Biol Trace Elem Res (2011) 139:1–9
DOI 10.1007/s12011-010-8637-x
Effects of Multivitamin/Mineral Supplementation
on Trace Element Levels in Serum and Follicular Fluid
of Women Undergoing in Vitro Fertilization (IVF)
Mehmet Okan Özkaya & Mustafa Nazıroğlu &
Cihan Barak & Murat Berkkanoglu
Received: 25 October 2009 / Accepted: 1 February 2010 /
Published online: 24 February 2010
# Springer Science+Business Media, LLC 2010
Abstract We investigated effects of multivitamin/mineral supplementation on element
levels in serum and follicular fluid of women undergoing IVF. We used three groups in this
study. The first group was used as an age-matched and nonpregnant control (n=13). Group
2 (n=30) constituted the IVF group and women in the third group who were undergoing
IVF also received a multivitamin/mineral tablet daily for 45 days. Follicular fluid and serum
selenium and zinc levels and follicular fluid copper levels were lower in IVF patients than
in controls although follicular fluid aluminum and iron levels were higher in IVF patients
than in controls. However, follicular fluid and serum aluminum, copper, zinc and selenium
levels, and serum magnesium levels were higher in the multivitamin/mineral group than in
the IVF group although follicular fluid iron levels were lower in the multivitamin/mineral
group than in the IVF group. In conclusion, we observed that copper, zinc, and selenium in
serum and follicular fluid decreased in women undergoing IVF. Multivitamin/mineral
supplementation in serum and follicular fluid of women undergoing IVF normalized the
trace element levels.
Keywords Zinc . Selenium . Copper . IVF . Human fertilization . Follicular fluid
M. O. Özkaya
Department of Obstetrics and Gynecology, Faculty of Medicine, Suleyman Demirel University,
Isparta, Turkey
M. Nazıroğlu (*)
Department of Biophysics, Medical Faculty, Suleyman Demirel University, Morfoloji Binasi, Cunur,
32260 Isparta, Turkey
e-mail: mnaziroglu@med.sdu.edu.tr
C. Barak
Department of Chemistry, Science Institute, Balıkesir, Turkey
M. Berkkanoglu
IVF Center of Antalya Private Hospital, Antalya, Turkey
2
Özkaya et al.
Abbreviations
FSH
Follicle stimulating hormone
GSH-Px Glutathione peroxidase
hCG
Human chorionic gonadotrophin
IVF
In vitro fertilization
ROS
Reactive oxygen species
SOD
Superoxide dismutase
Introduction
There has been special interest in effects of dietary trace element deficiencies on
physiological functions including reproduction particularly [1, 2]. Severe dietary deficiencies of trace elements including copper, selenium, and zinc are commonly seen in patients
with IVF [1–3].
A significant, but largely neglected, factor, causing infertility is poor nutrition. Good
nutrition is particularly important for DNA synthesis, because most of its essential
components are derived from the diet. Moreover, several enzymes involved in DNA
synthesis are zinc or vitamin B dependent [2, 3]. DNA synthesis is important for the
development of oocytes. Animal studies have demonstrated that deficiencies of vitamin A,
C, and D result in diminished fertility [4, 5]. Despite their suspected impact on female
reproduction and environmental prevalence, concentrations of these elements within the
follicular fluid have not been investigated thoroughly [2].
Furthermore, the diet is the source of exogenous antioxidant vitamins (vitamin A, C, and
E) and trace elements (selenium, zinc, and copper) and they are also essential for oocyte
maturation and rupture. These antioxidants protect DNA and other important molecules
from oxidative damage, which would otherwise induce apoptosis. For example, zinc plays
critical roles in biological membrane stabilization, protein synthesis, and nucleic acid
metabolism and growth of normal tissue and as a cofactor of several enzyme systems [6].
The antioxidant role of zinc and copper is mainly due to the presence of Cu–Zn superoxide
dismutase (SOD) [6]. Selenium is an essential trace element for human health. The function
of selenium as an essential element in animals and humans are attributed to about 12 known
mammalian selenoproteins, thioredoxin reductases, selenoproteins P and W, and phospholipid hydroperoxide, contain selenocysteine, that is specifically incorporated through a
unique cotranslational mechanisms [7]. Selenium is also a cofactor for glutathione
peroxidase (GSH-Px), an important antioxidant enzyme for removing lipid hydroperoxides
and hydrogen peroxide [7]. Catalase contains zinc and copper as cofactors. It also catalyzes
the reduction of hydrogen peroxide to water [8]. Magnesium provides of stabilization of
DNA, RNA, and ribosomes and also activates approximately 300 enzymes including those
in energy metabolism and reactive oxygen species (ROS) production. In addition,
magnesium plays a protective role in the developing fetus brain [9]. Calcium has a basic
function in neurotransmitter secretion and magnesium blocks the entrance of calcium into
cells [10]. Enzymatic and nonenzymatic antioxidants are also essential for inhibition of free
radical production related to oocyte maturation and rupture [11–13]. However, whether a
multivitamin/mineral mixture supplementation modulates infertility-induced element
changes in the serum and follicular fluid of patients with IVF is currently unknown and
is therefore the basis of this study.
We investigated the effects of multivitamin/mineral supplementation on the concentrations of various minerals and vitamins in serum and follicular fluid of patients with IVF.
Role of Element in Patients with IVF
3
Subjects and Methods
Control and Patients
A total of 56 paired samples of follicular fluid and serum were obtained from women who
underwent IVF at the Antalya and Isparta IVF centers of Turkey. Also, 13 paired samples of
follicular fluid and serum were obtained from healthy control subjects. The study protocol was
approved by the local Ethics Committee of the Medical Faculty, Suleyman Demirel University,
Isparta, Turkey, by protocol number June, 2009:17. Informed consent for experimental use of
serum and follicular fluids was obtained from all patients and controls. The mean age±SD of
the untreated and treated patients was 28.8±3.2 years (range of 22–43 years) and 30.7±
4.5 years (a range of 22–43 years), respectively whereas the mean age±SD of healthy untreated
subjects was 32.2±5.2 years, with a range of 25–43 years. Healthy control subjects and treated
and untreated patients were all nonsmokers and free from major medical illness including
hypertension; all were interested in becoming pregnant. Patients were excluded if they had
myoma, adenomyosis, a congenital uterine anomaly, ovarian tumors, or if they used estrogens,
progesterone, androgens, or chronic use of any medications.
Study Groups
We used three groups in this study. The first group was used as control (n=13) who
received a placebo (candy). Group 2 (n=30) constituted the IVF group and they also
received the placebo only. The third group (n=26) daily received orally a multivitamin/
mineral tablet (Megadyn Pronatal Film Tablet, Mecom Medical Health Product Inc,
Istanbul, Turkey) for 45 days before serum and follicular fluid collection. The contents of
the multivitamin/mineral tablet are shown in Table 1.
Table 1 Vitamins and Mineral Content in a Multivitamin Tablet
Vitamin A (retinol)
4,000 IU
Vitamin B1 (thiamin)
1.6 mg
Vitamin B2 (riboflavin)
Vitamin B6 (pyridoxine)
1.8 mg
2.6 mg
Vitamin B12 (cyanocobalamine)
4 mcg
Vitamin C (ascorbic acid)
100 mg
Vitamin D2 (ergocalsiferol)
500 IU
Vitamin E (α-tocopherol acetate)
15 mg
Vitamin H (D-biotin)
0.2 mg
Calcium D-pantothenate
10 mg
Folic acid
Nicotinic acid
0.8 mg
19 mg
Calcium
125 mg
Iron
60 mg
Magnesium
100 mg
Phosphor
125 mg
Copper
1 mg
Manganese
1 mg
Zinc
7.5 mg
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Özkaya et al.
IVF Stimulation Protocol and Follicular Fluid Aspiration
A staff nurse randomized the patients at initiation of stimulation using a computergenerated list. All patients from both groups were instructed to take oral contraceptive pills
(Ginera Schering, Germany) once daily for 21 days.
After the confirmation of pituitary down-regulation on day 3 of the cycle by sonographic
detection of a linear endometrium and suppressed ovaries (no antral follicles 0.10 mm) and
serum estradiol levels of 50 pg/ml, human chorionic gonadotropin (hCG) stimulation with
recombinant follicle stimulating hormone (FSH; Gonal F, Serono, Turkey) was commenced. The initial FSH dose was 150 IU/day for high responders, 225–300 IU/day for
intermediate responders and 450 IU/day for low responders. The initial dose for ovarian
stimulation was based upon ovarian reserve indicators, which included the number of antral
follicles on day 3 of a previous, basal spontaneous cycle. The dose was adjusted on day 6 if
needed and thereafter in accordance with the patient’s response as indicated by her estradiol
level and number of developing follicles. The hCG administration continued until at least
two follicles 0.17 mm in diameter were detected when 10,000 IU hCG was administered
followed 35 h later by transvaginal ultrasound-guided oocyte retrieval.
Blood and Follicular Fluid Collection and Preparation
Care was taken to completely aspirate each follicle to one tube. Each follicle was aspirated
separately and follicular fluid containing the oocyte was collected. Immediately after
removal of the oocyte, each follicular fluid was centrifuged at 500×g for 10 min at +4°C to
remove cellular components and the supernatant was kept frozen on ice.
Venous blood (5 ml) was taken from the antecubital vein, using a monovette system of
blood collection, into tubes without anticoagulant but protected against light, after an
overnight fast. Serum samples were obtained from the blood samples by centrifugation at
1,500×g for 10 min at +4°C.
Serum (2 ml) and follicular fluid (3 ml) samples were stored at −30°C for <1 month
pending measurement of trace element. For each blood and follicular sample, two blank
samples of highly purified water (element content <0.01) were collected using the same
tools/equipments (e.g., gloves, syringes etc.) in order to allow determination of background
‘noise’ (lower detection limit).
Apparatus
The ICP-AES system used was a Perkin-Elmer Optima 3100 XL emission spectrometer equipped
with the Perkin-Elmer AS 90 plus autosampler and was controlled by a computer. The plasma
operating conditions used in the ICP system were 253.6 nm wavelength, 151/min plasma gas
flow rate, 0.5 argoncarrier flow rate, plus 21/min sample flow and rates. The peristaltic pump was
a Watson Marlow 323 SD model. The microcolumn was a glass tube (0.7−10 cm, Aldrich C
3669) packed with modified anion-exchange resin (1 g). Transport lines consisted of 1.25 mm i.d.
poly tetrafluoroethylene tubing.
Reagents
All reagents were of analytical reagent grade and deionized water was used throughout.
Stock solutions of copper, zinc, selenium aluminum, iron, magnesium, and calcium were
prepared by taking appropriate amounts of standards in nitric acid solution. Working
Role of Element in Patients with IVF
5
solutions were prepared immediately before use. Adjustment of pH was made with buffer
(acetic acid, boric acids, and their potassium salts). Double-distilled deionized water was
used in the current study. All glassware used was washed with 10% nitric acid for 1 day and
rinsed with deionized water before use.
Copper, Zinc, Iron, Calcium, Magnesium, and Aluminum Analysis
Copper, zinc, iron, calcium, magnesium, and aluminum levels were estimated by atomic
absorption spectrophotometry and the ICP-AES system after digestion with nitric acid and
hydrogen peroxide as described in a previous study [14]. Serum samples (0.5 ml) were
slowly digested with 3 ml nitric acid for 12 h. The samples were then digested in
microwave oven for 5–10 min. Finally, they were allowed to cool at room temperature for
1 h. Before 0.5 ml hydrogen peroxide (30%) was added.
Selenium Analysis by Hydride Technique
Temperature programs of flow injection mercury/hydride analyses for selenium determinations in serum and follicular fluid of patients with IVF are shown in Table 2. Palladium
solution (200 mg/l) was used as a matrix modifier in selenium measurements in the frozen
serum samples. Selenium in the serum samples measured directly without previous acid
digestion and appropriate dilution (usually 1:1) was made only with acid (HCl). In selenium
analysis we used 0.2% NaBH4 in 0.05% NaOH as the reducing agent. Graphite furnace
programs for selenium measurement in serum samples are described in Table 2. Samples
and standards to be analyzed for selenium were treated with an equal volume of HCL
followed by heating at 90°C for 20 min. After pre-reduction the solution may be diluted
without the risk of re-oxidation from Se+4 to Se+6. Each analysis was repeated three times.
Statistical Analyses
All results are expressed as means±SD. Significance among values for control subjects,
patients, and treatment groups was assessed with Student’s t test. Data were analyzed using
the SPSS statistical program (version 9.05 software, SPSS Inc. Chicago, IL, USA). p values
of less than 0.05 were regarded as significant.
Results
Follicular fluid element levels of the various groups of controls and patients with IVF are
shown in Table 3. Follicular fluid selenium (p<0.05) and zinc (p<0.01) levels were
Table 2 Temperature Programs
of Flow Injection Mercury/Hydride Analyses for Selenium
Determinations in Serum and
Follicular Fluid of Patients with
IVF
Parameters
Integration time (s)
15
Lamp
HCL
Slit (nm)
0.2
Wavelength (nm)
196
Cell temperature
900°C
Carrier solution
10% (v/v) HCl
Reducing solution
0.2% NaBH4 in 0.05% NaOH
6
Özkaya et al.
Table 3 The effects of Multivitamin/Mineral Supplementation on Follicular Fluid Element Levels in
Patients with IVF (Mean±SD)
Parameters
Control (n=13)
IVF (n=30)
IVF+SUPPL (n=26)
Copper (μg/l)
450.8±48.2
407.9±73.6
838.0±29.6***
Zinc (μg/l)
545.8±49.3
307.6±11.5**
464.9±78.6****
Selenium (μg/dl)
6.02±0.2
5.03±0.1*
Aluminum (μg/dl)
21.58±1.5
33.61±5.2*
1208.3±68.3
1415.5±60.9*
9.51±0.6
9.32±0.5
9.58±0.5
56.86±4.4
52.48±4.8
57.31±2.6
Iron (μg/l)
Magnesium (mg/dl)
Calcium (mg/dl)
6.05±0.1****
65.82±3.3***
948.1±41.0*****
*p<0.05 versus control; **p<0.01 versus control; ***p<0.01 versus IVF group; ****p<0.05 versus IVF
group; *****p<0.001 versus IVF group
significantly lower in the IVF patients than in controls although follicular fluid iron and
aluminum levels were significantly (p<0.05) higher in patients with IVF than in the
controls. However, copper (p<0.01), zinc (p<0.05), selenium (p<0.05), and aluminum
levels (p<0.01) in follicular fluid were significantly higher in the treatment group than in
the untreated IVF group although iron levels were significantly (p<0.001) lower in the
treatment group than in the IVF group. Calcium and magnesium levels in patients with IVF
were not increased by the multivitamin/mineral complex supplementation.
Serum element levels of the three groups are shown in Table 4. Serum copper, zinc, and
selenium levels were significantly (p<0.05) lower in patients with IVF than in controls,
whereas serum copper, zinc, selenium, magnesium, and aluminum levels were significantly
(p<0.05 and p<0.01) higher in the treatment group than in the untreated IVF group. Iron
and calcium levels in patients with IVF were not increased by the multivitamin/mineral
supplementation.
Discussion
We detected the presence of copper, zinc, aluminum, iron, magnesium, calcium, and
selenium in the follicular fluid of patients with IVF. There are few reports on metal levels in
follicular fluid. Recently, Silberstain et al. [2] investigated the average concentrations of
Table 4 The effects of Multivitamin/Mineral Supplementation on Serum Element Levels in Patients with
IVF (Mean±SD)
Parameters
Control (n=13)
IVF (n=30)
IVF+SUPPL (n=26)
Copper (μg/l)
84.74±6.22
79.38±6.59*
85.96±4.33**
Zinc (μg/l)
87.15±5.71
68.47±11.41*
83.76±6.67***
Selenium (μg/dl)
6.65±0.54
Aluminum (μg/dl)
28.81±2.37
30.77±2.18
5.91±0.51*
38.67±4.32**
6.74±0.85**
Iron (μg/l)
49.10±4.01
45.23±3.73
45.40±3.97
Magnesium (mg/dl)
1.57±0.13
1.59±0.21
2.12±0.34**
Calcium (mg/dl)
8.30±0.57
8.31±1.07
8.96±0.65
*p<0.05 versus control; **p<0.05 versus IVF group; ***p<0.01 versus IVF group
Role of Element in Patients with IVF
7
elements in 33 follicular fluid samples and they found similar element levels in other
human tissues and fluids. Also the Siberstein study reported that calcium and magnesium
are the most abundant metals, followed by copper, zinc, and iron (present in concentrations
of hundreds of ppb). The following metals namely lithium, selenium, aluminum, and
manganese were found in trace amounts. Lithium, cadmium, barium, titanium, and bismuth
were not detected (<0.2 ppb) in our studies of follicular fluid and blood of patients with
IVF. Similarly, Zha et al. [15] reported the presence of zinc and manganese in follicular
fluid of non-professionally exposed.
Serum copper levels were lower in patients with IVF than in controls. Copper is an
essential element in biological systems. The biological functions of copper are intimately
related to its redox properties as a transition metal. Redox cycling between Cu2+ and Cu1+
can catalyze the production of highly toxic hydroxyl radicals [16].
In the current study, follicular fluid iron levels were higher in patients with IVF than in
controls. Iron levels were lower in multivitamin/mineral supplemented groups than in
untreated IVF groups. It is well known that oxidative stress enhances non-heme iron
absorption. Oxidative stress during oocyte maturation and rupture may influence
biomarkers of iron status through its inflammatory effects on metabolism [11–13], or the
continuous exposure to ischemia/reperfusion in IVF may produce a degree of hypoxia that
leads to increased hemoglobin concentrations as an adaptive response [17].
Zinc is a micronutrient abundantly present in meat and seafood. Zinc serves as a cofactor for
more than 80 metalloenzymes involved in DNA transcription and protein synthesis [6].
Because DNA transcription is a major part of germ cell development, zinc is likely to be
important for reproduction [1]. Furthermore, zinc finger proteins are implicated in the genetic
expression of steroid hormone receptors [3], and zinc also has antioxidant properties [18]. In
the current study, zinc levels were lower in patients with IVF than in controls although zinc
levels increased in multivitamin/mineral supplemented groups. Studies on the effects of zinc
deficiency in women with IVF are scarce. Ng et al. [10] investigated zinc levels in follicular
fluid of 33 women undergoing IVF in Singapore and did not observe any correlation between
follicular fluid zinc concentration and follicle volume. Jameson [19] reported longstanding
infertility in seven, normal sexually developed women with celiac disease. These women all
had normal menstrual cycles, but low serum zinc levels. Soltan and Jenkins [20] measured
plasma zinc levels in 48 infertile and 35 control women and found no differences between
these two groups. Ronaghy and Halsted [21] reported two women aged 19 and 20 years,
suffering from nutritional dwarfism with delayed sexual maturation. These women had no
breast tissue or pubic hair and had infantile external genitalia with extremely low blood
plasma and erythrocyte zinc levels. After zinc supplementation, these women experienced
their first menstrual period and developed breast tissue as well as pubic hair growth.
Selenium levels were lower in IVF patients than in controls. The relationship between
fertility and selenium status and Se-dependent GSH-Px enzyme activities has been
examined in just a few studies that, in general, found lower serum and follicular fluid
concentrations to be associated with increased infertility rate. Paszkowski et al. [22] found
that 112 IVF patients had lower serum and follicular fluid selenium levels than controls,
due mostly to a lower intake of selenium. Reduced levels of GSH-Px are reported in the
follicular fluid of women with unexplained infertility [23]. Yang et al. [24] found higher
levels of oxidant, hydrogen peroxide in fragmented embryos compared with nonfragmented
embryos, while Paszkowski et al. [22] reported the elevated consumption of antioxidants,
which suggests increased ROS levels, during incubation of poor quality of embryos.
However, there is also evidence to suggest that antioxidant vitamins in follicular fluid act
on the ovary to modify its function [12].
8
Özkaya et al.
Follicular fluid zinc and selenium were significantly lower in IVF patients than in
controls. Serum zinc levels were higher in the treatment group than in untreated IVF
patients. To our knowledge, there is no publication on zinc levels in patients with IVF.
However, our results are in agreement with studies conducted on genetically manipulated
mice where decrease in the litter size and number of litters per month with deficiency of
follicular fluid Zn–Cu–SOD activity has been reported [25, 26]. Intense metabolism of the
granulose cells together with high numbers of macrophages and neutrophil granulocytes in
the follicle wall at ovulation may represent a site of active free radical generation in
follicular fluid of IVF patients [27]. These free radicals can cause damage to biomolecules
including DNA, and thus be mutagenic and carcinogenic. Aerobic cells have developed
their own defense system in order to control the flux of free radicals. The Cu–Zn–SOD
enzyme is the first line of defenses in oxidative stress. The Cu–Zn–SOD converts
superoxide into hydrogen peroxide. Hydrogen peroxide can be transformed into water and
oxygen by selenium-dependent GSH-Px enzyme. In a recent study, we observed that
antioxidant vitamin were lower in patients with IVF than in control although lipid
peroxidation levels were higher in patients with IVF than in control [28]. Hence, decreases
of follicular fluid and serum zinc and selenium levels, and serum copper levels as a
component of antioxidant enzymes attributed to increased levels of free radical production.
In conclusion, the results of the current study show that follicular fluid zinc and
selenium, and serum zinc, copper, and selenium levels were lower in patients with IVF than
in control. However, follicular fluid copper, selenium, and aluminum levels, and serum
copper, zinc, selenium, aluminum, and magnesium levels were higher in IVF+vitamin+
mineral group when compared to the IVF group. Hence, a combination of these
micronutrients provides a better effect on accumulation of the elements in follicular fluid.
Acknowledgment The authors wish to thanks Prof. Dr. Hakan Kaya (IVF center, Isparta Private Hospital,
Turkey) for helping samples collection and Dr. Peter Butterworth of King’s College, London for assistance
with the presentation of the manuscript and correction of the English language.
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