Basil C. Tarlatzis1 and Christina Vaitsopoulou1
(1)
1st Department of Obstetrics/Gynecology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
Basil C. Tarlatzis
Email: basil.tarlatzis@gmail.com
3.1 Introduction
Ovarian stimulation constitutes an integral part of fertility treatment with the different assisted reproduction techniques (ART). As documented in other areas of medicine, patients’ response to medication depends on their genomic profile. Hence, in order to understand the variations in ovarian response to stimulation between different women, it is necessary to examine the genomics of ovarian and endometrial function. This will allow to predict more accurately low, normal, or high responders and adopt the stimulation protocol accordingly.
The aim of this chapter is to review the involvement of different genes in follicular, oocyte and endometrial development, and their relevance to ovarian stimulation.
3.2 Follicular Development and Angiogenesis
In humans and large mammals, the follicles grow after antrum formation until they become gonadotropin dependent and enter a phase of rapid terminal development [1]. Gonadotropin dependence is acquired at a given follicular diameter. Below this diameter, the small antral follicles constitute a pool of gonadotropin-responsive follicles, which is the reserve for ovulation. This is a dynamic reserve, since it is emptied by the entry of follicles in the follicular waves of terminal development mediated by the follicle-stimulating hormone (FSH) gene expression and renewed by the continuous growth of smaller follicles.
3.3 FSH, LH, and Their Receptors
In the female reproductive system, angiogenesis is a process essential for normal tissue growth and plays a crucial role in follicular growth and the selection of the ovulatory follicle [2]. In the ovary, blood vessel formation facilitates the delivery of many substances, including oxygen, nutrients, and FSH to follicles. A capillary network around each follicle is necessary for follicles to grow beyond the secondary stage, which contains multiple layers of granulosa and theca cells. As the follicle develops, endothelial cells are recruited to the theca cell layer at the adjacent ovarian stroma. Endothelial cell proliferation is maintained in healthy tertiary follicles to support the expansion of the theca vasculature. In contrast, follicular atresia is associated with inadequate development and regression of the theca vasculature. Increased vascularity is a determining factor in the establishment of follicular dominance. Consequently, angiogenic factors are of increasing interest in ovarian physiology.
FSH and luteinizing hormone (LH) gene products are pituitary glycoproteins essential for normal gonadal function [3]. They regulate gonadal growth, differentiation, endocrine function, and gametogenesis. The effects of FSH and LH are mediated through binding to specific cell surface receptors, FSHR, and LHR, respectively. The presence of FSHR and LHR mRNA in denuded oocytes and preimplantation embryos from zygotes to blastocysts has been demonstrated, indicating a possible role for gonadotropins in the resumption of meiosis and early embryonic development.
FSHR and LHR are G-protein-coupled receptors, which span the plasma membrane seven times and transduce the biological action of FSH and LH, using cyclic AMP (cAMP) as the main intracellular second messenger [3]. The FSHR gene contains a single large exon, which encodes the transmembrane and intracellular domains, and nine smaller exons, which encode the extracellular domain.
Ovarian response to FSH stimulation depends on the FSH genotype. Factors proposed to affect ovarian response to FSH are the distribution of FSH isoforms and single nucleotide polymorphisms [3]. The role of two distinct FSHR variants, Thr307/Asn680 and Ala307/Ser680, in ovarian response to FSH in women undergoing controlled ovulation induction has been investigated by Loutradis et al. [3], who examined the prevalence of Ser680Asn polymorphisms of the FSHR gene. According to the results, good responders carry more often the Asn/Ser genotype. This finding may reflect a better and more rapid ovarian response to exogenous stimulation, possibly due to a more efficient FSHR. The Ser/Ser variant might be related to higher serum FSH levels, while the Asn/Ser, with lower serum FSH levels. However, Mohiyiddeen et al. [4] demonstrated that FSHR genotype does not predict metaphase II oocyte output or fertilization rates in ICSI patients.
Signaling mediated by the LHR gene expression is important for patients’ response to exogenous gonadotropins (i.e., hCG) administered during controlled ovarian hyperstimulation (COH), and inter-individual variability in LHR activity could significantly impact the outcome [5]. O’Brien et al. [5] found that insLQ polymorphism (rs4539842) is not associated with patients’ response to COH and it is not a predictor for ovarian hyperstimulation syndrome (OHSS). However, they associated the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) rs4073366 polymorphism with the OHSS during COH and found that the improved function conferred to LHR by this polymorphism in vitro possibly is not reflective of the situation in the ovaries.
3.4 Oocyte Development and Quality
Human cumulus cells express long pentraxin 3 (PTX3), which is a member of complex superfamily of multifunctional proteins characterized by a cyclic multimeric structure [6]. PTX3 is highly conserved in evolution, and its protein is present in human cumulus matrix, suggesting that this molecule is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorushyaluronan-rich extracellular matrix. Moreover, a higher relative abundance of PTX3 mRNA in cumulus cells from fertilized oocytes has been detected compared with cumulus cells from unfertilized oocytes, indicating that PTX3 is a possible marker for oocyte quality [6].
Granulosa cells (GC) are the most important somatic cells for determining the final size of preovulatory follicles [7]. Luteinized GC can proliferate, and the telomerase activity (TA) of luteinized GC may predict the clinical outcome of IVF treatment. However, telomerase activity seems to be more significant for predicting the outcome of IVF treatment than telomere length (TL) in granulosa cells. Telomeres are the physical ends of eukaryotic chromosomes. They consist of a 5- to 15-kb-long tandem repeat hexanucleotide sequence (TTAGGG)n that protects the ends of the double-stranded DNA. Hence, telomeres play an essential role in the maintenance of chromosomal stability and cell viability. There is evidence that TL is longer in the oocytes of women who become pregnant than in those of women who fail to become pregnant after IVF treatment [7]. In addition, TL can predict oocyte development. Telomeric DNA deficiency is associated with genomic instability in somatic cells and plays a role in the development of aneuploidies commonly found in female germ cells and human embryos.
In GC of preantral follicles, NFIA, a transcription factor involved in the control of cell growth in humans and model systems, and HIF1A, an interacting protein that activates the transcription of target genes involved in energy metabolism, angiogenesis, and apoptosis, are over-expressed [8].
Luteinization of granulosa cells is initiated by the LH surge or the addition of LH or human chorionic gonadotropin (hCG). Nevertheless, it has been shown that the removal of granulosa cells from the follicle causes spontaneous luteinization in the absence of LH [9]. Therefore, luteinization is a differentiation pathway programmed before antral formation, and the only way follicles can escape this procedure is by inhibitory factors. Such inhibitors may be present in follicular fluid or may come directly from the oocyte itself [9]. The LH surge is able to remove such inhibitory factors to disrupt connections between granulosa cells and the oocyte and to induce genes that facilitate luteinization.
One molecule that may have a role in the prevention of luteinization is activin A [9], a dimeric glycoprotein and member of the transforming growth factor (TGF)-β superfamily. Human chorionic gonadotropin (hCG) and activin A have opposite effects on the luteinized granulosa cells involved in luteal formation, with hCG increasing luteinization to a more luteal phenotype [9]. Activins are necessary for follicular granulosa cell proliferation, FSHR regulation, FSH-induced aromatase expression, decreased theca cell androgen production and increased oocyte maturation. As activin A appears to have a positive role in the follicle and a negative role in the corpus luteum, its activity appears to be suppressed at the follicular–luteal transition [9].
Other luteinization inhibitors are the bone morphogenetic proteins (BMPs) [2]. The BMP cytokine system plays a crucial role in folliculogenesis and angiogenesis. The BMP cytokines are growth factors belonging to the transforming growth factor β superfamily. In the ovary, BMP cytokines act as luteinization inhibitors by suppressing LHR gene expression in GC. Of the BMP cytokines, BMP-7 is most highly expressed in the theca cell layer in the ovarian follicles. According to Akiyama et al. [2], BMP-7 can induce vascular endothelial growth factor (VEGF)-A mRNA and protein expression in human GC. In many species, VEGF, which is detected in the granulosa and theca layer of secondary follicles, is recognized as an important factor in the recruitment of a vascular network to the theca layer [2]. During the process of follicular development, the vasculature of the follicle is limited to the theca layer outside the basement membrane. Therefore, it is likely that follicles create a gradient of angiogenic factors to stimulate vascularization toward the basement membrane, maximizing the supply of oxygen, nutrients, and hormones to GC. Furthermore, in endothelial cells, BMP-7 can increase the number of cells, accelerate tube formation, and increase the VEGF receptor mRNA. Thus, endothelial cells could be stimulated to form vasculature by BMP-7 via two distinct mechanisms: induction of VEGF expression in GC and increased sensitivity of endothelial cells to VEGF [2].
3.5 Follicular Development and Signaling Pathways
VEGF promotes early folliculogenesis. Bonnet et al. [8] identified the overexpression of members of VEGF pathway, including VEGF-A and NRP1 (a VEGF receptor), in GC and overexpression of FLT1 (another VEGF receptor) in oocytes. These gene expressions suggest a role for the VEGF pathway in GC-oocyte and GC-GC crosstalks [8]. The VEGF pathway may protect GC against atresia. Atresia is an important process in folliculogenesis and concerns the majority of the follicles. Apoptosis is found in the oocytes of primordial follicles and progressively extends to GC of growing follicles. Bonnet et al. characterized the expression of different genes of the BCL2 family either in oocytes (BCL2L1, BCL2L10, BCL2L11 [BIM], BCL2L14 [BCLG]), or in GC (BCL2 BCL2L2, BOK). For example, the BCL2L1 gene plays a crucial role in the survival of germ cells. BCL2L10 may play other roles related to cell cycle control and oocyte maturation.
Oogenesis begins with the migration of primordial germ cells into the gonadal ridges and their proliferation within ovarian nests or cysts [1]. Then primary oocytes are developed, meiotic prophase starts, and primordial follicles are formed, each one consisting of a primary oocyte arrested at the diplotene stage of prophase I of meiosis. The primordial follicles may begin to grow immediately or after a gap depending on the species, or they become quiescent.
The reserve of primordial follicles seems to determine the ovarian activity of the adult [1]. In fact, the regulation of germ and somatic cell survival, proliferation, and differentiation involves the same factors and molecular mechanisms from the formation of the primordial follicles up to the stage when the follicles become gonadotropin dependent and enter terminal development. Two main signaling pathways play major roles in all these processes. The first one is the PTEN/PI3K/PDPK1 (previously known as PDK1)/AKT1 (previously known as AKT or PKB) signaling pathway, which regulates germ cell survival, follicular growth activation, and follicle growth [1]. This pathway is activated by various hormones, growth factors, and cytokines. Among them, insulin, insulin-like growth factors (IGF), and KIT ligand are crucial for the survival and differentiation of germ and somatic ovarian cells. The second important signaling pathway involves SMAD transcription factors, which are activated by factors of the transforming growth factor-B (TGFB) super family (i.e., BMP and AMH for the SMAD1/5/8 pathway) and the TGFB and activins for the SMAD2/3 pathway [1]. It orchestrates the formation and development of follicles under the control of oocyte (bone morphogenetic protein 15 [BMP15], growth differentiation factor-9 [GDF9])- and somatic cell (BMP2, BMP4, AMH, activins)-derived factors. BMP15, like other genes, is not expressed in the oocyte until the primary follicle stage and is involved in the transition from primary to secondary follicles [8]. However, βFGF, GDF 9, and BMP 4 are involved in the transition from primordial to primary follicles [8].
Various mutations in genes encoding the ligands, receptors, or signaling effectors of the PTEN/PI3K/PDPK1/AKT1 or the SMAD signaling pathways can accelerate the exhaustion rate of the ovarian reserves and cause premature ovarian insufficiency (POI) [1]. Mutations in some factors of the TGFβ super family affect the transition of growing follicles between the two follicular reserves. In humans, various mutations in BMP15 and, to a lesser extent, in GDF9 and INHA have been found to be associated with POI, while genetic variants of AMH and its receptor AMHRII are associated with different ages at menopause, confirming the importance of these factors for the lifespan of the ovarian reserves [1]. Antimullerian hormone (AMH) gene is the best endocrine marker of the population of small antral follicles in humans because AMH expression in female mammals is strictly restricted to granulosa cells of growing follicles, while the granulosa cells of the largest preantral and the small antral healthy growing follicles express the highest amounts of AMH in the ovaries [1].
The Notch signaling pathway contributes to cell communication by influencing cell proliferation, differentiation, and apoptosis, as mentioned by Bonnet et al. [8]. Notch is involved in GC proliferation through NOTCH1/ CNTN1 binding. According to Tanriverdi et al. [10], the Notch genes encode transmembrane receptors that are highly conserved evolutionarily and modulate cell proliferation, differentiation, and survival. The Notch signaling pathway is initiated by a receptor-ligand interaction between two neighboring cells [10]. Cleavage of the receptors occurs after Notch receptors bind to their ligands. The intracellular domain of the receptor releases and translocates to the nucleus, where Notch forms transcriptional complexes with transcription factors of the CSL family (C promoter binding factor 1/suppressor of hairless/Lag-1). Notch genes are actively expressed by cumulus cells during folliculogenesis [10]. However, Notch is not released by oocytes and atretic follicles. Tanriverdi et al. [10] showed that Notch signaling proteins (Notch1, Notch2, Notch3, Notch4, Jagged 1, and Jagged 2) can be an indicator for understanding the ovarian response in ovulation induction.
Androgen signaling is crucial for normal folliculogenesis. Androgens’ physiological functions are mediated through androgen response element (ARE)-dependent genomic actions and via membrane-initiated non-genomic signaling [11]. During primordial follicle recruitment, androgens induce expression of KIT ligand. Also, the androgen receptor (AR)-induced PI3K/AKT pathway, through modulation of FOXO3 and GDF9, may be involved in primordial follicle recruitment. In the preantral stage of follicular development, androgens, through a synergistic interaction between the nuclear and extranuclear signaling, regulated by a common adaptor protein called paxillin, induce the expression of a micro-RNA in granulosa cells, which contribute to follicular survival by inhibiting pro-apoptotic protein levels and preventing follicular atresia [11]. Androgens also increase FSHR and intracellular cAMP levels that enhance the sensitivity of preantral follicles toward FSH actions. Moreover, androgens stimulate the expression of key steroidogenic enzymes, aromatase P450 and P450 side chain cleavage enzyme in a mechanism mediated by the induction of an orphan nuclear receptor, liver receptor homolog 1 (LRH1) [11]. In addition, androgens contribute to estradiol (E2) synthesis, which probably plays a role in controlling the primordial follicle pool [8]. All these actions together promote preantral follicle growth and transition to antral stage [11]. In peri-ovulatory GC, androgens can induce the expression of Cox2 and Areg genes and thereby can directly influence the ovulatory process [11].
Bonnet et al. [8] identified the overexpression of LXRB, FXRA, and RXRA genes in GC preantral stages. RXR is a retinoid X receptor that binds as heterodimers (LXR/RXR, FXR/RXR, etc.) and becomes transcriptionally active only in the presence of a ligand. LXRs are activated by oxysterols [generated by intermediates of steroid hormone and cholesterol synthesis (CYP21A1, STAR, P450scc products)] and regulate ovarian steroidogenesis at antral stages [8]. FXRs are activated by bile acid, sterol, and different lipids. Other growth factors and hormones that have been shown to regulate primordial follicle assembly are connective tissue growth factor (CTGF), tumor necrosis factor alpha (TNFa), members of the brain derived neurotrophic factor (BDNF) / NTRK2 neurotrophin signaling pathway and kit ligand (KITL) and GDF9 [12]. Evidence suggests that fibroblast growth factor-2 (FGF-2) may also be a regulator of follicle assembly [12].
Vos et al. [13] indicated the presence of matrix metalloproteinases (MMPs) MMP-14 and MMP-2 during human ovarian follicular development from the primordial follicle to the tertiary follicle and corpus luteum, and MMP-2 is present in the follicular fluid. Proteins of the matrix metalloproteinase (MMP) family are involved in degrading the extracellular matrix in normal physiological processes, such as embryonic development, reproduction and tissue remodeling [13]. Most MMPs are secreted as inactive pro-proteins, which are activated when cleaved by extracellular proteinases. MMP-14 (former MT1-MMP) is a member of the membrane-type MMP (MT-MMP) subfamily [13]. These proteins are expressed at the cell surface rather than secreted and contain a transmembrane domain. Apart from functioning as a gelatinase itself, MMP-14 also cleaves pro-MMP-2 (72 kD) into its active 66 kD form.
Elizur et al. [14] found that elevated levels of the Fragile X Mental Retardation 1 gene (FMR1) mRNA in granulosa cells are associated with low ovarian reserve in women carriers of the FMR1 premutation, while Peprah [15] tried to explain the decreased fertility in these women. Fragile X Syndrome (FXS) is caused by hypermethylation of the expanded CGG repeats adjacent to exon 1 of the FMR1 [15]. The expanded CGG repeats can be categorized as common, intermediate, premutation, and full mutation alleles. Common alleles usually contain 6–40 CGG repeats, which are stable and usually do not expand upon transmission from parent to offspring. Intermediate alleles containing 41–60 CGG repeats have variable expansion risks, whereas premutation alleles (i.e., 55–199 CGG repeats) are usually unmethylated and can expand to the full mutation (e.g., > 200 CGG repeats) upon transmission from parent to offspring [15]. FMR1 premutation carriers also have disorders associated with ovarian function including loss of fertility and hypoestrogenism. As Peprah [15] mentioned, premutation alleles with 59–99 CGG repeats are associated with an increased risk of ovarian dysfunction in female carriers. Additionally, the length of the CGG repeats contributes to the variations observed in age of ovarian dysfunction resulting in a loss of reproductive capacity [15].
Huang et al. [16] demonstrated that fractalkine, a chemokine produced at the sites of inflammation and a major regulatory protein for leukocyte recruitment and trafficking expressed in human ovary and luteinizing GC together with CX3CR1, can increase the biosynthesis of progesterone in a dose-dependent manner by enhancing transcript levels of key steroidogenic enzymes but without affecting estradiol (E2) production [16]. CX3CR1 is a seven-transmembrane-spanning G-protein-coupled receptor expressed on monocytes, natural killer (NK) cells, and some lymphocyte subpopulations and is also expressed in human granulosa cells. Higher expression of fractalkine was found in luteinizing granulosa cells than in granulosa cells in the follicular phase [16]. According to the results, fractalkine is important for the ovary luteinizing process as autocrine/paracrine factor. The finding that fractalkine could increase hCG-stimulated progesterone production may have clinical relevance in some reproductive endocrine diseases, such as corpus luteum function defect and polycystic ovary syndrome, with insufficient progesterone secretion, which may result in menstrual disorders and miscarriage [16].
3.6 Progesterone and Its Receptors
Progesterone (P4) is a steroid hormone produced by the ovary, and its secretion depends on the ovary’s gonadotropin stimulation and physiological status, as mentioned by Peluso [17]. Granulosa cells, thecal/stromal cells, and luteal cells secrete P4 from the ovary, at different levels. P4 acts at the hypothalamus–pituitary axis to regulate gonadotropin secretion and mating behavior at the mammary gland to stimulate its development and at the uterus. P4 inhibits the development of ovarian follicles during the estrous cycle and pregnancy, prevents apoptosis of granulosa and luteal cells, and plays a major role in regulating steroidogenesis, mitosis, and apoptosis [17].
The progesterone receptor (PR) gene, located on chromosome 11q22–23, comprises eight exons and seven introns (A–G) [18]. One PR polymorphic variant, PROGINS, consists of a 320-bp PV/HS-1 Alu insertion in intron G and two single nucleotide polymorphisms (SNPs): (1) SNP-G3432T (mRNA nucleotide counting, NCBI: X51730) affects exon 4 and causes an amino acid substitution (V660L, which does not affect translocation of PGR-A and PGR-B to the nucleus) and (2) SNPC3764T affects exon 5 and is a silent mutation (H770H). PROGINS might modify the risk for several benign and malignant gynecological disorders, and according to Romano et al. [18], the PROGINS polymorphism of the human PR diminishes the response to progesterone.
Two nuclear P4 receptors (PGR-A and PGR-B) have been identified, which function as transcription factors according to Peluso [17]. Apart from the PR receptors, other receptors may be involved in mediating P4’s actions in granulosa cells before the gonadotropin surge. For example, GABAA receptors can bind P4 and its metabolites. Although GABAA receptor subunits are present within the ovary, they do not appear to transduce P4’s biological effects in granulosa cells because GABAA inhibitors do not block P4’s actions in granulosa cells [17]. Similarly, P4 binds to the ovarian glucocorticoid receptor, which is expressed in both granulosa and luteal cells, but it is not involved in the anti-apoptotic and anti-mitotic actions of P4. A third possibility could involve the oxytocin receptor (OXTR), because P4 can displace oxytocin binding to its own receptor and thereby attenuate its action in the uterus. Although various receptors can bind P4, this binding cannot account for the majority of its actions within granulosa and luteal cells [17]. These mechanisms involve rapid responses after the P4 binding to either PR that localizes at or near the plasma membrane, to a family of membrane progestin receptors (MPRa, MPRb, and MPRc), to a membrane complex composed of serpine 1 mRNA binding protein (SERBP1) and progesterone receptor membrane component 1 (PGRMC1).
3.7 Endometrial Development and Receptivity
During the proliferative phase, endometrium is stimulated by high levels of E2, and after ovulation in the early secretory phase, it is the target of low but rising levels of P4 and E2 [19]. Thus, genes regulated in early secretory endometrium (ESE), as compared to proliferative endometrium (PE), may be regulated by E2 and P4. Genes upregulated in late proliferative endometrium (LPE), as compared to menstrual endometrium, include oviductal glycoprotein-1, connexin-37, olfactomedin-1, SFRP4, while downregulated genes include MMPs-1, −3, and −10, IL-1b, IL-8, −11, inhibin bA, and SOX4 [19]. E2 treatment of human endometrial cells can result in upregulation of N-cadherin. However, N-cadherin can be downregulated in early secretory endometrium (ESE) compared to PE, suggesting that N-cadherin expression is inhibited by P4. ESE is characterized by inhibition of cellular mitosis, in contrast to the mitotic activity that occurs in PE. Furthermore, ESE is biosynthetically active, likely in preparation for embryonic implantation [19].
As Giudice [19] mentioned, the mid-secretory phase is the most well-characterized phase of the cycle with regard to gene expression analysis. Upregulated genes in mid secretory (MSE), compared to early secretory endometrium (ESE), are related to the cellular differentiation and cell–cell communications that underlie receptivity to embryonic implantation. These include the processes of cell adhesion, suppression of cell proliferation, regulation of proteolysis, metabolism, growth factor and cytokine binding and signaling, immune and inflammatory responses [19]. Striking upregulation has been observed with genes encoding secreted proteins, cytokines, and genes involved in detoxification mechanisms. Immune gene highly upregulated in MSE vs. ESE is CXCL14, a chemokine also known as breast and kidney expressed chemokine (BRAK), which recruits monocytes in the setting of inflammation and without inflammation, and it may be a major recruiter of monocytes and other cell types to the endometrium during the implantation window. Also, leukemia inhibitory factor (LIF) is highly upregulated, and in some women with infertility and repetitive miscarriage, low levels of LIF in MSE have been reported, as they have point mutations in the coding region of the LIF gene [19].
Among the most highly downregulated genes in MSE, compared to ESE, are the secreted frizzled related proteins (SFRP), olfactomedin 1, the progesterone receptor (PR), PR membrane component 1, ER-a, MUC-1, 17bHSD-2, and MMP-11 [19]. In addition, the transition from mid-secretory to late secretory endometrium in the absence of embryonic implantation is characterized by P withdrawal and preparation for desquamation of the tissue and menstruation. Accordingly, gene expression profiling reveals changes in genes involved in the extracellular matrix, the cytoskeleton, cell viability, smooth muscle contraction, hemostasis, and transition in the immune response to include an inflammatory response [19].
Moreover, the endometrium highly expresses the tumor suppressor p53 (a transcription factor with an N-terminal transactivation domain, a core DNA-binding region, and a C-terminal tetramerization domain) [20]. In addition, p53 activates genes, such as the pro-apoptotic Bax, NOXA, and PUMA (members of the Bcl-2 family), the forkhead transcription factor FOXO1 and promyelocytic leukemia zinc finger protein (PLZF). FOXO transcription factors are critical mediators in cell fate decisions in response to growth factors, hormonal and environmental cues, and they share striking functional homologies with p53. Both are involved in the control of cell cycle arrest and the induction of apoptosis. Promyelocytic leukemia zinc finger protein (PLZF) is a progesterone-inducible anti-proliferative factor that confers resistance to apoptosis and participates in endometrial stromal cell fate decision toward the end of the menstrual cycle [20].
According to Tapia et al. [21], at the secretory phase in the human endometrium, the mRNA levels of the complement system molecules C4b-binding protein (C4BP) and adipsin (complement component factor D, CFD) increase. It is postulated that the complement system might provide the uterine cavity with immunity against bacterial infection. In this sense, C4BP may protect the embryo where an increased expression of an inhibitor of complement system activation could reduce the chance of a misdirected complement attack to the embryo [21]. By contrast, adipsin may have a non-complement function in the female reproductive tract. Adipsin is necessary for the production of embryotrophic factor-3 (ETF-3), which stimulates embryo development. Thus upregulation of adipsin in human endometrium may assist the embryo during the implantation process as shown for other chemokines in the endometrium [21].
As it is described by Tapia et al. [21], several downregulated genes are associated with cell cycle regulation, like cyclin B1 (CCNB1), which binds to p34 (cdc2) to form the mitosis promoting factor during G2 phase. In human secretory phase endometrium, CCNB1 decreases compared to the proliferative phase. Moreover, CCNB1 may play an important role in proliferation and differentiation of the endometrial tissue under steroidal regulation. Furthermore, cellular retinol binding protein-2 (CRABP2) is a cytosolic protein that binds retinoic acid (RA) with high affinity [21]. The CRABP2 transcript decreases from the proliferative to the secretory phase of the human endometrium at the time of embryo implantation, which might suggest that RA signaling is required to be silenced, since it shuttles RA to the RA receptors in the cell nucleus.
Both endometrial receptivity and blastocyst implantation are regulated by cytokines and growth factors [21]. The cytokine endothelin-3 (EDN3) and fibroblast growth factor receptor-1 (FGFR1) are transcripts consistently downregulated in the endometrium during the window of implantation. FGFR1 and its transcript are significantly higher in proliferative than in secretory human endometrium. Fibroblast growth factor 2 (FGF-2) promotes endometrial stromal proliferation, and ovarian steroid hormones modulate its synthesis and function in endometrial cells.
An unusual leukocyte subpopulation of CD16(−) natural killer (NK) cells infiltrates the stromal areas of the human cycling endometrium [22]. The density of endometrial CD16(−) NK cells is low in the proliferative phase but rises after ovulation during the early to midsecretory phase. These NK cells are shed with other endometrial cells during menstruation, but their number increases in the endometrium when embryo implantation occurs. It is supported that the postovulatory rise of endometrial NK cells results from selective extravasation of circulating peripheral blood (PB) CD16(−) NK cells [22]. One of the key molecules involved in this event is interleukin 15 (IL-15), a cytokine/chemokine that is uniquely expressed in the human endometrium, exhibits a chemotactic activity for PB CD16 (−) NK cells and attenuates their binding capacity to dermatan sulfate, the major CD62L ligand expressed on human uterine microvascular endothelial cells (HUtMVECs). HUtMVECs bear a membrane-bound form IL-15 under the influence of ovarian steroids, which may be favorable for preventing downregulation of CD62L on PB CD16(−) NK cells and facilitating their initial contact with HUtMVECs [22].
3.8 Conclusions
The aforementioned genes contribute, either independently or through interactions among them, to the regulation of folliculogenesis and oogenesis, as well as to the development of endometrial receptivity. Moreover, they seem to be involved in ovarian response to stimulation. Hence, they are obvious candidates to be explored as putative molecular markers for women undergoing ovarian stimulation for ART. Particularly, the study of combinations of these genes appears to be very interesting in order to increase their predictive value. This information could provide a pharmacogenomic approach to ovarian stimulation, in the context of personalized medicine, which could increase the efficiency and the safety of the procedure. Hence, it would enable to individualize ovarian stimulation by selecting the appropriate dose for each woman aiming to achieve an optimal response while, at the same time, minimizing the risks for reduced or excessive follicular development.
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