الفهرس 
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Abstract
Recurrent spontaneous abortion represents a disorder in obstetric practice with an estimated prevalence between 0.5 and 2%. Many etiological factors have been proposed, but most of them remain controversial. Next to genetic, structural, endocrine, metabolic and immunological factors, lifestyle factors such as smoking and consumption of alcohol or coffee have been associated with recurrent spontaneous abortion. The risk of
spontaneous abortions associated with exposure to endogenous or exogenous substances may be modified by the genetic variation in individual metabolic detoxification activities,
thus in the phase I/phase II balance. One considering factor of recurrent spontaneous abortion is environmental pollution. The strongest evidence of environmental contaminant exposures interfering with healthy reproductive function in adult female is xenobiotic as PAHs.
Thus the objectives of the present study is to detect the role of lycyclic aromatic hydrocarbon pollutants in idiopathic recurrent spontaneous abortion.
Thus, it is necessary to carry out health risk from the exposure of PAHs in
environment to human through:
1. Estimation of urinary metabolites of PAHs to detect the degree of exposure.
2. Determination of oxidative stressors through the measurement of lipid peroxide product as malondialdehyde (MDA), antioxidant enzymes as, glutathione-S-transferase
(GST) and catalase (Cat).
3. Determination of female sex hormones as FSH, progesterone.
4. Determination of the major products of DNA oxidation 8-hydroxy-2-deoxyguanosine
level.
5. Determination of the guardian of the genome and the cellular gatekeeper p53 level.
The present study was conducted on 76 subjects divided into 4 groups:
group I: Consisted of 18 control subjects.
group II: Included of 24 aborted women two times only.
group III: Comprised of 18 aborted women three times only.
group IV: Comprised of 16 aborted women more than three times.
Measurement of OH-PAHs in human urine is a method to assess recent individual
exposure to PAHs, in particular, when multiple routes of exposure have to be taken into account. Urinary metabolites of PAHs reflect a more accurate estimation of the quality of the actual PAH intake compared to ambient air measurements because they estimate the
internal dose.
The herein results manifested that there are high quantities of the metabolites ( 1-
Naphthol and 2- Naphthol) in the urine of the aborted women more than that of control
group.
Thus, naphthalene is considered an ubiquitous environmental carcinogen that is released by all sorts of industrial, domestic and natural burning processes leading to a considerable background exposure of the general population. Naphthalene is metabolized to more than 30 different metabolites. Among them 1- and 2-hydroxynaphthalene are used as urinary biomarkers of occupational exposure to naphthalene. 1- and 2-Naphthol levels in urine were also found to be suitable for human biomonitoring of the naphthalene exposure in environmental medicine. Urinary naphthols, 1- and 2-naphthol, have been suggested as route-specific biomarkers for exposure to airborne polycyclic aromatic hydrocarbons.
Many literatures identify PAHs as environmental endocrine disruptors. PAHs may act as antiestrogens by binding with the aryl hydrocarbon receptor, leading to induction of aryl hydrocarbon-responsive genes that result in a broad spectrum of antiestrogenic responses, or PAHs may act as antiestrogens by ntagonistically binding the estrogen receptor (ER). PAHs also have been reported to act as weak estrogens by binding the ER.
In humans, the Aryl hydrocarbon receptor (AhR) is localized in liver, lungs, kidneys, placenta, lymphocytes, ovary, and breast. AhR/ARNT complex activation is tissue-specific and depends on co-regulators present in different cell types. The activation of AhR by endogenous or exogenous compounds leads to its nuclear translocation and transcriptional activation. Unliganded AhR is associated with cytoplasmic protein complex consisting of molecular chaperone hsp90, the co-chaperone p23 and immunophiline-like protein XAP2.
Upon ligand binding, AhR migrates into the nucleus, thanks to unmasking of nuclear localization sequences in the NH2 terminal region of AhR, it dimerizes with Arnt. This heterodimer activates transcription of target genes through binding to specific DNA motifs termed xenobiotic responsive elements (XRE) or dioxin responsive elements (DRE), initiating a chain of events leading to chromatin remodeling, formation of pre-initiation complexes, and elongation of RNA transcripts by Pol II . The canonical target genes of AhR pathway include genes coding some phase I and II XMEs, such as CYP1A1,
CYP1A2, CYP1B1, Glutathione-S-transferase, UGT1A6, ALDH3A1 or NQO1.
Exposure to environmentally relevant concentrations of TCDD induces a significant reduction of FSH and LH during the preovulatory period in rats, strongly suggesting that the reproductive toxicity of TCDD can in part be related to a dysregulation of the hypothalamo-hypophyseal axis by mechanism(s) not yet completely understood. The observation that treatment with exogenous gonadotropin-releasing hormone (GnRH) partially overcomes the blockage of preovulatory surges of LH and FSH after TCDD exposure may indicate insufficient production in and/or release of GnRH from the hypothalamus as a result of TCDD action to the central nervous system (CNS). This TCDD-induced inhibition of gonadotropin surges has been explained by a decreased responsiveness of the hypothalamus to the positive feedback of estrogens, without affecting preovulatory serum estrogen levels. This hypothesis is confirmed by the observation that tenfold higher than physiological serum concentrations of estrogens completely reverse the TCDD effects on gonadotropin secretion.
This latter effect can be blocked by the partial estrogen antagonist tamoxifen providing further evidence for a functional relationship between both the aryl hydrocarbon
and estrogen-mediated signaling pathways. Members of the AhR signaling pathway are expressed in the preoptic area of the brain (POA), a region known to control reproductive functions.The distribution pattern of AhR gene expression closely overlaps with that of glutamic acid ecarboxylase (GAD), the enzyme necessary for gamma-aminobutyric acid (GABA) synthesis.
The abovementioned results is in agreement and represented a very good explanation to the present results which elucidated the significant decrease in the level of FSH in the sera of aborted women than that of normal control group.
It was provided evidence that at least of the effect of TCDD in reducing progesterone
secretion was very likely through a direct action on the activity of the enzyme, which
converts cholesterol to pregnenolone. Luteal cytochrome p450scc activity (assayed with
25-OH as a substrate) was decrease to 50% of control progesterone. This effect was
intensifying after the addition of TCDD with aminoglutethimide, the inhibitor of p450scc
to 5.9% of control progesterone.
It was concluded, that TCDD exposure of luteal cells isolated from mature porcine
corpora lutea decreases progesterone secretion by reduction of the activity of
mitochondrial enzymes, which converts cholesterol into pregnenolone. TCDD action in
luteal cells is not an ER-, but is an AhR-dependent, mechanism.
Therefore, the results of the current study provide evidence that the exposure of
aborted women to PAHs induced high significant decrease in the level of their serum
progesterone level than that of control group.
The tumor suppressor p53 gene is known as “the guardian of the genome”. It plays a
crucial role in maintaining genomic stability and tumor prevention. The p53 protein
responds to a wide variety of stresses, such as DNA damage, telomere shortening, hypoxia,
aberrant oncogene activation, or even nutrient depravation. Functioning as a sequencespecific
transcription factor activated by these stresses, p53 initiates a transcriptional
program that can lead to apoptosis cell cycle arrest or senescence in cells. This contributes
to tumor suppression by either preventing or repairing genomic damage or eliminating
potentially oncogenic clones of cells. p53 is most commonly expressed in the germ cells
and it functions in the surveillance of damaged germ cells to eliminate defective offspring
from the population. Therefore, the first functions of the p53 protein were to prevent
developmental defects in the germ line.
The p53-mediated apoptosis is involved in a number of mechanisms related to
human reproduction, such as ovarian cell death for its homeostasis and formation and
development of the placenta. The p53 is expressed in the trophoblast at all stages of
gestation, but with an increase in the first quarter a critical period for embryo selection. In
addition, p53 regulates genes in the trophoblast cell invasion process, which involves
degradation and remodeling of the extracellular matrix of the uterus. However, despite
being an integral part of many reproductive functions, apoptosis may be pathological and
damaging during reproduction in certain scenarios. Increased apoptosis in the maternalfetal
interface has been associated with several reproductive disorders including
preeclampsia and intrauterine growth restriction. Furthermore, studies in mice clearly
suggest an interaction between increased p53 expression, excessive apoptosis and pregnancy loss. Further investigations have shown that the p53 protein is highly expressed
in human placentas of abnormal pregnancies.
It was hypothesize that with a p53 Arg/Arg background there is an enhanced
induction of apoptosis, contributing to post-implantation selection, which is directly
reflected in intra-uterine survival. This mechanism may be functioning as a post-zygotic
selection step, since the majority of RPLs seem to be due to a high rate of embryo
aneuploidies. Additional support for this hypothesis is provided by the study of Norimura
et al. which shows that p53+/+ pregnant mice exposed do X-rays exhibited a higher rate of
apoptosis, reduced survival of abnormal fetus, and a higher rate of death and abortion of
normal fetus.
The low expressions of angiogenesis- and apoptosis-related genes are associated with
RPL. Abnormal apoptosis may cause pregnancy loss during human pregnancy. It is well
known that p53, the guardian of the genome, is a stress response protein that functions
primarily as a tetrameric transcription factor which regulates a large number of genes in
response to various stresses, including oncogene activation and DNA damage. p53 is
involved in both the pro-survival response of cell cycle arrest and DNA repair, and the prodeath
response of apoptosis. If a mutation occurs, p53 may not only lose its normal
functions, but also gain new abilities that promote tumorigenesis.
It was indicated that the AhR. Arnt-independent pathway involves p53. Activation of
p53 occurs in response to a number of cellular stresses, including DNA damage, and leads
to the activation of several genes whose product trigger cell cycle arrest, apoptosis, or
DNA repair. Several PAH metabolites possess high mutagenic activity that can induce p53
through their DNA-damaging activity.
Likewise, the results of the present study indicated that human p53 level was upregulated
in the sera of the aborted women than that of control group.
The p53 gene induces cell growth retardation, apoptosis, and affects cell
differentiation and DNA repair.
Recently, it was found that p53 is also involved in spontaneous abortion. Thus,
polymorphism of p53 codon 72 increases the incidence of recurrent miscarriage. It was
found that expression of p53 in villus tissue of women with RSA was significantly higher
than in control individuals. It can be speculated that overexpression p53 promotes
apoptosis and, thereby, plays an important role in RSA.
There are reports of higher Bax expression in the trophoblast, endometrial, and
matrix and decidual cells of early spontaneous abortion, and Bax is also overexpressed in
placental tissue in pregnancies complicated with preeclampsia or diabetes. In our study,
both CDKN1A and Bax were expressed at significantly higher levels suggesting that p53
induces apoptosis by activating both these signaling proteins.
Oxidative stress has also been implicated as an important cause of recurrent
pregnancy loss. Loss of antioxidant defenses has been shown to be associated with
recurrent pregnancy loss. Biochemical markers of ROS-induced membrane damage such
as lipid peroxidation products, reach high levels immediately before abortion. It has been
proposed that an oxidant/antioxidant imbalance is associated with pregnancy loss.
Oxidative stress is implicated in first trimester miscarriage from premature placental
perfusion of maternal oxygenated blood and accompanying ROS into the early embryonic
environment. Early embryo development occurs in a low oxygen state, and it is not until
the tenth to twelfth week of gestation that maternal blood begins to gradually infiltrate the
intervillous space of the yet fully developed placenta. The limited oxygen environment is
thought to act as a protective mechanism against the deleterious and teratogenic effects of
ROS on early stem cells at a time of extensive cell division. This early hypoxic
environment also plays a vital physiological role in placental cell type differentiation
switching from proliferative villous cytotrophoblasts into invasive extra-villous
trophoblasts (EVT) important in spiral artery remodeling. At the end of the first trimester,
oxygen tension rises sharply which coincides with the infusion of oxygenated maternal
blood into the placenta and triggers an apoptotic cascade that serves to establish the
definitive discoid placenta. However, in 70% of early miscarriage cases EVT invasion is
insufficient allowing for the premature onset of maternal intra-placental circulation and its
consequential burst of ROS on the conceptus. When OS develops too early in pregnancy it
can impair placental development and/or enhance syncytiotrophoblastic degeneration,
culminating in pregnancy loss.
The syncytiotrophoblastic deterioration and OS that occur as a result of abnormal
placentation may explain the heightened sensitivity of syncytiotrophoblasts to OS during
The 1st trimester, and could contribute significantly to idiopathic RPL. In keeping with this
idea, plasma lipid peroxides and GSH have been observed in increased levels, in addition
to decreased levels of vitamin E and β-carotene in patients with RPL.
Therefore the results of the herein study shows that circulating levels of oxidative
stress biomarker (MDA) was strikingly significantly higher in aborted women as
compared to control group. The present results show that systemic oxidative stress, of
which lipid peroxidation represents a major manifestation, plays an important role in
recurrent spontaneous abortion. Since, MDA is a byproduct of lipid peroxidation, thus an
elevation in MDA levels may reflects an overproduction of lipid peroxides and/or impaired
antioxidant defense mechanism which associated with panic decrease in the GSH and
catalase enzymatic levels in the aborted women group as compared with control group,
Another study showed significantly low levels of the antioxidant enzymes GPx, SOD, and
catalase in patients with idiopathic RPL, in addition to increased MDA levels.
BP-7,8-dione was also found to be a potent mutagen of p53 in vitro but only under
redox cycling conditions, leading to G to T transversions and a mutational pattern
reminiscent of that seen in lung cancer patients. The mutations were abolished by ROS
scavengers, suggesting that they were ROS-mediated.
The p53 was a potential mediator of pregnancy. Some alleles of p53 and its negative
regulator, MDM2, are under positive evolutionary selection. Significant decreases in
embryonic implantation, pregnancy rate and litter size were observed in mating’s with
p53−/− female mice but not with p53−/− male mice. p53 can regulate both basal and
inducible transcription of leukemia inhibitory factor (LIF) in mice. These suggest that p53
could have a new function in reproduction. The p53 tumor suppressor gene contains 11
exons, located on chromosome 17p13. The codon 72 polymorphism (rs1042522) is located
in exon 4 with CGC to CCC transition, leading to an arginine to proline amino acidic
substitution in amino-acid position 72.
Once implanted, the embryo continues to survive and grow by stimulating its own
blood supply through angiogenesis. A good exchange between fetus and mother is
necessary to ensure the normal fetal growth, therefore cell arrest in cytotrophoblasts and
blood vessels can result in imbalances of cell differentiation leading to miscarriage.
Significant differences in the codon 72 polymorphic form of p53 gene might affect the
biological activity of p53. The Arg72 variant of p53 protein is markedly better than the
Pro72 form in inducing apoptosis and suppressing cellular transformation, while the Pro72
form appears to induce a higher level of G1 arrest than the Arg72 form, the lower level of
apoptosis in p53 Pro allele carriers might cause misguided growth of cells or tissues
leading to miscarriage. The increasing cells arrested at the G1 checkpoint might cause
inadequate trophoblastic growth leading to abortion. G1 arrest or the potential to resist
apoptosis might be the explanation that Pro/Pro carriers are more associated with RPL than
with Arg allele carriers.
Leukemia inhibitory factor (LIF) is the cytokine produced and secreted by the
endometrial glands of uterus, regulating implantation through its influence on the luminal
epithelium and stromal decidualization, but also its influence on reproductive tract cells
such as leukocytes and glandular epithelium, during the pre-implantation phase of
pregnancy. Sufficient uterine LIF protein is an essential condition for implantation, and the
LIF production in fertile women was greater than in the infertile women has been reported.
p53 plays a significant role in maternal reproduction, through the regulation of LIF. Loss
of p53 decreased both the level and function of LIF in uteri. In the absence of p53, low LIF
is produced, the uterus does not become adequately receptive and implantation of embryos
is impaired. Arg72 was clearly more active than p53 Pro72 in transactivating LIF. Pro72
may alter the activity of p53, and in turn result in the reduced uterine LIF levels and
decreased implantation rates in women.
The findings of the present study proved that the aborted women were exposed to
high levels of PAHs induced panic oxidative DNA damage which in turn produce high
significant levels of (8-OHDG) associated with up regulation of p53. These findings were
in agreement with a lot of studies.
Oxidation of DNA occurs normally but increases with exposure to oxidizing agents.
Guanosines are susceptible to oxidation, and this reaction can lead to G:C → T:A
mutations that could have serious consequences. These oxidized bases are recognized and
excised by DNA repair machinery. 8-OHdG is excreted in urine and, as such, provides an
assessment of general oxidative stress throughout the body. So 8-hydroxy-2′-
deoxyguanosine (8-OHdG) becomes a biomarker for oxidative stress.
The abovementioned findings confirm and through lights on the results of the present
study which emphasized that there was a high significant positive correlation between (8-
OHdG) and PAHs, MDA, GST and p53, While there was a negative correlation with GSH,
CAT, FSH and progesterone; meanwhile there was a high significant positive correlation
between p53 and PAHs, MDA, GST and p53 associated with a significant negative
correlation with GSH, CAT, FSH and progesterone.
Future well designed large studies might be necessary to validate this association in
different populations incorporated with environmental factors in the susceptibility of
recurrent pregnancy loss.