- Email: [email protected]
MicroRNAs related to androgen metabolism and polycystic ovary syndrome
Accepted Manuscript MicroRNAs related to androgen metabolism and polycystic ovary syndrome Anja E. Sørensen, Pernille B. Udesen, Marie Louise Wissing, Anne Lis M. Englund, Louise T. Dalgaard PII:
S0009-2797(16)30224-1
DOI:
10.1016/j.cbi.2016.06.008
Reference:
CBI 7724
To appear in:
Chemico-Biological Interactions
Received Date: 14 February 2016 Revised Date:
25 May 2016
Accepted Date: 3 June 2016
Please cite this article as: A.E. Sørensen, P.B. Udesen, M.L. Wissing, A.L.M. Englund, L.T. Dalgaard, MicroRNAs related to androgen metabolism and polycystic ovary syndrome, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.06.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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MicroRNAs Related to Androgen Metabolism and Polycystic Ovary Syndrome
T. Dalgaard1. 1
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Anja E. Sørensen1,3, Pernille B. Udesen2, Marie Louise Wissing2, Anne Lis M. Englund2 and Louise
Department of Science and Environment, Roskilde University, Roskilde, Denmark; 2Fertility
Clinic Region Sjaelland, Holbaek Hospital, Holbaek, Denmark, 3The Danish Diabetes Academy,
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Odense University Hospital, Odense C, Denmark. Address correspondence to:
1, DK-4000 Roskilde, Denmark.
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Louise T. Dalgaard, Department of Science and Environment, Roskilde University, Universitetsvej
Email: [email protected] . Phone: +45 46 74 27 13.
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Word count: Abstract: 247, text: 3965, Figures: 2, Tables: 3.
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Abstract Polycystic ovary syndrome (PCOS) is a frequent endocrine disorder in women. PCOS is associated with altered features of androgen metabolism, increased insulin resistance and impaired fertility. Furthermore, PCOS, being a syndrome diagnosis, is heterogeneous and characterized by polycystic ovaries, chronic
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anovulation and evidence of hyperandrogenism, as well as being associated with chronic low-grade inflammation and an increased life time risk of type 2 diabetes. A number of androgen species contribute to the symptoms of increased androgen exposure seen in many, though not all, cases of PCOS: Testosterone, androstenedione, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS), where the quantitatively highest amount of androgen is found as DHEAS. The sulfation of DHEA to DHEAS depends
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on a number of enzymes, and altered sulfate metabolism may be associated with and contribute to the pathogenesis of PCOS.
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MicroRNAs (miRNAs) are small, non-coding RNAs that are able to regulate gene expression at the posttranscriptional level. Altered miRNA levels have been associated with diabetes, insulin resistance, inflammation and various cancers. Studies have shown that circulating miRNAs are present in whole blood, serum, plasma and the follicular fluid of PCOS patients and that these might serve as potential biomarkers and a new approach for the diagnosis of PCOS. In this review, recent work on miRNAs with respect to PCOS will be summarized. Our understanding of miRNAs, particularly in relation to PCOS, is currently at a very early stage, and additional studies will yield important insight into the molecular mechanisms behind
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this complex and heterogenic syndrome.
Keywords: Polycystic ovary syndrome, microRNA, androgens, DHEA, DHEAS, ovarian steroid metabolism, biomarker
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Abbreviations: DHEA dehydroepiandrosterone, DHEAS dehydroepiandrosterone sulfate, PCOS polycystic ovary syndrome, T2D type 2 diabetes mellitus, Abbreviations: CYP Cytochrome P450, scc side chain cleavage, Fdx Ferredoxin, FdR Ferredoxin reductase, AR androgen receptor, SF steroidogenic factor, SULT sulfotransferase, DENND DENN/MADD Domain Containing, StAR Steroidogenic Acute Regulatory Protein, CYB5B Cytochrome B5 Type B, PAPS 3'-Phosphoadenosine 5'-Phosphosulfate, HSD Hydroxy steroid dehydrogenase , AKR Aldo-Keto Reductase Family.
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1. Introduction to Polycystic Ovary Syndrome and androgen production
1.1. Polycystic Ovary Syndrome: Definitions and clinical presentation, heterogeneity
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Polycystic ovary syndrome (PCOS) is a common endocrine, reproductive and heterogeneous disorder with a prevalence among premenopausal women between 7-15% depending on the definition used 1. The etiology of PCOS is not well characterized and diagnosis is based on reproductive and endocrinological characteristics such as clinical and/or biochemical hyperandrogenism, anovulationand polycystic ovarian morphology with the exclusion of other pituitary, androgenic or adrenal disorders1. The leading cause of
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anovulatory infertility is PCOS and, moreover, PCOS is also associated with an increase in pregnancy complications. Furthermore, PCOS is also associated with obesity, insulin resistance and pancreatic β-cell dysfunction contributing to an increased lifetime risk of developing type 2 diabetes (T2D) and cardiovascular
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diseases1. However, at present time there is no consensus of which method that is the most effective for assessing the risk. International guidelines about assessment and management of PCOS vary. In Australian Guidelines2
National
(ref:
https://jeanhailes.org.au/contents/documents/Resources/Tools/PCOS_evidence)based_guideline_for_assessm ent_and_management_pcos.pdf recommends to measure blood pressure annually and a total lipid profile every
second
year
in
all
women
with
PCOS.
In
Danish
National
Guidelines3
(http://static1.squarespace.com/static/5467abcce4b056d72594db79/t/561cb676e4b09f2277ba139d/14447222
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94970/PCOS_rev2015_finalrev%5B1%5D.pdf ) it is recommended to do this only in women with risk factors of cardiovascular disease. Therefore, today it may be difficult to guide women and health professionals about PCOS. A possible biomarker of diabetes and cardiovascular disease might help to identify the women with PCOS and increased risk of cardiovascular disease and diabetes. The altered
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androgen levels and increased levels of insulin in women with PCOS are inextricably linked together. Understanding this connection and the mechanisms behind could be the key to a more exact diagnosis of
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PCOS.
1.2. Metabolic and fertility consequences of PCOS The diverse nature of PCOS is reflected in the existence of three different although overlapping definitions for the diagnosis of PCOS and substantial controversy still exists on the matter (Figure 1). After an international meeting at the U.S. National Institute of Health (NIH) in 1990, the diagnosis of PCOS required clinical and/or biochemical evidence of hyperandrogenism in the presence of chronic anovulation. Ovarian morphology was not part of the NIH-1990 criteria4. With advances in ovarian imaging, polycystic ovarian morphology was added as a criterion after a joint European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) meeting convened in Rotterdam in 2003. The so-called Rotterdam criteria required two of the three features (hyperandrogenism, chronic 3
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anovulation and polycystic ovaries) to be present and place equal diagnostic importance to each of the three features5. Nevertheless, a compromise between the two pre-existing criteria was made in 2006 by the Androgen Excess Society (AES) arguing that hyperandrogenism is an essential and cardinal feature of PCOS and should always be included in the diagnosis of PCOS together with either chronic anovulation or
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polycystic ovarian morphology6. Common for all of the diagnostic criteria is the exclusion of other disorders presenting with similar features such as Cushing’s syndrome, adrenal hyperplasia and androgen-secreting
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neoplasms to name a few.
Figure 1: Diagnostic criteria and possible phenotypes for polycystic ovary syndrome. Hyperandrogenism as defined either based on biochemical or clinical characteristics. NIH: National Institutes of Health. AES: Androgen Excess Society.
The metabolic and reproductive phenotype of a PCOS patient depends on the diagnostic criteria used, but is also influenced by ethnicity, race, life stage and body composition. It is possible to distinguish between four main phenotypes of PCOS with the Rotterdam criteria embracing all of them: The reproductive and metabolic consequences range from the more severe phenotype (hyperandrogenism, anovulation with or
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without polycystic ovaries), also sometimes referred to as ‘classic PCOS’, to a more mild phenotype (polycystic ovaries and anovulation)7 (Figure 1). A close association between insulin resistance accompanied with compensatory hyperinsulinemia and hyperandrogenism exist, which is partly due to the fact that insulin stimulates androgen production in the
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ovaries as well as lowers the production of sex hormone binding globulin (SHBG) produced by the liver, which will increase the amount of bioavailable free testosterone8,9. The majority of PCOS women are insulin resistant partly independent of body weight10 Normo-androgenic PCOS women, who are anovulatory, tend to be less insulin resistant than women with the classic PCOS phenotype (reviewed by 11).
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1.3. Hyperandrogenism in PCOS; etiology and environmental factors
Family-based studies, twin studies, genome-wide association studies and fetal programming studies all implicate a genetic origin of hyperandrogenism and PCOS, in which environmental components also may be
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involved. Intrauterine growth retardation, exposure to excess androgens during gestation, exposure to environmental factors such as a sedentary lifestyle combined with poor dietary intake and a lack of exercise may later in life trigger and play a part in the development of PCOS (reviewed by 12). The detrimental effect of obesity is evident from studies of age-matched obese and lean PCOS women showing a higher degree of hyperandrogenism in the obese PCOS women13. Lifestyle intervention and weight loss often improves their metabolic and reproductive profile.
The source(s) of excessive androgen levels in PCOS are not completely resolved. Elevated androgen levels
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could arise from abnormal steroidogenic activity within the ovary, enhanced activity of the P450 steroidogenic enzymes involved in androgen production, defects in cortisol metabolism and/or increased secretion of androgens from the adrenal glands14. Studies of the hypothalamic–pituitary–adrenal function in women with PCOS have found that circulating ACTH levels are similar to those found in controls15–17.
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(horrocks, ACHT..,(clin end), 1982)( Azziz, R, Adrenal androgen excess,1998)(lanzone,corticotropin…, fert. Steril (1995), Women with PCOS and adrenal precursor androgen (APA) excess demonstrates hypersecretion of adrenocortical products in response to ACTH. In addition to androgens, a hypersecretion
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of pregnenolone, 17-hydroxyprenolone, 11-deoxycortisol and cortisol have been observed. Meanwhile there is a general agreement of that APA excess in women with PCOS not is caused by adrenal steroidogenic defects16,18. (Glintborg, sign.. 2005) (Azziz, R, Adrenal androgen excess,1998)
1.4. Androgen and steroid hormone metabolism in the developing ovarian follicle Androgen biosynthesis occurs predominately in steroidogenic tissues such as the ovaries and in the adrenal cortex. Excess of circulating endogenous androgens such as testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA), and the DHEA sulfate metabolite (DHEAS) is often measured for
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diagnosis of PCOS. The ovary is a complex endocrine organ and it is dependent on two anterior pituitary peptide hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These two hormones are under the control of yet another hormone, the gonadotropin-releasing hormone (GnRH) secreted from the hypothalamus in a pulsatile fashion. LH secretion is more dependent on GnRH than FSH possible explaining
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the increased LH to FSH ratio observed in PCOS. Within each follicle the oocyte is surrounded by an inner layer of granulosa cells and an outer layer of theca cells. Both of these ovarian cell types are steroidogenically active throughout their lifespan although granulosa cells first achieve this function during the antral follicle development stage. Granulosa cells are able to respond to FSH and gradually to LH during the menstrual cycle while theca cells only express LH receptors.
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Both theca and granulosa cells together with LH and FSH are required for the production of androgens coining the two-cell-two-gonadotropin compartment theory. Theca cells, under the influence of LH, express three key steroidogenic enzymes CYP11A, 3β-HSD and CYP17, which are required for the de novo
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conversion of the steroid precursor cholesterol into androgens. Hydroxylation followed by cleavage of the cholesterol side chain by the mitochondrial CYP11A enzyme yields pregnenolone that can easily diffuse out of the mitochondria. Once converted to pregnenolone, it may be further metabolized into 17βhydroxypregnenolone or progesterone by CYP17 or 3β-HSD, respectively. The CYP17 enzyme converts 17β-hydroxypregnenolone into dehydroepiandrosterone (DHEA). In humans, DHEA is the major steroid precursor with the adrenal glands being responsible for up to 97% of circulating DHEA. However, DHEA has a limited diagnostic use in PCOS because of its diurnal variation, its inter-subject variation and stress-
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induced increases. Therefore, the sulfated, more stable and biological inactive form (DHEAS) is preferred when assessing adrenal from ovarian androgen production19. PCOS is often considered a disorder with a predominately ovarian androgen production but approximately 50% of women with PCOS also have elevated serum DHEAS20. The enzyme responsible for the sulfation of DHEA into DHEAS is DHEA
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sulfotransferase (DHEA-ST; SULT2A1) and the amount of DHEAS is a combination of the biosynthesis of DHEA together with the activity of SULT2A121. Almost all of the circulating DHEAS is produced by the adrenal glands19. The level of DHEAS in the serum is increased in women with the classic anovulatory
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PCOS showing features of hyperandrogenism22. Another role of DHEA is as precursor for androstenedione converted by 3β-HSD; the main androgen secreted by the theca cells and the most important precursor of dihydrotestosterone and testosterone. Androstenedione diffuse across the basal lamina into the granulosa cells where it can be converted into the primary female sex hormone 17β-estradiol by the enzyme aromatase (CYP19) under the control of FSH23.
2. MicroRNAs: Pervasive post-transcriptional regulators
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2.1. MicroRNA biogenesis and mechanism of action MicroRNAs (miRNAs) are small, non-coding, single-stranded, endogenous regulatory RNA molecules consisting of ∼22 nucleotides. They modulate the expression of target genes on a post-transcriptional level by binding to the 3’untranslated region (3’UTR) of target mRNAs thereby inhibiting their translation and/or
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inducing their degradation24. More than 2800 miRNAs have been identified in Homo sapiens so far although the function of most of them remains to be determined25. The putative larger miRNA precursor transcripts can be found either within introns of both non-coding and coding transcripts or in exonic regions of the genome. Some miRNA genes possess their own promotor, others share a promotor with the host genes in which they reside. RNA polymerase II or III is responsible for the transcription of the miRNA into the initial
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long stem-loop structured primary miRNA (pri-miRNA). Within the nucleus, the pri-miRNA is cleaved by the enzyme Drosha yielding a shorter (app. 70 nt) hairpin-structured pre-miRNA which can be exported to the cytoplasm though the exportin5 complex for further maturation. Subsequently, the enzyme Dicer cleaves
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the pre-miRNA to form a double-stranded RNA duplex containing the mature miRNA and a passenger stand. The mature miRNA is loaded onto the RNA-induced silencing complex (RISC) by Argonaute (Ago) proteins
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whereby miRNAs can exert its actions on target mRNAs24 (Figure 2).
Figure 2: The canonical miRNA biogenesis pathway. MiRNA genes are located either in introns, are intergenic or polycistronic. The primary miRNA (Pri-miRNA) is transcribed by polymerase II (or polymerase III) and is cleaved by the microprocessor complex Drosha-Pasha (DGCR8) in the nucleus to generate pre-cursor miRNA (pre-miRNA). Pre-miRNA is transported to the cytoplasm by Exportin 5, it is further cleaved to its mature length (~22 nt) by the RNase III enzyme Dicer
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in complex with the double-stranded RNA-binding protein TRBP. Argonaute proteins (AGO2-4) unwind the miRNA duplex and facilitate incorporation of the guide strand into the RNA-induced silencing complex (RISC). AGO then guides the RISC miRNA assembly to target mRNAs through cleavage, translational repression and translocation to P-bodies, whereas the
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passenger strand is degraded.
3. MicroRNAs controlled by androgens or altered in PCOS 3.1. MicroRNAs found dysregulated in PCOS follicular fluid
Ovarian follicular fluid, comprised of blood plasma components, hormones and ovarian cell secretions,
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serves as an important environment for the development and maturation of the oocyte26. Recent studies indicate that dysregulated miRNAs may play a part in ovulatory dysfunction observed in PCOS. Analysis of
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microRNAs isolated from follicular fluid from PCOS women has identified several differently expressed miRNAs27,28. MiRNAs are both present in the supernatant and in microvesicles of the follicular fluid27. Among the up-regulated miRNAs was miRNA-9, - 18b, -32, -34c and -135a. Based on Target gene analysis, possible target genes related to the PCOS phenotype, including roles in carbohydrate metabolism, beta-cellfunction and steroid synthesis28, were identified. Another study found miR-132 and miR-320 down-regulated in PCOS27 compared to healthy controls. The two studies used different PCOS diagnostic criteria possible accounting for the discrepancies. Further, the finding of down-regulated miR-320, contrasts the results from
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a study by Yin et al.29, which demonstrated that miR-320 was upregulated in follicle fluid and granulosa cells from women with PCOS compared to controls. The study also reported hyperandrogenemia and downregulation of E2F1/SF-1 proteins, which inhibits estradiol release in the follicular fluid and GCs of PCOS patients. They thereby hypothesized that the high levels of miR-320 could be the partial reason for hyperandrogenemia in PCOS patients. However, it has not been possible to consistently reproduce these
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findings in other studies (Table 1).
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It is known that advanced maternal age has been associated with an altered follicular fluid composition30 and age-associated miRNAs have been isolated from follicular fluid from women undergoing IVF treatments31. While miRNA-21 was present in the follicle fluid from younger women, three different miRNAs were upregulated in older women (miR 99b-3p, miR-134 and mir-109b). Whether a similar pattern for these miRNAs could be found in women with PCOS, has at present time not been investigated, however miR-29a, miR-132 and miR-193b levels were negatively associated with age in another study32. In conclusion, comparing the limited number of studies of miRNA in follicle fluid, the miRNA profile varies. This could be due to different methods, age of the women or inclusion criteria, but could also illustrate the heterogeneous nature of PCOS.
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3.2. Circulating serum microRNAs associated with PCOS and/or hyperandrogenism Definition of PCOS as a syndrome includes a large group of women with different clinical manifestations. To date no single criterion is sufficient for the diagnosis. As circulating in serum, miRNA could serve as biomarker. A number of MiRNAs have been reported to be differently expressed in serum of women with
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PCOS compared to women without PCOS. One study reported miR-93 and miR-223 significantly upregulated in women with PCOS33. A target gene analysis revealed peroxisome proliferator receptor (PPAR) activity as a pathway possibly regulated by miR-223. PPAR has been shown to be important in insulin resistance and hyperandrogenism, though this study did not show any correlation between miR-223 and testosterone levels or insulin resistance33,34. Long et al. (2014)35 found significantly increased levels of
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miR-30c, miR-222 and miR-146a, whereas others (miR-16, miR-19a, miR-24, miR-106b and miR-186) were borderline significantly increased (P-values: 0.058-0.086). MiR-320 were near significant decreased (p=0.078), in line with the findings by Sang et al. (2013)27. Long et al. further found strong and positive
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correlation between miR-222 and serum insulin levels, whereas miR-146a was negatively correlated with serum testosterone. A third study, reported another five miRNAs significantly upregulated: let-7i3pm, miR5706, miR-4463, miR-3665 and miR-638 while four miRNAs were downregulated miR-124-3p, miR-128, miR-29a-3p and let-7c36. These results are not in line with previous data, and it is possible that the discrepancy could be caused by the size of the verification cohort (n=9), whereas other studies included 2568 women with PCOS.
Though the contradictory results regarding the miRNA profile of PCOS, many of the differently expressed
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miRNAs are involved in steroid metabolism. Research have shown that, not only excess androgens, but also other imbalances in steroid metabolism affect the follicle development and ovulation and these imbalances might be regulated by microRNAs.
Androgens (testosterone and dihydrotestosterone) regulate follicular atresia by inducing the expression of
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miR-125b in both mouse granulose, human KGN granulosa tumor cells and human granulosa cells from women undergoing in vitro fertilization37. MiR-125b targets pro-apoptotic proteins which suppress follicular atresia and thereby promotes follicle development. This could indicate that a critical balance exists between
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the essentiality of androgens in normal follicular fluid and the adverse effects of the androgen excess in women with PCOS.
Functional studies also support the regulatory role of miRNA in steroid metabolism: 80 different miRNAs were tested on human granulosa cells and release of progesterone, testosterone and estradiol was assessed. Numerous miRNAs reduced progesterone, testosterone and estradiol, but only miR-107 was found to increase the release of testosterone38. This specific miRNA was also found significantly upregulated in granulosa cells of women with PCOS39. Further, it is well-known that sulfation of steroid hormones can inactive these. Altered sulfate metabolism, in part by actions of miRNAs could, hypothetically, be associated with and contribute to the pathogenesis of
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PCOS. Indeed, mutations in PAPSS2 cause androgen excess and may contribute to PCOS features40. Inactivation of xenobiotics through sulfation increases water-solubility, hence facilitating their excretion, while sulfation of steroid hormones is also inactivating. The donor of the sulfate group can be 3’phosphoadenosine 5’-phosphosulfate (PAPS), the sulfur-containing amino acids L-methionine (L-Met) and
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L-cysteine (L-Cys) or macromolecules that undergo normal degradation by sulfatases within lysosomes23. Sulfation occurs in the cytosol or in the membranes of the Golgi apparatus of the cell. Sulfate homeostasis may by altered by age, time of the day, a woman’s gestational period and certain types of diseases which consequently may lead to a defective sulfation of substrates.
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3.3. A possible miRNA network with targets in the ovarian steroid metabolism
Ovarian steroid metabolism is regulated at the cellular level in that the granulosa cells lining the follicle produce estrogens derived from androgens produced by the more peripherally located theca-cells. The
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enzymes responsible for catalysing distinct reactions in the pathways leading to androgen and estrogen production are therefore also in many cases restricted to either theca or granulosa cells (listed in Table 2, with their most important roles). Also genes not directly involved in steroid hormone synthesis, but rather with roles in lipid transport or cellular signalling have been shown to be important for controlling ovarian androgen and estrogen metabolism23. Variants near or in the DENND1A gene has been reproducibly associated with the PCOS syndrome in genome-wide association studies41,42. The actual cellular function of the protein encoded by DENND1A is not well characterized, although over-expression of a variant
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(DENND1A V2) in vitro could recapitulate cardinal features of hyperandrogism in human theca-cells43. In order to identify miRNAs with a possible involvement in ovarian androgen production, we performed searches for miRNAs targeting selected genes involved in androgen synthesis or metabolism of ovarian steroidogenic cells (Table 2). The updated TargetScanHuman database44 was used to extract all miRNAs
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conserved in mammals for the particular genes (Table 2) and miRNAs with three or more targets and found in follicular fluid32 and in human granulosa cells45 were listed suggesting a network of miRNAs targeting ovarian steroid hormone metabolism (Table 3). No studies of human theca cell miRNA expression was
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identified in the published literature or in online data repositories. Reassuringly, of the identified miRNAs possibly targeting transcripts involved in androgen synthesis, miR-24 and miR-151 were reported to be suppressed in follicular fluid from PCOS patients. Moreover, miR-151 levels correlated with serum free testosterone32, while miR-24 and miR-19 is reported to reduce testosterone release of cultured human ovarian cells38. Moreover, estradiol release was decreased in culture human ovarian cells transfected with miR-24, miR-150 and miR-151, further emphasizing that these miRNAs may be important for ovarian function. However, it is not yet clear, what the physiological and pathophysiological regulation of these miRNAs is or what their combined effect is.
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It will be interesting to investigate the impact of each of these miRNAs on the regulation of the individual predicted transcripts in this network. Moreover, since at least some of the miRNAs in the network appear to be altered in PCOS patient follicular fluid, it is also relevant to examine the extent to which altered levels of these miRNAs are reflected in changed gene-expression in PCOS patient granulosa cells. There is firm
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evidence that CYP19A1 (aromatase) activity as well as mRNA levels are decreased in PCOS patients, which is associated with the decreased size of follicles46,47 and CYP19A1 mRNA is a predicted target of miR-19,
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miR-199 and miR-186, however it is not known if these miRNAs are increased in PCOS patient samples.
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Table 2. Selected genes important for ovarian production of steroid hormone metabolites
Steroidogenic cells Steroidogenic cells Steroidogenic cells Steroidogenic cells Steroidogenic cells
SULT2A1
Steroidogenic cells
Fdx1 Ferredoxin FdxR Fdx reductase NR5A1/SF1
Steroidogenic cells Steroidogenic cells Steroidogenic cells
AR
Steroidogenic cells
CYP17A1/P450c17
High in Theca, low in granulosa cells High in Theca, low in granulosa cells High in theca cells, low in granulosa cells Theca cells
HSD3B2/3βHSD2 DENND1A V2
Granulosa cells Granulosa cells
Granulosa cells
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HSD17B1/17βHSD1
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HSD17B5/AKR1C3 HSD17B6/3αHSD CYP19A1/P450aro/ Aromatase
Entry point for steroidogenesis Cholesterol -> Pregnenolone Generates activated sulfate groups Generates activated sulfate groups Electron donor co-factor Electron donor co-factor Mitochondrial cholesterol transfer protein, promotes androgen synthesis Sulfates DHEA to DHEAS, decrease testosterone synthesis CYP450 enzyme co-factor CYP450 enzyme co-factor Transcriptional regulator of enzymes involved in steroid synthesis Androgen receptor, mediates cellular responses to androgens 17OH-Pregnenolone -> DHEA 17OH-Progesterone -> Androstenedione Androstenediol -> Testosterone
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PAPSS1/ PAPS synthase 1 PAPSS2/ PAPS synthase 2 POR/P450 oxidoreductase CYB5B/CYB5B StAR/StAR
Role in androgen metabolism
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CYP11A1/P450scc
Ovarian expression Cell type and level Steroidogenic cells
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Gene /protein abbreviation
Cellular growth factor signaling
Androgen synthesis Androstenedione -> Testosterone Backdoor to DHT, 5alfa reductase Generates Estrone in GC by FSH stimulation Androstenedione -> Estrone Testosterone -> Estradiol Generates estradiol in GC by FSH stimulation Estrone -> Estradiol
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Abbreviations: GC granulosa cell, CYP Cytochrome P450, scc side chain cleavage, DHT dihydrotestosterone, FSH follicle stimulating hormone, Fdx Ferredoxin, FdR Ferredoxin reductase, AR androgen receptor, SF steroidogenic factor, SULT sulfotransferase, DENND DENN/MADD Domain Containing, StAR Steroidogenic Acute Regulatory Protein, CYB5B Cytochrome B5 Type B, PAPS 3'-Phosphoadenosine 5'-Phosphosulfate, HSD Hydroxy steroid dehydrogenase , AKR Aldo-Keto Reductase Family 14,23,43,48.
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Table 3. A possible miRNA network targeting ovarian steroid metabolism
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miRNA miR-302 family/ /372 Genesymbol /373 miR-199 /520 miR-150 miR-324 miR-151 miR-24 miR-19 miR-212 /3129 miR-186 DENND1A x x x x x x StAR x x x x x CYB5B x x x x x x FDX1 x x x x FDXR x x Sult2A1 x x x x SF1 x PAPSS2 x x x CYP19A1 x x x AR x x x x HSD17B6 x MiR-302, -372, -373, -520, -151, -24, -19 and -199: The mature miRNA species belongs to the 3’ arm of the precursor. MiR150, -324,-212,-3129 and -186: The mature miRNA species belongs to the 5’ arm of the precursor. Common miRNAs putatively targeting selected mRNAs encoding proteins important for ovarian steroidogenesis were identified in TargetScanHuman, release 7.0, using searches for miRNAs conserved in mammals targeting each of the genes listed in Table 2. MiRNAs in common between 3 or more genes were identified using Venn diagrams.
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3.4. Can circulating or follicular microRNAs be used clinically for PCOS diagnosis or prognosis of fertility treatment? PCOS is a syndrome, which encompasses a broad spectrum of signs and symptoms of ovarian dysfunction. As a syndrome, there is no single diagnostic criterion sufficient for the clinical diagnosis. The clinical signs
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of PCOS can vary over time, which complicates the task of diagnosing PCOS in a clinical setting. Further, women with PCOS are in greater risk of developing type 2 diabetes and cardiovascular diseases10,49, which underline the need for a correct and reliable diagnosis. Circulating miRNAs could serve as biomarkers as they are easily accessible. MiRNAs in follicular fluid could also serve as a marker during fertility treatment. Unfortunately, of the presently published studies of miRNAs in serum of women with PCOS a common
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conclusion is that it is possible to distinguish between controls and women with PCOS, however, the three studies report different results33,35,36. Long et al (2014)35 found as mentioned, miR-30c, miR-222 and miR146 upregulated and assessed the value of the miRNAs’ ability to discriminate women with PCOS from
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healthy controls by establishing receiver operator characteristics (ROC-curves). The combination of the three miRNAs yielded the largest area under the ROC-curve, and was suggested as diagnostic markers. Another study (by Sathyapalan et al (2015))33 concluded, also based on ROC-curve analysis, that miR-93 could serve as biomarker supporting a positive clinical diagnosis of PCOS. A combination of miRNAs did not improve the diagnostic potential compared to miR-93 alone in this study. The third study did not assess the value of their results as diagnostic markers 36, however, there was no overlap in the reported differentially expressed miRNAs of this study with the other two published studies. The reason for the different results obtained in
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different studies is not known, but may be due to ethnic differences in studied populations, different inclusion criteria, age, body weight of study participants, as well as different assay methods. Clearly, there is a need for larger, well controlled studies examining miRNA profiles in serum of PCOS patients and control subjects in order to gain a consensus. MiRNAs in follicular fluid are also suggested as biomarkers and
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similar to reported studies investigating serum miRNA levels there is great discrepancy between studies, which is likely due to inconsistencies in methods, inclusion criteria and probably different ethnicity and
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heterogeneity of the patients.
4. Conclusions and perspectives Many different miRNA species have been implicated in the pathophysiology of PCOS and regulation of ovarian steroid hormone metabolism. However, currently there is limited knowledge of the impact of single or combined sets of co-regulated miRNAs on ovarian androgen and estrogen metabolism and how these may contribute to PCOS.
A large number of miRNAs have been associated with altered levels in serum or follicular fluid from PCOS patients compared with fertile control patients. Whether these sets of miRNA could serve as biomarkers for 14
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guidance of treatment or has prognostic value is currently not established, but as more studies are published it is expected that at least a proportion of the reported miRNAs can be validated. Extracellular miRNAs are localized in exosomes, which markedly stabilize them and provide protection from extracellular nucleases. Thus, miRNAs as a molecular class offers specific advantages as possible biomarkers. However, in order to
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make full use of the potential of miRNAs as biomarkers, more studies with carefully controlled design and sample preparation are necessary. PCOS being a clinically heterogeneous disorder is likely to have heterogeneous etiology. Thus, it is possible that miRNAs could be used to identify subtypes of PCOS patients, for which different optimal treatment options exist, leading to personalized treatment
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With contrasting studies being published regarding the expression and regulatory role of miRNAs in ovarian function as well as levels of miRNA in serum and follicular fluid, it is imperative that large replication studies are performed to identify the specific miRNAs with robust roles and regulation. Because miRNAs
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often exist in families, an increasingly important approach will be to modulate levels of entire miRNA families or co-regulated miRNAs together as well as separately in order to establish their roles in the intact tissue or organ. As it is known that many miRNAs act most strongly at the level of translational inhibition, investigations of mRNA expression arrays may have low power to reveal the true impact of regulated microRNAs on the PCOS phenotype. It would be ideal to have proteomic data from granulosa and theca cells of PCOS patients versus control subjects, but currently no such studies have been published.
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Moreover, since many miRNAs in general appear to have roles in stress response modulation, whereas they may be dispensable in the normal physiological situation it is important to investigate the proper models of metabolic, inflammatory or hormonal stress in order to elucidate their natural functions. An important note is that ovarian function and regulation is highly species specific, and several human specific miRNAs have
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been described45 that appear to be granulosa cell specific. Thus, it is possible that more, yet undescribed, miRNAs are expressed in specific cell types of the ovary. It is likely that more miRNAs with roles in ovarian hormonal metabolism and impact on development of PCOS exist, but are still to be identified and
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characterized.
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Table 1. List of microRNAs observed in polycystic ovary syndrome (PCOS) and involved in androgen metabolism or function
miR-9
Detected in Tissue/Cell Follicular fluid Granulosa cells
miR-18b
Follicular fluid Granulosa cells
Human
miR-21
Follicular fluid Granulosa cells
Human Mouse Rat
miR-24
Follicular fluid
Human
miR-27b
Whole blood
Human
miR-29a
Follicular fluid
Human
Promotes progesterone release Inhibits testosterone and estradiol release Anti-apoptotic. Increased after FSH exposure; inconclusive testosterone response Negatively correlated with serum estradiol Positively correlated with testosterone Positively correlated with DHEAS
miR-30c
Serum Granulosa cells
Human Rat
Increased after FSH exposure
miR-103
Whole blood Granulosa cells
Human
miR-107
Granulosa Cells
Human
miR-132
Follicular fluid Granulosa cells
Human Mouse Rat
Follicular fluid Granulosa cells Serum Plasma Follicular fluid Granulosa cells
Human
miR-146a miR-151
Follicular fluid
miR-155
Serum Granulosa cells Serum Follicular fluid
miR-222
Observation in PCOS
Human
Inhibits testosterone release
Increased in PCOS
28,38,50
28,38,50
Increased in PCOS
31,38,51–53
Decreased in PCOS
32
Increased in PCOS.
52
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Increased in PCOS
Decreased in PCOS
32
Increased in PCOS
35,53
Increased in PCOS
38,52
Increased in PCOS
38,39
Decreased in PCOS
27,38,50,54
Increased in PCOS
28,38,50
Reduces progesterone, estradiol and testosterone release
Increased in PCOS
27,35,38
Human
Negatively correlated with serum total and free testosterone
Decreased in PCOS
32
Human
Inhibits testosterone release
Increased in PCOS
38,50,52
Human
Increases estradiol secretion
Increased in PCOS
27,35,55,56
Human Mouse
Induces GCs proliferation Increases estrogen release
Inconsistent effects on estradiol secretion. Increases testosterone Negatively correlated with FSH in PCOS patients
Differentially expressed in follicular fluid Decreased serum expression in PCOS. Inconsistent expression in follicular fluid in PCOS; Increased expression granulosa cells in PCOS
Human
Follicular fluid Granulosa cells
miR-320
Serum Follicular fluid Granulosa cells
Human
miR-383
Follicular fluid Granulosa cells
Human
miR-518
Follicular fluid
Human
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miR-224
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Reported Function(s)
Promotes progesterone release Inhibits estradiol release Positively correlated with testosterone Increases testosterone release Increases estradiol secretion Reduces progesterone and testosterone release Reduces progesterone and testosterone release
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miR-135a
Species
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microRNA
Enhances the release of estradiol from GCs by CYP19A1 Positively correlated with total and free testosterone and androstenedione in PCOS patients
28,57,58
27,29,32,35,59
Increased in PCOS
28,29,60
Increased in hyperandrogenic PCOS
32
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Highlights for: MicroRNAs Related to Androgen Metabolism and Polycystic Ovary Syndrome
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Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of the reproductive age PCOS is clinically diverse presenting with infertility, hyperandrogenemia and the metabolic syndrome A network of microRNAs may target mRNAs involved in steroid hormone synthesis or metabolism Circulating or follicular microRNAs could be used as biomarkers for patient stratification Specific microRNAs may be involved in disease pathology, however more research is needed
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S0009-2797(16)30224-1
DOI:
10.1016/j.cbi.2016.06.008
Reference:
CBI 7724
To appear in:
Chemico-Biological Interactions
Received Date: 14 February 2016 Revised Date:
25 May 2016
Accepted Date: 3 June 2016
Please cite this article as: A.E. Sørensen, P.B. Udesen, M.L. Wissing, A.L.M. Englund, L.T. Dalgaard, MicroRNAs related to androgen metabolism and polycystic ovary syndrome, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.06.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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MicroRNAs Related to Androgen Metabolism and Polycystic Ovary Syndrome
T. Dalgaard1. 1
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Anja E. Sørensen1,3, Pernille B. Udesen2, Marie Louise Wissing2, Anne Lis M. Englund2 and Louise
Department of Science and Environment, Roskilde University, Roskilde, Denmark; 2Fertility
Clinic Region Sjaelland, Holbaek Hospital, Holbaek, Denmark, 3The Danish Diabetes Academy,
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Odense University Hospital, Odense C, Denmark. Address correspondence to:
1, DK-4000 Roskilde, Denmark.
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Louise T. Dalgaard, Department of Science and Environment, Roskilde University, Universitetsvej
Email: [email protected] . Phone: +45 46 74 27 13.
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Word count: Abstract: 247, text: 3965, Figures: 2, Tables: 3.
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Abstract Polycystic ovary syndrome (PCOS) is a frequent endocrine disorder in women. PCOS is associated with altered features of androgen metabolism, increased insulin resistance and impaired fertility. Furthermore, PCOS, being a syndrome diagnosis, is heterogeneous and characterized by polycystic ovaries, chronic
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anovulation and evidence of hyperandrogenism, as well as being associated with chronic low-grade inflammation and an increased life time risk of type 2 diabetes. A number of androgen species contribute to the symptoms of increased androgen exposure seen in many, though not all, cases of PCOS: Testosterone, androstenedione, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS), where the quantitatively highest amount of androgen is found as DHEAS. The sulfation of DHEA to DHEAS depends
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on a number of enzymes, and altered sulfate metabolism may be associated with and contribute to the pathogenesis of PCOS.
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MicroRNAs (miRNAs) are small, non-coding RNAs that are able to regulate gene expression at the posttranscriptional level. Altered miRNA levels have been associated with diabetes, insulin resistance, inflammation and various cancers. Studies have shown that circulating miRNAs are present in whole blood, serum, plasma and the follicular fluid of PCOS patients and that these might serve as potential biomarkers and a new approach for the diagnosis of PCOS. In this review, recent work on miRNAs with respect to PCOS will be summarized. Our understanding of miRNAs, particularly in relation to PCOS, is currently at a very early stage, and additional studies will yield important insight into the molecular mechanisms behind
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this complex and heterogenic syndrome.
Keywords: Polycystic ovary syndrome, microRNA, androgens, DHEA, DHEAS, ovarian steroid metabolism, biomarker
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Abbreviations: DHEA dehydroepiandrosterone, DHEAS dehydroepiandrosterone sulfate, PCOS polycystic ovary syndrome, T2D type 2 diabetes mellitus, Abbreviations: CYP Cytochrome P450, scc side chain cleavage, Fdx Ferredoxin, FdR Ferredoxin reductase, AR androgen receptor, SF steroidogenic factor, SULT sulfotransferase, DENND DENN/MADD Domain Containing, StAR Steroidogenic Acute Regulatory Protein, CYB5B Cytochrome B5 Type B, PAPS 3'-Phosphoadenosine 5'-Phosphosulfate, HSD Hydroxy steroid dehydrogenase , AKR Aldo-Keto Reductase Family.
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1. Introduction to Polycystic Ovary Syndrome and androgen production
1.1. Polycystic Ovary Syndrome: Definitions and clinical presentation, heterogeneity
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Polycystic ovary syndrome (PCOS) is a common endocrine, reproductive and heterogeneous disorder with a prevalence among premenopausal women between 7-15% depending on the definition used 1. The etiology of PCOS is not well characterized and diagnosis is based on reproductive and endocrinological characteristics such as clinical and/or biochemical hyperandrogenism, anovulationand polycystic ovarian morphology with the exclusion of other pituitary, androgenic or adrenal disorders1. The leading cause of
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anovulatory infertility is PCOS and, moreover, PCOS is also associated with an increase in pregnancy complications. Furthermore, PCOS is also associated with obesity, insulin resistance and pancreatic β-cell dysfunction contributing to an increased lifetime risk of developing type 2 diabetes (T2D) and cardiovascular
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diseases1. However, at present time there is no consensus of which method that is the most effective for assessing the risk. International guidelines about assessment and management of PCOS vary. In Australian Guidelines2
National
(ref:
https://jeanhailes.org.au/contents/documents/Resources/Tools/PCOS_evidence)based_guideline_for_assessm ent_and_management_pcos.pdf recommends to measure blood pressure annually and a total lipid profile every
second
year
in
all
women
with
PCOS.
In
Danish
National
Guidelines3
(http://static1.squarespace.com/static/5467abcce4b056d72594db79/t/561cb676e4b09f2277ba139d/14447222
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94970/PCOS_rev2015_finalrev%5B1%5D.pdf ) it is recommended to do this only in women with risk factors of cardiovascular disease. Therefore, today it may be difficult to guide women and health professionals about PCOS. A possible biomarker of diabetes and cardiovascular disease might help to identify the women with PCOS and increased risk of cardiovascular disease and diabetes. The altered
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androgen levels and increased levels of insulin in women with PCOS are inextricably linked together. Understanding this connection and the mechanisms behind could be the key to a more exact diagnosis of
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PCOS.
1.2. Metabolic and fertility consequences of PCOS The diverse nature of PCOS is reflected in the existence of three different although overlapping definitions for the diagnosis of PCOS and substantial controversy still exists on the matter (Figure 1). After an international meeting at the U.S. National Institute of Health (NIH) in 1990, the diagnosis of PCOS required clinical and/or biochemical evidence of hyperandrogenism in the presence of chronic anovulation. Ovarian morphology was not part of the NIH-1990 criteria4. With advances in ovarian imaging, polycystic ovarian morphology was added as a criterion after a joint European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) meeting convened in Rotterdam in 2003. The so-called Rotterdam criteria required two of the three features (hyperandrogenism, chronic 3
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anovulation and polycystic ovaries) to be present and place equal diagnostic importance to each of the three features5. Nevertheless, a compromise between the two pre-existing criteria was made in 2006 by the Androgen Excess Society (AES) arguing that hyperandrogenism is an essential and cardinal feature of PCOS and should always be included in the diagnosis of PCOS together with either chronic anovulation or
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polycystic ovarian morphology6. Common for all of the diagnostic criteria is the exclusion of other disorders presenting with similar features such as Cushing’s syndrome, adrenal hyperplasia and androgen-secreting
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neoplasms to name a few.
Figure 1: Diagnostic criteria and possible phenotypes for polycystic ovary syndrome. Hyperandrogenism as defined either based on biochemical or clinical characteristics. NIH: National Institutes of Health. AES: Androgen Excess Society.
The metabolic and reproductive phenotype of a PCOS patient depends on the diagnostic criteria used, but is also influenced by ethnicity, race, life stage and body composition. It is possible to distinguish between four main phenotypes of PCOS with the Rotterdam criteria embracing all of them: The reproductive and metabolic consequences range from the more severe phenotype (hyperandrogenism, anovulation with or
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without polycystic ovaries), also sometimes referred to as ‘classic PCOS’, to a more mild phenotype (polycystic ovaries and anovulation)7 (Figure 1). A close association between insulin resistance accompanied with compensatory hyperinsulinemia and hyperandrogenism exist, which is partly due to the fact that insulin stimulates androgen production in the
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ovaries as well as lowers the production of sex hormone binding globulin (SHBG) produced by the liver, which will increase the amount of bioavailable free testosterone8,9. The majority of PCOS women are insulin resistant partly independent of body weight10 Normo-androgenic PCOS women, who are anovulatory, tend to be less insulin resistant than women with the classic PCOS phenotype (reviewed by 11).
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1.3. Hyperandrogenism in PCOS; etiology and environmental factors
Family-based studies, twin studies, genome-wide association studies and fetal programming studies all implicate a genetic origin of hyperandrogenism and PCOS, in which environmental components also may be
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involved. Intrauterine growth retardation, exposure to excess androgens during gestation, exposure to environmental factors such as a sedentary lifestyle combined with poor dietary intake and a lack of exercise may later in life trigger and play a part in the development of PCOS (reviewed by 12). The detrimental effect of obesity is evident from studies of age-matched obese and lean PCOS women showing a higher degree of hyperandrogenism in the obese PCOS women13. Lifestyle intervention and weight loss often improves their metabolic and reproductive profile.
The source(s) of excessive androgen levels in PCOS are not completely resolved. Elevated androgen levels
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could arise from abnormal steroidogenic activity within the ovary, enhanced activity of the P450 steroidogenic enzymes involved in androgen production, defects in cortisol metabolism and/or increased secretion of androgens from the adrenal glands14. Studies of the hypothalamic–pituitary–adrenal function in women with PCOS have found that circulating ACTH levels are similar to those found in controls15–17.
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(horrocks, ACHT..,(clin end), 1982)( Azziz, R, Adrenal androgen excess,1998)(lanzone,corticotropin…, fert. Steril (1995), Women with PCOS and adrenal precursor androgen (APA) excess demonstrates hypersecretion of adrenocortical products in response to ACTH. In addition to androgens, a hypersecretion
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of pregnenolone, 17-hydroxyprenolone, 11-deoxycortisol and cortisol have been observed. Meanwhile there is a general agreement of that APA excess in women with PCOS not is caused by adrenal steroidogenic defects16,18. (Glintborg, sign.. 2005) (Azziz, R, Adrenal androgen excess,1998)
1.4. Androgen and steroid hormone metabolism in the developing ovarian follicle Androgen biosynthesis occurs predominately in steroidogenic tissues such as the ovaries and in the adrenal cortex. Excess of circulating endogenous androgens such as testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA), and the DHEA sulfate metabolite (DHEAS) is often measured for
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diagnosis of PCOS. The ovary is a complex endocrine organ and it is dependent on two anterior pituitary peptide hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These two hormones are under the control of yet another hormone, the gonadotropin-releasing hormone (GnRH) secreted from the hypothalamus in a pulsatile fashion. LH secretion is more dependent on GnRH than FSH possible explaining
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the increased LH to FSH ratio observed in PCOS. Within each follicle the oocyte is surrounded by an inner layer of granulosa cells and an outer layer of theca cells. Both of these ovarian cell types are steroidogenically active throughout their lifespan although granulosa cells first achieve this function during the antral follicle development stage. Granulosa cells are able to respond to FSH and gradually to LH during the menstrual cycle while theca cells only express LH receptors.
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Both theca and granulosa cells together with LH and FSH are required for the production of androgens coining the two-cell-two-gonadotropin compartment theory. Theca cells, under the influence of LH, express three key steroidogenic enzymes CYP11A, 3β-HSD and CYP17, which are required for the de novo
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conversion of the steroid precursor cholesterol into androgens. Hydroxylation followed by cleavage of the cholesterol side chain by the mitochondrial CYP11A enzyme yields pregnenolone that can easily diffuse out of the mitochondria. Once converted to pregnenolone, it may be further metabolized into 17βhydroxypregnenolone or progesterone by CYP17 or 3β-HSD, respectively. The CYP17 enzyme converts 17β-hydroxypregnenolone into dehydroepiandrosterone (DHEA). In humans, DHEA is the major steroid precursor with the adrenal glands being responsible for up to 97% of circulating DHEA. However, DHEA has a limited diagnostic use in PCOS because of its diurnal variation, its inter-subject variation and stress-
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induced increases. Therefore, the sulfated, more stable and biological inactive form (DHEAS) is preferred when assessing adrenal from ovarian androgen production19. PCOS is often considered a disorder with a predominately ovarian androgen production but approximately 50% of women with PCOS also have elevated serum DHEAS20. The enzyme responsible for the sulfation of DHEA into DHEAS is DHEA
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sulfotransferase (DHEA-ST; SULT2A1) and the amount of DHEAS is a combination of the biosynthesis of DHEA together with the activity of SULT2A121. Almost all of the circulating DHEAS is produced by the adrenal glands19. The level of DHEAS in the serum is increased in women with the classic anovulatory
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PCOS showing features of hyperandrogenism22. Another role of DHEA is as precursor for androstenedione converted by 3β-HSD; the main androgen secreted by the theca cells and the most important precursor of dihydrotestosterone and testosterone. Androstenedione diffuse across the basal lamina into the granulosa cells where it can be converted into the primary female sex hormone 17β-estradiol by the enzyme aromatase (CYP19) under the control of FSH23.
2. MicroRNAs: Pervasive post-transcriptional regulators
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2.1. MicroRNA biogenesis and mechanism of action MicroRNAs (miRNAs) are small, non-coding, single-stranded, endogenous regulatory RNA molecules consisting of ∼22 nucleotides. They modulate the expression of target genes on a post-transcriptional level by binding to the 3’untranslated region (3’UTR) of target mRNAs thereby inhibiting their translation and/or
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inducing their degradation24. More than 2800 miRNAs have been identified in Homo sapiens so far although the function of most of them remains to be determined25. The putative larger miRNA precursor transcripts can be found either within introns of both non-coding and coding transcripts or in exonic regions of the genome. Some miRNA genes possess their own promotor, others share a promotor with the host genes in which they reside. RNA polymerase II or III is responsible for the transcription of the miRNA into the initial
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long stem-loop structured primary miRNA (pri-miRNA). Within the nucleus, the pri-miRNA is cleaved by the enzyme Drosha yielding a shorter (app. 70 nt) hairpin-structured pre-miRNA which can be exported to the cytoplasm though the exportin5 complex for further maturation. Subsequently, the enzyme Dicer cleaves
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the pre-miRNA to form a double-stranded RNA duplex containing the mature miRNA and a passenger stand. The mature miRNA is loaded onto the RNA-induced silencing complex (RISC) by Argonaute (Ago) proteins
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whereby miRNAs can exert its actions on target mRNAs24 (Figure 2).
Figure 2: The canonical miRNA biogenesis pathway. MiRNA genes are located either in introns, are intergenic or polycistronic. The primary miRNA (Pri-miRNA) is transcribed by polymerase II (or polymerase III) and is cleaved by the microprocessor complex Drosha-Pasha (DGCR8) in the nucleus to generate pre-cursor miRNA (pre-miRNA). Pre-miRNA is transported to the cytoplasm by Exportin 5, it is further cleaved to its mature length (~22 nt) by the RNase III enzyme Dicer
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in complex with the double-stranded RNA-binding protein TRBP. Argonaute proteins (AGO2-4) unwind the miRNA duplex and facilitate incorporation of the guide strand into the RNA-induced silencing complex (RISC). AGO then guides the RISC miRNA assembly to target mRNAs through cleavage, translational repression and translocation to P-bodies, whereas the
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passenger strand is degraded.
3. MicroRNAs controlled by androgens or altered in PCOS 3.1. MicroRNAs found dysregulated in PCOS follicular fluid
Ovarian follicular fluid, comprised of blood plasma components, hormones and ovarian cell secretions,
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serves as an important environment for the development and maturation of the oocyte26. Recent studies indicate that dysregulated miRNAs may play a part in ovulatory dysfunction observed in PCOS. Analysis of
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microRNAs isolated from follicular fluid from PCOS women has identified several differently expressed miRNAs27,28. MiRNAs are both present in the supernatant and in microvesicles of the follicular fluid27. Among the up-regulated miRNAs was miRNA-9, - 18b, -32, -34c and -135a. Based on Target gene analysis, possible target genes related to the PCOS phenotype, including roles in carbohydrate metabolism, beta-cellfunction and steroid synthesis28, were identified. Another study found miR-132 and miR-320 down-regulated in PCOS27 compared to healthy controls. The two studies used different PCOS diagnostic criteria possible accounting for the discrepancies. Further, the finding of down-regulated miR-320, contrasts the results from
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a study by Yin et al.29, which demonstrated that miR-320 was upregulated in follicle fluid and granulosa cells from women with PCOS compared to controls. The study also reported hyperandrogenemia and downregulation of E2F1/SF-1 proteins, which inhibits estradiol release in the follicular fluid and GCs of PCOS patients. They thereby hypothesized that the high levels of miR-320 could be the partial reason for hyperandrogenemia in PCOS patients. However, it has not been possible to consistently reproduce these
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findings in other studies (Table 1).
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It is known that advanced maternal age has been associated with an altered follicular fluid composition30 and age-associated miRNAs have been isolated from follicular fluid from women undergoing IVF treatments31. While miRNA-21 was present in the follicle fluid from younger women, three different miRNAs were upregulated in older women (miR 99b-3p, miR-134 and mir-109b). Whether a similar pattern for these miRNAs could be found in women with PCOS, has at present time not been investigated, however miR-29a, miR-132 and miR-193b levels were negatively associated with age in another study32. In conclusion, comparing the limited number of studies of miRNA in follicle fluid, the miRNA profile varies. This could be due to different methods, age of the women or inclusion criteria, but could also illustrate the heterogeneous nature of PCOS.
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3.2. Circulating serum microRNAs associated with PCOS and/or hyperandrogenism Definition of PCOS as a syndrome includes a large group of women with different clinical manifestations. To date no single criterion is sufficient for the diagnosis. As circulating in serum, miRNA could serve as biomarker. A number of MiRNAs have been reported to be differently expressed in serum of women with
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PCOS compared to women without PCOS. One study reported miR-93 and miR-223 significantly upregulated in women with PCOS33. A target gene analysis revealed peroxisome proliferator receptor (PPAR) activity as a pathway possibly regulated by miR-223. PPAR has been shown to be important in insulin resistance and hyperandrogenism, though this study did not show any correlation between miR-223 and testosterone levels or insulin resistance33,34. Long et al. (2014)35 found significantly increased levels of
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miR-30c, miR-222 and miR-146a, whereas others (miR-16, miR-19a, miR-24, miR-106b and miR-186) were borderline significantly increased (P-values: 0.058-0.086). MiR-320 were near significant decreased (p=0.078), in line with the findings by Sang et al. (2013)27. Long et al. further found strong and positive
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correlation between miR-222 and serum insulin levels, whereas miR-146a was negatively correlated with serum testosterone. A third study, reported another five miRNAs significantly upregulated: let-7i3pm, miR5706, miR-4463, miR-3665 and miR-638 while four miRNAs were downregulated miR-124-3p, miR-128, miR-29a-3p and let-7c36. These results are not in line with previous data, and it is possible that the discrepancy could be caused by the size of the verification cohort (n=9), whereas other studies included 2568 women with PCOS.
Though the contradictory results regarding the miRNA profile of PCOS, many of the differently expressed
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miRNAs are involved in steroid metabolism. Research have shown that, not only excess androgens, but also other imbalances in steroid metabolism affect the follicle development and ovulation and these imbalances might be regulated by microRNAs.
Androgens (testosterone and dihydrotestosterone) regulate follicular atresia by inducing the expression of
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miR-125b in both mouse granulose, human KGN granulosa tumor cells and human granulosa cells from women undergoing in vitro fertilization37. MiR-125b targets pro-apoptotic proteins which suppress follicular atresia and thereby promotes follicle development. This could indicate that a critical balance exists between
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the essentiality of androgens in normal follicular fluid and the adverse effects of the androgen excess in women with PCOS.
Functional studies also support the regulatory role of miRNA in steroid metabolism: 80 different miRNAs were tested on human granulosa cells and release of progesterone, testosterone and estradiol was assessed. Numerous miRNAs reduced progesterone, testosterone and estradiol, but only miR-107 was found to increase the release of testosterone38. This specific miRNA was also found significantly upregulated in granulosa cells of women with PCOS39. Further, it is well-known that sulfation of steroid hormones can inactive these. Altered sulfate metabolism, in part by actions of miRNAs could, hypothetically, be associated with and contribute to the pathogenesis of
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PCOS. Indeed, mutations in PAPSS2 cause androgen excess and may contribute to PCOS features40. Inactivation of xenobiotics through sulfation increases water-solubility, hence facilitating their excretion, while sulfation of steroid hormones is also inactivating. The donor of the sulfate group can be 3’phosphoadenosine 5’-phosphosulfate (PAPS), the sulfur-containing amino acids L-methionine (L-Met) and
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L-cysteine (L-Cys) or macromolecules that undergo normal degradation by sulfatases within lysosomes23. Sulfation occurs in the cytosol or in the membranes of the Golgi apparatus of the cell. Sulfate homeostasis may by altered by age, time of the day, a woman’s gestational period and certain types of diseases which consequently may lead to a defective sulfation of substrates.
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3.3. A possible miRNA network with targets in the ovarian steroid metabolism
Ovarian steroid metabolism is regulated at the cellular level in that the granulosa cells lining the follicle produce estrogens derived from androgens produced by the more peripherally located theca-cells. The
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enzymes responsible for catalysing distinct reactions in the pathways leading to androgen and estrogen production are therefore also in many cases restricted to either theca or granulosa cells (listed in Table 2, with their most important roles). Also genes not directly involved in steroid hormone synthesis, but rather with roles in lipid transport or cellular signalling have been shown to be important for controlling ovarian androgen and estrogen metabolism23. Variants near or in the DENND1A gene has been reproducibly associated with the PCOS syndrome in genome-wide association studies41,42. The actual cellular function of the protein encoded by DENND1A is not well characterized, although over-expression of a variant
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(DENND1A V2) in vitro could recapitulate cardinal features of hyperandrogism in human theca-cells43. In order to identify miRNAs with a possible involvement in ovarian androgen production, we performed searches for miRNAs targeting selected genes involved in androgen synthesis or metabolism of ovarian steroidogenic cells (Table 2). The updated TargetScanHuman database44 was used to extract all miRNAs
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conserved in mammals for the particular genes (Table 2) and miRNAs with three or more targets and found in follicular fluid32 and in human granulosa cells45 were listed suggesting a network of miRNAs targeting ovarian steroid hormone metabolism (Table 3). No studies of human theca cell miRNA expression was
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identified in the published literature or in online data repositories. Reassuringly, of the identified miRNAs possibly targeting transcripts involved in androgen synthesis, miR-24 and miR-151 were reported to be suppressed in follicular fluid from PCOS patients. Moreover, miR-151 levels correlated with serum free testosterone32, while miR-24 and miR-19 is reported to reduce testosterone release of cultured human ovarian cells38. Moreover, estradiol release was decreased in culture human ovarian cells transfected with miR-24, miR-150 and miR-151, further emphasizing that these miRNAs may be important for ovarian function. However, it is not yet clear, what the physiological and pathophysiological regulation of these miRNAs is or what their combined effect is.
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It will be interesting to investigate the impact of each of these miRNAs on the regulation of the individual predicted transcripts in this network. Moreover, since at least some of the miRNAs in the network appear to be altered in PCOS patient follicular fluid, it is also relevant to examine the extent to which altered levels of these miRNAs are reflected in changed gene-expression in PCOS patient granulosa cells. There is firm
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evidence that CYP19A1 (aromatase) activity as well as mRNA levels are decreased in PCOS patients, which is associated with the decreased size of follicles46,47 and CYP19A1 mRNA is a predicted target of miR-19,
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miR-199 and miR-186, however it is not known if these miRNAs are increased in PCOS patient samples.
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Table 2. Selected genes important for ovarian production of steroid hormone metabolites
Steroidogenic cells Steroidogenic cells Steroidogenic cells Steroidogenic cells Steroidogenic cells
SULT2A1
Steroidogenic cells
Fdx1 Ferredoxin FdxR Fdx reductase NR5A1/SF1
Steroidogenic cells Steroidogenic cells Steroidogenic cells
AR
Steroidogenic cells
CYP17A1/P450c17
High in Theca, low in granulosa cells High in Theca, low in granulosa cells High in theca cells, low in granulosa cells Theca cells
HSD3B2/3βHSD2 DENND1A V2
Granulosa cells Granulosa cells
Granulosa cells
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HSD17B1/17βHSD1
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HSD17B5/AKR1C3 HSD17B6/3αHSD CYP19A1/P450aro/ Aromatase
Entry point for steroidogenesis Cholesterol -> Pregnenolone Generates activated sulfate groups Generates activated sulfate groups Electron donor co-factor Electron donor co-factor Mitochondrial cholesterol transfer protein, promotes androgen synthesis Sulfates DHEA to DHEAS, decrease testosterone synthesis CYP450 enzyme co-factor CYP450 enzyme co-factor Transcriptional regulator of enzymes involved in steroid synthesis Androgen receptor, mediates cellular responses to androgens 17OH-Pregnenolone -> DHEA 17OH-Progesterone -> Androstenedione Androstenediol -> Testosterone
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PAPSS1/ PAPS synthase 1 PAPSS2/ PAPS synthase 2 POR/P450 oxidoreductase CYB5B/CYB5B StAR/StAR
Role in androgen metabolism
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CYP11A1/P450scc
Ovarian expression Cell type and level Steroidogenic cells
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Gene /protein abbreviation
Cellular growth factor signaling
Androgen synthesis Androstenedione -> Testosterone Backdoor to DHT, 5alfa reductase Generates Estrone in GC by FSH stimulation Androstenedione -> Estrone Testosterone -> Estradiol Generates estradiol in GC by FSH stimulation Estrone -> Estradiol
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Abbreviations: GC granulosa cell, CYP Cytochrome P450, scc side chain cleavage, DHT dihydrotestosterone, FSH follicle stimulating hormone, Fdx Ferredoxin, FdR Ferredoxin reductase, AR androgen receptor, SF steroidogenic factor, SULT sulfotransferase, DENND DENN/MADD Domain Containing, StAR Steroidogenic Acute Regulatory Protein, CYB5B Cytochrome B5 Type B, PAPS 3'-Phosphoadenosine 5'-Phosphosulfate, HSD Hydroxy steroid dehydrogenase , AKR Aldo-Keto Reductase Family 14,23,43,48.
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Table 3. A possible miRNA network targeting ovarian steroid metabolism
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miRNA miR-302 family/ /372 Genesymbol /373 miR-199 /520 miR-150 miR-324 miR-151 miR-24 miR-19 miR-212 /3129 miR-186 DENND1A x x x x x x StAR x x x x x CYB5B x x x x x x FDX1 x x x x FDXR x x Sult2A1 x x x x SF1 x PAPSS2 x x x CYP19A1 x x x AR x x x x HSD17B6 x MiR-302, -372, -373, -520, -151, -24, -19 and -199: The mature miRNA species belongs to the 3’ arm of the precursor. MiR150, -324,-212,-3129 and -186: The mature miRNA species belongs to the 5’ arm of the precursor. Common miRNAs putatively targeting selected mRNAs encoding proteins important for ovarian steroidogenesis were identified in TargetScanHuman, release 7.0, using searches for miRNAs conserved in mammals targeting each of the genes listed in Table 2. MiRNAs in common between 3 or more genes were identified using Venn diagrams.
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3.4. Can circulating or follicular microRNAs be used clinically for PCOS diagnosis or prognosis of fertility treatment? PCOS is a syndrome, which encompasses a broad spectrum of signs and symptoms of ovarian dysfunction. As a syndrome, there is no single diagnostic criterion sufficient for the clinical diagnosis. The clinical signs
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of PCOS can vary over time, which complicates the task of diagnosing PCOS in a clinical setting. Further, women with PCOS are in greater risk of developing type 2 diabetes and cardiovascular diseases10,49, which underline the need for a correct and reliable diagnosis. Circulating miRNAs could serve as biomarkers as they are easily accessible. MiRNAs in follicular fluid could also serve as a marker during fertility treatment. Unfortunately, of the presently published studies of miRNAs in serum of women with PCOS a common
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conclusion is that it is possible to distinguish between controls and women with PCOS, however, the three studies report different results33,35,36. Long et al (2014)35 found as mentioned, miR-30c, miR-222 and miR146 upregulated and assessed the value of the miRNAs’ ability to discriminate women with PCOS from
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healthy controls by establishing receiver operator characteristics (ROC-curves). The combination of the three miRNAs yielded the largest area under the ROC-curve, and was suggested as diagnostic markers. Another study (by Sathyapalan et al (2015))33 concluded, also based on ROC-curve analysis, that miR-93 could serve as biomarker supporting a positive clinical diagnosis of PCOS. A combination of miRNAs did not improve the diagnostic potential compared to miR-93 alone in this study. The third study did not assess the value of their results as diagnostic markers 36, however, there was no overlap in the reported differentially expressed miRNAs of this study with the other two published studies. The reason for the different results obtained in
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different studies is not known, but may be due to ethnic differences in studied populations, different inclusion criteria, age, body weight of study participants, as well as different assay methods. Clearly, there is a need for larger, well controlled studies examining miRNA profiles in serum of PCOS patients and control subjects in order to gain a consensus. MiRNAs in follicular fluid are also suggested as biomarkers and
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similar to reported studies investigating serum miRNA levels there is great discrepancy between studies, which is likely due to inconsistencies in methods, inclusion criteria and probably different ethnicity and
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heterogeneity of the patients.
4. Conclusions and perspectives Many different miRNA species have been implicated in the pathophysiology of PCOS and regulation of ovarian steroid hormone metabolism. However, currently there is limited knowledge of the impact of single or combined sets of co-regulated miRNAs on ovarian androgen and estrogen metabolism and how these may contribute to PCOS.
A large number of miRNAs have been associated with altered levels in serum or follicular fluid from PCOS patients compared with fertile control patients. Whether these sets of miRNA could serve as biomarkers for 14
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guidance of treatment or has prognostic value is currently not established, but as more studies are published it is expected that at least a proportion of the reported miRNAs can be validated. Extracellular miRNAs are localized in exosomes, which markedly stabilize them and provide protection from extracellular nucleases. Thus, miRNAs as a molecular class offers specific advantages as possible biomarkers. However, in order to
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make full use of the potential of miRNAs as biomarkers, more studies with carefully controlled design and sample preparation are necessary. PCOS being a clinically heterogeneous disorder is likely to have heterogeneous etiology. Thus, it is possible that miRNAs could be used to identify subtypes of PCOS patients, for which different optimal treatment options exist, leading to personalized treatment
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With contrasting studies being published regarding the expression and regulatory role of miRNAs in ovarian function as well as levels of miRNA in serum and follicular fluid, it is imperative that large replication studies are performed to identify the specific miRNAs with robust roles and regulation. Because miRNAs
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often exist in families, an increasingly important approach will be to modulate levels of entire miRNA families or co-regulated miRNAs together as well as separately in order to establish their roles in the intact tissue or organ. As it is known that many miRNAs act most strongly at the level of translational inhibition, investigations of mRNA expression arrays may have low power to reveal the true impact of regulated microRNAs on the PCOS phenotype. It would be ideal to have proteomic data from granulosa and theca cells of PCOS patients versus control subjects, but currently no such studies have been published.
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Moreover, since many miRNAs in general appear to have roles in stress response modulation, whereas they may be dispensable in the normal physiological situation it is important to investigate the proper models of metabolic, inflammatory or hormonal stress in order to elucidate their natural functions. An important note is that ovarian function and regulation is highly species specific, and several human specific miRNAs have
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been described45 that appear to be granulosa cell specific. Thus, it is possible that more, yet undescribed, miRNAs are expressed in specific cell types of the ovary. It is likely that more miRNAs with roles in ovarian hormonal metabolism and impact on development of PCOS exist, but are still to be identified and
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characterized.
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Aziz, M.; Berthelsen, P.; Christiansen, H.; Clausen, H.; Dahl, K.; Engelbrechtsen, L.; Houmann, I.; Jónsdóttir, F.; Ervin, K.; Kjær Mandrup, M.; et al. Polycystisk Ovariesyndrom (PCOS); 2015. https://static1.squarespace.com/static/5467abcce4b056d72594db79/t/561cb676e4b09f227 7ba139d/1444722294970/PCOS_rev2015_finalrev[1].pdf
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Burghen, G. A.; Givens, J. R.; Kitabchi, A. E. Correlation of Hyperandrogenism with Hyperinsulinism in Polycystic Ovarian Disease. J. Clin. Endocrinol. Metab. 1980, 50, 113–116.
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Legro, R. S.; Kunselman, a R.; Dodson, W. C.; Dunaif, a. Prevalence and Predictors of Risk for Type 2 Diabetes Mellitus and Impaired Glucose Tolerance in Polycystic Ovary Syndrome: A Prospective, Controlled Study in 254 Affected Women. J. Clin. Endocrinol. Metab. 1999, 84, 165– 169.
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Table 1. List of microRNAs observed in polycystic ovary syndrome (PCOS) and involved in androgen metabolism or function
miR-9
Detected in Tissue/Cell Follicular fluid Granulosa cells
miR-18b
Follicular fluid Granulosa cells
Human
miR-21
Follicular fluid Granulosa cells
Human Mouse Rat
miR-24
Follicular fluid
Human
miR-27b
Whole blood
Human
miR-29a
Follicular fluid
Human
Promotes progesterone release Inhibits testosterone and estradiol release Anti-apoptotic. Increased after FSH exposure; inconclusive testosterone response Negatively correlated with serum estradiol Positively correlated with testosterone Positively correlated with DHEAS
miR-30c
Serum Granulosa cells
Human Rat
Increased after FSH exposure
miR-103
Whole blood Granulosa cells
Human
miR-107
Granulosa Cells
Human
miR-132
Follicular fluid Granulosa cells
Human Mouse Rat
Follicular fluid Granulosa cells Serum Plasma Follicular fluid Granulosa cells
Human
miR-146a miR-151
Follicular fluid
miR-155
Serum Granulosa cells Serum Follicular fluid
miR-222
Observation in PCOS
Human
Inhibits testosterone release
Increased in PCOS
28,38,50
28,38,50
Increased in PCOS
31,38,51–53
Decreased in PCOS
32
Increased in PCOS.
52
SC
Increased in PCOS
Decreased in PCOS
32
Increased in PCOS
35,53
Increased in PCOS
38,52
Increased in PCOS
38,39
Decreased in PCOS
27,38,50,54
Increased in PCOS
28,38,50
Reduces progesterone, estradiol and testosterone release
Increased in PCOS
27,35,38
Human
Negatively correlated with serum total and free testosterone
Decreased in PCOS
32
Human
Inhibits testosterone release
Increased in PCOS
38,50,52
Human
Increases estradiol secretion
Increased in PCOS
27,35,55,56
Human Mouse
Induces GCs proliferation Increases estrogen release
Inconsistent effects on estradiol secretion. Increases testosterone Negatively correlated with FSH in PCOS patients
Differentially expressed in follicular fluid Decreased serum expression in PCOS. Inconsistent expression in follicular fluid in PCOS; Increased expression granulosa cells in PCOS
Human
Follicular fluid Granulosa cells
miR-320
Serum Follicular fluid Granulosa cells
Human
miR-383
Follicular fluid Granulosa cells
Human
miR-518
Follicular fluid
Human
EP
miR-224
AC C
Reference s
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Reported Function(s)
Promotes progesterone release Inhibits estradiol release Positively correlated with testosterone Increases testosterone release Increases estradiol secretion Reduces progesterone and testosterone release Reduces progesterone and testosterone release
M AN U
miR-135a
Species
TE D
microRNA
Enhances the release of estradiol from GCs by CYP19A1 Positively correlated with total and free testosterone and androstenedione in PCOS patients
28,57,58
27,29,32,35,59
Increased in PCOS
28,29,60
Increased in hyperandrogenic PCOS
32
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Highlights for: MicroRNAs Related to Androgen Metabolism and Polycystic Ovary Syndrome
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Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of the reproductive age PCOS is clinically diverse presenting with infertility, hyperandrogenemia and the metabolic syndrome A network of microRNAs may target mRNAs involved in steroid hormone synthesis or metabolism Circulating or follicular microRNAs could be used as biomarkers for patient stratification Specific microRNAs may be involved in disease pathology, however more research is needed
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