**4. Discussion**

Current understanding of placenta association with sPTL-IM leading to PTB is strikingly limited, which further limits our ability to accurately predict the occurrence of this event. Despite great strides in neonatal care, infant survival rates remained low partly due to sPTL-IM nonetheless. This imparts a significant risk of various childhood diseases/disorders among premature infants. Thus, placenta proteome analyses of sPTL-IM allowed the identification of potential biomarkers and the exploration of the underlying molecular pathophysiology related to placenta insufficiency leading to this syndrome, where the study employing placenta proteome on this syndrome is still limited. As such, this report is designed to demonstrate the feasibility of using placenta proteome to study its association to sPTL-IM while making the initial findings widely available. In essence, we have identified 12 potentially important protein differences in the placenta of sPTL-IM.

Vimentin was presumed to play a vital role in anchoring and positioning organelles in the cytosol, maintenance of cell integrity, and stabilization of cytoskeletal interactions [22, 23]. Downregulation of vimentin was observed in the obese placenta as well as in human-aborted placenta tissues [24, 25]. A deficiency of this protein could result in the dramatic reorganization of vimentin filaments in the cytoplasm, leading to morphological changes and cellular fragmentation and ultimately destabilizes the overall cytoplasm architecture which might lead to PTL [23]. Meanwhile, the remaining half of the identified proteins that fell into this functional group includes cytoskeletal and cytoskeleton-associated proteins namely Tmod-3, septin 2, and CAPNS1 (the fetal side of placenta tissues) (**Figure 2**). Studies also demonstrated that pregnancy maintenance is regulated by various cytoskeletal and cytoskeleton-associated proteins [26, 27]. Tmod-3 is one of the components of membrane-associated tropomyosin-filamentous actin (TM-F-actin) networks, which is responsible for the polarization of plasma membranes of epithelial cell and sarcoplasmic reticulum membranes of skeletal muscle [28, 29]. This protein is also involved in endothelial cell migration, erythroblast enucleation and erythroblast-macrophage adhesion in erythropoiesis, and insulin-stimulated GLUT4 trafficking in adipocytes [30–32]. Moreover, Tmod-mediated stabilization of TM-F-actin is critical for membrane morphology, mechanics, and physiology [29]. It is thus conceivable that alteration in Tmod-3

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levels may contribute to the deregulation of actin filaments of the placenta.

vesicle fusion processes [33, 34].

cardiac defects and hemorrhages [35].

fetal side of the placenta.

Septin is known to play roles in vesicle trafficking, cytoskeletal remodeling, and apoptosis through the organization of actin-myosin contractile ring [33]. It regulates ring contraction or the attachment of actin-myosin contractile ring to the plasma membrane [34]. Septin was also proposed to have additional functions in mammals such as cell surface organization and

CAPNS1 is a regulatory subunit that is involved in calpain heterodimers (calpain-1 and calpain-2) stabilization and operation. CAPNS1 proteolytic activities had been reported to involve in various cellular functions including apoptosis, proliferation, migration, adhesion, and autophagy. *Capns1* deficiency was reported to be the factor of calpain-1 and -2 zero expression that resulted in embryonic lethality around embryonic day 10.5 due to possible

Collectively, alterations in the expression of proteins involved in the intermediate filaments and cytoskeletal network can lead to significant reorganization of the placenta cytoskeletal, as well as defects in placenta endothelial cell migration and tubule formation. Such cytoskeleton rearrangement and defects in endothelial cell signals transduction and function may be contributing factors to the characteristic disorganization of myofiber in placentas, where tissue structural capacity for stretch was altered, leading to sPTL-IM. This cause-and-effect interpretation is consistent with the higher incidence of PTL during multiple versus single gestations [36]. One possible justification for more proteins from this functional group was dysregulated in fetal compared to maternal which could be that the fetal involved significantly in the process of cytoskeleton reorganization. This observation is consistent with the findings by Tonami et al. [37], which have shown that microtuble-stabilizing protein is highly expressed in the

It is possible that limitations exist in the 2D-GE technology. This is due to the presence of proteins that are extremely acidic and basic, hydrophobic, large molecular weights, and low copy numbers [16]. Furthermore, limitations were observed when identifying and quantifying proteins as the protein spots need to be excised out from the 2D gels and digested with proteases.

The differentially expressed proteins identified from sPTL-IM can be generally classified into four functional classes, which were (1) structural/cytoskeletal components, (2) proteins involved in immune response, (3) proteins with fetal and placenta development properties, and (4) proteins involved in anticoagulation cascade.

### **4.1. Structural or cytoskeletal components**

A majority of the identified proteins were proteinaceous components of structural or cytoskeletons (**Figure 2**) which is not surprising since the cytoskeletal proteins are known as major regulators of the endothelial function and signaling processes [17]. A total of six proteins were grouped under this function. Half of the proteins that fall into this class were intermediate filaments namely cytokeratin 8, cytokeratin 1, and vimentin (**Figure 2**). Cytokeratin 8 was found to be differentially expressed in both the fetal and maternal sides from the sPTL-IM placenta while the expression of cytokeratin 1 and vimentin was observed only at the fetal side. Commonly, keratin types I and II copolymerize to form intermediate filaments in epithelia [18], and vimentin form type III filaments in endothelial cells [19]. As the placenta is a highly vascularized system of epithelial membranes, the presence of these components is expected.

Dysregulation of keratin expression has been reported in many pregnancy disorders. Studies had showed that dysregulation of keratins 7, 8, 18, and 19 expressions was associated with pregnancy disorder, with the results contradicting each other about up- or downregulation of these keratins [20, 21]. The reason for this discrepancy is still not certain, and it may be explained by the severity of cases, unhealthy women might be from different backgrounds, or there is a more complex effect of this pregnancy complication on placenta intermediate filaments. Most importantly, changes in keratin expression level might influence cell architectural properties such as cell polarity and shape.

Vimentin was presumed to play a vital role in anchoring and positioning organelles in the cytosol, maintenance of cell integrity, and stabilization of cytoskeletal interactions [22, 23]. Downregulation of vimentin was observed in the obese placenta as well as in human-aborted placenta tissues [24, 25]. A deficiency of this protein could result in the dramatic reorganization of vimentin filaments in the cytoplasm, leading to morphological changes and cellular fragmentation and ultimately destabilizes the overall cytoplasm architecture which might lead to PTL [23].

**4. Discussion**

12 Electrophoresis - Life Sciences Practical Applications

Current understanding of placenta association with sPTL-IM leading to PTB is strikingly limited, which further limits our ability to accurately predict the occurrence of this event. Despite great strides in neonatal care, infant survival rates remained low partly due to sPTL-IM nonetheless. This imparts a significant risk of various childhood diseases/disorders among premature infants. Thus, placenta proteome analyses of sPTL-IM allowed the identification of potential biomarkers and the exploration of the underlying molecular pathophysiology related to placenta insufficiency leading to this syndrome, where the study employing placenta proteome on this syndrome is still limited. As such, this report is designed to demonstrate the feasibility of using placenta proteome to study its association to sPTL-IM while making the initial findings widely available. In essence, we have identified 12 potentially

It is possible that limitations exist in the 2D-GE technology. This is due to the presence of proteins that are extremely acidic and basic, hydrophobic, large molecular weights, and low copy numbers [16]. Furthermore, limitations were observed when identifying and quantifying proteins as the protein spots need to be excised out from the 2D gels and digested with proteases.

The differentially expressed proteins identified from sPTL-IM can be generally classified into four functional classes, which were (1) structural/cytoskeletal components, (2) proteins involved in immune response, (3) proteins with fetal and placenta development properties,

A majority of the identified proteins were proteinaceous components of structural or cytoskeletons (**Figure 2**) which is not surprising since the cytoskeletal proteins are known as major regulators of the endothelial function and signaling processes [17]. A total of six proteins were grouped under this function. Half of the proteins that fall into this class were intermediate filaments namely cytokeratin 8, cytokeratin 1, and vimentin (**Figure 2**). Cytokeratin 8 was found to be differentially expressed in both the fetal and maternal sides from the sPTL-IM placenta while the expression of cytokeratin 1 and vimentin was observed only at the fetal side. Commonly, keratin types I and II copolymerize to form intermediate filaments in epithelia [18], and vimentin form type III filaments in endothelial cells [19]. As the placenta is a highly vascularized system of epithelial membranes, the presence of these components is expected. Dysregulation of keratin expression has been reported in many pregnancy disorders. Studies had showed that dysregulation of keratins 7, 8, 18, and 19 expressions was associated with pregnancy disorder, with the results contradicting each other about up- or downregulation of these keratins [20, 21]. The reason for this discrepancy is still not certain, and it may be explained by the severity of cases, unhealthy women might be from different backgrounds, or there is a more complex effect of this pregnancy complication on placenta intermediate filaments. Most importantly, changes in keratin expression level might influence cell architec-

important protein differences in the placenta of sPTL-IM.

and (4) proteins involved in anticoagulation cascade.

**4.1. Structural or cytoskeletal components**

tural properties such as cell polarity and shape.

Meanwhile, the remaining half of the identified proteins that fell into this functional group includes cytoskeletal and cytoskeleton-associated proteins namely Tmod-3, septin 2, and CAPNS1 (the fetal side of placenta tissues) (**Figure 2**). Studies also demonstrated that pregnancy maintenance is regulated by various cytoskeletal and cytoskeleton-associated proteins [26, 27].

Tmod-3 is one of the components of membrane-associated tropomyosin-filamentous actin (TM-F-actin) networks, which is responsible for the polarization of plasma membranes of epithelial cell and sarcoplasmic reticulum membranes of skeletal muscle [28, 29]. This protein is also involved in endothelial cell migration, erythroblast enucleation and erythroblast-macrophage adhesion in erythropoiesis, and insulin-stimulated GLUT4 trafficking in adipocytes [30–32]. Moreover, Tmod-mediated stabilization of TM-F-actin is critical for membrane morphology, mechanics, and physiology [29]. It is thus conceivable that alteration in Tmod-3 levels may contribute to the deregulation of actin filaments of the placenta.

Septin is known to play roles in vesicle trafficking, cytoskeletal remodeling, and apoptosis through the organization of actin-myosin contractile ring [33]. It regulates ring contraction or the attachment of actin-myosin contractile ring to the plasma membrane [34]. Septin was also proposed to have additional functions in mammals such as cell surface organization and vesicle fusion processes [33, 34].

CAPNS1 is a regulatory subunit that is involved in calpain heterodimers (calpain-1 and calpain-2) stabilization and operation. CAPNS1 proteolytic activities had been reported to involve in various cellular functions including apoptosis, proliferation, migration, adhesion, and autophagy. *Capns1* deficiency was reported to be the factor of calpain-1 and -2 zero expression that resulted in embryonic lethality around embryonic day 10.5 due to possible cardiac defects and hemorrhages [35].

Collectively, alterations in the expression of proteins involved in the intermediate filaments and cytoskeletal network can lead to significant reorganization of the placenta cytoskeletal, as well as defects in placenta endothelial cell migration and tubule formation. Such cytoskeleton rearrangement and defects in endothelial cell signals transduction and function may be contributing factors to the characteristic disorganization of myofiber in placentas, where tissue structural capacity for stretch was altered, leading to sPTL-IM. This cause-and-effect interpretation is consistent with the higher incidence of PTL during multiple versus single gestations [36].

One possible justification for more proteins from this functional group was dysregulated in fetal compared to maternal which could be that the fetal involved significantly in the process of cytoskeleton reorganization. This observation is consistent with the findings by Tonami et al. [37], which have shown that microtuble-stabilizing protein is highly expressed in the fetal side of the placenta.

## **4.2. Proteins involved in immune response**

Immunomodulatory proteins were also identified in placenta tissues from pregnant women with sPTL-IM. These include S100-A9, IGKC (fetal side of placenta tissues), and ANXA1 (maternal side of placenta tissues) (**Figure 1C**). Inflammation has been attributed to the occurrence of PTL in human [38]. This is possible because the presence of inflammatory cytokines cause cervical ripening and the maturation of the chorioamnion, leading to labor [38]. In addition, Pereira et al. also observed the presence of calgranulins and annexins in cervical-vaginal fluid from patients with spontaneous PTL [39].

Ferritin is a multimeric protein that consisted of two subunits: ferritin heavy chain (FTH) and FTL, which share about 50% sequence homology and is mainly found in the villous stroma of the placenta [55]. FTL has been reported to play a critical role in iron storage as well as regulation of intracellular iron homeostasis and inflammatory response [56, 57]. Mutation in the *FTL* has also been demonstrated to cause neuroferritinopathy and neurodegeneration [58, 59]. On the other hand, anti-inflammatory activities were observed from FTL overexpression where Fan et al. [56] reported inhibition on lipopolysaccharide (LPS)-induced transcription of anti-inflammatory factor at the feto-maternal interface of the placenta leading to PTL [60]. Therefore, it is possible that the production of inflammatory mediators induced by LPS at feto-maternal interface of the placenta can contribute to FTL degradation and synergized to increase intracellular free iron. This leads to increased oxidative stress. Furthermore, elevated placenta oxidative stress was thought to play a key role in stimulating the release of factors,

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

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Only one identified protein was classified in this group. Protein PP4-X (also known as annexin IV (ANX4)) was downregulated in sPTL-IM placentas when compared to the control placentas at the fetal side (**Figure 1C**). This protein has been known to be involved in multiple cellular functions including endocytosis, exocytosis, ion fluxes, and apoptosis [62, 63]. This protein is also one of the important components of biological cascades regulating coagulation which makes it an important factor for the maintenance of pregnancy [63]. Placenta structural abnormality and dysfunction in IUGR fetuses were reported to be caused by reduction in two members of annexins (ANX4 and ANX8) [64]. Furthermore, coagulative disorder and antiphospholipid antibody syndrome have been associated with an increased incidence of pregnancy complications, including PTL [65]. Several cases of increased polymorphism rate in genes related to coagulation have also been associated with PTB [66]. Therefore, it is conceivable that alterations in ANX4 levels may contribute to a failure of placenta anticoagulation

Interestingly, more proteins were differentially expressed in the fetal side than in the maternal side of the placenta tissues obtained from pregnant women who experienced sPTL-IM (**Tables 2** and **3**); this interpretation is consistent with the signals from the fetus where they are capable of initiating the onset of parturition process [67]. The work by Reinl and England [67] demonstrated a possible interplay between placenta corticotrophin-releasing hormone and fetal surfactant protein-A that drives parturition in human. Moreover, another study indicated that nuclear receptor coactivators 1 and 2 regulate the transcription of surfactant

The underlying mechanisms leading to sPTL-IM are not fully understood. This leads to limited prediction and prevention of sPTL-IM due to its complex pathophysiological process. Placenta was an excellent biological tissue that might facilitate the discovery of potential biomarkers as well as to assess dysfunction in the framework of sPTL-IM. Moreover, sPTL-IM

and structural abnormality, leading to impaired utero placenta development.

further leading to maternal endothelium activation [61].

protein-A and consequently the induction of labor [68].

**5. Conclusion**

**4.4. Proteins involved in anticoagulation cascade**

S100-A9 (also known as calgranulin B) is a calcium-binding protein that regulates inflammatory and immune responses which function extracellularly as an antimicrobial agent [40, 41]. Various studies have reported significantly elevated levels of calgranulins A and B in amniotic fluid from intra-amniotic-infected women [41], as well as in vaginal fluid from 86% of patients who were presented with PTL without obvious evidence of genital tract infection or inflammation [42]. Women in labor secondary to intra-amniotic inflammation have also been found to have increased *S100-A8* and *S100-A9* in amniotic fluid and maternal blood [41, 43]. Moreover, calgranulin B can interact directly with β2 integrins in forming "activated receptor" epitopes and plays a role in cytoskeletal reorganization [44].

IGKC functions as an antigen-binding, immune regulation, and disposal of immune complexes [45]. Several lines of evidence suggested that during late pregnancy, recruitment of circulating maternal innate and adaptive leukocytes into the cervix and/or myometrium, and then to the decidua interface via chemotactic cascades induces a pro-inflammatory state which leads to labor [46].

ANXA1 is a calcium-dependent phospholipid-binding protein that is involved in cell proliferation, differentiation, mediating inflammation, apoptosis, and maintenance of pregnancy [42, 47, 48]. It has been reported that *ANXA1* deficiency leads to changes in the inflammatory response and the anti-inflammatory effects of glucocorticoids [48]. Moreover, anti-inflammatory effects by the potent inhibition of neutrophil trafficking were observed from mice that were administered with ANXA1 [49]. A decrease of ANXA1 expression can result in the liberation of phospholipase A2 activity, making more arachidonic acid (precursor of prostaglandins) available for prostaglandin generation [50], which is crucial in uterine contraction and cervical ripening.

Therefore, it remains possible that the premature activation of this inflammatory pathway can lead to a breakdown of feto-maternal tolerance and play a role in the induction of labor, which subsequently can result in spontaneous PTL.

### **4.3. Proteins with fetal and placenta development**

Identified proteins that are associated to this function were Prdx 3 and FTL where both were identified from the fetal side of placenta tissues (**Figure 1C**).

Prdx 3 is a mitochondria antioxidant enzyme that was known to involve in apoptosis and response to oxidative stress [51]. Previous studies have shown that a lower expression of Prdx 3 in human placenta tissues triggered oxidative stress and caused dysfunction of mitochondria in trophoblast cells [52]. From our results, the downregulation of this protein in placentas of sPTL-IM patients suggested the stimulation of fatty acid cascade, such as arachidonic acid and cell cycle arrest by oxidative stress [53, 54]. Both processes are potentially synergistic mechanisms that contribute to preterm contractions, cervical dilatation, and PTL.

Ferritin is a multimeric protein that consisted of two subunits: ferritin heavy chain (FTH) and FTL, which share about 50% sequence homology and is mainly found in the villous stroma of the placenta [55]. FTL has been reported to play a critical role in iron storage as well as regulation of intracellular iron homeostasis and inflammatory response [56, 57]. Mutation in the *FTL* has also been demonstrated to cause neuroferritinopathy and neurodegeneration [58, 59]. On the other hand, anti-inflammatory activities were observed from FTL overexpression where Fan et al. [56] reported inhibition on lipopolysaccharide (LPS)-induced transcription of anti-inflammatory factor at the feto-maternal interface of the placenta leading to PTL [60]. Therefore, it is possible that the production of inflammatory mediators induced by LPS at feto-maternal interface of the placenta can contribute to FTL degradation and synergized to increase intracellular free iron. This leads to increased oxidative stress. Furthermore, elevated placenta oxidative stress was thought to play a key role in stimulating the release of factors, further leading to maternal endothelium activation [61].

#### **4.4. Proteins involved in anticoagulation cascade**

Only one identified protein was classified in this group. Protein PP4-X (also known as annexin IV (ANX4)) was downregulated in sPTL-IM placentas when compared to the control placentas at the fetal side (**Figure 1C**). This protein has been known to be involved in multiple cellular functions including endocytosis, exocytosis, ion fluxes, and apoptosis [62, 63]. This protein is also one of the important components of biological cascades regulating coagulation which makes it an important factor for the maintenance of pregnancy [63]. Placenta structural abnormality and dysfunction in IUGR fetuses were reported to be caused by reduction in two members of annexins (ANX4 and ANX8) [64]. Furthermore, coagulative disorder and antiphospholipid antibody syndrome have been associated with an increased incidence of pregnancy complications, including PTL [65]. Several cases of increased polymorphism rate in genes related to coagulation have also been associated with PTB [66]. Therefore, it is conceivable that alterations in ANX4 levels may contribute to a failure of placenta anticoagulation and structural abnormality, leading to impaired utero placenta development.

Interestingly, more proteins were differentially expressed in the fetal side than in the maternal side of the placenta tissues obtained from pregnant women who experienced sPTL-IM (**Tables 2** and **3**); this interpretation is consistent with the signals from the fetus where they are capable of initiating the onset of parturition process [67]. The work by Reinl and England [67] demonstrated a possible interplay between placenta corticotrophin-releasing hormone and fetal surfactant protein-A that drives parturition in human. Moreover, another study indicated that nuclear receptor coactivators 1 and 2 regulate the transcription of surfactant protein-A and consequently the induction of labor [68].
