Implantation is considered a bidirectional interaction between the embryo and the endometrial surface, including adhesion, invasion, and angiogenesis. This complex process occurs over approximately a 3–5-day interval during receptivity of the endometrium. This phase is essential for attaching the trophectoderm layer and subsequent invading and vascularization of the embryo. The limited duration of uterine receptivity for implantation during the mid-luteal phase is defined as the “window of implantation” (WOI), with a specific gene expression profile appropriate for embryo attachment and accommodation. Failure of implantation is the leading cause of pregnancy loss in assisted reproductive technology (ART), and it mainly stems from either impaired endometrial receptivity or low-quality embryos. These two are the pivotal causes of unsuccessful implantation, miscarriage, and RIF in people who undergo IVF (Simon and Laufer, 2012; Lessey and Young, 2019; Enciso et al., 2021). Maternal causes of the RIF include anatomical abnormalities, thrombophilia, infection, genetic alteration, immunological factors, and endometrial receptivity (Simon and Laufer, 2012; Bashiri et al., 2018; Makrigiannakis et al., 2021). Studies have demonstrated that RIF patients suffer from relatively lower endometrial receptivity at the time of embryo transfer (Ruiz-Alonso et al., 2013).

A broad spectrum of etiologies are involved in RIF, but the exact mechanisms are not properly understood (Simon and Laufer, 2012; Bashiri et al., 2018). So, looking for further molecular mechanisms that participate in implantation failure is crucial. Meanwhile, microRNAs (miRNAs) are potential modulators of the involved signaling pathways in embryo implantation (Galliano and Pellicer, 2014; Zhou et al., 2020).

miRNAs are 18–25 non-coding nucleotide chains that act as post-transcriptional gene modifiers. miRNAs are substantially involved in modulating normal cells’ development, including cellular differentiation, proliferation, apoptosis, embryo early development, embryo-endometrial communication, endometrium receptivity, implantation, decidualization, and placenta formation (Galliano and Pellicer, 2014; Kolanska et al., 2021). Studies have shown that, in the bidirectional communication between the embryo and the endometrium, both secrete particular microRNAs, which can be taken up by the other party and influence the implantation process (Liang et al., 2017). These secreted miRNAs are rather stable and accessible in embryo culture and uterine fluid, making them potential non-invasive biomarkers to confirm the embryo quality.

Several studies demonstrated that impaired miRNA expression contributes to RIF. Both overexpressed and under-expressed endometrial miRNA patterns have been reported in the mid-secretory phase of RIF patients compared to fertile women (Revel et al., 2011). The expression of endometrial microRNAs is different in impaired endometrium during WOI. Previous studies have revealed unique irregular expression of 105 miRNAs in individuals with RIF (Shi et al., 2017). There was also a positive correlation between upregulation of these miRNAs and successful implantation 24 h after frozen embryo transfer. Downregulation of miR-198, miR-522, and miR-891a has been shown in implantation failure (Parks et al., 2014). Kuokkanen et al., (2010) have shown higher expression levels of 12 miRNAs, including miR-30d, during the mid-secretory phase of the menstrual cycle (Kuokkanen et al., 2010). Additionally, endometrial and serum expression of miR-203, miR-31, miR-30b, and miR-30d were notably unregulated during the implantation period (Kresowik et al., 2014). Other studies have also shown that microRNA polymorphisms significantly differ between individuals with RIF and fertile controls (Lee et al., 2019). So, miRNAs can be used as a regulator of the implantation process.

MicroRNA-30d acts as a crucial coordinator for embryo-maternal cross-talks and regulates plenty of gene expressions involved in human embryonic implantation. Analyzing miRNA expression patterns showed significant upregulation of miR-30d in the epithelial layer of receptive human endometrium in the early mid-secretory phase versus pre-receptive endometrium (Altmäe et al., 2013; Moreno-Moya et al., 2014). Human endometrium secretes free form or exosome-coated miR-30d in the embryo fluid (EF), where the pre-implantation embryo can take it up via the trophectoderm layer (Figure 1). Also, treating mice embryos with miR-30d resulted in increased embryo adhesion rate, whereas using a specific miR-30d inhibitor led to a reduced adhesion rate. Additionally, the adhesion molecules such as integrin beta-3, integrin alpha-7, and cadherin-5 were upregulated following mir-30d treatment (Vilella et al., 2015). It was also shown that miR-30d in mice and humans had the same targets to extend the results to humans (Vilella et al., 2015).

Balaguer et al. showed the correlation between maternal miR-30d deficiency and diminished endometrial receptivity markers, which led to decreased implantation rates and impaired fetal development in mice (Balaguer et al., 2019). Furthermore, miR-30d transfection in the epithelial cell line of human endometrium induced the expression of abundant mRNAs and proteins associated with embryo adhesion, implantation, and development (Moreno-Moya et al., 2014).

In another study in 2021, it was demonstrated that miR-30d-5p expression levels were considerably decreased in women with RIF compared to average fertile women (Zhao et al., 2021). miR-30d-5p is also possibly responsible for SOCS1 downregulation, which ultimately inhibits the activation of the LIF-induced STAT3 pathway. In addition to embryo adhesion, miR-30d enhances endometrial angiogenesis by inhibiting MYPT1 and consequent VEGF activation (Lin et al., 2017; Zhao et al., 2021). Therefore, the impact of miR-30d and associated pathways on implantation can improve the outcome.

Upregulation of specific molecular pathways involved in human embryo implantation has been detected in WOI. Adhesion molecules such as integrin beta-3, cadherin-5, L-selectin, and mucin 1 (MUC-1) play critical roles in apposition and adhesion. Growth factors such as insulin-like growth factor 1 (IGF), heparin-binding epidermal growth factor (HB-EGF), and vascular endothelial growth factor (VEGF) are essentially required to develop a normal vascular network. Additionally, inflammatory responses enhance endometrial receptivity during WOI through inflammatory cytokines, including IL-1, IL-6, and leukemia inhibiting factor (LIF), which regulate fetal-maternal interactions during pregnancy and are essential for embryo implantation, trophoblast growth, and differentiation (Trolice and Amyradakis, 2012). LIF/gp130/STAT pathway is also crucial for embryo implantation as it is pivotal in angiogenesis. LIF acts through activation of gp130/STAT3 signals and subsequent VEGF expression. LIF glycoprotein expression level increases during the WOI in the secretory phase of normal fertile women. On the contrary, in RIF patients, the LIF level in the secretory phase was decreased (Hambartsoumian, 1998). Furthermore, endometrial and blood levels of LIF were concordantly decreased in RIF patients, followed by pregnancy failure (Comba et al., 2015).

There was also a significant decrease in both protein and mRNA levels of LIF and gp130 in most women with unexplained infertility and RIF at the proliferative and the secretory phases (Hambartsoumian, 1998; Tawfeek et al., 2012; Zhao et al., 2021). Heterozygous mutation of the LIF gene results in reduced LIF activity and may be the infertility cause in some women (Giess et al., 1999). Moreover, LIF/JAK/STAT3 pathway is involved in angiogenesis in the endometrium and placenta via inducing VEGF expression. LIF also positively impacts integrin expression, and VEGF per se induces integrin expression, so they are crucial in inducing the adhesion (Alfer et al., 2017) (Figure 1).

As shown in Figure 1, activation of LIF/JAK/STAT3 is also involved in promoting invasion via increasing the expression of MMPs (Suman et al., 2013). Upregulation of the JAK/STAT3 pathway negatively impacts SOCS1 expression as well. SOCS1 expression is increased in the endometrium of the RIF patients. In a recent study in 2021, decreased LIF and p-STAT3 protein levels in the RIF patients were detected, which may represent the critical role of this pathway for successful embryo implantation. The negative correlation between SOCS1 and miR-30d-5p has been demonstrated in the mentioned study. Decreased levels of miR-30d in the RIF group reduce STAT3 and phosphorylation of JAK, while SOCS1 is significantly increased, representing that the SOCS1 gene is the target of miR-30d, which participates in embryo implantation (Zhao et al., 2021). The reduced angiogenesis by inhibition of the LIF/JAK/STAT3 pathway can be compensated by miR-30d upregulation. miR-30d is one of the angio-miRs and promotes angiogenesis via MYPT1/c-JUN/VEGFA pathway (Lin et al., 2017).

VEGF is an essential element in the embryo-endometrium reciprocal interactions. VEGF promotes vascularization and improves blastocyst adhesion, implantation, and growth capability. Furthermore, endometrial angiogenesis is stimulated by the embryo via the production of active VEGF-A, which can induce vessel formation and consequent placental development. It has been shown that VEGF-A gene mutation leads to embryo loss and impaired placental development, and other developmental anomalies (Carmeliet et al., 1996; Ferrara et al., 1996; Kapiteijn et al., 2006; Guo et al., 2021). VEGF gene polymorphisms correlate with an increased risk of RIF (Jung et al., 2016; Shim et al., 2018). It has also been reported that VEGF expression was significantly reduced in women with RIF (Hannan et al., 2011; Lash et al., 2012). Therefore, impaired vascularity in the endometrium of the group with unexplained subfertility during the mid-late follicular phase has been demonstrated (Raine-Fenning et al., 2004). This diminished blood perfusion may lead to pathological hypoxia, impairing endometrial receptivity in infertile patients. However, 2%–5% oxygen concentration at the initial steps of embryo development is known as physiological hypoxia, and it is not life threatening.

“Hypoxia-inducible factor 1 (HIF-1)” is overexpressed in tissues with low oxygen concentrations and acts as a hypoxia-sensitive transcription factor. HIF-1 modifies cell adaptation to hypoxic conditions as it is upregulated even in basic levels of physiological hypoxia (Moeinabadi-Bidgoli et al., 2021). Additionally to normal embryo development, which occurs in a physiologic hypoxic environment, HIF-1 also improves embryo’s survival in pathological hypoxic environments (Dunwoodie, 2009). Moreover, endometrial HIF-1α expression is upregulated in RIF women, owing to the possible hypoxic microenvironment in these patients’ endometrium (Xu et al., 2011; Chen et al., 2016). This overexpression possibly promotes local angiogenesis to overcome hypoxia-induced consequences. Studies have shown that vascularization at implantation sites is promoted by HIF-1α expression via inducing VEGF gene expression (Guo et al., 2021). Eventually, error-free function of the implantation-related signaling pathways seems indispensable for a successful pregnancy.

In 2019, (Balaguer et al., 2019) demonstrated that the miR-30d deficiency negatively impacts endometrial receptivity and fetal growth. There was a significant difference in mRNA levels of receptivity markers such as COX2, LIF, MSX1, MSX2, estrogen, and progesterone just in the early stages of implantation in miR-30d deficient murine compared to the wild type (WT). miR-30d deficiency during the WOI lowers the expression of adhesion molecules in the blastocysts such as integrin beta-3, integrin alpha-7, and cadherin-5 and subsequently impairs embryo implantation. Additionally, impediment of maternal miR-30d transfer to the embryo led to a decreased implantation rate. The absence of miR-30d also led to fetal and placental development impairment. They showed that the pretreatment of miR-30d knocked out (KO) embryos with miR-30d analogs recovered impaired implantation in both WT and KO groups (Balaguer et al., 2019). Also, transiently miR-30d transfection into in vitro cultured human endometrial epithelial cells (hEECs) positively activated genes associated with the reproductive system’s function (Moreno-Moya et al., 2014). So, using miR-30d seems to help improve the implantation outcome. miR-30d can be transferred via exosomes to reach the implantation microenvironment (Liang et al., 2017).

As mentioned earlier, extracellular vesicles (EVs) are involved in the reproductive process, including implantation and embryo development. Exosomes are small EVs found in both uterine fluid and embryo culture mediums (Andronico et al., 2019). Indeed, there is a bidirectional embryo-maternal communication through these secreted exosomes, whereby endometrial cells and the embryo take up one another’s exosomes (Figure 1). Meanwhile, endometrial exosomes are reported to be intensely engaged in facilitating the optimized condition for embryo implantation. (Blázquez et al., 2018) demonstrated that EVs derived from endometrial mesenchymal cells (EV-endMSCs) positively impacted embryo total cell number, development, and hatching (Blazquez et al., 2018). As mentioned before, EV miRNAs are essential regulators of the implantation process. In fact, exosomes are delivery vehicles for transferring miRNAs (Andronico et al., 2019). Due to exosomes’ limited size capacity, hybrid exosomes were used to load larger plasmids in a recent study (Lin et al., 2018). Afterward, these hybrid exosomes are capable of carrying CRISPR/dCas9 as a potential option to regulate miR-30d expression (Lin et al., 2018).

Activation of the miR-30d gene via CRISPR/dCas9 can be considered a novel treatment for implantation impairment. Nuclease-deactivated Cas9 (dCas9) can be used for cellular re-programming, such as activating silent endogenous genes or over-expression and down-expression of specific genes. Since dCas9 endonuclease activity is suppressed, it is an effective gene-editing tool for activating or repressing gene expression without breaking the DNA. CRISPR/dCas9 can be a more powerful activator via fusing dCas9 proteins to a tetramer of VP16 to make a hybrid protein called dCas9‐VP64 (Chavez et al., 2015). This hybrid combination requires a suitable transfer system as well. There are many targeted gene delivery systems, including viruses, liposomes, and membrane-derived vesicles such as exosomes. In order to avoid triggering immunogenic responses, exosomes will be administered. Delivery of the CRISPR/dCas9 with exosome–liposome hybrid nanoparticles is an effective way to transfer large-sized plasmid DNA and overcome the small size of exosomes (Lin et al., 2018; Nazerian et al., 2021).

Five groups will be considered to evaluate the efficacy of enhancement in the miR-30d expression on the implantation: 1- The first group consists of the female mice that mate with the male mouse and will be pregnant. 2- The second group consists of the female mice housed with vasectomized males. 3- The third group consists of female mice that receive blastocyst of IVF procedure without any gene manipulation. 4- The fourth group consists of female mice that received blastocyst of IVF procedure and were incubated with the liposome-exosome hybrids lacking episomal vectors. 5- The last group consists of female mice that receive blastocyst of IVF procedure that were genetically manipulated by trophoblast-specific gene delivery as discussed previously. 4 days before embryo transfer (ET), in the third, fourth, and the fifth groups, female mice will be housed with vasectomized males to become pseudopregnant, which is necessary for pregnancy. Then ET will be performed by the non-surgical embryo transfer method.