„GRIGORE. T. POPA” UNIVERSITY OF MEDICINE AND PHARMACY OF IASI
EXPERIMENTAL RESEARCH REGARDING THE
INFLUENCE OF VARIOUS FORMS OF OXIDATIVE
STRESS UPON THE FEMININ GENITALIA - summary of the doctoral thesis -
SCIENTIFIC COORDINATOR,
University Professor Doctor Carmen Lăcrămioara ZAMFIR
PHD(c),
Ana Maria CEFĂLAN (HALICIU)
IASI
- 2017 -
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CONTENT
GENERAL PART
Chapter I: Oxidative stress – General considerations .......... Eroare! Marcaj în document nedefinit.
I.1. Evolution of the notion of oxidative stress .... Eroare! Marcaj în document nedefinit.
I.2. Action mechanisms ........................................ Eroare! Marcaj în document nedefinit.
I.2.1. Reaction of the oxygen reactive species with the proteins .Eroare! Marcaj în document nedefinit.
I.2.2. SRO reaction with the carbohydrates . Eroare! Marcaj în document nedefinit.
I.2.3. SRO reaction with the nucleic acids ... Eroare! Marcaj în document nedefinit.
I.2.4. Reaction of the oxygen reactive species with the lipidsEroare! Marcaj în document nedefinit.
I.3. Types of oxidative stress and their impact upon the tissular level Eroare! Marcaj în document nedefinit.
I.4. Oxidants.......................................................... Eroare! Marcaj în document nedefinit.
I.5. Antioxidants ................................................... Eroare! Marcaj în document nedefinit.
Chapter II: Morphology of the female genitalia .................... Eroare! Marcaj în document nedefinit.
II.1. Female external genitalia organs .................. Eroare! Marcaj în document nedefinit.
II.2. Female internal genitalia organs .................... Eroare! Marcaj în document nedefinit.
II.2.1. Vagina ................................................ Eroare! Marcaj în document nedefinit.
II.2.2. Uterus ................................................. Eroare! Marcaj în document nedefinit.
II.2.3. Fallopian tubes ................................... Eroare! Marcaj în document nedefinit.
II.2.4. Ovaries ............................................... Eroare! Marcaj în document nedefinit.
Chapter III: Effects of the oxidative stress at the female genitalia level
III.1. Idiopathic sterility ........................................ Eroare! Marcaj în document nedefinit.
III.2. Primary dysmnenorrhea................................ Eroare! Marcaj în document nedefinit.
III.3. Follicular ovarian cysts................................. Eroare! Marcaj în document nedefinit.
III.4. Ovarian cancer .............................................. Eroare! Marcaj în document nedefinit.
III.5. Pelvic inflamatory disease ............................ Eroare! Marcaj în document nedefinit.
III.6. Endometriosis ............................................... Eroare! Marcaj în document nedefinit.
III.7. Menopause .................................................... Eroare! Marcaj în document nedefinit.
III.8. Premature births, miscarriage, eclampsia ..... Eroare! Marcaj în document nedefinit.
PERSONAL PART
Chapter IV: Research motivation, scope and objectives ................................................................... 7
Chapter V: Material and metode.......................................................................................................... 9
V.1. Material .......................................................... Eroare! Marcaj în document nedefinit.
V.2. Working methods .......................................... Eroare! Marcaj în document nedefinit.
V.2.1. Histopathological and imunohistochemical explorationEroare! Marcaj în document nedefinit.
V.2.1.1. Handling the sampled tissuesEroare! Marcaj în document nedefinit.
V.2.1.2. Colouring with hematoxylin-eosinEroare! Marcaj în document nedefinit.
V.2.1.3. Imunihistochemical study chimicEroare! Marcaj în document nedefinit.
V.2.2. Determining methods of the oxidative biomarkersEroare! Marcaj în document nedefinit.
V.2.2.1. Determining the malondialdehyde (MDA)Eroare! Marcaj în document nedefinit.
V.2.2.2. Determining the superoxide dismutasis (SOD)Eroare! Marcaj în document nedefinit.
V.2.2.3. Determining the catalase (CAT)Eroare! Marcaj în document nedefinit.
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V.2.2.4. Determining the glutation peroxidase (GPx)Eroare! Marcaj în document nedefinit.
V.3. Handling and statistical interpretation of the obtained dataEroare! Marcaj în document nedefinit.
Chapter VI: Results ...................................................... Eroare! Marcaj în document nedefinit.
VI.1. Evaluation of the animals’ weight in the experimental groupsEroare! Marcaj în document nedefinit.
VI.2. Evaluation of the relative uterus and ovaries weight for the experimental groupsEroare! Marcaj în document nedefinit.
VI.3. Evaluation of the biomarker attesting the lipidic peroxidase (MDA)Eroare! Marcaj în document nedefinit.
VI.3.1. MDA ................................................ Eroare! Marcaj în document nedefinit.
VI.3.2. MDA correlation with weight .......... Eroare! Marcaj în document nedefinit.
VI.4. Evaluation of the antioxidant enzymes ........ Eroare! Marcaj în document nedefinit.
VI.4.1. SOD ................................................. Eroare! Marcaj în document nedefinit.
VI.4.2. SOD correlation with weight .......... Eroare! Marcaj în document nedefinit.
VI.4.3. CAT ................................................. Eroare! Marcaj în document nedefinit.
VI.4.4. CAT correlation with laboratory animals’ weightEroare! Marcaj în document nedefinit.
VI.4.5. CAT correlation with MDA ............. Eroare! Marcaj în document nedefinit.
VI.4.6. CAT correlation with SOD .............. Eroare! Marcaj în document nedefinit.
VI.4.7. GPx evaluation ................................. Eroare! Marcaj în document nedefinit.
VI.4.8. GPx correlation with weight ........... Eroare! Marcaj în document nedefinit.
VI.4.9. GPx correlation with MDA.............. Eroare! Marcaj în document nedefinit.
VI.4.10. GPx correlation with SOD ............. Eroare! Marcaj în document nedefinit.
VI.4.11. GPx correlation with CAT ............. Eroare! Marcaj în document nedefinit.
VI.5. Histopathologic examination .............. Eroare! Marcaj în document nedefinit.
Chapter VII: Discutions ............................................... Eroare! Marcaj în document nedefinit.
Chapter VIII: Conclusions .......................................... Eroare! Marcaj în document nedefinit.
REFERANCES ………………………………………………………………………………..121
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INTRODUCTION
In the last decades, a much circulated notion is that of the oxidative stress. It is
defined as the disequilibria between the oxidants and the antioxidants, in favour of the
oxidants, having the destructive and pathogenic potential. The oxidant substances are
normally formed during the aerobic metabolism duration, their quantity increasing in certain
conditions. In such circumstances, the antioxidant physiological mechanisms, which
inactivate the oxygen reactive species remove the altered molecules and repair de lesions, can
prove to be insufficient.
From the physiological point of view, the oxidation stress s can be defined as an
excessive bio-availability of the oxygen reactive species, which is the net result of disturbing
the balance between the ROS production and destruction, with late influence upon the
antioxidant defence.
In the last two decades, numerous studies have been published containing information
regarding the theory of the oxidative stress. In any case, despite the large proof which
supports the fact the oxygen reactive species are produced at the cellular level cellular and
can produce cellular destruction, a direct causal relation between the oxidative stress and
aging has still not been clearly established.
The conditions which determine the apparition of the oxidative stress are an important
physiologic and evolutionary factor which modulates the adaptive answer of the organisms.
The individuals in a population are permanently submitted to the action of various stress s
factors. The answer to stress s is, in itself, a naturally adaptive process which allows the body
to function and maintain or to restore the homeostasis. But the answer to stress can be
inadequate, exaggerated, resulting in adverse consequences for the human body and for the
individual’s biological status (1).
A perturbation of the balance between the pro-oxidants and antioxidants in favour of
the first determines the apparition of the oxidative stress, with known secondary effects.
These effects refer to the oxidative changed, determined by the action of the pro-oxidants
upon the biomolecules. Despite all this, not all the secondary effects caused by the oxidative
stress are directly of an oxidative nature. Moreover, the secondary effects upon the
biomolecules can result in the changes similar to the oxidative stress, such as the ions level
(2, 3). Generally, the oxidative stress can result through the excess production of oxidants,
diminished antioxidants concentrations diminish, depletion of the essential metallic co-factors
in the diet, such as selenium, magnesium or zinc, which can activate the antioxidant enzymes
and through the failure of the immune systems (4).
The biological systems are exposed to the SRO endogenously formed (oxygen
reactive species are intracellularly produced through the reduction-oxidation reactions) or
which results from the external environment (for example, ionising ion-irradiations are the
source for the oxygen reactive species). These radicals have harming effects upon the
organism’s cells when they accumulate because they bring about lesions upon the proteins,
lipids, carbohydrates and nucleic acids.
In the physiological conditions, the organism benefits from the anti-oxidation systems
which removes the free radicals produced in excess (they are known under the name of free
radical scavengers), so that only when they fall out of balance, the equilibrium between the
quantity of freely-produced radicals and the quantity of antioxidants (either by producing too
many free radicals, either by decreasing the antioxidant capacity), SRO can become deadly
for the organism, thus explaining their implication, not only in aging, but also in a large
number of diseases (5, 6).
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SRO are involved in the pathogeny of more than 100 disorders of the human species,
through the aggression upon all types of DNA bio-molecules, proteins, carbohydrates, lipids
and upon the cellular metabolism without being able to mention which was the primary target
of the oxidative stress in the DNA degradations appear before the detectable changes of the
peroxidation of their lipids and the protein oxidative degradations – these being dependent
upon the sensibility of the determination method (7).
It is considered that the lesions determined by the oxidative stress can be classified in:
DNA affectation, oxidation of the poly-non-saturated fat acids in the lipids (lipoid
peroxidization) and oxidation of the amino-acids in the proteins.
Oxidative stress can be produced by various exogenic or endogenic factors which act
upon the production-destruction balance of the oxygen free radicals. The perturbation of this
balance can be produced by the ionising radiations, ultraviolet radiations, atmosphere
pollutants, due to tobacco consumption, different drugs, among which the chemotherapies
play a central role, but also by modifying the circadian rhythm.
The endogen and exogenic factors which changes the circadian rhythm determines, at
the same time, the apparition of the oxidative stress through the change of the antioxidant
enzymes’ activity and that of the antioxidants with the low molecular weight. Recent studies
have shown substantial proof for these changes, suggesting the importance of limiting the
circadian rhythm perturbation with the aim of avoiding the excessive oxidative stress. The
glutathione peroxidase activity and, consequently that of the glutathione reductase, follows
the rhythm of releasing the melatonin within the circadian rhythm. Another clue for an
implication of the melatonin within the redox control process is proven by the increased
affinity of the citosolic chinon reductasis 2, previously considered a receptor of melatonin.
Recent data indicate a role of the melatonin in the inhibition of the formation of the
mitochondrial radicals, a function which can directly intervene in the process of antioxidative
defence. The rhythmical changes within the process of oxidative destruction of the proteins
and lipids have also been reported (8).
The circadian rhythm is influenced by the day-night cycle so that the organism can
synchronise the external conditions with the internal metabolism in order to allow for the
temporal separation of the incompatible metabolic processes. The activation and monitoring
of the response paths of the oxidative stress are energy dependent. Since oxygen reactive
species can be used as important secondary messangers during the stress time, it can be
advantageous for the homeostasis that the ROS is in close connection with the daily cycles of
light-darkness. Moreover, since the photosynthesis is influenced by the sun, the ROS levels
will fluctuate during the day, and the bodies will develop various systems in order to face up
the period increase of their toxic products. It was proven that the circadian rhythm
coordinates the homeostasis of the oxygen reactive species and the transcriptional answer.
The chemotherapies, ever more used nowadays during the oncological treatment, even
in young ages, determines the adverse effects also upon the tissues which are not considered a
target, altering the quality of life of the patients during and after finishing the treatment. The
oxidative stress directly or indirectly caused by the chemotherapy agents is one of the
mechanisms at the basis of the anti-cancerous drugs’ toxicity at the level of the healthy
tissues. A good understanding of the oxidative stress’ mechanism upon the normal tissues can
be essential for the improvement of the strategies for preventing or attenuate the toxicity of
the chemotherapy agents without compromising their therapeutic value.
The majority of the chemotherapies agents proved to induce the oxidative stress,
including cyclophosphamide, cisplatin, fluorouracil, mitomycin or bleomycin. Certain
therapeutic agents, such as the alkylate agents, produce the oxidative stress at the level of the
healthy tissues, causing injuries at this level. Although these chemotherapies agents improve
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the survival rate of the patients, the oxidative stress launched at the level of the normal tissues
is a significant adverse effect and decreases the patients’ quality of life.
The cellular antioxidants and the antioxidant enzymes play a vital role within the
process of removing the excess-formed free radicals within the oxidative stress, maintaining
the balance of the redoc cellular status’ balance within normal conditions. The oxygen
reactive species or the nitrogen one form following the antioxidant cellular chemotherapy
treatment and lead to oxidative changes and to the inactivity of the antioxidant enzymes. This
cascade of events modifies the redox cellular status towards the direction of the oxidative
stress. The oxygen reactive species produced as response to chemotherapy decrease the
cellular reserve of glutathione and, thus, accentuate the oxidative stress.
The cyclophosphamide is a chemotherapy from the alkylating agents’ group and is
one of the most used cytostatic of all times, constituting a true cytostatic model. Presently, we
witness an ever increased usage of it in the oncologic pathology, including the paediatric one.
Cyclophosphamide is metabolized in the liver, through hydroxylation by the P-450
cytochrome, in two active components: 4-hidroxide cyclophosphamide and aldofosfamide.
The first component is metabolised in the lived in non-toxic products, released by the
kidneys, and the aldofosfamide is non-enzymatically converted in the tissues in mostar
fosforamide alkylates and acrolein. The acrolein can determine urotoxicity, while the Mostar
fosforamide is a bi-functional alkylate considered an active component. The
cyclophosphamide can be used in mono or polychemotherapy.
The cyclophosphamide is a chemotherapy drug frequently used in clinic for treating
the malignant and non-malignant tumours. Its metabolites can interact with the cellular
macromolecules, such as the proteins, membrane lipids, RNA or DNA, and can induce
apoptosis.
Given the numerous present studies, the cyclophosphamide has proven to be involved
in ovotoxicity, even causing infertility in women. The reproductive function presents
deteriorations given the rapid depletion of the ovocitary reserve mediated by the apoptotic
cells and given the ovarian atrophy with the extinction of the due primordial follicles. The
apoptosis, which is an essential physiological process in the physiology of the ovarian
function and in this organ’s development, becomes toxic when the ovary is exposed to
cyclophosphamide. Also, the toxic metabolics of the cyclophosphamide interfere upon the
intracellular antioxidant system, playing an important role in the detoxing of the oxygen
reactive species by the endogen or exogenic antioxidant molecules, such as SOD, the lipid
peroxidation defending at the membrane cellular level.
The treatment with cyclophosphamide causes temporary amenorrhoea or permanent
ovary insufficiency in women. The cyclophosphamide destroys the primordial and primary
ovarian follicles, but also the preantral and antral follicles. At the level of the primordial
follicles and the small primary follicles, the cyclophosphamide seems to affect the oocytes
through apoptotic destruction since in the mature follicles, the cyclophosphamide induces the
apoptosis of the granular cells, followed by the death of the oocytes. The cyclophosphamide-
induced apoptosis is preceded by an increased level of ROS, a low level of glutathione and
the reduction of the redox potential. It was proven that the depletion of the reduced
glutathione mediates the apoptosis induced by the cyclophosphamide through the activation
of the oxidative stress.
The substances which neutralise the possible negative effect of the free radicals are
grouped in the antioxidant defence system. This system comprises an increased number of
elements being as diverse as the free radicals, the cells containing a variety of the capable
substances which annihilate the multiple radical species especially in order to insure a
maximum protection (9, 10).
There are two defence mechanisms against the oxygen reactive species:
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➢ the primary defence mechanism which includes the antioxidants which can be
enzymatic (for example the superoxide dismutase, catalase, glutathione
peroxidases) and, respectively, the non-enzymatic antioxidants (for example,
vitamin E, vitamin C, carotenoids, bilirubin, uric acid, glutathione and others)
which have the role of neutralising the free radicals produced in the body thus
preventing their attack upon the cellular components (proteins, lipids,
carbohydrates and nucleic acids).
➢ The secondary defence mechanism which includes the lipolytic enzymes,
phospholipids, proteolytic enzyme, endonuclease, glycosylated, which discharge proteins,
lipids and nucleic acids injured by the other oxygen reactive species (11,12,13).
RESEARCH MOTIVATION, SCOPE AND OBJECTIVES
The implications of the oxidative stress upon the female genitalia shapes an extended
and varied pathology, and which has at its foundation the same oxidation action link, namely
the inhibition of the proteic synthesis, the alteration of their lipids and the DNA degradation.
All the component segments of the female genital tract are affected (the dysfunctions at the
vaginal, ovarian, tube, uterus level, endometriosis, embryopathy, syndrome of the polycystic
ovary, preeclampsia, miscarriage, etc.) (14).
Within this context, it is frequently accused the association of the oxidative stress with
an ever more increased number of chemotherapy agents; if the oxidative action mechanisms
and those associated with the chemotherapy are accurately described, the way in which they
superimpose and their repercussions upon the main functional landmarks of the female
genitalia are insufficiently known and described in the speciality literature.
Thus, cyclophosphamide – one of the most important chemotherapies in the treatment
of numerous forms of cancer in the genital area (breast, ovarian, endometrial cancer)
generates a profound change of the reproduction female potential, the infertility being
frequently evoked as one of the most significant adverse effects; its action mechanism
presupposes the DNA destruction and the multiplication of the cellular apoptosis and through
the excess production of oxygen reactive species within the induced oxidative stress. Its
toxicity determines the local aggressiveness of the free radicals often limits its usage on the
long term, despite the good results obtained in the oncologic therapy (15).
The influence which the biorhythms, especially the circadian rhythm, exercise upon
the organism represents a starting point for numerous theories and controversies which
punctuate the multiple connotations linked to their implication in the unfolding of the vital
functions. The rhythmicity is induced upon all the cells through well determined mechanisms,
coordinated at the cerebral central level and which, in mammals, has presumed the adaptation
of the light/dark, day/night cycle; this adaptation has generated an adjustment of all the links
of the hypothalamus-pituitary-suprarenal axis within the context of the synchronic
functioning of the similar cellular population.
The circadian rhythm, genetically determined and configured in every specie, imposes
a precise coordination mediated through the intermediation of some extremely sensible
connections, of the feedback type, between the various centres which insure the perception,
identification and integrity of the changes which took place within the surrounding
environment. The variations of the light stimuli have a major influence upon maintaining the
initial coordination of the circadian rhythm, adjusting the organism’s reactions. In the case of
the female genitalia, since its functionality follows its own biorhythm, which is not found in
other systems of the human body, a much more complex analysis is imposed, which would
properly go through and evaluate all the stages involved (16).
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Presently, we witness at the apparition of new disorders which are determined, for
example, by the so-called inversion of the circadian rhythm, through the exposure during
night to the artificial light given the working conditions. This fact brings about a
multiplication of the increase in the cancer at the genital area level.
The so-called internal clock, which controls the rhythmical activity of each system, is
permanently adapted to the environmental conditions; the perturbation of the circadian
rhythm and the de-synchronisation of the functioning rhythm could be correlated with the
installation of the same oxidative stress, generating the apparition of an extremely large
palate of disorders of the female genitalia (17).
No matter the sub-layer which insures the genesis and evolution of the oxidative
stress, the antioxidants can represent a direct therapeutic helper involved in the shaping of an
efficient treatment, with the decrease of the oxidative stress’ effects. Most of the clinical
studies on this issue cannot offer a unilateral appreciation and a clear interpretation of the
therapy significance which includes the antioxidants.
Presently, there appears also a double positioning linked to the real potential of the
antioxidants to adequately modulate and protect the female genitalia from the dysfunctions
generated by the oxidative stress: there are opinions which sustain their administration,
considering that they have a significant contribution in the reduction of the oxidative stress,
but there are also opinions which claim that the antioxidants can release mechanism which
decrease the oxidative stress, but, at the same time, modify also the reactivity of the tumour
cells to the involved chemotherapy agent’s action, process which implicitly leads to the
increase of their degree of resistance to therapy. In the pathologies correlated with the
oxidative stress, a prolonged time period has been refuted with regard to any association of
the antioxidants (18).
The reduced number of studies analysing these aspects plead for a thorough study and
a clear and detailed analysis of the oxidation mechanisms through which the chemotherapy,
but also the alteration of the circadian rhythm determines the female infertility. Moreover, the
implication of the antioxidants in the therapy for the female genitalia disorder must be re-
evaluated and correctly positioned.
All these motivated aspects for the selection of the doctoral theme, in the condition in
which the etiopathogenesis of a considerable number of female genitalia disorders continue to
present unclear aspects, the oxidative stress being blamed as an important causing factor.
Our experimental research has the following objectives:
Shaping an experimental model using laboratory animals for the reproduction of the
oxidative stress;
Emphasizing the existence of the correlation between the oxidative stress and the
administration of the chemotherapy-type of medication;
Emphasizing the existence of the correlation between the perturbation of the
circadian rhythm and the installation of the oxidative stress;
The experimental evaluation of the amplitude of the oxidative stress’s effects in
each of the two types of stress considered in the study;
The experimental evaluation of some effects for some antioxidants upon the
oxidative stress.
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MATERIAL AND METHODS
For the analysis of the oxidative stress and its effects, we used 8 groups of 10 adult
female rats, the Wister breed, with an initial weight between 200 and 220 g. The animals
were kept in individual cages, in identical laboratory conditions (temperature comprised
between 22 ± 2◦C, humidity 40 %, a controlled circadian rhythm of 12 h light/12h darkness),
in the first week, in order to accommodate them to the new environmental conditions. All the
animals have received the same standardised food and ad libitum water. The entire
experiment took place while respecting all the criteria and standards imposed by the Ethics
Commission of the “Grigore T. Popa” University of Medicine and Pharmacy of Iasi, as well
as the norms foreseen in the Guidelines on the Care and Use of Animals for Scientific
Purposes, National Advisory Committee for Laboratory Animal Research, 2004.
The animals were weighted for the whole duration while the experiment was carried
through, for supervising the weight and their evolution at the same time with the installation
of the oxidative stress. Moreover, weekly, we have evaluated the relative weight of the uterus
and ovaries collected from 2 rats in each group.
At the end of the 4 weeks of work, blood was sampled from the retro-orbitary plexus,
following which the animals were euthanized in a separate room through the by
administering intraperitoneal 100 mg/kg ketamine, associated with xylazin, 10 mg/kg. This
stage of the experimental study was carried on while respecting the procedures in forced
which foresee the causing of a fast and painless death.
The ovaries and uterus, together with the vaginal segment were sampled from each
animal and were separately weighted, and then fragments from each organ were sampled in
order to be examined.
RESULTS AND DISCUSSIONS
Our experimental study has aimed to emphasize the implications of the oxidative
stress upon the female genitalia, this causing an extended and varied pathology, which has at
its basis the same oxidative action links, namely the inhibition of the proteic synthesis,
degradation of the DNA and the alteration of the lipids. Although the impact of the SRO
excess at this level is more difficult to emphasize, more and more speciality studies plead for
an increase in the various oxidative mechanisms which intervene in the pathogenesis of the
disorders in the genital area disorders and question the contribution of the antioxidants in the
modulation of their evolution and extension.
The reproduction is a fundamental process which allows the living organisms to keep
their offspring and to evolve through the transmission of the genes. Despite this, the
metabolism, which has sustained the sexual steroidal hormons, is powerfully activated during
the spermatogenesis, indicating the fact that the reproductive system in itself generates high
levels of ROS. During the male DNA transfer to the female partner, the spermatozoids are
exposed to high oxygen concentrations. Thus, the reproduction as a whole is a process which
is severely exposed to oxidative stress in various stages (19).
The oxidative stress was involved on a large scale as origin for several issues
affecting the female reproductive health, which, in its turn, influences the ease of conception,
maintains a healthy pregnancy and the genetic changes transmitted to the offspring.
Following the action of the oxidative stress dysfunctions appear at the ovarian,
vaginal, uterus, tubal level, but also endometriosis, embryopathy, the polycystic ovary
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syndrome, preeclampsia and miscarriage; the heterogeneity, non-uniform distribution on the
age segments, in the case of the female fertile life and especially the compromise of the
female functions impose a re-evaluation of all the components of the oxidative stress and
especially its forms of action (20).
Under normal conditions, the antioxidants converted ROS in H2O in order to prevent
the overproduction of ROS. There are two types of antioxidants in the human organism: the
enzymatic and non-enzymatic antioxidants. Moreover, the enzymatic antioxidants, known as
natural antioxidants, neutralise the ROS produced in excess and prohibit the deterioration of
the cellular structure. The enzymatic antioxidants are composed from the superoxide
dismutase, catalase, glutathione peroxidases and glutathione reductasis, which cause also the
reduction of the oxygenated water in water and alcohol (21). The non-enzymatic antioxidants
are also known as synthetic antioxidants or food supplements. The complex antioxidant
system of the organism is influenced by the food intake of the antioxidant vitamins and
minerals, such as vitamin C, vitamin E, selenium, zinc, taurine, hypo-taurine, glutathione,
beta-carotene and carotene (22).
Within our experimental study, during the first work stage, we have evaluated the
general status of the laboratory animals included in the study, monitoring their weight in
order to appreciate the general state not only during repose, but also during the stress
conditions. Thus, we were able to observe that there were significant differences during the 7
groups. Thus, reported to the witness group, the CP group recorded a continuous decrease
and significant weight loss from one week to another, marking the most drastic reduction of
the weight in the groups used.
Our experimental model has proved the fact that the LE group was influenced by the
perturbation of the circadian rhythm; presently, the accent is placed ever more upon the
correlation which is established between the variable changes of the circadian rhythm and the
apparition of the oxidative stress, as a direct consequence of these processes in which the
organism can no longer accommodate to the environment and becomes ever more slightly
aggressed by the excess of the free radicals.
In our study we have observed the fact that both antioxidants used exercise a direct
and real influence upon the general status of the organism. Vitamin E intervenes in the
process of reducing the lipid peroxidation, and its antioxidant potential allows for the
restauration to a large degree of a normal evolution dynamics for the general state of the
organism. Vitamin E acts not only upon the process of forming in excess the SRO, but also
upon the existing SRO level, launching the initiation of some redox links capable of
modulating not only locally but also from a distance sequences of the antioxidant defence
(23).
On the other hand, the zinc acts upon the reduction of the SRO excess, protecting the
cellular membranes and those of lipid peroxidation (24). It is an extremely powerful
antagonist of the catalytic properties of the iron and copper, the two metals being recognised
for their active involvement within the redox mechanisms (25).
Our experiment has shown also changes of weight with regard to the uterus, with a
significant reduction compared to the control group, in the CP, CP + vit. E group and in the
LE group, a fact which will be reflected upon the fertility of the used laboratory animals. In
the case of the ovaries, the relative weight has decreased in the CP, CP + vit. E group and, in
a lesser extent in the LE group. Although both types of oxidative stress caused the uterus’
weight decrease and also that of the ovaries, the more significant changes were produced by
the administration of CP. Moreover, the most efficient antioxidant for the modulation of this
process was vitamin E.
In the experiment which we carried on, in order to sketch a complete picture of the
effects of the free radicals from the oxidative stress, for each of the 7 batches in our study we
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carried on a biochemical evaluation of the biomarkers, the most useful in the interpretation of
the intensity and the involvement of the oxidative stress in the alterations of the female
genitalia: MDA, as loyal indicator of the lipid peroxidation, as well as the antioxidants,
representatives of the SOD, catalases and GPx.
Malondialdehyde is a marker of the oxidative stress which results from the lipid
peroxidation of the poly-non-saturated fatty acids (26). This aldehyde is a powerful toxic
molecule and must be considered as an important marker for their lipid peroxidation. Their
interaction with the DNA and the proteins is frequently considered potentially mutagenic and
aterogenic (27).
In our study, determining the MDA has presented distinct variations in the analysed
groups; thus, in the control group, in the dynamics, the average MDA values did not present
significant changes. The rest of the groups presented distinct variations of the MDA. For the
CP group, the average MDA level recorded significant changes from one week to the other.
Generally, it was accepted the fact that the accentuated lipid peroxidation represents one of
the toxic effects of the CP administration.
At the level of the groups which received CP but also antioxidants, we could observe
a slight initial increase in the MDA level, following which a continuous decrease of the
MDA, until the end of the experiment. Vitamin E determined a slightly more accentuated
decrease of the MDA than the ZnCl2, one of the possible explanations of this fact being the
interference of the vitamin E with the reactions which unfold during the lipid peroxidation,
thus blocking the SRO formation, but, overall, the effects of the two antioxidants used were
similar in amplitude as the reduction of the oxidative stress’ effects.
The LE group presented a significant increase of the MDA; the prolonged exposure to
the light determines the change of the circadian rhythm, which eases the adaptation to normal
environment conditions. The recent data mention the fact that any change to the exposure to
the light stimuli attract a pile of events which, in the end, will accentuate the cellular
vulnerability to the oxidative-type of alterations; it is believed that the light presents a major
significance as the trigger to the alterations of the circadian rhythm, through the
intermediation of the retinal-hypothalamic system, with direct intervention of some signals
from the neuronal and umoral level. Due to this reason, we have chosen the exposure to the
prolonged light as the so-called stress or circadian, for the inducement of the oxidative type
of stress (28).
If in the case of the chemotherapy with CP, its oxidative action mechanism is
elucidated, in the case of the circadian rhythm disturbance, there exist even more question
marks linked to the connection between the oxidative stress. This can appear as a
consequence of the collapse of antioxidant protection factors under the influence of the
disturbance of the circadian rhythm or it might be the result of the chaotic and continuous
generation of SRO, which comes out of the control of the metabolic links already eroded by
the disturbance of the organism’s biorhythms (29).
Our study has revealed the fact that the circadian stress has determined a direct
change of the MDA values compared to the witness group, their progressive increase being
correlated with the persistence of the circadian stress. The majority of the cells have the
capacity to repair the effects of the moderate oxidative stress, but at the same time with the
accentuation of the oxidative attack, the cells die and an extremely diversified pathology
appears.
In our experiment we have aimed at also the way in which it intervenes and evolves
the system of the most significant antioxidant enzymes. The biochemical analysis of the SOD
activity has allowed us to observe the fact that this enzyme has presented the variations in the
analysed groups, during the 4 weeks of the experiment. The average values of the SOD have
significantly decreased in the 2nd and 3rd weeks within the control group, in order to return to
12
the same average level during the 4th week. Within the CP group, the SOD average level has
recorded a continuous decrease, from the beginning until the end of the experiment, from one
week to another, a fact which sustains the oxidative action mechanism of the CP, as well as
the decrease of the antioxidant activity, in the organism’s attempt to surpass the stress
conditions through the intermediation of its own antioxidant system. In the conditions in
which the most accentuated decrease of the SOD took place in the CP group, we can consider
that the defence mechanism is almost surpassed by the intensity of the oxidative stress (30).
Within the CP group, it was received at the same time also the vitamin E, following a
moderate decrease of the SOD activity from the beginning of the experiment, the level of
SOD has registered a significant increase, a similar evolution being observed also in the CP +
ZnCl2 group, with a slight decrease of the average values towards the group which has
received the vitamin E. Thus, we were able to observe that both antioxidants used by us in
this experiment have allowed for an increase of the SOD.
Within the LE group, the SOD activity has recorded a decrease, but a higher decrease
compared to that determined in the CP., and the groups to which it was administered the
vitamin E or ZnCl2, has increased. The smallest average value of the SOD was registered in
the CP group, and the highest at the LE + vit. E group. There is data in the recent speciality
literature which confirms that an SOD increase can be interpreted within the context of the
successive increase of the fatty acids concentration, the phospholipids, glucose and
cholesteryl esters, through the stimulation of the direct metabolic connection for the
activation of these compounds (31).
Within our experiment, we have carried on also the biochemical dosage of the
catalases activity, the important marker of the antioxidant activity. Within the CP group it is
to be observed a marked decrease of the catalases activity ever since the first week of
administration, the decrease which has a progressive evolution until the last week of the
experiment. Within the groups which were administered the antioxidant in association with
CP, following a slight decrease in the first week, it follows the increase of the catalases
activity, but without surpassing the initial average value. Within the LE group, it is to be
found a more accentuate decrease, while the LE+vit. Group the activity of the catalases
progressively increases, without surpassing the initial average value.
Our study has evaluated also the GPx activity, which is in close connection with the
presence of the reduced glutathione (GSH) as donor of the hydrogen ions. Glutathione is the
main cellular mediator of the redox system and is synthetized at the cytosol level from the L-
glutamate, L-cysteine and wisteria. GSH participates in a large number within the
detoxification reaction forming the disulphate glutathione, which is again converted in GSH
through the action of the glutathione reductasis, through the consumption of NADPH. This
enzyme is involved in various polymorphs, and the decreased activity can compromise the
GSH concentration and can lead to embryopathy (32).
During the observation, the CP group is remarked through the significant decrease of
the GPx activity. Within the CP groups in association with the antioxidants, the GPx activity
increases, not reaching the initial basic values. This data, in association with the data from the
experimental studies, show the protective character of the antioxidants upon the oxidative
stress launched by the CP administration.
Within the LE group, it is recorded the significant decrease of the GPx activity
beginning with the first week of the study, decrease which is maintained until the end of the
experiment. In the LE group, to which it was administered also the antioxidant, the GPx
increases. Finally, the lowest average value of the GPx activity was found in the CP group,
and the highest value is to be found in the CP + ZnCl2 group and the LE + vit. E group,
slightly more decreased when compared to the witness group.
13
The histopathological examination of the segments of the genital path has revealed the
existence of a correlation between the form of the oxidative stress used in our experiment and
the resulting tissue alterations.
Thus, at the level of the vaginal mucosa, at the CP group we were able to observe the
apparition and the multiplication of the mucosa cells with vacuolated appearance in the
superficial layers of the epithelium; the large number of cell layer with mucus content, the
abundance of the cellular layers upon which this epithelium is composed, the disappearance
of the keratin pellicle shown in normal conditions upon the vaginal epithelium surface, as
distinct area of demarcation for the superficial mucigene, specific for this area in rats, support
our case, in the conditions in which the pregnancy is absent or during the proestrus phase, an
alteration which has no connection with the cyclical changes which happen at this level. We
have considered that this process is generated by the CP cytotoxicity, which infers with the
normal redox circuits and causes the apparition of the oxidative stress (33). CP generates a
succession of degenerative and necrosis processes, followed by a significant gonadal atrophy.
In our experiment, we have observed that the CP + vit. E groups and the CP + ZnCl2
respectively have presented a degree of vaginal atrophy, but the accumulation of the
mucigene cells at the epithelium level was considerably reduced when compared to the CP
group; the two antioxidants have offered a relative protection for the vaginal mucosa.
In the case of the LE group, we have observed the complete absence of the mucigene
cells upon the surface of the vaginal epithelium, the entire vaginal mucosa maintaining its
initial configuration, without significant changes. The prolonged exposure to light did not
determine the alterations obvious at the vaginal level.
In our study, for both types of stress, the antioxidants used have considerably
improved the changes which took place at the genitalia level, the analysed biochemical
markers sustaining this observation.
In our experimental study, the histopathologic examination of the uterine tissue
fragments sampled from the CP group has emphasized the existence of some changes at the
uterine mucosa level. The epithelium atrophy of the endometrial glands, as well as the
frequent cellular apoptosis dispersed at the surface epithelium level were the most frequent.
Similar changes were observed also at the level of the LE group. The administering of
vitamin E and of the ZnCl2 was followed by a reduction of their alterations.
In our study, the histopathological examination of the ovary has revealed a series of
changes correlated with the nature of the induced oxidative stress. The CP effect has caused a
series of changes at the level of the CP group; the ovarian morphology emphasizes the almost
full disappearance of the primordial ovarian follicular, of the great follicles and the luteal
bodies, in parallel with the significant multiplication of the atresia follicular.
The examination of the ovaries in the CP + ZnCl2 group has emphasized, just like in
the CP + vit. E group, an improvement of the effects produced by the CP at the level of the
ovarian morphology. The fact that the zinc is missing the toxicity and, moreover, in the
conditions of oxidative stress produces cysteine-rich metalotioneine, the potential to
neutralise the OH radicals and to inhibit the production of the inflammatory cytokine imposes
as an election antioxidant in the case of this oxidative stress form. We were able to observe
the reparation of the primordial follicular at the periphery of the ovary’s cortical and the re-
establishment of the normal proportion between the ovarian follicles types.
Upon analysing the ovaries sampled from the LE group we observed that the changes
in the ovary’s structure presupposed the diminishing follicular reserve, the disappearance of
the primordial follicles and of the luteal bodies, and the addition of vitamin E or of the
ZnCl2.
14
Another work stage of our study aimed at the exploration of the immune-histo-
chemistry exploration of the alterations produced by the oxidative stress through the usage of
some specific antibodies, by emphasizing the localisation of the oxidative stress impact.
One of this, 8-OHdg, is considered as being the most loyal marker which allows for
the evaluation of the DNA alterations produced by the oxidative stress. The mechanism
through which the oxidative stress influences the DNA is that of allowing the release of some
massive SRO quantities, which are highly instable and which have a special ability to interact
with the DNA; once the antioxidant defence links are crossed, the SRO will initiate a
succession of events which progressively link and lead to the frequently irreversible
deterioration of the DNA (34).
In this study, from the comparative analysis of the immune-histo-chemical study of 8-
OHdg for the vaginal segment and the uterine one corresponding to the induced oxidative
stress through the administration of CP, we were able to observe an intense positive immune-
expression at the level of the two mucosa. The vaginal epithelium and the uterine one have
reacted to the stress and they allowed us to consider its intensity following the CP effects was
highly increased.
The LE group presented in its turn a positive immune-expression for 8-OHdg; a stable
circadian rhythm, with exposure 12/24 hours to light and, respectively, darkness allows the
normal unfolding of the reproduction function, by maintaining unchanged the circuits which
process the adaptation to the environmental conditions, the melatonin being one of the main
vectors involved in this process.
In the present study, the prolonged exposure to light acts as stress or, by mobilising
the SRO which acts specifically, and produces the typical lesions at the female genitalia level.
The rat groups which received at the same time the antioxidants have shown a much
more reduced expression of the 8-OHdg. Both vitamin E and the zinc have increased the
immune-expression 8-OHdg, reducing the intensity of the oxidative stress. Although it was
initially considered that the association of the antioxidants in the case of the pathologies in
which the oxidative stress is acknowledged as being the pathogen vector it does not have the
effect in the case of female genitalia, our study proves through the bio-chemical and with
regard to the immune-histo-chemical evaluation study the fact that the antioxidants improve
the considerable effects of the oxidative stress.
Our results, in accordance with other experimental studies, suggest the existence of a
powerful oxidative mechanism, capable of determining the changes which in the end
determine the DNA alteration. These alterations will produce the indirect and unequal
modulation of the genes’ expression which will train an instability of the corresponding gene
profile implicitly, an abnormal cellular proliferation. The vaginal epithelium cells and
respectively, those from the endometrial epithelium, have promptly reacted to the
administration of this antibody through a reaction which sustains the presence of the
oxidative lack of equilibrium through a reaction which supports the presence of the oxidative
lack of equilibrium, a fact which can affect later on the expression of the various genes which
have a role in maintaining and coordinating the cellular and specific functions.
Another marker of the oxidative stress used was the 4-HNE, the result of the
phospholipidic peroxidation. Its evaluation allowed us to sample the level of the oxidative
stress at the cellular level, in the conditions in which 4-HNE is a very loyal indicator of the
SRO concentration; given its importance, presently it is the most studied of all the products
resulting following the lipid peroxidation (35).
The immuno-histo-chemical expression of the 4-HNE, which indicates to us the
intensity of the oxidative stress, had a high positive level for the CP and LE groups, the two
forms of induced oxidative stress correlated with the increased level of lipid peroxidation,
15
detected through the immune-histo-chemical evaluation of the 4-HNE and emphasized in the
level of the vaginal mucosa and the uterine one.
In the case of the perturbation of the circadian rhythm through the chronical exposure
to light, we were able to observe also a positive intense immune-expression of the 4-HNE, at
the level of the vaginal and uterus mucosa, similar to the one present in the CP group; the
increase of the duration of exposure to the light stimulus has caused the considerable
reduction of the local antioxidant device and the accentuation of the lipid peroxidation, and
especially the antioxidants present, in their turn, a secretion which respects the principles of
the cyclic rhythm.
The supplementing with antioxidants, not only vitamin E but also ZNCl2, was
followed by the decrease of the 4-HNE level, which immunohistochemically expression has
become faintly positive compared to the CP and LE groups.
AMH is one of the markers which allows us the appreciation of the ovarian function
and, in normal conditions, it is produced and expressed at the level of the granular cells of the
primary, preantral and antral follicular in an early stage, but it is not expressed in the
primordial, mature and atresia follicular (36).
In the experiment we have carried on during the doctoral thesis, the AMH immuno-
expression analysis at the level of the control group has allowed us to identify the reactivity
of the granular cells and the oocyte in normal conditions, the AMH level being increased in
the granular cells of the primary, preantral, small dimension antral follicular.
We have not perceived the presence of AMH at the atresia follicular level, these
observations being correlated with the data from the speciality literature, which mentions the
absence of AMH in this type of follicular during the fertile life of the female, but becoming
active once the menopause is installed.
In our study, the AMH immuno-expression at the level of the ovaries sampled from
the CP group has revealed a different dynamic from that encountered in the control group, the
CP aggressiveness for all types of ovarian follicles being known as one of the most important
limitations of its usage in the long-term treatments.
Compared to other chemotherapy agents, CP is one of the alkylate agents which
determines the irreversible follicular alterations; not only the oocyte but also the granular
cells are affected by the CP’s action, its ototoxic action being more evidently found of the
primary, as well as the preantral follicular level.
Within the LE group, the AMH immuno-expression was similar to that in the CP
group, highly positive at the level of the atresia follicles; the oxidative stress induced by the
perturbation of the circadian rhythm has caused a decrease in the AMH level of the currently
developing follicles. This fact might be correlated with the AMH’s own variation of the
circadian type; although much more reduced compared to the classic sexosteroids, any factor
contributing to the derangement of the AMH’s own secretion rhythm will cause a series of
effects which, in the end, lead to the destabilization of the ovarian follicular population.
The oxidative stress in the gynaecological environment is a probable mediator of
conception, yet direct proof is required in order to confirm this hypothesis. The generation of
ROS is a consequence of the active metabolic cells and it is probable that the legal of the
oxidative stress threshold to insure the promotion of conception.
The best available proofs suggest that a varied diet, with the regular usage of the
multivitamins, limited with regard to caffeine and alcohol and the maintenance of a healthy
body weight, can promote fertility. Prospective studies regarding the evaluation of the diet
and the collection of biological samples before conception are necessary for the true adequate
of the relation between the oxidative stress, the food factors and the female fertility.
Oxidative stress is associated with the decrease of female fertility in the animal
models and in vitro, yet until presently the relations in women have not been directly studied.
16
The exposures associated with the oxidative stress and with the proof which influences the
calendar and the maintenance of a viable pregnancy includes the pregnancy complications
(for example, preeclampsia), extreme body weights, alcohol, tobacco and caffeine. The
contribution of the antioxidants nutrients, including the usage of the multivitamins, influences
the generation of oxygen reactive species and can play a beneficial role in the female fertility.
The antioxidants can be administered when the specific etiology cannot be
identified as in the idiopathic infertility because there is no other treatment based upon proof
of idiopathic infertility, and the reports indicate the presence of OS. The strategies for
overtaking the in vitro OS conditions and for the balancing between the in vivo and in vitro
environments can be used in ART in order to successfully treat infertility.
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