**1. Introduction**

With their1 2020 book, *The Force of Nonviolence: An Ethico-Political Bind*, feminist political philosopher Judith Butler joins a long tradition of nonviolent resistance that includes proponents such as Leo Tolstoy, Albert Einstein, Henry David Thoreau, Te Whiti o Rongomai III, Martin Luther King Junior, Václav Havel, Alice Paul, Rosa Parks, and others, noticeably Mahatma Gandhi. Many social justice movements have been inspired by the ideal and strategy of nonviolent resistance. For example, according to one estimate, between 1966 and 1999, "nonviolent civic resistance played a critical role in fifty [out] of sixty-seven transitions from authoritarianism" (Ackerman and DuVall 2000). In addition, in their book, Chenoweth and Stephan conclude both that "historically, nonviolent resistance campaigns have been *more effective* in achieving their goals than violent resistance campaigns" (Chenoweth and Stephan 2013, p. 220; emphasis added) and that, since "civil resistance campaigns are more successful than violent campaigns at overcoming barriers to participation" (Ibid, p. 28), they produce *more stable outcomes* in the long run.

In contrast, although "violent insurgencies captured power in some cases", the human costs in terms of casualties were very high. Moreover, "the conditions in these countries after the conflict ended have been overwhelmingly more repressive than in transitions driven by nonviolent civic pressure" (Ibid, p. 60). Typical for post-violent-conflict societies were widespread retaliatory violence, lack of respect for human rights and lack of respect for minority rights (Ibid, p. 60). Gene Sharp's influential 1973 three-volume opus, *The Politics of Nonviolent Action* (Sharp 1973), provides a theoretical foundation for nonviolent action by linking it with a specific understanding of power. Different methods of nonviolent action that he discusses include nonviolent protest, social noncooperation, economic boycotts, strikes, political noncooperation and nonviolent intervention, and he explains that adversaries can be nonviolently affected or changed through conversion, persuasion, accommodation and coercion.

**Citation:** Du Toit, Louise, and Jana Vosloo. 2021. When Bodies Speak Differently: Putting Judith Butler in Conversation with Mahatma Gandhi on Nonviolent Resistance. *Religions* 12: 627. https://doi.org/10.3390/ rel12080627

Academic Editor: Daniel H. Levine

Received: 4 July 2021 Accepted: 3 August 2021 Published: 10 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The specific contribution of Butler's book against this backdrop is to make a philosophical case for the use of nonviolent resistance toward oppressive regimes, without turning it into an absolute prohibition on violence or self-defence (Butler 2020, p. 23). Their argument is rooted in their understanding of the human subject as constituted in discursive and performative ways—an understanding they partly take from Michel Foucault, and partly develop more concretely in terms of the performative body. Butler's argument for nonviolence is therefore embedded in their earlier work on subject formation, and the in/ability to appear (and be recognised) in the social or public world as a moral and political subject—themes that run through their whole oeuvre, as we will show.

Even though Butler's book title references Gandhi's concept of *Satyagraha* (meaning love force, truth force or soul force),2 and they evoke him explicitly a handful of times (Butler 2020, pp. 16, 21, 181, 201), Gandhi's own thinking does not feature much in the book. The point of this article is to go beyond the book's purview and place Butler and Gandhi in conversation on the topic of nonviolence. Although the abovementioned sources are important for addressing the common assumption that nonviolent tactics as a strategic choice are naïve, misplaced, ineffective or implausible (Chenoweth and Stephan 2013, p. 17), the focus of this article is on a more fundamental, i.e., ontological, level. In quite different yet complementary ways, Gandhi and Butler argue that the imperative for nonviolent resistance aimed at social transformation flows from a certain relational ontology, a certain perspective on the world and human co-existence. The aim of this article is to draw out the most salient implications of such a conversation for the right of public assembly and contemporary cultures of protest globally.

The ontology that Butler places at the root of nonviolence is a secular social ontology of the constituted, performative, exposed subject, coupled with a radically egalitarian imagination, as is discussed. In his turn, the vision that underlies Gandhi's nonviolence is a faith-based ontology that sees the force of love as a cosmic principle greater or more powerful than violent force. It is important to remain faithful to the very different vocabularies that these two thinkers employ; nevertheless, it is mutually illuminating and fruitful for a systematic philosophical exploration of the nature of nonviolent resistance, we claim, to place them in conversation with each other, on four central themes related to nonviolent resistance. The conversation between Gandhi and Butler is structured according to these themes: (i) the ontological roots of the nonviolent imperative; (ii) their rejection of an instrumental view of violence; (iii) nonviolent resistance seen as communicative action; and (iv) nonviolence viewed as a way of life. The discussion emphasises the continuities and complementarities between the two thinkers but also indicates where tensions remain. We conclude the article with some concrete suggestions that flow from this discussion for social protest and public assembly.

#### **2. Gandhi and Butler on Nonviolent Resistance**

## *2.1. The Ontological Roots of the Nonviolent Imperative*

Butler's social ontology is best understood as being inspired by Foucault, who is a key critic of the "constituting" subject of western modernity. Against this influential view of the human subject as sovereign, as pre-existing its social conditions and as fully transparent to itself and governed by reason alone, Foucault posits a "constituted", i.e., a historically contingent subject, who is formed or moulded into a self through everyday social practices, dominant discourses and knowledge formations, and the power relations that infuse these. "Objectifying knowledge practices" and "processes of subjectivation" or of self-formation all represent a form of power that transforms human beings into the subjects required (and recognised) by the dominant, anonymous structures at work in modern societies. Thus, writes Foucault in "The Subject and Power" (Foucault 1982, p. 781), "[t]his form of power applies itself to immediate everyday life which categorizes the individual, marks him by his own individuality, attaches him to his own identity, imposes a law of truth on him which he must recognize and which others have to recognize in him." There is therefore no presocial or prediscursive, or natural subject.

Instead, we find ourselves always already embedded in, shaped by, measured, called forth and named by a force field of norms embedded in language and in other material manifestations, ranging from singular bodies to institutions, to, nowadays, the Anthropocene planet itself. Butler largely adopts this Foucauldian understanding of the subject but focuses it on the notion of bodily performativity or the animate body. They define their key notion of performativity as "not a singular or deliberate 'act', but, rather, as the reiterative and citational practice[s] by which discourse produces the [material] effects that it names" (Butler 1993, p. 2). Butler (2015, p. 28) in their later work noted that "performativity characterises first and foremost that characteristic of linguistic utterances that in the moment of making the utterance makes something happen or brings the phenomenon into being".

Their notion of performativity thus equally inverts the modernist logic where action proceeds from a unified, pre-existing, rational and free mind. Performativity instead posits that people become subjects, e.g., gendered subjects, through the performance of desires, acts, speech and gestures that cite or reiterate the dominant (heterosexual) gendered and gendering norms (see their *Gender Trouble*, Butler 1990). An important point that Butler takes over from Foucault (1982, p. 778) is that we become subjects partly by objectifying ourselves as objects of knowledge and power; we are at once object and subject of these ongoing processes. It is by inserting ourselves through bodily gestures, expressions, motilities and so forth into the force field of norms that constitutes or moulds recognisable (gendered) subjects, and thus by "passing", that we come to recognise ourselves and be recognised by others, as properly gendered subjects. Our actions are themselves attuned to, or aligned with, discursive meanings. When this attuning translates into bodily habits, then discursive norms have been successfully materialised in the world. This understanding of performativity moreover extends far beyond gendering processes to encompass the subject as a whole. Butler's social ontology therefore entails that we are constituted as subjects in an inescapably relational, social and discursive manner. We only appear as legible persons or subjects to the extent that we align our sense of self and our behaviour (speech and action) with the social scripts and norms that shape personhood. Dominant norms and social recognition thus work together to give shape, form and content to individual subjects.

However, and this is key, Butler's social ontology does not lead them to an understanding of the self as fully socially or externally determined (as they explain in detail in *Giving an Account of Oneself*, Butler 2005). Key here is that abstract norms as deposited in language, are not only pervasive and powerful, but at the same time are unstable and ephemeral, and fully dependent for their endurance upon the ongoing, largely habitual obedience or docility of bodies that reiterate, repeat and re-cite them. Since it is obedient or imitative, citational actions and habits bring selves and other social realities into being and sustain them and make them matter and materialise in the human world; therefore, action is also the key to resistance against oppression. When bodies perform or act *differently*, by transgressing or otherwise challenging, disrupting, misquoting, parodying or queering the norms, then they can over time change, or subvert, the norms themselves. Furthermore, new sets of norms can shape radically different selves and institutions into being over time.

Butler's social ontology does not lead to a deterministic view of the self, as we have seen, but neither does it allow for change to be driven from somewhere beyond the social world. There is no self, and there are no hermeneutic resources on the radical outside of (or beyond) the discursive force field which has constituted us as subjects. Butler's commitment to nonviolence as preferred change strategy thus stems from their understanding of the deeply relational, communicative nature of the self. We only come into being as subjects or persons through our constant exposure to the norms embedded in discourse, action and institutions, and to others, who (we) need to recognise us as such.

Exposure to norms and others is a prerequisite for being shaped or moulded into a person; at the same time, it renders us vulnerable. This is because of what Butler calls "normative violence", or the violence of the norm (Butler 2004b): dominant norms are always embedded in, and serving, dominant power relations. On a basic level, Butler's

social ontology implies that every person is in principle, constitutively, equally vulnerable because of the pervasive ways in which we are all exposed to, and dependent upon, social recognition in conjunction with dominant norms. On another level, however, vulnerability is differentially distributed, and the distinction between our shared ontological vulnerability (precariousness) and politically wrought vulnerability (precarity) is important here (Butler 2009). The potential for violence lies in the precise way in which norms distribute the terms and conditions of subject-formation. Simply put, where heterosexism, patriarchy and racism are dominant norms embedded in discourse and institutions, and in practices of recognition and knowledge production, and thus in self-understandings, there, bodies marked as gender nonconforming, female and/or black, are likely to struggle to appear as "proper" subjects and citizens, both to themselves and to others.

This is precisely the kind of situation where Butler would evoke the force of nonviolent resistance, which is exerted where and when "a social and political practice [is] undertaken in concert, culminating in a form of resistance to systemic forms of destruction coupled with a commitment to world building that honours global interdependency ... and equality" (Butler 2020, p. 21). We want to draw attention to a number of elements in this quote. First, note the element of collective action3 ("undertaken in concert") in which shared resistance is expressed towards a situation that is read as systemically destructive. Moreover, this collective resistance is performed against the normative violence exerted by the dominant social system, which prevents some human beings from appearing as subjects and citizens in public spaces. For Butler, then, wherever there is performative resistance against oppression, one can discern a struggle against socially induced precarity or the destructive unequal distribution of the chance of becoming (social) subjects. Butler (2020, p. 10) therefore argues that "nonviolence requires a critique of egological ethics as well as of the political legacy of individualism in order to open up *the idea of selfhood as a fraught field of social relationality*" (emphasis added). Nonviolent resistance illuminates not only the direct suffering or grief carried by groups and individuals that struggle to appear as social subjects but also shows how social mechanisms operate in order to pre-emptively "disappear" that suffering as suffering, to render those lives publicly "ungrievable".4 As Ruti (2017, p. 97) points out, Butler "deftly demonstrates [that] one of the ruses of power is to delimit the domain of grievability so that—under normal circumstances—we are prevented from mourning the suffering (or death) of those deemed different from, or inferior to ourselves". Grievability therefore does not only play a role at the end of a life: whether the loss of any particular life would register socially and publicly as a loss or not is already inscribed in the multiple ways in which people's lives are either materially and symbolically supported or not (Butler 2020, p. 59).

Note also from the quote above that the destruction (violence) identified in the system is opposed in nonviolent resistance not by yet another form of destruction but rather by a force of a different nature—by the power that springs from "a commitment to world building that honours interdependency and equality". The commitment to world building and respect for interdependency is rooted in Butler's social ontology, which is wary of processes of individualisation that turn the oppressed into the main cause of their own oppression and thereby obliterate or erase the largescale social complicity that is always needed for systemic oppression, as discussed. They relate equality with interdependence as follows: "Equality is ... a feature of social relations that depends for its articulation on an increasingly avowed interdependency—letting go of the body as a 'unit' in order to understand one's boundaries as relational and social predicaments: including sources of joy, susceptibility to violence, sensitivity to heat and cold, and tentacular yearnings for food, sociality, and sexuality" (Butler 2020, p. 45). This quote helps us to understand why Butler proposes that a radically egalitarian imagination must inform nonviolent resistance and provides much of its countervailing force or impetus. For Butler (2020, p. 16), this radically egalitarian vision lies on the level of the social imaginary and springs from the understanding that each person is dependent, "or formed and sustained in relations of depending upon, and being depended upon". Thus, "social interdependency characterizes life, and ... violence [is] an attack on that interdependency". Therefore, the infliction of harm (violence) requires a forgetfulness of interdependency, and the egalitarian imagination by contrast increasingly avows and reckons with it, also or especially, in how it performs its demands for social inclusion and change. The shape or nature of performative resistance must be compatible with the affirmation of equal grievability.

Active, collective resistance to destructive (bond-denying and -defying) norms must therefore take the form of bond-affirming ("world-building") yet transgressive action; the performance must be at once relational and resistant, resisting *through* relations. Put differently, in our activist attempts to change socially embedded norms, we must remain ever mindful of their very nature. Recall that norms only have power over us because and insofar as we repeat and uphold them in our everyday actions. In that sense, every norm that holds sway does so through a pervasive yet often silent social pact. This is why all lasting and effective change in the shared world depends upon new ways of acting, in concert, combined with a respect for fundamental equality. In addition, why those very ways of acting must be respectful of the ontological sociality or interdependence, or the very worldliness, of all meaning making, including subject-formation. The *force* of nonviolence as conceived by Butler lies in the fact that as collective transgressive action, it remains steadfastly social, communicative, relational and committed to "living and sustainable [and sustaining] bonds" (Butler 2020, p. 15). Concerted transgressive action therefore demonstrates and reveals at once the social nature and power of existing norms and their dependence upon obedient repetition, as well as their fundamental changeability.

Gandhi's ontological orientation takes another, yet comparable, route to nonviolent resistance. Butler (2020, p. 181) comments on what Gandhi calls "the law of love", which he views as "a higher law than that of destruction". True to Butler's social ontology, they state that his stance "may not rest upon a discoverable law" but rather function only rhetorically. However, Gandhi does not intend for the law of love to be a mere rhetorical device. In *Hind Swaraj* (CWMG (The Collected Works of Mahatma Gandhi. Gandhi 1958) vol. 10, p. 84), Gandhi explains that the [human] universe would disappear without "the force of love and pity5 [which] is infinitely greater than the force of arms". He writes, "[h]istory is really a record of every *interruption* of the *even* working of the force of love or of the soul ... History, then, is a record of an interruption in the course of nature. Soul-force, being natural, is not noted in history" (CWMG 10, p. 90; emphases added). He gives the example of two brothers who overcome their mutual animosity as a story that would not go down in history. Soul-force or love force (*Satyagraha*) works evenly, constantly and unnoticed in the background, *building the world*, which is why the world as such is based "not on the force of arms but on the force of truth or love". Similar to Butler, then, Gandhi also derives his nonviolent imperative from a vision of the human world as consisting of a fabric of social bonds, woven, built and continuously sustained through the force and labour of love and pity or empathy.6 In contrast with Butler for whom the force of nonviolence derives purely from social bonds, Gandhi's ontology is however a religious one: truth force or soul force is an "indefinable mysterious power that underlies everything"; it is also called "God", by Gandhi.7 He sees love force, truth force, or God, as "purely benevolent", for "in the midst of death, life persists, in the midst of untruth, truth persists, in the midst of darkness, light persists". Therefore, if one aligns oneself with love force, one sides with and taps into the stronger force in the universe. For Gandhi, then, there is a force in the world, in the cosmos and in nature, that is a force for good and that exists independently of the human world but which underpins and sustains also the social bonds in the human world. This is why he claims, "as long as there is even a handful of men true to their pledge [of *Satyagraha*], there can be only one end to the struggle and that is victory" (Gandhi 1928, p. 116). Drawing from this quotation, and because Gandhi understands nonviolence as tapping into a divine source, one might deduce that Gandhi is more optimistic about the effectiveness of nonviolence than the secular Butler. However, later in his life, Gandhi also experienced many setbacks and failures of his nonviolent tactics, most spectacularly with conflicts between Hindus and Muslims in India. He was thus finally not naïve about the

limits of nonviolence and conceded cases in which the force of violence was needed (see Note 10). However, we think the larger point which he shares with Butler is to draw our attention to the *limits of violence* as an instrument for decisive and lasting social change, and to open up more strategic space for nonviolent tactics.8

Gandhi's nonviolence therefore comes from a different place than that of Butler but ends up being very similar in its practical implications.9 Because *Satyagraha* is attuned with the love force, which is God, it aims for "the conquest of the adversary" purely through persuasion, since it cannot be reconciled with causing him harm. For Gandhi, his faith position implies that "hatred is a positive breach of the ruling principle" of *Satyagraha*, and therefore, it has no place within nonviolent resistance—it would oppose and thereby dilute or completely thwart this alternative force. Moreover, because love force or God is an ontological force, it (or He) is also latently present in one's adversary—there is always the chance that the opponent can (learn to) tap into their own capacity for love and pity.10 The adversary or opponent is never the enemy whom one is allowed to hate this is very similar to Butler's idea that nobody should be socially construed as finally ungrievable. The temptation to construe another person as ungrievable and therefore violable is particularly strong within the frame of self-defence, as we see below. For Gandhi, for the *Satyagrahi* to indulge in having enemies would be a failure of faith in the force of love. Instead of aiming to harm the adversary, therefore, *Satyagraha* aims for and "postulates the conquest of the adversary by suffering in one's own person" (Gandhi 1928, p. 124). Where hatred and violence attempt to either eliminate the opponent altogether or to coerce them into obedience, through the infliction of harm or threat of harm, love force instead aims for the opponent to change their own perspective, to learn something and to connect with their own capacity for love and empathy. With their notions of vulnerability, precarity and grievability, we have seen how Butler positions suffering as a central element that is evoked and activated through collective resistance. Similarly, yet differently, Gandhi sees "the potency of suffering" as "key to *Satyagraha"* (Ibid, p. 18). At first glance, their attitudes towards suffering as a key element of nonviolent resistance may appear quite different, as Butler sees hidden suffering as being brought to light and simultaneously resisted through collective nonviolent action, whereas Gandhi sees the collective nonviolent action or *Satyagraha* itself as necessarily entailing bodily suffering. We show why we regard their positions on this point as complementary rather than contradictory, under theme (iii) below.

For Gandhi, based on his faith-based ontology, nonviolence cannot be treated as a mere instrument that might be discarded and replaced with violence as a supposedly more efficient means. Hatred and harm cannot be a viable alternative to love and truth. This leaves us with the interesting implication that Gandhi's nonviolence is not in the first place or only a moral demand but fundamentally ontological and strategic.11 In order to "see" the force of love as stronger than the force of violence and hatred, as Gandhi does, one needs a kind of countercultural imagination.12 At least in many scholarly circles in the west, violence is viewed as ingrained and inevitable, an inherent part of agonistic politics, and the ideal of nonviolence is viewed as naïve and utopian. Nevertheless, Gandhi might be described as sharing with Butler their alternative, "egalitarian imaginary" (Butler 2020, p. 24), which insists upon foregrounding the human capacity for love and pity and which foregrounds a similar relational ontology. In addition, similar to Butler's understanding of the social construction of subjects, Gandhi has a keen insight into how everybody's understanding is a finite function of their time, their social group and their upbringing. A vivid example is when he decided not to hold then President of the Transvaal Republic, Paul Kruger's, blatant racism against him, seeing that Kruger had only ever read the Old Testament and regarded Indians as the "descendants of Esau" (Gandhi 1928, pp. 56–57). Long before scholars such as Chenoweth and Stephan (2013) did the research to back up the empirical claim, Gandhi's faith position led him to reject violent resistance as a relatively ineffectual short-cut (CWMG 25, p. 424) to social change, rooted in a lack of faith. This critical view of violence, which shares many elements with that of Butler, is discussed next.


*Article* **Human Placental Transcriptome Reveals Critical Alterations in Inflammation and Energy Metabolism with Fetal Sex Differences in Spontaneous Preterm Birth**

**Yu-Chin Lien 1,2 , Zhe Zhang 3 , Yi Cheng 4 , Erzsebet Polyak 4 , Laura Sillers 2 , Marni J. Falk 4 , Harry Ischiropoulos 2 , Samuel Parry 1,5 and Rebecca A. Simmons 1,2,\***

**Abstract:** A well-functioning placenta is crucial for normal gestation and regulates the nutrient, gas, and waste exchanges between the maternal and fetal circulations and is an important endocrine organ producing hormones that regulate both the maternal and fetal physiologies during pregnancy. Placental insufficiency is implicated in spontaneous preterm birth (SPTB). We proposed that deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability during pregnancy may ultimately result in SPTB. To explore our hypothesis, we performed a RNA-seq study in male and female placentas from women with SPTB (<36 weeks gestation) compared to normal pregnancies (≥38 weeks gestation) to assess the alterations in the gene expression profiles. We focused exclusively on Black women (cases and controls), who are at the highest risk of SPTB. Six hundred and seventy differentially expressed genes were identified in male SPTB placentas. Among them, 313 and 357 transcripts were increased and decreased, respectively. In contrast, only 61 differentially expressed genes were identified in female SPTB placenta. The ingenuity pathway analysis showed alterations in the genes and canonical pathways critical for regulating inflammation, oxidative stress, detoxification, mitochondrial function, energy metabolism, and the extracellular matrix. Many upstream regulators and master regulators important for nutrient-sensing and metabolism were also altered in SPTB placentas, including the PI3K complex, TGFB1/SMADs, SMARCA4, TP63, CDKN2A, BRCA1, and NFAT. The transcriptome was integrated with published human placental metabolome to assess the interactions of altered genes and metabolites. Collectively, significant and biologically relevant alterations in the transcriptome were identified in SPTB placentas with fetal sex disparities. Altered energy metabolism, mitochondrial function, inflammation, and detoxification may underly the mechanisms of placental dysfunction in SPTB.

**Keywords:** placenta; transcriptome; spontaneous preterm birth; bioenergetic metabolism; interactome; fetal sex disparity

### **1. Introduction**

Preterm birth (delivery before or at 37 weeks of gestation) is the leading cause of morbidity and mortality in newborn infants worldwide [1]. Fifteen million babies are born

**Citation:** Lien, Y.-C.; Zhang, Z.; Cheng, Y.; Polyak, E.; Sillers, L.; Falk, M.J.; Ischiropoulos, H.; Parry, S.; Simmons, R.A. Human Placental Transcriptome Reveals Critical Alterations in Inflammation and Energy Metabolism with Fetal Sex Differences in Spontaneous Preterm Birth. *Int. J. Mol. Sci.* **2021**, *22*, 7899. https://doi.org/10.3390/ijms22157899

Academic Editors: Hiten D. Mistry and Eun Lee

Received: 16 June 2021 Accepted: 20 July 2021 Published: 23 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

prematurely annually, resulting in an excess of one million deaths. Infants who survive preterm birth often have serious and lifelong health problems, including lung disease, vision loss, and neurodevelopmental disorders. Spontaneous preterm birth (SPTB) remains a significant and poorly understood perinatal complication. SPTB includes the preterm spontaneous rupture of membranes, cervical insufficiency, and preterm labor. While the exact etiology remains unknown, many factors may contribute to SPTB, including placental dysfunction, abnormal cervical remodeling, uterine distension, vascular disorders, and chorioamnionitis [2,3].

During pregnancy, the placenta facilitates nutrient transport and gas exchange and supports the growth and development of the fetus. It also produces and releases hormones into the maternal and fetal circulation to regulate uterine functions, the maternal metabolism, and fetal growth and development. Therefore, a well-functioning placenta is crucial for normal gestation. Placental dysfunction is associated with preeclampsia and fetal growth restriction. Emerging evidence suggests that placental insufficiency is also associated with a significant proportion of preterm births, especially early preterm births, as well as those complicated by chorioamnionitis [4–6]. The placenta protects the fetus against infections, toxins, xenobiotic molecules, and maternal diseases [7]. The placenta also produces a wide variety of metabolites, many of which are involved in energy production [8,9]. In our previous metabolomic analysis of placenta samples obtained from women with SPTB, we observed a significant elevation in the levels of amino acids, prostaglandins, sphingolipids, lysolipids, and acylcarnitines in SPTB placentas compared to term placentas [10], which suggests an imbalance between the supply capacity and metabolic demands in SPTB placentas.

Fetal sex plays an important role in pregnancy complications and perinatal outcomes.  A higher incidence of preterm birth is also observed among women carrying male fetuses. Although the underlying mechanism for the increased preterm birth rate of male newborns is still unclear, a potential more proinflammatory intrauterine milieu generated by male placentas may partially contribute to the increased incidences [15–17].

We hypothesized that deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability throughout the course of pregnancy may ultimately result in SPTB. To test this hypothesis, we assessed the transcriptomes in both male and female placenta samples obtained from women with spontaneous preterm deliveries. We also integrated the transcriptomic data with our previously published metabolomic data [10] to assess the interactions of the altered genes and metabolites. Therefore, the aim of this study was to elucidate the underlying mechanisms for placental insufficiency and dysfunction, especially metabolic changes and sex differences, which will lead to a better understanding of the etiology of prematurity and the development of preventative treatments.

### **2. Results**

### *2.1. Clinical Characteristics*

There were no differences in maternal ages between term and spontaneous preterm birth (SPTB) placenta samples (Table 1). Of note, none of the preterm or term placental samples were from mothers with preeclampsia or gestational diabetes, and none of the women received low-dose aspirin for the prevention of preeclampsia. All of the women contributing placenta samples presented in labor with either a preterm premature rupture of membranes (PPROM), premature rupture of membranes (PROM), or cervical dilation. Thirteen women had preterm labors, and none of the women who labored at term received a betamethasone treatment prior to delivery. Three of the women with preterm labors received 17-hydroxy progesterone and five received vaginal progesterone for prematurity prevention. None of the women with term delivery received supplemental progesterone. A greater percentage of women with preterm labor received antibiotics, with a primary indication of PPROM or Group B—streptococcus (GBS) prophylaxis; however, only one

woman with preterm labor was diagnosed with chorioamnionitis. Chronic medications administered to women with preterm deliveries included albuterol and inhaled corticosteroids for asthma in three women, psychotropic medications in two women, and antihypertensive drugs in four women. The chronic medications documented for women with term deliveries included albuterol in one woman and ferrous sulfate medication in one woman.

**Table 1.** Demographics of the study cohort.

ND: not determined.

### *2.2. Global Assessment of Transcriptome Profiles in Placentas*

To investigate the genes and novel pathways that are disrupted in placentas from SPTB, the gene expression profiles of placental tissues from eight male preterm cases with a mean gestational age (GA) of 29.4 weeks, seven male term controls with a mean GA of 39.7 weeks, eight female preterm cases with a mean GA of 32.1 weeks, and eight female controls with a mean GA of 39.5 weeks were assessed by RNA-Seq. The power calculation using a false-positive rate of 0.05 (two-sided) and power of 80% to target a two-fold change indicated that six samples were sufficient to determine the significant differences between the groups. One male control and one female preterm cases were considered as outliners due to the potential contamination of other cell types and were excluded from further analysis of the differential gene expressions. The principal component analysis (PCA plot, Figure 1) indicated that the transcriptome profiles for males and females were readily distinguishable. Preterm and term birth groups also separated, suggesting significant differences in the transcriptome profiles of preterm and term placentas. Genes were considered differentially expressed, with an FDR (*q*-value) ≤ 0.05. The comparison of male SPTB vs. male term deliveries yielded a total of 724 differentially expressed genes, with 347 upregulated and 377 downregulated (Figure 2a and Supplemental Table S1) (*q* ≤ 0.05 vs. term controls; *q*-value ≤ 0.05 was considered significant). Interestingly, the difference of the transcriptome profiles between female SPTB and female term deliveries was much smaller than that of the male transcriptomes. Only 66 differentially expressed genes were identified in the comparison of female SPTB vs. term, with 28 upregulated and 38 downregulated (Figure 2b and Supplemental Table S2). Most differentially expressed genes in each comparison were unique, suggesting a different molecular basis for placenta dysfunction and possibly preterm births of males vs. females. Five differentially expressed genes were identified in both male and female SPTB compared with their term controls, including four upregulated genes, *ASB4, CMAS, KATNBL1*, and *PRR9*, and one downregulated gene, *SLC28A1* (Supplemental Table S3). Differences in the expression between SPTB and

term placentas of these genes are likely due to gestational age effects and unlikely to be associated with preterm births.

**Figure 2.** Volcano plots identifying differentially expressed genes with an FDR (*q*-value) < 0.05. Male SPTB placentas compared with term placentas (**a**). Female SPTB placentas compared with term placentas (**b**). Male term placentas compared with female term placentas (**c**). Male SPTB placentas compared with female SPTB placentas (**d**).

To determine the possible effect of gestational age (GA) on the differences in the gene expressions, we took two approaches. First, we compared our SPTB transcriptomes with the human placenta studies conducted by Eidem et al. [18] and Brockway et al. [19], who identified 37 and 170 GA-specific candidate genes, respectively. We also compared the SPTB transcriptomes with our proteomics data on four placentas from term deliveries and four placentas from elective second trimester terminations (Supplemental Table S4). In the proteomic dataset, 4711 proteins were identified, and 953 proteins were differentially expressed in term compared to second trimester placentas (proteins associated with blood were eliminated) (Supplemental Table S5). Together, we identified 54 GA-specific candidate genes in male SPTB placental transcriptome and five candidate genes in female SPTB placental transcriptome (Supplemental Table S6). These GA-specific candidate genes were removed from the datasets prior to the ingenuity pathway analysis.

### *2.3. Differences in the Transcriptome Profiles between Male and Female Placentas*

Comparing male with female transcriptomes from term placentas, 319 differentially expressed genes were identified, with 177 upregulated and 142 downregulated in male compared to female placentas (Figure 2c and Supplemental Table S7). Thirty-nine differentially expressed genes were either X- or Y-chromosome-linked. In the comparison of male and female SPTB transcriptomes, in addition to 36 sex chromosome-associated genes, 144 differentially expressed genes were identified, with 105 upregulated and 39 downregulated in male compared to female STPB placentas (Figure 2d and Supplemental Table S8). These genes regulated at least 20 canonical pathways (Supplemental Table S9). Only 29 differentially expressed genes overlapped between the STPB and term groups when comparing male with female placentas.  The expression of *ICAM2, MTNR1B*, and *HIST1H3H* were lower in male placentas from both preterm and term births compared with female placentas. In contrast, the expression levels of *AADACL3, RNR1*, and *RNR2* were higher in male placentas.

**Table 2.** Overlay of differentially expressed genes comparing male and female placentas at both SPTB and term births.

### *2.4. IPA Identifies Multiple Pathways That Are Altered in Male SPTB Placentas*

To identify the molecular pathways that may contribute to placenta dysfunction and, possibly, SPTB, an ingenuity pathway analysis (IPA) was used to map differentially expressed genes into functional networks. An IPA analysis of 670 non-GA-associated differentially expressed genes in male SPTB placentas revealed 65 canonical pathways that were altered in preterm births. As predicted by the activation z-score, the top canonical pathways activated in male SPTB included the NRF2-mediated oxidative stress response, xenobiotic metabolism pathways, and estrogen receptor signaling (Table 3). The top

pathways inhibited in male SPTB were VDR/RXR activation and the antiproliferative role of the transducer of ERBB2 (TOB) in T-cell signaling (Table 3).

In addition to the identification of altered canonical pathways, IPA disease and a biological function analysis also revealed more than 65 genes that were differentially expressed in male SPTB placentas, such as *RBP4, VEGFA, EREG, PLA2G2A, CRH, PRL*, and *LEP*, which regulated the metabolic processes, particularly lipid and fatty acid metabolism (Supplemental Table S10). This was consistent with our previous metabolomics study that showed a marked elevation of multiple acylcarnitine species and significantly decreased the fatty acid oxidation in SPTB placentas [10].

### 2.4.1. Inflammatory Signaling and Oxidative Stress Pathways Are Activated

Many environmental exposures, including infections, during pregnancy increase the production of mediators of oxidative stress and abnormal metabolism, which may lead to

spontaneous preterm births [20]. Several inflammatory signaling and detoxification pathways were altered in male SPTB placentas, including xenobiotic metabolism pathways, the NRF2-mediated oxidative stress response, glutathione-mediated detoxification, superoxide radical degradation, transforming growth factor-β (TGF-β) signaling, antiproliferative role of TOB in T-cell signaling, glucocorticoid receptor signaling, and PI3K signaling (Table 3). Many of these pathways were predicted to be activated in male SPTB placentas, consistent with the presence of inflammation and oxidative stress.

Xenobiotic metabolizing enzymes and transporters play critical roles in the metabolism, elimination, and detoxification of harmful xenobiotics and toxic endogenous compounds in the placenta via nuclear receptors, including the constitutive active receptor (CAR), pregnane X receptor (PXR), and aryl hydrocarbon receptor (AHR) [21]. These three nuclear receptor superfamilies were predicted to be activated in male SPTB placentas (z-score = 2.71, 2.11, and 2.45, respectively). Twelve genes comprising these pathways were altered in male SPTB placentas, including *GSTA1, GSTM5*, *MGST1,* and *CHST2* (Supplemental Table S11).

Consistent with previous studies showing that oxidative stress is associated with SPTB [22,23], glutathione-mediated detoxification and the nuclear factor erythroid 2-like 2 (NRF2)-mediated oxidative stress response were both disrupted in male SPTB placentas. Eleven genes comprising these pathways were altered in male SPTB placentas, including *CAT, PRKCA,* and *SOD3* (Supplemental Table S11).

The IPA Tox analysis, which links gene expression to clinical pathology endpoints, further identified the genes directly contributing to mitochondrial dysfunction in male SPTB placentas, including *CAT, BTG2*, *BCL2*, and *GSTA1* (Supplemental Table S12). These genes regulate the processes such as mitochondrial transmembrane potential, permeability transition of mitochondria, depolarization of mitochondria, and reactive oxygen species detoxification.

Both TGF-β and TOB play critical roles in maintaining a normal pregnancy, and low levels of TGF-β are associated with an increased risk of preterm birth [24–26]. Both TGF-β and TOB signaling were predicted to be inhibited in male SPTB placentas (z-score = −0.82 and −2.00, respectively). *TGFB1*, *TGFBR1*, *INHBA*, *INHA*, and *CCNE1* were examples of differentially expressed genes comprising these pathways (Supplemental Table S11). Further, underscoring TGF-β1 0 s importance in SPTB placentas, its downstream signal transducers SMAD3 and SMAD4 were all predicted as inhibited regulators in male SPTB placentas. They regulate the differential expression of more than 90 genes in our dataset (Figure 3a).

Interestingly, glucocorticoid receptor signaling was altered in male, but not female, SPTB placentas. Glucocorticoids activate a number of physiologic pathways in the placenta and are also critical for fetal organ development and survival during pregnancy and parturition [27]. Previous studies have shown that term female placentas have a higher glucocorticoid receptor expression compared to term male placentas [28]. Twenty-seven genes comprising this pathway were differentially expressed, including *FGG, IL5RA, KRT24, KRT5*, and *PRL* (Supplemental Table S11).

Phosphoinositide 3-kinase (PI3K) signaling modulates the immune system during pregnancy [29]. The disruption of PI3K signaling leads to an unbalanced adaptation of the maternal innate immune system to gestation and increases the fetal mortality [30]. We found that PI3K signaling was activated in male SPTB placentas, including the differentially expressed genes *CD180*, *PIK3AP1*, and *PLCD3* (Supplemental Table S11).

Cumulatively, these data suggest that the key pathways regulating oxidative stress and the ability of the placentas to handle toxins are altered in preterm birth placentas.

### 2.4.2. Metabolic Pathways Are Altered in SPTB Placentas

The IPA analysis identified multiple metabolic canonical pathways that were altered in male SPTB placentas, including amino acids, sphingomyelins, vitamin B6, purines, and myo-inositol (Table 3). Pathways regulating histidine, sulfite oxidation, and urea cycle metabolism were disrupted, including the differentially expressed genes *CPS1, ARG2*, and

*MTHFD1* (Supplemental Table S13). Sulfite oxidation, catalyzed by mitochondrial intermembrane space enzyme sulfite oxidase, is associated with oxidative stress in the placenta.

**Figure 3.** Ingenuity Pathway Analysis® (IPA)-annotated mechanistic network or differentially expressed genes regulated by critical upstream regulators. Mechanistic network regulated by TGFB1, SMAD3, and SMAD4 (**a**). Differentially expressed genes regulated by the PI3K complex (**b**), SMARCA4 (**c**), CDKN2A (**d**), and TP63 (**e**). Orange-filled and blue-filled shapes indicate predicted activation and inhibition, respectively; red-filled and green-filled shapes indicate increased and decreased expressions, respectively; orange-red lines indicate activation; blue lines indicate inhibition; yellow lines indicate findings inconsistent with the state of downstream activity; grey lines indicate that the effect was not predicted.

Sphingomyelin synthase (*SGMS1*) and sphingomyelin phosphodiesterase 2 (*SMPD2)*, two genes involved in sphingomyelin metabolism, were downregulated in male SPTB placentas (Supplemental Table S13). Sphingomyelins are plasma membrane components, as well as signaling sphingolipids. An altered distribution of sphingomyelin and other sphingolipid species has been shown to play an important role in preeclampsia [31–33]. Sphingomyelin can also be degraded into phosphocholine and ceramide via SMPD2 [34]. Reduced levels of SMPD2 result in decreased levels of ceramides which is associated with impaired trophoblast syncytialization [35]. Ceramides also act as lipid secondary messengers and influence oxidative stress via regulating the expression and activity of antioxidant enzyme manganese-dependent superoxide dismutase (MnSOD) [36].

Pyridoxal 50 -phosphate (PLP), an active form of vitamin B6, acts as a coenzyme in the metabolism of amino acids, lipids, carbohydrates, and one-carbon units. PLP also functions as an antioxidant to prevent free radical generation and lipid peroxidation, modulates mitochondrial function, and regulates the immune system [37,38]. Five genes regulating PLP metabolism were differentially expressed in male SPTB placentas, including *DAPK1* and *PNPO* (Supplemental Table S13). These metabolic changes were consistent with our metabolomics study in SPTB placentas that metabolites of amino acids and sphingolipids were altered in the SPTB placentas [10]. Furthermore, purine metabolism is altered in male SPTB placentas, which was also observed in the placentas from an intrauterine inflammation preterm birth mouse model [39].

Several signaling pathways regulating general energy metabolism were altered in male SPTB placenta, including c-AMP-mediated signaling, signal transducer and activator of the transcription 3 (STAT3) pathway, Janus kinase 2 (JAK2) signaling, and PI3K signaling. c-AMP signaling is important for the differentiation and function of trophoblasts and placentas and is the major route to trigger trophoblast fusion [40]. It also interacts with protein kinase A (PKA) and MAPK signaling and plays a critical role in glucose and lipid metabolism [41]. c-AMP signaling was predicted to be activated in male SPTB placentas (z-score = 1.51). Twelve genes regulating the c-AMP signaling pathway were differentially expressed, including *ADCY1, PDE6H, ADORA2B*, and *CNGA1* (Supplemental Table S13). Not only is PI3K signaling key in immune system functions, it is also a key regulator of glucose and lipid metabolism and oxidative stress through modulating mitochondrial respiratory chain activity, oxidative phosphorylation, and mitochondrial integrity [42–44]. Via PI3K signaling, the placenta can fine-tune the supply of maternal nutrient resources to the fetus [45]. Nine genes comprising this pathway were differentially expressed (Supplemental Table S13). The PI3K complex was also predicted as an activated upstream regulator with a z-score of 2.35. It regulates expression changes of 24 genes in male SPTB placentas (Figure 3b).

These results suggest that the pathways that regulate key metabolic functions of the placenta, including fatty acids and glucose, are altered in SPTB placenta.

### 2.4.3. Retinoids, Vitamin D, and PPAR Signaling Is Disrupted

Nuclear receptors are a superfamily of transcription factors that can bind to DNA directly and regulate the gene expression upon binding to their ligands. The IPA analysis revealed that multiple nuclear receptor signaling pathways were altered in male SPTB placentas, including retinoid X receptors (RXR), peroxisome proliferator-activated receptors (PPARs), vitamin D receptor (VDR), thyroid hormone receptor (TR), and PXR (Table 3). A total of 23 genes comprising these pathways were differentially expressed, including *IGFBP1, IL1RL1, WT1, ADCY1, TGFB1,* and *GSTA1* (Supplemental Table S14). In our study, VDR/RXR activation was predicted to be inhibited in male SPTB placentas with a z-score of −1.34, and PPARα/RXRα activation was predicted to be activated with a z-score = 1.26.

### 2.4.4. Extracellular Matrix and Cell Adhesion Are Disrupted

The extracellular matrix (ECM) is important for the architecture of placental stroma, which supports trophoblasts and provides the environment for a healthy pregnancy. Placentas from pregnancies complicated by preeclampsia exhibit peri-villous coagulation and villous fibrosis, resulting from the overproduction of ECM in the connective tissue [46,47]. Multiple pathways regulating the extracellular matrix were altered in male SPTB placentas, including the Wnt/Ca+ pathway, fibrosis/stellate cell activation, the inhibition of matrix metalloproteases, and the epithelial–mesenchymal transitional pathway (Table 3). Thirtytwo genes comprising these pathways were differentially expressed, including *PDE6H, COL11A2, COL24A1, LEP, IL1RL1, PROK1,* and *MMP12* (Supplemental Table S15).

Cell–cell and cell–ECM communication are important in coordinating proliferation and differentiation during placenta development. Cell adhesion molecules, including transmembrane receptor integrins, can facilitate cell–cell and cell–ECM adhesion [48,49]. Pathways regulating cell adhesion were altered in male SPTB placentas, including epithelial adherens junction signaling, integrin signaling, and gap junction signaling (Table 3). Twenty-five genes comprising these pathways were differentially expressed, including *NOTCH3*, *ITGAD*, *ITGB6*, *MYLK3*, *ADCY1*, and *HTR2B* (Supplemental Table S15).

### 2.4.5. Estrogen Receptor Signaling Is Activated

Interestingly, estrogen receptor signaling was predicted to be activated in male SPTB placentas with a z-score of 1.89 but was unaltered in female SPTB placentas (Table 3). Eighteen genes comprising this pathway were differentially expressed, including *ADCY1*, *LEP*, *MMP12*, *PROK1*, and *VEGFA* (Supplemental Table S16). Estrogen receptor signaling plays a critical role in trophoblast differentiation, placental function, and fetal development and modulates placenta and fetal communication [50–52]. Estrogen also regulates placental angiogenesis via modulating the expression of VEGF, angiopoietin-1, and angiopoietin-2 [53]. Estrogen receptor alpha and beta expression in the placenta are altered in preeclampsia and intrauterine growth restriction [54].

### *2.5. Canonical Pathways Altered in Female SPTB Placentas*

An IPA analysis of 61 non-GA-associated differentially expressed genes in female SPTB placentas revealed nine canonical pathways that were altered in preterm births (Table 4). Most of these are critical pathways regulating the metabolism and nutrient sensing, including PKA signaling, insulin-like growth factor 1 (IGF-1) signaling, Gprotein-coupled receptor (GPCR)-mediated nutrient sensing, α-adrenergic signaling, and cholecystokinin/gastrin-mediated signaling. PKA pathway activation plays a major role in steroidogenic gene regulation in human placentas [55]. PKA is also located in mitochondria and regulates mitochondrial protein phosphorylation [56]. IGF-1 plays a critical role in fetal and placenta growth and development [57,58]. Interestingly, maternal IGF-1 and IGF1R polymorphisms are associated with preterm birth [59] and the expression of IGF binding proteins (IGFBPs) are altered in placentas from idiopathic spontaneous preterm births [21]. α1-Adrenergic signaling stimulates the placenta blood flow, and dysregulation of this pathway has been implicated in placenta ischemia-induced hypertension [60]. Finally, cholecystokinin is one of the most highly upregulated genes in early placentas from women who later developed preeclampsia compared with women who experienced a normal pregnancy; however, its role in the placenta has not been investigated [61]. *CHD5*, *GH1*, *PRKAR1A*, *HIST1H1A*, *PYGM*, *YWHAZ*, and *CCK* were the differentially expressed genes regulating these pathways (Supplemental Table S17).

**Table 4.** Top canonical pathways altered in female SPTB placentas.

The IPA disease and biological function analysis also revealed that 17 genes in female SPTB placentas that regulate lipid, protein, and carbohydrate metabolism, including *CCK*, *GH1*, *APOC3*, *SULT1E1*, *TGM3*, and *ASB4* (Supplemental Table S18), were differentially expressed.

Overall, our data indicate that nutrient sensing and lipid, protein, and carbohydrate metabolism are disrupted in female SPTB placentas.

### *2.6. Upstream Regulators and Regulatory Networks Regulating Nutrient-Sensing, Metabolic, and Mitochondrial Function Are Altered in SPTB Placenta*

The ingenuity pathway analysis can identify upstream regulators that mediate changes in the gene expression. The top upstream regulators identified in male SPTB placenta are listed in Table 5. The activated regulators in male SPTB placentas include SMARCA4, RAF1, and JUN. The important inhibited regulators include PHB2, α-catenin, TP63, and CDKN2A. Many of these upstream regulators are involved in glucose and lipid metabolism, nutrient-

sensing, and mitochondrial function. SMARCA4 is part of the large ATP-dependent chromatin remodeling complex SNF/SWI. It regulates the transcription of many genes for fatty acid and lipid biosynthesis [62,63]. In addition, SMARCA4 also plays a role in trophoblast stem cell renewal and placenta development [64]. SMARCA4 was predicted as an activated regulator (z-score = 3.20) and modulated the expression changes of 28 genes in male SPTB placentas (Figure 3c). Oncoprotein c-Jun (JUN) regulates the cell cycle and apoptosis, and the expression is altered in the placenta in pregnancies complicated by preeclampsia [65]. JUN was predicted as an activated regulator (z-score = 2.21) and regulates expression changes in 31genes in male SPTB placentas. Cyclin-dependent kinase inhibitor 2A (CDKN2A) regulates cell senescence and was predicted as an inhibited upstream regulator, and the expression of 15 of its downstream targets was disrupted (Figure 3d). Attenuation of the senescence program occurs in IUGR human placentas, and a knockdown of the CDKN2A expression results in functional and morphological abnormalities in murine placenta [66]. Tumor protein p63 (TP63) was predicted as an inhibited regulator (z-score = −2.01), modulating the expression changes of 25 genes in male SPTB placentas (Figure 3e). It regulates cytotrophoblast differentiation and fusion [67].

**Table 5.** Top upstream regulators altered in male SPTB placentas.