The association between dietary inflammatory index and C-reactive protein in plasma and semen with semen quality: A cross-sectional study.

Background: Infertility affects couples worldwide, with male factors being responsible for half of all cases. Objective: This study aimed to investigate the relationship between dietary inflammatory index (DII) and levels of C-reactive protein (CRP) in plasma and semen with the quality of semen in infertile males. Materials and Methods: In this cross-sectional study, 88 infertile men referring to Besat hospital, Tehran, Iran from December 2021-November 2022 were enrolled. A detailed questionnaire requesting information, and a 168-item semiquantitative food frequency questionnaire, were completed by participants. A food frequency questionnaire was used to calculate the DII. Additionally, semen and blood samples were collected from each participant for semen analysis and CRP-level assessment. Statistical analyses were performed to explore the association between DII and CRP levels with sperm quality. The correlation between DII and serum/semen CRP, besides assessing nutrients in each DII quartile group, was also explored. Results: A significant difference was observed between different DII quartiles considering sperm motility (p = 0.006) and morphology (p = 0.014). Post hoc study revealed a significant difference between the 1st and 2nd quartiles and the 1st and 4th quartiles of DII regarding sperm motility (p = 0.011, and 0.017 respectively) and a significant difference between the 1st and 2nd quartiles of DII considering sperm morphology (p = 0.009). A statistically significant inverse correlation was also observed between DII and sperm motility (p = 0.017). Carbohydrates and ß-carotenes were significantly different between the 4 DII quartiles (p = 0.043 and p = 0.026, respectively). Finally, no significant correlation was observed between DII and CRP levels in blood and semen (p > 0.05). Conclusion: The findings suggested a notable correlation between DII and semen quality; however, no significant association were observed between DII and CRP levels in blood and semen.

Bisphenol-S Influence on Oxidative Stress and Endocrine Biomarkers of Reproductive System: A Systematic Review and Meta-Analysis.

Background: Bisphenol-S (BPS), as a new human public health concern, was introduced to the plastic industry by BPA-free labeled products following the restrictions of Bisphenol-A (BPA) as a safe alternative. However, recent research has revealed a controversial issue. In this regard, the present study aimed to review the relationship between BPS exposure and reproductive system dis/malfunction. Methods: PubMed and other databases were searched up to January 2021. The standard mean difference (SMD) with a 95% confidence interval (CI) was calculated for the main parameters using the random-effects model. Finally, 12 studies with 420 subjects were included in this research. Forest plot, meta-regression, and non-linear dose-response effect were calculated for each parameter by random-effects model. Results: Based on the results of in vitro assessment, a significant increase was found in the oxidative stress parameters, including superoxide dismutase (SMD: 0.63, 95% CI: 0.321, 0.939), thiobarbituric acid reactive substances (SMD: 0.760, 95% CI: 0.423, 1.096), and reactive oxygen species (SMD: 0.484, 95% CI: 0.132, 0.835). In addition, the hormonal assessment revealed a significant decrease in male testosterone concertation (SMD: -0.476, 95% CI: -0.881, -0.071). Moreover, in vivo examination revealed a significant decrease in hormonal parameters, such as female testosterone (SMD: -0.808, 95% CI: -1.149, -0.467), female estrogen (SMD: -2.608, 95% CI: -4.588, -0.628), female luteinizing hormone (SMD: -0.386, 95% CI: -0.682, -0.089), and female follicle-stimulating hormone (FSH) (SMD: -0.418, 95% CI: -0.716, -0.119). Besides, linear and non-linear correlations were detected in the main parameters. Conclusion: In conclusion, based on the current meta-analysis, BPS was suggested to be toxic for the reproductive system, similar to the other bisphenols. Moreover, a possible correlation was indicated between oxidative and hormonal status disruption induced by BPS in male and female reproductive systems dis/malfunction.

Human Placental Mesenchymal Stem Cell-derived Exosomes in Combination with Hyperbaric Oxygen Synergistically Promote Recovery after Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a critical medical condition during which sensorimotor function is lost. Current treatments are still unable to effectively improve these conditions, so it is important to pay attention to other effective approaches. Currently, we investigated the combined effects of human placenta mesenchymal stem cells (hPMSCs)-derived exosomes along with hyperbaric oxygen (HBO) in the recovery of SCI in rats. Ninety male mature Sprague-Dawley (SD) rats were allocated into five equal groups, including; sham group, SCI group, Exo group (underwent SCI and received hPMSCs-derived exosomes), HBO group (underwent SCI and received HBO), and Exo+HBO group (underwent SCI and received hPMSCs-derived exosomes plus HBO). Tissue samples at the lesion site were obtained for the evaluation of stereological, immunohistochemical, biochemical, molecular, and behavioral characteristics. Findings showed a significant increase in stereological parameters, biochemical factors (GSH, SOD, and CAT), IL-10 gene expression and behavioral functions (BBB and EMG Latency) in treatment groups, especially Exo+HBO group, compared to SCI group. In addition, MDA levels, the density of apoptotic cells and gliosis, as well as expression of inflammatory genes (TNF-α and IL-1ß) were considerably reduced in treatment groups, especially Exo+HBO group, compared to SCI group. We conclude that co-administration of hPMSCs-derived exosomes and HBO has synergistic neuroprotective effects in animals undergoing SCI.

Accelerated wound healing using three-dimensional amniotic membrane scaffold in combination with adipose-derived stem cells in a diabetic rat model.

The most important factors in the non-optimal healing of diabetic wounds are the lack of a suitable scaffold in the wound site for the migration and replacement of cells, as well as the lack of blood supply and effective growth factors in the wound site. Herein we investigated whether a bioengineered micro-porous three-dimensional decellularized amniotic membrane-scaffold (DAMS) in combination with adipose-derived stem cells (ASCs) could promote healing in ischemic wounds in diabetic type 1 rat. The diabetic animals were randomly divided into non-treated (untreated group), engraftment by DAMS (DAMS group), transplanted by ASCs (ASC group), and DAMS in combination with ASCs (DAMS+ASC group). Stereological, immunohistochemical, molecular, and tensiometrical assessments were performed on post-surgical days 7, 14, and 21. We found that the rate of wound closure, the volumes of new epidermis and dermis, the numerical density of fibroblasts and blood vessels, the numbers of proliferating cells and collagen deposition as well as biomechanical properties of the healed wounds were significantly higher in the treatment groups in comparison to the untreated group, and were the highest in DAMS+ASC ones. The transcripts for TGF-ß and VEGF genes were significantly upregulated in all treatment regimens compared to the untreated group and were the highest for DAMS+ASC group. This is while expression of TNF-α and IL-1ß as well as cell density of neutrophils decreased more significantly in DAMS+ASC group as compared with other groups. Overall, it was found that using both DAMS engraftment and ASC transplantation has more impact on diabetic wound healing.

Bovine oocyte developmental competence and gene expression following co-culturing with ampullary cells: An experimental study.

BACKGROUND: There is no sufficient information on the impact of bovine ampullary oviductal epithelial cells (BAOECs) on in vitro oocyte maturation competence and gene expression. OBJECTIVE: This study aimed to examine the oocyte developmental competence following co-culturing with a monolayer of fresh and frozen-thawed ampullary cells. MATERIALS AND METHODS: Bovine cumulus-oocyte complexes (COCs) were distributed into three groups: control group; where in COCs were cultured in cell-free media for 24 hr and FML and FTML groups in which the COCs were cultured in maturation media for 18 hr and then transferred into a media containing fresh and frozen-thawed BAOECs monolayer, respectively (BAOECs were extracted from the oviducts of slaughtered cattle and were then cultured freshly or frozen-thawed) for a further 6 hr. After 24 hr, the expanded COCs were evaluated for nuclear maturation, fertilization rate, and gene expression (GDF9, StAR, CASP3, and FSHr). RESULTS: Nuclear maturation rate in the FTML group was significantly higher than the control group (p = 0.02). The fertilization rate of FTML group was significantly higher than the control and FML groups (p = 0.05 and p = 0.03, respectively). In terms of gene expression, GDF9 were upregulated in the presence of the BAOECs during the last 6 hr of the in vitro maturation (p < 0.001). Furthermore, the expression of the StAR gene in the FTML group was higher than the other groups (p = 0.02). CONCLUSION: Ampullary cells co-culturing (especially frozen-thawed cells) for in vitro maturation of bovine oocytes yields encourages the results and demonstrates the beneficial effect of co-culture on gene expression and developmental competence.

Protective effects of hesperetin on the quality of sperm, apoptosis, lipid peroxidation, and oxidative stress during the process of cryopreservation: An experimental study.

BACKGROUND: Hesperetin is a bioflavonoid compound, largely used in Chinese traditional medicine and found plenty in citrus fruits. Hesperetin has beneficial effects against different diseases. The sperm cryopreservation process is a common method that is used in infertility laboratories. It has been reported that during the cryopreservation process, the quality of sperm is significantly reduced. OBJECTIVE: To investigate the effect of hesperetin on the quality of human spermatozoa during the cryopreservation process. MATERIALS AND METHODS: In this experimental study, 22 sperm sample of normozoospermia men who reffered to the infertility department of the Shariati Hospital (Tehran, Iran) Between October and November 2019 were collect and divided in to three groups as: 1) fresh, 2) control (frozen-thawed group without treatment), and 3) treatment group as frozen-thawed samples supplemented with 20 µM hesperetin. Motility, Viability, morphology, Apoptotic-like changes, intracellular H 2 O 2 , intracellular O2 - , and lipid peroxidation (LPO) was measured. RESULTS: Hesperetin treatment during the cryopreservation process of human sperm significantly improved the viability, motility, and morphology rates of the spermatozoa after frozen-thawed process in control group (p < 0.01). In addition, it significantly reduced the reactive oxygen species (ROS) level, LPO level and increased the percentage of viable sperm cells with intact plasma membrane (p < 0.01) after frozen-thawed process. CONCLUSION: Hesperetin can improve the quality of human sperm and also protect human sperm against reactive oxygen species, LPO, and apoptosis during the cryopreservation-thawing process.

Coenzyme Q10 enhances remyelination and regulate inflammation effects of cuprizone in corpus callosum of chronic model of multiple sclerosis.

Multiple Sclerosis (MS) is a chronic, progressive demyelinating disease of the central nervous system that causes the most disability in young people, besides trauma. Coenzyme Q10 (CoQ10)-also known as ubiquinone-is an endogenous lipid-soluble antioxidant in the mitochondrial oxidative respiratory chain which can reduce oxidative stress and inflammation, the processes associated with demyelination in MS. Cuprizone (CPZ) intoxication is a well-established model of inducing MS, best for studying demyelination-remyelination. In this study, we examined for the first time the role of CoQ10 in preventing demyelination and induction of remyelination in the chronic CPZ model of MS. 40 male mice were divided into four groups. 3 group chewed CPZ-containing food for 12 weeks to induce MS. After 4 weeks, one group were treated with CoQ10 (150 mg/kg/day) by daily gavage until the end of the experiment, while CPZ poisoning continued. At the end of 12 weeks, tail suspension test (TST) and open field test (OFT) was taken and animals were sacrificed to assess myelin basic protein (MBP), oligodendrocyte transcription factor-1 (Olig1), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) by real-time polymerase chain reaction (real-time PCR) and total antioxidant capacity (TAC) and superoxide dismutase (SOD) by Elisa test. Luxol fast blue (LFB) staining was used to evaluate histological changes. CoQ10 administration promoted remyelination in histological findings. MBP and Olig-1 expression were increased significantly in CoQ10 treated group compare to the CPZ-intoxicated group. CoQ10 treatment alleviated stress oxidative status induced by CPZ and dramatically suppress inflammatory biomarkers. CPZ ingestion made no significant difference between normal control group and the CPZ-intoxicated group in TST and OFT. CoQ10 can enhance remyelination in the CPZ model and potentially might have same effects in MS patients.

Effect of feeding male mice with palm, fish, and sunflower oils on sperm characteristics and sex ratio of offspring.

Several studies have indicated that feeding female animal with fatty acids could result in alteration of offspring sex ratio; however, there is little information available regarding the effect of feeding male animal with fatty acids on sex ratio of offspring. The aim of current study was to investigate the effect of paternal feeding with n-3 and n-6 polyunsaturated fatty acids (PUFAs) on sperm characteristics and sex ratio of offspring in mice. Male mice received 2.50% of palm oil (control), fish oil (n-3 PUFAs) or sunflower oil (n-6 PUFAs) for two months. Sperm kinematics and viability were assessed using computer-assisted sperm analyzer and eosin-Y staining, respectively. Additionally, female mice were randomly introduced to males of three experimental groups to determine reproductive parameters including litter size, conception rate and sex ratio. Fish oil enhanced sperm concentration, motility and viability, whereas sunflower oil decreased sperm concentration. Nevertheless, progressive motility, velocity, linearity and straightness of sperms were not affected by source of fatty acids. Although supplementation with fish oil resulted in male-biased sex ratio, palm and sunflower oils did not impact sex ratio. Besides, source of fatty acids failed to influence conception rate and litter size. In conclusion, the present study provided evidence for the impact of paternal fish oil consumption on sex ratio of offspring for the first time. Moreover, it showed beneficial effect of n-3 PUFAs and adverse effects of n-6 PUFAs on seminal parameters.

Comparison of Epigenetic Modifier Genes in Bovine Adipose Tissue-Derived Stem Cell Based Embryos, as Donors, with In Vitro and Parthenogenesis Embryos.

OBJECTIVE: Regarding that undifferentiated mesenchymal stem cells, as donor cells, require less epigenetic reprogramming, possibility of using bovine adipose tissue-derived stem cells (BASCs) with low level of DNMTs and HDACs expression was evaluated. MATERIALS AND METHODS: In this experimental study, we examined gene expression of epigenetic modifiers including DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and histone deacetylases (HDAC1-3), as well as protein levels of histone H3 acetylation at lysine 9 (H3K9ac) and POU5F1 (also known as OCT4) at two stages of preimplantation development among in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) groups. RESULTS: The results revealed that developmental competence of IVF embryos was higher than SCNT embryos (P<0.05). In the PA and SCNT groups, DNMT1, HDAC2 and HDAC3 mRNA were overexpressed (P<0.05), and proteins levels of H3K9ac and POU5F1 were reduced at 6-8 cells and blastocyst stages compared to IVF (P<0.05). The mRNA expression of DNMT1 and HDAC1 and proteins levels of POU5F1 and H3K9ac were significantly different between SCNT and PA groups (P<0.05) in both developmental stages (except HDAC1 in blastocyst stage). CONCLUSION: The SCNT embryos derived from BASCs have endured considerable nuclear reprogramming during early embryo development. Comparison of PA and SCNT blastocysts demonstrated that HDAC1 and DNMT1 may attribute to developmental competence variability of bovine embryos.

Long-term administration of high-dose methylphenidate-induced cerebellar morphology and function damage in adult rats.

BACKGROUND AND AIM: Stated in previous studies, physicians are typically prescribing methylphenidate (MPH), commonly known as Ritalin, for children diagnosed with attention deficit hyperactivity disorder (ADHD). Nevertheless, researchers have not still understood mechanisms of this stimulant medication. Research has also found an association between apoptosis signaling pathway, neurological disorder, as well as treatment targets for neurological diseases. Therefore, the present study investigated effects of 3-week Ritalin oral (20 mg/kg) administration versus vehicle therapy on cerebellar morphology and function in adult male rats. MATERIALS AND METHODS: A total number of 30 adult male rats were randomly but equally divided into control and treatment groups. In fact, the treatment group was administered by Ritalin at doses of 20 mg/kg for 21 days and the control group only received saline. At the end of weeks 1, 2, and 3 following drug treatment, rotarod performance test was fulfilled. Once the study ended, tissues of the cerebellum were separated; then, inflammation parameters (i.e. tumor necrosis factor [TNF- α] and interleukin 1 beta [IL-1ß]), pro-apoptotic genes (that is, bcl-2-associated X [bax] and caspase-8 proteins), along with histological changes were analyzed. RESULTS: According to the findings, Ritalin with the high dose of 20 mg/kg could remarkably enhance the levels of bax and caspase-8 genes compared with those in the control group (p < 0.05). It should be noted that treatment with Ritalin could significantly increase TNF-α and IL-1ß levels in isolated cerebellar cells (p < 0.05). Moreover, 20 mg/kg of Ritalin decreased mean volumes of granular layer, white matter, as well as molecular layers. It also reduced the number of Purkinje cells compared with those in control rats. In addition, lower coordination movement was observed in the group receiving Ritalin. CONCLUSION: Data analysis showed that chronic treatment with increased dose of Ritalin could possibly lead to neuroinflammation and neurodegeneration in the cerebellum of adult rats.

Niacin improves maturation and cryo-tolerance of bovine in vitro matured oocytes: An experimental study.

BACKGROUND: Nicotinic acid (niacin) is a broad-spectrum lipid-modifying agent that has potent antioxidant properties and reduces the production of lipid peroxidation. OBJECTIVE: The purpose of the present study was to investigate the maturation, embryo development and cryo-tolerance merit, and levels of malondialdehyde (MDA), total oxidant status, and total antioxidant capacity following the supplementation of bovine oocytes maturation medium with different concentrations of niacin. MATERIALS AND METHODS: Immature cumulus-oocyte complexes were cultured in tissue culture medium-199 maturation media supplemented with 0, 100, 200, and 400 µM niacin under a standard in vitro culture condition. After 24 hr of culture, the nuclear maturation rate was assessed. Then, two groups of immature cumulus-oocyte complexes were cultured in TCM-199 either with or without 400 µM niacin and evaluated for embryo development. Also, matured cumulus-oocyte complexes in both groups were frozen using a standard vitrification procedure. After vitrification, oocytes were warmed in two steps and evaluated for embryo development. In addition, the level of total antioxidant capacity, total oxidant status, and MDA were measured. RESULTS: The results indicated that although the treatment with 400 µM niacin increased in vitro nuclear maturation (87.6±5.3), it did not improved the embryo development to the blastocyst stage. Higher cleavage and blastocyst rates were observed in vitrified oocytes that were cultured with supplemented 400 µM niacin compared to the control group (without niacin) (53.6±2.7 and 10.6±1.6 vs. 46.2±4.1 and 6.3±2.4, respectively). Also, the addition of 400 µM niacin to the maturation media could decrease MDA levels after maturation. CONCLUSION: Niacin could improve the quality of in vitro embryo production (IVP) embryos and tolerance of bovine oocytes to vitrification.

Investigation of the association of idiopathic male infertility with polymorphisms in the methionine synthase (MTR) gene.

OBJECTIVE: Spermatogenesis is a complex process that is regulated by a number of genes, some of which are involved in folate-dependent 1-carbon metabolism. Methionine synthase (encoded by MTR) is a key enzyme participating in this pathway. This study aimed to investigate the relationship of the MTR 2756A > G polymorphism with idiopathic male fertility in the Iranian population. METHODS: The participants of this study included 100 men with idiopathic infertility and 100 healthy men as the control group. Genotyping of MTR 2756A > G was performed using the polymerase chain reaction and restriction fragment length polymorphism technique. The obtained data were analyzed using SPSS ver. 20.0 with a level of confidence of p< 0.05. RESULTS: The frequencies of the A and G alleles at this locus were 77% and 23% in infertile patients and 84% and 16% in the control group, respectively. The frequencies of the GG, GA, and AA genotypes were 5%, 36%, and 59% in the infertile patients versus 3%, 27%, and 70% in the control group, respectively. No significant difference was observed in any genetic models. CONCLUSION: In general, the findings of this study suggest that the MTR 2756A > G single-nucleotide polymorphism is not a predisposing factor for idiopathic infertility in men.

Detection of Polymorphisms in MTHFD1 G1958A and Its Possible Association with Idiopathic Male Infertility.

PURPOSE: The role of male infertility is important in human infertility pathology. Spermatogenesis is a complex developmental process which is regulated by a number of genes. Methylenetetrahydrofolate dehydrogenase1 (MTHFD1) is involved in the synthesis of purine, pyrimidine, and methionine. The aim of this study was to identify the MTHFD1, G1958A polymorphism and its association with idiopathic male infertility in Iranian population. MATERIALS AND METHODS: This case-control study was conducted on 200 Iranian men, 100 cases with idiopathic infertility (experimental group) and 100 normal men (control group). The subjects were assessed for the MTHFD1 G1958A polymorphism, using the polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP). The chi-square test was used to determine the association between MTHFD1 G1958A polymorphism and male infertility, using SPSS software. P ? 0.05 was considered significant. RESULTS: Totally, the frequency of A allele and AA homozygous genotype was found 51% and 47.3% respectively, with 52.5% and 30% in the experimental group versus 42% and 21% in control group. There was a statistically significant correlation between the frequencies of A allele (95 % CI = 1.028- 2.265, OR = 1.526, p = 0.035) and AA homozygous (% CI = 0.995- 4.494, OR = 2.114, 95 p = 0.05) genotype with the MTHFD1 G1958A polymorphism (P ? 0.05). CONCLUSION: These results suggest that the polymorphism in MTHFD1 G1598A gene could be considered as an important genetic disorder associated with the etiology of male infertility.

Supplementation of L-carnitine during in vitro maturation of mouse oocytes affects expression of genes involved in oocyte and embryo competence: An experimental study.

BACKGROUND: Oocyte developmental competence is one of the key factors for determining the success rate of assisted reproductive technique. OBJECTIVE: The aim of the current study was to investigate the effect of L-carnitine (LC) supplementation during in vitro maturation (IVM), on preimplantation embryo development and expression of genes involved in embryo competence derived from oocytes selected with brilliant cresyl blue (BCB) test. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) were obtained from NMRI mice ovaries. COCs were stained with BCB and then BCB+ (colored cytoplasm) oocytes cultured in IVM medium supplemented with 0.3 or 0.6 mg/ml LC. COCs untreated with LC were used as control. Fertilization rate and blastocyst development rate were determined after in vitro fertilization. In addition, quantitative reverse transcriptase polymerase chain reaction was used to measure relative genes expression related with development (Ccnb1, Mos, Ces5, and Dppa2) and apoptosis (Bax and Bcl-xL) in oocytes and embryos. RESULTS: Oocytes treated with both LC concentrations showed higher blastocyst development rate compared with untreated oocytes (p<0.01). Moreover, fertilization rate was increased in oocytes treated with 0.6 mg/ml LC (p<0.01). Treatment of oocytes with both LC concentrations increased (p<0.01) the level of Ccnb1 mRNA in MII oocytes. The two-cell stage embryos and blastocysts derived from LC-treated oocytes (0.6 mg/ml) showed increased the expression levels of Dppa2 and Bcl-xl mRNA, respectively (p<0.01). CONCLUSION: The results of the present study show that adding of LC to the IVM medium of BCB+ oocytes can ameliorate reproductive success following in vitro fertilization.

DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs).

OBJECTIVE: Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT). We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs) would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and histone deacetyltransferses (HDAC1, HDAC2, HDAC3) in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4) and acetylation of H3K9 (H3K9ac) in BADSCs cultures and different passages in vitro. MATERIALS AND METHODS: In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR), and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3), 5 (P5) and 7 (P7). RESULTS: The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. CONCLUSION: Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages.