‫مجلة جامعة كرميان‬ Journal of Garmian University ‫طؤظاري زانكؤي طةرميان‬

https://doi.org/10.24271/garmian.4
Role of Hormones in the Regulation of Antioxidants in
Female Infertility
Ayad F. Palani
Department of Chemistry, College of science, University of Garmian.
E-mail: ayad.palani@garmian.edu.krd

Abstract
Many diseases including infertility were found to be affected by oxidative stress (OS) situation. New
relationships between Hormones and OS are proposed recently. The aim of this study is to investigate
the role of reproductive hormones (Luteinizing hormone [LH] and Follicle-stimulating hormone
[FSH]) in the regulation of OS status in infertile women. Study groups consist of (42) infertile and
[19] healthy women both are in the reproductive age, blood samples were taken and serum separated
by centrifugation and kept at (-45Cº) until analysis. The following biochemical tests were done for
each serum sample including [LH, FSH, Catalase, Glutathione, L-carnitine and Malondialdehyde].
Results were statistically analyzed by ANOVA table and values were considered significant at the
level (p ≤ 0.5). Results of this study showed a significant increase in LH and Malondialdehyde level
and a significant decrease in catalase levels of infertile women compared with fertile women. And an
increase but not significant were seen in FSH and L-carnitine level, while no significance seen in the
level of glutathione.
Key words: oxidative stress, hormonal regulation, oocyte maturation, lipid peroxidation

Introduction
Infertility is the most concerned human health problem affecting about 10-15% of all
married couples [1]. It is defined as inability of a sexually active, non-contracepting couple to
achieve conception in one year [2]. Statistically female factors are responsible for 40%-50% of
infertility cases while the others are due to male factor as well as combined female/male
factors and unexplained infertility [3]. It is very important to identify the factors which affect

female fertility, in the considering in the biochemical factors there is a growing evidence of
possible role of free radicals and oxidative stress (OS) in infertility [4]. OS has been
demonstrated in many of the causes of infertility, such as endometriosis, plycystic ovarian
disease, unexplained infertility, tubal infertility, and recurrent pregnancy loss [5].
OS occurs due increase ROS production and low antioxidant defense. Biological systems
provided with complex enzymatic and non-enzymatic antioxidants system. Enzymatic
antioxidants include :( superoxide dismutase [SOD], Catalase [CAT] and glutathione reductase
[GRD]/peroxidase (GPX]) [6]. GSH is the most important non-enzymatic antioxidant, it is a
major cellular antioxidant. Low GSH levels impairs the cellular antioxidant defense systems,
in addition to L-carnitine, uric acid, etc. [7].

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ROS include: superoxide [O2•ˉ] anion, hydrogen peroxide [H2O2], peroxyl [ROO•] and
hydroxyl [OH•] radicals have potential implications in reproductive biology [8]. It is proposed
that ROS have role in many different reproductive processes such as: oocyte maturation,
fertilization, luteal regression, and endometrial shedding [9]. ROS have a positive role in
physiology only in small amount, but when ROS exceed the normal level it become harmful
and cause many pathologies [10], the presence of antioxidants counteract the effects of these
reactive species, body needs sufficient antioxidants power to protect itself, OS results when
antioxidants defense decreased and it has been implicated in many pregnancy-associated
problems [11], due to its role in the damage to oocytes in developing follicles, ovulation,
implantation ,and impairment of luteal support to pregnancy [12], [5].

ROS attack each cell component including lipids causing peroxidation of membrane
phospholipids, destroying cell membrane and eventually cell death [13]. Lipids breakdown
result in the formation of various oxidatively modified toxic products like MDA [10].

Hormones contribute in the cellular generation of free radicals and have a role in the OS
status [14]. New relationships between Hormones and OS are proposed recently. It has been
demonstrated the activities of Antioxidant enzymes have been shown to be regulated by
nutrients and hormones [15], and the levels of several hormones (e.g., growth hormone and
prolactin) have a correlation with different antioxidant enzyme activities in different tissues
[16], Also a relationship between reproductive hormones and plasmatic total antioxidant
capacity have been observed [17], [18].

The aim of this study is to estimation of the levels of antioxidants and lipid peroxidation
[LPO] in infertile women, and to investigate the effect of reproductive hormones (Luteinizing
hormone [LH] and Follicle-stimulating hormone [FSH]) on the regulation antioxidants and
LPO levels, and find the role and the correlation of these parameters on female infertility.

Materials and Methods

The population of this study consists of (42) infertile women who attended to [Sherai
Naqib hospital for obstetrics and gynecology] in kalar city for infertility evaluation and 19
healthy women, both are in the reproductive age (32.6 ±7.4 and 29.8±7.9 years) with normal
body weight (72.85±13.21 and 72.40±12.23 kg) for infertile and fertile women respectively.
Women who have not pregnant for at least 1 year were and their partners have normal semen
parameters and who have normal menstrual cycle were considered as unexplained infertility,
any other causes of infertility included disorder of menstrual cycle were excluded. No history
of chronic illness (e.g. diabetes, high blood pressure, anemia etc.), smoking or alcohol intake

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‫مجلة جامعة كرميان‬ Journal of Garmian University ‫طؤظاري زانكؤي طةرميان‬

was included. Blood samples (4-5 ml) were drawen using sterile syringes and collected in gel
containing tubes in order to enhance blood clotting and for better separation of serum, then
serum separated and preserved in different 4 polyethylene eppedorf tubes in a deep freeze (-
45 C°) for further biochemical analysis. The samples were thawed at room temperature only
once at the time of assay.

A. Estimation of Hormones
Hormones were estimated using ELISA technique based on the principle of a solid phase
enzyme-linked immunosorbent assay. Thawed serum samples have been assayed for LH and
FSH using (MonoBind, USA) kits. A series of standards and serum samples were added to
specific wells and then 100 μL of enzyme conjugate were added to all wells and incubated for
60 min, then wells were washed 3 times and 100 μL of substrate were added and incubated
for 15 min. 50 μL of stop solution added and a yellow color formed and read at 450 nm.
Hormone level is calculated automatically by the microplate reader and results expressed as
mIU/mL.

B. Estimation of Catalase activity

CAT activity were estimated according to Aebi method [19] depending on the ability of
H2O2 to decompose by the action of CAT to produce H2O and O2.The decrease in the
absorbance in the UV region per time is corresponded to CAT activity. 2.0 ml of substrate (10
pmol/ml of H2O2 in 50 mmol/l sodium-potassium phosphate buffer, pH 7.0) was incubated
with 100 µl serum. The decomposition of H2O2 was followed directly for 2 min by the
decrease in absorbance at 240 nm and results were calculated and expressed as k/ml.

C. Estimation of L-carnitine
L-carnitine were estimated using (My Biosource, USA) assay kit. This assay employs the
quantitative sandwich enzyme immune-assay technique. 100 µL of Standards and diluted
samples were pipetted into the wells and incubated at 37° for 2 hrs. After removing any
unbound substances, 100 µL biotin-conjugated antibody specific for L-carnitine is added to
the wells. Wells washed and 100 µL avidin conjugated [HRP] is added to the wells and
incubated at 37° for 1 hour. Following a wash 90 µL substrate solution is added to the wells.
The color development is measured at 450 nm and results expressed as ng/mL.

D. Estimation of Glutathione (GSH)

Glutathione level measured according to the method of Moron et al. [20], based on the
reaction of GSH with 5,5-dithiobis-2-nitro-benzoic acid (DTNB) from (Sigma Aldrech,
Germany) at pH 8.0. The optical density of yellow color measured at 412 nm. 0.1 mL of

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‫مجلة جامعة كرميان‬ Journal of Garmian University ‫طؤظاري زانكؤي طةرميان‬

serum samples were added to 0.9 mL of (0.6 mmol DTNB) reagent and 10 µL of
trichloroacetic acid (Labtech, Australia), mixtures were incubated at 25°C for 5 min. after
centrifugation the absorbance of yellow color measured and the results were calculated from a
glutathione standard curve and expressed as mmol/mL.

E. Estimation of Lipid Peroxidation

LPO were estimated by reaction of thiobarbituric acid (TBA) from (Qulaikems, India)
with Malondi-aldehyde (MDA) according to Botsoglou et al. [21]. In the presence of heat and
acid, MDA reacts with TBA to produce a pink color complex. 0.1 ml of were added to 0.9 ml
distilled water and 0.5 ml of TBA and trichloroacetic acid reagent were added, tubes
incubated for 1 hour in boiling water bath. Resulting color read at 535 nm MDA expressed as
(nmol/ml).

F. Statistical analysis
The statistical were analyzed using Minitab software program. Significance of difference
in mean was analyzed by ANOVA. Correlations with values of p ≤ 0.05 used to indicate a
statistical significance.

Results and Discussion

Results of this study represented in (Table-1) showed a significant increase in LH and
MDA level and a significant decrease in CAT level of infertile women compared with fertile
women. Results agree with Hussain et al. [13], and Pyari et al. both reported an increase in
MDA level and decrease in CAT level in infertile women [4], and disagree with (Mohan and
Sultana) who reported low LH level in infertile women compared to fertile [22]. The
following figure shows the differences in the assayed biochemical parameters between
infertile and fertile groups.

Fig.1: Means ±SD of biochemical parameters for infertile and fertile women

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‫مجلة جامعة كرميان‬ Journal of Garmian University ‫طؤظاري زانكؤي طةرميان‬

Table-1: Means ±SD of serum biochemical parameters for infertile and fertile women
Infertile Women Fertile Women
Biochemical parameter P value
n=[46] n=[19]
LH (mIU/mL) 9.32±6.69 3.70±3.39 0.001
FSH (mIU/mL) 4.81±3.30 2.90 ± 1.41 n.s
CAT (k/L) 1.57 ± 0.48 1.04 ± 0.40 0.008
L-carnitine (ng/ml) 69.03 ± 23.42 48.78 ±17.62 n.s
GSH (mmol/mL) 0.42 ±0.20 0.54 ± 0.19 n.s
MDA (nmol/mL) 124.30 ± 37.91 88.75 ± 20.75 0.05
n.s : non-significant difference at p ≤ 0.05

LH levels in infertile were higher than in fertile women (9.32±6.69 and 3.70±3.39
mIU/mL) respectively. While there were no difference in FSH levels between infertile and
fertile women (4.81±3.30 and 2.90 ± 1.41 mIU/mL) respectively. Although there are no
emersion of the ssociated symptoms (e.g. irregular menstrual cycle), raised LH with normal
FSH suggests polycystic ovarian syndrome PCOS [23].About 20-25% of normally
menstruating women have PCOS. Thus, it is possible that some apparently normal women
with normal ovulatory cycles low fertilization rate, this may be considered as pre PCOS [24].
The ovulatory process is initiated by the midcycle flux of LH hormone that induces
substantial biochemical, molecular, and cellular changes, culminating in the release of a
mature ovum. Ovary response to LH that is essential for ovulation is subsequent to the
expression of a set of specific genes such as prostaglandin synthase (II), the expression of
prostaglandin synthase (II) is associated with ROS production [25], thus it can be deducted
that high LH levels are associated with increase of ROS production [26]. Excess ROS
production in the ovarian follicles resulting in disruption of redox state and enhance OS
situation [27]. These ROS by targets DNA, lipids, proteins and can cause apoptosis and cell
death in the ovarian follicles and granulosa cells via damaging mitochondria ad consequently
effecting the ovulation and causing infertility [6], [28].

ROS are neutralized by a complex antioxidant defense system consisting of enzymes such
as catalase, superoxide dismutase and glutathione peroxidase/reductase, and numerous non-
enzymatic antioxidants [29].

(Mancini et al. 2009) proposed a role of systemic hormones in the regulation of

antioxidants [30], and (Pajovic and Saicic 2008) suggested the role sexual hormones in the
expression of endogenous antioxidant enzymes [31]. Several hormones have been reported
(e.g. growth hormone and prolactin) to have a correlation with different antioxidant enzyme
activities in different tissues [8]. LH acts as a modulator of gene expression of antioxidant
enzymes [31] via intermediary role in the realization of the effects of ovarian steroids on the
activity of antioxidative enzymes [32]. Elevation of LH is to enhance catalase synthesis so as
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to control the level of ROS, through modulation of gene expression of the antioxidant enzyme
(catalase) [33]. This alternation in the expression of catalase increase catalase activity (1.57 ±
0.48 and 1.04 ±0.40 k/mL) as results showed for infertile compared to fertile women
respectively. The antioxidant enzymes neutralize the toxic effect of ROS and protect the
oocyte and embryo [12]. Low antioxidant defense in the follicular fluid increase the
generation of ROS result in decline fertilization rates [34].

No significant differences seen in GSH levels (0.42 ±0.20 and 0.54 ±0.19 mmol/mL). The
absence of differences in GSH levels between groups is due to that GSH role in the oocyte is
improve male prenucleus formation, normal fertilization and embryo development [35]. As
the infertility here occurs due to oocyte disturbance a step before male sperm fusion into the
egg which the role of GSH begins.

The elevation of MDA in serum of infertile women (124.30 ±37.91 nmol/mL) as compared
to fertile women (88.75±20.75 nmol/mL) is due to peroxidation of oocyte membrane lipid.
High levels of lipids [saturated and unsaturated fatty acids] found in the mammalian oocytes
structure and in the energy metabolism during oocyte maturation [36], [37]. LH mediated OS
induce oxidation of lipids, LPO in the follicle is injurious for oocyte, and cause loss of oocyte
membrane viability which leads to premature oocyte death and infertility [36].

Conclusion
In conclusion, LH has a double edge sword effect in female fertility via enhancing the
production of ROS and increasing enzymatic antioxidant activity through alteration of the
expression of enzymatic antioxidant (catalase). Consequences of slightly increase in LH level
returns in oxidative stress condition which give rise to destroy lipids in the oocyte membrane
leading to loss membrane viability and oocyte premature death.

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