EPIGENETICS IN REPRODUCTIVE MEDICINE

I. Basic concepts of epigeneticsDespite the fact that every cell in a human body contains the same genetic material, not every cell looks or behaves the same. Long nerve cells stretch out the entire length of an arm or a leg; cells in the retina of the eye can sense light; immune cells patrol the body for invaders to destroy. How does each cell retain its unique properties when, in its DNA-containing nucleus, it has the same master set of genes as every other cell? The answer is in the epigenetic regulation of the genes [1].

All cells in the human body carry the same DNA complement, which originates from a single cell at conception. Highly orchestrated epigenetic mechanisms facilitate the complex patterning required to ensure normal human development and support stable regulation of appropriate patterns of gene expression in diverse cell types. Epigenetic mechanisms define mitotically heritable differences in gene expression potential without altering the primary DNA sequence. These mechanisms are highly regulated by a large number of proteins that establish, read, and erase specific epigenetic modifications, thereby defining where and when the transcriptional machinery can access the primary DNA sequences to drive normal growth and differentiation in the developing embryo and fetus. Several types of epigenetic marks work in concert to drive appropriate gene expression. These include DNA methylation at CpG dinucleotides, covalent modifications of histone proteins, ncRNAs, and other complementary mechanisms controlling higher order chromatin organization within the cell nucleus [2].

1. DNA methylationDNAmethylation is typically associated with gene silencing through binding of methylation-sensitive DNA binding proteins and/or by interacting with various modifications of histone proteins that modulate access of gene promoters to transcriptional machinery . In eukaryotic species, DNA methylation involves transfer of a methyl group to the cytosine of the CpG dinucleotide .The vast majority of mammalian DNA methylation occurs at CpG dinucleotides. CpGs are distributed non randomly in the genome. They are concentrated in genomic regions called CpG islands ranging in size from 200 bp to several kilobases. These CpG islands, often unmethylated, are typically located within gene promoters of actively transcribed housekeeping genes and tumor suppressor genes [2].

Specific proteins, such as DNA methyltransferases, can establish or maintain DNA methylation patterns. Studies in mice demonstrate that DNA methyltransferases are essential for normal embryonic development. DNA methylation is established de novo by the DNA methyltranferase (DNMT) enzymes DNMT3a and DNMT3band maintained through mitosis primarily by the DNMT1 enzyme. DNMT1 is the primary maintenance methyltransferase with a high affinity for hemimethylated DNA. Its primary function is to copy the methylation patterns during replication. DNMT1o, one developmental stage-specific isoform of DNMT1, is an oocyte-derived protein that enters nuclei at the eight-cell stage of early embryos and has an essential role in maintenance of epigenetic marks [2].

DNA demethylation is also critical during primordial germ cell (PGC) and early embryo development. This can occur via passive demethylation that is associated with cell division or via active demethylation using excision repair mechanisms. The active pathway requires hydroxylation of the 5-methylcytosine to 5-hydroxymethylcytosine by the enzymes TET1 and TET2, followed by deamination by AID and APOBEC1 before base or nucleotide excision repair. All the enzymes in this pathway are expressed in mouse PGCs, suggesting a role in gametic epigenetic reprogramming [2].

2. Histone ModificationThe basic unit of chromatin consists of an octamer of histone proteins, two each of H2A, H2B, H3, and H4. DNA wraps around this core, which provides structural stability and the capacity to regulate gene expression (Fig. 1). Each core histone within the nucleosome contains a globular domain and a highly dynamic N-terminal tail extending from the globular domains (Fig. 1). Histone proteins have tails that can have a number of post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, proline isomerization, citrullination, butyrylation, propionylation, and glycosylation. Recently published data from the ENCODE Project Consortium analyzed 11 such histone post-translational modifications including acetylation and methylation, which mark active and repressive chromatin, as well as modifications associated with transcription. Assessing various histone modifications in a number of tissues, that data set identified different chromatin states including inactive, bimodal, and active, each of which has different functional properties. Bimodal states, in which a combination of active and repressive marks are present in the chromatin of a promoter region of a gene, facilitate rapid changes in gene expression, as might be expected during early development, when differentiation and specification occur [2].

Chromatin assembly and disassembly are highly orchestrated processes that are coordinated by histone chaperones and ATP dependent chromatin remodeling complexes. Histone chaperones promote chromatin assembly by preventing non-specific histone-DNA interactions while also promoting the correct histone-DNA interactions [3].3. Regulatory ncRNAsRegulatory ncRNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), and long ncRNAs (lncRNAs) play important roles in gene expression regulation at several levels: transcription, mRNA degradation, splicing, and translation. SiRNAs are double-stranded RNAs (dsRNA) that mediate post-transcriptional silencing, in part by inducing heterochromatin to recruit histone deacetylase complexes [2].MiRNAs comprise a novel class of endogenous, small (1824 nucleotides in length); single-stranded RNAs generated from precursor RNA cleaved by two RNA polymerase III enzymes DROSHA and DICER to produce mature miRNA. These miRNAs can control gene expression by targeting specific mRNAs for degradation and/or translational repression. They can also control gene expression by recruiting chromatin-modifying complexes to DNA through binding to DNA regulatory regions, thereby altering chromatin conformation (33, 34). Expression of miRNA in human blastocysts correlates with maintenance of pluripotency in embryo development [2].

LincRNAs, a subset of lncRNA, exhibit high conservation across different species. They have been shown to guide chromatin-modifying complexes to specific genomic loci, thereby participating in the establishment of cell typespecific epigenetic states. In embryonic development, expression of lncRNAs, regulated by the pluripotent transcription factors OCT4 and NANOG, facilitates cell lineagespecific gene expression (38). LincRNAs also play an important role in developmental processes such as X-chromosome inactivation and genomic imprinting [2].

II. Epigenetics and InfertilityFactors Associated with the Infertility Phenotype

Several studies have identified different genetic and epigenetic factors as being involved with the onset and progression of the infertility phenotype. However, possible perturbed mechanisms involved in regulating gene expression in the disease state are rather poorly understood. The classic nature versus nurture argument on whether ones environment rather than genetic makeup could influence the onset of infertility and other complex disorders has been reviewed by various studies [4].

Epigenetic Factors Influencing the Infertility PhenotypeSeveral histone deacetylases and demethylases have recently been identified and attributed with regulation of chromatin state. However, their functional role and association with diseased states is only just beginning to be understood, particularly those factors affecting male and female infertility. Post translational modifications of histone tails by factors including histone chaperones and methyltransferases are involved in the proper regulation of gene expression. Due its dynamic nature and plasticity, the landscape of chromatin can be altered, rendering a region of the genome active or inactive. This altered state of packaging renders certain regions of the genome more accessible to transcription machinery (euchromatin) and are marked by DNA hypomethylation, RNA Pol II, and covalent histone modifications such as histone 3 trimethylated at lysine 4 (H3K4me3) and the histone variant H2A.Z. Inactive/repressed regions are known to be associated with DNA hypermethylation, histone 3 trimethylated at lysine 27 (H3K27me3), and SUZ12 (part of the polycomb group complex, PcG) [4].

Due to this, the main focus of recent epigenetic research has focussed on discovering new factors involved in altering chromatin state and further looking at its involvement in diseased and normal tissue. Recent studies have identified a critical role for the JHMD2A (Jumonji C domain-containing histone demethylase 2A) histone demthylase in male infertility, obesity, and spermatogenesis. Using knockout mice as models, these studies identified a critical role for JHMD2A in the regulation and expression of two genes, protamine 1 (OMIM #182880, Prm1) and transition nuclear protein 1 (OMIM #190231, Tnp1) involved in the condensation and proper packaging of chromatin in the male sperm. A higher degree of spermDNA compaction has previously been attributed to the increased presence of highly basic protamine proteins compared to histones in chromatin, a deficiency of which has been associated with infertility in mice. Identification of other regulatory mechanisms involved in the recruitment of factors, in addition to JHMD2A, involved in the deposition of histones along with others affecting transcriptional activity of genes involved in infertility will increase our understanding of mechanisms involved in both perturbed and normal states [4].

a. Epigenetics and Female InfertilityInfertilityThe World Health Organization defines the term primary infertility as the inability to bear any children, whether this is the result of the inability to conceive a child, or the inability to carry a child to full term after 12 months of unprotected sexual intercourse. Primary infertility is sometimes known as primary sterility. However, in many medical studies, the term primary infertility is only used to describe a situation where a couple is not able to conceive [5].

Secondary infertility is defined as the inability to have a second child after a first birth. Secondary infertility has shown to have a high geographical correlation with primary infertility. Fecundity describes the ability to conceive after several years of exposure to risk of pregnancy. Fecundity is often evaluated as the time necessary for a couple to achieve pregnancy. The World Health Organization recommends defining fecundity as the ability for a couple to conceive after two years of attempting to become pregnant [5].

The terms infertility and infecundity are often confused. Fertility describes the actual production of live offspring, while fecundity describes the ability to produce live offspring. Fecundity cannot be directly measured, though it may be assessed clinically. Typically, fecundity may be assessed by the time span between a couples decision to attempt to conceive and a successful pregnancy [5].

EpigeneticsThe epigenetic mechanism of action suggests that environmental factors alter how a gene is expressed, but without directly changing the DNA sequence. Epigenetics is the study of inherited changes in phenotype (factors that account for appearance) that are not directly related to, nor explained by changes in our DNA pattern. For this reason, this field of study is known as "epi," the greek root for "above," indicating that a change has occurred that is not directly related to the genetic code, but above it somehow. In epigenetics, non-genetic causes are considered responsible for different expressions of phenotypes. Or, termed in a different way, epigenetics describes changes in the expression of our genes that are not caused by alterations in the DNA sequence. Essentially, a different factor accounts for the change in gene expression [5].

Exogenous, or environmental components may affect gene regulation and thus, potentially, subsequent expression in the phenotype. Changes to gene expression induced by environmental contaminants can be permanent or transient. Research has shown that epigenetic changes may in fact be reversed [5].

b. Epigenetics and male infertilityAberrant epigenetic regulation, male infertility and embryonic developmentIt is crucial that proper regulation of epigenetic processes is maintained throughout spermatogenesis to not only ensure proper sperm function, but also proper embryonic development. It has been found that the sperm epigenetic environment plays a role in establishing epigenetic marks in the embryo, thus aberrant epigenetic regulation in spermatogenesis has a profound effect on both male fertility and embryonic development [6].1. DNA methylationImproper DNA methylation of various genes has been implicated in abnormal semen parameters, as well as several instances of male factor infertility. This aberrant methylation can occur globally or be limited to one specific locus. A study by Houshdaran et al. demonstrated that poor sperm concentration, motility and morphology were associated with broad DNA hypermethylation across a number of loci. Four of these sequences PAX8, NTF3, SFN and HRAS were single copy sequences unique to non-imprinted genes. Moreover, the repetitive element Satellite 2 was also found to be hypermethy-lated. The authors proposed that hypermethylation of these loci results from the improper erasure of already established methylation marks rather than aberrant de novo methylation following epigenetic reprogramming. The data from this study suggest that methylation defects present outside of imprinted loci may be a key factor in some cases of infertility. Recent research has identified a critical role for the JHMD2A (Jumonji C-terminal containing histone demethylase 2A) histone demethylase in male infertility, obesity and spermatogenesis. Studies using knock-out mice models identified a critical role for JHMD2A in the regulation and expression of protamine 1 and transition nuclear protein 1, both of which are critical for DNA condensation during chromosomal packaging in sperm. The lack of proper DNA packaging in sperm has been associated with infertility in mice. It is possible that aberrations of a number of other proteins regulating the activities of the proteins involved in DNA compac-tion could cause infertility [6].

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate pathway that catalyzes the reduction of 5,10- methylenetetrahydrofolate to 5-methylenetetrahydrofolate. The enzyme maintains bioavailability of methionine so it can be converted to S-adenosylmethionine, a methyl donor for a variety of substrates including DNA. To better characterize the role of MTHFR in spermatogenesis and male fertility, Khazamipour et al.compared the methylation status of the MTHFR gene promoter in the testes of men with non-obstructive azoospermia to men with obstructive azoospermia without defects in spermatogenesis. It was found that 53% of men with non-obstructive azoospermia had hypermethylation of the MTHFR promoter, whereas none of the men with obstructive azoospermia exhibited hypermethylation of this region. These statistically significant data indicate that MTHFR hypermethylation is a specific epigenetic aberration and may strongly contribute to certain cases of male infertility. Interestingly, a study by Kelly et al. demonstrated that adminis-tration of betaine during pregnancy, nursing and post-weaning can indeed improve testicular histology and fertility .Wu et al., in a very recent study concluded that hypermethylation of MTHFR gene promoter in sperm was associated with idiopathic male infertility. The authors demonstrated that the number of patients with hypermethylation was three times to that of control individuals [6].

2. Genome imprintingAppropriate establishment of genomic imprints is critical to the maintenance of fertility. Indeed, both paternal and maternal imprinting defects have been identified in several groups of men experiencing male factor infertility. Substantial work has been directed towards confirming the presence of these imprinting abnormalities. A study by Houshdaran et al. demonstrated that poor semen parameters were linked with increased DNA methylation at several differentially methylated loci, more than likely due to a defect in methylation erasure during epigenetic reprogramming. These loci included PLAG1, a maternally imprinted gene, DIRAS3 and MEST. An additional study by Kobayashi et al. examined seven imprinted genes, including the paternally imprinted GTL2 and H19 loci, in 97 infertile men. They found that 14.4% of the patients studied exhibited abnormal paternal imprinting and 20.6% exhibited abnormal maternal imprinting. Donors with normal sperm count and motility were methylated at the H19 locus, whereas one patient with moderate oligospermia and three patients with severe oligosper-mia showed no methylation of the locus. Similarly, healthy donors displayed methylation at the GTL2 locus while two patients with moderate oligospermia and four with severe oligospermia displayed no methylation. Further sequencing of both loci concurrently showed that one patient with severe oligospermia exhibited a defect in both the H19 and GTL2 loci. Moreover, five of the ten patients with severe oligospermia and three of the eight patients with moderate oligospermia displayed aberrant methylation of maternally imprinted loci and differentiallymethylated regions [6].

Poplinski et al. compared the methylation status of the H19/ IGF2 imprinting control region 1 (ICR1) and MEST loci in 33 healthy donors and 148 men with idiopathic infertility. Normozoospermic men displayed high methylation at the H19/IGF2 ICR1 locus and low methylation at the MEST locus, while low methylation of the H19/IGF2 ICR1locus and high methylation of the MEST locus was found to be associated with low sperm concentration. The H19/IGF2 ICR1locus of men with idiopathic infertility was 89.6% methylated with less than 40 million sperm in comparison to 95.9% methylated in normal donors. As methylation of this locus increased, sperm count increased linearly. Moreover, decreased methylation of H19/IGF2 ICR1locus directly correlated with decreased sperm motility. Data from two different studies by Marques et al. also indicated that some oligozoospermic patients and secretory azoospermic patients with hypospermatogenesis exhibit loss of methylation at H19. Similarly, in 2010 Boissonnas et al. found that many patients with teratozoospermia and oligoasthenoteratozoospermia exhibited hypomethylation at variable CpG islands at the H19 locus. Hypermethylation at MEST was more strongly linked with poor sperm quality than hypomethylation at H19/IGF2 ICR1. This hypermethylation of MEST was observed in samples from infertile men with less than 40% sperm motility and less than 5% normal sperm morphology. Men with idiopathic infertility exhibited MEST methylation of 9.6% in comparison to 4.3% in controls. Furthermore, increased methylation of MEST linearly correlated with decreased sperm motility. These data are supported by the findings of a study by Marques et al [6].

3. Nuclear protein transitioningThe exchange of protamines for histones is a crucial step in the process of spermatogenesis, causing the DNA to be tightly wrapped for efficient transmission of nuclear material to the oocyte upon fertilization. It is known that the Prm1 to Prm2 ratio (P1/P2 ratio) is strictly maintained and regulated. Indeed, deviation from the standard ratio of 0.81.2 has been shown to lead to infertility. A change in either direction of this ratio adversely affects semen quality and DNA integrity. Patients with abnormally depressed or elevated P1/P2 ratios are characterized by poor sperm concentration, motility and morphology as well as decreased fertilization capabilities. Studies suggest that the most common cause of infertility by aberrant protamine exchange is an increase in the P1/P2 ratio caused by a decrease in Prm2 levels, although improper regulation of Prm1 levels has also been implicated in some cases of male infertility. Furthermore, a study by de Yebra et al. showed that men with a higher P1/P2 ratio were also more likely to have lower total protamine levels and higher intermediate protein levels. Moreover, this same study additionally demonstrated that some infertile men completely lack Prm2 in their sperm nuclei. There have been no cases reported of fertile men with severely altered P1/P2 ratios; indeed, it appears to be a characteristic limited exclusively to certain infertile males. Interestingly, a link between abnormal protamine incorporation and aberrant genomic imprinting has recently been discovered. Hammoud et al. found that infertile males with abnormal protamines exhibited statistically significant hypermethylation at the imprinted loci KCNQ1, LIT1 and SNRPN. Moreover, this study also demonstrated that these patientsshowed hypomethylation of the H19 locus [6].

As discussed previously, hyperacetylation of histone H4 is required in the transition from histones to protamines. This step decreases the affinity of the interaction between the sperm histones and DNA to allow the exchange for transition proteins to occur. A study by Sonnack et al. showed that men exhibiting qualitative and/or quantitative infertility have significantly decreased levels of histone H4 acetylation associated with impaired spermatogenesis. In the seminiferous tubules of men with round spermatid maturation arrest, only approximately 60% of spermatids were immunopositive for this hyperacetylation and many were multinucleated. Moreover, infertile men withqualitatively normal spermatogenesis exhibited approximately 90% immunopositive spermatids and infertile men with qualitatively abnormal spermatogenesis exhibited approximately 75% immunopositive spermatids. This contrasts significantly with the almost all hyperacetylated round spermatids in fertile men. Interestingly, spermatocytes in the seminiferous tubules of men with round spermatid maturation arrest exhibited an additional signal, indicating that early hyperacetylation of histone H4 may lead to premature nuclear protein transitioning and subsequent infertility [6].

4. Chromosome structureThe global structure of chromatin is known to affect gene expression by modulating which regions are available to be accessed by transcription factors and other transcriptional proteins and which are not. This feature of epigenetic regulation becomes particularly important when considering normally expressed genes that are crucial for proper spermatogenesis and subsequent oocyte fertilization. It has now been established that an increase in total heterochromatic variants is strongly linked to some cases of male factor infertility. Indeed, it has been demonstrated that there is an increase in the frequency of chromosomal variants, from 32.55% to 58.68%, in infertile men compared to controls. The large polymorphic variation 9hq+, located in centromeric heterochromatin on chromosome 9, was shown to increase in frequency from 4.25% in controls to 14.69% in men with severe infertility. It is thought that this rise in heterochromatic regions, not only on chromosome 9 but amongst many chromosomes, may down-regulate normally active genes. Indeed, polymorphic variations on the Y chromosome have been implicated in male infertility based on this reasoning. All the major genes/loci known to be epigenetically different in some infertile individuals are listed in Table 2 [6].

III. Epigenetics and PCOS1. miRNALittle is known about the roles of miRNAs during follicular development, steroidogenesis and in PCOS. Several studies on miRNA expression have been done on intact ovaries (chicken, mice, pig, sheep and cattle), as well on the different ovarian components, such as granulosa cells (mice, pig, horses and human), theca cells, follicular fluid (humans, cattle and mares), cumulus cells (mare), cumulus-oocyte complexes (COCs) (cattle) and corpora lutea (cattle) [7].

The possible modes of action for miRNA within the pathophysiology of PCOS have only been sparsely investigated, and thus far, only a few miRNA-PCOS studies exist (see Table 1).

It might be possible that identified PCOS susceptibility genes, such as DENND1A, which interestingly also encodes miR-601, could result in genetic and epigenetic factors overlapping, thus influencing miRNA target specificity. A pilot study investigating global methylation in twenty PCOS women and 20 BMI- and age-matched controls, using peripheral leukocyte DNA, showed no significant differences in the median global DNA methylation percentages. Despite the negative result, epigenetics may still play a part in PCOS pathogenesis, since GC and ovary gene-expression could be tissue specifically epigenetically modified in PCOS. Thus, PCOS is genetically complex with a large degree of heterogeneity and is considerably influenced by environmental and genetic cues, one of these being microRNAs [7].

2. Serum/Plasma miRNA Biomarkers for PCOSPresent abundantly in serum, miRNAs could serve as a non-invasive biomarker for PCOS, as they have been shown to be stable in serum, are resistant to nuclease activity and are easy to detect. It is not known specifically how miRNAs enter serum or whether the miRNAs present in serum are disease-specific, since serum is a result of different components secreted by various tissues and cells, and identifying their cellular origin can be difficult. Currently, several other biomarkers in the serum of PCOS women are used for diagnostic purposes, e.g., luteinizing hormone (LH) and androgen concentrations, as well as follicle-stimulating hormone (FSH) [7].

A recent case-control study investigating 12 PCOS patients, 12 healthy females and 12 male controls, subdivided further based on BMI levels, revealed that obesity significantly reduced the expression of four miRNAs selected for evaluation in whole blood: miR-21, miR-27b, miR-103 and miR-155 in control women and men, but tending to show an increase in expression in PCOS women. Further analysis of their hormone profile showed a positive correlation between serum free testosterone levels and miR-21, miR-27b and miR-155. Perhaps, the elevated free testosterone found in the PCOS samples could partly explain the observed increase of these miRNAs. Further, bioinformatics analysis and target gene analysis revealed that miR-21, miR-27b, miR-103 and miR-155 could be involved in hormone metabolism, as well as reproductive cellular processes [7].

Using miRNA arrays, the expression of serum miRNAs in patients with PCOS compared to age-matched controls has been evaluated . Following an initial miRNA profiling based on a relative two-fold change in expression levels, nine miRNAs (miR-222, miR-16, miR-19a, miR-106b, miR-30c, miR-146a, miR-24, miR-186 and miR-320) were chosen for further analysis. The expression levels for eight of the miRNAs were upregulated in serum from PCOS patients, whereas miR-320 displayed decreased expression in the PCOS subjects. However, following Q-PCR validation of the nine miRNAs expression in the entire study population (n = 68 PCOS, n = 68 controls), only miR-222, miR-146a and miR-30c remained significantly increased in the PCOS patients. Sensitivity and specificity analysis, using receiver operating characteristic (ROC) curves and area under the curve (AUC), revealed that a combination of the three miRNAs was able to distinguish between the PCOS and controls. In addition, correlation analysis adjusted for age and BMI showed that miR-222 strongly correlated positively with serum insulin levels in PCOS women. Interestingly, upregulated expression levels of miR-222 have also been associated with type 2 diabetes and gestational diabetes mellitus. Further, miR-146a correlated negatively with serum testosterone in PCOS women. Decreased miR-146 has been linked to inflammation and insulin resistance in T2D individuals. An interesting observation made by Long et al. was that most of the miRNAs present and differentially expressed in ovarian tissue from PCOS women were not released into the blood and, therefore, were not altered in PCOS serum [7].

In conclusion, identification of distinct miRNAs present within the circulation would prove a useful tool for diagnosis and perhaps treatment of PCOS. Comparing the miRNA profile of PCOS patients to healthy controls reveals that miRNAs might contribute to the pathogenesis. Indeed, miR-21, miR-27b and miR-103 are associated with PCOS, as well as metabolic features, such as obesity, T2D, low-grade inflammation and adipogenesis dysfunction. Furthermore, insulin sensitivity and the suppression of androgens have been associated with miR-222 and miR-146a, respectively. Profiling of serum miRNAs does not necessarily reflect the more local changes within the ovary, and the functional role and significance of miRNAs in blood from PCOS patients still need to be determined [7].

3. MicroRNAs as Biomarkers for PCOS Based on Follicular Fluid ContentComparing the miRNAs found in follicular fluid to the miRNAs found in the bloodreveals the common occurrence of miR-186, miR-21, miR-155, miR-103, miR-19a and miR-16, although with different expressions levels and significance associated with PCOS. Moreover, the miRNA profile of follicular fluid varies between studies, highlighting that PCOS is a complex and heterogenic syndrome. Interestingly though, increased expression of miR-146 was found in serum from PCOS patients and also in follicular fluid by Roth et al. and Sang et al., respectively. Adding to this, miR-222 and miR-24 were also found to be highly expressed in follicular fluid, as well as identified in PCOS serum. A differential expression, albeit in different directions, was observed for miR-320. Taken together, this still warrants further studies [7].

4. Possible Role for miRNA in the Abnormal Follicular Development and Function in PCOSMany different theories have been brought forth in an attempt to explain the mechanisms responsible for the impaired ovulation, abnormal follicular development and excessive follicle formation commonly found in women with PCOS, but with varying results. An altered appearance and function of granulosa cells (GCs) with respect to FSH, LH and androgens has been proposed. Defects in steroidogenesis by the theca cells (TCs) and increased activation of primordial follicles, abnormal expression of anti-Mllerian hormone, increased follicle survival and/or a decreased apoptosis rate have also been reported. Many of the factors involved in these processes are still unknown and the mechanisms unestablished [7].

Taken together, altered expression of ovarian miRNAs might play a role in the processes determining the fate of granulosa cells (proliferation and differentiation vs. apoptosis), and this might lead to the hyper-proliferating granulosa cells, as seen in PCOS. The roles and mechanisms of miR-224, miR-320 and miR-383 in GCs during folliculogenesis in general and in PCOS remain unknown [7].

IV. Epigenetics and Endometriosis

1. DNA methylationRecently it has been shown that DNMT1, DNMT3a and DNMT3b are over expressed in endometriotic tissue. These findings are likely to provoke new ideas regarding the origin and aetiology of endometriosis. For example, over-expression of these enzymes would be expected to alter global DNA expression in endometriotic cells. DNA microarray analysis of endometriotic tissue supports this expectation since a substantial number of genes display significantly altered expression patterns. Another possible explanation for these observed irregularities in gene expression may be due to abnormalities in the regulation and function of the major transcription factor, NF-B, in endometriosis (for review see Guo 2007 ). While this may be so, the introduction of epigenetics into the fray offers new insights into the origin and progression of disease, such as the combined effect of disrupting traditional and epigenetic regulators of transcription. In support of this notion Wren et al demonstrated that epigenetic mechanisms such as histone modifications, methylation and acetylation may play a role in the aetiology of endometriosis. However, results from microarray studies provide an unusual paradox. The majority of studies found almost the same number of down regulated genes as there were up regulated in ectopic endometrial tissue. For example, Kao et al reported 91 genes significantly over expressed and 115 under expressed. Similarly Eyster et al 2002 and Eyster et al 2007 reported more genes over expressed in ectopic endometrial tissue. Yet enhancement of DNMT function in endometriosis should lead to increased levels of DNA methylation hence, increasing the number of silenced or down regulated genes. This raises the question as to how a system can be in place where global gene expression in endometriotic cells should be down-regulated by over active methylation, and yet the evidence clearly shows many genes are up-regulated. However, it should be noted that Burney et al reported a higher frequency of under expressed genes in the eutopic endometrium of women with endometriosis vs disease free controls. A possible explanation to this paradox will be discussed in section 3.6. Of course it must be considered that not all genes are epigenetically regulated, genetic mechanisms are likely to play a significant role in aberrant gene expression in endometriosis [8].

2. Epigenetic Modification of Steroid Synthesis and Receptors in EndometriosisThe deregulation of DNMTs is not the only evidence supporting the hypothesis that epigenetics plays a major role in endometriosis. Izawa et al demonstrated that the expression of the cytochrome p450 aromatase enzyme (CYP19) is dependent on the methylation status of its promoter by treating endometriotic cells with the demthylating agent 5-aza-deoxycytidine and observing the fold change in aromatase mRNA expression. Current studies have reported either a weak or no association between polymorphisms of the aromatase gene and endometriosis that could account for the observed over expression of aromatase in endometriosis. Those studies that have associated certain aromatase polymorphisms with endometriosis have been criticised for faulty data analysis or non reproducible results [8].Aromatase is a key enzyme involved in the synthesis of estrogen and plays a crucial role in the pathogenesis of endometriosis. With the exception of two studies aromatase is reported to be highly up regulated in endometriotic cells whilst being nearly undetectable in normal endometrium. The importance of aromatase in the pathology of endometriosis is aptly demonstrated by the use of aromatase suppressing drugs for the treatment of the disease. This class of drugs, although with limited clinical data, have shown to be effective in the symptomatic treatment of endometriosis. Aromatase is normally expressed in a cyclic fashion throughout the menstrual cycle in eutopic endometrium however, expression levels are consistently elevated in endometriotic cells. If the over-expression is initiated by hypomethylation of the promoter and maintained by aromatase activating cytokines such as IL-6, IL-11 and TNF, all of which have been shown to be dysregulated in endometriosis , a consequence would be over-expression of aromatase leading to increased synthesis of estrone, which is converted to estradiol, a potent estrogenic factor that initiates a number of pathways leading to the proliferation and survival of endometriotic cells. The conversion of estrone to estradiol is catalysed by the enzyme 17-hydroxysteroid dehydrogenase type 1 (17HSD I), which is reportedly up regulated in endometriosis. The increased activation of aromatase in endometriotic cells leads to a self sustaining positive feedback loop for estradiol production, whereby prostaglandin E2 (PGE2) activity induces the up regulation of aromatase leading to increased estradiol levels. In turn, this leads to the up regulation of the cycloxygenase-2 enzyme (COX-2) resulting in the formation of more PGE2, an important factor in the pathology of endometriosis thus the cycle becomes self perpetuating (Figure 25) [8].

Aberrant methylation of the aromatase promoter is not the only factor altering gene expression due to epigenetic alterations in endometriosis. Regulation of aromatase is mediated by steroidogenic factor-1 (SF-1) which is the aromatase enhancer, and chicken ovalbumin upstream promoter transcription factor (COUP-TF) which is its repressor. Indeed, SF-1 has recently been shown to be over-expressed in endometriotic cells. An explanation for the apparent over-expression of SF-1 has been proposed by Xue et al who observed hypomethylation of the CpG island near to its promoter region. The discovery of epigenetic modifications in the promoters of aromatase and its enhancer SF-1 provide some understanding of the establishment of the reported positive feedback loop. As with mutations, once these epimutations are established, they are retained throughout each cellular division, ensuring the survival of the endometriotic cells [8].

It is not only the synthesis of estrogen that is affected by aberrant methylation in endometriosis. In order for estrogen to mediate its mitogenic effects within the cell it must first bind to its receptor, of which there are two variants, estrogen receptor (ERA) and estrogen receptor (ERB) coded for by separate genes. Cells that over express estrogen receptors are highly sensitive to estrogenic effects. Such cell types include breast and ovarian cancer cells which, as with endometriotic cells, possess an enhanced proliferative capacity. Recent studies have shown that the mRNA of one isoforms of the estrogen receptor (estrogen receptor 2 gene, encoding estrogen receptor ) is over expressed in endometriosis. This apparent over-expression was found to result from hypomethylation of the CpG islands in the promoter of the ESR2 gene. ERB is important as it is known to regulate several genes involved in signal transduction, cell cycle progression and apoptosis, however in contrast to endometriosis several studies have shown ERB to be down regulated in ovarian cancer and it thought that loss of ERB expression may induce malignant transformation. It is also important to note that several endocrine disrupters though to be risk factors for endometriosis mediate signalling cascades via ERB. Therefore, not only does epigenetic modification lead to enhanced estrogen production but it also leads to increased sensitivity towards estrogen and estrogen-like compounds in endometriotic cells, resulting in a self sustaining endometriotic cell population [8].

Due to abnormal estrogen synthesis and metabolism observed in endometriosis, progestogenic agents are commonly administered to women with the disease in order to suppress endometriotic cellular proliferation by down regulating estrogen production and acting as an anti-inflammatory agent. The efficacy of progestogens in relieving persistent pain symptoms associated with endometriosis is relatively poor, and a reported 9% of women are completely unresponsive to progestogen treatment. The relative inefficiency or total lack of response to progestogenic treatment has thus, led some to conclude that endometriotic cells are somehow resistant to the effects of progesterone. Evidence for this comes from studies of the progesterone receptors in endometriotic cells. As with ER, there are two progesterone receptor isoforms, PR-A and PR-B. Unlike the ER, these encode as splice variants of the same gene and each has distinct functions and distinct levels of expression in the eutopic endometrium, depending on the phase of the menstrual cycle. PR-B is a transcriptional activator for several genes containing a PR-B dependent promoter. PR-A, on the other hand is a transcriptional repressor for PR-B and ER. Studies have shown that PR-B expression is absent, and only very low levels of PR-A are expressed in endometriotic cells, offering some explanation for progesterone resistance in endometriosis. Additionally, Wu et al showed that the aberrant hypermethylation of the PR-B promoter, reduces its expression to an almost silenced state. PR-A and PR-B, although coded by the same gene, have distinct promoters, thus it may be possible that aberrant methylation of the PR-A promoter may be responsible for its reduced expression. Conversely Wu et al suggested that alteration of PR-A expression may not be due to altered methylation of its promoter, but is likely due to other as yet, unknown mechanisms. Nevertheless, it is reasonable to conclude that there are epigenetic mechanisms by which estrogen production is both enhanced and unopposed in endometriosis (Figure 26) [8].

3. HOXA10 in EndometriosisHOX are a family of genes containing homeobox domains that act as transcription factors essential for regulating genes associated embryonic development. Several members of the HOX gene family play crucial roles during embryogenesis, for example HOXA9, HOXA10, HOXA11 and HOXA13 are involved with the development of the female reproductive tract, and unlike the majority of HOX genes they are expressed into adulthood. The involvement of HOXA10 in the development of the uterus affords specific interest with regards to endometriosis since any aberrant expression of HOXA10 may result in abnormalities, in either the function or morphology of the uterus. HOXA10 expression is reportedly down regulated in patients with endometriosis, perhaps reflecting the findings that patients with endometriosis are more likely to present with anatomical complications of the reproductive tract. HOXA10 under-expression in endometriosis patients may also explain the associated subfertility observed in these patients since HOXA10 along with HOXA11 are responsible for successful implantation of the embryo [8].

The origin of the down regulation of the HOXA10 gene in endometriosis was investigated by Wu et al 2005 and Kim et al 2007. Wu et als study screened eutopic endometrium of women with endometriosis, whereas Kim et al screened eutopic endometrium from baboons with experimentally induced endometriosis. Both studies identified hypermethylation in the promoter region of the HOXA10 gene. However, these studies examined only methylation patterns of HOXA10 in the eutopic endometrium of endometriosis cases and controls, but did not examine the methylation status of HOXA10 in ectopic endometrium. Wu et als study was also confined only to women with stage III-IV endometriosis. Therefore, definite conclusions regarding the aberrant expression of HOXA10 in endometriotic tissue in humans cannot be made. However, a study by Lee et al 2009 using a murine model of experimentally induced endometriosis, found inducing endometriosis led to methylation dependant changes in HOXA10 expression in eutopic endometrium. This essentially turns current thinking on its head, as it has long been thought eutopic endometrium dictates the fate and function of ectopic endometrium (via polyclonal origin of ectopic endometrium from refluxed eutopic cells) not, as Lee et al demonstrated, the other way around. Although it may be that interplay of signalling exists between the two cell types, the mechanism by which ectopic endometrium can influence epigenetic alteration of eutopic endometrium remains to be elucidated [8].

Interestingly, further study has reported that HOXA10 hypomethylation can be induced by in-utero exposure to diethylstilbestrol (DES), a known endocrine disruptor. The ramifications of DES exposure are discussed further in section 3.5. The effect of altered HOXA10 expression in eutopic endometrium is obvious in terms of uterine morphology and embryonic implantation. What the biological significance of HOXA10 down regulation, in endometriotic tissue may be remains speculative. Some clues may arise from microarray study of HOXA10 knockdown cells, which reported HOXA10 as a regulator of hundreds of genes involved in a variety of cellular processes. Of particular interest was the finding that HOXA10 knockdown led to a 5.78 fold increase in the CYP19 (aromatase) gene, the significance of which is discussed in section 3.2. It is also important to note that HOXA10 and HOXA11 are progesterone responsive genes and members of the HOX family themselves regulate a number of other genes including IGFBP-1 and integrins [8].

Nevertheless, the altered expression of HOXA10 in women with endometriosis may provide an explanation for one of the most puzzling aspects of endometriosis, which is, if retrograde menstruation is near universal, why do only a fraction of women develop endometriosis? It may be that HOXA10 aberrations result in improper development of the uterus. This is supported by the observed uterine anatomical abnormalities in women with endometriosis such as increased frequencies of septate uterus and the reported decreased elasticity of the reproductive organs. Both aspects are thought to increase the volume of menstrual reflux in some women, overwhelming the immune system which is thus unable to remove all the refluxed endometrial cells [8].

4. Epigenetics and the EnvironmentEpigenetics provides a link between genotype and the environment, and how exposures to different environmental, pharmacological and dietary elements can translate into heritable changes in gene expression. During early mammalian development the methylome is stripped and then re-applied in order to start development from a blank state in which methylation errors are removed, for this reason it was thought that alteration of epigenetic marks such as methylation patterns could not effect subsequent generations. If epigenetic marks were heritable then the complex phenotypic consequences they encode, which may include disease phenotypes, must be heritable also. DNA methylation by DNMTs is dependent on methyl donors such as S-adenosylmethionine (SAM), the major methyl donor, which is synthesised as part of the methionine cycle. The formation of this cycle is, in turn, dependent on dietary factors such as folic acid, vitamin B12, choline and betaine. Animal studies have shown that restricting dietary methyl donors produces a reduction in DNA methylation, and in some cases, increased risk of developing tumours, indicating that epigenetic aberrations mediated by dietary changes can result in complex disease phenotypes. The sparse epidemiological studies which have reported on the influence of diet and endometriosis suggest that a diet high in fruit and green vegetables and low in meat and alcohol consumption is protective against developing endometriosis, however some findings were inconsistent. From an epigenetic perspective a diet high in fruit and green vegetables would provide a significant source of methyl donors, which may protect against demethylation induced genetic instability during foetal development. Alcohol consumption has been shown to alter histone acetylation and methylation patterns which may account for the observed estrogenic effect of alcohol [8].

V. Epigenetics and Fibroids

1. DNA MethylationDeoxyribonucleci acid methylation that occurs at the C5 position of cytosine, resulting in 5-methylcytosine (5mC), mostly within CpG dinucleotides , is involved in various developmental processes by silencing, switching, and stabilizing genes. Deoxyribonucleci acid hypomethylation and imbalanced expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) are found in human uterine leiomyoma compared with the adjacent myometria. In addition, the aberrant DNA hypomethylation is found in the distal promoter region of ER-a (21188 to 2790) in the uterine leiomyoma compared with the myometrium. A restriction landmark genomic scanning profile study found 29 aberrant methylation spots(10 methylated and 19 demethylated) in leiomyoma compared with myometrium. This study also found that DNMT1 and DNMT3A mRNA expression levels are higher in leiomyoma compared with myometrium. Recently, Maekaya et al identified 14 hypomethylated genes (FAM9A, CPXCR1, CXORF45, TAF1, NXF5, VBP1, GABRE, DBX53, FHL1, BRCC3, DMD, GJB1, AP1S2, and PCD11X) and one hypermethylated gene (HDAC8) located on the X chromosome in uterine leiomyomas. Most recently, Navarro et al found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma vs. normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. Interestingly, they found three known tumor suppressors genesKLF11, DLEC1, and KRT19withhypermethylation, mRNA repression, and protein expression in leiomyoma. These results indicate a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma [10].

2. Histone ModificationHistone modification is the second most important epigenetic factor that has a critical role in regulation of gene expression. Histones proteins can be modified in many ways in their N-terminal tail, including acetylation, phosphorylation, methylation, ubiquitylation, sumoylation, and adenosine diphosphate (ADP) ribosylation, deamination, and proline isomerization. Histone deacetylase 6 (HDAC6) is a regulatory factor in the endocrine traffic network and possesses histone deacetylase activity and represses transcription. Wei et al examined the HDAC6 expressions and its pathogenic role in the uterine leiomyoma. They found a regular pattern of increasing HDAC6 and ER-a expression in leiomyoma samples.

It is well known that epigenetic modifications are acquired during development and play a central role in cellular differentiation and normal tissue and organ function in adulthood. However, during crucial stages of development, environmental exposures can alter genome state related to differentiation programming of cells or organs, thus promoting disease susceptibility in later life. A recent study reported that through nongenomic effects on developing uterus during developmental reprogramming, environmental Es recruit the epigenetic regulator EZH2 and reduce the levels of repressive histone mark H3K27me3 in chromatin and promote uterine tumorigenesis [10].

3. MicroRNAMicroRNAs are a novel class of small nonprotein-coding RNAs that regulate a high number of biological processes by targeting mRNAs for cleavage or translational repression. Studies have shown that several miRNAs, including let7, miR-21, miR-93, miR-106b, and miR-200 and their predicted target genes, are significantly dysregulated in uterine leiomyoma compared with normal myometrium. Additionally, miRNA expression seems to be strongly associated with tumor size and race. Pan et al reported that miR-21 is overexpressed in leiomyomas, with specific elevation during the secretory phase of the menstrual cycle in women who received depot-medroxyprogesterone acetate and oral contraceptives, but decreased owing to gonadotropin releasing hormone agonists (GnRHa) therapy. Recently Zavadil et al examined global correlation patterns between altered miRNA expression and the predicted target genes in uterine leiomyomas and matched myometria. They found that numbers of dysregulated miRNAs are inversely correlated with their targets at the protein level. Patterns of inverse association of miRNA with mRNA expression in uterine leiomyomas revealed an involvement of multiple candidate pathways, including extensive transcriptional reprogramming, cell proliferation control, mitogen-activated protein kinase (MAPK), transforming growth factor (TGF)-b, WNT, Janus kinase/signal transducers and activators of transcription signaling, remodeling of cell adhesion, and cellcell and cellmatrix contacts. More recently, Fitzgerald et al reported that elevated leiomyoma miR-21 levels are predicted to decrease programmed cell death 4 (PDCD-4) levels, thus leiomyomas differ from other tumors in which loss of PDCD-4 is associated with tumor progression [10].

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