Chemicals

Analytical grade reagents: n-hexane, methanol, acetonitrile, 1-naphthol, 2-naphthol, and β-glucuronidase from Helix pomatia (G7017) were procured from Sigma Chemicals (St Louis, USA). OxiSelect BPDE Protein Adduct ELISA Kit was purchased from Cell Biolabs, Inc. (St; 7758 Arjons Drive, San Diego, USA). The follicle stimulating hormone (FSH) and P4 levels were estimated using the Stat Fax ELISA unite (from Awareness' technology, USA) by ELISA kit from Glory Science Co. (ltd, Del Rio, USA). Test kits for the estimation of malondialdehyde (MDA), catalase, glutathione-S-transferase (GST), and reduced glutathione (GSH) were procured from Biodiagnostics Co. (29 Taahreer st; Dokki Giza, Egypt). A test kit for the estimation of creatinine was procured from Diamond Diagnostic Co. (30175 Hannover, Germany).

Study area and design

This case study involving women with recurrent spontaneous abortions and their corresponding control counterparts was carried out in the coastal area of Alexandria using a random sampling method. Informed consent was sought and obtained from the volunteers before recruitment into the study after the ethical committee of Medical Research Institute, Alexandria University (IORG 0008812) approved the study protocol. The study was conducted in compliance with ethical principles guiding research with human subjects in accordance with the Helsinki declaration and subsequent revisions.

Selection of subjects

The participants in this study were recruited from El-Shatby Maternity Hospital, Alexandria University, and comprised a total of 76 healthy women aged 20–35 years. The control population was made up of 18 fertile women with at least one living child after successful full-term delivery, and who had no history of recurrent spontaneous abortion. The test subjects were made up of 58 women who had experienced at least 2 successive unexplained recurrent spontaneous abortions up to 20 weeks gestational age.

A semi-structured questionnaire was administered to all participants in the study for the history of past and present ailments with an emphasis on toxoplasmosis, rubella, and herpes simplex. Women with endocrine disorders (diabetes mellitus & thyroid disorders), uterine abnormalities, hypertension, liver diseases, urinary tract insult, residents of industrial areas, and smokers were all excluded from the study.

Sample collectionUrine samples

Spot urine samples (10 ml) were collected from all subjects of the study, stored at − 20 °C, and used for the estimation of PAH metabolites in urine (1-naphthol and 2-naphthol).

Blood samples

Whole blood samples (8 ml) were collected from all subjects of the study and distributed as follows; 3 ml was dispensed into an anticoagulant container EDTA for the estimation of glutathione and catalase enzyme activities; 5 ml was dispensed into the plain container without anticoagulant, allowed to clot and retract and then centrifuged at 4000 rpm for 10 min to extract serum for the estimation of benzo[a]pyren-7,8-dihydrodiol-9,10-epoxide-albumin adduct (BPDE-albumin), MDA, FSH and P4. The red cells were washed once with 10 volumes cold saline followed by the addition of 4 volumes of cold deionized water to get red cell pellets. The red cell storm was centrifuged at 500 g at 4 °C for 10 min. The supernatant was collected and stored at − 70 °C until the time of estimation of GST.

Mussel samples

Two mussel species, Donax trunculus and Andar aduloii were collected from the Mediterranean Sea “Alexandria coast” for the estimation of tissue PAH content. Mussel samples were opened raw, and the flesh was scraped out of the shell with a stainless-steel scalpel. The gills and digestive glands of the mussels were dissected out and then stored at -80 °C for determination of PAHs.

Laboratory methodsDetermination of PAH concentration in mussel samples

The following PAHs; Naphthalene (Naph), acenaphthylene (Acy), acenaphthene (Ace), flourene (Flo), phenanthrene (Phe), anthracene (Ant), fluoranthene (Flu), pyrene (Pyr), chrysene (Chy), benzo[g,h, i]perylene (BghiP), dibenzo[a,h]anthracene (DahA), Indeno[1,2,3-cd]pyrene (IcdP), benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF), benzo[b]fluoranthene (BbF) and benzo[a]anthracene (BaA) were estimated in two species of mussels Donax trunculus and Andara duloii. PAH extraction was performed as follows: freshly collected mussel samples from each species were dissected and the soft tissue from 30 to 40 individual bivalves was pooled together and dried in an open oven at 50 °C. Five grams of the dried samples were extracted in methanol with a Soxhlet extractor for 8 h followed by a lipid saponification step with the addition of potassium hydroxide and distilled water to the mixture and Soxhlet reflux extraction was continued for additional 2 h. Then, the methanolic part was extracted 3 times with n-hexane. The n-hexane fraction extracts were dried with anhydrous sodium sulfate and concentrated under a rotary evaporator. Subsequentially, concentrated samples were cleaned and fractioned on a chromatographic column (silica gel, aluminum oxide, and anhydrous sodium sulfate) and finally sample elution from the column was performed by adding an n-hexane to elute saturated aliphatic fraction (F1) and a mixture of n-hexane and dichloromethane (9:1) to elute hydrocarbon fraction (F2). Nitrogen gas was used to concentrate the samples and 2 µl of each sample and PAH standards were subjected to GC–MS analysis. The response factor of the individual PAH compounds to the internal standard was measured and calculated at least 3 times in the beginning, in the middle, and at the end for each batch of GC injections (10 samples). The method detection limits for each PAH compound ranged from 0.3 to 1.1 ng g − 1 of wet weight. According to previous reports [18], the extracted PAHs were identified and quantified using Gas chromatography, (Agilent technologies 1200 series), at the National Institute of Oceanography &Fisheries, Alexandria. The 16 priority PAHs were identified and quantified by comparing their retention time with a mixture of PAH standards.

Biochemical analysisDetermination of plasma catalase enzyme activity

Catalase enzyme activity was determined by the spectrophotometric method [19]. Briefly, 50 µl of the sample was mixed with 0.5 ml of phosphate buffer and 0.1 ml of hydrogen peroxide and, incubated for one minute at 37 °C. Then chromogen inhibitor and peroxidase enzymes were added. The mixture was incubated for 10 min at 37 °C. Sample blank, standard blank, and standard samples were performed for quality control. All samples were measured at 510 nm by spectrophotometer. The analysis principle relies on the Catalase enzyme reacting with a known quantity of H2O2, and the reaction was stopped with a catalase inhibitor after one minute.

The remaining H2O2 reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP) in the presence of hydrogen peroxide (HRP) to form a colored complex whose absorbance is inversely proportional to the concentration of catalase in the sample.

Catalase activity was determined according to the following equation.

$$}\,\,\,\,\,\,\frac}}}}\,\,\,*1000$$

Determination of erythrocyte glutathione-S-transferase (GST)

The Biodiagnostic assay test kit estimates total GST activity (cytosolic and microsomal) by measuring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH. The conjugation is associated with increased absorbance at 340 nm, with the rate of increase in direct proportion to the GST activity in the sample [20]. Briefly: 50 µl of the sample was mixed with phosphate buffer (1 ml), and GSH (0.1 ml) and incubated for 5 min at 37 °C. Then, CDNB was added, and the mixture was incubated for 5 min at 37 °C. Then, the reaction was terminated by adding trichloroacetic acid. Finally, the sample was centrifuged at 3000 rpm for 5 min and the absorbance of the sample was measured at 340 nm against blank. GST activity was calculated according to the following equation.

$$}\,\,\, = \frac \,}}}\,\times \,\frac}\,\,}}}}}}$$

Where: 0.0096 is the molar extinction coefficient of CDNB at 340 nm.

Estimation of malondialdehyde (MDA)

Estimation of MDA was done using the thiobarbituric acid (TBA) assay. MDA formed from the breakdown of polyunsaturated fatty acid serves as a convenient index for determining the extent of the peroxidation products in the body. TBA reacts with MDA in an acidic medium at a temperature of 95 °C for 30 min to form the TBA reactive product. The absorbance of the resultant pink product can be measured at 534 nm. MDA in the sample react with TBA to give a red colored (MDA-TBA) whose absorbance was measured at 534 nm. Briefly, TBA was added to 200 µl of the serum and the reaction mixture was heated to 95 °C for 30 min in a temperature-controlled heating block. The reaction was stopped by placing the reaction mixture on ice after which the concentration of MDA in the sample was read at 534 nm [21]. MDA concentration was measured according to the following equation:

$$}\,\,\,\,\frac}\,}}}}\,}}}\,\times\,\,10$$

where: 10, concentration of standard.

Estimation of reduced glutathione (GSH)

The modified standard Ellman’s method was employed in the estimation of GSH in which, the tested serum (100 µl) was added to Ellman’s reagent (5,5’-dithiobis-2-nitrobenzoic acid (DNTB)). The reduced chromogen is directly proportional to GSH concentration, and its absorbance can be measured at 405 nm [22]. GSH concentration was calculated according to the following equation:

$$}\,\, = \,\,\frac \, \times 30}}\, = \,A_ \times\,\,66.66$$

where: 30 is the concentration of standard and 0.45 is the absorbance of standard.

Determination of serum Progesterone (P4) hormone

Serum P4 estimation was done using the enzyme-linked immunosorbent assay method which is based on the principle of the specific interaction between antigens and their corresponding antibodies. The absorbance of the color complex formed after a competitive reaction between biotinylated antibody, native antigen, and enzyme-antigen conjugate for a limited number of antibody binding sites is proportional to the concentration of P4 in the sample [23]. Briefly, microplates’ wells for each serum reference, control, and patient specimen were formatted to be assayed in duplicate. 25 µl of the sample was pipetted into the provided microwell plate from the manufacturer’s ELISA kit, then, 50 µl of P4 enzyme was added and the plate was gently steered for 10–20 s. Progesterone biotin reagent was added to all wells and the plate was gently mixed by stirring for additional 10 s. The plate was incubated for 60 min at room temperature. Then The contents of the microplate were discarded by decantation or aspiration followed by a washing step with a washing buffer three times. Then the substrate solution was added to each well and incubated for 20 min at room temperature, and the reaction was terminated by adding a stop solution. Finally, the absorbance of all samples was measured at 450 nm in a microplate reader. The concentration for each unknown was obtained from the standard curve.

Determination of serum follicle stimulating hormone (FSH)

Estimation of FSH utilizes the principle of specific interaction between antigens and their corresponding antibodies The absorbance of the color complex formed after a competitive reaction between biotinylated antibody, native antigen, and enzyme-antigen conjugate for a limited number of antibody binding sites is proportional to the concentration of FSH in the sample [24]. Briefly, microplates' wells for each serum reference, control, and patient specimen were formatted to be assayed in duplicate. 25 µl of the sample was pipetted into the provided microwell plate from the manufacturer’s ELISA kit, then, 100 µl of FSH enzyme was added and the plate was gently steered for 10–20 s. The plate was incubated for 60 min at room temperature. Then the contents of the microplate were discarded by decantation or aspiration followed by a washing step with a washing buffer three times. Then the substrate solution was added to each well and incubated for 15 min at room temperature, and the reaction was terminated by adding a stop solution. Finally, a microplate reader measured the absorbance of all samples at 450 nm (using a reference wavelength of 620–630 nm to minimize well imperfections).

Determination of serum BPDE-albumin adduct

BPDE-albumin adduct measurement was carried out by immunoassay detection method. Bovine serum albumin (BSA) standards and samples were added into 96 wells of ELISA plates and incubated at 37 ℃ for 2 h. Anti-BPDE-1 antibody was used to probe for the BPDE-albumin adducts present in the standard and sample, followed by horse radish peroxide (HRP) conjugated secondary antibody. The concentration of BPDE-albumin in the sample is determined from a standard curve prepared from predetermined BPDE-BSA standards [25]. Briefly, 100 µl of the sample or reduced/BPDE-BSA standards were added to the 96-well albumin binding plate. All were incubated at 37 ºC for at least 2 h or 4 °C overnight. Wells were washed with PBS three times and then wells were taped on an absorbent pad. Then the diluted anti-BPDE-I antibody was added to all wells and incubated for 1 h at room temperature on an orbital shaker followed by adding the diluted secondary antibody-HRP conjugate to the wells. Substrate solution was added and incubated for 2–3 min. Finally, the reaction was stopped by adding a stop solution, and each well’s absorbance was measured at 450 nm in a microplate reader. The reduced BSA standard was used as an absorbance blank.

Determination of 1-naphthol and 2-naphthol

The estimation of 1-naphthol and 2-naphthol was performed by high-performance liquid chromatography (HPLC). Briefly: Urine samples were enzymatically hydrolyzed with 30 µl of β-glucuronidase and sulfatase for 16 h at 37 ℃ in a shaking water bath. After hydrolysis, 5 ml of acetonitrile was added and mixed for 10 s. The samples were centrifuged at 1000 rpm for 10 min. A 20 µl of supernatant was injected into the HPLC (Agilent technologies 1200 series). The mobile phase used was acetonitrile–water (35:65%) + 100 µl acetic acid per liter solvent, at a flow rate of 1 ml/min. the excitation and emission wavelengths were 227 nm and 430 nm respectively and the fluorescence detector. The column used was Eclipse XDB-C18 (Made in the USA) 150 mm X 4.5 mm. Quantification of the 1-naphthol and 2-naphthol in the urine sample is obtained by standard curve by measuring different concentrations of the reference standard, and then the curve plotted by instrument data analysis program and the final concentration of the 1-naphthol in the urine sample expressed as µmol/mol creatinine from the following equation:

$$\begin& }}\mu }\\&\quad = \,\,\frac}}}}\,\times \,\frac \end$$

where: 144.17 is molecular weight of α-naphthol and β-naphthol.

Determination of urine creatinine

Estimation of urine creatinine was done using a modified Jaffe’s reaction method. Principally, creatinine in the sample reacts with alkaline picrate within a specific time interval to avoid interferences to form a colored complex whose absorbance is proportional to the concentration of creatinine in the sample [26]. Briefly, diluted urine samples were mixed with alkaline picrate and the absorbance (A1) after 30 s and after 120 s (A2) of the sample addition was measured at 492 nm. Creatinine concentration was calculated according to the following equation:

$$\begin } &= \,\,\frac}\,}}}}\,}}}\\&\quad \times \,}\, \times \,50 \end$$

where: 50 is the dilution factor.

Statistical analysis

Data were analyzed using IBM SPSS software package version 20.0. The Shapiro–Wilk test and the D’Agstino test were used to test for normality in the distribution of quantitative variables. Vision test was done with histogram and QQ plot. Quantitative data were described using mean and standard deviation for normally distributed data while abnormally distributed data were expressed using median, minimum, and maximum. For normally distributed data, analysis of variance and post hoc tests were used to determine variations among multiple groups’ means. For abnormally distributed data, Mann–Whitney and Kruskal Wallis test were used to compare multiple groups’ means. The spearman test was used to study the correlation between different parameters. The significance of the obtained results was judged at the 5% level.