1. Introduction

Breast cancer (BC) is a malignant growth that initiates in the lobules/ducts lining cells within the breast glandular tissue and its global prevalence continued an upward trend [1, 2]. In 2020, >2 million newly diagnosed BC patients and about 700,000 BC-related deaths occurred globally, contributing to both great psychological and economic costs and influencing the long-term quality of life [3]. BC is heterogeneous with specific traits that affect predictive and prognostic outcomes of cases after and before menopause [4]. The disparities in cancer features between old and young females may be associated with risk factors variations such as ethnicity or race, reproductive history and body size [4].

Although menopause does not cause BC, risk of developing BC elevates as a woman ages [5]. A female who experiences menopause after 55 years old has an elevated risk of uterine, breast and ovarian tumors [5]. Longer estrogen exposure elevates a female’s risk of BCs [6]. Thus, due to hormonal considerations, females who have experienced natural menopause are approximately twice as likely to develop BC [5]. In different age groups, cancer features and biological biomarkers play an important role in predicting outcomes [4]. Moreover, it is very important to evaluate factors significantly impact BC outcomes in postmenopausal BC patients [7].

About 80% of newly found cases in BC are hormone-dependent. It has been claimed that these hormones promote the formation and spread of tumors [8]. In contrast to the well-established relation between reproductive hormone levels and postmenopausal BC risk, it is unknown how a single circulating hormone detection can predict subsequent BC risk [9]. In this regard, early research suggests that luteinizing hormone (LH) may play a part in carcinogenesis. This hormone regulates cell migration and invasion in BC cells with functioning LH receptors [10]. Prolactin (PRL) plays a major part in BC development and other hormonally sensitive tumours, including endometrial, ovarian, lung and pancreatic cancers, in addition to stimulating the growth of mammary glands [11]. Also, a meta-analysis’s findings showed a favourable correlation between BC development and higher PRL levels [12]. Moreover, progesterone (PRG) also may have a function in BC etiology, and, to cure or prevent BC, there is interest in decreasing progesterone activity [13]. In the past, mechanistic studies implicate PRG in BC development, whereas limited data have not provided strong support for a risk association with blood levels [13]. Recently there is notable clinical and experimental evidence that PRG, at its physiological levels and alone, is incapable of develop BCs, thus its standing as a “carcinogenic” hormone is undeserved [14]. From another hand, PRG natural chemical structure is quite different from chemically synthetic progestins, which, at the cell level, causes different actions [15]. Compared to synthetic progestins, a meta-analysis including 86,881 postmenopausal females found that that the use of natural PRG was related to a markedly lower BC risk [16]. Despite all of this, and regarding disease progression, it is suggested that PRG may promotes pre-neoplastic BC progression in the mature breast epithelium by stimulation of cyclical mammary stem cell proliferation or occult tumor initiating cells [13]. Moreover, it is suggested that BC progression is a result of PRG/progesterone receptor (PR) signaling and a switch from paracrine to autocrine proliferation regulation [13].

From another hand, follicle-stimulating hormone (FSH) and its receptor (FSHR) have an important function in several tumors, including ovarian [17], endometrial [18] and prostate [19] cancer. Via activating adenylyl cyclase and subsequent elevated cyclic adenosine monophosphate levels, FSH-FSHR induces cancer cell proliferation, differentiation and metastasis [20]. Although BC tissues do not express FSHR, elevated FSH levels have been linked to a markedly worse prognosis in BC cases [21].

The aim of this study is to assess the role of FSH, LH, PRL and PRG hormones blood levels in BC development in postmenopausal Egyptian women. Also, this study aimed to determine whether these reproductive hormones impact BC aggressiveness features, including late tumor stages, high grades, large size, lymph node invasion, distant metastasis and the negative expression of progesterone receptor, estrogen receptor and human epidemic growth factor receptor-2 (HER2) protein.

2. Material and methods

3. Results

3.2 Reproductive hormones and BC severity

There was no significant association between levels of PRL and PRG and BC advanced features (Table 3). In contrast, both FSH and LH (Table 4) elevated circulating levels were significantly (p < 0.05) associated with the disease progression including tumor late stages, lymph node invasion, high grades, large size and negative estrogen receptor status. Although high FSH levels were not associated (p > 0.05) with negative progesterone receptor nor HER2 statues, these elevated levels were associated (p = 0.048) with triple negative subtype. However, LH elevated levels were associated with patients with negative HER2 (Table 4). Moreover, both FSH (Fig. 1A,B) and LH (Fig. 1C,D) were significantly correlated with CEA and CA 15.3 in postmenopausal BC patients.

Table 3.Impact of PRL and PRG levels on BC progression in postmenopausal patients. Data were expressed as median (inter quartile range).

Categories	Prolactin (ng/mL)	p value	Progesterone (ng/mL)	p value
Primary tumor stage
	Early stage (T1–T2)	4.1 (2.4–7.2)	0.2649	1.2 (0.9–2.4)	0.9250
	Late stage (T3–T4)	3.2 (2.3–4.2)	1.1 (0.85–3.2)
Lymph node invasion
	Negative (N0)	3.8 (2.2–7.2)	0.8961	1.1 (0.84–2.6)	0.9143
	Present (N1)	3.4 (2.4–5.7)	1.2 (0.9–3.2)
Metastasis
	Negative (M0)	3.6 (2.4–7.2)	0.3181	1.1 (0.9–2.5)	0.2566
	Present (M1)	3.4 (2.2–3.9)	2.2 (0.9–3.4)
Tumor histological grade
	Low grade (G1–G2)	2.9 (2.0–5.2)	0.0789	1.5 (0.9–2.6)	0.5007
	High grade (G3)	3.7 (2.8–7.9)	1.1 (0.84–3.6)
Tumor size
	Small (≤2 cm)	3.5 (2.3–6.9)	0.6614	1.3 (0.9–2.5)	0.6669
	Large (>2 cm)	3.6 (2.4–5.4)	1.0 (0.84–3.6)
Progesterone receptor
	Negative	3.4 (2.2–4.5)	0.3714	1.1 (0.8–2.5)	0.4847
	Positive	3.8 (2.4–7.2)	1.1 (0.9–3.6)
Estrogen receptor
	Negative	3.7 (2.2–4.4)	0.7910	0.9 (0.7–1.6)	0.0201
	Positive	3.3 (2.4–6.9)	1.3 (0.9–3.6)
HER2
	Negative	3.2 (2.2–7.0)	0.5672	1.1 (0.84–2.5)	0.8144
	Positive	3.7 (2.5–6.1)	1.2 (0.9–3.4)
	Triple negative	3.0 (2.2–4.1)	0.1370	1.5 (0.7–2.5)	0.7590
	Other subtypes	3.6 (2.4–7.2)	1.1 (0.9–3.2)
HER2: human epidemic growth factor receptor-2.

Table 4.Impact of FSH and LH levels on BC progression in postmenopausal patients. Data were expressed as median (inter quartile range).

Categories	FSH (µIU/mL)	p value	LH (µIU/mL)	p value
Primary tumor stage
	Early stage (T1–T2)	13.1 (6.2–22.6)	0.0010	8.2 (4.1–11.0)	0.0019
	Late stage (T3–T4)	25.3 (16.1–33.7)	12.4 (8.9–19.9)
Lymph node invasion
	Negative (N0)	13.2 (6.1–21.9)	0.0069	6.4 (3.9–10.6)	0.0021
	Present (N1)	23.4 (12.9–30.7)	11.5 (9.0–17.4)
Metastasis
	Negative (M0)	20.1 (12.2–29.8)	0.2763	10.3 (5.6–15.1)	0.7303
	Present (M1)	23.0 (10.3–28.8)	9.4 (5.7–12.3)
Tumor histological grade
	Low grade (G1–G2)	13.7 (10.3–26.4)	0.0218	6.9 (4.2–11.1)	0.0050
	High grade (G3)	24.9 (13.8–32.5)	11.5 (9.3–17.4)
Tumor size
	Small (≤2 cm)	11.3 (5.3–18.9)	0.0025	5.1 (2.9–8.4)	0.0001
	Large (>2 cm)	23.4 (13.7–30.3)	12.5 (9.4–19.9)
Progesterone receptor
	Negative	18.7 (11.0–31.4)	0.7073	10.2 (7.1–17.1)	0.3635
	Positive	18.9 (11.8–27.5)	10.0 (4.2–13.3)
Estrogen receptor
	Negative	28.4 (17.1–36.2)	0.0169	12.5 (9.2–20.8)	0.0115
	Positive	14.9 (10.9–27.0)	9.4 (4.5–12.6)
HER2
	Negative	18.9 (11.9–30.5)	0.4544	11.4 (8.14–19.0)	0.0461
	Positive	18.2 (10.9–27.5)	8.2 (4.2–12.5)
	Triple negative	29.3 (19.6–36.1)	0.0480	11.4 (7.5–15.8)	0.4180
	Other subtypes	17.2 (11.3–27.5)	10.0 (5.2–13.6)
FSH: follicle-stimulating hormone; LH: luteinizing hormone; HER2: human epidemic growth factor receptor-2.

Fig. 1.Correlation between FSH and both (A) CEA (r = 0.508) and (B) CA 15.3 (r = 0.439) serum levels revealed a significant (p < 0.001) association in postmenopausal breast cancer patients. Similarly, LH was significantly correlated with (C) CEA and (D) CA 15.3 (r = 0.263 and 0.271, respectively). CEA: carcinoembryonic antigen; CA 15.3: cancer antigen 15.3; FSH: follicle-stimulating hormone; LH: luteinizing hormone.

4. Discussion

BC is a heterogeneous disorder with varied response to therapy, behavior, molecular features and morphological appearances [23]. Recently, molecular targeted therapy, treatment and individualized diagnosis for BC cases have become topics of intense research [24]. How to enhance BC patients’ long-term prognosis and the overall survival rate has increasingly become the focus of clinicians [24]. It is, therefore, vital to evaluate risk factors associated with BC severity to improve or prevent the disease prognosis.

The prognostic importance of FSH, LH, PRL and PRG reproductive hormones in BC was investigated in this research. These hormones levels were evaluated in postmenopausal BC patients and healthy controls. Among BC patients, the association between circulating hormones levels with BC aggressiveness behavior was also assessed.

As the exclusion criteria in this study included all patients with chronic diseases, such as liver and kidney diseases, the platelet count, haemoglobin levels, red and white blood cells counts, as well as liver and kidney function tests did not change significantly (p > 0.05) between patients and controls. Although there was no significant (p > 0.05) difference in these hormones levels between BC cases and healthy controls, both FSH and LH blood levels were significantly related to BC progression including tumor late stages, lymph node invasion, high grades, large size and negative estrogen receptor status. LH elevated levels were also associated with negative HER2 expression. Elevated levels of FSH were associated (p = 0.048) with triple negative subtype. Moreover, both of them were significantly correlated with CEA and CA 15.3 in postmenopausal BC patients. Conversely, in each PRL and PRG there was no significant (p > 0.05) association with these tumor features.

Maintained levels of these hormones after menopause can be explained in the light of some evidence that have been previously confirmed in previous studies. Ovarian aging earliest hormonal evidence is the selective FSH increase owing to decreasing inhibin B (granulosa cell number marker) levels [25]. With the ovulatory cycles loss and the decrease in ovarian function, gonadotropin-releasing hormone (GnRH) pulses occur more commonly [26] while decreasing estradiol concentrations permit elevates in both GnRH [27] and in its related gonadotropin responses [28]. After menopause in the medial basal hypothalamus, some autopsy studies suggest an elevate in GnRH expression [29]. This effect is mediated by a decrease in the inhibitory neuropeptide, dynorphin, and an increase in the stimulatory neuropeptides, kisspeptin and neurokinin B [30]. Moreover, in postmenopausal women, FSH and LH disappearance half-life is prolonged as a consequence of changes in these 2 glycoprotein hormones isoform composition with the estradiol loss [31, 32].

Several reproductive factors are related to BC risk, potentially through a hormonal pathway [33]. In Shi study, they evaluated whether or not high blood FSH causes great severity of BC of both pre- and post-menopausal patients [34]. Compared to benign breast tissue vessel, they found that FSH receptor was highly expressed on endothelium of BC vessel [34]. Also, it was reported that FSH promoted the angiogenesis on human umbilical vein endothelial cells (HUVEC) as it elevated matrix metalloproteinase expression, activated phosphorylation of a wide range of kinases and calcium influx and stimulated HUVEC invasion, migration and proliferation [34]. Their clinical investigation revealed a linear positive association between microvessel density of BC and FSH level [34].

In accordance with our results, Sanchez et al. [35] found in T-47D BC cells that both FSH and LH alter the expression of genes implicated in invasion, motility and adhesion via their receptors activation. They reported that LH affected the expression of varied genes involved in tumor biology of these BC cells [35]. In in vivo rat model of BC progression, they found a direct association between these gonadotrophins and the tumor growth extent and, thus, they highlighted that these hormones could potentially associate with BC progression, particularly in post-menopausal cases who typically have high gonadotrophin levels [35]. From another hand, Sheng et al. [18] findings suggested that FSH stimulates endometrial cancer metastasis and proliferation.

At the cellular level, BC development is impacted by the cytokines and hormones secreted by the ovary and the placenta (reproductive factors) [36]. The majority of BCs (about 80% of new cases) depend on hormones [8, 36]. In this setting, these hormones are known to promote cancer development and progression [10]. By controlling many kinases that activate actin cytoskeletal proteins, LH hormone can regulate cell invasion and migration in BC cells that express its functional receptors (LHR) [10]. Via rapid extra-gonadal LHR signaling to the Rous sarcoma virus oncoprotein (Src)/Focal Adhesion Kinase/paxillin pathway, Mondaca et al. [10] showed that LH induces paxillin phosphorylation and this process results in the phosphorylation/activation of the nucleation promoter factors cortactin and N-Wiskott-Aldrich syndrome protein (N-WASP). As a consequence, Arp2/3 complexes induce actin polymerization, required to promote cell invasion, migration and adhesion, so stimulating tumor cells metastatic spread [10]. Similarly, some studies have reported the impact of FSH receptors (FSHRs) on various BC cell lines, such as T-47D, MCF-7, MDA-MB-231 and SK-BR-3. These cancerous cells proliferation and survival may be enhanced by FSH [37]. Moreover, FSH, as an estrogen main regulator, may related to estrogen dysregulation, further influencing the BC pathogenesis. In postmenopausal patients, interactions between estrogen and FSH may explain BC treatment complexity [37].

Preoperatively elevated serum levels of CEA and CA 15.3 have suggested by previous studies to be significantly related to BC severity in the multivariate analysis including advanced stage, axillary node metastasis and large tumor size. Also, compared to those with normal levels, BC cases with elevated CEA and CA 15.3 levels exhibited a worse BC prognosis, even in stage-matched analysis [38, 39]. In this study, both FSH (r = 0.508 (p = 0.0001); r = 0.439 (p = 0.001), respectively) and LH (r = 0.263 (p = 0.040); r = 0.271 (p = 0.042), respectively) elevated levels were significantly correlated with CEA and CA 15.3 in postmenopausal BC patients.

Traditionally, BC is assorted in the light of the presence of some receptors (progesterone (PR), estrogen (ER), human epidemic growth factor receptor-2 (HER2)) [40]. Molecular classification based on immunohistochemistry reveals a close association of these receptors with BC prognosis. In BC patient, this step is considered as a standard regimen for selecting the most useful approaches [41]. The initial BC predictive molecular biomarkers are PR and ER as cases with positive receptors generally possess high hormone therapy sensitivity [41]. Also, another oncogene related to BC progression is the gene coding HER2, and an abnormality in HER2 expression has close relation with BC development, onset, metastasis and invasion [41]. Another important preliminary result of this study that needs further evaluation and future studies is that elevated FSH and LH levels were associated with negative estrogen receptor and HER2 expression.

Despite our important result, some limitations including retrospective nature and single-center cohort may exist. Also, this study, owing to limited resources, lake adjustment for some potential confounding variables that may influence fertility hormones including menopause timing, age, adiposity and behavioral factors. Thus, further multicenter studies including additional cohort are required to validate these observations.

5. Conclusions

Our findings endorse that FSH and LH elevated levels may have extragonadal effects on BC progression and poor outcomes. Therefore, we encourage initiatives to integrate the measurement and management of such hormones into the follow-up of BC patients. Especially in post-menopausal females with high gonadotrophins circulating levels, it may provide new perspectives for therapeutic drugs.

Availability of data and materials

Data supporting the reported results are available from the corresponding author upon request.

Author contributions

MAA—conceptualized the study. MAA, SSM, MAEAE, AA—investigation and experimental work. NHA, MMES—supervision and validation of the study. AA—samples providing and collection. All authors read and approved the final manuscript.

Ethics approval and consent to participate

According to the 1975 Helsinki Declaration’s ethical principles, the study protocol was accepted by the Ethics and Scientific Committees of Mansoura University (R.21.o7.1376). Clinicopathologic information and serum samples were obtained from each patient prior to the initiation of any particular therapies, following informed consent.

Acknowledgment

Authors want to thank the staff of Surgical Oncology Department, Mansoura Oncology Centre, Faculty of Medicine, Mansoura University, Mansoura, Egypt for their kind assistances in samples collection.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.