**1. Introduction**

There is a rapidly increasing interest in placentation in obstetric adverse outcome, neonatal and adult life health after infertility, and its related therapies. Abnormal placentation is a common finding in the infertile population, even among those couples conceiving spontaneously after a period of infertility. A higher risk of preterm birth (PTB) and low birth weight (LBW) has been found in these pregnancies [1]. Moreover, abnormal placentation and obstetric complications such as PTB, preeclampsia (PE), and fetal growth restriction (FGR) have been associated with endometriosis, a common factor of infertility [2]. Another maternal condition at risk for the development of placental anomalies, commonly associated with infertility, is polycystic ovary syndrome (PCOS), in which the prevalence of gestational diabetes mellitus (GDM) is significantly increased [3]. Of note, GDM is an independent risk factor for the onset of placental disorders with altered structure, function and hypertrophic growth of the organ [4].

**Citation:** Manna, C.; Lacconi, V.; Rizzo, G.; De Lorenzo, A.; Massimiani, M. Placental Dysfunction in Assisted Reproductive Pregnancies: Perinatal, Neonatal and Adult Life Outcomes. *Int. J. Mol. Sci.* **2022**, *23*, 659. https:// doi.org/10.3390/ijms23020659

Academic Editor: Micheline Misrahi

Received: 23 November 2021 Accepted: 5 January 2022 Published: 8 January 2022

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An important actual focus in reproductive medicine, neonatology and population health is that up to 6% (range between 0.2% and 6.4%) of European births is conceived by assisted reproductive technology (ART) [5] and concerns existing health conditions of individuals born following ART. ART is a group of in vitro techniques used to treat moderate and severe infertility, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), frozen embryo transfer (FET), oocyte donation (OD), blastocyst culture, intrauterine insemination, and preimplantation genetic testing for aneuploidy (PGT-A). Unfortunately, each of these techniques may represent a possible confounding factor to the identification of a precise relationship between ART procedures and obstetric or neonatal outcomes, including children and their adulthood health. Many studies report an evident increase of obstetrical risk and perinatal complications with ART, especially for hypertensive disorders of pregnancy (HDP), from gestational hypertension to eclampsia [6–13]. A recent meta-analysis of 50 cohort studies including 161,370 ART and 2,280,241 spontaneously conceived singleton pregnancies found increased risks for several obstetric complications. Significant worst outcomes regarded pregnancy-induced hypertension, placenta previa, abruption, antepartum hemorrhage, oligohydramnios, cesarean delivery, PTB, very low birth weight (VLBW), LBW and perinatal mortality and morbidity [14].

The role of the placenta in these conditions has been demonstrated in several studies. It is well documented that ART can be associated with changes in placental morphology and structure, growth dynamics, imprinted and non-imprinted genes, and other aspects regulating placentation [15]. Several studies demonstrate that incidence of placenta previa was significantly higher in ART than in spontaneous conception (OR 3.14, 95% CI [8]; RR 3.71, 95% CI [16]). Moreover, the placentas from ART pregnancies presented a significantly greater weight and higher placental weight-to-birth weight ratio [17,18]. Some observations indicate that placentas of pregnancies obtained by ART have an increased placental thickness and higher incidence of hematomas [19]. It has been also demonstrated that in children conceived by ART the frequency of imprinting disorders is higher than expected in the general population. ART is associated with increased risk of epigenetic alterations influencing gene expression and DNA methylation in early development and in the placenta, eventually triggering diseases and affecting long-term health [20–22]. These findings support the hypothesis of a primary ART responsibility for adverse perinatal outcomes with evident suspicions for anomalies of placentation, although fertility treatments themselves are often associated with impaired placentation and related adverse pregnancy outcomes [1]. Infertile couples who did not undergo ART showed an increased risk of adverse obstetric outcomes, such as placental abruption, fetal loss and GDM [23].

Based on these observations, the main question to address is whether placental alterations (from macroscopic to molecular level) observed in treated infertile patients are the result of ART or if they originate from infertility itself. A deeper investigation of the underlying molecular basis of reduced placental functions in infertility condition and especially after ART treatment is then needed.

In this review, we provide a summary of the most recent literature on placental dysfunction associated with obstetric, perinatal outcomes in singletons born after NON-ART and ART treatments. ART and infertility may be the cause of common dysregulated pathways, including changes in trophoblast invasion, environmental conditions, vascular defects, chronic inflammation, and oxidative stress. Each of them can be prevalent or coexist with others with different and common molecular pathways in a grading and timing that can configurate many clinical features, leading often to neonatal–adult and maternal consequences. This review is based on systematic reviews (SRs), large cohort studies, meta-analyses, and genetic, epigenetic and molecular studies.

#### **2. Abnormal Placentation and ART: Molecular Factors and Involved Signaling** Abnormal placentation may present in a variety of phenotypes, severity, clinical conditions and consequences as the result of several types of infertility treatments and

**2. Abnormal Placentation and ART: Molecular Factors and Involved Signaling**

*Int. J. Mol. Sci.* **2022**, *22*, x FOR PEER REVIEW 3 of 20

Abnormal placentation may present in a variety of phenotypes, severity, clinical conditions and consequences as the result of several types of infertility treatments and techniques used in ART. For these reasons, it becomes difficult to linearly relate placental influence to obstetrical and perinatal (or neonatal) outcomes after ART. techniques used in ART. For these reasons, it becomes difficult to linearly relate placental influence to obstetrical and perinatal (or neonatal) outcomes after ART. An altered expression of factors and molecules involved in proper placental development, leading to impaired trophoblast invasion and subsequent reduced vascular

An altered expression of factors and molecules involved in proper placental development, leading to impaired trophoblast invasion and subsequent reduced vascular remodeling and placenta hypoperfusion, sustain several clinical conditions leading to obstetric and perinatal risks often found in ART pregnancies, such as PE, FGR and placenta previa or accrete (Figure 1) [24–28]. Syncytiotrophoblast stress has been associated with a dysregulated expression of placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFLT-1) [29,30]. Circulating levels of the anti-angiogenic factor sFLT-1 are increased, and those of PlGF are decreased even before the onset of the clinical symptoms of PE and FGR [31–35]. The increased ratio sFLT-1:PlGF is thought to contribute to the systemic endothelial response and correlate with the severity of FGR and PE. A recent study indicates that the release of sFLT-1 from the placenta is regulated by the epidermal growth factor receptor (EGFR) pathway and the mitochondrial electron transport chain and its downstream pathways, both significantly increased in preeclamptic placentas [36]. The inhibition of these signaling pathways significantly reduces sFLT-1 release from primary cytotrophoblast cells [36]. Vrooman demonstrated in the rat model that an embryo culture from the 1-cell to blastocyst stage increased levels of sFLT-1 together with placental overgrowth, reduced fetal weight, and lower placental DNA methylation [37]. remodeling and placenta hypoperfusion, sustain several clinical conditions leading to obstetric and perinatal risks often found in ART pregnancies, such as PE, FGR and placenta previa or accrete (Figure 1) [24–28]. Syncytiotrophoblast stress has been associated with a dysregulated expression of placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFLT-1) [29,30]. Circulating levels of the anti-angiogenic factor sFLT-1 are increased, and those of PlGF are decreased even before the onset of the clinical symptoms of PE and FGR [31–35]. The increased ratio sFLT-1:PlGF is thought to contribute to the systemic endothelial response and correlate with the severity of FGR and PE. A recent study indicates that the release of sFLT-1 from the placenta is regulated by the epidermal growth factor receptor (EGFR) pathway and the mitochondrial electron transport chain and its downstream pathways, both significantly increased in preeclamptic placentas [36]. The inhibition of these signaling pathways significantly reduces sFLT-1 release from primary cytotrophoblast cells [36]. Vrooman demonstrated in the rat model that an embryo culture from the 1-cell to blastocyst stage increased levels of sFLT-1 together with placental overgrowth, reduced fetal weight, and lower placental DNA methylation [37].

**Figure 1.** Molecules regulating proper placental development, whose dysregulation is involved in an abnormal placental development. NO, nitric oxide; VEGF, vascular endothelial growth factor; GATA3, GATA binding protein 3; PlGF, placental growth factor; sFLT-1, soluble fms-like tyrosine kinase 1; sEndoglin, soluble endoglin; pEGFL7, placental epidermal growth factor-like domain 7; cEGFL7, circulating epidermal growth factor-like domain 7; ELA, elabela. **Figure 1.** Molecules regulating proper placental development, whose dysregulation is involved in an abnormal placental development. NO, nitric oxide; VEGF, vascular endothelial growth factor; GATA3, GATA binding protein 3; PlGF, placental growth factor; sFLT-1, soluble fms-like tyrosine kinase 1; sEndoglin, soluble endoglin; pEGFL7, placental epidermal growth factor-like domain 7; cEGFL7, circulating epidermal growth factor-like domain 7; ELA, elabela.

Over the last decades, several other factors have been demonstrated to be altered in pregnancy disorders associated with abnormal placentation (e.g., soluble endoglin (sEndoglin), PlGF and epidermal growth factor-like domain 7 (EGFL7)), with the aim to create a panel of markers to allow an earlier and more precise diagnosis of PE. sEndoglin has been shown to be upregulated in abnormal placenta, typical of FGR and PE, and released into the maternal blood, where it acts as antiangiogenic factor by inhibiting Over the last decades, several other factors have been demonstrated to be altered in pregnancy disorders associated with abnormal placentation (e.g., soluble endoglin (sEndoglin), PlGF and epidermal growth factor-like domain 7 (EGFL7)), with the aim to create a panel of markers to allow an earlier and more precise diagnosis of PE. sEndoglin has been shown to be upregulated in abnormal placenta, typical of FGR and PE, and released into the maternal blood, where it acts as antiangiogenic factor by inhibiting transforming growth factor-beta (TGF-β) signaling in the vasculature. sEndoglin markedly increased,

beginning 2 to 3 months before clinical manifestations of PE [38,39]. EGFL7, originally discovered as a largely endothelial restricted gene, has been recently demonstrated to be expressed in the placenta and involved in placental angiogenesis and trophoblast migration [40–42]. Altered EGFL7 expression is associated with abnormal placentation and systemic maternal endothelial dysfunction, observed in PE [40,43,44]. Maternal treatment with nitric oxide (NO) donors increases placental EGFL7 levels and improves maternal hemodynamic state and perinatal outcome [45,46]. In the maternal circulation, endothelial dysfunction and abnormal hemodynamic state are associated with increased levels of EGFL7, which return to control levels after NO treatment [44]. Moreover, EGFL7 dosage in maternal circulation allows to discriminate between PE and FGR [43]. Although there are no data correlating EGFL7 with ART, dosage of its circulating levels could help identify ART-associated abnormal placentation.

In FGR and placental insufficiency, the levels of the hormone melatonin are significantly reduced, and this decrease is correlated with proinflammatory activities of tumor necrosis factor alpha (TNF-α), interleukine-1beta (IL-1β), and IL-6 [47]. Melatonin is an antioxidant factor and an anti-inflammatory agent [48,49]. Several studies support the production of this hormone in the ovary as a whole [50,51], the granulosa cells, including those making up the cumulus oophorus [52,53], and the oocyte [54]. In women undergoing ART, melatonin significantly increased the implantation rate [55]; a similar result was obtained in women affected by PCOS undergoing intrauterine insemination [56]. Melatonin crosses the cell membrane, thus interacting with intracellular molecules via different signaling pathways and displaying scavenger functions [57]. Melatonin upregulates the primary implantation receptors, ErbB1 and ErbB4, and significantly reduces intracellular ROS in mouse blastocysts (increasing the embryo total antioxidant capacity) and promotes mitosis of the inner cell mass and trophectoderm cells [58]. Recently, it has been demonstrated that the supplementation of culture medium with melatonin (10−<sup>9</sup> mol/L) improves the growth of mouse parthenogenetic embryo potentially by promoting cell cycle progression [59]. Despite all these functions, melatonin is not present in culture media used in ART. We could speculate that the addition of melatonin to the ART media may be beneficial.
