Serum magnesium and calcium levels in infertile women during a cycle of reproductive assistance

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1 Magnesium Research 17; 3 (): ORIGINAL ARTICLE Serum magnesium and calcium levels in infertile women during a cycle of reproductive assistance Elena Grossi 1,, Sara Castiglioni 3, Claudia Moscheni 3, Patrizio Antonazzo 1,, Irene Cetin 1,, Valeria Maria Savasi 1, 1 Unit of Obstetrics, Gynecology, Department of Biomedical, Clinical Sciences, ASST Fatebenefratelli Sacco, Hospital L. Sacco via GB Grassi 74, Milan, Italy; Centre for Fetal Research Giorgio Pardi; 3 Departments of Biomedical and Clinical Sciences Luigi Sacco, Italy Correspondence: Elena Grossi. University of Milan, ASST Fatebenefratelli Sacco Via G. B. Grassi, 74, 157 Milan, Italy <elena_grossi@virgilio.it> Abstract. Magnesium (Mg) and calcium (Ca) are essential cations for women s preconception health. It is well known that, in blood, the concentration of ionized form of these two cations is temporally altered during menstrual cycle, suggesting a correlation between sex steroid hormones and serum calcium and magnesium levels. Evidence from literature suggests that in assisted reproductive technology increasing estrogens during ovarian hyperstimulation may also modulate serum magnesium and calcium levels. Therefore, we first examined total serum magnesium and calcium levels during follicular phase in a large population of infertile patients who underwent intrauterine insemination (IUI). The results were compared to a group of fertile women. Successively, we studied the total serum magnesium and calcium concentrations in infertile patients before and after ovarian hyperstimulation for in vitro fertilization (IVF). Results highlight that total serum concentration of magnesium and calcium does not seem altered in infertile women. During stimulation with gonadotropins, the values of the two cations do not change significantly in ovarian-stimulated women. However, we found a downward trend in the total magnesium and calcium levels in relation to the rising estrogens. Key words: infertility, intrauterine insemination, in vitro fertilization, estrogen, total serum magnesium level, total serum calcium level doi:1.1684/mrh In recent years, infertility in couples desiring a baby has increased dramatically. In 9, a report by the European society of human reproduction and embryology (ESHRE) regarding European data on assisted reproduction technology (ART) was published [1]. The report shows in Europe, a continuing increase of the number of treatment cycles. More than half a million of treatment cycles have been reported in 9 in Europe. Clinicians are continually committed to study the causes of infertility and the possible treatment to resolve it. In addition, the international federation of gynecology and obstetrics (FIGO) published in 15 a summary of recommendations on preconception nutrition. FIGO calls for increased awareness of the impact of women s nutrition on their health and their child s health, underlining the importance of not only food quantity but also food quality, in particular the micronutrients []. In our research, we have focalized on the following two minerals: calcium and magnesium. Calcium is important for a woman s preconception health, particularly for vascular function, muscle contraction, nerve transmission, and glandular secretion of hormones. Calcium is mobilized from the maternal skeleton during pregnancy for fetal bone growth and development. Therefore, 35 To cite this article: Grossi E, Castiglioni S, Moscheni C, Antonazzo P, Cetin I, Savasi VM. Serum magnesium and calcium levels in infertile women during a cycle of reproductive assistance. Magnes Res 17; 3(): doi:1.1684/mrh.17.41

2 E. GROSSI, ET AL. adequate intake is important to ensure adequate skeleton stores. Low calcium intake is implicated as hypertensive disorders during pregnancy []. Magnesium is the most common metal ion involved in the functioning of enzymes and acting as a cofactor in over 6 enzymatic reactions and as an activator for an additional enzymes [3]. The World Health Organization has stated that subclinical deficiencies of magnesium among the population prevail in both developed and developing countries [4]. A number of recent studies showed that the vast majority of European and United States population has a magnesium intake that falls below the daily-recommended amount. A percentage of subclinical magnesium deficiency between.5% and 15% in otherwise healthy women [5, 6] has been reported. Such datum is of importance, as magnesium deficiency may impact a number of conditions related to women s health. In particular, in infertile patients, relationship between total serum magnesium and calcium levels and ovarian stimulation is not well understood, as few articles have explored it in the literature. The most important article related to this aspect was published ten years ago by O Shaughnessy et al. and focused on the relationship between sex steroid hormones and the concentrations of ionized magnesium and calcium in the blood of patients who are undergoing in vitro fertilization [7]. This analysis was limited to 14 patients. O Shaughnessy et al. suggested that with each increment in estrogen level, a decrease in ionized Mg level occurred, and as the progesterone rose in the blood, the ionized Ca + /Mg + ratio increased. The main aim of our study was to evaluate total serum magnesium and calcium levels in a population of women who had diagnosis of infertility and underwent intrauterine insemination (IUI). We compared results in infertile population to total serum magnesium and calcium levels in a control group of fertile women. Successively, we evaluated if serum estrogen levels modulate the blood levels of circulating total magnesium and calcium in a group of hyperstimulated patients undergoing in vitro fertilization (IVF). Milan from November 14 to November 15. We decided to design the following two studies: the first one was an observational case-control study including a group of infertile women who underwent IUI and a control group of fertile women. Inclusion criteria for this study were the following: no relevant systematic disease, body mass index 3 kg/m, normal karyotype and normal follicular stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), thyroid-stimulating hormone (TSH), and tubal patency. For magnesium and calcium measurements, blood samples were collected in the initial follicular phase (during nd and 5 th days of menstrual cycle), prior to ovarian hyperstimulation. The same laboratory performed all the dosages. The second study that we planned was a prospective observational study including patients undergoing IVF and who met the inclusion criteria described above. These patients underwent controlled ovarian hyperstimulation (COH). Follicle monitoring began on day 3 of their cycle. Baseline transvaginal ultrasound (7. MHz, model General Electric company, Wauwatosa, WI, USA.) confirmed that there were no ovarian cysts or follicles >1 mm in mean diameter. Recombinant FSH was administrated until transvaginal ultrasound confirmed the presence of follicles 14 mm. Simultaneously, administration of gonadotropin-releasing hormone (GnRH) antagonist was started subcutaneously among the patients. Exogenous gonadotropin administration was discontinued and human chorionic gonadotropin (hcg) was administrated when one, two, or more of the developing follicles reached >18 mm mean diameter. Micronized progesterone (6 mg) was then administrated vaginally or orally after ovarian pick-up and continued throughout the luteal phase. In this second group, we measured total magnesium and calcium levels in each woman during follicular phase before the 3 rd day of menstrual cycle and at the end of administration of gonadotropins before the injection of 1, IU hcg (human Chorionic Gonadotropins; Msd Italia Srl, Italy) for trigger. Materials and methods The study was performed in our third level fertility centre situated in hospital Luigi Sacco in Hormone and cation assays The venous samples of all patients were analyzed in our reference laboratory. 36

3 Serum Mg and Ca in assisted reproductive technology The serum concentrations of progesterone and 17 -estradiol were analyzed using an automated electro chemo luminescence in immunoassay (ECLIA Elecsys/Cobas ; Roche Diagnostics International Rotkreuz, Switzerland) method. Total serum calcium and magnesium levels were analyzed using an integrated system named Architect ci8 from Abbott Diagnostics (Abbott Park, Illinois, USA). Serum calcium concentration was measured by coloring calcium with Arsenazo III dye; calcium-dye complex was analyzed by a spectrophotometric method. Serum magnesium concentration was determined by an enzymatic method; magnesium functioned as a cofactor during the enzymatic reaction with isocitrate dehydrogenase. The normal reference ranges for our laboratory are as follows: total serum Mg level between 1.8 and.4 mg/dl and total serum Ca level between 8.5 and 1.5 mg/dl. Statistical analysis Where appropriate, data were presented as mean ± standard deviation (SD). The paired Student s test was used to ascertain significance between means of total Mg and Ca levels without hormone ovarian stimulation and before the pick-up. A p <.5 was defined as statistically significant. Results Regarding the observational study, the two groups of patients were comparable for all the characteristics. In the group of infertile women, we enrolled one hundred and two patients who underwent IUI and met all inclusion criteria. In this group, mean age of the women was 36 ± 4.9 years (range 19-46) and mean BMI was 3 ± 3. kg/m (range ). Mild asthenoteratozoospermia and HIV or HCV infection among men were the main infertility causes for majority of the couples (n = 6) and unexplained infertility was the second main cause (n = 4). In the control group, we enrolled fifteen fertile women. Mean age of the women was 35.7 ± 6.9 years (range 19-45) and all selected women presented normal weight, with nobody having endocrine diseases. Mean total serum magnesium concentration was 1.95 ±.1 mg/dl (range ) in the control group. Mean total serum magnesium concentration was 1.99 ±. mg/dl (range ) among infertile women. There was no statistically significant difference between the two groups of patients. With regard to calcium analysis, there was no statistically significant difference between the two groups of patients, because we found a mean total calcium level of 8.8 ±. (range ) mg/dl in the control group and mean serum calcium concentration of 9.3 ±.3 (range ) mg/dl in the infertile group. Figures 1 and show the distribution of total magnesium and calcium values among infertile women according to laboratory reference control range. Evaluating the graphs, we can note that 15 patients (14.7%) presented hypomagnesemia. Only two patients (1.9%) had hypocalcemia. Regarding the prospective study, we included twenty-five patients who underwent IVF and met all inclusion criteria. Mean age of the women was 36.6 ± 5. (range 9-4) years and mean BMI was 1.9 ±.8 (range ) kg/m. Sixteen (64%) couples were concerned by severe male factor infertility and nine (36%) couples by unexplained infertility cause. Mean dose of gonadotropins administered was 5 UI/day for a minimum of 1 days to a maximum of 14 days (total dose range UI). Mean total serum magnesium concentration among women at the beginning of stimulation (T1) was. ±. mg/dl and estrogen concentration was 84.5 ± 4.3 pg/ml. Mean total serum magnesium value among women at the end of stimulation (T) was 1.9 ±.1 mg/dl and estrogen level was 45.4 ± 975. pg/ml. The estrogens levels vary importantly due to different doses of gonadotropins administered and the variable capacity of granulosa cells to produce estrogens. The difference among the mean total magnesium concentrations was not statistically significant (p =.8), but a downward trend was found. A similar trend was observed between mean total serum calcium levels, but less remarkable, with 9.3 ±.4 versus 9. ±.4 mg/dl for low and high estrogen levels, respectively (p =.4). Figure 3 shows total serum magnesium values according to estrogens levels before and after COH. 37

4 E. GROSSI, ET AL. 3 Total magnesium (mg/dl) Mg =.4 mg/dl Mg = 1.8 mg/dl Patients (n) Figure 1. Distribution of patients total serum magnesium values within normal reference ranges. 1 Ca = 1.5 mg/dl Total calcium (mg/dl) Ca = 8.5 mg/dl Patients (n) Figure. Distribution of patients total serum calcium values within normal reference ranges. Figure 4A shows the mean total serum Mg levels in all patients before and after COH, at lowest and highest concentrations of estrogen. Figure 4B shows the mean total serum Ca levels in all patients before and after COH. Discussion An extensive literature search has revealed scanty data regarding the modification of calcium and magnesium concentrations in infertile women and the possible influence by estrogen levels on the concentrations of these two cations. The number of couples with infertility problems has steadily grown over the last years due to various reasons and, as a consequence, the number of IVF cycles has also increased worldwide. Considering this picture, it seems important to know if in infertile women there are different levels of magnesium and calcium compared to fertile women and if their concentrations change during ovarian stimulation. 38

5 Serum Mg and Ca in assisted reproductive technology 3 Total Magnesium (mg/dl) Serum Estrogen (pg/ml) Before COH After COH Figure 3. Total magnesium values at T1 (before ovarian hyperstimulation) and at T (after ovarian hyperstimulation) To our knowledge, this is the only one record describing both total serum magnesium and total calcium levels in such groups of numerous infertile women. Results of the first part of our study highlight that the total serum concentrations of magnesium and calcium do not seem altered in a wide infertile women population during early follicular phase. Calcium and magnesium values are within the control reference range for majority of the patients. A possible clinical limitation of our study is the cause of infertility among the examined population. The majority of women were healthy and their infertility cause was related to their partner due to asthenoteratozoospermia and/or viral infection. We have excluded infertility causes linked to ovarian function impairment. In addition, overweight patients were not included, as we know that metabolic syndrome and obesity may lead to magnesium deficiency [8]. Guerrero et al. revealed that low magnesium level was a frequent finding among participants with metabolic syndrome and showed a strong and independent relationship between metabolic syndrome and decreased serum magnesium level [9]. For these reasons, we have decided to exclude overweight participants to avoid possible bias. Our results allow us to rule out that total magnesium and calcium values were altered in infertile women with a normal BMI. This information was not evidence in the published literature so far. The results of the second part of our study support the hypothesis that during gonadotropins stimulation the concentrations of two cations do not change significantly in stimulated women. However, we observed a downward trend in the total levels of magnesium and calcium in relation to rising estrogen levels. Our results are in agreement with the only two available studies presented in the literature regarding this field [7, 1]. One is by O Shaughnessy et al. [7] that demonstrated a correlation between estrogens and ionized Mg in fourteen infertile women. During the increase in estrogens levels due to ovarian hyperstimulation, there was a significant decrease in ionized Mg. The authors suggest that serum estrogen levels in women modulate the blood levels of circulating ionized magnesium. The second paper was published by Muneyyirci- Delale et al. [1] and reported that healthy women in reproductive age presented recurring menstrual cycling change of ionized Mg and cyclic alterations in the ionized Ca to Mg ratio in their serum. These changes in serum concentrations of cations could affect the vasculature, synaptic transmission, and excitation-secretion coupling leading to the well-known premenstrual 39

6 E. GROSSI, ET AL. A.15 Total magnesium (mg/dl) P = B Before COH After COH Total calcium (mg/dl) P = Before COH After COH Figure 4. A) Mean total serum magnesium value ± SD of all patients before and after COH. B) Mean total serum calcium value ± SD of all patients before and after COH. syndromes during the luteal phase in women deficient in Mg. It is not clear if the decrease in ionized serum magnesium concentration is a result of intracellular movement of the Mg + used in some enzymatic reactions, as suggested by Facchinetti et al. [11] for normal young women. We know that the serum magnesium levels decrease during pregnancy and we know that estrogens, produced by the placenta, have very high levels during pregnancy. We could assume that there could be a relationship between estrogen and serum magnesium levels; when estrogen levels increase, serum magnesium levels reduce. According to the recent study of Tabrizi et al., [1] about 13% of the studied population of Iranian pregnant women had hypomagnesemia. The measurement of magnesium levels during the three trimesters of pregnancy indicated a slight decrease in serum magnesium level during the second trimester of pregnancy, but it was not significant. Low serum magnesium status during pregnancy could be the consequence of active transport of element into the fetus due to high estrogen levels in these patients. On the contrary, the level of calcium during all trimesters of pregnancy underwent insignificant changes. This could be explained by the increasing intestinal calcium absorption, decreasing urinary calcium excretion and increased rate of bone turnover during pregnancy. Given that the majority of women during childbearing age do not meet the recommended dietary allowance (RDA) for magnesium [], it is unlikely that the increased demand for magnesium during pregnancy and lactation is being met. Considering the slight decline of total serum magnesium and calcium levels in women during COH, we suggest screening of infertile patients for calcium and magnesium levels before the start of a treatment cycle of ovarian hyperstimulation, 4

7 Serum Mg and Ca in assisted reproductive technology preferably during follicular phase of menstrual period. We recommend this measurement especially if other factors influencing these cation drops, such as overweight, coexist. Further studies are required to better define the role of calcium and magnesium in conditions such as infertility and ovarian hyperstimulation for IVF. Acknowledgements This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The authors declare no conflicts of interest in preparing this article. Disclosure Financial support: none. Conflict of interest: none. References 1. Ferraretti AP, Goossens V, Kupka M, et al. The European IVF-monitoring (EIM), consortium for the European society of human reproduction and embryology (ESHRE). Assisted reproductive technology in Europe, 9: results generated from European registers by ESHRE. Hum Reprod 13; 8: Hanson MA, Bardsley A, De-Regil LM, et al. The international federation of gynaecology and obstetrics (FIGO) recommendations on adolescent, preconception, and maternal nutrition: think nutrition first. Int J Gynaecol Obstet 15; 131: S Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM. Metal ions in biological catalysis: from enzyme databases to general principles. J Biol Inorg Chem 8; 13: World Health Organization. Calcium and magnesium in drinking-water: public health significance. Geneva, Switzerland: World Heath Organization, 9 (194 p.). 5. Kieboom BC, Kiefte-de Jong JC, Eijgelsheim M, et al. Proton pump inhibitors and hypomagnesaemia in the general population: a population-based cohort study. Am J Kidney Dis 15; 66: Schimatschek HF, Rempis R. Prevalence of hypomagnesaemia in an unselected German population of 16 individuals. Magnes Res 1; 14: O Shaughnessy A, Muneyyirci-Delale O, Nacharaju VL, Dalloul M, Altura BM, Altura BT. Circulating divalent cations in asymptomatic ovarian hyperstimulation and in vitro fertilization patients. Gynecol Obstet Invest 1; 5: Farhanghi MA, Mahboob S, Ostadrahimi A. Obesity induced magnesium deficiency can be treated by vitamin D supplementation. J Pak Med Assoc 9; 59: Guerrero-Romero F, Rodriguez-Moran M. Low serum magnesium levels and metabolic syndrome. Acta Diabetol ; 39: Muneyyirci-Delale O, Nacharaju VL, Dalloul M, Altura BM, Altura BT. Sex steroids hormones modulate serum ionized magnesium and calcium levels throughout the menstrual cycle in women. Fertil Steril 1998; 69: Facchinetti F, Borella P, Valentini M, Fiorini L, Genazzani AR. Premenstrual increase of intracellular magnesium levels in women with ovulatory, asymptomatic menstrual cycles. Gynecol Endocrinol 1988; : Tabrizi FM, Pakdel FG. Serum level of some minerals during three trimesters of pregnancy in Iranian women and their newborns: a longitudinal study. Indian J Clin Biochem 14; 9: