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 was:-
Firstly, the assessment of occurrence and distribution of PAHs in mussels of Alex Coast, to identify the origin of PAHs in the Alex Coast, and conduct an environmental risk assessment to evaluate the possibility of occurrence of adverse ecological effects to benthic species due to the exposure to PAHs concentrated in the Alex Coast sediments.
The present results of the study revealed that there were high concentration of many PAHs detected in the tissues of two species of mussels collected from Alex Coast which may supposed to be at big risk for human health.
Secondly, the investigation of the possible association between exposure to PAHs in the environment and female idiopathic recurrent spontaneous abortion 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.
3. Determination of female sex hormones as FSH, progesterone.
4. Determination of the albumin adducts of benzo[a] pyrene.
The present study was conducted on 76 subjects who were 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.
Urinary excretions mainly contain (in measurable quantities) metabolites of PAH with small molecular weight such as naphthalene, phenanthrene and pyrene. Especially exposure assessment of humans to naphthalene has gained interest in environmental health since this most volatile PAH was classified as possible human carcinogen by several national and international agencies and the external naphthalene exposure in the environment of the general population is reported to be high in comparison with other PAH compounds.
Naphthalene is metabolized to more than 30 different metabolites. Among them 1- and 2-hydroxynaphthalene (naphthol) have been 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.
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.
B[a]P is metabolically activated to 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), an ultimate carcinogenic metabolite known to bind covalently to proteins in humans as albumin. Assessment of BPDE-albumin adducts has been proposed as a long-term biomarker of exposure to PAHs and the adduct lead to changes in protein structure and/or functions. Protein changes accumulate and reach a level sufficient for pathway failure and pathway failure leads to cell damage and toxicity.
Protein adducts are good candidates for biomarkers, to diagnose chemical poisoning, examine exposure to toxicants, clinically monitor drugs and evaluate pharmacological responses to a therapy. Adducts with serum albumin and hemoglobin are most commonly studied as biomarkers, because these two proteins are abundant in the blood, have multiple modifiable amino acids, and at least serum albumin has a highly conserved sequence among species which facilitates the translation of protein adduct studies from animals to humans. Protein adducts have some advantages over other types of biomarkers.
Protein adducts are usually stable. They are formed via nucleophilic substitution, in which reactive xenobiotics are often metabolized to electrophilic agents, which attack nucleophilic atoms (especially O, N and S) at side chains of amino acid residues. Protein adducts are generally long-lived and accumulative, compared to small molecules and their metabolites, because protein adducts have the same lifetime as the protein, e.g. 3-4 weeks for human serum albumin and 17 weeks for hemoglobin in erythrocytes. Protein adducts also have a longer lifetime than DNA adducts, because they are not actively repaired.
Serum albumin (SA) has been chosen from among the serum proteins because of its abundance and because its role as a carrier of fatty acids, endobiotics and xenobiotics increases the probability that it will bind and form covalent adducts with ultimate carcinogens.
Most of the drugs crossing the human placenta diffuse passively. In this case, placental blood flow, pH of maternal and fetal blood, physicochemical characteristics of the compounds, and protein binding determine a drug’s ability to cross placental membranes. Transplacental transfer of genotoxins in humans has been demonstrated by the presence of carcinogen-macromolecule adducts in either umbilical cord blood proteins or DNA isolated from fetal tissues.
The human placenta contains a wealth of enzymatic machinery responsible for both phase I and phase II reactions. However, data on the contribution of placental biotranformations in the conversion of xenobiotics into potential toxic metabolites are very scarce. Compared with the liver, placental metabolism seems to be minor and does not limit the extent of xenobiotic passage across the placenta.
The aforementioned findings confirmed and represented excellent interpretation to the present results which exhibited that there was an increase in the level of serum albumin adduct in the serum of aborted groups than that of normal control group.
Several studies identified 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 antagonistically binding the estrogen receptor (ER). PAHs also have been reported to act as weak estrogens by binding the ER.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsive to both natural and man-made environmental compounds. AhR and its nuclear partner ARNT are expressed in the female reproductive tract in a variety of species and several indications suggest that the AhR might play a pivotal role in the physiology of reproduction.
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 (X associated protein “XAP2”). Upon ligand binding, AhR migrates into the nucleus, unmasking of nuclear localization sequences in the NH2 terminal region of AhR, 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. Nevertheless, the list of AhR-regulated genes has grown considerably over the recent years and it includes also a variety of genes unrelated to xenobiotic metabolism such as genes encoding proteins involved in proliferation/apoptosis control (TGF- , TGF- 2, Bax, p27kip1), cytokines (IL-1 , IL-2) or proteins forming transcription factor complexes (c fos, Jun-B, c-Jun, Jun D). The products of these AhR-regulated genes are involved in metabolism of xenobiotic compound, as well as of many endogenous substances, like steroid hormones.
The glutathione-S-transferase (GST) families of enzymes, being important members of phase II detoxification pathways, catalyse the conjugation of a variety of electrophilic substances to glutathione, facilitating their elimination from the body. Usually, the foreign substances activated by the phase I reactions are acted upon by the GST enzymes.
GST enzymes are believed to play a crucial role in female reproduction as suggested by their presence in placenta and ovarian follicles in excessive amounts. Among the various GST enzymes, GSTP1 is reported to be the predominant isoform in placenta, suggesting a possible role for this enzyme in pregnancy. An association between RPL and GST gene polymorphisms was suggested by recent reports.
The aforementioned, findings represented excellent interpretation to the present results which demonstrated the high significant increase in the enzymatic activity of glutathione-S-transferase aborted women when compared with that of normal control group.
AhR-mediated actions can affect the regulation of the hypothalamo-pituitary-gonadal (HPG) axis by altering the secretion pattern of preovulatory follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion in primed female rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or related AhR ligands. 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. 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.
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.
Progesterone is a sex steroid hormone involved in the regulation of the menstrual cycle, pregnancy, and embryogenesis. 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.
The best-characterised mechanism for the action of TCDD involves the cloned AhR, a cytosolic receptor that binds TCDD acts as a transcription factor to regulate gene expression. It showed that toxic and biochemical effects associated with exposure to TCDD are mediated via initial binding to the cytosol, the aryl hydrocarbon receptor protein present in target tissue and organs.
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.
PAHs have also shown to significantly decrease the accumulation of trophoblast stem cells in the early placenta thereby limiting their differentiation into other cell types vital for hormone synthesis and ongoing placental development, a process that could contribute to defective deep placentation (DDP). Direct prenatal exposure to airborne PAHs has been associated with fetal growth restriction (FGR) with an increased exposure-related risk in the first trimester. Secondary (Phase II) metabolizing enzymes are required to further detoxify reactive PAH-metabolites in which their inefficient clearance results in prolonged exposure leading to sustained cytotoxicity and mutagenicity. Phase II enzymes include glutathione-s-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), NAD(P)H-dependent quinone oxidoreductase-1 (NQO1), and aldehyde dehydrogenase-3(ALDH3).
The placenta is a vital organ of pregnancy that serves as a maternal-fetal connection through which nutrient, O2, and hormone exchanges occur. It also provides protection and immunity to the developing fetus. In humans, normal placentation begins with proper trophoblastic invasion of the maternal spiral arteries and is the key event that triggers the onset of these placental activities. The placental vasculature undergoes changes to ensure optimal maternal vascular perfusion. Prior to the unplugging of the maternal spiral arteries by trophoblastic plugs, the state of low O2 tension in early pregnancy gives rise to normal, physiological hypoxia. During this time, the syncytiotrophoblast is devoid of antioxidants, and thus, remains vulnerable to oxidative damage. Between 10 and 12 weeks of gestation, the trophoblastic plugs are dislodged from the maternal spiral arteries, flooding the intervillous space with maternal blood. This event is accompanied by a sharp rise in O2 tension, marking the establishment of full maternal arterial circulation to the placenta associated with an increase in ROS, which leads to OS.
At physiological concentrations, ROS stimulate cell proliferation and gene expression. Placental acclimation to increased O2 tension and OS at the end of the 1st trimester up-regulates antioxidant gene expression and activity to protect fetal tissue against the deleterious effects of ROS during the critical phases of embryogenesis and organogenesis. Amongst the recognized placental antioxidants are heme oxygenase (HO)-1 and -2, Cu,Zn-SOD, catalase, and GPx. If maternal blood flow reaches the intervillous space prematurely, placental OS can ensue too early and cause deterioration of the syncytiotrophoblast. This may give rise to a variety of complications including miscarriage, recurrent pregnancy loss, and preeclampsia, amongst others.
Overwhelming placental OS has been proposed as a causative factor of spontaneous abortion. As mentioned earlier, placentas of normal pregnancies experience an oxidative burst between 10 and 12 weeks of gestation. This OS returns to baseline upon the surge of antioxidant activity, as placental cells gradually acclimate to the newly oxidative surroundings. In cases of miscarriage, the onset of maternal intraplacental circulation occurs prematurely and sporadically between 8 and 9 weeks of pregnancy in comparison to normal continuous pregnancies.
The activity of serum prolidase, a biomarker of extracellular matrix and collagen turnover, has been observed to be decreased in patients with early pregnancy loss. Its levels were also shown to negatively correlate with increased OS, possibly accounting for the heightened placental vascular resistance and endothelial dysfunction secondary to decreased and dysregulated collagen turnover. Decreased activity of serum paraoxonase/arylesterase- a major determinant of high-density lipoprotein (HDL) antioxidant status- was noted in patients with early pregnancy loss. A negative correlation with lipid hydroperoxide was also observed in these patients, indicating their high susceptibility to lipid peroxidation. Oxidative stress can also affect homeostasis in the ER. Persistence of endoplasmic OS can further sustain ER stress, eventually increasing decidual cell apoptosis and resulting in early pregnancy loss. Decreased detoxification ability of GPx may occur in the setting of Se deficiency, which has been linked to both spontaneous abortion and recurrent pregnancy loss. Apoptosis of placental tissues may result from OS induced inflammatory processes triggered by a variety of factors. Several etiologies may underlie improper initiation of maternal blood flow to the intervillous space; yet it may be through this mechanism by which both spontaneous and recurrent pregnancy loss occur.
It has been suggested that the maternal uterine spiral arteries of normal pregnancies may involve uterine natural killer (NK) cells as a regulator of proper development and remodeling. Angiogenic factors are known to play key roles in the maintenance of proper spiral artery remodeling. Thus, the involvement of uterine NK cells in RPL has been supported by the early pregnancy findings of increased levels of angiogenic factors secreted by uterine NK cells, as well as increased in vivo and in vitro endothelial cell angiogenesis induced by uterine NK cells in patients with RPL. Women experiencing RPL have also been noted to have increased endometrial NK cells, which were positively correlated to endometrial vessel density. Accordingly, it has been suggested that an increase of uterine NK cells increases pre-implantation angiogenesis, leading to precocious intra-placental maternal circulation, and consequently, significantly increased OS early in pregnancy. 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.
The findings of the herein study shows that circulating levels of oxidative stress biomarker malondialdehyde was strikingly significantly higher in aborted women when compared with 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, malondialdehyde (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 when compared with that of 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.
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 serum albumin adduct, PAHs, MDA, and GST, while there was a 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.