In the normal process of implantation blastocysts attach to the decidua, which stimulates cytotrophoblast differentiation into the villous and extravillous trophoblasts [1]. EVT migrate through the decidua into the underlying third of the myometrium and maternal vasculature, forming placental bed, and initiate spiral artery remodeling [2, 3]. Placenta Accreta Spectrum (PAS) disorders fall within the group of Placental Implantation Abnormalities (PIA) [4]. Clinically, it featuresabnormal adherence of the placenta to the uterine wall and the absence or/and defective decidua [5].Recently, the subgroups of placenta accreta, increta and percreta were re-defined into gradesfrom 1 to 3, respectively, according to the degree of accreta invasiveness. Histologically:

• grade 1 (accreta), the placental villi directly attach to the myometrium
• grade 2 (increta), placental villi intervene with the myometrial fibers or extend deep into the uterine vasculature.
• grade 3 (percreta) features villous tissue/trophoblasts within or even penetrating the serosa; in severe cases, they also invade the bladder wallor other pelvic organs.

EVT are assumed to undergo epithelial-to-mesenchymal transition (EMT) which enhances migratory capacity and invasiveness [6]. However, their EMT- mediated invasion ceases beyond the early second trimester [7] which limit the trophoblast invasion to the early stages of the pregnancy [8]. In PAS, EVT seem to maintain EMT features even later in pregnancy[9, 10]. Activation of EMT has been proposed as a crucial dedifferentiation step from benign noninvasive to malignant phenotypes of epithelial cancer cells, cancer invasion and metastasis [11, 12]. As the EVT invade into the decidua, they secreteregulatory factors such as proteolytic enzymes, which dissolve extra cellular matrix. Secreted activators act on proteinase and matrix metalloproteinase present in the decidua. Crosstalk between trophoblasts and decidua ensures controlled yet sufficient invasion [2]

Decidua secretes pro-invasive factors such us IL-1ß, IL-6. LIF, IL-11, IL-8, IL-15, IP-10, RANTES, Eotaxin, IL-7 and anti-invasive factors IL-10 and IL12 [13, 14]. IL-10 and IL-12 regulate MMP-2, MMP-9 and TIMP-1 expression, respectively [15]. MMP-2 and -9 occur to be the most prominent proteinases secreted by the EVT [16]. Immunohistochemical analysis of MMP2 expression of placenta percreta tissue sections resulted in higher expression of MMP-2, supporting the invasion of the placenta [17], yet this could not be noted at the mRNA level in placenta accreta cases [18]. Probably, as the placenta accreta features lower severity of invasion compared to placenta percreta, thus the expression levels of MMP-2 differ between the grades of invasion. In cancer, higher MMP-2, but also MMP-9 activity was linked not only to tumor cell invasion, but also to angiogenesis [19-23].

Recently, an analysis of the immune cell population in PASreveled decreased uNK cells, CD4+ T cells, Fox3P+ Tregs, and rare B cells with higherimmune cell infiltration rates in the placental bed [350,351]. On the other hand, Schwedeet al. reported increased Fox3P+ cell count and decreased counts of immature non-activatedCD209+ DCs [352].

To determine the differentially expressed genes of the placental bed microenvironment between PAS and control.

FFPE tissue sections of 20 samples were microdisected with LCM. RNA was isolated, libraries for bulk RNAseq were created and sequenced. Raw data was processed with nf-core/rnaseq’pipeline in its default settings to derive gene counts.

Study design PAS: areas of the myometrium with trophoblasts Ctrl: areas of decidua and myometrium with invaded trophoblasts CtrlMus: areas of myometrium without trophoblasts

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