The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future Factors Involved in Embryo Implantation Success

*Almena López-Luna, Dinorah Hernández-Melchor, Leticia Ramírez-Martínez and Esther López-Bayghen*

## **Abstract**

In the field of assisted reproductive technology, endometrial receptivity is a crucial aspect that affects implantation rates in *in-vitro* fertilization procedures; in fact, impaired endometrial receptivity has been identified as the rate-limiting step for favorable pregnancy outcomes once factors regarding embryo quality have been optimized. The endometrium is a dynamic tissue that undergoes proliferative and secretory changes in each menstrual cycle, acquiring a short and transient period of embryo receptivity known as the Window of Implantation. Precise embryo-endometrial synchrony is necessary to achieve a successful pregnancy, and it involves complex and multifactorial processes related to morphological, biochemical, and genetic changes. On that behalf, defining the receptive window of each patient for personalized embryo transfer is a current goal. Here, we review different indicators of endometrial receptivity throughout the menstrual cycle, spotlighting the opening of the window of implantation: classical histological and biochemical markers, genetic factors, leading-edge transcriptomic signatures and miRNA profiles, and novel features such as the microbiome and secretome. Understanding the molecular mechanisms behind endometrial receptivity will facilitate the optimization and improvement of infertility treatments.

**Keywords:** endometrial receptivity, embryo implantation, menstrual cycle, window of implantation, decidualization

## **1. Introduction**

The field of assisted reproductive technology (ART) has grown significantly in use and understanding over the past few decades, nevertheless, the rates of successful pregnancies in *in-vitro* fertilization (IVF) procedures are still relatively low. Impaired endometrial receptivity (ER) has been identified as the rate-limiting step for favorable pregnancy outcomes once all other factors, including the acquisition and selection of the best quality embryo(s), have been optimized. Correct and synchronized maturation of the endometrial tissue is essential for embryo

**Figure 1.**

*Endometrial dynamics throughout the menstrual cycle. This picture was modified from Servier Medical Art under the Creative Commons License 2018.*

implantation [1, 2]. The endometrium is a dynamic tissue that undergoes proliferative and secretory changes in each menstrual cycle. Throughout most of this cycle, the endometrium remains "non-adhesive" to embryos and it only acquires a short and transient period of embryo receptivity known as the "window of implantation" (WOI) [3, 4]. In humans, during a natural cycle, the endometrium becomes receptive 6 to 8 days after ovulation and it remains receptive for approximately 24–48 h, this time is assumed to occur between days 20 and 24 of a regular menstrual cycle [2, 5] (**Figure 1**). The cyclic fluctuations of the endometrium are coordinated by the ovarian hormones estrogen (E2) and progesterone (P4); a finely balanced signaling process mediated by these hormones defines the WOI. The menstrual cycle is divided in two phases: proliferative or follicular, and secretory or luteal [6]. During the proliferative phase (PP), E2 from the maturing follicle allows the elongation of the spiral arteries and the proliferation of endometrial stromal cells (EnSCs) and glands [7]. Afterwards, during the secretory phase (SP), P4 from the corpus luteum induces secretory changes; the endometrium thickens and it acquires a receptive phenotype that will support blastocyst attachment [8–10]. During the Mid-Secretory Phase (MSP) circulating P4 induces EnSCs to undergo decidualization [11]. Decidualization is the transformation of the endometrial stroma into a dense cellular matrix known as the decidua, this process initiates during the SP in the stroma and, if pregnancy occurs, it progresses into the development of the decidua which will in turn form the maternal placenta [12]. The optimization of the endometrium to support embryo implantation is a complex and multifactorial process that involves morphological, biochemical, and genetic changes [13]. ER is a key aspect that affects implantation rates in IVF procedures considering that a precise embryo-endometrial synchrony is completely necessary to achieve a successful pregnancy [4, 14]. Thus, understanding the molecular mechanisms behind ER will facilitate the optimization and improvement of infertility treatments.

### **2. Factors involved in endometrial receptivity**

#### **2.1 Evaluation of endometrial morphology for receptivity assessment**

Morphological changes during the endometrial cycle generate markers that have been used over decades to assess receptivity, such as histological evaluation of a biopsy and ultrasound examination of the endometrium. Endometrial biopsies are now

**13**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

considered to provide little clinically relevant information [15, 16]. Additionally, the formation of pinopodes was thought to show potential as a clinical marker to assess ER [17]. However, the presence of pinopodes was demonstrated not only during the WOI but also in the post-receptive endometrium, precluding in this way its use as a marker of ER [17, 18]. On the other hand, ultrasound examination is a routinely used technique in IVF procedures [9, 19]. This non-invasive technique is based on the interpretation of a medical ultrasound of the endometrium. Various ultrasonographic parameters have been proposed as pregnancy predictors, such as endometrial thickness, volume, and blood flow patterns. The most commonly used is endometrial thickness [20, 21]. Due to differences in stimulation protocols, sonographic approaches, and difficulties in obtaining a standard sagittal view of the uterus, discrepancies in the cut-off value of endometrial thickness to achieve pregnancy arise [22]. Generally, it is considered that a minimum of 6–8 mm in endometrial thickness is necessary for a successful pregnancy [23–25]. Nevertheless, case reports have described pregnancy establishment despite an endometrial thickness of no more than 4 mm [26, 27]. Threedimensional (3D) sonography assesses ER by considering endometrial thickness, volume, and angiogenic dynamics. The endometrial volume of fewer than 2 ml has been shown to decrease pregnancy rates significantly [19, 28, 29]. Another evaluated criterion is an endometrial pattern, which can be classified as triple-line, intermediate, or homogenous [30]. Among these, the triple-line pattern has been suggested to reflect ER [24, 31] broadly. Finally, the impact of ovarian stimulation on ER has yet to be determined. Abnormal hormone concentrations the due to stimulation protocols during IVF might affect endometrial morphology and thereby ER [32]. Comprehensively, although morphological elements are important components of receptivity, there is still no consensus on the extent in which they can be used as WOI predictors.

Endometrial genetic abnormalities can lead to implantation failure due to dysregulation of critical processes such as trophoblast invasion and angiogenesis. Here, we discuss common genetic abnormalities that have been analyzed to determine their role in implantation failure (**Table 1**). Parental chromosomal abnormalities such as mutations and translocations should be considered relevant in the efficacy

*2.2.1 Angiogenetic factors: vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), TP53 tumor suppressor (TP53), murine double minute 2 (MDM2), herpes virus-associated ubiquitin-specific protease* 

Successful pregnancy is dependent on adequate placental circulation and fetal vasculature. The development of a normal vascular network during implantation, embryo development, and placentation requires cooperation between different cell types and various growth factors. *VEGF* is a potent angiogenic factor that plays an essential role in embryo implantation/development. Four *VEGF* polymorphisms have been reported to affect VEGF activity and expression increasing aberrations in vascular formation and/or function. The polymorphism −1154G/A located in the promoter region has been associated with RPL [33], RSA [34, 35], and RIF [36, 37]. Moreover, a meta-analysis showed that genotypes −2578C/A, −634G/C, and 936C/T increase the risk of RSA as well [38]. Furthermore, *eNOS,* which is expressed in the terminal chorionic villous vessels, is important for vascular nitric oxide (NO) production to supply nutrients to the fetus. Only the eNOS Glu298Asp polymorphism has been shown to be significantly associated with RPL [39]. Additionally, successful trophoblast invasion requires the induction of paracrine apoptotic reactions to

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

**2.2 Genetic factors involved in ER**

of improving reproductive outcome.

*(HAUSP)*

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*

considered to provide little clinically relevant information [15, 16]. Additionally, the formation of pinopodes was thought to show potential as a clinical marker to assess ER [17]. However, the presence of pinopodes was demonstrated not only during the WOI but also in the post-receptive endometrium, precluding in this way its use as a marker of ER [17, 18]. On the other hand, ultrasound examination is a routinely used technique in IVF procedures [9, 19]. This non-invasive technique is based on the interpretation of a medical ultrasound of the endometrium. Various ultrasonographic parameters have been proposed as pregnancy predictors, such as endometrial thickness, volume, and blood flow patterns. The most commonly used is endometrial thickness [20, 21]. Due to differences in stimulation protocols, sonographic approaches, and difficulties in obtaining a standard sagittal view of the uterus, discrepancies in the cut-off value of endometrial thickness to achieve pregnancy arise [22]. Generally, it is considered that a minimum of 6–8 mm in endometrial thickness is necessary for a successful pregnancy [23–25]. Nevertheless, case reports have described pregnancy establishment despite an endometrial thickness of no more than 4 mm [26, 27]. Threedimensional (3D) sonography assesses ER by considering endometrial thickness, volume, and angiogenic dynamics. The endometrial volume of fewer than 2 ml has been shown to decrease pregnancy rates significantly [19, 28, 29]. Another evaluated criterion is an endometrial pattern, which can be classified as triple-line, intermediate, or homogenous [30]. Among these, the triple-line pattern has been suggested to reflect ER [24, 31] broadly. Finally, the impact of ovarian stimulation on ER has yet to be determined. Abnormal hormone concentrations the due to stimulation protocols during IVF might affect endometrial morphology and thereby ER [32]. Comprehensively, although morphological elements are important components of receptivity, there is still no consensus on the extent in which they can be used as WOI predictors.

#### **2.2 Genetic factors involved in ER**

Endometrial genetic abnormalities can lead to implantation failure due to dysregulation of critical processes such as trophoblast invasion and angiogenesis. Here, we discuss common genetic abnormalities that have been analyzed to determine their role in implantation failure (**Table 1**). Parental chromosomal abnormalities such as mutations and translocations should be considered relevant in the efficacy of improving reproductive outcome.

*2.2.1 Angiogenetic factors: vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), TP53 tumor suppressor (TP53), murine double minute 2 (MDM2), herpes virus-associated ubiquitin-specific protease (HAUSP)*

Successful pregnancy is dependent on adequate placental circulation and fetal vasculature. The development of a normal vascular network during implantation, embryo development, and placentation requires cooperation between different cell types and various growth factors. *VEGF* is a potent angiogenic factor that plays an essential role in embryo implantation/development. Four *VEGF* polymorphisms have been reported to affect VEGF activity and expression increasing aberrations in vascular formation and/or function. The polymorphism −1154G/A located in the promoter region has been associated with RPL [33], RSA [34, 35], and RIF [36, 37]. Moreover, a meta-analysis showed that genotypes −2578C/A, −634G/C, and 936C/T increase the risk of RSA as well [38]. Furthermore, *eNOS,* which is expressed in the terminal chorionic villous vessels, is important for vascular nitric oxide (NO) production to supply nutrients to the fetus. Only the eNOS Glu298Asp polymorphism has been shown to be significantly associated with RPL [39]. Additionally, successful trophoblast invasion requires the induction of paracrine apoptotic reactions to

*Modern Medical Genetics and Genomics*

*under the Creative Commons License 2018.*

**Figure 1.**

implantation [1, 2]. The endometrium is a dynamic tissue that undergoes proliferative and secretory changes in each menstrual cycle. Throughout most of this cycle, the endometrium remains "non-adhesive" to embryos and it only acquires a short and transient period of embryo receptivity known as the "window of implantation" (WOI) [3, 4]. In humans, during a natural cycle, the endometrium becomes receptive 6 to 8 days after ovulation and it remains receptive for approximately 24–48 h, this time is assumed to occur between days 20 and 24 of a regular menstrual cycle [2, 5] (**Figure 1**). The cyclic fluctuations of the endometrium are coordinated by the ovarian hormones estrogen (E2) and progesterone (P4); a finely balanced signaling process mediated by these hormones defines the WOI. The menstrual cycle is divided in two phases: proliferative or follicular, and secretory or luteal [6]. During the proliferative phase (PP), E2 from the maturing follicle allows the elongation of the spiral arteries and the proliferation of endometrial stromal cells (EnSCs) and glands [7]. Afterwards, during the secretory phase (SP), P4 from the corpus luteum induces secretory changes; the endometrium thickens and it acquires a receptive phenotype that will support blastocyst attachment [8–10]. During the Mid-Secretory Phase (MSP) circulating P4 induces EnSCs to undergo decidualization [11]. Decidualization is the transformation of the endometrial stroma into a dense cellular matrix known as the decidua, this process initiates during the SP in the stroma and, if pregnancy occurs, it progresses into the development of the decidua which will in turn form the maternal placenta [12]. The optimization of the endometrium to support embryo implantation is a complex and multifactorial process that involves morphological, biochemical, and genetic changes [13]. ER is a key aspect that affects implantation rates in IVF procedures considering that a precise embryo-endometrial synchrony is completely necessary to achieve a successful pregnancy [4, 14]. Thus, understanding the molecular mechanisms behind ER will

*Endometrial dynamics throughout the menstrual cycle. This picture was modified from Servier Medical Art* 

facilitate the optimization and improvement of infertility treatments.

**2.1 Evaluation of endometrial morphology for receptivity assessment**

Morphological changes during the endometrial cycle generate markers that have been used over decades to assess receptivity, such as histological evaluation of a biopsy and ultrasound examination of the endometrium. Endometrial biopsies are now

**2. Factors involved in endometrial receptivity**

**12**


**15**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

*PT53* Codon 72 Pro rs1042522 Homozygote genotype is associated with

*PTGS2* G-765C rs20417 Association with implantation failure

*TFF3* rs225361 A/G rs11701143 Homozygous genotype is associated

*VEGF* G-1154A rs1570360 Homozygote A/A genotype associated

**Gene Polymorphism rs code Relevance References** *PR* H770H-C/T No association [54] G/T—Val660Leu rs1042838 More prevalent in women with

Codon 72 Arg rs1042522 Homozygote genotype is associated with

unexplained infertility

RPL [41, 75], IRM [76] and RIF [37, 40]

RIF

susceptibility

with less live births before their first spontaneous abortion

with RSA [34, 35], RPL [33], and RIF [36, 37]

No associated [71]

V660L No association [54]

rs225361 A/G rs11701143 No association [66] rs11701143 T/C rs225361 Associated with idiopathic RSA [61] rs11701143 T/C rs225361 No association [66] rs225439 G/A rs225439 No association [61, 66] ros533093 C/T rs533093 No association [61, 66] rs77436142 G/C rs7743614 No association [61, 66]

C-2578A rs699947 No associated [34] G-634C rs2010963 No associated [34] C936T rs3025039 No associated [34]

[65]

[37, 40, 41, 75, 76]

[75]

[55]

[61]

[33–37]

secrete proteases capable of digesting the endometrial extracellular matrix (ECM) [36]. TP53 is a potent regulator of apoptosis, cell cycle, angiogenesis, and embryonic development. A TP53 polymorphism at codon 72, encoding either proline or arginine, was reported to alter the TP53 activity and affect human fertility [40]. The Arg72 variant has been shown to induce higher apoptotic activity than Pro72. Therefore, Pro72 variant might cause inadequate trophoblast invasion, increasing the risk of RPL [41] and RIF [37, 40]. In this manner, women with Pro/Pro genotype have a higher risk of RPL than women with the Arg/Arg or Arg/Pro genotypes. Following, MDM2 and HAUSP regulate TP53. MDM2 binds to TP53 to degrade it through poly-ubiquitination, blocking its ability to function as a transcription factor. The *MDM2* SNP309 is a functional SNP that increases MDM2 expression levels and attenuates TP53 pathway. HAUSP, on the other hand, acts as a specific deubiquitinase for TP53, the A allele has a significant association with infertility in young patients (<35 years) but not in the older patients, similarly to the *MDM2* SNP309 G allele. Those observations suggest that *MDM2* and *HAUSP* may be involved in the regula-

*Probably implicated mutations in implantation failure, studies are listed even when no association was found, rs code* 

tion of human fertility through the regulation of TP53 [40].

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

*is mentioned whenever it is reported.*

*Genetic abnormalities involved in implantation failure.*

**Table 1.**


*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*

*Probably implicated mutations in implantation failure, studies are listed even when no association was found, rs code is mentioned whenever it is reported.*

#### **Table 1.**

*Modern Medical Genetics and Genomics*

*HPA1* HPA1 a/b

(L33P)

**Gene Polymorphism rs code Relevance References**

*eNOS* VNTR (B/A) Association to the risk of RPL [77]

Glu298Asp Homozygote genotype T/T is associated

*F2* G20210A rs1799963 No association [58, 60, 62,

*F5* G1691A, Leiden rs6025 No association [57, 58, 62,

*F8* V34L More prevalent in women with RPL [42, 58]

*FGB* G-455A rs1800790 No association [57, 58, 62,

*GPIIIa* C1565T No association [57] *HAUSP* rs1529916 G/A rs1529916 Allele A is associated with RIF [40]

*LIF* C715A No associated [72]

*MDM2* T309G rs2279744 Allele G is associated with RIF [40] *MTHFR* A1298C rs1801131 No association [58, 62–64] C677T rs1801133 More frequent in women with

*MUC4* VNTR No association [70]

*MUC1* VNTR Women with unexplained infertility

*PAI-1* 4G/5G rs1799889 More prevalent in women with RIF [63]

H1299R (R2) No association [58, 62, 63] Y1702C No association [58, 62, 63]

G3400A More frequent in nulligravid women [72, 73] G3424A No associated [72] T1414G rs929271 G/G genotype is associated with RIF [40, 74]

> unexplained infertility [64], RPL [42, 58], and RSA [60]

might have susceptibility to implantation failure due to small *MUC1* allele size

and RPL [42, 58]

in women with RPL

with risk of IRM

which P allele may be the risk and X allele its guard factor

Heterozygous genotype is more frequent in women with RSA in the first trimester [57] and women with RPL [42]

More frequent in women with RIF [60]

No association [62, 63]

No association [58, 62, 63]

No association [57, 62, 63]

No association [68, 69]

No association [62]

No associated [78]

[33]

[39]

[44]

63]

[42, 57]

63]

63]

[42, 58–60]

[67]

[42, 58, 63]

*APOE* Heterozygous genotype is more frequent

*ESR1* IVS1-397T>C rs2234693 Related to unknown thin endometrium in

IVS1-351A>G rs9340799

**14**

*Genetic abnormalities involved in implantation failure.*

secrete proteases capable of digesting the endometrial extracellular matrix (ECM) [36]. TP53 is a potent regulator of apoptosis, cell cycle, angiogenesis, and embryonic development. A TP53 polymorphism at codon 72, encoding either proline or arginine, was reported to alter the TP53 activity and affect human fertility [40]. The Arg72 variant has been shown to induce higher apoptotic activity than Pro72. Therefore, Pro72 variant might cause inadequate trophoblast invasion, increasing the risk of RPL [41] and RIF [37, 40]. In this manner, women with Pro/Pro genotype have a higher risk of RPL than women with the Arg/Arg or Arg/Pro genotypes. Following, MDM2 and HAUSP regulate TP53. MDM2 binds to TP53 to degrade it through poly-ubiquitination, blocking its ability to function as a transcription factor. The *MDM2* SNP309 is a functional SNP that increases MDM2 expression levels and attenuates TP53 pathway. HAUSP, on the other hand, acts as a specific deubiquitinase for TP53, the A allele has a significant association with infertility in young patients (<35 years) but not in the older patients, similarly to the *MDM2* SNP309 G allele. Those observations suggest that *MDM2* and *HAUSP* may be involved in the regulation of human fertility through the regulation of TP53 [40].

### *2.2.2 Apolipoprotein E (APOE)*

Due to the increase in total cholesterol levels during pregnancy, *APOE* plays a crucial role in lipid metabolism. *APOE* has three alleles in the long arm of chromosome 19 at position 13.2: ε2, ε3, and ε4. Individuals harboring the allele ε4 have higher cholesterol levels than the ones carrying the ε3/ε3 allele, whereas levels in those with the ε2 allele are lower [42].

### *2.2.3 Estrogen receptor α (ESR1)*

*ESR1* is a ligand-activated transcription factor essential for sexual development and reproductive function; its dysregulation leads to the development of various diseases such as cancer, cardiovascular disease, and inflammation, among others [43]. Due to alternative splicing of mRNA, it possesses three isoforms: ERαΔ3, ERα36, and ERα46 [43]; in a study performed by Yuan and Le [44], the polymorphisms rs2234693 and rs9340799 were related to the uncommonly thin endometrium.

#### *2.2.4 Leukemia inhibitor factor (LIF)*

LIF is an important implantation factor that promotes proliferation, invasion, and differentiation; its expression is regulated by the transcription factor tumor protein TP53 (TP53). Few studies have found a correlation between LIF gene polymorphism and reproductive capacity, Kang et al. demonstrated that SNP in the 3'UTR of the LIF (rs929271) gene is associated with infertility [40].

#### *2.2.5 Mucin 1 (MUC1), Mucin 4 (MUC4)*

MUC1 is an anti-adhesion molecule secreted by human endometrial epithelium, it has been suggested that its expression prevents the adherence of blastocyst to the endometrium. Interestingly, MUC1 must be locally removed in a paracrine fashion at the implantation site during the WOI to allow contact between the embryo and the endometrium, making it an important factor in determining ER [45–48]. *MUC1* is a highly polymorphic gene that differs in the size of the region carrying the O-glycosylation sites: the variable number tandem repeat region (VNTR), which can go from 20 to 125 repeats [49]. Similarly, MUC4 is a greatly expressed mucin in endometrial epithelium [50], its gene is highly polymorphic and it contains a VNTR region that can go from 145 to 395 repeats [51]. Although its role in human infertility has not been fully explored, studies in other species have suggested that it plays a role in embryo implantation [52, 53].

#### *2.2.6 Progesterone receptor (PR)*

The PROGINS complex are three mutations in the *PR* gene that may be associated with unexplained infertility and implantation failure: a 306 bp insert in intron G of the dT2 allele in *PR*, the mutated alleleV660L, and guanine to thymine substitution in exon 4, resulting in a valine to leucine change in the hinge region of PR, and a cytosine to thymine substitution at exon 5 [54].

#### *2.2.7 Prostaglandin-endoperoxide synthase 2 (PTGS2)*

*PTGS2* is a key enzyme involved in the conversion of arachidonic acid to prostaglandins (PGs). The −765G>C SNP mutation in the promoter region of *PTGS2* upstream the transcriptional start site in the putative Sp1 site can cause alterations in Sp1 binding

**17**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

[55]. Accordingly, the hypermethylation of the NF-IL6 site within the *PTGS2* promoter results in elevated gene expression in eutopic endometrium in endometriosis [56].

*2.2.8 Thrombolytic factors: coagulation factor II (F2), coagulation factor V (F5), coagulation factor XIII a chain (F13A1), methylenetetrahydrofolate reductase* 

Thrombophilia, the predisposition for thrombosis, has been shown to be a risk factor for successful pregnancy due to impaired vascularization at the time of implantation. Therefore, the possible association between early pregnancy loss and polymorphisms at coagulation factors and thrombolytic genes responsible for inherited or acquired thrombophilia has been investigated. The coagulation factor II SNP G20210A in the 3′-untranslated region of *F2* causes elevated prothrombin in plasma, leading to enhanced blood coagulation [57]. Furthermore, factor V Leiden mutation, G1691A, is a single nucleotide substitution in the *F5* gene that results in reduced clearance of factor Va due to its blocked inactivation by activated protein C, increasing the risk of thrombosis [42, 57]. Also, factor XIII V34L polymorphism is a guanine to thymine substitution in exon 2 of *F13A1* that leads to a valine for leucine change in residue 34; this SNP leads to reduced susceptibility to fibrinolysis and influences fibrin degradation [57]. Moreover, MTHFR is the rate-limiting enzyme in the methyl cycle. C677T polymorphism causes a substitution of valine for alanine, resulting in a thermolabile variant of the enzyme with reduced catalytic activity; combined with the SNP AC1298C, it is associated with hyper-homocysteinemia, a risk factor for venous and arterial thrombosis [42, 58–60]. Additionally, PAI-1 is a key regulatory element in the fibrinolysis cascade, it is believed to control proteolysis and remodeling of maternal tissue during trophoblast invasion. The 4G/5G polymorphism is located 657 bp upstream from the start site of transcription within the *PAI-1* promoter and results in an allele with decreased transcriptional activity [42].

TFF3 is a mucin-associated peptide co-expressed in mucus cells that acts as a mitogen to promote epithelial cell migrations and mediates epithelial repair after damage. The SNP rs11701143 is located in the promoter region of *TFF3* within the regulatory region of the transcription binding site, whereas rs225361 is an intron variant located within a regulatory region. The exact function of both SNPs remains

The immune system plays a major role in the process of implantation and pregnancy maintenance [62]. During decidualization, endocrine processes transform uterine fibroblasts into cells that can produce hormones, growth factors, and matrix components to support embryo implantation [63, 64]. Furthermore, tolerance of the immune maternal system is required in pregnancy to avoid rejection of the semiallograft or allograft embryo and for its successful implantation [65]. The decidua is a privileged site for immune tolerance; a large number of molecules and immune cell types participate in this process, leukocytes, macrophages, T lymphocytes, and dendritic cells comprise around 30 to 40% of the cells within the decidual stroma in early pregnancy. Among leukocytes, uterine natural killer (uNK) cells are activated and they significantly increase during decidualization (65–70%) [66, 67]. Increases in uNK cells denote three main functions in the endometrium: regulation of placental and trophoblast growth by cytokines [68, 69], local immunomodulation [70, 71], and

*(MTHFR), plasminogen activator inhibitor-1 (PAI-1)*

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

*2.2.9 Trefoil factor 3 (TFF3)*

to be elucidated [61].

**2.3 Immunological factors contributing to ER**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*

[55]. Accordingly, the hypermethylation of the NF-IL6 site within the *PTGS2* promoter results in elevated gene expression in eutopic endometrium in endometriosis [56].

## *2.2.8 Thrombolytic factors: coagulation factor II (F2), coagulation factor V (F5), coagulation factor XIII a chain (F13A1), methylenetetrahydrofolate reductase (MTHFR), plasminogen activator inhibitor-1 (PAI-1)*

Thrombophilia, the predisposition for thrombosis, has been shown to be a risk factor for successful pregnancy due to impaired vascularization at the time of implantation. Therefore, the possible association between early pregnancy loss and polymorphisms at coagulation factors and thrombolytic genes responsible for inherited or acquired thrombophilia has been investigated. The coagulation factor II SNP G20210A in the 3′-untranslated region of *F2* causes elevated prothrombin in plasma, leading to enhanced blood coagulation [57]. Furthermore, factor V Leiden mutation, G1691A, is a single nucleotide substitution in the *F5* gene that results in reduced clearance of factor Va due to its blocked inactivation by activated protein C, increasing the risk of thrombosis [42, 57]. Also, factor XIII V34L polymorphism is a guanine to thymine substitution in exon 2 of *F13A1* that leads to a valine for leucine change in residue 34; this SNP leads to reduced susceptibility to fibrinolysis and influences fibrin degradation [57]. Moreover, MTHFR is the rate-limiting enzyme in the methyl cycle. C677T polymorphism causes a substitution of valine for alanine, resulting in a thermolabile variant of the enzyme with reduced catalytic activity; combined with the SNP AC1298C, it is associated with hyper-homocysteinemia, a risk factor for venous and arterial thrombosis [42, 58–60]. Additionally, PAI-1 is a key regulatory element in the fibrinolysis cascade, it is believed to control proteolysis and remodeling of maternal tissue during trophoblast invasion. The 4G/5G polymorphism is located 657 bp upstream from the start site of transcription within the *PAI-1* promoter and results in an allele with decreased transcriptional activity [42].

## *2.2.9 Trefoil factor 3 (TFF3)*

*Modern Medical Genetics and Genomics*

those with the ε2 allele are lower [42].

*2.2.4 Leukemia inhibitor factor (LIF)*

*2.2.5 Mucin 1 (MUC1), Mucin 4 (MUC4)*

role in embryo implantation [52, 53].

and a cytosine to thymine substitution at exon 5 [54].

*2.2.7 Prostaglandin-endoperoxide synthase 2 (PTGS2)*

*2.2.6 Progesterone receptor (PR)*

*2.2.3 Estrogen receptor α (ESR1)*

Due to the increase in total cholesterol levels during pregnancy, *APOE* plays a crucial role in lipid metabolism. *APOE* has three alleles in the long arm of chromosome 19 at position 13.2: ε2, ε3, and ε4. Individuals harboring the allele ε4 have higher cholesterol levels than the ones carrying the ε3/ε3 allele, whereas levels in

*ESR1* is a ligand-activated transcription factor essential for sexual development and reproductive function; its dysregulation leads to the development of various diseases such as cancer, cardiovascular disease, and inflammation, among others [43]. Due to alternative splicing of mRNA, it possesses three isoforms: ERαΔ3, ERα36, and ERα46 [43]; in a study performed by Yuan and Le [44], the polymorphisms rs2234693

LIF is an important implantation factor that promotes proliferation, invasion, and differentiation; its expression is regulated by the transcription factor tumor protein TP53 (TP53). Few studies have found a correlation between LIF gene polymorphism and reproductive capacity, Kang et al. demonstrated that SNP in the

MUC1 is an anti-adhesion molecule secreted by human endometrial epithelium, it has been suggested that its expression prevents the adherence of blastocyst to the endometrium. Interestingly, MUC1 must be locally removed in a paracrine fashion at the implantation site during the WOI to allow contact between the embryo and the endometrium, making it an important factor in determining ER [45–48]. *MUC1* is a highly polymorphic gene that differs in the size of the region carrying the O-glycosylation sites: the variable number tandem repeat region (VNTR), which can go from 20 to 125 repeats [49]. Similarly, MUC4 is a greatly expressed mucin in endometrial epithelium [50], its gene is highly polymorphic and it contains a VNTR region that can go from 145 to 395 repeats [51]. Although its role in human infertility has not been fully explored, studies in other species have suggested that it plays a

The PROGINS complex are three mutations in the *PR* gene that may be associated with unexplained infertility and implantation failure: a 306 bp insert in intron G of the dT2 allele in *PR*, the mutated alleleV660L, and guanine to thymine substitution in exon 4, resulting in a valine to leucine change in the hinge region of PR,

*PTGS2* is a key enzyme involved in the conversion of arachidonic acid to prostaglandins (PGs). The −765G>C SNP mutation in the promoter region of *PTGS2* upstream the transcriptional start site in the putative Sp1 site can cause alterations in Sp1 binding

and rs9340799 were related to the uncommonly thin endometrium.

3'UTR of the LIF (rs929271) gene is associated with infertility [40].

*2.2.2 Apolipoprotein E (APOE)*

**16**

TFF3 is a mucin-associated peptide co-expressed in mucus cells that acts as a mitogen to promote epithelial cell migrations and mediates epithelial repair after damage. The SNP rs11701143 is located in the promoter region of *TFF3* within the regulatory region of the transcription binding site, whereas rs225361 is an intron variant located within a regulatory region. The exact function of both SNPs remains to be elucidated [61].

#### **2.3 Immunological factors contributing to ER**

The immune system plays a major role in the process of implantation and pregnancy maintenance [62]. During decidualization, endocrine processes transform uterine fibroblasts into cells that can produce hormones, growth factors, and matrix components to support embryo implantation [63, 64]. Furthermore, tolerance of the immune maternal system is required in pregnancy to avoid rejection of the semiallograft or allograft embryo and for its successful implantation [65]. The decidua is a privileged site for immune tolerance; a large number of molecules and immune cell types participate in this process, leukocytes, macrophages, T lymphocytes, and dendritic cells comprise around 30 to 40% of the cells within the decidual stroma in early pregnancy. Among leukocytes, uterine natural killer (uNK) cells are activated and they significantly increase during decidualization (65–70%) [66, 67]. Increases in uNK cells denote three main functions in the endometrium: regulation of placental and trophoblast growth by cytokines [68, 69], local immunomodulation [70, 71], and control of trophoblast invasion [69]. Furthermore, trophoblast cells play a major role in immune tolerance since these cells do not express major histocompatibility complex (MHC) class I (HLA-A and HLA-B) or class II molecules; ensuring that maternal T cells with αβ receptors cannot mount a classic cytotoxic attack against fetal paternal alloantigens. The trophoblast also protects itself by expressing Fas ligand (Fas L), which is important in the elimination of maternal reactive T cells by apoptosis induction [72–74]. Other important component of this process is T-regulatory (Treg) cells; these cells are essential for immunosuppression, prevention of autoimmunity, and maternal tolerance to the fetus [75–78]. Treg cells have been shown to be locally enriched in decidua during early normal pregnancy [79]. Furthermore, Forkhead box P3 (Foxp3) is a master regulator of Treg cell development, function, and differentiation [80]. Expression of *FOXP3* was reduced approximately two-fold in endometrial biopsies of infertile women, implicating that the impaired differentiation of uterine T-cells into the Treg phenotype is a key determinant of fertility [81, 82]. On the other hand, helper T cells (CD4+) facilitate embryo implantation by regulating endometrial differentiation; they secrete interleukins and interferons that establish the implantation microenvironment. Successful pregnancy is dependent upon Th1/Th2 balance [83]. While Th1 cytokines are harmful for pregnancy, Th2 cytokines favor fetal growth and regulate uterine expression of fatty acid amide hydrolase (FAAH), LIF, and trophoblast release of human chorionic gonadotropin (hCG), which are known to play important roles during implantation [84–86]. Piccinni et al. demonstrated that T cells from decidua of women with a miscarriage show predominantly Th1-type cytokines with decreased Th2-type [84, 87, 88]. Finally, P4 and E2 mediate the downregulation of the maternal immune system [89]. P4 stimulates decidual proliferation; therefore, pregnancy results in an upregulation of P4 receptors on activated lymphocytes among placental cells and decidual CD56+ cells. In the presence of sufficient P4, these cells express progesterone-induced blocking factor (PIBF), a mediator that exerts substantial anti-abortive activities.

## **2.4 Biochemical markers involved in ER**

We review molecular markers involved in the decidualization process that could be suitable as makers to assess ER.

## *2.4.1 Homeobox A10 (HOXA10)*

HOXA10 is a transcription factor member of the homeobox family, known to be involved in the genetic control of embryonic development and in the regulation of the adult female reproductive tract [12, 90]. HOXA10 regulates downstream target genes that lead to endometrium development and receptivity acquisition [91], such as insulin-like growth factor binding protein 1 (*IGFBP1*) [12, 92, 93], genes of the Wnt pathway (reviewed by Sonderegger et al. [94], integrin β3 (*ITGB3*) [12, 90, 92], and empty spiracles homolog 2 (*EMX2*) [12, 95]. HOXA10 is regulated by P4 in a dosedependent manner [12, 90, 91]. The expression of HOXA10 is low during the PP and it rapidly increases in the MSP [12, 90]. The diminished expression of HOXA10 in endometria of women with recurrent pregnancy loss (RPL) [96], adenomyosis [97], endometriosis [92], polycystic ovary syndrome (PCOS) [98], and idiopathic infertility [99] indicates that this gene could be essential for fertility [12].

#### *2.4.2 Heparin-binding epidermal growth factor-like growth factor (HB-EGF)*

HB-EGF is a member of the epidermal growth factor (EGF) family, it is expressed in the human uterus at the time of implantation and its expression is under steroidal hormone control [100–103]. The transmembrane form is associated

**19**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

with cell adhesion and migration, it allows communication with the blastocyst by acting as a chemoattractant [101, 104]. HB-EGF expression is low in the PP and increases in the ESP immediately prior to the WOI, after which its levels decrease [101–103, 105]. Also, mRNA levels are low in pregnant endometrium and high in placental tissues at an early stage of development, suggesting that the HB-EGF ligand not only potentiates the health and survival of the peri-implantation embryo, but also induces the progression of its development [104]. HB-EGF stimulates epithelial expression of key endometrial proteins that are important biomark-

LIF is a member of a cytokine family with functional redundancy that includes interleukin 6 (IL6), oncostatin (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin 1 (CT1). They regulate proliferation, differentiation, and cell survival in different cellular systems [106]. LIF acts on cells by binding to the heterodimeric LIF receptor (LIFR), which consists of two transmembrane proteins, LIFR and glycoprotein 130 (gp130). LIFR activates several signaling pathways including the JAK/STAT, MAPK, and P13-kinase pathways, whereas gp130 participates in the activation of STAT1, STAT3, and STAT5B [107]. LIF induces the expression of cytokines and other regulatory molecules that could serve to regulate preimplantation development and embryo implantation [106–108]. LIF is one of the most important cytokines for receptivity during the WOI, the expression of LIF and LIFR reaches its highest level during the WOI in the MSP, LSP, and in early pregnancy in both surface and glandular epithelial cells under the influence of P4 [106, 107, 109, 110]. LIF can also be detected in decidual leukocytes, which are abundant at the implantation site; interestingly, LIF expression is low in women with unexplained infertility [106, 107, 111]. LIF also plays a crucial role in the regulation of fetal-maternal interactions during pregnancy, this cytokine mediates uterine receptivity through autocrine/paracrine interactions by binding to LIFR on the luminal epithelium to permit blastocyst attachment [106], but also regulates trophoblast function and

Integrins are ubiquitous cell adhesion molecules involved in maintaining normal tissue morphology and participate in cell–cell and cell-substrate interactions [100, 110, 112, 113]. In the human endometrium, integrins are involved in early embryo-endometrial interactions [90]. ITGB3 subunit is present after the ESP and its expression extend into the pregnancy [112, 114]. It has been reported that healthy fertile women show higher ITGB3 expression than patients with unexplained infertility [46, 96, 113–115]. Moreover, its dysregulation appears to characterize two distinct pathophysiological conditions that involve distinct mechanisms of defective ER: Type I and Type II. Type I defect is an out-of-phase endometrium with negative ITGB3 subunit expression, portrays a shifted WOI, and hormonal inadequacy or responsiveness is implicated, on the other hand, Type II defect is an "in-phase" endometrium with negative ITGB3 subunit expression and connotes the complete loss of the WOI. Furthermore, ITGB3 is expressed in EnSCs and endometrial glands with the highest levels in the MSP to LSP, suggesting a role in the regulation of endometrial function and implantation [115, 116]. Due to its temporal distribution and the effects of implantation when it is not present, ITGB3 is a useful molecular marker to assess ER. ITGB3 is regulated in the endometrium through a molecular mechanism

via sex steroid signaling where HOXA10 acts as an intermediary [90, 96].

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

*2.4.3 Leukemia inhibitory factor (LIF)*

vascular formation in the placenta [109].

*2.4.4 Integrin β3 (ITGB3)*

ers of the WOI, including LIF, HOXA10, and ITGB3 [100].

#### *The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*

with cell adhesion and migration, it allows communication with the blastocyst by acting as a chemoattractant [101, 104]. HB-EGF expression is low in the PP and increases in the ESP immediately prior to the WOI, after which its levels decrease [101–103, 105]. Also, mRNA levels are low in pregnant endometrium and high in placental tissues at an early stage of development, suggesting that the HB-EGF ligand not only potentiates the health and survival of the peri-implantation embryo, but also induces the progression of its development [104]. HB-EGF stimulates epithelial expression of key endometrial proteins that are important biomarkers of the WOI, including LIF, HOXA10, and ITGB3 [100].

## *2.4.3 Leukemia inhibitory factor (LIF)*

*Modern Medical Genetics and Genomics*

control of trophoblast invasion [69]. Furthermore, trophoblast cells play a major role in immune tolerance since these cells do not express major histocompatibility complex (MHC) class I (HLA-A and HLA-B) or class II molecules; ensuring that maternal T cells with αβ receptors cannot mount a classic cytotoxic attack against fetal paternal alloantigens. The trophoblast also protects itself by expressing Fas ligand (Fas L), which is important in the elimination of maternal reactive T cells by apoptosis induction [72–74]. Other important component of this process is T-regulatory (Treg) cells; these cells are essential for immunosuppression, prevention of autoimmunity, and maternal tolerance to the fetus [75–78]. Treg cells have been shown to be locally enriched in decidua during early normal pregnancy [79]. Furthermore, Forkhead box P3 (Foxp3) is a master regulator of Treg cell development, function, and differentiation [80]. Expression of *FOXP3* was reduced approximately two-fold in endometrial biopsies of infertile women, implicating that the impaired differentiation of uterine T-cells into the Treg phenotype is a key determinant of fertility [81, 82]. On the other hand, helper T cells (CD4+) facilitate embryo implantation by regulating endometrial differentiation; they secrete interleukins and interferons that establish the implantation microenvironment. Successful pregnancy is dependent upon Th1/Th2 balance [83]. While Th1 cytokines are harmful for pregnancy, Th2 cytokines favor fetal growth and regulate uterine expression of fatty acid amide hydrolase (FAAH), LIF, and trophoblast release of human chorionic gonadotropin (hCG), which are known to play important roles during implantation [84–86]. Piccinni et al. demonstrated that T cells from decidua of women with a miscarriage show predominantly Th1-type cytokines with decreased Th2-type [84, 87, 88]. Finally, P4 and E2 mediate the downregulation of the maternal immune system [89]. P4 stimulates decidual proliferation; therefore, pregnancy results in an upregulation of P4 receptors on activated lymphocytes among placental cells and decidual CD56+ cells. In the presence of sufficient P4, these cells express progesterone-induced blocking factor

(PIBF), a mediator that exerts substantial anti-abortive activities.

ity [99] indicates that this gene could be essential for fertility [12].

*2.4.2 Heparin-binding epidermal growth factor-like growth factor (HB-EGF)*

HB-EGF is a member of the epidermal growth factor (EGF) family, it is expressed in the human uterus at the time of implantation and its expression is under steroidal hormone control [100–103]. The transmembrane form is associated

We review molecular markers involved in the decidualization process that could

HOXA10 is a transcription factor member of the homeobox family, known to be involved in the genetic control of embryonic development and in the regulation of the adult female reproductive tract [12, 90]. HOXA10 regulates downstream target genes that lead to endometrium development and receptivity acquisition [91], such as insulin-like growth factor binding protein 1 (*IGFBP1*) [12, 92, 93], genes of the Wnt pathway (reviewed by Sonderegger et al. [94], integrin β3 (*ITGB3*) [12, 90, 92], and empty spiracles homolog 2 (*EMX2*) [12, 95]. HOXA10 is regulated by P4 in a dosedependent manner [12, 90, 91]. The expression of HOXA10 is low during the PP and it rapidly increases in the MSP [12, 90]. The diminished expression of HOXA10 in endometria of women with recurrent pregnancy loss (RPL) [96], adenomyosis [97], endometriosis [92], polycystic ovary syndrome (PCOS) [98], and idiopathic infertil-

**2.4 Biochemical markers involved in ER**

be suitable as makers to assess ER.

*2.4.1 Homeobox A10 (HOXA10)*

**18**

LIF is a member of a cytokine family with functional redundancy that includes interleukin 6 (IL6), oncostatin (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin 1 (CT1). They regulate proliferation, differentiation, and cell survival in different cellular systems [106]. LIF acts on cells by binding to the heterodimeric LIF receptor (LIFR), which consists of two transmembrane proteins, LIFR and glycoprotein 130 (gp130). LIFR activates several signaling pathways including the JAK/STAT, MAPK, and P13-kinase pathways, whereas gp130 participates in the activation of STAT1, STAT3, and STAT5B [107]. LIF induces the expression of cytokines and other regulatory molecules that could serve to regulate preimplantation development and embryo implantation [106–108]. LIF is one of the most important cytokines for receptivity during the WOI, the expression of LIF and LIFR reaches its highest level during the WOI in the MSP, LSP, and in early pregnancy in both surface and glandular epithelial cells under the influence of P4 [106, 107, 109, 110]. LIF can also be detected in decidual leukocytes, which are abundant at the implantation site; interestingly, LIF expression is low in women with unexplained infertility [106, 107, 111]. LIF also plays a crucial role in the regulation of fetal-maternal interactions during pregnancy, this cytokine mediates uterine receptivity through autocrine/paracrine interactions by binding to LIFR on the luminal epithelium to permit blastocyst attachment [106], but also regulates trophoblast function and vascular formation in the placenta [109].

## *2.4.4 Integrin β3 (ITGB3)*

Integrins are ubiquitous cell adhesion molecules involved in maintaining normal tissue morphology and participate in cell–cell and cell-substrate interactions [100, 110, 112, 113]. In the human endometrium, integrins are involved in early embryo-endometrial interactions [90]. ITGB3 subunit is present after the ESP and its expression extend into the pregnancy [112, 114]. It has been reported that healthy fertile women show higher ITGB3 expression than patients with unexplained infertility [46, 96, 113–115]. Moreover, its dysregulation appears to characterize two distinct pathophysiological conditions that involve distinct mechanisms of defective ER: Type I and Type II. Type I defect is an out-of-phase endometrium with negative ITGB3 subunit expression, portrays a shifted WOI, and hormonal inadequacy or responsiveness is implicated, on the other hand, Type II defect is an "in-phase" endometrium with negative ITGB3 subunit expression and connotes the complete loss of the WOI. Furthermore, ITGB3 is expressed in EnSCs and endometrial glands with the highest levels in the MSP to LSP, suggesting a role in the regulation of endometrial function and implantation [115, 116]. Due to its temporal distribution and the effects of implantation when it is not present, ITGB3 is a useful molecular marker to assess ER. ITGB3 is regulated in the endometrium through a molecular mechanism via sex steroid signaling where HOXA10 acts as an intermediary [90, 96].

Other identified markers that are important for decidualization in the human endometrium include *PR*, particularly its encoded isoform progesterone receptor A (PR-A), homeobox A11 (*HOXA11*), *PTGS2*, *MUC1,* and interleukin 11 receptor (*IL11R*) [12].

#### **2.5 Transcriptomic signature to determine the WOI**

Microarray technology has been widely used to determine the transcriptomic profile of the endometrium by analyzing the expression of large batches of genes at different stages of the menstrual cycle. The most representative and commercially available test in this regard is perhaps the Endometrial Receptivity Array (ERA), developed in 2009 by Diaz-Gimeno et al., this test identifies the unique transcriptomic signature of the receptive endometrium by analyzing 238 differentially expressed genes, predicting the WOI for personalized embryo transfer (pET) [108]. Various research groups have analyzed changes in gene expression during the different phases of the endometrial cycle using microarray-based technologies [117–120], however, due to differences on results, unanimity about the main genes to be analyzed to determine the WOI has not been reached. Factors that contribute to the disagreement among studies results include differences on experimental design, utilized probes, sample acquisition day, sample size, collection method, and the application of distinct statistical analyses. Nevertheless, some genes have been reported to be expressed similarly in more than one work, here, we present a compilation of the expression profiles of those candidate genes in the human endometrium (**Table 2**).

### **2.6 miRNAs involved in ER**

Micro-RNAs (miRNAs) are small, single-stranded, non-protein-coding RNA sequences of ~18–25 nucleotides in length that play an important post-transcriptional regulatory role in gene expression [121, 122] by targeting mRNAs for cleavage or transcriptional repression [123]. More than two decades have passed since the initial discovery of miRNAs in *Caenorhabditis elegans* by Lee et al. [124]; since then, great progress has been made in the understanding of miRNAs: what they are, how are synthesized, how regulate gene expression, and how they are involved in the formation and progression of pathological disorders. Extracellular miRNAs have been ubiquitously detected in body fluids [125]. Therefore, the presence and stability of miRNAs in biological fluids have advocated their potential as noninvasive biomarkers. Nevertheless, the identification of reliable miRNA biomarkers with reproducible profiles has been a challenge, and their diagnostic promise has remained a work in progress since they have still not entered the clinical field [126]. Nonetheless, given that miRNAs are differentially expressed in the endometrium across the menstrual cycle [127–131], several studies have been conducted to explore their role in ER [131–136]. **Table 3** presents a summary of these studies.

#### **2.7 The endometrial secretome as a potential tool to ascertain ER**

The aim to develop alternative non-invasive strategies to provide accurate receptivity assessment has drawn assiduity to the endometrial secretome, which is based in the identification of factors secreted by cells or tissues at a particular time in either physiological states or pathological conditions [137], including proteins, lipids, and metabolites. Therefore, the analysis of differentially present molecules in the uterine cavity at different time points of the menstrual cycle could potentially help to identify the WOI and to diagnose uterine pathologies. Sample collection of endometrial fluid (EF) collection in the peri-implantation period is an easy procedure performed with minimally invasive tools that could easily be implemented

**21**

**Table 2.**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

**Gene Functional category Expression profile References**

Claudin 4 [*CLDN4*] Cell adhesion ESP to MSP ↑ [73, 85, 93, 94,

Endothelin 3 [*EDN3*] Vasoactive substances ESP to MSP ↓ [73, 92, 93,

Cell cycle PP to MSP

IL15 precursor [*IL15*] Immune response ESP to MSP ↑ [73, 85, 92–94,

Immune response ESP to MSP

Apoptotic pathways PP to ESP

*Genes expressed in human endometrium and its expression profiles at the different phases of the endometrial cycle. A compilation of the genes exhibiting the same expression profile in more than one work is presented, regardless of the* 

ESP to MSP MSP to LSP

PP to ESP ESP to MSP

PP to ESP ESP to MSP ESP to LSP

ESP to MSP MSP to LSP

MSP to LSP

ESP to MSP MSP to LSP

MP to LPP PP to ESP MSP to LSP

MSP to LSP LSP to MP MP to PP

PP to ESP ESP to MSP

MSP to LSP MP to LPP

ESP to MSP

PP to ESP ESP to MSP MSP to LSP

Signal transduction PP to ESP ↑ [92, 101]

↑ ↑ ↓

↑ ↑

↑ ↑ ↓

↑ ↑ ↑

ESP to MSP ↑ [73, 86, 96]

ESP to MSP ↑ [73, 92–94,

↑ ↑

↑ ↑

↑ ↑ ↓

↑ ↑ ↑ ↓

↓ ↓

↓ ↑ ↑

↑ ↑

↑ ↑ ↑

ESP to MSP ↓ [73, 88, 91, 93,

ESP to MSP ↑ [85, 93, 94, 98]

[73, 86, 91–95]

[85, 92, 96]

[73, 85, 92, 94, 96, 97, 99]

[73, 92–94, 97, 101]

[55, 64, 94, 102,

96, 97]

100]

103]

104]

96]

[87, 94] [73]

96]

[86, 87]

[87, 88]

[73, 88, 92, 93]

[73, 85, 91–93, 96, 97, 101]

[73, 94, 95]

[73, 94]

Annexin 4 [*ANX4*] Apoptosis PP to MSP

trafficking

cytokines

pathway

Immune modulators/

Response to stress and oxidoreductase activity

Anatomical structure development

Anatomical structure development

Tissue remodeling and blastocyst invasion

Anatomical structure development

development

Osteopontin [*SPP1*] Cell adhesion PP to SP

Degradation of the extracellular matrix

Homeobox A10 [*HOXA10*] Transcription factor ESP to MSP

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

Decay-accelerated factor

Growth arrest and DNAdamage-inducible protein

Glutathione peroxidase 3

Inhibitor of DNA binding 4, dominant negative helix– loop helix protein [*ID4*]

Insulin-like growth factor binding protein 1 [IGFBP-1]

Mitogen-activated protein kinase kinase kinase 5 [*MAPKKK5*]

Matrix metalloproteinase 26

Msh homeobox homologs

Olfactomedin 1 [*OLFM1*] Anatomical structure

Natural cytotoxicitytriggering receptor 3

Sex-determining region Y-box 4 [*SOX4*]

Tissue inhibitor metalloproteinase 3 [*TIMP-3*]

*differences among studies.*

*Expression profiles of genes involved in ER.*

[*DAMMP26*]

1,2 [*MSX1*]

[*NCR3*]

[DAF]

[*GADD45*]

[*GPX3*]

Apolipoprotein D [*APOD*] Cholesterol transport and

Dickkopf-1 [*DKK1*] Regulation of WNT signaling

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*


*Genes expressed in human endometrium and its expression profiles at the different phases of the endometrial cycle. A compilation of the genes exhibiting the same expression profile in more than one work is presented, regardless of the differences among studies.*

#### **Table 2.**

*Expression profiles of genes involved in ER.*

*Modern Medical Genetics and Genomics*

**2.6 miRNAs involved in ER**

**2.5 Transcriptomic signature to determine the WOI**

Other identified markers that are important for decidualization in the human endometrium include *PR*, particularly its encoded isoform progesterone receptor A (PR-A), homeobox A11 (*HOXA11*), *PTGS2*, *MUC1,* and interleukin 11 receptor (*IL11R*) [12].

Microarray technology has been widely used to determine the transcriptomic profile of the endometrium by analyzing the expression of large batches of genes at different stages of the menstrual cycle. The most representative and commercially available test in this regard is perhaps the Endometrial Receptivity Array (ERA), developed in 2009 by Diaz-Gimeno et al., this test identifies the unique transcriptomic signature of the receptive endometrium by analyzing 238 differentially expressed genes, predicting the WOI for personalized embryo transfer (pET) [108]. Various research groups have analyzed changes in gene expression during the different phases of the endometrial cycle using microarray-based technologies [117–120], however, due to differences on results, unanimity about the main genes to be analyzed to determine the WOI has not been reached. Factors that contribute to the disagreement among studies results include differences on experimental design, utilized probes, sample acquisition day, sample size, collection method, and the application of distinct statistical analyses. Nevertheless, some genes have been reported to be expressed similarly in more than one work, here, we present a compilation of the expression profiles of those candidate genes in the human endometrium (**Table 2**).

Micro-RNAs (miRNAs) are small, single-stranded, non-protein-coding RNA sequences of ~18–25 nucleotides in length that play an important post-transcriptional regulatory role in gene expression [121, 122] by targeting mRNAs for cleavage or transcriptional repression [123]. More than two decades have passed since the initial discovery of miRNAs in *Caenorhabditis elegans* by Lee et al. [124]; since then, great progress has been made in the understanding of miRNAs: what they are, how are synthesized, how regulate gene expression, and how they are involved in the formation and progression of pathological disorders. Extracellular miRNAs have been ubiquitously detected in body fluids [125]. Therefore, the presence and stability of miRNAs in biological fluids have advocated their potential as noninvasive biomarkers. Nevertheless, the identification of reliable miRNA biomarkers with reproducible profiles has been a challenge, and their diagnostic promise has remained a work in progress since they have still not entered the clinical field [126]. Nonetheless, given that miRNAs are differentially expressed in the endometrium across the menstrual cycle [127–131], several studies have been conducted to explore their role in ER [131–136]. **Table 3** presents a summary of these studies.

**2.7 The endometrial secretome as a potential tool to ascertain ER**

The aim to develop alternative non-invasive strategies to provide accurate receptivity assessment has drawn assiduity to the endometrial secretome, which is based in the identification of factors secreted by cells or tissues at a particular time in either physiological states or pathological conditions [137], including proteins, lipids, and metabolites. Therefore, the analysis of differentially present molecules in the uterine cavity at different time points of the menstrual cycle could potentially help to identify the WOI and to diagnose uterine pathologies. Sample collection of endometrial fluid (EF) collection in the peri-implantation period is an easy procedure performed with minimally invasive tools that could easily be implemented

**20**


**23**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

**Methodology Results Targets**

Compared with the WOI, 9 differentially expressed miRNAs were identified in the EPP, 8 in the LPP, 6 in the ESP and 4 in the LSP. MiR-30d was the most differentially secreted maternal miRNA in the EF during the WOI

With a 2-fold threshold: 105 miRNAs were differentially expressed: 93 **↑** and 12 **↓**. After raw signal value correction, 15 were found to be significantly different. 10 **↑** [hsa-miR-374a-5p, 145-5p, 30b-5p, 196b-5p, 199a-5p, 199b-5p, 449a, 424-5p, 125b-5p, 21-5p] and 5 **↓** [hsa-miR-1207-5p, 4306, 572, 5739, 6088]

**Predicted:** Cell cycle and endocrine processes

**Predicted:** TAM analysis: miR-30 family, human embryonic stem cell regulation, epithelialmesenchymal transition, and miRNA tumor suppressors. Network regulatory analysis: 176 miRNA-mRNA interactions. The top core mRNA were ABP1, AQP3, ASS1, and TIMP3, the top core miRNAs were has-miR-4668-5p, 429, and 5088

in ART procedures. Proteomics of EF has already rendered valuable information regarding ER; Casado-Vela et al. identified 803 proteins in EF aspirates using three different proteomic strategies [138]. Additionally, Boomsma et al. [139] analyzed endometrial secretions prior to embryo transfer from 210 women undergoing IVF to determine differences in cytokine profiling at the time of implantation, finding a negative and a positive association of monocyte chemo-attractant protein-1 (MCP-1) and IFN-γ-inducible 10 kDa protein (IP-10) levels and implantation, respectively. Lipidomics, on the other hand, seems to have rendered slight information on receptivity [140], nevertheless, a study performed by Berlanga et al. [141] and followed by Vilella et al. [142, 143] carried out lipidomic analyses of EF from patients at different stages of their menstrual cycle, they determined a significant increase in Prostaglandin E2 (PGE2) and Prostaglandin F2α (PGF2α) between days 19 and 21, coincident with the WOI. In a recent study performed by Durairaj, Aberkane et al. [144], the contribution of EnSCs to failed implantation was examined by analyzing the secretome profile of EnSCs cultures *in-vitro*. From there, they encountered that secretome profiles of pregnant women are less divergent in implantation-positivecultures particularly in Day 0 (undifferentiated cells), suggesting that endometrial defects linked to reproductive failure could be more prominent in the PP, a phase that is commonly thought to be not relevant for ER studies. This research group also demonstrated that the secretome of undifferentiated EnSCs compromises blastocyst development. Finally, they determined that a deficiency of endometrial mesenchymal stem-like cells (MSCs) could lead to aberrant EnSC function and implantation failure. Overall, this study remarks the importance of progenitor cell populations in

*Studies conducted to analyze miRNA expression profiles during the menstrual cycle as potential biomarkers of the WOI. A summary of the differentially expressed miRNAs and predicted targets found in recent studies is presented. Its* 

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

**Samples:** EF [n = 20] at different phases of the menstrual cycle of healthy women. Timing of sample collection: EPP, LPP, ESP, WOI, and LSP **Technique:** Microarray, 866 miRNAs evaluated

**Samples:** Endometrial biopsies from the WOI [5–7 days after ovulation]: 7 from RIF group and 5 from control group [infertile patients that delivered after one transfer attempt] **Technique**: Microarray Technology

*relevance regarding the role of miRNAs is also addressed.*

*Studies of miRNA-profiling during the WOI.*

**Reference and relevance**

[130] Differential miRNAs across cycle. Release of miRNAs into the EF and its uptake by the embryo

[135] Shows a significant different expression of miRNAs in the WOI of RIF patients that may contribute to impaired ER

**Table 3.**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*


*Studies conducted to analyze miRNA expression profiles during the menstrual cycle as potential biomarkers of the WOI. A summary of the differentially expressed miRNAs and predicted targets found in recent studies is presented. Its relevance regarding the role of miRNAs is also addressed.*

#### **Table 3.**

*Modern Medical Genetics and Genomics*

**Samples:** Endometrial biopsies LPP [n = 4] and MSP [n = 4] **Technique:** Microarray Technology

**Samples:** Secretory endometrium; 12 fertile women versus 11 women with RIF **Technique:** TaqMan miRNA array cards

**Samples:** Infertile women. Receptive [LH + 7, n = 5] vs. prereceptive [LH + 2, n = 5] in natural cycles. Receptive [hCG + 7, n = 5] vs. prereceptive [hCG + 4, n = 5] in stimulated IVF cycles **Technique:** Next-gen seq., 626 miRNAs evaluated

**Samples:** Endometrial samples on oocyte retrieval day from healthy ovum donors [n = 50]. EnSCs [n = 20] isolated and cultured **Technique:** miRNA PCR array, 704 miRNAs + specific miRNA-200b primers evaluated

**Samples:** Receptive MSP [LH + 7, n = 4] vs. prereceptive ESP [LH + 2, n = 5] endometrial biopsies from 9 healthy fertile women **Technique:** Microarray, 723 human and 76 human viral miRNAs evaluated

**Methodology Results Targets**

49 differentially expressed miRNAs: 12 **↑** in the MSP [miR-214, 503, 134, 450, 382, 376A, 369-5p, 222, 370, 542-3p, 105, and 127] and 12 **↓** in LPP [miR-210, 193a-3p, 345, 29b, 29c, 30b, 204, 203, 582-5p, 30d, 200c, and 31]

13 differentially expressed miRNAs: 10 **↑** [miR-23b, I45, 99a, 27b, 652, 139-5p, 195, 342-3p, 150, and 374b] and 3 **↓** [miR-32, 628-5p, and 874]

20 differentially expressed miRNAs in natural cycles: 8 **↑** [miR-30d, 30b, 30b, 31, 21, 193a-5p, 193a3p, 203] and 12 **↓** [miR-33a, 452, 125b, 455-3p, 455-5p, 483-5p, 143, 100, 504, 424, 424, 503]. 22 differentially expressed miRNAs in stimulated cycles: 19 **↑** [miR-187, 708, 433, 320a, 320b, 34c-5p, 320c, 320d, 485- 5p, 574-5p, 375, 23b, 423-5p, 193b, 34b, 503, 424, 455-5p, 483-5p] and 3 **↓** [miR-886-5p, let-7f, let-7a]

43 differentially expressed miRNAs: 26 **↑** [miR-95, 888, 936, 1185, 518f, 548 k, 593, 486-5p, 29c, 449b, 300, 371- 5p, 1224-3p, 891a, 365, 541, 409-5p, 33b, 154, 376a, 133a, 218–2, 22, 614, 369-3p, 185] and 17 **↓** [miR-146a, 155, 181b, 181a, 135b, 181d, 200c, 141, 182, 429, 483-3p, 200a, 96, 183, 9, 30a, 126] miR-95 **↑** by P4 and E2 + P4; miR-96 and miR-135b both **↓** by E2 or P4

4 significantly expressed different miRNAs in receptive samples: 2 **↑** [hsa-miR-30b and 30d] and 2 **↓** [hsa-miR-494 and 923] Suggests 12 genes that could serve as a new panel for ER: CAST, CFTR, DPYSL2, FI IR, FGFR2, LIF, MTFI, NPAS2, P4HA2, PPARGCIA, TACC2, RAB40B

**Predicted:** Cell cycle pathways in the LPP. Wnt signaling pathway in the PP. **Validated:** Decreased transcripts of predicted targets [cyclins, CDKs, and E2F3]

**Predicted:** Wnt signaling, adherents junctions, p53 signaling, cell adhesion, and cell cycle pathways. **Validated:** Decreased transcripts of N-cadherin, H2AFX, NTN4, and SFRP4. Wnt and cell cycle pathways **↓** in RIF-IVF

**Predicted:** Cell cycle, transport, cell adhesion, cell death, and metabolism

**Predicted:** Growth factors, interleukins, ECM remodeling enzymes. • Top pathways by **↓** miRNAs: axon guidance, adherents junction, actin cytoskeleton regulation, ErbB [EGFR] signaling, and renal cell carcinoma. • Top pathways by **↑** miRNAs: actin cytoskeleton regulation, adherent junction, axon guidance, Wnt signaling, and MAPK

**Predicted:** Transcription, cell proliferation, and apoptosis. Involvement in pathways such as axon guidance, Wnt/β-catenin, ERK/MAPK, TGF-β, p53 and leukocyte extravasation. They identified *SEPT7, CRMP1, SLC44A1, HES1, FXR2,* and *TNF144B* as genes that interact with genes *MIR30B* and *MIR30D*

**Reference and relevance**

[132] Shows distinct miRNA profiles in LPP and MSP

[134] Evaluates differentially expressed miRNAs in RIF-IVF patients

[154] Makes comparison between natural vs. stimulated cycles. Suggests that ovarian stimulation may shift the WOI

[136] Provides miRNA signature of EnSCs during decidualization *in vitro*

[133] Provides miRNA signature of fertile human endometria: receptive vs. prereceptive

**22**

*Studies of miRNA-profiling during the WOI.*

in ART procedures. Proteomics of EF has already rendered valuable information regarding ER; Casado-Vela et al. identified 803 proteins in EF aspirates using three different proteomic strategies [138]. Additionally, Boomsma et al. [139] analyzed endometrial secretions prior to embryo transfer from 210 women undergoing IVF to determine differences in cytokine profiling at the time of implantation, finding a negative and a positive association of monocyte chemo-attractant protein-1 (MCP-1) and IFN-γ-inducible 10 kDa protein (IP-10) levels and implantation, respectively. Lipidomics, on the other hand, seems to have rendered slight information on receptivity [140], nevertheless, a study performed by Berlanga et al. [141] and followed by Vilella et al. [142, 143] carried out lipidomic analyses of EF from patients at different stages of their menstrual cycle, they determined a significant increase in Prostaglandin E2 (PGE2) and Prostaglandin F2α (PGF2α) between days 19 and 21, coincident with the WOI. In a recent study performed by Durairaj, Aberkane et al. [144], the contribution of EnSCs to failed implantation was examined by analyzing the secretome profile of EnSCs cultures *in-vitro*. From there, they encountered that secretome profiles of pregnant women are less divergent in implantation-positivecultures particularly in Day 0 (undifferentiated cells), suggesting that endometrial defects linked to reproductive failure could be more prominent in the PP, a phase that is commonly thought to be not relevant for ER studies. This research group also demonstrated that the secretome of undifferentiated EnSCs compromises blastocyst development. Finally, they determined that a deficiency of endometrial mesenchymal stem-like cells (MSCs) could lead to aberrant EnSC function and implantation failure. Overall, this study remarks the importance of progenitor cell populations in

the endometrium that supports the acquisition of receptivity and raises the prospect of screening the endometrium before the initiation of an ART procedure.

#### **2.8 The microbiome as a novel aspect of ER**

Historically, the uterus was assumed to be free of bacteria as the fetal environment was considered to be physiologically sterile [145], this notion implies that the neonate's microbiome is acquired only during and after birth. Although recent research still supports this conception [146], others have characterized upper genital tract microbiota [147–150], suggesting that the endometrial and vaginal microbiota not be identical [151]. What is more, the study by Moreno et al. [147, 152], which defined the microbiota in the EF as *Lactobacillus*-dominated (LD) or non-*Lactobacillus*-dominated (NLD), suggested that the presence of an NLD microbiota in a receptive endometrium was associated with a significant decrease in implantation, pregnancy, ongoing pregnancy, and live birth rates. Nevertheless, they acknowledge that in the absence of pathological signs, an NLD microbiota could be considered normal since *Lactobacillus*-deficient communities have been identified in the genital tract of otherwise healthy asymptomatic women [153]. This conception sets the stage for further research of the human microbiome, expands the possibilities to assess individualized receptivity based on the endometrial microbiome, and opens the door to explore targeted therapies for an altered endometrial microbial habitat.

## **3. Remarks**

This review encloses different aspects of ER, spotlighting the opening of the WOI. Altogether, this compilation could aid in the development of new clinical practices that define an individual's receptive window for pET to improve ART results ultimately.

Nowadays, ER is the rate-limiting step in successful ART procedures that end up in pregnancy and child delivery. The endometrial tissue is a ponderous element in fertility; it constitutes the soil in which a viable embryo will implant to achieve progeny. The attainment of ER involves an extensive assortment of genetic and biochemical mechanisms that must integrate in a parallel manner. Understanding of this process as an entity is still insufficient; nevertheless, the surge of new technologies is contributing in the deciphering of receptivity mechanisms and in search of novel biomarkers that could serve to detect the WOI. Notably, although most studies focus on individual genetic mutations, a more comprehensive view of the parental genetics is needed to determine whether an endometrium is adequate for embryo transfer before the initiation of an ART procedure. Due to ambiguous or non-conclusive results in search of genetic predispositions of endometrialassociated infertility, it would be controversial to provide genetic counseling currently. Perhaps, in the future, massive sequencing could help to provide insights into the importance of single and multiple genetic mutations to establish a receptive or non-receptive profile. If assertive, this profile could be applied as an endometrial pre-implantation parental test to improve the rates of healthy pregnancies and live births. With this in mind, in our opinion, the best short-term approaches towards detecting or improving ER are the transcriptome, microbiome, and miRNA signatures, all achievable using the power of NGS. This review encloses different aspects of ER, spotlighting the opening of the WOI. Altogether, this compilation could aid in the development of new clinical practices that define an individual's receptive window for pET to improve ART results ultimately.

**25**

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

**Abbreviations and nomenclatures**

ART assisted reproductive tech CNTF ciliary neurotrophic factor

EGF epithermal growth factor EMX2 empty spiracles homolog 2 eNOS endometrial nitric-oxide synthase

EnSCs endometrial stromal cells EPP early-proliferative phase ER endometrial receptivity ERA endometrial receptivity array

ESP early-secretory phase ESR1 estrogen receptor 1

F2 coagulation factor II F5 coagulation factor V F8 coagulation factor 8 FAAH fatty acid amide hydrolase

Fas L Fas ligand FGB β fibrinogen Foxp3 Forkhead box P3 gp130 glycoprotein 130 GPIIIa glycoprotein IIIa

HOXA10 homeobox A10 HOXA11 homeobox A11

HSCORE histological score

IL6 interleukin 6

ITGB3 integrin β3

MiRNA microRNA MP menstrual phase

IL11R interleukin 11 receptor

IVF in-vitro fertilization LD lactobacillus-dominated LH luteinizing hormone LIF leukemia inhibitory factor

LPP late-proliferative phase LSP late-secretory phase

MDM2 murine double minute 2

F13A1 coagulation factor XIII A chain

hCG human chorionic gonadotropin

HPA1 human platelet alloantigens 1

IP-10 IFN-γ-inducible 10 kDa protein

LIFR leukemia inhibitory factor receptor

MCP-1 monocyte chemo-attractant protein-1

MHC major histocompatibility complex

HAUSP herpesvirus-associated ubiquitin-specific protease

IGFBP1 insulin-like growth factor binding protein 1

HB-EGF heparin-binding epithermal growth factor-like growth factor

3D three-dimensional ApoE apolipoprotein E

CT1 cardiotrophin 1 E2 estrogen

ECM extracellular matrix EF endometrial fluid

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*

## **Abbreviations and nomenclatures**

*Modern Medical Genetics and Genomics*

metrial microbial habitat.

**3. Remarks**

results ultimately.

**2.8 The microbiome as a novel aspect of ER**

the endometrium that supports the acquisition of receptivity and raises the prospect

Historically, the uterus was assumed to be free of bacteria as the fetal environment was considered to be physiologically sterile [145], this notion implies that the neonate's microbiome is acquired only during and after birth. Although recent research still supports this conception [146], others have characterized upper genital tract microbiota [147–150], suggesting that the endometrial and vaginal microbiota not be identical [151]. What is more, the study by Moreno et al. [147, 152], which defined the microbiota in the EF as *Lactobacillus*-dominated (LD) or non-*Lactobacillus*-dominated (NLD), suggested that the presence of an NLD microbiota in a receptive endometrium was associated with a significant decrease in implantation, pregnancy, ongoing pregnancy, and live birth rates. Nevertheless, they acknowledge that in the absence of pathological signs, an NLD microbiota could be considered normal since *Lactobacillus*-deficient communities have been identified in the genital tract of otherwise healthy asymptomatic women [153]. This conception sets the stage for further research of the human microbiome, expands the possibilities to assess individualized receptivity based on the endometrial microbiome, and opens the door to explore targeted therapies for an altered endo-

This review encloses different aspects of ER, spotlighting the opening of the WOI. Altogether, this compilation could aid in the development of new clinical practices that define an individual's receptive window for pET to improve ART

Nowadays, ER is the rate-limiting step in successful ART procedures that end up in pregnancy and child delivery. The endometrial tissue is a ponderous element in fertility; it constitutes the soil in which a viable embryo will implant to achieve progeny. The attainment of ER involves an extensive assortment of genetic and biochemical mechanisms that must integrate in a parallel manner. Understanding of this process as an entity is still insufficient; nevertheless, the surge of new technologies is contributing in the deciphering of receptivity mechanisms and in search of novel biomarkers that could serve to detect the WOI. Notably, although most studies focus on individual genetic mutations, a more comprehensive view of the parental genetics is needed to determine whether an endometrium is adequate for embryo transfer before the initiation of an ART procedure. Due to ambiguous or non-conclusive results in search of genetic predispositions of endometrialassociated infertility, it would be controversial to provide genetic counseling currently. Perhaps, in the future, massive sequencing could help to provide insights into the importance of single and multiple genetic mutations to establish a receptive or non-receptive profile. If assertive, this profile could be applied as an endometrial pre-implantation parental test to improve the rates of healthy pregnancies and live births. With this in mind, in our opinion, the best short-term approaches towards detecting or improving ER are the transcriptome, microbiome, and miRNA signatures, all achievable using the power of NGS. This review encloses different aspects of ER, spotlighting the opening of the WOI. Altogether, this compilation could aid in the development of new clinical practices that define an individual's receptive

of screening the endometrium before the initiation of an ART procedure.

**24**

window for pET to improve ART results ultimately.



**27**

**Author details**

and Esther López-Bayghen<sup>2</sup>

Genética SC, INGENES, Mexico

provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Almena López-Luna1,2, Dinorah Hernández-Melchor1,2, Leticia Ramírez-Martínez2

1 Laboratorio de Investigación y Diagnóstico Molecular, Instituto de Infertilidad y

2 Departamento de Toxicología, Cinvestav-IPN, Ciudad de México, Mexico

\*

\*Address all correspondence to: ebayghen@cinvestav.mx

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future…*

*DOI: http://dx.doi.org/10.5772/intechopen.80452*

*The Genetic and Biochemical Blueprint of Endometrial Receptivity: Past, Present, and Future… DOI: http://dx.doi.org/10.5772/intechopen.80452*

## **Author details**

*Modern Medical Genetics and Genomics*

MSP mid-secretory phase

MUC1 Mucin 1 MUC4 Mucin 4

OSM oncostatin P4 progesterone TP53 tumor protein 53

PGE2 prostaglandin E2 PGF2α prostaglandin F2α PGs prostaglandins

PP proliferative phase PR progesterone receptor PR-A progesterone receptor A RIF repeated implantation failure RPL recurrent pregnancy loss RSA recurrent spontaneous abortion SNP single-nucleotide polymorphism

SP secretory phase

TFF3 trefoil factor 3 Treg T-regulatory cells uNK uterine natural killer

MSCs mesenchymal stem-like cells

NGS next generation sequencing NLD non-lactobacillus-dominated

PAI-1 plasminogen activator inhibitor-1 PCOS polycystic ovarian syndrome PCR polymerase chain reaction pET personalized embryo transfer

PIBF progesterone-induced blocking factor

TAM tool annotations human miRNAs

VEGF vascular endothelial growth factor VNTR variable number tandem repeats

WOI window of implantation

MTHFR methylenetetrahydrofolate reductase

**26**

Almena López-Luna1,2, Dinorah Hernández-Melchor1,2, Leticia Ramírez-Martínez2 and Esther López-Bayghen<sup>2</sup> \*

1 Laboratorio de Investigación y Diagnóstico Molecular, Instituto de Infertilidad y Genética SC, INGENES, Mexico

2 Departamento de Toxicología, Cinvestav-IPN, Ciudad de México, Mexico

\*Address all correspondence to: ebayghen@cinvestav.mx

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[23] Noyes N et al. Factors useful in predicting the success of oocyte donation: A 3-year retrospective analysis. Fertility and Sterility. 2001;**76**(1):92-97

[24] Friedler S et al. The role of ultrasonography in the evaluation of endometrial receptivity following assisted reproductive treatments: A critical review. Human Reproduction Update. 1996;**2**(4):323-335

[25] Khalifa E et al. Sonographic appearance of the endometrium: The predictive value for the outcome of in-vitro fertilization in stimulated cycles. Human Reproduction. 1992;**7**(5):677-680

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[29] Raga F et al. Assessment of endometrial volume by threedimensional ultrasound prior to embryo transfer: Clues to endometrial receptivity. Human Reproduction. 1999;**14**(11):2851-2854

[30] Zhao J, Zhang Q, Li Y. The effect of endometrial thickness and pattern measured by ultrasonography on pregnancy outcomes during IVF-ET cycles. Reproductive Biology and Endocrinology. 2012;**10**:100

[31] Zhao J et al. Endometrial pattern, thickness and growth in predicting pregnancy outcome following 3319 IVF cycle. Reproductive Biomedicine Online. 2014;**29**(3):291-298

[32] Fatemi HM, Popovic-Todorovic B. Implantation in assisted reproduction: A look at endometrial receptivity. Reproductive Biomedicine Online. 2013;**27**(5):530-538

[33] Coulam CB, Jeyendran RS. Vascular endothelial growth factor gene polymorphisms and recurrent pregnancy loss. American Journal of Reproductive Immunology. 2008;**59**(4):301-305

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**28**

*Modern Medical Genetics and Genomics*

[1] Lessey BA. The role of the endometrium during embryo implantation. Human Reproduction. Human Reproduction Update.

[11] Lockwood CJ et al. The role of decidualization in regulating endometrial hemostasis during the menstrual cycle, gestation, and in pathological states. Seminars in Thrombosis and Hemostasis.

[12] Lu Z, Hardt J, Kim JJ. Global analysis of genes regulated by HOXA10 in decidualization reveals a role in cell proliferation. Molecular Human Reproduction. 2008;**14**(6):357-366

[13] Jabbour HN et al. Endocrine regulation of menstruation. Endocrine

[14] Fox C et al. Local and systemic factors and implantation: What is the evidence? Fertility and Sterility.

[15] Murray MJ et al. A critical analysis of the accuracy, reproducibility, and clinical utility of histologic endometrial dating in fertile women. Fertility and Sterility. 2004;**81**(5):1333-1343

[16] Coutifaris C et al. Histological dating of timed endometrial biopsy tissue is not related to fertility status. Fertility and Sterility. 2004;**82**(5):1264-1272

[17] Nikas G. Endometrial receptivity: Changes in cell-surface morphology. Seminars in Reproductive Medicine.

[18] Quinn CE, Casper RF. Pinopodes: A questionable role in endometrial receptivity. Human Reproduction Update. 2009;**15**(2):229-236

[19] Zhang T et al. The role of threedimensional power Doppler ultrasound parameters measured on hCG day in the prediction of pregnancy during in vitro

Reviews. 2006;**27**(1):17-46

2016;**105**(4):873-884

2000;**18**(3):229-235

1998;**4**(5):448-458

2007;**33**(1):111-117

[2] Diedrich K et al. The role of the endometrium and embryo in human implantation. Human Reproduction

[3] Strowitzki T et al. The human endometrium as a fertility-determining factor. Human Reproduction Update.

[4] Teh WT, McBain J, Rogers P. What is the contribution of embryo-endometrial asynchrony to implantation failure? Journal of Assisted Reproduction and Genetics. 2016;**33**(11):1419-1430

[5] Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Human Reproduction Update.

[6] Reed BG, Carr BR. The normal menstrual cycle and the control of ovulation. In: De Groot LJ et al., editors. South Dartmouth (MA): Endotext; 2000

[7] Cha J, Sun X, Dey SK. Mechanisms

successful pregnancy. Nature Medicine.

[8] Mahajan N. Endometrial receptivity array: Clinical application. Journal of Human Reproductive Sciences.

of implantation: Strategies for

[9] Heger A, Sator M, Pietrowski D. Endometrial receptivity and its predictive value for IVF/ICSI-outcome. Geburtshilfe und Frauenheilkunde.

[10] Beier HM, Beier-Hellwig K. Molecular and cellular aspects of endometrial receptivity.

2012;**18**(12):1754-1767

2015;**8**(3):121-129

2012;**72**(8):710-715

2000;**15**(Suppl 6):39-50

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Update. 2007;**13**(4):365-377

2006;**12**(5):617-630

2006;**12**(6):731-746

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on implantation success or failure. American Journal of Obstetrics and Gynecology. 2016;**215**(6):684-703

Chapter 3

Abstract

embryo evaluation.

1. Introduction

37

in vitro fertilization, karyotype

Aneuploidy Rates Inversely

Correlate with Implantation

during In Vitro Fertilization

Procedures: In Favor of PGT

Elizabeth Schaeffer, Leonardo Porchia, Almena López-Luna,

Aneuploidy, the hold of an abnormal number of chromosomes that differs from the normal karyotype, is a recognized leading cause of miscarriage and congenital disabilities. In human gametes and embryos, aneuploidy rates are prevalent, and these rates increase with advanced maternal age; additionally, it has been suggested that hormonal stimulation for achieving in vitro fertilization (IVF) protocols further increases aneuploidy rates. Although about 65% of chromosomally abnormal embryos culminate in spontaneous miscarriages, there is still evidence of live births harboring crucial aneuploidies. Furthermore, although some frequent aneuploidies are consistent, others differ between countries, making it harder to focus on a specific set of anomalies but vital to focus regionally on those more prevalent. Preimplantation genetic testing (PGT) is a highly endorsed technique in assisted reproductive treatments to evaluate possible embryo aneuploidies, genetic defects, and congenital disorders. On this subject, this study shows that IVF aneuploidy rates in embryo cohorts of high morphological quality are inversely associated with implantation rates. In its entirety, this study reinforces the utility of PGT for

Keywords: aneuploidy, preimplantation genetic testing, embryo implantation,

Aneuploidy is defined as a chromosome number that is not an exact multiple of the usually haploid number [1]. The terms haploid and diploid that describe single (n) and double (2n) chromosome sets in cells originate from the Greek terms haplóos meaning single and diplóos meaning double. The term ploidy was subsequently derived to describe the total chromosome content of cells. Consequently, the term euploid refers to a chromosome with an exact multiple of the haploid number [2]. Human body cells (somatic cells) are diploid, carrying two complete sets of chromosomes: one set of 23 chromosomes from their father and one set of 23 chromosomes from their mother; the two sets combined provide a full complement

Dinorah Hernández-Melchor and Esther López-Bayghen

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[151] Mitchell CM et al. Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women. American Journal of Obstetrics and Gynecology. 2015;**212**(5):611. e1-611.e9

[152] Moreno I, Franasiak JM. Endometrial microbiota-new player in town. Fertility and Sterility. 2017;**108**(1):32-39

[153] Ravel J et al. Vaginal microbiome of reproductive-age women. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(Suppl 1):4680-4687

[154] Sha AG, Liu JL, Jiang XM, Ren JZ, Ma CH, Lei W, Su RW, Yang ZM. Genome-wide identification of microribonucleic acids associated with human endometrial receptivity in natural and stimulated cycles by deep sequencing. Fertil Steril. 2011;**96**(1):150-155 e155

## Chapter 3

*Modern Medical Genetics and Genomics*

2009;**8**(10):4622-4632

2009;**24**(6):1427-1435

2015;**12**(4):237-243

3):S271-S275

[140] Demiral I et al. Genomic, proteomic and lipidomic evaluation of endometrial receptivity. Turkish Journal of Obstetrics and Gynecology.

of human endometrial fluid aspirate. Journal of Proteome Research.

on implantation success or failure. American Journal of Obstetrics and Gynecology. 2016;**215**(6):684-703

[148] Aagaard K et al. The placenta harbors a unique microbiome. Science Translational Medicine.

Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene. Peer

[150] Franasiak JM et al. Endometrial microbiome at the time of embryo transfer: Next-generation sequencing of the 16S ribosomal subunit. Journal of Assisted Reproduction and Genetics.

[151] Mitchell CM et al. Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women. American Journal of Obstetrics and Gynecology. 2015;**212**(5):611.

2014;**6**(237):237ra65

[149] Verstraelen H et al.

Journal. 2016;**4**:e1602

2016;**33**(1):129-136

[152] Moreno I, Franasiak

2017;**108**(1):32-39

JM. Endometrial microbiota-new player in town. Fertility and Sterility.

[153] Ravel J et al. Vaginal microbiome of reproductive-age women. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(Suppl 1):4680-4687

[154] Sha AG, Liu JL, Jiang XM, Ren JZ, Ma CH, Lei W, Su RW, Yang ZM. Genome-wide identification of microribonucleic acids associated with human endometrial receptivity in natural and stimulated cycles by deep sequencing. Fertil Steril. 2011;**96**(1):150-155 e155

e1-611.e9

[139] Boomsma CM et al. Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF. Human Reproduction.

[141] Berlanga O et al. How endometrial secretomics can help in predicting implantation. Placenta. 2011;**32**(Suppl

[143] Vilella F et al. PGE2 and PGF2alpha concentrations in human endometrial fluid as biomarkers for embryonic implantation. The Journal of Clinical Endocrinology and Metabolism.

stromal cell secretome precedes embryo implantation failure. Molecular Human

[145] Ansbacher R, Boyson WA, Morris JA. Sterility of the uterine cavity. American Journal of Obstetrics and Gynecology. 1967;**99**(3):394-396

[146] Perez-Munoz ME et al. A critical assessment of the "sterile womb" and "in utero colonization" hypotheses: Implications for research on the pioneer infant microbiome. Microbiome.

[147] Moreno I et al. Evidence that the endometrial microbiota has an effect

[142] Vilella F, Ramirez LB, Simon C. Lipidomics as an emerging tool to predict endometrial receptivity. Fertility and Sterility. 2013;**99**(4):1100-1106

2013;**98**(10):4123-4132

[144] Peter Durairaj RR et al. Deregulation of the endometrial

Reproduction. 2017;**23**(8):582

**36**

2017;**5**(1):48
