Don’t Monkey With My Hormones! by Jeffrey Dach MD

This article is part two of a series. For Part One Click Here

Hormone Monkey Studies

Hormone Studies from the Primate Center at Winston Salem are quite revealing and allow us to answer questions about different effects of various hormone formulations.

Left image: Macaque Monkeys,  courtesy of wikimedia commons (link)

If you are an avid reader of these pages, then you know I often quote the 2008 French Cohort Study by Fournier demonstrating safety of bioidentical hormones.  This study showed that bioidentical hormone users in France (using Estradiol and Progesterone) had same breast cancer risk as non-hormone users.(1)  In other words, there was no increased risk of breast cancer with this combination of bioidentical hormones.

Here is the  Data from Table 3 from the French Cohort study (Fournier 2008):

Hormone Combination             Relative Risk of Invasive Breast Cancer (1)
Non-User                                                              1.0
Estrogen (Estradiol) alone                                      1.29  (p=0.73 non-significant)
Estrogen (estradiol) / Progesterone (Bioidentical)    1.0
Estrogen (Estradiol) / Medroxyprogesterone            1.48

Adding MedroxyProgesterone (MPA)

When the added progestin is  Medroxyprogesterone (MPA), there is increased breast cancer risk (RR=1.48).  However, there is no increased risk with the addition of progesterone (RR=1.0).  Confirmatory findings were seen in the WHI study (First Arm) which also showed increased invasive breast cancer with the combination of Premarin and Medroxyprogesterone (MPA) also called Prempro.

Estrogen Alone

Now that we have the 11 year follow-up data of the Second Arm, Premarin-Alone (CEE horse estrogen) study of the Women’s Health Initiative, we can ask some more questions.  The Premarin-Alone Arm showed a 20-27% decrease in breast cancer  compared to placebo.

Why Less Cancer with Premarin-Alone, and More with Estradiol Alone ?

One might ask the obvious question, “Why is Premarin-Alone associated with decreased breast cancer risk compared to non-hormone users, while Estradiol-alone (bioidentical) is associate with a 29% increase in invasive breast cancer in the French Cohort study (p=0.73 non-significant)?

Monkey Studies Explain the Findings

The answer comes from Dr Wood at the the primate center in Winston Salem in a 2008 report using Macaque monkeys. (2)  They compared Premarin and Estradiol in a monkey model, and found a highly significant 259-330% increase in breast cell proliferation in Estradiol treated monkeys compared to controls.  The Premarin (CEE ) treated monkeys however, had far less cell proliferation, only 75% was noted using the KI-67 antigen test of cell proliferation.

This difference in cell proliferation, indicating a stonger estrogenic effect with Estradiol, explains why estradiol alone (French Cohort) is associated with increased breast cancer risk, while Premarin (WHI second arm) is associated not.  Premarin has less proliferative effect on breast tissue than does Estradiol as demonstrated in monkeys, and in the differing breast cancer rates in human studies.

Why Not Use Bi-EST, Rather than Estradiol  ?

So this underscores the importance of adding progesterone to an estradiol-alone regimen, such as the Vivelle-Dot or Climara Patch which contain Estradiol Alone.  Post-menopausal women given an estrogen-alone regimen because of prior hysterectomy may be at increased risk of breast cancer and would do better to either add progesterone to their program, or switch to the Bi-Est/ Progesterone combination.  We preferentially use a Bi-Est/ Progesterone combined cream to make sure both hormones are taken together in the proper ratio to prevent endometrial hyperplasia and reduce breast proliferation.  We also make sure every hormone-user takes Iodine supplements which are breast cancer preventive.

Why is Bi-Est Superior to Estradiol Alone ?

Bi-Est is a combination of Estradiol with Estriol, usually in a ratio of 20% Estradiol, and 80% estriol.  Studies show that Estriol is associated with less breast proliferation, and is thought to be preventive of breast cancer. (7-13)

How Does Estriol (E3) Prevent Breast Cancer ?

Estriol and the Estrogen Receptor Beta.

As it turns out, basic science has given us important answers here.  There are actually two estrogen receptors, ER- Alpha, and ER-Beta.  ER- Alpha is associated with breast cell proliferation, while ER-Beta with suppression of proliferation and prevention of breast cancer.  Estradiol (E2) binds equally to both receptors, whereas Estriol (E3) binds preferentially to ER-Beta, explaining its protective effect. (21-24)

Bi-Est is Safer then Estradiol Alone

Armed with this information about Estriol (E3), we can now confidently say that the Bi-Est combination (containing 80% Estriol) with Progesterone is the safest HRT regimen one can possibly devise.    This in combination with Iodine supplementation (Iodoral), and Vitamin D supplementation makes this program even safer.

Protective Effects of Progesterone Compared with Harmful Effects of Medroxyprogesterone – Breast Cell Proliferation

Another question we might ask is:

“How can we explain why Medroxyprogesterone (MPA) causes increased breast cancer, while (bioidentical) progesterone does not”

Dr. Wood published a primate hormone study in 2007 in which Medroxyprogesterone (MPA) was compared to Progesterone, finding Estradiol plus Medroxyprogesterone (MPA) significantly increased breast cell proliferation using Ki67 markers.  However,  Estradiol with Progesterone did not increase cellular proliferation. (3)   Previous primate studies by Cline in 1998 showed the same increased breast cell proliferation with addition of Medroxyprogesterone (MPA) to Premarin treated monkeys. (5)

Dr Cline’s conclusion (5):

“These findings indicate that progestogens(MPA) may exacerbate, not antagonize mammary gland proliferation induced by estrogen (Premarin) replacement therapy“

This protective effect of Progesterone (bioidentical) was confirmed in two double blind human studies by Chang and Foidart in which addition of Progesterone reduced the proliferative effects of estrogen, thereby explaining its cancer preventive effect.(19,20)  Breast proliferation is the underlying factor in increasing cancer risk.  Inhibiting breast proliferation prevents breast cancer.

This has been part two of a series. For Part One click here:

Articles with related interest:

The Safety of Bio-Identical Hormones

The Importance of BioIdentical Hormones

Bioidentical Hormones Prevent Arthritis

Bioidentical Hormone Estrogen Prevents Heart Disease

Morning Rounds With Steven Economou MD

Waking Up from the Synthetic Hormone Nightmare

Jeffrey Dach MD
7450 Griffin Road, Suite 190
Davie, Fl 33314
954-792-4663

Links and References :

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2346534/?tool=pubmed
J Clin Oncol. 2008 March; 26(8): 1260–1268.
Use of different postmenopausal hormone therapies and risk of histology- and hormone receptor-defined invasive breast cancer
Agnès Fournier,1 Alban Fabre,1 Sylvie Mesrine,1 Marie-Christine Boutron-Ruault,1 Franco Berrino,2 and Françoise Clavel-Chapelon1*

Conclusion
The increase in risk of breast cancer observed with the use of CHTs other than estrogen+progesterone and estrogen+dydrogesterone seems to apply preferentially to ER+ carcinomas, especially those ER+/PR−, and to affect both ductal and lobular carcinomas.

In the present analysis, the use of estrogen+progesterone was not significantly associated with the risk of any breast cancer subtype, though we found trends of increasing risks with increasing duration of use for lobular and ER+/PR− carcinomas. The RR associated with estrogen+dydrogesterone was significantly above one for lobular breast cancer. Use of estrogen+other progestagens was associated with increases in risk of both ductal and lobular carcinomas, and of ER+/PR+ and ER+/PR− carcinomas.

Progestagens may act on breast tissue through their interactions with steroid receptors, growth factors, and oncogenes, and with the cell-cycle and estrogen-metabolizing enzymes.45 Because they differ in their chemical structure, metabolism, pharmacokinetics, and potency, it is reasonable to expect them to induce different responses in the breast.46 However, in vitro data are conflicting, possibly because of variations in the experimental conditions.45,47 Therefore, in vivo studies are of particular interest. Some studies found that the proliferation of breast epithelium increased during the luteal phase of the menstrual cycle.48,49

However, in vivo, progesterone has been found to oppose the proliferative effects of estradiol on breast tissue of pre- and postmenopausal women.50,51 The contrary has been found for medroxyprogesterone acetate (MPA) in postmenopausal women52 or surgically postmenopausal macaques.53 In such a study on macaques, compared to placebo, estradiol+MPA resulted in significantly greater proliferation in lobular and ductal breast epithelium, while estradiol+micronized progesterone did not.54 These studies support our findings suggesting that, when combined with an estrogen, progesterone may have a safer risk profile in the breast than some other progestagens.

2008  Dr Wood, Estradiol Compared to Premarin –  standard doses of CEE may result in less  estrogen-induced epithelial proliferation in the breast compared with E2.

2) http://www.ncbi.nlm.nih.gov/pubmed/18520696

Menopause. 2008 Sep-Oct;15(5):890-8.
Comparative effects of oral conjugated equine estrogens and micronized 17beta-estradiol on breast proliferation: a retrospective analysis.  Wood CE, Clarkson TB, Chen H, Veenstra TD, Xu X, Scott L, Cline JM. Departments of 1Pathology/Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1040, USA.

To evaluate the effects of oral conjugated equine estrogens (CEE) and micronized 17beta-estradiol (E2) on breast proliferation in a postmenopausal primate model.
DESIGN: Data from nine studies were analyzed retrospectively. The primary outcome measure was breast epithelial proliferation determined by immunolabeling for the Ki67 antigen. Other measures included progesterone receptor expression and endometrial thickness (as surrogate markers of systemic estrogen exposure) and urinary estrogen metabolite profile. All CEE doses were given at the human equivalent of 0.625 mg/day (n = 281), whereas E2 was given at the human equivalent of 1.0 mg/day or less (n = 131).
RESULTS: Oral CEE(Premarin) resulted in a modest overall increase in breast epithelial proliferation of 75% that reached significance at P < 0.05 compared with placebo in one of four parallel-arm studies.

In contrast, oral E2(estradiol) resulted in a more substantial increase in breast epithelial proliferation of 259% (all studies) to 330% (parallel-arm studies only) that reached significance at P < 0.05 in all five E2 studies evaluated. Breast epithelial expression of progesterone receptor, a widely used marker of estrogen receptor activity, and endometrial thickness showed similar increases after treatment with CEE and E2 (P < 0.05 in all available studies). Relative amounts of urinary methoxyestrogens and the 2-hydroxyestrogen-to-16 alpha-hydroxyestrone ratio were higher after CEE compared with E2 treatment (P < 0.05 for all).
CONCLUSIONS: This retrospective analysis of oral estrogen effects in postmenopausal macaques suggests that standard doses of CEE may result in less estrogen-induced epithelial proliferation in the breast compared with E2.

2007 Estradiol plus wither Progesterone or Medroxyprogesterone.  Increase breast proliferation with E2 plu MPA, but no increased proliferation with E2 and Progetsterone

3) http://www.ncbi.nlm.nih.gov/pubmed/16841178
Breast Cancer Res Treat. 2007 Jan;101(2):125-34.
Effects of estradiol with micronized progesterone or medroxyprogesterone acetate on risk markers for breast cancer in postmenopausal monkeys. Wood CE, Register TC, Lees CJ, Chen H, Kimrey S, Cline JM. Department of Pathology/Section on Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1040, USA.

The addition of the synthetic progestin medroxyprogesterone acetate (MPA) to postmenopausal estrogen therapy significantly increases breast cancer risk. Whether this adverse effect is specific to MPA or characteristic of all progestogens is not known. The goal of this study was to compare the effects of oral estradiol (E2) given with either MPA or micronized progesterone (P4) on risk biomarkers for breast cancer in a postmenopausal primate model. For this randomized crossover trial, twenty-six ovariectomized adult female cynomolgus macaques were divided into social groups and rotated randomly through the following treatments (expressed as equivalent doses for women):

(1) placebo;
(2) E2 (estradiol)(1 mg/day);
(3) E2 + P4 (progesterone)(200 mg/day); and
(4) E2 + MPA (medroxyprogesterone)(2.5 mg/day).

Hormones were administered orally, and all animals were individually dosed. Treatments lasted two months and were separated by a one-month washout period. The main outcome measure was breast epithelial proliferation, as measured by Ki67 expression.

Compared to placebo, E2 + MPA resulted in significantly greater breast proliferation in lobular (P < 0.01) and ductal (P < 0.01) epithelium, while E2 + P4 did not. Intramammary gene expression of the proliferation markers Ki67 and cyclin B1 was also higher after treatment with E2 + MPA (P < 0.01) but not E2 + P4. Both progestogens significantly attenuated E2 effects on body weight, endometrium, and the TFF1 marker of estrogen receptor activity in the breast. These findings suggest that oral micronized progesterone has a more favorable effect on risk biomarkers for postmenopausal breast cancer than medroxyprogesterone acetate.

2005 Overview of Monkey Studies

4) http://www.ncbi.nlm.nih.gov/pubmed/15883111
Maturitas. 2005 May 16;51(1):64-74.
Controversies about HRT–lessons from monkey models.
Clarkson TB, Appt SE.Source  Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1040, USA.

Lessons from monkey models contribute significantly to a better understanding of the controversies in reconciling the differences in postmenopausal hormone treatment outcomes between observational and randomized trial data. Monkey studies brought attention to premenopausal estrogen deficiency with resulting premature coronary artery atherosclerosis.

Recently, those monkey studies were confirmed for premenopausal women in the NHLBI-sponsored Women’s Ischemia Syndrome Evaluation (WISE) Study. Monkey studies have provided convincing evidence for the primary prevention of coronary artery atherosclerosis when estrogens are administered soon after the development of estrogen deficiency. Equally convincing are the data from monkey studies indicating the total loss of these estrogens beneficial effects if treatment is delayed for a period equal to six postmenopausal years for women. An attempt has been made using the monkey model to identify the hormone treatment regimen most effective in preventing the progression of coronary artery atherosclerosis. By a substantial margin, the most effective approach is that of using estrogen containing oral contraceptive during the perimenopausal transition, followed directly by hormone replacement therapy postmenopausally. Because of similarities between human and nonhuman breast, monkeys have had a major role in clarifying controversies surrounding the breast cancer risk of estrogen only versus estrogen plus progestin therapies. The results of monkey studies suggest little or no effects of estrogen(Premarin) only treatment; whereas, estrogen (Premarin)+progestin clearly increases breast cancer risk.

1998 Dr Cline – Macaques Monkeys- Breast proliferation increased by addition of MPA to Premarin.

5) http://www.ncbi.nlm.nih.gov/pubmed/9598869
Breast Cancer Res Treat. 1998 Apr;48(3):221-9.
Effects of conjugated estrogens, medroxyprogesterone acetate, and tamoxifen on the mammary glands of macaques. Cline JM, Soderqvist G, von Schoultz E, Skoog L, von Schoultz B. Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27157-1040, USA.
The purpose of this work was to examine the mammary glands of adult, ovariectomized female cynomolgus macaques (Macaca fascicularis) in a long-term study of the effects of hormone treatments on chronic disease.

Treatments included conjugated equine estrogens (CEE), medroxyprogesterone acetate (MPA), CEE+MPA, and tamoxifen. Doses were scaled from those given women. Treatments were given in the diet for three years, followed by necropsy and tissue collection. Endpoints evaluated included glandular histology, histomorphometry, and immunohistochemical detection of the proliferation marker Ki-67, estrogen receptor (ER), and progesterone receptor (PR) in mammary epithelial cells.

Major findings were as follows: CEE (Premarin) induced PR expression and focal to diffuse lobuloalveolar proliferation.

Proliferation was increased by the addition of MPA, but was not induced by MPA alone. Tamoxifen induced ER and PR but not Ki-67 expression or glandular hyperplasia. Neoplasms were not seen. These findings indicate that progestogens(MPA) may exacerbate, not antagonize mammary gland proliferation induced by estrogen replacement therapy, and that tamoxifen has both estrogen agonist and antagonist effects on sex steroid receptor expression in the normal primate breast.

1999 – Benign breast biopsies from 86 post menopausal women

6) http://www.ncbi.nlm.nih.gov/pubmed/10599719
J Clin Endocrinol Metab. 1999 Dec;84(12):4559-65.
Hormone replacement therapy with estrogen or estrogen plus medroxyprogesterone acetate is associated with increased epithelial proliferation in the normal postmenopausal breast. Hofseth LJ, Raafat AM, Osuch JR, Pathak DR, Slomski CA, Haslam SZ. Department of Physiology, Michigan State University, East Lansing 48824, USA.

The relative effects of postmenopausal hormone replacement therapy (HRT) with estrogen alone vs. estrogen+progestin on breast cell proliferation and on breast cancer risk are controversial. A cross-sectional observational study was carried out to examine the proliferative effects of HRT with estrogen or estrogen plus the progestin, medroxyprogesterone acetate, in breast tissue of postmenopausal women.

Benign breast biopsies from 86 postmenopausal women were analyzed with antiproliferating cell nuclear antigen (anti-PCNA) and Ki67 antibodies to measure relative levels of cell proliferation. Epithelial density and estrogen and progesterone receptor status were also determined. The women were categorized either as users of: 1) estrogen (E) alone;(Premarin)
2) estrogen+medroxyprogesterone acetate (E+P); or
3) no HRT.

Compared with no HRT, the breast epithelium of women who had received either E+P or E alone had significantly higher PCNA proliferation indices, and treatment with E+P had a significantly higher index (PCNA and Ki67) than treatment with E alone.

Breast epithelial density was significantly greater in postmenopausal women treated with E and E+P, compared with no HRT. Thus, the present study shows that postmenopausal HRT with E+P was associated with greater breast epithelial cell proliferation and breast epithelial cell density than E alone or no HRT. Furthermore, with E+P, breast proliferation was localized to the terminal duct-lobular unit of the breast, which is the site of development of most breast cancers. Further studies are needed to assess the possible association between the mitogenic activity of progestins and breast cancer risk.

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Estriol

7) www.ncbi.nlm.nih.gov/pubmed/9577246
Altern Med Rev. 1998 Apr;3(2):101-13.
Estriol: safety and efficacy. Head KA. Free FIll Text
Abstract While conventional hormone replacement therapy provides certain benefits, it is not without significant risks. Estriol has been found to provide some of the protection without the risks associated with stronger estrogens. Depending upon the situation, estriol may exert either agonistic or antagonistic effects on estrogen. Estriol appears to be effective at controlling symptoms of menopause, including hot flashes, insomnia, vaginal dryness, and frequent urinary tract infections. Results of research on its bone-density-maintaining effects have been contradictory, with the most promising results coming from Japanese studies. Estriol’s effect on cardiac risk factors has also been somewhat equivocal; however, unlike conventional estrogen prescriptions, it does not seem to contribute to hypertension. Although estriol appears to be much safer than estrone or estradiol, its continuous use in high doses may have a stimulatory effect on both breast and endometrial tissue.

8) www.ncbi.nlm.nih.gov/pubmed/3119187
Cancer. 1987 Dec 15;60(12):2873-81.
Antimammary carcinogenic activity of 17-alpha-ethinyl estriol.
Lemon HM.Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68105.
Abstract Both initiation and promotion of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis were inhibited by prophylactic therapy for 1 to 7 months using 17-alpha-ethinyl-estriol in doses as low as 1.0 microgram/d administered to intact virgin female Sprague-Dawley rats at 35 to 65 days of age. Administration of 638-micrograms single or multiple doses 2 to 3 weeks before DMBA induced a 75% to 85% reduction in cancer incidence after 1 year (P less than 0.001). When treatment was begun 2 weeks after DMBA, 1.0 microgram/d infused for 84 days resulted in a 44% reduction in incidence, with higher-dose, more prolonged therapy achieving a 73% reduction, equal to the reduction in carcinoma incidence observed after ovariectomy. Biopsies of nontumorous mammary glands showed a positive correlation between prelactational lobuloalveolar hyperplasia, hormone dose, and reduction in incidence of mammary carcinoma. Similar treatment with 17-alpha-ethinyl-estradiol-17B and diethylstilbestrol did not inhibit the 90% to 100% incidence of carcinoma observed in DMBA-treated control rats, and induced lactational hyperplasia in mammary gland biopsies. Continuous ethinyl estriol infusion subcutaneous (sc) in 2.5 to 7.5 micrograms daily dosage significantly increased uterine weights by as much as 10% to 46% after 2 to 4 weeks. At the time of mammary neoplasm development when rats were necropsied, no significant difference was observed in uterine weights between rats receiving 638 micrograms/mo in a readily soluble pellet implant, and uterine weights of control rats. Ethinyl estriol given seven times monthly in 638-micrograms bolus doses was more inhibitory of mammary carcinogenesis than estriol after a year (P less than 0.1 greater than 0.05). Short-term intermittent administration of ethinyl estriol to young nulliparous women may offer a method of simulating the differentiating effect of pregnancy on mammary tissues, increasing durable resistance to carcinogenesis.

9) www.ncbi.nlm.nih.gov/pubmed/6996403
Acta Endocrinol Suppl (Copenh). 1980;233:17-27.
Pathophysiologic considerations in the treatment of menopausal patients with oestrogens; the role of oestriol in the prevention of mammary carcinoma.
Lemon HM.At menopause, several abnormalities in oestrogen metabolism have been reported, which may increase the likelihood of cancer development in the breast or uterus following oestrone or oestradiol-17 beta supplementation. Occult hypothyroidism reduces the rate of oestrogen inactivation by C2 hydroxylation, and 15-20% of women have low rates of C16 hydroxylation to oestriol. Reduced sex hormone binding globulin concentration occurs in association with obesity, thereby increasing the biologically active unbound fraction of oestradiol in plasma. Since oestriol undergoes minimal metabolism after absorption, does not bind to sex hormone binding globulin, and has an anti-oestradiol action by decreasing the duration of nuclear binding of oestradiol-receptor proteins, it is less likely to induce proliferative changes in target organs of cancer-prone women than oestrone or oestradiol. Intermittent non-conjugated oestriol treatment has demonstrated the most significant anti-mammary carcinogenic activity of 22 tested compounds as well as anti-uterotropic activity in intact female Sprague Dawley rats fed either of two dissimilar carcinogens (7, 12 dimethylbenz(a) anthracene, procarbazine) and followed for their natural life span. The protective effect was specific for mammary carcinomas only and has been decreased in rats with a 20% increase in growth curves. Clinical experience thus far with oral oestriol therapy of post-menopausal women has indicated little hazard of cancer development.

10) jama.jamanetwork.com/article.aspx?articleid=357461
JAMA. 1978 Jan 2;239(1):29-30. Estriol, the forgotten estrogen? Follingstad AH.

10A) www.ncbi.nlm.nih.gov/pubmed/97100
Front Horm Res. 1977;5:155-73.
Clinical and experimental aspects of the anti-mammary carinogenic activity of estriol.
Lemon HM. Abstract Intermittent implantation of 600–1,300 microgram estriol subcutaneously beginning 48 h before oral administration of 7,12-dimethylbenz(a)anthracene or procarbazine prevents development of 80–90% of carcinomas of the breast occurring during the natural life span of the intact female Sprague-Dawley rat. Some estriol precursors were less inhibitory of breast cancer development among 23 other estrogens and androgens, progestins and glucocorticoids tested. More frequent or lower estriol doses than 100–200 microgram/kg/24 h every 2 months were less inhibitory of breast carcinogenesis. No other types of neoplasms were reduced in incidence by estriol implants, which also reduced uterine weights by 20–25%. Intermittent substitution of estriol for estrone or estradiol in the nuclear receptor complexes of target cells probably accounts for these observations, which resemble the effect of castration in reducing breast cancer incidence. Human studies indicate excellent tolerance for oral estriol doses of 10–200 microgram/kg/24 h, which may correct subnormal estriol/estrone + estradiol urinary quotients associated with elevated risk of breast carcinogenesis in epidemiologic investigations.

11 )  cancerres.aacrjournals.org/content/35/5/1341
Cancer Res. 1975 May;35(5):1341-53.
Estriol prevention of mammary carcinoma induced by 7,12-dimethylbenzanthracene and procarbazine.
Lemon HM. Abstract The concentration of estrogenic, androgenic, progestational, and adrenocortical steroid hormones in body fluids of mature intact Sprague-Dawley female rats was increased by s.c. implantation of 5 to 7 mg NaCl pellets containing 1 to 20% steroid 48 hr before administration p.o. of either 7,12-dimethylbenz(a) anthracene or procarbazine. The incidence of rats developing one or more mammary carcinomas in each treated group was compared to that ovserved in simultaneously treated groups receiving only the carcinogen, steroid, or no treatment whatsoever, with weekly observation of all rats until palpably growing tumors were biopsied and proven carcinomatous or until death occurred from other causes determined by autopsy. A total of 105 untreated or steroid-implanted rats followed to death (234 to 256 days median observation) developed no breast carcinomas. Rats fed either of the carcinogens developed initial evidence of breast carcinoma, after 136 to 156 days median observation, in 51 to 57% of 318 total treated rats. Nonbreast carcinomas and sarcomas developed in 5 to 10% of the carcinogen-treated rats.

12) www.ncbi.nlm.nih.gov/pubmed/6773286
Acta Endocrinol Suppl (Copenh). 1980;233:45-50.
The significance of oestriol in the management of the post-menopause.
Tzingounis VA, Aksu MF, Greenblatt RB.
Abstract Oestrogen replacement therapy relieves many post-menopausal symptoms and has been successfully employed clinically for this purpose for more than four decades. Recently the alleged relationship between oestrogens and cancer has stimulated a re-evaluation of an old oestrogen preparation, oestriol (E3). The dosages of E3 employed appear to vary considerably, and the need was felt to establish the dosage on a scientific basis. Accordingly in the study reported here E3 was administered in various dosages (2, 4, 6, and 8 mg/d) to 52 symptomatic post-menopausal women as oestrogen replacement therapy for a six-month period. Assays of follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestrone (E1) and oestradiol (E2) were performed before and during therapy and vaginal cytology, cervical mucus and endometrial studies were performed during the period of administration. The clinical effectiveness of E3 was found to be directly related to dosage. E3 did not induce endometrial proliferation and proved a poor suppressor of FSH and LH. The ability of oestriol to relieve vasomotor instability and to improve vaginal maturation without inducing notable side effects is sufficient reason for it to be included in the management of the post-menopausal syndrome.

12) www.ncbi.nlm.nih.gov/pubmed/6789033
Maturitas. 1981 Mar;3(1):47-53.
Oestrogens, gonadotropins and prolactin after intra-vaginal administration of oestriol in post-menopausal women.
Keller PJ, Riedmann R, Fischer M, Gerber C.
Abstract Serum total oestrone, 17 beta-oestradiol and oestriol concentrations and FSH, LH and prolactin values were measured radioimmunologically in post-menopausal women before and after intra-vaginal application of 0.5 mg oestriol. While oestrone and oestradiol were not altered, there was a 3100% increase in the mean oestriol values within 1 or 2 h; pre-treatment levels were again reached 8 h later. Both gonadotropins were moderately decreased, the serum prolactin values appeared to be slightly elevated. Repeated intra-vaginal application of oestriol resulted in a significant rise of the mean serum oestriol levels while the other oestrogens remained unchanged. The same was true for FSH and LH, a considerable negative feedback was therefore excluded. Again there seemed to be a slight rise of the prolactin secretion. It was concluded that intra-vaginal administration of oestriol is a most suitable local and systemic oestrogen replacement therapy, which is more effective than the oral regimen.
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1996 Dr Cline -Monkeys

14) http://www.ncbi.nlm.nih.gov/pubmed/8572040
Am J Obstet Gynecol. 1996 Jan;174(1 Pt 1):93-100.
Effects of hormone replacement therapy on the mammary gland of surgically postmenopausal cynomolgus macaques. Cline JM, Soderqvist G, von Schoultz E, Skoog L, von Schoultz B. Source Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1040, USA.

Our purpose was to define the proliferative response and receptor status in the mammary glands of surgically postmenopausal macaques given hormone replacement therapy, equivalent for monkeys to that given women.

STUDY DESIGN:Surgically postmenopausal adult female cynomolgus macaques (Macaca fascicularis) were given either :

no treatment (n = 26),
conjugated equine estrogens (n = 22), or
combined therapy with conjugated equine estrogens and medroxyprogesterone acetate (n = 21).

Drugs were administered in the diet, at doses equivalent on a caloric basis to 0.625 mg per woman per day for conjugated equine estrogens and 2.5 mg per woman per day for medroxyprogesterone acetate, for 30 months.

Mammary gland proliferation was assessed subjectively and by morphometric and stereologic means. Estrogen receptor and progesterone receptor content and proliferation were studied by immunohistochemistry.

RESULTS: In this model combined therapy with conjugated equine estrogens and medroxyprogesterone acetate induced greater proliferation than did conjugated equine estrogens alone. The percentage of estrogen receptor-positive cells was decreased in the conjugated equine estrogens plus medroxyprogesterone acetate group. The percentage of progesterone receptor-positive cells was increased by treatment with conjugated equine estrogens alone.

CONCLUSION: These results indicate a proliferative response of mammary gland epithelium to therapy with conjugated equine estrogens plus medroxyprogesterone acetate in postmenopausal macaques. The clinical implication of this finding may be a greater risk for development of breast neoplasms in women receiving combined hormone replacement therapy.

Progesterone Reduces Breast Cell Proliferation Caused By Estrogen

19) www.ncbi.nlm.nih.gov/pubmed/7890063
Fertil Steril. 1995 Apr;63(4):785-91.
Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo.
Chang KJ, Lee TT, Linares-Cruz G, Fournier S, de Ligniéres B.
Source National Taiwan University Hospital, Taipei.
To study the effect of E2 and P on the epithelial cell cycle of normal human breast in vivo.
DESIGN: Double-blind, randomized study. Topical application to the breast of a gel containing either a placebo, E2, P, or a combination of E2 and P, daily, during the 10 to 13 days preceding breast surgery.
PATIENTS : Forty premenopausal women undergoing breast surgery for the removal of a lump. MAIN OUTCOME MEASURES. Plasma and breast tissue concentrations of E2 and P. Epithelial cell cycle evaluated in normal breast tissue areas by counting mitoses and proliferating cell nuclear antigen immunostaining quantitative analyses.
RESULTS: Increased E2 concentration increases the number of cycling epithelial cells. Increased P concentration significantly decreases the number of cycling epithelial cells.
CONCLUSION: Exposure to P for 10 to 13 days reduces E2-induced proliferation of normal breast epithelial cells in vivo.

20) www.ncbi.nlm.nih.gov/pubmed/9591509
Fertil Steril. 1998 May;69(5):963-9.
Estradiol and progesterone regulate the proliferation of human breast epithelial cells.
Foidart JM, Colin C, Denoo X, Desreux J, Béliard A, Fournier S, de Lignières B.
Source University of Liège, Belgium.
To study the effects of estradiol and progesterone on the proliferation of normal human breast epithelial cells in vivo.
DESIGN: Double-blind randomized study.
SETTING: Departments of gynecology and of cell biology at a university hospital.
PATIENT(S): Forty postmenopausal women with untreated menopause and documented plasma FSH levels of >30 mIU/mL and estradiol levels of <20 pg/mL.
INTERVENTION(S): Daily topical application to both breasts of a gel containing a placebo, estradiol, progesterone, or a combination of estradiol and progesterone during the 14 days preceding esthetic breast surgery or excision of a benign lesion.
MAIN OUTCOME MEASURE(S): Plasma and breast tissue concentrations of estradiol and progesterone. Epithelial cell cycles were evaluated in normal breast tissue by counting mitoses and performing quantitative proliferating cell nuclear antigen immunolabeling analyses.
RESULT(S): Increasing the estradiol concentration enhanced the number of cycling epithelial cells, whereas increasing the progesterone concentration significantly limited the number of cycling epithelial cells.
CONCLUSION(S): Exposure to progesterone for 14 days reduced the estradiol-induced proliferation of normal breast epithelial cells in vivo.
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21) cancerres.aacrjournals.org/content/64/1/423.long
Cancer Res. 2004 Jan 1;64(1):423-8.
Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest.
Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL, Leitman DC.
Source Department of Obstetrics, University of California, San Francisco, CA
Abstract Studies indicate that estrogen receptor (ER) alpha mediates breast cancer-promoting effects of estrogens.

The role of ERbeta in breast cancer is unknown. Elucidating the role of ERbeta in the pathogenesis of breast cancer is important because many human breast tumors express both ERalpha and ERbeta. We show that adenovirus-mediated expression of ERbeta changes the phenotype of ERalpha-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERalpha. In contrast, introducing ERbeta into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERbeta inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21(Cip1) and p27(Kip1), which leads to a G(2) cell cycle arrest.

These results demonstrate that ERalpha and ERbeta produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ER beta-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

22) www.ncbi.nlm.nih.gov/pubmed/20494112
Biochem Biophys Res Commun. 2010 May 21;396(1):63-6.
The role of estrogen receptor beta (ERbeta) in malignant diseases–a new potential target for antiproliferative drugs in prevention and treatment of cancer.
Warner M, Gustafsson JA. Source  Center for BioSciences, Department of BioSciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.
Abstract The discovery of ERbeta in the middle of the 1990s represents a paradigm shift in our understanding of estrogen signaling. It has turned out that estrogen action is not mediated by one receptor, ERalpha, but by two balancing factors, ERalpha and ERbeta, which are often antagonistic to one another. Excitingly, ERbeta has been shown to be widespread in the body and to be involved in a multitude of physiological and pathophysiological events. This has led to a strong interest of the pharmaceutical industry to target ERbeta by drugs against various diseases. In this review, focus is on the role of ERbeta in malignant diseases where the anti proliferative activity of ERbeta gives hope of new therapeutic approaches.

23) www.ncbi.nlm.nih.gov/pubmed/21479371
Mol Med Report. 2008 Jan-Feb;1(1):15-20.
Is it necessary to control the level of estrogen receptor α and β activation in postmenopausal hormone replacement therapy in order to achieve the optimal outcome? (Review). Zhu BT. Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center,
Abstract : Endogenous estrogens exert an array of biological actions on women, many of which are mediated by the estrogen receptors (ERs) α and β. Results from our recent studies suggest that the human ERα and ERβ systems are differentially activated under different physiological conditions. In non-pregnant young women, the ERα system is preferentially activated over the ERβ system, mainly by estrone (E1) and its major oxidative metabolite, 2-hydroxy-E1. These two estrogens are among the quantitatively major estrogens present in young women, and have approximately 4-fold preferential activity for ERα over ERβ. During pregnancy, however, there is a preponderance of activation of ERβ over ERα conferred by various pregnancy estrogens such as estriol and other D-ring derivatives of 17β-estradiol (E2). These estrogens have an up to 18-fold preference for binding to ERβ than for ERα, and some of them are produced in unusually large quantities. Given this new information, it is hypothesized that the estrogens ideal for female hormone replacement therapy (HRT) would be those which produce a hormonal condition mirroring that found in non-pregnant young women rather than in pregnant women. Endogenous estrogen derivatives, such as the sulfated conjugates of E1, may be among the ideal candidates for achieving this clinical purpose. In comparison, Premarin, the most commonly-used HRT containing a mixture of conjugated estrogens isolated from pregnant mare’s urine, is less suitable because several of its estrogenic components can produce a strong preferential over-stimulation of the human ERβ signaling system.

24) endo.endojournals.org/content/147/9/4132.long
Endocrinology. 2006 Sep;147(9):4132-50. Epub 2006 May 25.
Quantitative structure-activity relationship of various endogenous estrogen metabolites for human estrogen receptor alpha and beta subtypes: Insights into the structural determinants favoring a differential subtype binding.
Zhu BT, Han GZ, Shim JY, Wen Y, Jiang XR. Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Basic Pharmaceutical Sciences, College of Pharmacy, 700 Sumter Street, Columbia, South Carolina 29209,

To search for endogenous estrogens that may have preferential binding affinity for human estrogen receptor (ER) alpha or beta subtype and also to gain insights into the structural determinants favoring differential subtype binding, we studied the binding affinities of 74 natural or synthetic estrogens, including more than 50 steroidal analogs of estradiol-17beta (E2) and estrone (E1) for human ER alpha and ER beta. Many of the endogenous estrogen metabolites retained varying degrees of similar binding affinity for ER alpha and ER beta, but some of them retained differential binding affinity for the two subtypes. For instance, several of the D-ring metabolites, such as 16 alpha-hydroxyestradiol (estriol), 16 beta-hydroxyestradiol-17 alpha, and 16-ketoestrone, had distinct preferential binding affinity for human ER beta over ER alpha (difference up to 18-fold). Notably, although E2 has nearly the highest and equal binding affinity for ER alpha and ER beta, E1 and 2-hydroxyestrone (two quantitatively predominant endogenous estrogens in nonpregnant woman) have preferential binding affinity for ER alpha over ER beta, whereas 16 alpha-hydroxyestradiol (estriol) and other D-ring metabolites (quantitatively predominant endogenous estrogens formed during pregnancy) have preferential binding affinity for ER beta over ER alpha. Hence, facile metabolic conversion of parent hormone E2 to various metabolites under different physiological conditions may serve unique functions by providing differential activation of the ER alpha or ER beta signaling system. Lastly, our computational three-dimensional quantitative structure-activity relationship/comparative molecular field analysis of 47 steroidal estrogen analogs for human ER alpha and ER beta yielded useful information on the structural features that determine the preferential activation of the ER alpha and ER beta subtypes, which may aid in the rational design of selective ligands for each human ER subtype.

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