**1. Introduction**

Early placental development is one of the main factors determining perinatal fetal growth and postnatal fetal and maternal health. In humans, blastocyst implantation is an invasive process that occurs 7–9 days after fertilization [1]. Rapidly proliferating cytotrophoblast cells (CTBs) are the progenitor trophoblast cells which proliferate as well as differentiate into different trophoblast lineages throughout gestation [2]. If the balance between proliferation and differentiation of CTBs is dysregulated, it can result in severe disorders including preterm birth, intrauterine growth restriction (IUGR), and preeclampsia [3,4]. These pregnancy related disorders affect about a third of human pregnancies [5].

In sheep, the blastocyst hatches out of the zona pellucida at day 8–9 and is surrounded by a single layer of mononuclear cells called trophectoderm (TE) [6]. Instead of invading the uterus, the hatched blastocyst elongates from day 11–16 due to rapid proliferation of trophoblast cells and adopts a filamentous shape comprised of mainly extraembryonic trophoblast cells [7–9]. Conceptus elongation is critical for implantation, placentation, and successful establishment of pregnancy in sheep [10–12]. Reduced conceptus elongation and compromised placental function in domestic ruminants is one of the main causes of embryonic mortality resulting in reduced fertility [13–15]. Rapid trophoblast proliferation is an important phenomenon during early stages of pregnancy in both humans and domestic ruminants. The molecular mechanisms involved in regulating trophoblast proliferation and invasion are not well understood. Therefore, exploring the genes involved in sheep trophectoderm elongation can help to better understand the reasons for reduced fertility in domestic ruminants and to improve the diagnosis and treatment of various pregnancy-related disorders in humans.

Trophoblast proliferation and differentiation is an intensively regulated process, and the role of several genes in placental development has been studied using various in vivo and in vitro models [16–20]. The pluripotency factor LIN28 is a highly conserved RNA binding protein which is expressed in placenta and has two paralogs, LIN28A and LIN28B [21,22]. It is usually described as a protooncogene due to its ability to regulate and stabilize oncogenes at the post-transcriptional level in tumor cells [23,24]. It also inhibits the biogenesis of lethal-7 (*let-7)* miRNAs in mammalian cells by binding pri-*let-7* and pre*-let-7* [25–30]. LIN28 is low and *let-7* miRNAs are high in differentiated cells and adult tissues, hence *let-7* miRNAs are considered markers of cell differentiation [31–33]. *Let-7* miRNAs reduce the expression of different proliferation factors either by directly targeting their mRNA or through chromatin-dependent pathways by targeting the ARID3B-complex, which is comprised of AT-Rich Interaction Domain 3A (ARID3A), AT-Rich Interaction Domain 3B (ARID3B) and lysine demethylase 4C (KDM4C) [18,34]. We recently showed that term human placentas from IUGR pregnancies had reduced LIN28A and LIN28B and high *let-7* miRNAs compared to term human placentas from control pregnancies [18]. We further demonstrated that LIN28B is localized to cytotrophoblast cells in human placenta, and knockout of LIN28 in immortalized first trimester human trophoblast (ACH-3P) cells leads to an increase in *let-7* miRNAs, reduced expression of proliferation-associated genes, and reduced cell proliferation [18–20].

Insulin like growth factor 2 mRNA binding proteins (*IGF2BP1, IGF2BP2, IGF2BP3*), high mobility group AT-hook 1 (*HMGA1*), *ARID3B,* and MYC protooncogene (*c-MYC)* are all *let-7* miRNA targets with known roles in cell proliferation [18,35–41]. IGF2BPs are highly conserved RNA binding oncofetal proteins with three paralogs, IGF2BP1, IGF2BP2, and IGF2BP3 [42]. By binding different mRNAs, IGF2BPs decide the fate of those mRNAs by controlling their localization, stability, and translation [40]. Many studies have reported the role of IGF2BPs in cell proliferation, cell invasion, tumorigenesis, and embryogenesis [40–51]. IGF2BPs have also been found in sheep trophoblast cells suggesting their role in rapid proliferation of these cells [52]. HMGA1 promotes invasion of trophoblast cells and reduced levels of HMGA1 has been linked to pathogenesis of preeclampsia [53,54]. ARID3B binds with ARID3A and KDM4C to form the ARID3B-complex. The ARID3B-complex plays a vital role in cell proliferation by transcriptional regulation of stemness genes including *HMGA1*, *c-MYC*, vascular endothelial growth factor A (*VEGF-A*), and Wnt family member 1 (*WNT1*) [18,34,55–59]. ARID3B knockout in immortalized first trimester human trophoblast cells results in reduced proliferation of these cells [18]. The *c-MYC* protooncogene has been identified as a proliferation factor in human cytotrophoblast cells and its level is reduced when cytotrophoblasts differentiate into syncytiotrophoblast [60].

To date, the role of LIN28-*let-7* miRNA axis in trophoblast cells has not been studied in vivo. The aim of this study was to demonstrate the role of LIN28-*let-7* axis in the regulation of proliferation-associated genes in trophoblast cells in vivo. We used sheep as an in vivo model to generate trophectoderm specific knockdown of LIN28A or LIN28B by infecting day 9-hatched blastocysts with shRNA-expressing lentiviral particles. This way, only the trophoblast cells will be infected by the lentiviral particles [61–63] and any phenotype will be due to knockdown of LIN28A or

LIN28B in trophoblast cells. We hypothesized that the LIN28-*let-7* miRNAs axis plays an important role in sheep trophoblast cell proliferation and conceptus elongation by regulating the expression of genes associated with cell proliferation including *IGF2BP1, IGF2BP2, IGF2BP3*, *HMGA1*, *ARID3B,* and *c-MYC*.
