Effects of tamoxifen on the reproductive system of female breast

Tamoxifen (TMX) is regarded as standard treatment for Background: breast cancer (BC) patients‎. In recent years, several studies have reported gynecological side effects and due to TMX's estrogenic effects. Here, we evaluate the side effects of TMX on the ‎endometrium and ovaries of female BC patients. This was an ultrasound-based cohort study conducted in three Methods: oncology centers in Baghdad, Iraq. A total of ‎‎255 female patients were included, 140 premenopausal (PreM) and 115 postmenopausal (PostM), with estrogen receptor (ER)-positive BC using TMX adjuvant hormonal treatment for at least three months after surgery and adjuvant ‎chemo/radiotherapy.‎ Ultrasound (US) on the endometrium and ovaries of the women following ‎BC surgery/chemotherapy (baseline) and at 3, 6, 12, and 24 months following was performed‎. Data collected included age, menopausal status, co-morbid chronic illness and medications, including duration of TMX treatment. Presence of ovarian cyst was significantly higher in the PreM Results: ‎compared to PostM ‎women, while there were no significant differences for other gynecological findings.‎ At ‎baseline, endometrial thickness (ET) was significantly higher in the PreM compared to the PostM women. In both groups, women with increased ET became more frequent from baseline to 3 ‎months, from 3 to 6 ‎months, from 6 to 12 months, and from 12 ‎ to 24 months. At all time periods, ‎women with increased ET was ‎significantly higher in the PostM compared PreM women, resulting ‎in a risk of ET increase by 6 folds (ranging from 3 – ‎‎11 folds) ‎in PostM compared to PreM women. Longer duration of TMX is associated with increased ET. Conclusions: Duration of TMX did not appear to increase the risk of various gynecological outcomes, for example endometrial cancer rate was low. Finally, there was an increase in ET, which appeared to be six-folds higher in PostM compared to PreM women.‎


Introduction
Breast cancer (BC) is the second most prevalent cancer globally and the top cause of cancer-related deaths in women 1 . In Iraq, BC is the most prevalent cancer in the population. In 2012, BC occurred in 19.5% of all newly diagnosed cancer cases, and 34% of women with cancer 2 . Additionally, BC is the primary cause of cancer-related death in Iraqi women, causing 23.6% mortality among all women-related deaths).
Estrogen receptors (ER) have multiple functions that affect the reproductive, musculoskeletal and cardiac systems, and the central nervous system. Additionally, it has been suggested to be involved in the proliferation of BC tumors tissues 3 ; about 70-80% of BC cases are ER-positive. There are two classes of ER, α and β, and their activity is primarily regulated by estradiol (E 2 ) binding. Therefore, E 2 plays a vital role in the pathogenesis of BC through these receptors. Patients with these tumors are candidates for endocrine therapy after surgical removal of the primary tumor and treatment with ionizing radiation or cytotoxic chemotherapy 4 . The major strategy for BC treatment starts with surgery, chemotherapy, ionizing radiation therapy, endocrine (hormonal) therapy, and targeted therapy 5 .
Tamoxifen (TMX) is a selective ER modulator, which acts as an inhibitor for the effect of estrogen on breast tissues. Its side effects include vaginal dryness, vaginal discharge, flushes, cataracts, night sweats, thrombotic events, stroke, and rarely endometrial cancer and uterine cancer 6 . Many studies have examined the relationship between TMX use, duration of TMX use, whether female patients are premenopausal (PreM) or postmenopausal (PostM), and the development of gynecological side effects 7-14 . The present study aimed to identify if there is a relationship between TMX use and changes in the endometrium and ovaries of female breast cancer patients. In addition, the study aimed to classify if there are unreported gynecological side effects with TMX use, and whether menopausal status (PreM or PostM) leads to a different TMX effect on the endometrium and ovaries.

Study design
This is an ultrasound (US)-based cohort study, conducted in three oncology centers In Bagdad, Iraq. Female BC patients treated with TMX at dose of 20 mg/day as part of therapeutic regimen for the treatment of BC were included in the study. Abdominal US was used for the assessment of endometrial thickness (ET) and abnormalities in the endometrial cavity and ovaries.
All procedures performed in the study were in accordance with the ethical standards of the Institutional Research Committee at the College of Medicine, Baghdad University, who approved the study protocol (approval date: 3 rd December 2019; number: 2019/0234), and with the 1964 Helsinki declaration and its later amendments.
Written informed consent was obtained from all participants to be included in the study.

Study setting
The study was conducted at the three main oncology centers in Baghdad: Breast Cancer Center of Al-Elwia Teaching Hospital, Oncology Teaching Hospital at Baghdad Medical City, and Al-Amel Oncology Hospital. These oncology centers are teaching hospitals under the supervision of the College of Medicine, Baghdad University. Periods of recruitment were between December 2018 and May 2019. Data about patients (US findings) were obtained from their hospital records (each patient was followed-up for at least 6 months, with maximum duration of follow-up of 2 years).

Sample size calculation
A sample size of 256 women was calculated considering the power of 80%, the confidence level of 95% and a relative precision 5% and prevalence of ET of 12% in PreM and 10.6% in PostM based on a study by Lee et al. 2018 15 . Therefore, a 20% value was chosen, the following formula were used to calculate the estimated sample size: 2 4 . p q n d = Where, p is prevalence, q is (1 -p), and d is relative precision.

Participants
A total of 255 patients, 140 premenopausal and 115 postmenopausal female BC patients were included in the study.
We included female patients with ER-positive BC treated with TMX as an adjuvant hormonal treatment at a dose of 20 mg/day for at least three months after surgery and in addition to adjuvant chemo/radiotherapy.
We excluded patients who were on irregular treatment, patients with increased uterine thickness for any reason at baseline assessment, patients who had other gynecological or non-gynecological malignancies, patients with secondary BC and patients with incomplete or lost medical records.

Menopausal status
Hormonal levels and menstrual history were used for the assessment of menopausal status. A PreM woman was defined as a woman with regular menstrual cycles with follicular stimulating hormone (FSH) <40.0 mIU/mL. A PostM woman defined as a woman with amenorrhea longer than one year and FSH >40 mIU/mL on two sequential occasions at the time of diagnosis with BC 15 .

Data collection
All women that participated in the study were interviewed by the researchers; data were collected from them using a predesigned survey in a personal interview setting and from their medical records. The women were followed-up prospectively, and if the women had previous US reading it will be incorporated in study. Patient characteristics, US findings, and results of histopathological examination of endometrial biopsy whenever available were collected. The survey collected the following patient characteristics: age of the patient, menopausal status, co-morbid chronic illness and past medical history (e.g. hypertension, diabetes mellitus (DM)), duration of TMX use, side effects, and current used medications.

Abdominal ultrasonography
Abdominal US was used for the assessment of ET and abnormalities in the endometrial cavity and ovaries. Endometrial and ovarian assessment by US was performed before the start of hormonal therapy and alterations were periodically measured at an interval of three months. Assessment of the endometrial lining was done by calculating the maximum thickness from the outermost limits of the endometrial-myometrial juncture and the thickening was defined according to menopausal status as follows 16 : -PreM women with endometrial thickness ≥12 mm; -PostM women with endometrial thickness ≥ 5 mm.
Patients who were diagnosed with endometrial pathology were followed-up for six months to assess their status and dealt with accordingly. Patients diagnosed with ovarian cyst (> 30 mm on US) were further evaluated by cancer antigen 125 (CA-125); if normal, US examination was commenced every three months, if high CA-125, patients underwent further radiological assessment, including CT scan or MRI 15 .
Diagnostic hysteroscopy was done for the patients that showed abnormalities on US and biopsy was taken whenever indicated (when the US demonstrated uterine abnormalities or mass, and/or patients with abnormal uterine bleeding).

Statistical analysis
Chi-square test, Fisher's exact test, independent t-test, and paired t-test were used to compare between PreM and PostM groups. Logistic regression used to calculate the odds ratio (OR) and 95% confidence intervals. Linear regression analysis was performed to assess the relationship between different variables. SPSS 23.0.0 (Chicago, IL) and GraphPad Prism version 8.0.0 used to perform statistical analysis. P<0.05 was considered significant.

Results
In total, 255 women with BC were included in this study. Age range was 32 to 78 years, with mean age 50.4±9.0 years. The most common age group was 40-49 years (36.1%), followed by 50-59 years (32.5%). In total, 15.3% of the patients had DM, while 3.9% had hypertension (a higher frequency in the PreM compared to PostM women).
There was no significant difference in the history of hypertension, DM, use of medications (angiotensin-converting enzyme/ angiotensin receptor blocker, or metformin), cancer stage, and treatment duration of TMX between PreM and PostM women ( Table 1).
From US examinations, only the presence of ovarian cyst was significantly higher in the PreM compared to PostM women. Other US findings were not significant between groups (Table 2).
At baseline, ET was significantly higher in the PreM compared to the PostM group. In both groups, women with increased ET became more frequent from baseline to 3 months, from 3 months to 6 months, from 6 months to 12 months, and from 12 months to 24 months. At all time periods, the number of women with increased ET was significantly higher in the PostM compared with PreM group (Table 3; Figure 1).
ET was significantly higher in PostM compared to PreM women, resulting in an increased risk of endometrial thickening by 6 fold (OR: 6.000, 95%CI: 3.313 -10.867, p-value <0.001) in PostM compared to PreM women ( Figure 2). At baseline there was not a significant correlation between duration of TMX with ET; however from 3 months until 24 months after TMX therapy there was significant correlation between duration of TMX with EM thickness (Table 4).
There was no significant correlation between TMX treatment duration with any gynecological outcomes (Table 5).

Discussion
In the present study, mean ET after 24 months was 11.1±6.0 mm in PreM and 13.0±9.0 mm for PostM women (no significant difference between groups). This agreed with other studies 17,18 . In a retrospective study that examined 614 women with BC, 53 of them had history of TMX usage, and ET was significantly higher in women that received TMX (11 vs 6 mm in   Another significant finding in the present study was that the presence of ovarian cyst was significantly higher in PreM (27.9%) compared to PostM women (7.8%), which is in agreement with other studies 15,24 .

Study limitations
The retrospective nature of the study is a potential bias. In addition, the short duration of prospective follow-up (i.e. six months) also added limitation to the number of events observed the researcher.

Conclusions
Longer duration of TMX is associated with increased ET in Iraqi women with BC; however, the duration of TMX did not appear to increase the risk of various gynecological outcomes. In addition, endometrial cancer rate was low, and there was a high rate of ET, which appears to be six-folds higher in PostM compared to PreM women.

Data availability
Zenodo: Effects of Tamoxifen