, Fisher Reagent, USA, AC326600025), lithium chloride (LiCl, Sigma-Aldrich, USA, 13013), 2D quant kit (GE Healthcare, USA, 80-6483-56), glycerol (Sigma-Aldrich, USA, G5516), sodium dodecyl sulfate (SDS, Bio-Rad, USA, 1610302), bromophenol blue (Bio-Rad, USA, 1610404), pre-stained BLUeye (Gene Direx, USA, PM007-0500), acrylamide/bis mixed solution (37.5:1) (Nacalai tecque, Japan, 06121), wide-range gel preparation buffer (Nacalai tecque, Japan, 07831-94), N,N,N′,N′-tetramethylethylenediamine (TEMED, GE Healthcare, USA, 17-1312-01), ammonium persulfate (APS, Sigma-Aldrich, USA, 09913), agarose NA (GE Healthcare, USA, 17-0554- 01), glycine (Bio-rad, USA, 1610718), DNase I (New England Biolabs, UK, M0303 L), immobiline dry strip pH 3-10 non-linear, 13 cm (GE Healthcare, USA, 17-6001-15), dry strip cover fluid (GE Healthcare, USA, 17-1335-01), iodoacetamide (IAA, Bio-Rad, USA, 1632109), formaldehyde solution (Sigma-Aldrich, USA, F8775), silver nitrate (Sigma-Aldrich, USA, 209139), sodium carbonate (Sigma-Aldrich, USA, S7795), sodium thiosulfate pentahydrate (Sigma-Aldrich, USA, 217247), glacial acetic acid (Sigma-Aldrich, USA, 320099), ethanol (EtOH, Fisher Chemical, USA, A955-4), phosphoric acid (Fisher Chemical, USA, A260-500), ammonium sulfate (Fisher Chemical, USA, A702-500), Coomassie brilliant blue G-250 (Sigma, UK, 27815), acetonitrile (Fisher Chemical, UK, A998-212), acetic acid (Fisher Chemical, USA, A35-500), ammonium hydrogen citrate (Merck, UK, 101154), trifluoroacetic acid (TFA, Sigma-Aldrich, UK, 302031), and α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, UK, C8982).

Milli-q water (Milli-Q Integral 3 water purification system, Merck Millipore, USA) was used in preparing all the chemical solutions required.

#### **2.3. Tissue collection, processing, and storage**

To improve the homogeneity and comparability, placentas selected in this study were collected from pregnant women who were experiencing sPTL-IM or sTL-IM with prior history of spontaneous PTL-IM among Malay ethnicity only.

Placentas with attached fetal membranes and umbilical cords were collected within 10 min of delivery and thoroughly washed in ice-cold PBS to remove the blood. Placenta tissues were collected from six different locations from the side facing the basal plate (maternal side) as well as the side facing the chorionic plate (fetal side). Relative consistency was maintained during collection by selecting six closest locations between all collected placentas; a grid indicated with six locations was overlaid as a guide. Moreover, the site with calcium deposits, visible connective tissue, and blood clots were avoided. The chorion with adherent decidua was separated from the amnion using sterile forceps. All collected tissues were snapped frozen in liquid nitrogen and stored at −80°C until protein extraction.

Based on the histology evidence by a trained histologist, no evidence of histological infection in placenta parenchyma was reported in all samples.
