Impact of COVID-19 and vaccination on first and second trimester screening results.

COVID-19 has been shown to affect pregnant women. Since pregnant women are at risk of this infection, vaccination against COVID-19 has been suggested as an imperative way to diminish rate of COVID-19 in this population. In the current observational study, we have collected data of first and second trimester screening (FTS and STS) from pregnant women who were infected with SARS-CoV-2 and/or vaccinated against COVID-19 during their pregnancy, and compared this data with a group of control pregnant women. The cohort included 4612 and 2426 women referred for FTS and STS, respectively. There was no significant difference in median values of Pregnancy-associated plasma protein A (PAPP-A) and human chorionic gonadotropin-beta subunit (ßHCG) between infected women and controls. Moreover, these levels were not different between "Infected + vaccinated" and "Only vaccinated" groups. However, median values of PAPP-A and ßHCG were higher in "Infected + vaccinated" and "Only vaccinated" groups compared with "Infected" and "Control" groups (P < 0.001). Median values of unconjugated Estriol (uE3) and ßHCG markers were not different between "Only vaccinated" and "Control" groups, yet both markers were elevated in "Infected" and "Infected + vaccinated" groups compared with other groups. AFP values were higher in "Infected" group (P = 0.012). However, multiple of the median (MoM) and risk of open spina bifida (OSB) were not affected. Finally, median of calculated risk of trisomy 18 was lower in "Infected" and "Vaccinated" groups compared with controls (P = 0.007). Moreover, AstraZeneca and Sinopharm vaccines were associated with elevation of the calculated risk values of trisomy 21 and trisomy 18 (P < 0.001). While Sinopharm did not affect nuchal translucency (NT) and NT MoM (P = 0.13), AstraZeneca and Barakat increased and decreased these values, respectively (P values = 0.0027 and 0.015, respectively). Taken together, COVID-19 during pregnancy might be associated with some adverse obstetric outcomes. Besides, vaccination against this infection might affect the results of STS or FTS.

Incorporation of second-tier tests and secondary biomarkers to improve positive predictive value (PPV) rate in newborn metabolic screening program.

BACKGROUND: Nowadays, neonatal screening has become an essential part of routine newborn care in the world. This is a non-invasive evaluation that evaluated inborn errors of metabolisms (IEMs) using tandem mass spectrometry (LC-MS/MS) for the evaluation of the baby's risk of certain metabolic disorders. METHODS: This retrospective study was conducted on 39987 Iranian newborns who were referred to Nilou Medical Laboratory, Tehran, Iran, for newborn screening programs of IEMs. We incorporated second-tier tests and secondary biomarkers to improve positive predictive value (PPV). RESULTS: Statistical data were recorded via call interviewing in 6-8 months after their screening tests. The overall prevalence of IEM was 1:975. The mean age of all participants was 3.9 ± 1.1 days; 5.1% of participants were over 13 days and 7.7% were preterm or underweight. A total of 11384 (29.4%) of the cases were born in a consanguineous family. The type of delivery was the cesarean section in 8332 (51.3%) valid cases. The neonatal screening results had an overall negative predictive value (NPV) of 100% and the overall PPV of 40.2%. The false-positive rate was 0.15%. CONCLUSION: This study showed a high incidence of metabolic disease due to a high rate of consanguineous marriages in Iran and indicated that incorporation of second-tier tests and secondary biomarkers improves PPV of neonatal screening programs.

Expression Analysis of Aurora-C and Survivin, Two Testis-Specific Genes, in Patients with Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The high frequency of positive families shows the importance of public awareness and screening strategies in those families. Cancer/testis (CT) antigens such as Aurora-C and Survivin are a group of antigens expressed in various tumor types of human cancers. Therefore, the aim of this study was to investigate the expression of Aurora-C and Survivin genes in malignant and normal tissues and their correlation to clinicopathological characteristics. METHODS: Tumor samples were obtained from 33 patients and adjacent non-tumorous tissues from 7 patients were also used as control. Patients were diagnosed with various stages of colorectal cancer. The level of Aurora-C and Survivin genes were evaluated by using real-time quantitative Polymerase Chain Reaction. RESULTS: The expression pattern of Survivin and Aurora-C revealed significant changes in tumor tissues when compared with normal tissues (p < 0.05). Also, these expressions were associated with the grade of disease and tumor size. There was no significant relationship between the expression of Survivin and Aurora-C genes (p > 0.05). CONCLUSIONS: In conclusion, the overexpression of Aurora-C and Survivin genes may play an important role in the development of colorectal cancer and may play a potential role in cancer therapy.

Comparison of smear microscopy, culture, and real-time PCR for quantitative detection of Mycobacterium tuberculosis in clinical respiratory specimens.

BACKGROUND: As a rapid diagnostic technique, the real-time polymerase chain reaction (PCR) can detect Mycobacterium tuberculosis with a high sensitivity and specificity. However, further studies are needed to confirm it as a standard method. In this study, we evaluated the cyp141 gene for the detection and quantification of M. tuberculosis in respiratory specimens and compared the results with direct microscopy and culture. METHODS: Sputum samples (n = 247) were collected from patients of the different provinces of Iran. DNA was extracted from clinical specimens and H37Rv strain. After measuring the standard strain DNA concentration by NanoDrop and using the Avogadro number, the DNA was diluted 6 times in order to obtain 1 × 10(6) to 10 template copies. A Taqman probe was designed for detection of the target in a real-time PCR using the specific primers. RESULTS: Of 247 samples, 135 (55%) were culture-negative. Of 112 (45%) culture-positive samples, 88 were positive by both smear and culture and 24 were smear-negative but culture-positive. The real-time PCR enumerated 1.5E + 02 to 4.3E+ 03, 8.5E + 03 to 5.5E + 04, 7.2E + 04 to 1.1E + 06, and 1.2E + 06 to 8.1E + 07 M. tuberculosis cells in the specimens with smear-negative, 1-plus, 2-plus, and 3-plus codes, respectively. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the real-time PCR were 90.2% (101/112), 97.8% (132/135), 97.1%, and 92.3%, respectively. CONCLUSIONS: The overall sensitivity and specificity, the results in comparison with those of the Xpert MTB/RIF kit, and the good correlation with molecular and phenotypic methods, show that cyp141 could be a good target for the quantification of M. tuberculosis in sputum and possibly other clinical specimens.

Multicomponent siRNA/miRNA-loaded modified mesoporous silica nanoparticles targeted bladder cancer for a highly effective combination therapy.

Bladder cancer is one of the concerning urological malignant diseases in the world, which has a clinical need for effective targeted therapy. The development of nanotechnology-based gene delivery to bladder tumor sites is an effective strategy for targeted cancer therapy with low/no toxicity. With this view, in the present work, the mesoporous silica nanoparticles (MSNs) modified with c(RGDfK)-PLGA-PEG [c(RGDfK)-MSN NPs] were constructed for co-delivery of miR-34a and siPD-L1 within bladder cancer cells and tissues. Our findings showed that miR-34a is downregulated while PD-L1 is up-regulated in cell lines and animal studies. This nano-carrier is biocompatible in the serum environment and effectively protects miR-34a and siPD-L1 against serum degradation. However, we showed that c(RGDfK)-MSN NPs could simultaneously downregulate PD-L1 expression and up-regulate miR-34a in the T24 cells and T24 mice model and enhance anti-tumor effects both in vivo and in vitro. In conclusion, these findings presented new suggestions for improving targeted therapeutic strategies with specified molecular objectives for bladder cancer treatment.

The Effect of Alpha-Tocopherol on the Expression of Epidermal Growth Factor Receptor and Transforming Growth Factor Beta Genes in Three Developmental Stages of Echinococcus granulosus.

BACKGROUND: In recent decades platyhelminths have been used as model organisms to address some of the fundamental questions related to the growth and development of animal organisms. Epidermal Growth Factor Receptors (EGFR) and Transforming Growth Factor beta (TGF-beta) have a regulatory role in the growth and development of Echinococcus species. This study determined the effect of alpha-tocopherol on the expression of EGFR and TGF-beta genes in three in vitro developmental stages of E. granulosus. METHODS: E. granulosus protoscoleces were cultured in diphasic medium containing bovine serum and CMRL 1066. Three developmental stages of E. granulosus, i.e. invaginated protoscoleces, evaginated protoscoleces and three-proglottid worms, were treated by alpha-tocopherol (250 µg/ml for 36 h) and the expression of EGFR and TGF-beta genes were evaluated by using qPCR analysis. RESULTS: Intact protoscoleces were successfully developed to the segmented worms in diphasic culture media. Higher levels of both EGFR and TGF-beta gene expression were observed in the invaginated protoscoleces as well as the segmented worms in comparison to the non-treated controls. CONCLUSION: Administration of alpha-tocopherol to different developmental stages of E. granulosus significantly enhanced EGFR and TGF-beta expression in the parasite. Both oxidant and non-oxidant activities of alpha-tocopherol could explain the study findings. Overexpression of the genes could in turn enhance growth factor effects and facilitates the viability of the parasite.

Fine-tuning of routine combined first- trimester screening: The ratio of serum-free- beta-human chorionic gonadotropin (fß-hCG) to pregnancy-associated plasma protein-A (PAPP-A) could improve performance of Down syndrome screening program, a retrospective cohort study in Iran.

OBJECTIVES: To evaluate the performance of the current national screening policy for Down syndrome (DS) in Iran and suggest a more efficient protocol with a wealth of a large series of first-trimester screening (FTS) data obtained from Nilou medical laboratory. To fulfill this aim, detection rate (DR), positive screening rate (PSR), false negative rate (FNR) and odds of being affected given a positive results (OAPR) were calculated at different cutoff risk. In the latest update of DS screening program in Iran, there is no place for intermediate group to be further investigated. Next, we proposed a novel parameter namely the ratio of fß-hCG multiple of the median (MoM) value to PAPP-A MoM value to delicately categorize FTS results in a way that reduce FNR without imposing unnecessary anxious and extra money on most families. METHODS: The present investigation was conducted retrospectively on 197,210 pregnancies undergoing FTS for aneuploidies in Nilou medical laboratory, Tehran, Iran, from March 2015 to February 2016. RESULTS: Intermediate risk group is important as 23 out of 45 FN fell in the range 1:250 to 1:1100. By applying the proposed index, the ratio of fß-hCG MoM to PAPP-A MoM and subsequent decision about NIPT, 8 out of 23 FN cases in intermediate group could be detected. CONCLUSION: Compared with the current policy, our novel proposed approach had better performance and could be applied by the Iran National Health Service to improve the screening program guideline.

Non-invasive prenatal test to screen common trisomies in twin pregnancies.

OBJECTIVES: Recent years have witnessed a shift from invasive methods of prenatal screening to non-invasive strategies. Accordingly, non-invasive prenatal testing (NIPT) using cell-free fetal DNA in maternal plasma has gained a considerable deal of interest from both geneticists and obstetricians. Efficacy of this method in identification of common aneuploidies has been extensively assessed in singleton pregnancies. However, a limited number of studies have addressed the twin pregnancies. In this context, the present study is aimed at identification of the efficacy of NIPT in twin pregnancies. METHODS: NIPT was performed on twin pregnancies to screen trisomies 13, 18 and 21. Pregnant women referring to Nilou Clinical Laboratory between March 2016 and December 2018 were included in this research. RESULTS: In the current study, a total 356 twin pregnancies were screened in search for trisomies 13, 18 and 21. 6 cases exhibited positive NIPT results in which the presence of trisomies 13, 18 and 21 was confirmed by fetal karyotype in 1, 2 and 2 cases, respectively. One twin pregnancy showed normal karyotype. The combined false-positive rate for these trisomies was 0.28%. No false negative case was observed. The combined sensitivity and specificity of NIPT in twin pregnancies were 100 and 99.7%, respectively. CONCLUSION: The results of the current study verify the feasibility, sensitivity and specificity of NIPT in twin pregnancies.

Synthesizing, characterizing, and toxicity evaluating of Phycocyanin-ZnO nanorod composites: A back to nature approaches.

C-Phycocyanin pigment was purified from a native cyanobacterial strain using a novel consecutive multi-step procedure and utilized for the first time for the green synthesis of phycocyanin-zinc oxide nanorods (PHY-ZnO NRs) by a simple, low-cost and eco-friendly approach. The PHY-ZnO NRs were characterized using UV-vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, FTIR, SEM, TEM, differential scanning calorimetry (DSC), thermogravimetric (TGA), and EDX spectroscopy analysis. The UV-vis spectra showed an absorption band at 364 nm which is characteristic of ZnO nanoparticles (ZnONPs). The rod-shaped PHY-ZnO NRs observed in the TEM and SEM images had an average diameter size of 33 nm, which was in good agreement with the size calculated by XRD. The elemental analysis of PHY-ZnO NRs composition showed that three emission peaks of zinc metal and one emission peak of oxygen comprised 33.88% and 42.50%, respectively. The thermogram of PHY-ZnO NRs sample exhibited the weight loss of biosynthesized nanoparticles registered to be 3%, emphasizing the purity and heat stability of zinc oxide nanorods coated with phycocyanin pigment-protein. MTT assay indicated that PHY-ZnO NRs had a less cytotoxicity on fibroblast L929 compared to the ZnONRs-treated cells. A remarkable increase in ROS level was measured in cells treated with ZnO at final concentrations of 100, 200 and 500 µg/ml (78 ± 7, 99 ± 8 and 116 ± 11, respectively). When it comes to PHY-ZnO NRs, a protective effect for phycocyanin was detected which declined the level of ROS content as confirmed by fluorescent microscopy. The distinctive features of phycocyanin for surface functionalization of ZnO nanoparticles deserve to be deemed as a nano-drug candidate for further researches.

The Effect of Preimplantation Genetic Screening on Implantation Rate in Women over 35 Years of Age.

OBJECTIVE: Advanced maternal age (AMA) is an important factor in decreasing success of assisted reproductive technology by having a negative effect on the success rate of intra-cytoplasmic sperm injection (ICSI), particularly by increasing the rate of embryo aneuploidy. It has been suggested that the transfer of euploid embryos increases the implantation and pregnancy rates, and decreases the abortion rate. Preimplantation genetic screening (PGS) is a method for selection of euploid embryos. Past studies, however, have reported different results on the success of pregnancy after PGS in AMA. Investigating the pregnancy rate of ICSI with and without PGS in female partners over 35 years of age referred to infertility centers in Tehran. MATERIALS AND METHODS: In this randomized controlled trial, 150 couples with the female partner over age of 35 were included. Fifty couples underwent PGS and the remaining were used as the control group. PGS was carried out using fluorescent in situ hybridization (FISH) for chromosomes 13, 18, 21, X and Y. Results of embryo transfer following PGS were evaluated and compared with those in the control group. RESULTS: Implantation rates obtained in the PGS and control groups were 30 and 32% respectively and not significantly different (P>0.05). CONCLUSION: PGS for chromosomes 13, 18, 21, X and Y does not increase implantation rate in women over 35 years of age and therefore the regular use of PGS in AMA is not recommended.

Evaluation of circulating cellular DCLK1 protein, as the most promising colorectal cancer stem cell marker, using immunoassay based methods.

BACKGROUND: DCLK1, as the most potential colorectal cancer stem cell (CSC) marker has been the core of many recent investigations. However, no study has been performed to evaluate the circulating cellular DCLK1 protein (CCDP) that might reflect the presences of colorectal CSC in circulation. OBJECTIVES: We aimed to evaluate CCDP in the peripheral blood mononuclear cells (PBMCs) of colorectal cancer (CRC) patients applying immunoassay based methods including PLA, IPCR and ELISA in order to introduce the method of choice for clinical detection of CCDP. METHODS: PBMCs were extracted from blood samples of 58 CRC patients along with 58 blood samples of tumor free controls. Total protein of PBMC was extracted and the CCDP level was evaluated. The results of three applied immunoassay tests were compared and the best approach for clinical application was introduced, accordingly. In addition, the correlation of CCD Plevel with clincopathologic findings of CRC patients was assessed. RESULTS: The results of three immuneassay methods confirmed each other. Based on our finding, ELISA could be the most judicious method for clinical evaluation of CCDP considering its simplicity for clinical implications. Our results also showed a significant higher amount of CCDP in peripheral blood of CRC patients compared to control group which was also correlated with patients' clinicopathologic finding such as stage, grade and neoadjuvant history. CONCLUSION: CCDP could be applied for monitoring purposes in CRC patients. However, its application needs to be more elucidated in future investigations implementing larger samples.

Development of in-house multiplex real time PCR for human papillomavirus genotyping in Iranian women with cervical cancer and cervical intraepithelial neoplasia.

BACKGROUND: HPV related cervical cancer as one of the most common women cancers in developing countries. Regarding accessibility of commercial vaccines, any long or short term modality for integrating preventive immunization against HPV in a national program needs comprehensive information about HPV prevalence and its genotypes. The important role of selecting most accurate diagnostic technologies for obtaining relevant data is underlined by different assays proposed in the literature. The main objective of the present study was to introduce an in-house HPV typing assay using multiplex real time PCR with reliable results and affordable cost for molecular epidemiology surveys and diagnosis. MATERIALS AND METHODS: 112 samples of formalin fixed paraffin embedded tissues and liquid based cytology specimens from patients with known different grades of cervical dysplasia and invasive cancer, were examined by this method and the result were verified by WHO HPV LabNet proficiency program in 2013. RESULTS: HPV was detected in 105 (93.7%) out of 112 samples. The dominant types were HPV 18 (61.6%) and HPV 16 (42.9%). Among the mixed genotypes, HPV 16 and 18 in combination were seen in 12.4% of specimens. CONCLUSIONS: According to acceptable performance, easy access to primers, probes and other consumables, affordable cost per test, this method can be used as a diagnostic assay in molecular laboratories and for further planning of cervical carcinoma prevention programs.

Induction of Apoptosis and Growth Suppression by Homeobox Gene TGIFLX in Prostate Cancer Cell Line Lncap.

BACKGROUND: TGIFLX, a Homoproteins cluster member located on the X chromosome, has a critical role in male reproduction and prostate development. Previous studies have shown the erratic expression of TGIFLX gene in a large proportion of prostate tumors. However TGIFLX function in prostate development remains unknown. The purpose of this study was to evaluate the consequences of TGIFLX expression on prostate cancer cell lines (LNCaP). METHOD: Inducible Tet-On gene expression system was used with a regulatory capability by doxycycline induction. In this system, stable LNCaP cells with TGIFLX tet-on plasmid were able to induce TGIFLX expression by doxycycline treatment. TGIFLX gene expression was confirmed by RT-PCR. RESULTS: Induction of gene expression caused cell proliferation decrement and apoptosis increment in LNCaP TGIFLX cells compared with control cells (P<0.01). Also, by using PEGFPN1 plasmid protein in this study localization was shown in nucleus. The gene was cloned in the plasmid and transfected to LNcap cells with plasmid PEGFPN1 TGIFLX and the plasmid was PEGFPN1. The TGIFLX expression was confirmed by RT-PCR and fluorescent microscopy. CONCLUSION: TGIFLX expression demonstrated a tumor suppressor characterization in a prostatic cancer cell line with low grade of tumorigenicity (LNCaP). More cell lines with different level of tumorogenicity need to be investigated for further clarification of the TGIFLX gene function.

Differential expression of human homeodomain TGIFLX in brain tumor cell lines.

Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme (GBM) in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain (HD) transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expression vector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors.

Seminal molecular markers as a non-invasive diagnostic tool for the evaluation of spermatogenesis in non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is currently evaluated by the use of conventional histopathological methods. In some cases, focal spermatogenesis is present in the testes of patients with NOA which may be almost undetectable by routine histopathological examinations. Application of molecular markers in semen to predict the spermatogenesis status in the testis will emphasize the probability of finding sperm in NOA testis through further search using TESE or mTESE. Detection of germ cell-specific transcripts in semen is a signal of germ cells present in the testis. In this study, we used molecular methods to evaluate spermatogenesis status in azoospermic men. Semen samples were collected from 203 men with azoospermia. Total RNA was extracted from the semen precipitates. First-strand complementary deoxyribonucleic acid (cDNA) was synthesized by reverse transcriptase then, (RT)-PCRs were carried out using primers for testis stage-specific genes (DAZ, AKAP4, PRM1, and PRM2). Testicular tissue biopsies were used for evaluating spermatogenesis status in testis. Histopathological examination and LH, FSH, and testosterone level measurements (chemiluminescence assay) were performed. The presence of DAZ and PRM2 transcripts in semen significantly indicated the presence of spermatogonia and spermatids in the testicular tissues. Absence of all four markers in semen confirmed the histopathological results corresponding to sertoli cell only syndrome (SCO). Although TESE should not be excluded solely on this criteria, using PRM1, PRM2, AKAP4, and DAZ transcripts in semen would provide a non-invasive molecular diagnostic tool to better counsel patients before undergoing TESE.