**4. Metabolic and Cardiovascular Consequences of Placental Dysregulation in Mothers Following ART**

Studies demonstrated that vascular dysfunctions in IVF pregnancies were similar to those identified when babies were conceived naturally from mothers affected by PE [132,133]. Sundheimer and Pisarska explain that the size of the placental bed and successful trophoblast invasion and spiral artery remodeling determine maternal blood flow. With these premises, abnormal placentation associated with infertility can represent a consequent marker of overall health for both the mother and her offspring [134]. It has been well known for a long time that inadequate trophoblast invasion and poor spiral artery remodeling may cause reduced vascular perfusion in pregnancy [135].

Women of advanced maternal age are generally at increased risk of GDM, HDP, operative and cesarean deliveries [136,137]; however, those who conceive with the use of IVF are at increased risk of retained placenta, suggesting that placentation abnormalities may contribute to maternal morbidity, and this may be more pronounced in women with infertility [138].

Fertility treatments (IVF and NON-IVF combined) have been associated with an increased risk of severe maternal morbidity (SMM), defined as unexpected outcomes of labor and delivery that result in significant short- or long-term consequences to a woman's health after [139]. In a population-based cohort study of 114,409 singleton pregnancies with conceptions dating from 11 January 2013 to 10 January 2014 in Ontario, IVF was associated with an increased risk of SMM (rate 11.3/1000; aRR 1.89, 95% CI: 1.06–3.39) [140]. In this study, the authors also noted supra-additive effects of high body mass index (BMI) and IVF on the risk of PE and GDM. The link between fertility treatment and SMM was also confirmed in another cohort study conducted from Ontario between 2006 and 2012, demonstrating that women undergoing fertility treatment are at higher risk of SMM with a relative risk of 1.39 (95% confidence interval (CI) 1.23–1.56). Indicators of SMM were considered severe postpartum hemorrhage, maternal admission to an intensive care unit, puerperal sepsis, hysterectomy and cardiac conditions [141]. The American Heart

Association considers a history of PE or HDP as a major risk factor in these women for the development of cardiovascular diseases (CVD) [142].

In summary, cardiovascular and metabolic diseases derived from impaired placentation following infertility treatments may have long-term consequences on both the mother and the newborn.

### **5. Health Risk in Infancy as a Consequence of Placental Dysfunction Following ART**

According to the "developmental origins of health and disease theory" [143] and the Barker hypothesis of the developmental origins of chronic adult disease [144], placental dysfunction and abnormal fetal development may have long-term consequences on the neonate and his development, from childhood to adult life. A higher risk of diabetes mellitus and CVD were found in children whose mothers had PE and HDP, by long-term follow up observations [145,146]. A track of this condition is represented by the finding of reduced endothelial function in mothers and children after PE [147].

Evidence suggests, at least in animal studies, that IVF can promote endothelial dysfunction with increased vessel stiffness in offspring. This can induce arterial hypertension in vivo and an epigenetic origin of the condition has been suggested [132]. IVF causality is also suggested by the significantly elevated activity of enzymatic regulators in the cardiovascular system of IVF offspring [148]. Additionally, there is increasing evidence that offspring conceived by IVF displays a level of vascular dysfunction similar to that seen in children spontaneously conceived by mothers with PE [132,133]. Of note, in 2015 in a randomized case-control trial, antioxidant administration to IVF children was able to improve NO bioavailability and responsiveness of both systemic and pulmonary circulation [149], thus indicating that vascular dysfunctions induced by ART are reversible in young people.

Regarding the increased metabolic risk later in adult life, IVF-conceived mice showed decreased glucose tolerance, increased fasting glucose levels, and reduced insulin-stimulated protein kinase B (Akt) phosphorylation in the liver later in their adult life [150]. These findings are similar to those seen in humans. Some individuals conceived by ART have shown LBW, higher weight, height, and BMI in late infancy [151] and fasting insulin levels [152]. Adolescents conceived by ART showed significant differences in growth kinetics, glucose levels, and blood pressure in comparison with those conceived naturally by sub-fertile parents [153,154], suggesting that these alterations may be caused by ART procedures. Development delay at birth (probably caused by epigenetic alterations) and accelerated growth in late infancy, with metabolic alterations in adolescence, may predispose these individuals to chronic diseases such as obesity and type 2 diabetes [155,156]. An epigenetic programming of metabolism during prenatal and postnatal periods as a response to imprinting alterations that occurred during early embryonic development has been hypothesized [156,157].

### **6. Simultaneous Action of Factors Dysregulating Normal Placentation**

Infertility and its treatments, including ART and its various procedures (ICSI, blastocyst culture + FET + PGT-A with ovarian stimulation or hormonal preparation), may individually or synergistically participate in the dysregulation of embryo and placenta development. Consequently, it is probably impossible to separate the relative contribution of the many factors influencing embryo, placental, and newborn health when an infertile woman is treated with the many types of ART techniques. These techniques may represent confounding factors for a full understanding of previous studies and future ones to be designed.

Each technique may lead to important epigenetic changes and differential gene expression in the placenta [82,83] or damage the developing embryos with thermal, oxidative and mechanical stressing actions [82,98,158].

Pivotal reproductive hormones, such as human chorionic gonadotropin, progesterone and estradiol, are found at high concentrations at the maternal–fetal interface during vessel remodeling. This observation allows to hypothesize that such relevant vascular transformation may be under the influence of these hormones, which may control trophoblast migration [90,159]. A potential role in placental function dysregulation has been proposed for estrogens. In this respect, elevated estrogen exposure (as in controlled ovarian hyperstimulation) has been associated with higher rates of LBW and FGR [160].

Are perinatal adverse outcomes in infertile patients the consequence of ART or are they the consequence of the underlying infertility? A possible answer to this basic question may come from infertile couples conceiving spontaneously with no ART treatments; in these couples, a higher risk of PTB and LBW has been reported [1]. However, an increasing number of observations show vascular dysfunctions similar to those observed in PE in IVF offspring [132,133], and a common mechanism of action could be postulated.

The induction of defective methylation and consequent alteration of gene expression, which may impair placentation [85], can be caused by oxidative stress [161,162], as in endometriosis and by the altered hormonal milieu, i.e., supraphysiologic estradiol levels due to ovarian hyperstimulation [97]. Particular negative effects on embryo development may occur in PGT-A procedures, in which the epigenetic risk for long in vitro culture may be added to the trophectoderm mechanical stress. If the patient is also affected by PCOS, this adverse metabolic condition for the placenta would considerably increase the overall obstetric, perinatal and postnatal final risk [76,77]. All these factors could exert additive effects, leading to the pathologic condition.
