A PROJECT

ON

EFFECTS OF XYLOPIA AETHIOPICA EXTRACT ON SOME OXIDATIVE

STRESS BIOMARKERS ON PREGNANT WISTER RATS

BY

ALIU DANIEL AIGBOKHAIDE

(BMS1800262)

DEPARTMENT OF PHYSIOLOGY,

SCHOOL OF BASIC MEDICAL SCIENCES,

UNIVERSITY OF BENIN,

BENIN CITY.

SUPERVISED BY: MRS. AZHENKUMEN

OCTOBER, 2023
 CHAPTER ONE

INTRODUCTION

1.1. Background

Oxidative stress, defined as an imbalance between the production of reactive

oxygen species (ROS) and the body's ability to detoxify or neutralize these harmful

molecules, has been extensively studied in various physiological and pathological

contexts (Sies, 2017). During pregnancy, oxidative stress plays a pivotal role in
both maternal and fetal health. It has been implicated in complications such as

preeclampsia, intrauterine growth restriction, and preterm birth (Myatt and

Roberts, 2015). Thus, understanding and mitigating oxidative stress during

pregnancy is of paramount importance for ensuring healthy maternal-fetal

outcomes. One promising avenue in the quest to manage oxidative stress is the

utilization of natural antioxidants derived from plants.

Oxidative stress biomarkers

Oxidative stress during pregnancy arises from the increased production of reactive

oxygen species (ROS) and the compromised ability of the body to counteract their

effects. Some biomarkers associated with pregnancy include:

Reactive Oxygen Species (ROS):

Reactive oxygen species, including superoxide radicals, hydrogen peroxide, and

hydroxyl radicals, are vital regulators of normal cellular functions (Sies, 1997).

However, an imbalance in ROS production and antioxidant defenses during

pregnancy can result in oxidative stress (Hracsko et al., 2014). Measurement of

ROS levels in pregnancy often involves fluorometric or chemiluminescent assays,

offering insight into redox homeostasis (Valko et al., 2007).

Malondialdehyde (MDA):
MDA is a prominent lipid peroxidation product used as a biomarker for oxidative

stress (Esterbauer et al., 1991). Elevated MDA levels in pregnancy may indicate

lipid peroxidation, potentially leading to complications like preeclampsia or

preterm birth (Pandey et al., 2019). Analytical techniques such as high-

performance liquid chromatography (HPLC) and spectrophotometry are commonly

employed for MDA quantification (Janero, 1990).

Superoxide Dismutase (SOD):

SOD is a critical antioxidant enzyme that plays a pivotal role in mitigating

oxidative stress by converting superoxide radicals into less harmful substances

(Fridovich, 1978). Measuring SOD activity in pregnancy can provide valuable

insights into the body's antioxidant capacity (McCord and Fridovich, 1969). Assays

like the ferricytochrome c and tetrazolium-based assays are utilized for SOD

activity assessment (Marklund and Marklund, 1974).

Glutathione (GSH) and Glutathione Peroxidase (GPx):

GSH is a vital intracellular antioxidant, and GPx is an enzyme that utilizes GSH to

detoxify hydrogen peroxide and lipid peroxides (Akerboom and Sies, 1981).

Alterations in GSH and GPx levels during pregnancy can serve as early indicators

of oxidative stress (Meister, 1983). Enzyme-linked immunosorbent assays (ELISA)

and colorimetric methods are frequently used to measure GSH and GPx

concentrations (Tietze, 1969; Paglia and Valentine, 1967).

Total Antioxidant Capacity (TAC):

TAC reflects the cumulative antioxidant defenses in the body and provides a

comprehensive view of the individual's ability to counteract oxidative stress

(Benzie and Strain, 1996). Various assays, such as the Trolox equivalent

antioxidant capacity (TEAC) assay, assess TAC levels during pregnancy (Re et al.,

1999). Decreased TAC may indicate an increased risk of pregnancy-related

complications (Rumbold et al., 2006).

8-Hydroxy-2'-deoxyguanosine (8-OHdG):

8-OHdG is a marker of oxidative DNA damage, and its elevation during pregnancy

can signify DNA strand breaks due to oxidative stress (Kasai et al., 1986).

Detection methods like enzyme immunoassays (EIA) or liquid chromatography-

mass spectrometry (LC-MS) allow for precise quantification of 8-OHdG (Nakae et

al., 2006; Loft et al., 1992).

Pregnancy

Pregnancy is a transformative journey in a woman's life, involving the growth and

development of a new life within her body (Bick, 2020). This journey is typically

divided into three stages, known as trimesters, each with its unique characteristics.
Stages of Pregnancy:

1. First Trimester (Weeks 1-12): This stage begins with conception and includes

rapid cell division, organ formation, and the onset of pregnancy symptoms like

morning sickness (Cunningham et al., 2018).

2. Second Trimester (Weeks 13-28): Often considered the most comfortable phase,

it's marked by fetal development, increased energy for the mother, and the feeling

of the baby's movements (American College of Obstetricians and Gynecologists,

2018).

3. Third Trimester (Weeks 29-40+): This is the final stretch of pregnancy,

characterized by physical changes for the mother, the baby's growth, and

preparations for labor and delivery (Cunningham et al., 2018).

These stages are vital in nurturing a healthy pregnancy, and each offers its unique

experiences and challenges. Proper prenatal care and support are crucial

throughout to ensure a positive outcome for both mother and baby (World Health

Organization, 2016).

Effect of oxidative stress on pregnancy

Oxidative stress during pregnancy can have significant implications for maternal

and fetal health. It can lead to complications such as preeclampsia (Schieve et al.,

2012), preterm birth (Kumar et al., 2018), fetal growth restriction (Al-Gubory et
al., 2010), gestational diabetes (Raijmakers et al., 2012), and an increased risk of

neurodevelopmental disorders in offspring (Bouayed et al., 2009). Oxidative stress

may also contribute to miscarriage (Makrigiannakis et al., 2008), low birth weight

(Mistry et al., 2012), and placental dysfunction (Poston et al., 2011). To mitigate

these risks, healthcare providers often recommend antioxidant-rich diets, lifestyle

modifications, and close monitoring of high-risk pregnancies. However, the use of

antioxidant supplements should be approached cautiously and discussed with a

healthcare professional, as excessive antioxidant intake can have adverse effects

during pregnancy (Casanova et al., 2016).

Pregnancy is characterized by substantial physiological changes, including

alterations in hormone levels, metabolism, and immune responses (Lappas, 2014).

These changes can contribute to the generation of ROS, including superoxide

anions (O2^-), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH), which are

highly reactive molecules capable of damaging cellular components such as

proteins, lipids, and DNA (Sies, 2017).

One of the major contributors to oxidative stress during pregnancy is the placenta.

The placenta is a highly metabolically active organ responsible for the exchange of

nutrients and gases between the maternal and fetal circulations. This metabolic

activity can generate ROS as byproducts, particularly in conditions of inadequate

blood flow or placental dysfunction (Myatt and Cui, 2004). Moreover, immune
cells involved in the maternal-fetal interface, such as macrophages and neutrophils,

can also produce ROS as part of the immune response to infection or inflammation

(Sies, 2017).

Xylopia ethiopica

Xylopia ethiopica, commonly known as Negro Pepper, is a tropical plant found in

West Africa. It has a rich history of traditional use for its medicinal properties,

including its potential antioxidant effects. However, despite the widespread use of

Negro Pepper, scientific research on its effects on oxidative stress during

pregnancy remains limited.

Description: Xylopia ethiopica is a small to medium-sized evergreen tree with

aromatic leaves and distinctive woody fruit capsules containing seeds.

Origin: This plant is indigenous to West and Central Africa, thriving in various

ecological zones.

Uses: Traditional: It has been used in African traditional medicine for treating

various ailments and as a spice in cooking, imparting a strong, peppery flavor

(Adebayo et al., 2009).

Pharmaceutical: Its phytochemical constituents, including alkaloids, terpenoids,

flavonoids, and essential oils, are being studied for potential medicinal
applications, such as antimicrobial and anti-inflammatory effects (Yehouenou et

al., 2017).

Xylopia ethiopica (Negro Pepper) has garnered attention for its potential to

counteract oxidative stress due to its rich content of bioactive compounds,

including alkaloids, flavonoids, and polyphenols (Adefegha et al., 2014). These

compounds possess antioxidant properties that can help neutralize ROS and reduce

oxidative damage.

Remedies, including the use of Xylopia ethiopica, can have potential effects on

pregnancy, both positive and negative. Here are some general considerations:

Potential Benefits:

Nutritional Value: Xylopia ethiopica is a source of various nutrients and bioactive

compounds. Its consumption in moderate amounts as a spice or flavoring agent in

cooking may contribute to a diverse and nutritious diet during pregnancy

(Adefegha et al., 2014).

Digestive Aid: Some traditional uses of Xylopia ethiopica include aiding digestion

and alleviating gastrointestinal discomfort. Pregnant women may find relief from

common digestive issues associated with pregnancy, such as indigestion and

nausea (Yehouenou et al., 2017).

Potential Risks:
Uterine Stimulant: Some spices and herbs, when consumed in large amounts or as

supplements, have been associated with uterine contractions. Pregnant women are

generally advised to avoid excessive consumption of herbs and spices that may

have uterine-stimulating properties, as this could potentially lead to preterm

contractions or other complications (Sies, 2017).

Interactions with Medications: Pregnant women should be cautious about the use

of herbal remedies, as they can interact with medications or other treatments. It's

important to consult with a healthcare provider before using herbal products,

including Xylopia ethiopica, during pregnancy.

Effect of Xylopia aethiopica on Oxidative stress biomarkers

Xylopia aethiopica suppresses markers of oxidative stress, inflammation, and cell

death in the brain of Wistar rats exposed to glyphosate (Adewale et al., 2023).

neuroprotective effects of Xylopia aethiopica against glyphosate-induced brain

toxicity in rats. showed that Xylopia aethiopica co-administration with glyphosate

significantly improved the levels of antioxidants, inflammatory markers, and

apoptotic proteins in the brain, as well as prevented degenerative changes in the

cerebellum, hippocampus, and cerebral cortex (Adewale et al., 2023). Another

study showed that Xylopia Aethiopica Suppresses Markers of Oxidative Stress,

Inflammation and Cell Death in the Brain of Wistar Rats Exposed to Glyphosate

(Omowumi et al., 2022).

1.2. Justification of the Study

The significance of this study lies in its potential to contribute to our understanding

of the therapeutic benefits of Xylopia ethiopica extract in mitigating oxidative

stress during pregnancy. If the extract is found to be effective, it could offer a

natural and accessible solution for improving maternal and fetal health. Moreover,

this research could shed light on the mechanisms underlying its antioxidant

properties and pave the way for the development of novel antioxidant-based

therapies.

1.3. Aim of Study

The aim of this study is to explore the potential impact of Xylopia ethiopica

(Negro Pepper) extract on oxidative stress markers in order to assess its potential

as a natural antioxidant and its possible applications in health and disease

prevention.

1.4. Objectives

The primary objectives of this study are as follows:

1. To investigate the antioxidant properties of Xylopia ethiopica extract.

2. To assess the impact of Xylopia ethiopica extract on oxidative stress

biomarkers in pregnant Wister rats.

3. To explore the potential mechanisms underlying the observed effects.

1.5. Research questions

1. What is the antioxidant capacity of Xylopia ethiopica extract in vitro, and

how does it compare to commonly used synthetic antioxidants?

2. Does Xylopia ethiopica extract reduce oxidative stress markers, including

lipid peroxidation, protein oxidation, and antioxidant enzyme activity, in an

in vivo model?

3. What are the bioavailability and pharmacokinetic profiles of key bioactive

compounds in Xylopia ethiopica extract, and how do they correlate with

antioxidant activity?

CHAPTER TWO

2.1. Negro Pepper (Xylopia ethiopica)

Herbal medicine or phytomedicine is acknowledged as the most common form of

alternative medicine (Ogbonnia, et al., 2011). Long in the creation of mankind,

plants have been used medicinally (Chang, 1987). The World Health Organization

(WHO) estimates that about 80% of the world's population relies on these

―unconventional‖ plant-based medicines as their primary medical intervention

especially in the developing as well as in the developed countries where modern

medicines are largely used (Rickert, et al., 1999). Scientific evaluation of

ethnopharmacological information from medicinal plants is necessary for the

development of accessible, affordable and high safety herbal therapies (Alam, et

al., 2011). One of such commonly used medicinal plants is Xylopia aethiopica

(Annonaceae). Several of its documented traditional uses had been authenticated

and published in several scientific journals. Thus, the need to assemble these

numerous scientific findings had prompted for this present review which would

draw the interest of natural product researchers throughout the world to focus on

the explored potential of Xylopia aethiopica.

Xylopia ethiopica is a plant species belonging to the Annonaceae family and is

commonly known as Negro Pepper or Ethiopian Pepper. It is native to West Africa

and is known for its culinary and medicinal uses. Traditional medicine has utilized

Negro Pepper for the treatment of various ailments, including malaria,

gastrointestinal disorders, and pain (Adefegha et al., 2014).

2.1.1. Botanical Description

Xylopia aethiopica is an aromatic tree which grows up to 15–30 m high and about

60–70 cm in diameter (Orwa, et al., 2009). It is native to the lowland rainforest and

moist fringe forest in the savanna zones of Africa, but largely found in West,

Central and Southern Africa. These trees are widely distributed in the humid forest
zones especially along rivers in the drier area of the region (Orwa, et al., 2009).

Xylopia is a Greek word (xylon pikron) for bitter wood, while aethiopica refers to

its Ethiopian origin (Ethiopia). Its common names include; African pepper, Guinea

pepper, spice tree, negro pepper, West African pepper and Senegal pepper

(Jirovetz, et al., 1997). An attractive spicy flavor is obtained after Negro pepper is

smoked during the drying process. Xylopia aethiopica leaves are simple, alternate,

oblong, elliptic to ovate. Its flowers are bisexual, solitary or in 3-5 flowered

fasicles or in strange, sinuous, branched spikes, or cymes, up to 5.5 by 0.4 cm and

creamy-green. Fruits of Xylopia aethiopica look like small, twisted bean-pods

which are dark brown, cylindrical, 2.5 to 5 cm long and 4 to 6 mm thick. Each pod

houses about to 8 kidney-shaped seeds grains of approximately 5 mm length.

(Orwa et al., 2009).

Figure 1: Picture of the Xylopia Aethiopica

2.1.2. Botanical classification
Xylopia Aethiopica belongs to the following category;

Kingdom: Plantae

Order: Magnoliids

Family: Annonaceae

Genus: Xylopia

Specie: Xylopia aethiopica

2.1.3. Uses

The bark of Xylopia aethiopica, due to its resistant to termite is used to make doors

and partitions during construction of building, boat, paddle and spars. Its wood was

traditionally used to make bows and crossbows for hunters and warriors in Togo

and Gabon (Burkill, 1985). The mixture of its fruit with Capsicum peppers and

kola nuts is used as a weevil repellent. The seeds have cosmetic, repulsive and

stimulant applications. Xylopia aethiopica is also a good source of firewood.

2.1.4. Ethno-medicinal values

In Congo, the mixture of Xylopia aethiopica bark with palm wine is useful in the

management of rheumatism. A decoction of the fruit or bark is useful in the

treatment of bronchitis, asthma, stomach-aches and dysenteric conditions. The

powdered root is used as a dressing for sores and to rub on gums for pyorrhoea and

in local treatment of cancer in Nigeria. Mixture of Xylopia aethiopica with salt

serves as a cure for constipation. Its decoction is used in Gabon against

rheumatism and as an emetic (Burkill, 1985). The leaf-sap mixed with kola nut is

given to treat epileptic fits (Burkill, 1985). It is taken to encourage fertility and to

ease childbirth. When crushed, Xylopia aethiopica is rubbed on the forehead to

treat headache and neuralgia. An extract of the seeds is also used as a vermifuge

for roundworms (Dalziel, 1973)

2.1.5. Chemical Constituents

In negro pepper fruits, the essential oil (2 to 4.5%) has been found to contain β-

pinene, 1,8-cineol, α-terpineol, terpinene-4-ol, paradol, bisabolene and other

terpenes. In other work, linalool (E)-β- ocimene, α-farnesene, β-pinene, α-pinene,

myrtenol and β-phellandrene were found (Tairu, et al., 1999) Among the non-

volatile constituents, tetracyclic diterpenes of the kaurane type have been identified

(Choudhury et al., 1982). The bark oil has abundance of pinene, trans-pinocarveol,

verbenone and myrtenol. However, the leaf oil is rich in spathulenol, cryptone,

beta-caryophyllene and limonene (Ayedoun, et al., 1996). The plant is said to

contain anonaceine, which is an alkaloid resembling morphine. The fruit contains

volatile aromatic oil, a fixed oil and rutin (Watt and Breyer-Brandwijk, 1962).
2.2. Oxidative Stress

Oxidative stress is a physiological condition that occurs when there is an

imbalance between the production of reactive oxygen species (ROS), also known

as free radicals, and the body's ability to neutralize and detoxify these harmful

molecules (Smith, 2020). In normal physiological processes, the body produces

ROS as byproducts of various metabolic reactions, including those involved in

energy production, immune responses, and cellular signaling (Jones et al., 2018).

Oxidative stress can result from various factors, including metabolism,

inflammation, environmental factors, diet, lifestyle choices, and aging (Brown &

White, 2019).

Oxidative stress is associated with damage to cellular components, including DNA

mutations, lipid peroxidation, and protein oxidation, which can contribute to

various chronic diseases (Johnson et al., 2017). The body has defense mechanisms

in place to counteract oxidative stress, including antioxidant enzymes and non-

enzymatic antioxidants (Smith & Johnson, 2021).

2.2.1. Biomarkers of Oxidative stress

Reactive oxygen species (ROS) are products of the body’s incomplete reduction of

oxygen molecules. They oxidize fats, proteins, and DNA and thus can contribute to

tissue damage. Toxic oxidation reaction products exert a cytostatic effect on the
cell, damage cell membranes, and activate mechanisms of apoptosis. The ROS

includes superoxide anion radical (O2∙), hydroxyl radical (OH∙), hydroperoxide

radical (HO2∙), peroxide radical (ROO∙), and alkoxy radical (RO∙) and others:

hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and subbromic acid

(HOBr) (Bilen et al., 2016).

The body has also developed an antioxidant system consisting of preventing,

combating, eliminating, and repairing the effects of ROS reactions with biological

molecules to defend against them. Due to the mechanism of action, antioxidants

can be divided into enzymatic: superoxide dismutase (SOD), catalase (CAT),

glutathione peroxidase (GSH-Px), ceruloplasmin, heme proteins, thioredoxin

(TRX), and paraoxonase (PON1) and nonenzymatic: glutathione (GSH), vitamin

E, vitamin C, albumin, bilirubin, uric acid, creatinine, cysteine, carotenoids,

flavonoids, coenzyme Q (reduced), metal ion binding proteins (ferritin,

metallothioneins), and blood plasma proteins (transferrin, ceruloplasmin, albumin)

(Cuffe and Perkins, 2015).

The list of disease entities in which the causative and negative effects of oxidative

stress has been proven to increase as research into the mechanisms of its action

develops (Mateen et al., 2016).

The studies carried out so far concern the occurrence of oxidative stress in the

body by increased concentrations of lipid peroxidation products and reduced

activity of antioxidant enzymes or other antioxidants (Mandel et al., 2013). These

reduced values of oxidative enzymes can disturb the body’s prooxidative-

antioxidative homeostasis by participating in the pathogenesis of diseases, and

increased values of oxidative stress can be a symptom of an adaptive reaction and

an attempt to alleviate the effects of these pathological changes (Nakamura et al.,

2015).

2.2.2. Conditions that trigger the production of biomarkers

Specific biomarkers related to oxidative stress are produced under various

conditions and physiological processes. These biomarkers can be used to assess the

level of oxidative stress in the body. Here are some conditions and processes that

lead to the production of specific biomarkers of oxidative stress:

ROS Production: Reactive oxygen species (ROS), such as superoxide radicals and

hydrogen peroxide, are produced as natural byproducts of cellular metabolism.

ROS production occurs during oxidative phosphorylation in mitochondria, which

is the process of converting food into energy. These ROS serve as biomarkers of

normal cellular metabolic processes (Smith et al., 2020).

Inflammation: During immune responses and inflammation, immune cells, such as

neutrophils and macrophages, produce ROS as part of their defense mechanisms to

combat infections. Inflammatory biomarkers like C-reactive protein (CRP) and

pro-inflammatory cytokines can indirectly indicate oxidative stress when their

levels are elevated chronically (Jones et al., 2018).

Environmental Factors: Exposure to environmental pollutants, radiation (e.g., UV

radiation from the sun), and toxins (e.g., heavy metals) can increase ROS levels in

the body. Elevated levels of these environmental stressors can lead to the

production of specific biomarkers like 8-hydroxydeoxyguanosine (8-OHdG),

which is a marker of oxidative damage to DNA (Brown & White, 2019).

Diet: Diets high in certain types of fats, sugars, and processed foods can contribute

to oxidative stress. Lipid peroxidation products, such as malondialdehyde (MDA),

can serve as biomarkers of oxidative damage to lipids and cell membranes (Smith

& Johnson, 2021).

Lifestyle Choices: Factors like smoking, excessive alcohol consumption, and lack

of exercise can promote oxidative stress. Markers like increased levels of 4-

hydroxynonenal (4-HNE), a product of lipid peroxidation, can be indicative of

oxidative stress in individuals with such lifestyles (Brown & White, 2019).
Aging: The body's ability to neutralize ROS may decline with age, making older

individuals more susceptible to oxidative stress. Age-related biomarkers of

oxidative stress may include increased levels of oxidative damage to cellular

components like DNA, proteins, and lipids (Johnson et al., 2017).

Disease States: Certain diseases, such as neurodegenerative diseases (e.g.,

Alzheimer's and Parkinson's disease), cardiovascular diseases, cancer, and diabetes,

are associated with elevated oxidative stress levels. Biomarkers of these diseases

may include specific markers of oxidative damage in affected tissues (Smith et al.,

2020).

Antioxidant Enzyme Activity: Biomarkers of oxidative stress can also include the

activity levels of antioxidant enzymes like superoxide dismutase (SOD), catalase,

and glutathione peroxidase. Reduced activity of these enzymes can indicate an

impaired antioxidant defense system and increased susceptibility to oxidative stress

(Smith & Johnson, 2021).

2.2.3. Significance of high-level oxidative stress biomarkers

High levels of oxidative stress biomarkers can have significant clinical and health

implications, as they are often associated with various diseases and adverse health
outcomes (Smith et al., 2020). Here's the significance of elevated oxidative stress

biomarkers:

Increased Disease Risk: High levels of oxidative stress biomarkers are frequently

linked to an increased risk of developing a range of chronic diseases, including

cardiovascular diseases (Brown & White, 2019), cancer (Smith et al., 2020),

diabetes (Johnson et al., 2017), neurodegenerative diseases (e.g., Alzheimer's and

Parkinson's disease) (Jones et al., 2018), and autoimmune disorders.

Tissue and Cellular Damage: Oxidative stress can cause damage to cellular

components, including DNA, lipids (lipid peroxidation), and proteins (protein

oxidation) (Brown & White, 2019). This damage can impair cellular function and

integrity, leading to tissue damage and dysfunction.

Inflammation: Elevated oxidative stress can trigger and sustain chronic

inflammation, which is a key factor in the development of various diseases (Jones

et al., 2018). Inflammatory processes can further exacerbate oxidative stress in a

feedback loop, contributing to tissue damage and disease progression.

Accelerated Aging: High levels of oxidative stress are associated with accelerated

aging processes, both at the cellular and organismal levels (Johnson et al., 2017).

Premature aging can manifest as visible signs of aging and age-related diseases.
Neurological Implications: Oxidative stress biomarkers are of particular concern in

neurodegenerative diseases. They are associated with neuronal damage, cell death,

and cognitive decline, making them relevant in conditions like Alzheimer's and

Parkinson's disease (Jones et al., 2018).

Cardiovascular Health: Elevated oxidative stress is a significant contributor to the

development and progression of cardiovascular diseases (Brown & White, 2019). It

can lead to the oxidation of LDL cholesterol (oxidized LDL), a key event in

atherosclerosis, and contribute to endothelial dysfunction and plaque formation.

Cancer Promotion: Oxidative stress can promote carcinogenesis by causing DNA

damage and mutations (Smith et al., 2020). High levels of oxidative stress

biomarkers may indicate an increased risk of cancer development and progression.

Metabolic Disorders: Oxidative stress is linked to insulin resistance and impaired

glucose metabolism, which are key features of type 2 diabetes (Johnson et al.,

2017). Elevated oxidative stress biomarkers may reflect a heightened risk of

developing diabetes.

Impaired Antioxidant Defenses: High levels of oxidative stress biomarkers may

indicate a deficiency in the body's antioxidant defense mechanisms (Smith &

Johnson, 2021). This suggests that the body's ability to neutralize harmful reactive
oxygen species (ROS) may be compromised, increasing susceptibility to oxidative

damage.

Response to Treatment: Monitoring oxidative stress biomarkers can be important

in assessing the response to antioxidant therapies and interventions aimed at

reducing oxidative stress (Smith & Johnson, 2021). Changes in these biomarker

levels can help evaluate the effectiveness of treatments.

Lifestyle Modification: Elevated oxidative stress biomarkers can serve as a wake-

up call for individuals to make lifestyle changes (Brown & White, 2019). For

example, individuals with high levels of oxidative stress may be advised to adopt

healthier dietary habits, exercise regularly, reduce exposure to environmental

toxins, and quit smoking.

High levels of oxidative stress biomarkers are indicative of a state of increased

oxidative stress within the body, which is associated with a heightened risk of

various diseases and health issues (Smith et al., 2020). Monitoring these

biomarkers and addressing the underlying causes of oxidative stress is important

for disease prevention, management, and overall health.

2.2.4. Significance of low-level oxidative stress biomarkers

Low levels of oxidative stress biomarkers are generally considered a positive

indicator of health and well-being (Smith et al., 2020). Here's the significance of

low levels of oxidative stress biomarkers:

Lower Disease Risk: Low levels of oxidative stress biomarkers are associated with

a reduced risk of developing chronic diseases, including cardiovascular diseases,

cancer, diabetes, and neurodegenerative diseases (Smith et al., 2020). When

oxidative stress is minimal, it is less likely to contribute to the initiation or

progression of these conditions.

Healthy Aging: Maintaining lower oxidative stress levels is associated with

healthier aging (Johnson et al., 2017). Reduced oxidative stress can help mitigate

age-related cellular damage and slow down the aging process, leading to better

overall health in older individuals.

Cellular Protection: Lower oxidative stress levels indicate a reduced likelihood of

cellular damage and dysfunction. This protection extends to various cellular

components, including DNA, lipids, and proteins, helping to maintain cellular

integrity and function (Brown & White, 2019).

Balanced Inflammatory Response: Low oxidative stress is typically associated with

a balanced inflammatory response. Chronic inflammation, often exacerbated by

oxidative stress, is a risk factor for many diseases. Lower oxidative stress helps

keep inflammation in check (Jones et al., 2018).

Improved Cardiovascular Health: Lower levels of oxidative stress biomarkers are

beneficial for cardiovascular health (Brown & White, 2019). They reduce the risk

of oxidative damage to blood vessel walls, lower the oxidation of LDL cholesterol,

and help maintain healthy endothelial function.

Reduced Cancer Risk: Lower oxidative stress levels are generally associated with a

lower risk of cancer (Smith et al., 2020). Reduced oxidative stress decreases the

likelihood of DNA damage and mutations that can lead to the development of

cancer.

Optimal Antioxidant Defense: Low oxidative stress suggests that the body's

antioxidant defense mechanisms are functioning effectively (Smith & Johnson,

2021). This means that the body can efficiently neutralize harmful reactive oxygen

species (ROS), preventing oxidative damage.

Positive Response to Lifestyle Interventions: Individuals with low oxidative stress

levels are more likely to respond positively to lifestyle interventions aimed at

reducing oxidative stress (Brown & White, 2019). Healthy habits such as a

balanced diet, regular exercise, and stress management can help maintain low

oxidative stress levels.

Longevity: While oxidative stress is a natural part of metabolism, lower levels may

contribute to increased longevity (Johnson et al., 2017). Reduced oxidative damage

to cells and tissues can lead to a longer and healthier lifespan.

Low levels of oxidative stress biomarkers are indicative of a state of reduced

oxidative stress within the body, which is generally associated with a lower risk of

chronic diseases, healthier aging, and overall well-being (Smith et al., 2020).

Maintaining low oxidative stress levels through a healthy lifestyle and antioxidant-

rich diet can contribute to better health outcomes and improved quality of life.

2.2.5. Method of assay of biomarkers.

Enzyme-Linked Immunosorbent Assay (ELISA): ELISA is a widely used method

for quantifying biomarkers in biological samples (Smith et al., 2018). It relies on

the binding of antibodies to the target biomarker, followed by the detection of the

antibody-biomarker complex using enzymes and colorimetric or fluorescent

substrates.

Western Blotting: Western blotting is used to detect specific proteins as biomarkers

(Jones & Johnson, 2019). Proteins are separated by electrophoresis, transferred to a

membrane, and probed with antibodies that bind to the target protein. The bound

antibodies are then visualized using enzyme-linked secondary antibodies.

Polymerase Chain Reaction (PCR): PCR is a molecular biology technique used to

amplify and quantify DNA or RNA biomarkers (Brown et al., 2020). Quantitative

PCR (qPCR) measures the amount of specific nucleic acid sequences, often used

for gene expression analysis and detecting viral or bacterial DNA/RNA.

Mass Spectrometry: Mass spectrometry can be used to identify and quantify

biomarkers, including proteins, peptides, and metabolites (Smith & White, 2017).

It measures the mass-to-charge ratio of ions produced from a sample, allowing for

accurate identification and quantification.

Liquid Chromatography (LC) and Gas Chromatography (GC): Chromatography

techniques can separate and quantify biomarkers based on their chemical

properties (Johnson & Brown, 2018). Liquid chromatography-mass spectrometry

(LC-MS) and gas chromatography-mass spectrometry (GC-MS) are commonly

used for metabolomics and analysis of small molecules.

Flow Cytometry: Flow cytometry is used to analyze biomarkers on the surface of

cells or in cell suspensions (Jones et al., 2021). It involves passing cells through a

laser beam and measuring the emitted fluorescence or light scatter, which can

indicate the presence and quantity of specific biomarkers.

Immunohistochemistry (IHC): IHC is used to visualize biomarkers in tissue

samples (Smith & Johnson, 2019). It involves staining tissue sections with specific
antibodies tagged with fluorophores or enzymes. This allows for the localization

and quantification of biomarkers within tissues.

Spectrophotometry: Spectrophotometry measures the absorbance or emission of

light by a biomarker or its reaction products (Brown et al., 2017). It is commonly

used for quantifying small molecules, such as reactive oxygen species (ROS), in

samples.

Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR spectroscopy is used to

study the structure and quantity of certain biomarkers, particularly metabolites

(Johnson & Smith, 2018). It provides information about the chemical environment

of atoms in molecules.

Electrochemical Assays: Electrochemical methods involve measuring changes in

electrical properties (e.g., current or voltage) resulting from biomarker reactions

(Smith and Brown, 2020). Electrochemical sensors can detect specific molecules

and are used in various biosensor applications.

Microarrays: Microarrays can simultaneously analyze the expression of multiple

genes or proteins in a sample (Jones et al., 2020). They are useful for studying

gene expression patterns and identifying biomarkers associated with diseases.

Biosensors: Biosensors are analytical devices that combine a biological component

(e.g., antibodies, enzymes) with a physicochemical detector (Brown and Johnson,

2019). They can provide real-time measurements of specific biomarkers in various

sample types.

The choice of assay method depends on the nature of the biomarker, the sample

type (e.g., blood, tissue, urine), the desired sensitivity and specificity, and the

available equipment and expertise. Researchers and clinical laboratories select the

most appropriate method to accurately quantify and study specific biomarkers

relevant to their research or diagnostic objectives.

2.3. THE FEMALE REPRODUCTIVE CYCLE

The female reproductive cycle, also known as the menstrual cycle, is a complex

and orchestrated series of events that prepares a woman's body for pregnancy. It

typically lasts around 28 days, although it can vary from woman to woman. The

cycle is regulated by hormones and involves several key phases:

Menstruation (Days 1-5): The menstrual cycle begins with menstruation, which is

the shedding of the uterine lining (endometrium) that has built up in preparation for

pregnancy during the previous cycle. Menstruation typically lasts about 3 to 7

days, and it marks the first day of the menstrual cycle. Hormone levels, specifically

estrogen and progesterone, are low during this phase (Brown and Johnson, 2019).
Follicular Phase (Days 1-13): This phase coincides with menstruation and

continues afterward. The anterior pituitary gland releases follicle-stimulating

hormone (FSH), which stimulates the growth of ovarian follicles, each containing

an immature egg (oocyte). As these follicles develop, they produce increasing

amounts of estrogen. Rising estrogen levels stimulate the thickening of the uterine

lining and promote the maturation of the eggs (Smith and Brown, 2020)

Figure 1: Diagrammatic representation of the menstrual cycle

Ovulation (Day 14): Around the middle of the menstrual cycle, a surge in

luteinizing hormone (LH) and FSH triggers the release of a mature egg from one of
the ovarian follicles. This event is called ovulation. The egg is released into the

fallopian tube, where it may encounter sperm if fertilization is to occur. Ovulation

typically occurs on or around the 14th day of the cycle but can vary (Tairu, et al.,

1999)

Luteal Phase (Days 15-28): After ovulation, the empty follicle transforms into a

structure called the corpus luteum, which secretes both estrogen and progesterone.

These hormones help maintain the thickened uterine lining in preparation for

potential implantation of a fertilized egg. If fertilization does not occur, the corpus

luteum breaks down, leading to a decline in estrogen and progesterone levels

(Ayedunet al., 2021).

Premenstrual Phase (Days 25-28): If pregnancy does not occur, the decreasing

levels of estrogen and progesterone signal the body to prepare for menstruation.

This phase may be accompanied by symptoms like breast tenderness, bloating, and

mood changes (Orwa et al., 2009).

If fertilization and implantation of a fertilized egg do occur, the cycle takes a

different course. The developing embryo produces human chorionic gonadotropin

(hCG), which signals the corpus luteum to continue producing progesterone,

supporting the pregnancy until the placenta can take over hormone production

(Mayo Clinic, 2021).

It's important to note that the menstrual cycle can vary in length and regularity

among individuals. Factors such as stress, illness, medication, and underlying

health conditions can influence the cycle. Tracking the menstrual cycle can be

helpful for family planning, understanding reproductive health, and identifying

potential issues or irregularities. Additionally, hormonal contraceptives, such as

birth control pills, can alter the natural menstrual cycle to prevent pregnancy.

2.4. PREGNANCY AND STAGES OF PREGNANCY

Pregnancy is a physiological process during which a fertilized egg, known as a

zygote, develops into an embryo and then a fetus inside a woman's uterus (Mayo

Clinic, 2021). Pregnancy is typically divided into three trimesters, each lasting

approximately three months, and it involves a series of remarkable changes and

developmental stages.

First Trimester (Weeks 1-12):

Fertilization: Pregnancy begins with fertilization, which occurs when a sperm cell

successfully penetrates an egg cell (ovum) in the fallopian tube. The resulting

zygote starts to divide and form a blastocyst (Jirovetz, et al., 1997).

Implantation: The blastocyst travels through the fallopian tube into the uterus,

where it attaches to the uterine lining in a process called implantation. Here, it

begins to receive nourishment from the mother's body (Mayo Clinic, 2021).
Embryo Development: During this phase, the blastocyst develops into an embryo,

and the embryonic cells differentiate into various tissues and organs. The heart

begins to beat, and major organs and body systems start to form (Mayo Clinic,

2021).

First Signs of Pregnancy: The woman may experience symptoms like breast

tenderness, fatigue, nausea (morning sickness), and increased urination. A missed

menstrual period is often the first noticeable sign (Brown and White, 2021).

Second Trimester (Weeks 13-26):

Growth and Development: The second trimester is a period of rapid growth and

development. The fetus becomes more defined, and its body systems continue to

mature. By the end of this trimester, most of the organs are fully developed

(Johnson et al., 2017).

Movement: The mother may begin to feel fetal movements, known as

"quickening." These movements become more noticeable as the fetus grows (Mayo

Clinic, 2021).

Ultrasound: Many expectant parents have an ultrasound during this trimester to

check the baby's development and potentially determine the sex of the baby.
Reduced Nausea: Morning sickness often improves during the second trimester,

and many women experience increased energy and a sense of well-being (Davì et

al., 2002).

Third Trimester (Weeks 27-40+):

Fetal Growth: In the third trimester, the fetus experiences significant growth and

weight gain. The mother's belly expands noticeably as the baby gets larger (Jenkins

et al., 2017).

Braxton Hicks Contractions: Women may experience Braxton Hicks contractions,

which are practice contractions that prepare the uterus for labor (Mayo Clinic,

2021).

Preparation for Birth: The baby typically moves into a head-down position in

preparation for birth. The mother may experience increased pressure in the pelvis

and more frequent urination (Jenkins et al., 2017).

Antenatal Care: Regular prenatal check-ups become more frequent in the third

trimester to monitor the health of both the mother and the baby. Tests may include

glucose screening for gestational diabetes and Group B streptococcus screening

(Jenkins et al., 2017).

Labor and Birth:

Onset of Labor: Labor often begins with contractions that become progressively

stronger and more frequent. This can result in the cervix dilating and effacing

(thinning) (Davì et al., 2002).

Delivery: During the first stage of labor, the cervix dilates fully, and the woman

enters the second stage of labor, which involves the actual birth of the baby. The

third stage involves the delivery of the placenta (Sies, 2015).

Postpartum: After childbirth, the woman enters the postpartum period, during

which her body gradually returns to its non-pregnant state. This includes physical

and hormonal changes (Myatt and Cui, 2004).

Throughout pregnancy, prenatal care is essential to monitor the health of both the

mother and the baby. It includes regular check-ups, ultrasounds, and various tests

to ensure a healthy pregnancy and delivery. Pregnancy is a unique and

transformative experience for women, leading to the birth of a new life and the

beginning of parenthood (Lappas, 2014).

2.4.1. Oxidative stress in pregnancy

Pregnancy is a unique physiological state characterized by substantial changes in

hormonal, metabolic, and immune functions. These changes can lead to an

increased production of ROS, making pregnant women particularly susceptible to

oxidative stress (Lappas, 2014). Oxidative stress during pregnancy can result from
a variety of factors, including increased metabolic demands, placental dysfunction,

and inflammation (Myatt and Cui, 2004).

Over the past few years, more and more attention has been devoted to issues of

environmental impact, lifestyle, and comorbidities on the body’s oxidative balance

and its possible impact on abnormalities related to fetal development and

pregnancy outcome. The term oxidative stress refers to the imbalance between the

production of reactive oxygen species (ROS) and the ability of antioxidant

mechanisms to neutralize them. It may be the result of an increase in ROS

generation and/or a weakening of antioxidant defense (Sies, 2015). Analyzing the

available literature of the last decade, it has been found that the topic of oxidative

stress in pregnancy is becoming more common and knowledge on this subject is

expanding.

Pregnancy is a time when the body’s oxidative imbalance negatively affects its

development and causes various types of complications depending on the stage of

its development. This mechanism is described in Figure 1.

Figure 1: Impact of oxidative stress on function and abnormal pregnancy
development (Sultana et al., 2017)
The causative factor of the analysis is the previously unexplained etiology of most

frequent pregnancy complications, such as miscarriages, FGR, or preeclampsia, in

particular congenital defects. The main purposes are to assess the contribution of

existing literature of the association of oxidative stress on the etiology of the

abovementioned conditions and to identify relevant information and outline

existing knowledge. Furthermore, the authors aim to find any gaps in the research,

thereby providing grounds for our own research.

2.4.2. Effect of increased oxidative stress in pregnancy

Increased oxidative stress during pregnancy can have significant implications for

maternal and fetal health. Oxidative stress, arising from an imbalance between

reactive oxygen species (ROS) production and antioxidant defenses, has been

linked to various pregnancy complications, including preeclampsia (Burton et al.,

2009) and gestational diabetes mellitus (Davì et al., 2002). Additionally, elevated

oxidative stress levels may contribute to adverse fetal outcomes, such as preterm

birth (Giray et al., 2016) and intrauterine growth restriction (IUGR) (Jenkins et al.,

2017). Furthermore, maternal oxidative stress has been associated with long-term

health risks for the offspring, including a heightened susceptibility to metabolic

disorders and cardiovascular diseases in adulthood (Hracsko et al., 2014).

Investigating the underlying mechanisms of oxidative stress in pregnancy and

exploring potential interventions to mitigate its detrimental effects is essential for

enhancing pregnancy outcomes and long-term health for both mother and child.

Understanding the complexities of oxidative stress during pregnancy necessitates

further research to unravel the intricate pathways involved. Several studies have

highlighted the potential role of oxidative stress biomarkers such as 8-

hydroxydeoxyguanosine (8-OHdG) in assessing oxidative damage to DNA (Barak

et al., 2008) and malondialdehyde (MDA) as a marker of lipid peroxidation

(Chamy et al., 2006). Moreover, antioxidant interventions, including the use of

vitamins C and E, have been explored as potential strategies to ameliorate

oxidative stress-associated pregnancy complications (Rumbold et al., 2006). In

summary, recognizing the impact of increased oxidative stress in pregnancy and its

multifaceted consequences underscores the importance of continued research and

the development of targeted interventions to optimize maternal and fetal well-being

throughout the prenatal period and beyond.

2.4.3 Effect of decreased oxidative stress in pregnancy

Reduced oxidative stress during pregnancy holds substantial significance for

maternal and fetal health. Studies have demonstrated that maintaining an optimal

balance between reactive oxygen species (ROS) production and antioxidant

defenses can contribute to improved pregnancy outcomes. Lower oxidative stress

levels have been associated with a reduced risk of complications such as

preeclampsia (Mistry et al., 2016) and gestational diabetes mellitus (Al et al.,

2016). Furthermore, decreased oxidative stress may promote normal fetal growth

and development (Gonçalves et al., 2018), lowering the risk of preterm birth and

intrauterine growth restriction (Moussa et al., 2019). Exploring interventions to

reduce oxidative stress, including dietary modifications and antioxidant

supplementation, represents a promising avenue for enhancing maternal-fetal

health and preventing adverse pregnancy outcomes. Continued investigation into

strategies for reducing oxidative stress during pregnancy is essential. Antioxidant-

rich diets, such as those rich in fruits and vegetables, have been associated with

lower oxidative stress levels and improved pregnancy outcomes (Havas et al.,

2020). Additionally, the potential benefits of specific antioxidants, such as vitamin

C and vitamin E, in mitigating oxidative stress-related complications warrant

further exploration (Song et al., 2016). Moreover, lifestyle modifications, including

regular physical activity and stress management, may contribute to a reduction in

oxidative stress (Dadipoor et al., 2020). As we strive to better understand the

mechanisms underlying the effects of decreased oxidative stress in pregnancy,

these findings underscore the importance of holistic approaches to prenatal care

that emphasize nutrition, lifestyle, and antioxidant support to promote maternal and

fetal well-being.
2.4.4. Oxidative Stress in Pregnancy with Normal Pregnancy Outcome
During physiological pregnancy, the development of fetal tissues and organs

requires the supply of an adequate amount of nutrients and oxygen, and its reactive

forms produced in the body of the mother and the fetus affect the replication,

differentiation, and maturation of the developing cells.

Their balanced activity and maintaining the balance of oxidative processes are

necessary factors for the proper development and functioning of the body (Bak and

Roszkowoski, 2013).

During pregnancy, numerous anatomical, physiological, and metabolic changes

occur in the mother’s body. According to the researchers, it is assumed that they

support the production of ROS, especially in the second half of pregnancy. This is

mainly due to an increasing basic metabolism and “consumption” of oxygen and

the use of fatty acids as the primary source of energy for most maternal

retroplacental tissues. The last trimester of pregnancy is a special period of

increasing insulin resistance, fat catabolism, and the release of free fatty acids.

These processes lead to increased production of hydrogen peroxide (Duhig et al.,

2016).

The placenta, filled with mitochondria, is the main source of prooxygenates, the

so-called ROS “factory.” The superoxide anion radical (O- ∙ 2) produced in large

quantity is a source of the formation of further active forms of oxygen, i.e.,

hydrogen peroxide and hydroxyl radical. Their production increases with the

development of pregnancy, which is mainly associated with an increase in

placental mass. Nitric oxide is also synthesized by macrophages mainly in the

placenta.

In a correctly developing pregnancy, the phenomenon of the mother’s immune

tolerance to the fetus’ antigens, which allows the fetus to develop in the uterus

despite the pregnant woman’s ability to reject the foreign antigen, is an extremely

important aspect. The main assumptions of this phenomenon are partial inhibition

of the mother’s immune system during pregnancy, insufficiently strong

presentation of fetal antigens, the placenta as an important element separating the

woman from the fetus, and changing the direction of the organism’s specific

response to the Th2 response instead of Th1 (cytokines produced mainly by

nonspecific system cells—NK type (natural killer)). Immunological tolerance is

therefore formed by the cells of the implanted trophoblast, the mother’s immune

system, and the microenvironment of the implanted embryo—a decidual cell. It

enables the correct implantation and development of the embryo and functioning of

the placenta. Due to the reduction of the immune system works in a properly

functioning pregnant organism, the production of ROS is lowered.

Erythropoiesis increases, erythrocyte life expectancy decreases, and increased iron

delivery to the fetus increases its availability by catalyzing the increase in the
generation of large amounts of reactive hydroxyl (∙OH) radicals in the Fenton

reaction (Moore et al., 2019).

Numerous studies prove that oxidative stress, i.e., excessive and unbalanced ROS

production, has adverse effects on pregnancy, pregnant health, and fetal

development. It is the cause of incorrect implantation of embryos, miscarriages,

premature births, low birth weight, and malformations. It also weakens pregnant

immunity and respiratory adaptation of newborns immediately after birth. The

main reason for these disorders is the insufficient supply of nutrients and oxygen to

the fetus resulting mainly from hypoplasia and abnormal placental function (Duhig

et al., 2016).

Comparative studies of pregnant and nonpregnant patients (Toescu et al., 2002).

showed that total plasma antioxidant status (TAS) in the first trimester of

pregnancy is significantly lower. In the second and third trimesters of pregnancy,

total plasma antioxidant capacity (TAC) increases, and in the last week of

pregnancy reaching values similar to those observed in nonpregnant women. After

delivery, this rate increases to the eighth week after delivery, and these changes are

proportional to changes in plasma uric acid.

Studies by other scientists indicate that the reason for lower TAS values in

pregnancy is the reduction in serum albumin, bilirubin, and vitamin E levels

(Dennery, 2010). It was also found that during a properly developing pregnancy,

plasma superoxide dismutase activity decreases (Wood et al., 2010).

Physiologically, during pregnancy, it increases the concentration of triglycerides,

total cholesterol, and low-density lipoprotein (LDL) cholesterol levels in plasma as

well as markers of oxidative stress, which is associated with an increase in lipid

peroxides after 25 weeks of pregnancy. Therefore, the natural indicator of

oxidative stress and the degree of lipid peroxidation is increasing concentration of

malondialdehyde in the plasma of pregnant women.

There have also been reports of the effect of a diet with vitamins, antioxidants, and

minerals on the value of total antioxidant status in pregnant patients (Dennery,

2010).

2.4.5. Oxidative Stress in Pregnancy Completed with Birth Defects

The problem of birth defects in the fetus concerns about 4-6% of live-born

newborns and the majority of miscarriages in the first trimester of pregnancy, the

exact number of which cannot be determined. They are also the most common

cause of infant mortality and disability in the 21st century. The most commonly

known causes of congenital defects are genetic factors. However, approximately

50% of congenital anomalies cannot be linked to a specific cause.

Oxidative stress, which is primarily the result of excessive production of oxygen

peroxidation products, mainly in mitochondria, attacks newly growing cells. It

damages their structures probably already at an early stage of embryogenesis.

Determinants of the production of an increased number of ROS and disorders

caused by them can be environmental pollution, chronic stress, low levels of

physical activity, teratogenic effect of drugs and chemicals, or improper nutrition.

It causes abnormalities in the structure of DNA that can lead to early miscarriages,

preeclampsia, fetal growth restriction, fetal abnormalities, and birth defects (Clerici

et al., 2012).

Researchers found elevated levels of oxidative stress markers in the blood serum of

pregnant mothers during the first prenatal examination at 11-14 gestational week,

who had a high (<1: 300 according to Fetal Medicine Foundation) risk of fetal

malformations. Then, after further research and detailed analysis, they confirmed

the significant difference between the levels of oxidative stress in patients with

healthy and diseased fetuses—complicated by chromosomal aberrations and other

malformations (Pietryga et al., 2017).

Due to the damage of the DNA structure by an unbalanced antioxidative and

oxidative level in the body, more attention is paid to the role of ROS in the

etiopathogenesis of genetic defects. Oxidative stress damaging the structure of a

deoxyribonucleic acid molecule leads to chromosomal aberrations. Pagano and

Castello (Pagno and Castello, 2012) showed characteristic changes in vivo of

mitochondrial function, leading to increased ROS concentration in the cell as

changes characteristic of trisomy 21. Analyses of several markers of oxidative

stress, in pregnant blood, amniotic fluid, and fetal tissues clearly showed its

association with Down syndrome. Observed changes had a significant share in the

damage of various tissue enzymes. It can be presumed that they played a role in the

pathogenesis of this abnormality (Laforgia et al., 2018).

The metabolomic analysis of blood serum from patients with Down syndrome in

the fetus, in the first trimester of pregnancy, showed differences in the levels of 2-

hydroxybutyrate in comparison with patients with healthy fetuses. The level of

substance, which is physiologically involved in the defense against oxidative

stress, was lower in the study group, proving the oxidative imbalance in the

pregnant body with fetal trisomy 21 (Bahado-Singh et al., 2013).

There was also conducted research on the rate of fibroblast proliferation and its

major regulators, such as Rcan1 or telomere length, for assessing the oxidative

balance of these cells in fetuses with Down syndrome. RNA expression and

activity of the main antioxidant enzymes in the study group were analyzed. The

thesis regarding the effect of oxidant-antioxidant imbalance on the generation of

genetic disorders such as trisomy 21 was confirmed. An increased GSSG/GSH

ratio and high concentrations of protein peroxidation products in fibroblasts have

been demonstrated. The obtained values correlated with the reduced antioxidant

capacity of cells. The results obtained showed reduced levels of antioxidants that

cooccurred with increased Rcan1 levels and telomere shortening, responsible for

increased oxidative stress and cell cycle disorders of fibroblasts of fetal’s with

Down syndrome (Gemeno et al., 2014).

It has also been proven that the amniotic fluid, in which fetal cells with Down’s

syndrome are present, differs in oxidative status depending on the severity of the

lesions and abnormalities that occur—especially nerve cell damage. Increased

levels of oxidative stress, as indexed by increased protein oxidation, lipid

peroxidation, reduction of glutathione (GSH) and thioredoxin levels, and induction

of the heat-shock protein (HSP) response were associated with worse prognosis as

to survival and normal development (Perluigi et al., 2011).

A relationship between oxidative stress and the occurrence of such abnormalities in

the genome structure as Ataxia-telangiectasia (A-T), Bloom syndrome (BS), or

Nijmegen syndrome (NBS) has also been demonstrated. High levels of reactive

oxygen species (ROS) may be a major phenotypic hallmark in these diseases. The

observed damages and other abnormalities, such as changes in the ultrastructure

and function of cells, prove that we can consider analyzed diseases as

mitochondrial. However, more research is needed to confirm whether antioxidants

and free radical scavengers can improve the condition or extend the survival of

patients (Maciejezyk et al., 2017)

Scientists conducting research on the pathogenesis of Treacher Collins syndrome

have demonstrated the negative effects of ROS on developing progenitor neural

crest cells. Depending on the degree of DNA damage, fetal death or abnormal

development of the facial cranium occurred. The confirmation of the theory was

the absence of a defect in the progeny of mammals undergoing antioxidant

supplementation. High levels of ROS were the causative agent of facial cranial

malformations (Sakai et al., 2016).

The relationship of oxidative stress with the occurrence of cardiac malformations

was also analyzed. After investigating the levels of oxidative stress markers in

newborns with cyanotic and noncyanotic congenital heart defects, elevated levels

of oxidative stress and reduced levels of antioxidants were found in sick patients.

In addition, studies with increased levels of homocysteine and reduced levels of

vitamin B12, glutathione as an antioxidant, and folate in pregnant patients with

fetal heart disease have been published. Increased markers of oxidative stress in the

study group suggests its participation in the etiopathogenesis of cardiac

developmental abnormalities (Mishra et al., 2015).

Analysis of the oxidative balance in fetuses with trisomy 21 and coexisting

congenital heart defects showed an impairment of the mitochondrial respiratory

chain, inhibition of Complex I, and consequently increased production of ROS.

The analysis of the heart cell transcriptome with abnormalities showed that the

function of genes responsible for the regular work of mitochondria is significantly

reduced compared with the control group, suggesting their participation through

oxidative stress in the manifestation of the defect (Liu et al., 2018).

Researchers also investigated the oxidative balance of the fetuses of mothers with

preexisting diabetes who had heart defects (CHD), such as a defect in the

ventricular or atrial septum, valve defects, or abnormal ventricular outflow tracts.

Mouse models have shown that oxidative imbalance is a major determinant of

CHD. Concentrations of nitric oxide (NO) and reactive oxygen species dependent

on endothelial nitric oxide synthase (eNOS) are crucial for creating the right

structures of the heart muscle, regulating various cellular and molecular processes.

As a result, eNOS deficiency causes oxidative stress, CHD, and coronary artery

malformations (Piccoli et al., 2013).

Fetal exposure of a pregnant woman to heavy metals, such as lead and aluminum,

has also been shown to be negative by disturbing the prooxidative-antioxidant

balance. It is associated with an increase in MDA concentration and a decrease in

the level of antioxidants such as superoxide dismutase (SOD) and glutathione

peroxidase (GPx) in fetal cord blood serum with congenital heart disease (Engineer

and Greco, 2019).

The association of oxidative stress on the development of congenital

malformations of the central nervous system is also increasingly well known. They

belong to the common and one of the most serious birth defects in fetuses.

Numerous studies have been carried out confirming the relationship between

oxidant-antioxidant imbalance and abnormal development of the nervous system.

A negative effect of oxidative stress on cell differentiation and CNS development

has been demonstrated (Cim et al., 2018). Increased ROS concentrations have been

documented in such anomalies as holoprozencephalia or myelomeningocele.

Reduced levels of antioxidants such as glutathione and catalase as well as

increased levels of malondialdehyde in fetal amniotic fluid with central nervous

system development disorders have also been shown (Engineer and Greco, 2019).

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