Evaluation of the Concentration of Selected Elements in Patients with Cancer of the Reproductive Organs with Respect to Treatment Stage—Preliminary Study

(1) Background: The aim of this study was to assess the concentrations of selected elements in female patients with cancer of the reproductive organs, taking into account the stage of treatment. (2) Methods: The study sample consisted of 51 patients with advanced endometrial cancer and ovarian cancer, undergoing chemotherapy. The median age of the studied patients with endometrial cancer was 66.0 years (IQR: from 60.75 to 70.25), and with ovarian cancer―60.0 years (IQR: from 49.0 to 64.0). Each of the qualified women, after consent to participate in the study, had her blood drawn several times (before surgery, the first course of chemotherapy, the third course of chemotherapy, and the sixth course of chemotherapy) in order to determine serum levels of macro- and micronutrients (Na, Mg, Ca, Zn, P, Cu, Fe, Cd, Ni, and Sr). (3) Results: In the study group of patients with cancer of the reproductive tract, the concentrations of iron (<0.001), magnesium (0.038), sodium (0.014), and nickel (0.037) varied significantly over the course of the study. The analysis showed that the interaction between the stage of chemotherapy and the type of cancer had an effect on the concentrations of magnesium and cadmium (p < 0.05). (4) Conclusions: In the studied group of patients with ovarian and endometrial cancer, the applied chemotherapy significantly changed the concentrations of Fe, Na, and Ni, regardless of the type of tumor. Changes in Mg and Cd concentrations resulted from the interaction between the stage of chemotherapy and the type of cancer. The results of serum concentrations of selected elements in women with cancer of the reproductive organs may help understand the physiological changes resulting from the applied chemotherapy.


Introduction
Neoplastic diseases of the female reproductive organs are a serious health and social problem because the diagnosis of oncological disease, the commencement of therapy, and functioning after the disease change women's lives completely, often evoking feelings of insecurity, threat, and loss of femininity. These conditions are associated with a variety of both physical and emotional complaints. Oncological treatment (radiotherapy and chemotherapy) entails a number of complications. Consequently, all these aspects affect the quality of women's lives, both during the treatment and after its completion. The most common cancer of the female reproductive organs is endometrial cancer, which is usually

Materials and Methods
The study involved 51 patients treated in the Department of Gynecological Surgery and Gynecological Oncology of Adults and Adolescents, the Pomeranian Medical University in Szczecin. Informed consent was a prerequisite for participation in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Bioethical Commission (Approval no. KB-0012/81/18). Patients with advanced ovarian and endometrial cancer undergoing first-line chemotherapy and systemic treatment for recurrence were included in the study. For ovarian cancer, patients with primary cancer underwent surgery followed by chemotherapy, i.e., 6 courses of chemotherapy based on platinum analogs and paclitaxel, or, in the case of incomplete surgical treatment, additionally received 18 administrations of bevacizumab. For recurrent disease, the choice of chemotherapy depended on platinum sensitivity. Patients with endometrial cancer, in advanced cases, were treated with surgery followed by chemotherapy and radiotherapy. Chemotherapy regimens were based on platinum analogs and paclitaxel in the amount of 6 courses/cycles administered at 21-day intervals. Doxorubicin regimens were used in relapse.

Study Design
The research procedure was divided into three parts: Anthropometric measurements; • Serum biochemical analysis.
First, participants were asked about basic sociodemographic data (age, place of residence, employment status, education, and marital status) as well as menstruation, family history of cancer, medications taken, and physical activity. Anthropometric measurements were taken on an empty stomach, in light clothes, without shoes, after emptying the urinary bladder using an electronic scale with a height gauge. Based on the obtained data, the body mass index (BMI) was calculated-the range of 18.5-24.9 kg/m 2 was regarded as normal, overweight was defined as having a BMI in the range of 25.0-29.9 kg/m 2 , and obesity-BMI of 30 kg/m 2 and more. Following the study protocol, each of the qualified women, after consent to participate in the study, had her blood drawn several times (before surgery, the 1st course of chemotherapy, the 3rd course of chemotherapy, and the 6th course of chemotherapy). Venous blood (maximum 5.5 mL) was collected on an empty stomach (minimum eight hours from the last meal) using the Monovette system in order to determine serum levels of macro-and micronutrients (Na-the norm: 3104-3333 mg/L; Mg-the norm: 20-25 mg/L; Ca-the norm: 89-101 mg/L; Zn-the norm: 0.66-1.1 mg/L; P-the norm: 30-45 mg/L; Cu-the norm: 0.8-1.6 mg/L; Fe-the norm: 0.35-1.5 mg/L; Cd-the norm: 0.0049 mg/L; Ni-the norm: 0.002 mg/L; Sr-the norm: 0.05 mg/L). After obtaining the biological material, the blood was centrifuged. In total, 154 serum samples were obtained for further analysis, i.e., ovarian cancer (n = 81) and endometrial cancer (n = 73). The separated serum was kept frozen at −80 • C until analysis at the Department of Biochemistry and Medical Chemistry, Pomeranian Medical University in Szczecin.

Determination of Biochemical Parameters
Determinations of biochemical parameters were performed in a certified laboratory of the Pomeranian Medical University in Szczecin using commercial, standardized methods.

Analysis of Serum Element Concentrations
All samples were transferred into 1.5 mL microtubes and stored at −80 • C until processed. Samples were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, ICAP 7400 Duo, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a concentric nebulizer and a cyclonic spray chamber to determine their Ca, Zn, Cu, Fe, Na, Sr, P, Mg, Cd, and Ni content. Analysis was performed in radial and axial mode. The samples were thawed at room temperature and digested using the microwave digestion system MARS 5, CEM. The volume of the sample given to the research was 0.75 mL.
The samples were transferred to clean polypropylene tubes; 4 mL of 65% HNO 3 (Suprapur, Merck, Darmstadt, Germany) was added to each vial, and each sample was allowed 30 min pre-reaction time in the clean hood. After completion of the pre-reaction time, 1 mL of non-stabilized 30% H 2 O 2 solution (Suprapur, Merck, Darmstadt, Germany) was added to each vial. Once the addition of all reagents was complete, the samples were placed in special Teflon vessels and heated in microwaved digestion system for 35 min at 180 • C (15 min ramp to 180 • C and maintained at 180 • C for 20 min). At the end of digestion, all samples were removed from the microwave and allowed to cool to room temperature. In the clean hood, the samples were transferred to acid-washed 15 mL polypropylene sample tubes. A further 5-fold dilution was performed prior to ICP-OES measurement. The volume of 2 mL was taken from each digest. The samples were spiked with an internal standard to provide a final concentration of 0.5 mg/L Ytrium, 1 mL of 1% Triton (Triton X-100, Sigma, Darmstadt, Germany), and diluted to the final volume of 10 mL with 0.075% nitric acid (Suprapur, Merck, Darmstadt, Germany). The samples were stored in a monitored refrigerator at a nominal temperature of 8 • C until analysis.
Blank samples were prepared by adding concentrated nitric acid to tubes without sample and subsequently diluted in the same manner as described above.
Multi-element calibration standards (ICP multi-element standard solution IV, Merck and Phosphorus ICP Standard (AccuStandard, Inc., New Haven, CT, USA) were prepared with different concentrations of inorganic elements in the same manner as blanks and samples. Deionized

Statistical Analysis
All analyses in the present study were performed in MedCalc (ver.20.007, Ostend, Belgium) and R software (https://www.r-project.org, accessed on 16 February 2022). First, for clarity, raw measurements were presented separately to give the reader an overview of the data. In the case of values below the limit of detection (in particular for Cd, Ni, and Sr), the lowest measurable value was used in a dataset. This approach, however, was only used to present basic descriptive statistics and for correlation analyses by Spearman's method.
For further calculations, the data were log-transformed to conform to normality. We performed the repeated measures ANOVA with two corrections based on the estimates of sphericity by Greenhouse and Geisser or Huynh and Feldt as appropriate. Next, we utilized linear mixed models followed by the maximum likelihood ratio test to deal with missing data and to search for the interaction between the group (type of cancer) and the stage of therapy. Where necessary, Satterthwaite's method was used to obtain the p values. The dependent variable was the log concentration of a particular tested element, while the independent ones were the stage of therapy and the type of cancer (both fixed). The Patient ID was the only random variable. The statistical significance was set as p < 0.05.

Characteristics of the Participants
The study involved 51 women diagnosed with ovarian (n = 26, 52%) or endometrial (n = 24, 48%) cancer. The age of patients suffering from endometrial cancer was significantly higher compared to that of women with ovarian cancer (median: 66.0 years old; IQR: from 60.750 to 70.25 vs. median: 60.0 years; IQR: from 49.0 to 64.0, p < 0.0061). The women in both groups had their first period at the age of 14 years (p = 0.4546). The BMI of the participants remained stable over time (p = 0.864). However, in terms of weight status over time, we noticed that obesity was more prevalent in women with endometrial cancer. Other variables are shown in Table 1.

The Concentrations of the Tested Elements at the Surgery and during Chemotherapy
The basic descriptive statistics of the tested elements are shown in Table 2. In patients with ovarian cancer, an increase in Fe concentration was observed in the subsequent stages of treatment, as the mean level before the procedure oscillated around 1.1620 (SD = 0.6060), reaching higher values before the third and sixth cycle of chemotherapy, 2.2090 ± 1.1307 and 2.2000 ± 0.9382, respectively. The increase in Fe concentration in patients with uterine cancer was also evident with time; before surgery (1.175 ± 0.6189) vs. the third cycle (1.829 ± 0.8677) and the sixth cycle (1.987 ± 0.8455). The decrease in Mg concentration was observed especially in patients with uterine cancer: the initial mean value was 26.831 (SD =2.5907), whilst before the sixth cycle of chemotherapy, it decreased to 22.082 (SD =3.4344). Such a decrease was also noticed in patients with ovarian cancer but to a smaller extent. Electrolyte concentration alterations-especially in regard to lowering levels of Na and P along with the treatment-were demonstrated in both groups of patients. In turn, Ni concentrations increased along with the treatment in both groups. For further analysis, the data were log-transformed. We noticed that the concentrations of Fe (<0.001), Mg (0.038), Na (0.014), and Ni (0.037) differed significantly by the stage of therapy. The results are presented in Table 3.

The Concentrations of the Tested Elements by the Stage of Therapy and the Type of Cancer
In the linear mixed model, we were able to demonstrate the effect of the interaction between the stage of therapy and the type of cancer on the concentrations of Mg ( Figure 1) and Cd (Figure 2). In the case of both tested elements, the changes in concentrations observed along the study time points were significantly associated with cancer type. In the studied group of patients with cancer of the reproductive tract, serum magnesium (Mg) concentrations correlated with the cycle of chemotherapy and the type of cancer. However, due to the very limited number of raw data concerning Cd, these results should be treated with some caution. The details are shown in Table 4.
the studied group of patients with cancer of the reproductive tract, serum mag (Mg) concentrations correlated with the cycle of chemotherapy and the type of However, due to the very limited number of raw data concerning Cd, these results be treated with some caution. The details are shown in Table 4.

The Correlations between the Concentrations of the Tested Elements and the Stage of Therapy
In the following analysis, we treated the stage of therapy-being interval vari as quantitative data. We demonstrated that the concentration of Fe was po correlated with the stage of therapy irrespective of the type of cancer (Table 5).

The Correlations between the Concentrations of the Tested Elements and the Stage of Therapy
In the following analysis, we treated the stage of therapy-being interval variables-as quantitative data. We demonstrated that the concentration of Fe was positively correlated with the stage of therapy irrespective of the type of cancer (Table 5).

Discussion
Monitoring serum levels of elements can be a helpful diagnostic tool in the prevention and early detection of many neoplastic diseases [28]. Thus, the analysis of the concentrations of individual bioelements in the body may help objectivize the results of various studies. Many authors confirm the correlation between selected elements and specific disease states at certain stages of a woman's life. According to the current state of knowledge, macro-and microelements regulate the development and functioning of immune system cells. In turn, modulation of the immune response by these components may be an effective method in reducing the risk of disease and/or treating certain diseases, but their role in immune regulation and in pathologies of the immune system has not yet been fully understood [28]. It is very important to maintain the correct proportions between elements because both their deficiency and excess may lead to physiological disorders [29].
Numerous studies confirm that oxygen free radicals and associated inflammation play an important role in the multifactorial pathogenesis of ovarian cancer, in addition to genetic, epigenetic, and hormonal factors [30]. According to Roychoudhury's team, copper, by forming complexes with gonadoliberin, increases the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which may contribute to the development of ovarian cancer [31]. Gupte et al. demonstrated elevated copper levels in, among others, patients with ovarian and cervical cancer. Higher copper levels were directly related to cancer progression. According to the researchers, this element is involved in the angiogenesis of the tumor growth process [21]. Our study did not show any differences in serum copper concentrations between the studied patients, regardless of the diagnosed cancer (ovarian cancer vs. uterine cancer) and the stage of oncological treatment. On the other hand, the study by Atakul et al. demonstrated lower blood Cu and Zn concentrations as well as a lower Cu/Zn ratio in women with endometrial cancer compared to the healthy group. The results also confirm the correlation of Cu and Zn levels with age [32]. Rzymski et al. observed elevated cadmium (Cd) levels in neoplastic endometrial tissues, as well as altered Cu and Zn values [33]. Our results showed that cadmium levels were significantly affected by tumor type and the cycle of chemotherapy administered. In ovarian cancer, Cd levels increased in the third cycle of chemotherapy and then slightly decreased in the sixth cycle. In endometrial cancer, on the other hand, the concentration slightly increased in the first cycle of chemotherapy, then decreased in the third cycle, and remained at a similar level in the sixth cycle.
The influence of low magnesium concentrations on the treatment outcomes, prognosis, and survival of patients with advanced ovarian cancer undergoing chemotherapy is not fully understood [34]. The study by Castiglioni and Maier confirmed that serum magnesium levels in patients with precancerous lesions are decreased regardless of the therapy administered and correlate with the stage of cancer [35]. The results obtained by Yao et al. [36] showed that the concentration of magnesium decreased at the end of the first week of radiotherapy and after the introduction of chemotherapeutic agents that cause magnesium loss, such as cisplatin [37][38][39][40][41][42][43][44]. Among the side effects of chemotherapy, nephrotoxic implications and electrolyte disturbances, especially hypomagnesemia, are most commonly described [45]. In our research, the concentration of Mg correlated with the stage of treatment/cycle of chemotherapy. In the group of patients with endometrial cancer, the level of magnesium decreased in the first and third cycle of chemotherapy; then, it slightly increased during the sixth course. In patients with ovarian cancer, on the other hand, Mg levels decreased in the first and sixth cycles, while an increase was obtained in the third cycle.
Undoubtedly, the process of carcinogenesis may be influenced by changes in iron metabolism. In women, the level of this element depends on the period of life. In menstruating women, anemia and iron deficiency are associated with elevated levels of vascular endothelial growth factor (VEGF), which contributes to tumor angiogenesis. High concentrations of estrogens also play a negative role because free radicals produced in the metabolic cycle of estradiol contribute to the release of iron from ferritin, which, when unbound to protein, contributes to DNA damage. After menopause, the cessation of menstruation minimizes the loss of iron; however, overloading the body with this element may enhance the process of carcinogenesis as a result of accelerated production of reactive oxygen species (ROS) [46,47]. Another important and still unclear issue regarding Fe is the effect of cisplatin (CDDP)-based chemotherapy on the systemic balance of this metal. Yamashita and Nakamura's teams confirm an increase in serum Fe levels in patients undergoing cisplatin-based chemotherapy [48,49]. Changes in iron levels after the first day of high doses of cisplatin in cancer patients were indicated by Sartori et al. [50], who observed in these patients hypersideremia developing within 24 h [51]. A significant increase in iron levels found in patients with ovarian cancer at the end of a 5-day cisplatin course was shown in the study by Poller et al. [52]. We also observed a significant effect of the applied chemotherapy on the concentration of iron. An increase in Fe levels was observed in both ovarian and endometrial cancer patients during the third and sixth cycles of chemotherapy.
Electrolyte balance plays a vital role in the functioning of cells and organs. Patients with cancer are particularly vulnerable to electrolyte disturbances. The study by Omoyajowo et al. carried out to estimate the level of electrolytes in a group of patients with uterine cancer showed a significant increase in the concentrations of PO 4 3− (p < 0.05) and Na + (p < 0.0001). There was no significant difference in the concentration of K + in this study (p > 0.05). The authors argue that electrolyte imbalance determines the prognosis, diagnosis, and management of uterine cancer [53]. In our study, sodium levels in patients in both groups (ovarian cancer vs. endometrial cancer), compared to the results obtained before surgery, decreased significantly with subsequent cycles of chemotherapy.
Malignant neoplasms of female genital organs are diseases with a serious prognosis, taking a heavy toll on the body and its performance. While treating the underlying disease, i.e., cancer, it is also necessary to fight the side effects of cancer therapy. Further research on trace element monitoring may determine the effectiveness of cancer treatment and thus have a positive impact on treatment results and extend the survival time of patients.

Limitations
In our study, we did not perform a detailed analysis regarding the use of vitamin and mineral supplements by the studied patients. Our goal was to make a preliminary assessment of the concentrations of selected elements. We were afraid that the participants might hide the truth or provide unreliable information if they were asked more detailed questions about the vitamin preparations they were taking.

Conclusions
(1) In the studied group of patients with ovarian and endometrial cancer, the applied chemotherapy significantly changed the concentrations of Fe, Na, and Ni regardless of the type of tumor.
(2) Changes in Mg and Cd concentrations result from the interaction of the duration of chemotherapy and the type of tumor.
(3) The results of serum concentrations of selected elements in women with cancer of the reproductive organs may help understand the physiological changes resulting from the applied chemotherapy.