Introduction

Estrogens are master drivers of reproductive activity in women; controlling ovulation, zygote implantation, and mammary gland development [1,2,3]. Estrogens also have pleiotropic protective effects on non-reproductive tissues and organs and help to ensure optimal health of women during their childbearing years [4,5,6,7,8,9,10]. A women’s life can be divided into five main stages based on estrogen activity: childhood, puberty, reproductive life, perimenopause and postmenopause. Estrogen levels increase during puberty until menarche, when levels stabilize. They then vary in line with the monthly menstrual cycle until late perimenopause, when estrogen levels fall and stabilize. Menopause hormone treatments (MHTs), also known as hormone replacement therapy (HRT), are the most powerful treatments available for alleviating the symptoms associated with the cessation of estrogen production by the ovaries at menopause [11, 12]. However, these estrogen-based treatments are associated with an increased risk of breast cancer and estrogens also act as growth factors in estrogen receptor-positive (ER +) breast cancers, which account for 70% of all cases [13]. Therefore, there is an unmet medical need for a new generation of MHT with an improved benefit/risk profile for breast cancer in particular.

The ideal estrogen would display the following characteristics: 1) effective on menopause symptoms (in particular hot flushes, vulvo-vaginal atrophy, and osteoporosis); 2) neutral on mammary gland and breast cancer growth; 3) neutral on endometrial hyperplasia, which increases the risk of endometrial cancer; 4) cardioprotective against atherosclerosis and thromboembolism; 5) a favorable metabolic profile.

Accumulating data support the use of estetrol (E4), a natural fetal estrogen, for such clinical applications. E4 is currently approved by the US Food and Drugs Administration and by the European Medicines Agency as the estrogen component in a combined oral contraceptive and is under development for the treatment of symptoms due to menopause. It is also being evaluated for advanced endocrine-resistant breast cancer. In this review, we aim to define the extent to which E4 fulfills ‘the ideal criteria’, focusing in particular on its effects on the mammary gland and breast cancer. We will provide a brief summary of the clinical evidence associated with breast cancer risk and the uptake of exogenous estrogen-based formulations, followed by a review of E4 biology, E4 signaling pathways, and its actions on the mammary gland and breast cancer. We will then move on to discuss the potential benefits of E4 as a contraceptive, as a treatment for symptoms due to menopause, and for the treatment of endocrine-resistant breast cancer.

Estrogen-based Formulations and Breast Cancer Risk

Estrogens are widely used as a hormonal treatment for multiple therapeutic indications, in particular for contraception and in the treatment of symptoms due to menopause. Currently, 17β-estradiol (E2) and estriol (E3) are the most widely prescribed natural estrogens in combined oral contraceptives (COCs) and in MHT [11, 12]. Synthetic estrogenic molecules such as conjugated equine estrogen, ethinyl estradiol and E2-valerate are also used [12, 14].

Over the past two decades, the safety of COCs on breast tissue has been debated in the literature and overall, the risk of COC-associated breast cancer risk appears to remain low. However, there is limited evidence available as the incidence is low, studies are difficult, and require very large cohorts of patients [15,16,17]. Interestingly, the slight increase in risk that is observed, disappears ten years or more after treatment cessation [15], highlighting that estrogens promote preexisting breast cancer cell growth rather than inducing breast carcinogenesis. Moreover, the risk of ovarian, endometrial, and colorectal cancers is reduced for women using COCs [18], and such advantages may outweigh the potential negative effect of COCs on breast cancer risk for premenopausal women.

In 2002, for the first time, the Women’s Health Initiative (WHI) study [19] reported an association between MHT use and breast cancer risk. This study had unprecedented consequences on the prescription rates of MHTs, which subsequently decreased by 30% [20]. Between 2003 and 2011, several European and American epidemiological and observational studies including, the Million Women Study, the French E3N cohort, a study of Finnish women, and the European Prospective Investigation into Cancer and Nutrition (EPIC) studies, referenced breast cancer risk as a major side effect of MHTs [19, 21,22,23,24,25]. In 2012, the Cochrane meta-analysis confirmed the pro-tumoral risk of MHT [26] and in 2019, a long-term prospective study, with a mean follow-up period of 17.6 years, showed a correlation between MHT and breast cancer-associated mortality [27]. Finally, a meta-analysis of 58 studies, including 143,887 postmenopausal women with breast cancer and 424,972 women without breast cancer, was published by the Collaborative Group on Hormonal Factors in Breast Cancer [28] and again confirmed the correlation between MHT use and breast cancer risk. However, variations in the relative risk ratio were reported depending on modality (Table 1) and all MHT formulations, with the exception of estrogenic vaginal cream, were associated with an increased risk [28]. An excess risk of breast cancer was associated with both current or recent use (1–4 years) and long-term treatment. The incidence of mammary cancers correlated with treatment duration [23, 29] and decreases progressively after treatment cessation [21, 29]. The association between breast cancer and MHT use was higher for estrogen-progestogen combinations compared to estrogen-only formulations or placebo [12, 21, 24,25,26, 28]. Combinations with natural progesterone and dydrogesterone appeared to be safer [22] than those combined with synthetic progestins such as norethisterone acetate and medroxy-progesterone acetate (MPA), which potentiated the breast cancer risk [23, 25]. Both estrogenic and combined MHTs preferentially induced ER + breast cancer [28]. These observations support a role for MHTs in the potentiation of preexisting breast cancer cells rather than in the induction of carcinogenesis. In summary, the risk of breast cancer in MHT users was highest for oral estrogen-progestogen formulations used for more than 5 years.

Table 1 Menopause hormone therapy modality and associated relative risk of breast cancer [29]
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Together these data suggest that, if estrogens promote ER + breast cancer growth, exogenous administration of estrogen combined with a progestogen as a MHT in postmenopausal women could increase the risk of breast cancer. In western countries, MHTs are prescribed to almost 12 million women, highlighting the clinical need [28]. The following section aims to provide a critical overview of E4 and to define the potential advantages of E4 over conventional estrogens in the development of a new generation of MHT.

Estetrol Biology

There are four natural estrogens synthetized in humans, estrone (E1), E2, E3 and E4 (Fig. 1). E4 was discovered in 1965 by the team of Diczfalusy [30]. It is produced exclusively during pregnancy by the liver in both male and female fetuses [30, 31], through 15α- and/or 16α-hydroxylation of E2 or E3 [31]. E4 is detected from the 9th week of gestation in maternal urine and from the 20th week in the maternal plasma. Maternal plasma rates increase during pregnancy and reach 1 ng/ml (3 nM) by the second trimester. E4 fetal plasma levels at term are 12 times higher than those of the mother [32, 33]. Despite work in the 80 s that evaluated maternal E4 level as an index of pregnancy complications and fetus well-being [32, 34, 35], the physiological role of E4 during pregnancy still remains undefined.

Fig. 1
figure 1

Biochemical structures of natural estrogens. Schematic representation of endogenous estrogens, estrone (E1), 17β-estradiol (E2), estriol (E3) and estetrol (E4). Structural images were uploaded from ChemSpider (www.chemspider.com) with permission

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In humans, E4 is characterized by a high oral bioavailability of 90% [36], compared to 10% for E2. The human half-life of E4 is 28–32 h [36], compared to 90 min for E2 [14]. In rodents, the bioavailability of E4 is approximately 70% and its half-life is between 2 and 3 h [37]. Both absorption and oral bioavailability are dose-dependent and interindividual plasma variations after oral administration are low [36]. Taken together, the pharmacokinetic parameters of E4 make it suitable for oral use. The hepatic metabolism of E4 is slow in both humans and rodents and is similar across the two species. E4 metabolites are produced through conjugation, mainly methylation, (de)hydroxylation, glucuronidation and sulfation, they are inactive [32, 38] and are rapidly excreted in urine [32, 38,39,40]. The principal urine metabolite is the Ring D-monoglucuronide, but E4 can also be excreted in an unconjugated form that cannot be reciprocally converted back into E2 or E3 [35, 36].

E4 is selective for the estrogen receptors (estrogen receptor alpha [ERα] and estrogen receptor beta [ERβ]) and binds poorly to other nuclear receptors even at very high concentrations [38]. Although the binding affinity of E4 for both ERα and ERβ is moderate in comparison to E2 [38, 41] (Table 2), its binding affinity for ERα is almost 5 times higher than that of ERβ (Table 2). The interaction of E4 with ERα, at the ligand binding domain is similar to E2 [41]. There is currently no data available on potential binding of E4 to G-coupled protein estrogen receptor (GPER).

Table 2 Binding affinity of E2 and E4 for ERα and ERβ—equilibrium dissociation constant (nM) [38]
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Estetrol Signaling Pathways

In transgenic mouse studies, ERα has been shown to mediate most of the estrogenic actions in organs including the brain, endothelium, mammary gland, vagina, and uterus. ERα has also been shown to control processes such as atheroprotection, vasodilatation, nitric oxide synthesis, endothelial healing, and bone demineralization, and is also involved in the prevention of type-2 diabetes [7, 42,43,44,45,46,47,48,49]. Two distinct signaling pathways are associated with ERα activation (reviewed in [50]): the genomic/nuclear pathway [51] and the non-genomic/extra-nuclear/membrane-initiated steroid signaling (MISS) pathway [50, 52]. The genomic pathway is associated with the transcriptional activity of the nuclear form of the receptor, while the MISS pathway is induced by the membrane-anchored form of ERα or GPER and leads to rapid activation of intracellular signaling cascades (Fig. 2).

Fig. 2
figure 2

ERα signaling pathways. Schematic representation of the genomic pathway and the MISS pathway associated with E2-induced ERα signaling. Abbreviations: S118p, Serine 118 phosphorylated ERα; Co-Reg, co-regulators; TF, transcription factors

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The signaling pathways associated with E4 have been extensively studied in the endothelium in vivo and have been compared to E2-dependent pathways. In rats, E4 has been shown to stimulate vasodilation of arteries via ERα activation [53, 54]. Studies of transgenic mice with mutations in the transactivation function (AF), AF1 or AF2 domain, have revealed that E4 induces atheroprotective effects through the activation of the genomic pathway of ERα [41]. Moreover, E4 exerted a protective effect against neointimal hyperplasia [55], a phenomenon described as being genomic-ERα dependent [56]. E4 was also shown to reduce the effects of angiotensin II and to prevent hypertension in rodents through the genomic-ERα pathway [55]. As predicted by the binding affinity constant (Table 2), the potency of E4 required to activate the genomic-ERα pathway is 50–100 times lower than that of E2. In contrast to E2, E4 did not accelerate endothelial healing in vivo or enhance endothelial nitric oxide synthase (eNOS) activation ex vivo [41, 55]; both of which are actions that have been established to be dependent on the MISS pathway of ERα [44, 57]. In addition, E4 inhibited the action of E2 on endothelial healing and eNOS activation, suggesting E4 antagonizes the E2-induced MISS pathway of ERα [41]. These results support that in the endothelium, E4 is an agonist of the genomic-ERα pathway but an antagonist of the MISS pathway (Fig. 3).

Fig. 3
figure 3

Estrogen signaling pathways induced by E4-only treatment or a combination of E2 + E4

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E4 also acts through ERα to promote proliferation and growth of the mouse mammary gland [58], human ER + breast cancer cells [59, 60], patient-derived xenografts (PDX) from ER + breast tumors [60], and endometrial cancer cells [73]. Moreover, E4 treatment was also shown to decrease ovarian (E2) and gonadotropins (LH and FSH) hormone production [73]. The randomized phase II FIESTA study reported a favorable vaginal bleeding pattern and good cycle control associated with 15 mg E4/3 mg DRSP [74]. In addition, E4/DRSP treatment was also shown to be associated with favorable body weight control, well-being, high acceptability, and user satisfaction [75]. Since the combination of E4 and DRSP has been shown to have a neutral impact on breast cancer growth in vivo compared to E2 and progesterone [60], the use of an E4-based COC formulation could be advantageous in women who are concerned about the potential risk of breast cancer associated with conventional COCs. More interestingly, E4 did not increase the synthesis of sex hormone binging globulin (SHBG) [76,77,78] and had a limited effect on liver factors involved in coagulation, supporting a lower risk of cardiovascular side effects in comparison to E2 [55, 79]. This feature of E4 is possibly the most important advantage of E4-based formulations over conventional COCs.

Menopause Symptoms

Menopause is associated with the appearance of menopause symptoms, such as vasomotor symptoms (hot flushes and/or night sweats), genitourinary symptoms (vaginal dryness and atrophy, uterine bleeding and sexual dysfunction), urinary symptoms (incontinence and infections), mood change (irritability, anxiety, sadness, hyper-sensibility and depression), and cognitive disturbance [80, 81]. In addition, menopause is also associated with an increased risk of cardiovascular diseases, bone fractures related to osteoporosis, and metabolic complications such as type-2 diabetes [82,83,84,85]. Several preclinical and clinical studies have evaluated the impact of E4 on estrogen-sensitive tissues and organs related to menopause symptoms.

In an in vivo model of hot flushes in rats, E4 was shown to suppress vasomotor symptoms in a dose dependent manner [86]. E4 induced the synthesis of allopregnanolone in the brain of castrated rats, which is associated with body temperature regulation [87]. E4 also prevented E2-induced production of this molecule, highlighting the estrogenic and anti-estrogenic properties of E4 [88, 89]. Two clinical studies demonstrated that E4 (at a dose of 2–10 mg/day or 15 mg/day) effectively reduced the frequency and the severity of hot flushes in postmenopausal women [90, 91]. These results support a beneficial effect for E4 in controlling hot flushes.

In rodents, E4 promoted the proliferation of vaginal cells, increased vaginal weight and epithelium height [92], and induced vaginal cornification and maturation [93]. A randomized clinical study evaluating oral administration of E4 (at a dose of 2–40 mg/day) in postmenopausal women also revealed modifications to vaginal cytology, including a decrease in the percentage of parabasal cells and an increase in the quantity of superficial cells [90]. These preclinical and clinical studies show that E4 is associated with protective actions on the vagina and highlight a potential role for E4 in the prevention of vulvo-vaginal atrophy.

Interestingly, E4 attenuated brain injury in a neonatal rat model of hypoxic-ischemic encephalopathy, prevented oxidative stress and enhanced cell proliferation in primary hippocampal neuronal cell cultures in vitro, decreased early grey matter loss, and promoted neurogenesis and angiogenesis in vivo [94]. These results show that E4 presents neuroprotective properties and support that E4 could be investigated for the prevention of menopause symptoms related to cognitive function.

In another preclinical study, oral administration of E4 decreased levels of osteocalcin and increased bone density, mineral content, and bone strength in a dose dependent manner in ovariectomized rats [37]. In postmenopausal women, E4 also exerted a dose-dependent decrease in C-telopeptide and osteocalcine, markers of bone resorption and formation, respectively [70, 95]. At higher doses (20 and 40 mg), E4 stimulated bone formation, highlighting the potential use of E4 in the prevention of osteoporosis [70]. These results highlight promising clinical benefits and a potential role for E4 on bone fractures and osteoporosis risk.

Additional positive effects of E4 were also reported on menopause-associated risks, including metabolic disorders and cardiovascular issues. In mouse models, E4 reduced body weight gain, improved glucose tolerance, prevented obesity [96], and associated disorders such as atherosclerosis and steatosis [53]. Preclinical and clinical studies revealed that E4 has several potential vascular advantages (reviewed in [55]), including the prevention of angiotensin-II-dependent hypertension and neointimal hyperplasia, while having minimal impact on hemostasis, fibrinolysis, angiotensinogen, triglycerides, and cholesterol. Although E4 did not enhance eNOS action in murine adult aorta [41, 55], it favored flow-induced vasodilation [53, 54], an important vasculoprotective action of estrogen. In addition, E4 also prevented atherosclerosis in a dose-dependent manner in mice [41].

Importantly, preclinical and clinical studies demonstrated that E4 induces uterotrophic activity and acts as an agonist on the endometrium [41, 90]. It has also been shown to increase mouse uterine wet weight [37, 93, 97], epithelial proliferation, and height of the epithelium and stromal compartments [41] at a MHT therapeutic dose [60]. In rats, E4 increased the volume of luminal fluids, protein content, and the activity of alkaline phosphatase [97]; all markers of the estrogenic uterine response. Moreover, it induced major histological modifications, including the synthesis of the PR [98]. In postmenopausal women, E4 (10 mg/day) increased the thickness of the endometrium, highlighting its estrogenic action [90]. The addition of DRSP to E4 (5 or 10 mg/day) decreased E4-induced endometrial thickening in women [73]. These results support the addition of a progestogen to E4 formulations for MHT to protect the endometrium of non-hysterectomized women from hyperplasia and cancer.

Assessing the risk of breast cancer associated with E4 use in postmenopausal women is a long-term effort and can only be conducted with decades of patient follow-up. Nevertheless, as detailed in the previous section of this review, preclinical data obtained from several endocrine-sensitive breast cancer models, including PDX, revealed that E4 has a neutral impact on breast cancer growth and metastasis dissemination to the lung when used in mice at a dose (0.3 mg/kg/day) that corresponds to the steady-state obtained by once-a-day (15 mg E4/day) oral treatment in women. This neutral effect is not impacted by the addition of progesterone or DRSP [60]. These observations give E4-based formulations an advantage over E2-based MHTs and have important clinical implications as they highlight the possibility of develo** a combined estrogen, progestogen MHT that could have a positive impact on breast cancer risk.

Breast Cancer Treatment

Acquired endocrine resistance is a major cause of relapse in ER + breast cancer and therapeutic strategies to help overcome this are of the utmost importance. Several studies have suggested the potential of estrogen-based therapies in the treatment of advanced endocrine-resistant breast cancer. However, there is a reluctance to use E2 due to the potential for adverse effects, especially thromboembolism [99,100,101,102,103,104]. The pro-apoptotic properties of E4 on endocrine-resistant breast cancer demonstrated in vitro [79], support a potential role for E4 in the treatment of advanced endocrine-resistant breast cancer in postmenopausal women.

Conclusions and Perspectives

Of the natural estrogens, E4 is a unique native fetal estrogen with selective tissue actions that offers novel therapeutic opportunities for indications including, contraception, the treatment of symptoms due to menopause, and the treatment of endocrine-resistant advanced breast cancer.

The safety data for E4 in the breast are promising, nevertheless further progress in this field is expected, especially regarding the mechanisms of action of this natural estrogen on the mammary gland and breast cancer. A recent publication emphasized that, in addition to the genomic-ERα pathway [105], the MISS-ERα pathway also plays a role in promoting intercellular communication during mammary gland development [106]. Since E4 is characterized as an antagonist and an agonist of the MISS pathway depending on the tissue, it is important to fully characterize the molecular impact of E4 on mammary gland biology. In breast cancer, preclinical and clinical studies have shown that E4 formulations have both pro-tumoral and pro-apoptotic effects depending on the dose and whether or not it is combined with endogenous or exogenous E2. The molecular mechanisms leading to the anti-estrogenic action of E4 in particular, remain to be fully elucidated.

In conclusion, E4 does not meet every characteristic of the ideal estrogen. However, in comparison to other conventional estrogens, E4 does meet several important criteria. E4 could be considered as a friend of the mammary gland, even when combined to progesterone or DRSP to protect the endometrium of non-hysterectomized women, since it remains neutral on preclinical models of breast cancer at a dose that is effective at preventing hot flushes; a symptom of menopause that arises in 80% of postmenopausal women. However, at high dose, E4 remains a foe of the mammary gland highlighting the importance of exerting extreme caution when determining the dose required for management versus prevention of mammary side effects. E4 also displays cardioprotective features against atherosclerosis and has a limited impact on liver factors involved in coagulation, supporting a lower risk of thromboembolic events and thromboembolism. These features support a safer profile in terms of breast cancer risk and thromboembolism risk making E4 a safer estrogenic treatment option for women.