**2.2 Human endometrium transcriptomics**

The transcriptome reflects the genes that are actively expressed at any given time within a specific cell population or tissue [20]. Human endometrial receptivity transcriptome is a rather complex issue because the quantity of crucial genes that plays a main role in receptivity is still a debatable question. Despite so many publications that revealed hundreds of simultaneously up- and down-regulated genes, the number of selected genes usually differs from one publication to another.

The early search using mouse models started with a few identified genes of receptivity, such as leukemia inhibitory factor-LIF, Homeobox protein X3, genes of embryo response- Cyclooxygenase 2-COX 2; and decidualization -Interleukin 11 Receptor-IL-11R [20]. In 2003 from comparing the gene expression pattern of 375 human cytokines, chemokines, and related factors in receptive and prereceptive human endometrium identified IGF-1 (insulin-like growth factor-binding protein) as a new endometrial receptivity gene [21]. Furthermore, Zhang et al. proposed 148 receptivity biomarkers [22]. Tapia et al. suggested a list of 61 receptivity biomarkers [23]. Bhagwat et al. found 179 genes that have the potential to be called Receptivity Associated Genes [24]. In an enrichment analysis used to identify a meta-signature of highly presumed biomarkers of endometrial receptivity, a statistically significant meta-signature of 52 up-regulated and five down-regulated genes was identified [25]. The highest scores in receptive-phase endometrium reserved 5 up-regulated transcripts - GADD45A, SPP1, PAEP, GPX3 and MAOA. The five down-regulated transcripts receptivity-associated genes were SFRP4, EDN3, OLFM1, CRABP2 and MMP7 [22–24, 26, 27]. Interestingly, commercial Endometrial receptivity array (ERA test) by Igenomix [28, 29] shares 47 genes in common with the identified 57 putative receptivity biomarkers.

As the potential biomarkers for endometrial receptivity, many other molecules have been also studied - like mucin (MUC-1), trophinin, L-selectin, Wingless (Wnt) family members, etc. [30].

#### **2.3 Endometrial receptivity Array**

Endometrial receptivity array was developed and patented in 2009. The group of Garrido Gomez from Igenomix have developed a clinical algorithm with a computational predictor which test results are based on the expression analysis of 248 genes [29]. Expression profiling is accomplished by assaying mRNA levels with microarrays or next-generation sequencing technologies (RNA-seq), that allowed identification of the transcriptomic signature of the window of implantation [31]. The idea is to detect a specific point in time of endometrial cycle in which the WOI starts, allowing physicians to perform personalized embryo transfer (pET). The accuracy and consistency of the ERA test had been demonstrated in several trials, that showed that the ERA test is a reliable and reproducible method for determination of the exact time of the WOI that can be used with better results in comparison to histological dating of endometrial receptivity [32]. A pilot study was conducted by Igenomix of 17 RIF patients, who underwent oocyte donation and routine embryo transfer (ET) but were then treated with pET after the personalized diagnosis of their WOI. This study demonstrated that embryo-endometrial synchronization within an optimal time-frame increases the chances of success in an assisted reproductive treatment [33]. The same group showed that patients with at least three previous failed oocyte donation cycles, and IVF patients aged <40 years, with at least three failed IVF cycles with a receptive ERA diagnosis resulted in a 62.8% pregnancy rate [20]. Other groups also showed increased probability of having successful implantation and pregnancy after performed pET in accordance to the ERA result. Results in the Indian population revealed an endometrial factor in 27.5% of the RIF patients, which was significantly greater than 15% in the non-RIF group [17]. Increased percentages of non-receptive ERA test in women with RIF have been also demonstrated [17, 28, 34]. However, the data on the ability of the ERA test to improve the implantation chances in RIF patients are conflicting, with some studies showing no beneficial effect of the test [35, 36]. Also, some studies failed to demonstrated concordance between the ERA test and histological dating of the endometrial biopsies [37].

#### **2.4 Uterine microbiome**

Normal microbiome in healthy women primarily consists of hydrogen peroxideproducing Lactobacilli species [38]. During infancy the vaginal flora consists of aerobic and anaerobic bacterial populations, including Streptococcus and Staphylococcus, Prevotella and Enterobacteria species [39]. When puberty comes, the estrogen production causes glycogen to rise and pH to decrease with subsequent domination by Lactobacilli species. Microbiomes of all reproductive organs (vagina, cervix, Fallopian tubes, and ovaries) are significantly correlated [40]. It has been suggested that instead of a single most frequent microbiome, there are multiple core microbiomes: either dominated by variety of Lactobacillus species, or with a lower percentage of Lactobacilli and dominance of anaerobic bacteria [38].

Studies indicate that lower diversity in the microbiome show better outcomes [41–43]. It seems that gravid vaginal microbiome tends to be more stable and less diverse through all the period of pregnancy [44] with major change such as an increase in the dominance of four Lactobacillus spp. (*L. crispatus*, L. jensenii, L. gasseri, and *L. vaginalis*) and a decrease in the amount of anaerobic species [45].

Regarding the endometrial microbiome of women with RIF, Bacteroides and Proteobacteria seem to be the most represented [46]. Meta-analysis that was done in 2013 also proved that dysbiotic shifts are more frequent in subfertile population [47]. Also, a contamination from the transfer catheter tip by Enterobacteriaceae, Streptococcus, Staphylococcus, *Escherichia coli* and Gram-negative bacteria has a negative effect on implantation and pregnancy rates [48–52].
