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Understanding  the  effects  of  steroid  hormone  exposure  on  direct  gene  regulation  

 T.S.  Wiley1  and  J.T.  Haraldsen2  

 1Wiley Compounding Systems, Santa Fe, New Mexico, 87504, USA

2Department of Physics and Astronomy, James Madison University, Harrisonburg, Virginia, 22802, USA Steroid  hormones  have  been  widely  overlooked  as  controllers  of  gene  expression.  Through  the  mechanisms  of  gene  expression  (DNA  methylation,  histone  methylation,  and  RNAi),  we  discuss   the   impact   of   normal   reproductive   templates   on   the   pulsatility   and   amplitude   of  potential   gene-­‐regulating   treatment   protocols.   By   examining   the   interactions   of   estradiol  (E2)  and  progesterone  (P4)  in  women,  we  propose  that  changes  in  physiologic  reproductive  hormone  templates  of  exposure  and  timing  can  affect  fertility  and  even  cancer  through  the  silencing  or  amplification  of  gene  products;  such  as  P53  and  Bcl-­‐2   in  women.  We  propose  that  uncontrolled  hormone  levels,  due  to  aging  and/or  the  environment,  may  be  restored  to  a   normal   youthful   template   of   gene   expression   through   the   fluctuating   exogenous  application  of  E2  and  P4  that  mimic  the  normal  hormonal  milieu  of  reproductive  health.  We  hypothesize  that  this  may  lead  to  a  lower  risk  of  the  chronic  illnesses  of  aging  and  a  better  quality  of  life  in  patients  suffering  those  conditions.    

Introduction Steroid hormones are critical to the natural development of multicellular organisms and complex cellular functions for all planets, animals, and insects (1-4). Through the use of receptors and gene products, steroid hormones constantly control these regulatory systems using iterative hormone interactions (5,6). Since many diseases can be traced back to the uncontrolled or silenced gene expression (7), understanding the mechanisms by which steroid hormones control or modify gene products is paramount for the medical community. This is evident in the well-known use of corticosteroids to reduce inflammation by silencing inflammatory gene cascades (8,9).

Since the expression of regulatory genes is critical for the sustainability of life through the production of amino acids, enzymes, and proteins (REF), if there is an error in how and when specific genes are expressed, steroid hormones can either produce or reduce the levels of functionality down stream of all gene products throughout the body (7,10). This leads to body-wide disarray in all the cells’ ability to regulate all gene expression, which has been observed in cancer patients as well as other instances of chronic illnesses in aging (11), such as heart disease, diabetes, and neuro-degenerative states (12-16).

Although gene expression is controlled through many different mechanisms in the body, the three most prominent and recognized mechanisms today are through DNA (deoxyribonucleic acid) methylation (17), histone methylation and acetylation (18), and RNA (ribonucleic acid) interference (RNAi) (19). These mechanisms are critical for maintenance of gene function during the replication and transcription processes (17-19), where they control gene expression at different levels:

• DNA methylation controls genes through a direct tagging of specific cytosine bases on the DNA strand with a methyl group (CH3) (17).

• Histone methylation and acetylation through chromatin action allows histones to reduce or increase gene expression by literally stopping the transcription process. This acts as a locking mechanism on the DNA strand (18).

• RNAi is a completely different mechanism that involves the literal cutting and splicing of individual small pocket DNA strands (sDNA) to turn genes on and off or insert new material (19).

While these mechanisms differ in their methods, we show that all three of these mechanisms have a striking commonality; they are controlled primarily through interactions with steroid hormones.

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Since steroid hormones (i.e. estradiol (E2), progesterone (P4), testosterone (T), cortisol (C)) control many gene products (20-23), it is important to examine how the loss or disarray of this hormone product can be manifested as illness. By understanding the potentials of gene regulation within a cell, we can discuss the general mechanisms by which changing steroid hormone levels may have a distinct effect on the expression and repression of genes, which can in turn lead to an increased risk of the diseases of aging.

In this paper, we examine the connection of hormone production and deterioration, from aging and/or environmental changes, to the gene regulation through the various methods (cited above) of gene silencing and amplification. Since steroid hormones control the cellular regulation of proliferation and apoptosis, it is apparent that

the disruption of these hormones can systematically lead to diseases like diabetes, Alzheimer’s, and cancer through an increase or decrease via the methylation of DNA, uncontrolled histone modifications, and/or effects on the transcription of RNAi. Through an examination of specific hormonal processes, we elucidate the effects of aging and environmental cues on the production E2 and P4 that may ultimately disrupt the regulation of actual gene products. Through a detailed examination of the known literature in the field, we hypothesize that the pulsatility (exposure frequency) and amplitude (varying amounts) of steroid hormones will affect the gene expression via the three known mechanisms of DNA methylation, histone methylation and acetylation, and RNA interference.

In this paper, it is our intent to show how steroid hormones may be used to control these mechanisms of regulation through changes in exogenous dosing and timing of administration. Below, we provide a description of the steroid hormones, an overview of the gene expression mechanisms, and a discussion of the implications of pulsatility and amplitude in younger female reproductive templates, which varies significantly from the current standard of care replacement therapy, static non-varying dosing of synthetic hormone-like pharmaceuticals. Background The expression or repression of genes may happen in a number of ways. Currently, the known mechanisms for gene regulation being studied are via DNA methylation, Histone methylation and acetylation, and RNAi splicing (17-19). As mentioned above, these mechanisms provide specific actions on the genes to regulate gene products by altering the DNA transcription processes. It has been long ignored that steroid hormones production is the response to the environment that has a direct affect on all three mechanisms. This makes steroid hormones likely candidates for study in genomics and epigenetics. Steroid Hormones Steroid hormones are involved in all genetic, cellular, and bodily processes (2-4). They are growth and reproduction catalysts in bone, brain, heart, and reproductive organs (24). The production of estrogen in the body of a young female is critical for the regulation of functional gene control (25). Since estrogen, as a steroid hormone, affects gene expression (26), it is important to understand the

Figure   1   -­‐   (a)   The   normal   estradiol   and   progesterone   cycles  throughout  a  women’s  menstrual   cycle   (30).  The  day  12  peak  in  estradiol  correlates  to   increase   in  Bcl-­‐2  and  a  decrease  P53  gene   products,   while   the   day   21   peak   in   progesterone  correlates   to   a   decrease   in   Bcl-­‐2   and   an   increase   P53.   These  signal   the   proliferation   and   apoptosis   of   cells   throughout   the  cycle.  (b)  The  E2  and  P4  dosing  template  of  the  Wiley  Protocol  proposed   to   mimic   the   normal   cycle   using   bio-­‐identical  hormone   replacement   therapy   through   transdermal  application.

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possible methods by which steroid hormones act on these mechanisms.

Steroids travel through the blood using lipid carriers. This is critical for protein production within the cell that will, in turn, create hormone receptors for various other steroids (27). Once in a membrane receptor, the steroid will detach from the carrier and translocate through the cell membrane, where it can either directly access the nuclear promoter regions or bind to a non-nuclear receptor. Once the hormone-receptor complex has phosphorylated down to the nucleus (28), it will bind to those appropriate specific hormone response elements and promoter regions of the DNA strand. The interaction between the steroid hormone-receptor complex and binding sites causes the DNA signals to for messenger RNA (mRNA) transcription, which results in protein synthesis outside the nucleus, often other steroid and/or hormone receptors (29). All cellular and bodily functions are affected by the production of these proteins, which means that the conscious well being of a host is highly dictated by the amplitude and frequency of steroid hormones. The body is accustomed to a regular schedule for steroid hormone exposure (26). This is clearly evident in the estrogen and progesterone cycles of females across all species. The female menstrual cycle is a complex interaction of estrogen and progesterone through fluctuating repetitive patterns over 28 days (shown in Fig. 1(a)) (30,31). As a woman ages and her egg base declines, the normal patterns are disrupted and eroded leading to an erratic peri-menopausal state preceding menopause. In this state, literature verifies there are significant increases a woman’s risk of heart disease, diabetes, reproductive cancers, and osteoporosis (26). It is the ripening of eggs that creates the standard estradiol cycle in the reproductive template, which produces a large peak of estrogen at approximately the 12th days of a woman’s cycle (31,32). This surge of estrogen provokes a progesterone receptor from the nucleus of the cell as well as spikes the luteinizing hormone in the brain to instruct the ovary’s release of the egg. It is the ensuing degradation of the ruptured corpus luteum that begins the progesterone cycle (P4) at this time. P4 peaks on day 21 of this menstrual cycle. Should fertilization occur between days 19-22, the women will continue to experience even higher levels of progesterone in the now established pregnancy. However, if fertilization does not occur, apoptosis begins and the hormonal cues instruct the shedding of the uterine lining. This action of P4 blocks E2 receptors to assure repopulation on day 29/1 of the new cycle to restart the template.

To understand how the time and amount of hormonal exposure affects the overall expression of genes, we move now to discuss the cited mechanisms that control gene expression and how steroid hormones, especially estradiol, ties into these mechanisms. DNA Methylation The methylation of DNA is a process in which a methyl group (CH3) bonds to the 5 position of cytosine (C) only (33). This process is critical for the development of organisms due to its control over cellular differentiation, and its ability to alter gene expression over evolution.

The methylation occurs at cytosine sites that are connected to guanine (G) sites through the phosphate backbone, denoted as CpG (34), in the linear sequence of DNA bases. This is different than the hydrogen bonding that occurs between DNA base pairs (C-G and adenine (A) –thymine (T)). Methylation of non-CpG bases can occur, but has only been found, interestingly, in stem cells. The process of DNA methyltransferases (DNMT1 and DNMT3) transferring methyl groups to DNA using S-adenosyl methionine as a donor (illustrated in Figure 2), which is mitigated through the interactions of steroid hormones (17).

The percentage of methylated DNA in the body is known to vary more in the first stages of life, but seems to

= CpG

= CH3

Promoter Gene Anti-Oncogene

Norm

al C

ancerous

= S-adenosyl methionine

Figure  2  –  General  DNA  Methylation  mechanism.  The  process  of  DNA  methyltransferases  (DNMT1  and  DNMT3)  transferring  methyl  groups  to  DNA  using  S-­‐adenosyl  methionine  as  a  donor,  which   is   mitigated   through   the   interactions   of   steroid  hormones.      

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stabilizes quickly (34). Once stabilized, enzymes move along DNA and maintain the level of methylated cytosine sites throughout reproductive life. This is evident through epigenetic silencing and amplification of various genes (35,36).

Over the past decade, it has been discovered that hypermethylation (increased methylation) or hypomethylation (decreased methylation) can occur, specifically in the later stages of life when hormone production and interactions decline in both men (testosterone) and women (estrogen). Hypomethylation has been linked to the over-expression of oncogenes in cancer cells. However, in the literature, the exact controlling mechanism still remains unclear.

For example, in a 2001 paper by Hartsough et al (37), a clear link between DNA methylation and the proliferation of cancer cells was discovered. This is an indication of the distinct importance of the need to understanding these mechanisms. For breast cancer patients, the loss of steroid hormones is critical in the silencing of crucial gene products (38,39). The fact that the use of synthetic hormones does not alleviate this silencing factor is a note of great importance (40,41). The question must be asked, do synthetic hormones affect these mechanisms of regulation in any normal way? Histones Histones are proteins that affect the compaction and contraction of DNA using tightly wound bundles called nucleosomes. Histones, through chromatin, control the winding and unwinding of DNA for transcription controlling the expression of the gene (18). When DNA needs to be transcribed, the histones unravel, allowing the DNA transcription process to occur. In this way, histones play a critical role in gene regulation and expression.

Steroid hormones can control gene activation through methylation of the histone (similar to DNA methylation) or through acetylation of the histone in which an acetyl group (CH3O) is attached (illustrated in Figure 3). Therefore, the way steroid hormones control histone action is through methyl and acetyl group (18). The methylation and acetylation processes can stop the histone’s ability to unwind the DNA for transcription, which in turn stops gene expression. This steroid action effectively silences the histone itself and locks the DNA from transcription. When DNA wraps around a histone, that section of DNA becomes inaccessible and the gene is no longer active. Often, histone regulation is controlled through interactions with peptides hormones as well. However,

since histone methylation is dependent on interactions with methyltransferases (similar to DNA methylation), steroid hormone interactions are critical (42). RNA Interference (RNAi) RNA interference (RNAi) is the inactivation of genes during the transcription process (19,43,44). This occurs when microRNA (miRNA) and small interfering RNA (siRNA) bind to sections of messenger RNA (mRNA) and stops the production of the gene products, proteins and enzymes. The siRNA, which is can be as small as 22 nucleotides, are cut from longer double stranded RNA (dsRNA) by endoribonuclease enzymes called Dicers.

RNA silencing is a critical process in eukaryotic cells (plants, animals, and fungi)(45,46), because it can easily stop or enhance the production of vital gene products for functional or even evolutionary purposes. The silencing of gene products through RNAi is critical for an organism’s protection against viral infections or even cancer. The under or over expression of various gene products creates an instability in the cell. For more information on RNAi mechanisms, please refer to reference 47.

RNAi while capable of silencing and voicing genes is also (48,49), interestingly, instrumental in creating steroid production as well as, which, in turn, these

Figure   3   –   Histones   methylation   and   the   activation   of   DNA.  Histones  methylate  through  histone  methyltransferases  using  the   S-­‐adenosyl   methionine   as   a   donor,   which   places   methyl  groups   on   the  H3   and  H4  histones.  Acetylation   typically  uses  an  interaction  with  acetyl-­‐coenzyme  A  (18).

= CH3

= CH3O

Methylated)Histone)

Acetylated)Histone)

= S-adenosyl methionine

= Acetyl-Coenzyme A

Ac1ve)DNA)

Inac1ve)DNA)

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steroids may methylate by histones and regulate gene protein production in endless cascades. Hypothesis It is our hypothesis that gene expression is controlled, not just by the shear presence of steroid hormones in the blood, but by the frequency and amplitude of hormone production within the body. Steroid hormones are known to control the cell’s regulatory systems through modification of gene expression. It is also well known that all steroid hormones follow consistent rhythmic patterns of production driven by environmental cues. When these templates are disrupted by aging, pharmaceutical, or environmental interference, they may shutdown or reverse gene expression. We further hypothesize that this may lead to the diseases of cancer, heart disease, diabetes, or osteoporosis in aging. Below, we elucidate by example some of these effects of deregulated hormones, specifically in women (estradiol and progesterone). We also propose a basic genomic experiment that will confirm how the pulsatility and amplitude of hormone dosing can affect the activation of specific regulatory gene products such as BCL-2 and P53.

It is our belief that the deviation from the normal rhythmic template of exposure to steroid hormones in men and women can cause a regulatory chaos on the cellular level via the 3 mechanisms outlined above, which manifests itself in many ways including breast cancer and other peri-menopausal symptoms. Because of the nature of steroid hormones, static dosing of synthetic hormones-like drugs (the standard of care in Hormone Replacement Therapy) does not take into account natural rhythmic production, which provide cells with the proper signaling to produce gene products necessary for homeostasis.

Discussion: Genomic consequences of eroded estrogen cycles Since steroid hormones are critical to gene regulation, we focused on just estrogen as an example of the major consequences of eroded hormone cycles. The menstrual cycle in women produces greatly varying levels (amplitudes) of hormones (specifically estrogen and progesterone) in a pulsating fashion over 28 days. These hormones affect gene products through methylation of DNA or histones or through gene silencing via RNAi (Figures 2 and 3). Since the onset of peri-menopause signals wild fluctuations in estrogen production, it is reasonable to assume that the ensuing control of gene

expression also deeply affected adversely (shown in Fig. 4). The normal cycles of estrogen and progesterone manifest predictable regulatory gene expression through the repression or expression of genes like P53 and Bcl-2 and their gene products, which are timed throughout the cycle as well as initiating cascades of other functional gene products. As shown in figure 1(a), the normal cycle consists of an estrogen peak on day 12. This produces an increase in Bcl-2 (!) until day 12 and a decrease in P53 ("). On day 21, the progesterone peaks and commands the cells to begin apoptosis and signals a decrease in Bcl-2 (") by blocking E2 and an increase in P53 (!). This allows for regulation and control of the increase and decrease of cells all over the body.

The hormone cycle signals cells to proliferate and when apoptosis should occur for normal functioning. As a woman enters peri-menopause, these signals can be interrupted due to the body’s inability to produce normal estrogen and progesterone peaks. Therefore, the body’s clinical response is one of chaos, which produces hot flashes, mood swings, irritability, and loss of libido. However, physically the signals regulating proliferation and apoptosis have also deteriorated, which can lead to uncontrolled cell growth known as cancer or osteoporosis. Studies show that static replacement of hormones is unsuccessful in the long term in helping women with symptoms as well any amelioration of the disease process in aging. These studies have also shown that static dosing can accurately increase cardiac risk. Therefore, we conjecture that by following the body’s normal and

Chi

ldho

od

Pube

rty Peri-

menopause

Menopause

Reproductive Years

Estrogen Levels Relative Cancer Rate

0 60 50 40 12 13 Age (years)

Figure  4  –  An  illustration  of  the  estrogen  in  urine  and  relative  cancer  rate  as  a  function  of  age  [52].  As  age  increases  and  a  woman’s  estrogen  level  begins  to  decrease,  her  relative  cancer  risk  begins  to  increase  as  gene  expression  becomes  irregular.

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evolved rhythmic production template with bio-mimetic, bio-identical therapy, the body may regain the normal expression of gene products that may defend against cell abnormalities. We refer to the only bio-mimetic, bio-identical Hormone Replacement Therapy available (The Wiley Protocol®) (50), because it is such a regimen that takes these normal templates of reproductive fitness into account. The reestablishment of this well-established template of hormone production should control gene expression as well. Shown in figure 1(b), the Wiley Protocol® provides women with transdermal hormone therapy that consists of estrogen and progesterone provided topically. Recent unpublished studies of this regimen have demonstrated that women have an increase in sleep, energy, bone growth, libido, and overall quality of life with no increase in cancer incidence (51). Further clinical investigation is warranted. Proposed Clinical Experiment To examine how hormone regulation will control gene expression, we intend to examination how of the activation of gene products for various groups of women (young, peri-menopausal, post-menopausal) on different degrees of hormone replacement therapies (static, bio-mimetic, and none). The examination of various groups of women divided into youth, post-menopausal, and Wiley Protocol® will be subject to blood spot gene identification on days 12 and 21 of the normal cycle for young and Wiley Protocol® women. Post-menopausal women will follow chosen cycle days 12 and 21, respectively. Using the blood spot gene identification, we can examine the expression of P53 and BCL-2 for comparison to the expression in normally reproductive subjects. In particular, we expect that one should specifically look at the expression and repression of P53 and BCL-2. Conclusion Overall, we propose a hypothesis that steroid hormones affect gene expression through changes in both amplitude and frequency of dosing or exposure. A key example of this would be through the changing of hormones in women through menopause. Advances in hormone replacement therapy provide a wide range of testable parameters for this claim. We suggest the investigation of the expression of P53 and Bcl-2 in both peri- and post-menopausal women.

   

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References    

1. Geuns JMC. Steroid hormones and plant growth and development. Phytochemistry. 1978;17(1):1–14.

2. Beyer C. Estrogen and the developing mammalian brain. Anat Embryol. 1999;199(5):379–90.

3. Harris GW. Hormonal differentiation of the developing central nervous system with respect to patterns of endocrine function. 1970; 259:165-177.

4. Dubrovsky EB. Hormonal cross talk in insect development. Trends in Endocrinology & Metabolism. 2005 Jan;16(1):6–11.

5. Formby B, Wiley TS. Progesterone inhibits growth and induces apoptosis in breast cancer cells: inverse effects on Bcl-2 and p53. Ann Clin Lab Sci. 1998 Nov 1;28(6):360–9.

6. Evans RM. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–95.

7. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al. The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease. Science. 2006 Sep 29;313(5795):1929–35.

8. Stellato C. Post-transcriptional and Nongenomic effects of glucocorticoids. 2004;1:255-263.

9. Barnes PJ. How corticosteroids control inflammation: Quintiles Prize Lecture 2005. Br J Pharmacol. 2006 Jun;148(3):245–54.

10. Ringold GM. Steroid Hormone Regulation of Gene Expression. Annual Review of Pharmacology and Toxicology. 1985;25(1):529–66.

11. Münzer T, Harman SM, Sorkin JD, Blackman MR. Growth Hormone and Sex Steroid Effects on Serum Glucose, Insulin, and Lipid Concentrations in Healthy Older Women and Men. The Journal of Clinical Endocrinology & Metabolism. 2009 Oct;94(10):3833–41.

12. Hansen M, Hsu A-L, Dillin A, Kenyon C. New Genes Tied to Endocrine, Metabolic, and Dietary Regulation of Lifespan from a Caenorhabditis elegans Genomic RNAi Screen. PLoS Genet. 2005 Jul 25;1(1):e17.

13. Jiang N, Du G, Tobias E, Wood JG, Whitaker R, Neretti N, and Helfand SL. Dietary and genetic effects on age-related loss of gene silencing reveal epigenetic plasticity of chromatin repression during aging. Aging. 2013;5(11):813-824.

14. Iacopino AM, Christakos S. Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases. PNAS. 1990 Jun 1;87(11):4078–82.

15. Selvi RB, Kundu TK. Reversible acetylation of chromatin: Implication in regulation of gene expression, disease and therapeutics. Biotechnology Journal. 2009;4(3):375–90.

16. Rahman K. Studies on free radicals, antioxidants, and co-factors. Clin. Interv. Aging. 2007;2(2):219-236.

17. Das PM, Singal R. DNA Methylation and Cancer. JCO. 2004 Nov 15;22(22):4632–42.

18. Karlić R, Chung H-R, Lasserre J, Vlahoviček K, Vingron M. Histone modification levels are predictive for gene expression. PNAS. 2010 Feb 16;107(7):2926–31.

19. Hannon GJ. RNA interference. Nature. 2002 Jul 11;418(6894):244–51.

20. Ceschin DG, Walia M, Wenk SS, Duboé C, Gaudon C, Xiao Y, et al. Methylation specifies distinct estrogen-induced binding site repertoires of CBP to chromatin. Genes Dev. 2011 Jun 1;25(11):1132–46.

21. Liao X, Tang S, Thrasher JB, Griebling TL, Li B. Small-interfering RNA–induced androgen receptor silencing leads to apoptotic cell death in prostate cancer. Mol Cancer Ther. 2005 Apr 1;4(4):505–15.

22. Wabitsch M, Jensen PB, Blum WF, Christoffersen CT, Englaro P, Heinze E, et al. Insulin and Cortisol Promote Leptin Production in Cultured Human Fat Cells. Diabetes. 1996 Oct 1;45(10):1435–8.

23. Berger FG, Watson G. Androgen-Regulated Gene Expression. Annual Review of Physiology. 1989;51(1):51–65.

24. Lewis-Wambi JS, Jordan VC. Estrogen regulation of apoptosis: how can one hormone stimulate and inhibit? Breast Cancer Research. 2009 May 29;11(3):206.

25. Fata JE, Chaudhary V, Khokha R. Cellular Turnover in the Mammary Gland Is Correlated with Systemic Levels of Progesterone and Not 17β-Estradiol During the Estrous Cycle. Biol Reprod. 2001 Sep 1;65(3):680–8.

26. Gruber CJ, Tschugguel W, Schneeberger C, Huber JC. Production and Actions of Estrogens. New England Journal of Medicine. 2002;346(5):340–52.

27. Tsurufuji S, Sugio K, Sato H, Ohuchi K. A review of mechanism of action of steroid and non-steroid anti-inflammatory drugs. In: Willoughby DA, Giroud JP, editors. Inflammation: Mechanisms and Treatment. Springer Netherlands; 1981. p. 63–78.

28. Wierman ME. Sex steroid effects at target tissues: mechanisms of action. Advan in Physiol Edu. 2007 Jan 1;31(1):26–33.

29. Goldzieher, J, Castracane, V, Glob. libr. women's med.,(ISSN: 1756-2228) 2008; DOI 10.3843/GLOWM.10387

30. Fehring RJ, Schneider M, Raviele K. Variability in the Phases of the Menstrual Cycle. Journal of Obstetric, Gynecologic, & Neonatal Nursing. 2006;35(3):376–84.

31. Chiazze L, Jr., Brayer FT, Macisco JJ, Jr., Parker MP, et al. THe length and variability of the human menstrual cycle. JAMA. 1968 Feb 5;203(6):377–80.

32. Pauerstein CJ, Eddy CA, Croxatto HD, Hess R, Siler-Khodr TM, Croxatto HB. Temporal relationships of estrogen, progesterone, and luteinizing hormone levels

  8  

to ovulation in women and infrahuman primates. Am J Obstet Gynecol. 1978 Apr 15;130(8):876–86.

33. Iqbal K, Jin S-G, Pfeifer GP, Szabo PE. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3642–7.

34. Dodge JE, Ramsahoye BH, Wo ZG, Okano M, Li E. De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation. Gene. 2002 May 1;289(1–2):41–8.

35. Horvath S. DNA methylation age of human tissues and cell types. Genome Biology. 2013 Oct 21;14(10):R115.

36. Hannum G, Guinney J, Zhao L, Zhang L, Hughes G, Sadda S, et al. Genome-wide Methylation Profiles Reveal Quantitative Views of Human Aging Rates. Molecular Cell. 2013 Jan 24;49(2):359–67.

37. Hartsough MT, Clare SE, Mair M, Elkahloun AG, Sgroi D, Osborne CK, et al. Elevation of Breast Carcinoma Nm23-H1 Metastasis Suppressor Gene Expression and Reduced Motility by DNA Methylation Inhibition. Cancer Res. 2001 Mar 3;61(5):2320–7.

38. Lewis JS, Meeke K, Osipo C, Ross EA, Kidawi N, Li T, et al. Intrinsic Mechanism of Estradiol-Induced Apoptosis in Breast Cancer Cells Resistant to Estrogen Deprivation. JNCI J Natl Cancer Inst. 2005 Dec 7;97(23):1746–59.

39. Yang X, Yan L, Davidson NE. DNA methylation in breast cancer. Endocr Relat Cancer. 2001 Jun;8(2):115–27.

40. Stone A, Valdés-Mora F, Gee JMW, Farrow L, McClelland RA, Fiegl H, et al. Tamoxifen-induced epigenetic silencing of oestrogen-regulated genes in anti-hormone resistant breast cancer. PLoS ONE. 2012;7(7):e40466.

41. Xie R, Loose DS, Shipley GL, Xie S, Bassett RL, Broaddus RR. Hypomethylation-induced expression of S100A4 in endometrial carcinoma. Mod Pathol. 2007 Aug 3;20(10):1045–54.

42. Ceschin DG, Walia M, Wenk SS, Duboé C, Gaudon C, Xiao Y, et al. Methylation specifies distinct estrogen-induced binding site repertoires of CBP to chromatin. Genes Dev. 2011 Jun 1;25(11):1132–46.

43. Leung RKM, Whittaker PA. RNA interference: From gene silencing to gene-specific therapeutics. Pharmacology & Therapeutics. 2005 Aug;107(2):222–39.

44. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotech. 2004 Mar;22(3):326–30.

45. Lima WF, Prakash TP, Murray HM, Kinberger GA, Li W, Chappell AE, et al. Single-Stranded siRNAs Activate RNAi in Animals. Cell. 2012 Aug 31;150(5):883–94.

46. Baulcombe D. RNA silencing in plants. Nature. 2004 Sep 16;431(7006):356–63.

47. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet. 2014 Jan;15(1):7–21.

48. Ee PLR, He X, Ross DD, Beck WT. Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference. Mol Cancer Ther. 2004 Dec 1;3(12):1577–84.

49. Chittaranjan S, McConechy M, Hou Y-CC, Freeman JD, DeVorkin L, Gorski SM. Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi. PLoS Genet. 2009 Feb 13;5(2):e1000379.

50. Wiley TS, United States Patent #7,879,830. www.thewileyprotocol.com.

51. Taguchi J and Ridley C, Unpublished data, Clinical studies underway.

52. Hyde JS, DeLamater JD. Understanding human sexuality. New York: McGraw-Hill; 2011.