Cell and Tissue Journal

Abstract Introduction: Concerns about fossil energy costs, environmental deterioration, and energy security has created strong motivation for the research and development of routes to provide sustainable and renewable fuels. In recent years, the use of biomass to produce highly valued chemicals has attracted widespread attention. Lignocellulosic biomass, as a promising renewable resource for biofuel production, has distinct advantages in terms of economic and environmental benefits. The conversion of renewable raw materials to hydrocarbon fuels is an attractive alternative to fossil fuels from economic and environmental perspectives. The production process of lignocellulosic biomass mainly consists of biomass accumulation, biomass decomposition, simple sugars, and conversion of sugars to biofuel. One of the crucial steps for the economic success of lignocellulosic biofuels depends on the inhibition of competitive metabolism in microorganisms to achieve high productivity. To date, there has been a growing focus on the use of S. cerevisiae and E. coli as cell lines. These two cellular factories have well known advantages. They are genetically transmissible and several tools are available for genetic manipulation. In order to produce xylonate, the engineered xylose is first converted by a dehydrogenase into the intermediate xylonolactone, which is then slowly converted to xylonate in a nonenzymatic reaction.
Aim: The organic compound D-1,2,4-Butanetriol (BT) is a valuable chemical with wide-ranging applications in various fields such as pharmaceuticals, paper, polymer materials, and military applications. However, the chemical synthesis routes for BT have many drawbacks. By genetically modifying microorganisms, the metabolic pathway for producing many substances, including BT, can be engineered. When D-xylose is supplied to the bacterium, it is first converted into an intermediate compound called xylonolactone. This compound slowly converts into xylonate through a non-enzymatic reaction. To produce xylonate, the engineered bacteria receive xylose, which is initially converted by a dehydrogenase reaction catalysed by the xylose dehydrogenase enzyme into an intermediate compound, xylonolactone. Xylonolactone is slowly converted to xylonate in a nonenzymatic reaction. Xylonate is a five-carbon organic acid. Over the past few years, xylonate has increasingly been considered as an important chemical due to its potential as an important chemical component. Xylonate has many applications in the food, chemical, and pharmaceutical industries. Specifically, xylonate can act as a precursor for the synthesis of D-1,2,4-Butanetriol and as a concrete water reducing agent. E. coli was chosen as the target strain for genetic and metabolic engineering due to its fast growth in inexpensive culture media, the presence of two enzymes for BT synthesis, and product formation in less than 24 hours of fermentation. This study aimed to clone and express xylose dehydrogenase from Caulobacter vibrioides in E.coli.
Materials and Methods: At first, to access the bacterial gene sequence, the genome of the target bacterium was extracted. Then, to create a strain expressing the enzymes xylose dehydrogenase and xylonolactonase, the genes for these proteins were amplified from Caulobacter vibrioides CB1 and transferred into E. coli. For this purpose, the target genes were amplified using specifically designed primers via the Polymerase Chain Reaction (PCR) method and initially cloned into a pTZ57cloning vector and then subcloned into pET 26b expression vector. At the final step, the expression of the enzyme was assessed by SDS-PAGE, and the other confirmation was the reduction of NAD+ to NADH, which was used as an activity indicator of the enzyme, as investigated by a change in NADH absorbance at 340 nm.
Results: Confirmatory tests were performed to ensure the presence of the gene in the vectors (using restriction enzymes and colony PCR for gene amplification). The expression and activity of the enzyme were analyzed. The recombinant protein's presence was confirmed by SDS-PAGE for the xylose dehydrogenase gene, with a molecular weight of 52.2 kDa. The estimated expression level of the recombinant protein was approximately 25%.
Conclusion: The objective of this research was solely to establish the metabolic pathway for xylonate production in E. coli by surface expression of enzymes in this pathway (xylose dehydrogenase). The results obtained in this study confirm that half of the pathway is active at the cell surface, but further experiments are required to determine the precise production levels and complete the pathway.

Abstract Introduction:Cartilages are inherently avascular and have limited repair capacity after injury. If not treated properly, these injuries can lead to progressive joint destruction and diseases such as osteoarthritis. Current treatment methods, including surgery and cartilage transplantation, face limitations such as inadequate access to healthy tissue, graft rejection, and poor-quality cartilage formation. Therefore, finding new strategies to stimulate efficient cartilage regeneration has become a critical need in regenerative medicine.In recent years, exosomes derived from mesenchymal stem cells (MSCs) have emerged as a promising tool in tissue regeneration. These extracellular vesicles contain growth factors, miRNAs, and proteins involved in tissue repair that can transmit therapeutic signals to target cells. Glucosamine, a naturally occurring amino monosaccharide, is an important precursor in the synthesis of glycosaminoglycans (GAGs) and proteoglycans, which are major components of the extracellular matrix of cartilage. This compound is widely used as a dietary supplement or drug in the treatment of degenerative joint diseases such as osteoarthritis. As a common supplement in the treatment of joint diseases, glucosamine plays an important role in stimulating the synthesis of cartilage extracellular matrix, promoting the differentiation of stem cells into chondrocytes, and inhibiting cartilage destruction.


Aims: The aim of this study was to investigate the combined effect of mouse bone marrow stem cell-derived exosomes and glucosamine on the expression of cartilage-specific genes including Sox9, Acan, Col2a1, and Col10a1.
Materials and methods: Bone marrow mesenchymal stem cells were prepared from NMRI mice. The mice were euthanized by cervical dislocation, the femoral heads were removed, and the bone marrow contents were transferred into a cell culture flask using a syringe containing culture medium. The bone marrow cells were cultured and were ready for use after 3 to 5 passages. The cell supernatant was separated, and exosomes were extracted from the cell supernatant by successive rounds of centrifugation followed by ultracentrifugation. Exosomes were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The effects of exosomes and glucosamine on the expression of Sox9, Acan, Col2a1 and Col10a1 genes in mesenchymal stem cells were investigated in the presence of chondrogenic medium.
Results: The identification results from multiple characterization techniques confirmed the presence of exosomes with a size of approximately 100 nm. Real-time PCR results showed that the expression of Sox9, Acan and Col2a1 genes in stem cells treated with a combination of exosomes and glucosamine increased significantly compared to the other groups after 14 days of treatment, while the expression of the Col10a1 gene decreased significantly compared to the other groups.
Conclusion: This study demonstrated that the combination of exosomes derived from bone marrow mesenchymal stem cells and glucosamine can synergistically increase the expression of key genes in chondrogenic differentiation (Sox9, Acan and Col2a1) while reducing the expression of the Col10a1 gene (related to chondrocyte hypertrophy). These findings suggest the therapeutic potential of this combination in stimulating cartilage regeneration and preventing further degradation. Given these promising results, this approach should be further investigated in clinical studies as a novel therapeutic strategy for cartilage damage.

Abstract Introduction: Okra (Abelmoschus esculentus) is an annual culinary plant of Malvaceae family with high nutritional and medicinal values. This plant is widely cultivated in different regions of Iran as well as the world.
Aims This study investigated the fatty acid composition and antioxidant activity of seed oils extracted from seven okra cultivars, including: White, Sultani, Red, Velvet, Green, Texas, and Fawn. Material and methods: The mature and intact seeds of each cultivar were powdered and their oils were extracted using the modified Folch et al. method. The fatty acid methyl esters were obtained from seed oils using transesterification method. Then, the oils were injected to GC and GC/MS apparatus to detect their fatty acids profile. The DPPH method was applied to detect antioxidant activity of the extracted okra oils. Data was analyzed using SPSS and MVSP software. Results: The seed oils in all cultivars were comprised both saturated and unsaturated fatty acids, while, their total percentages varying among these cultivars. The highest total saturated fatty acid content (42.53%) was observed in the white cultivar, while the lowest (36.41%) was detected in the Sultani cultivar. Palmitic acid was the predominant saturated fatty acid in all the cultivars, with its highest (35.74%) and lowest (30.7%) percentages were found in the white and Texas cultivars, respectively. Stearic acid was the second most abundant saturated fatty acid in all examined oils, ranging from 3.69% (Sultani cultivar) to 4.94% (white cultivar). Some trace amount saturated fatty acids such as heptadecenoic, arachidic, behenic, and lignoceric acids were present in all the cultivars, with lauric acid had a restricted distribution and was only detected in a few cultivars, including: velvet, Texas, and white cultivars. Unsaturated fatty acids constituted 57.33% to 63.33% of the oils composition, with the lowest and highest percentages in white and Sultani cultivars, respectively. Linoleic acid was the primary unsaturated fatty acid in all the cultivars except white cultivar, where oleic acid was detected as predominated unsaturated fatty acid. The highest linoleic acid percentage (11.40%) was detected in red cultivar and the lowest (8.25%) in white cultivar. The amount of oleic acid ranged from 18.20% (red cultivar) to 49.30% (white cultivar). Some trace amount unsaturated fatty acids such as myristoleic, palmitoleic, trans-heptadecenoic, linolenic, gondoic, and erucic acids were present in all the cultivars. Additionally, all the cultivar had eicosadienoic acid in a trace amount, except for white cultivar. Phytochemical analysis using UPGMA dendrogram and PCA analysis divided these cultivars into two main clusters, confirming significant phytochemical diversity. It seems that change in the predominant unsaturated fatty acid, oleic acid in the white cultivar instead of linoleic acid. Additionally, the presence of palmitic acid as the dominant fatty acid in the velvet and white cultivars, were two main reasons for placement of white, green and velvet cultivars far from other cultivars. Antioxidant activity assessed by the DPPH method revealed IC50 values ranging from 689.23 µg/mL in green cultivar to 2551.12 µg/mL in Texas cultivar. Discussion: the seed oil profile of white cultivar is more different from the others. Moreover, the week or trace antioxidant activity was detected for the oils of diverse okra cultivars. These findings highlight the nutritional and industrial potential of okra seed oils. Additionally, the oil profile can strongly influence the antioxidant activity of the oils.

Abstract Introduction: Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide. 5-Fluorouracil is one of the commonly used chemotherapeutic drugs. The plant Artemisia absinthium has attracted attention as a potential herbal anticancer agent. This study investigated the effect of the methanolic extract of this plant on the expression of miR-34a and the apoptotic genes BAX, Caspase-3, and Caspase-9 in AsPC-1 pancreatic cancer cells.
Aims: This study aimed to evaluate the cytotoxic and pro-apoptotic effects of Artemisia absinthium methanol extract on pancreatic cancer cells. The research specifically investigated the molecular mechanism by analyzing expression changes in the tumor suppressor miR-34a and key apoptotic genes BAX, Caspase-3, and Caspase-9 to elucidate the extract's anti-cancer mode of action.
Materials and methods: To evaluate the cytotoxic effects of the plant extract on cancer cells, an MTT assay was performed to determine the viability and survival rate of the cells following treatment with various concentrations of the extract, the chemotherapeutic drug fluorouracil (5-FU), and the combined treatment of the extract and the drug. This assay measures cellular metabolic activity and allows quantification of live and dead cells after exposure to different treatments. Based on the obtained results, the IC₅₀ value for each treatment was calculated, representing the concentration at which 50% of the cells were inhibited or killed.
After determining the IC₅₀ value, cells were treated with concentrations equal to, lower, and higher than the IC₅₀ to further investigate the cytotoxic effects and the mode of cell death induced by the treatments. To distinguish between apoptotic and necrotic cell death, the Annexin V-FITC/PI assay was employed. This assay detects phosphatidylserine externalization on the cell membrane and enables differentiation between live, early apoptotic, late apoptotic, and necrotic cells.

In addition to the morphological and physiological assessments, molecular analyses were conducted to examine the expression levels of key apoptosis-related genes, including Caspase-3, Caspase-9, and BAX, as well as the regulatory microRNA miR-34a. Gene expression analysis was performed using Real-time PCR (qPCR).

Results: The results of the MTT assay demonstrated that the proliferation of AsPC-1 pancreatic cancer cells was inhibited by treatment with the extract of Artemisia absinthium and the chemotherapeutic drug fluorouracil (5-FU) in a concentration-dependent manner. As the concentration of each treatment increased, cell viability significantly decreased, indicating a marked cytotoxic effect of both the plant extract and the drug. Moreover, a possible synergistic effect between the extract and fluorouracil in suppressing. To determine the mode of cell death induced by these treatments, the Annexin V-FITC/PI assay was performed. The results revealed that a considerable proportion of treated cells underwent programmed cell death (apoptosis), while the percentage of necrotic cells remained relatively low. These findings suggest that the observed reduction in cell viability is mainly mediated through the activation of apoptotic pathways rather than necrosis. Furthermore, Real-time PCR analysis showed a significant upregulation in the expression of the regulatory microRNA miR-34a and the apoptosis-related genes Caspase-3, Caspase-9, and BAX in the treated groups compared to the control group.
Discussion: The obtained results suggest that Artemisia absinthium extract exerts its cytotoxic effect primarily through the induction of apoptosis rather than necrosis in AsPC-1 pancreatic cancer cells. The observed upregulation of miR-34a, Caspase-3, Caspase-9, and BAX implies activation of intrinsic apoptotic pathways. These findings are consistent with previous studies reporting pro-apoptotic properties of A. absinthium and other Artemisia species. Therefore, the extract may enhance the therapeutic response of pancreatic cancer cells when combined with conventional chemotherapeutic agents such as fluorouracil.
Conclusion: In conclusion, Artemisia absinthium extract demonstrated strong antiproliferative and apoptosis-inducing effects on AsPC-1 cancer cells in a dose-dependent manner. Its combination with fluorouracil produced a synergistic cytotoxic impact, significantly enhancing cell death through apoptotic signaling. The molecular findings support the potential of this extract as a complementary therapeutic agent. Further studies are recommended to explore its mechanisms and evaluate its efficacy in in vivo models of pancreatic cancer.

Abstract Aim: The aim of this study was to investigate different concentrations of iron oxide nanoparticles in and benzylaminopurine (BAP) bell pepper anther culture on the traits of callus formation, embryogenesis, regeneration and rooting.
Material and methods: The experiment was conducted as a factorial experiment in a completely randomized design under in vitro culture conditions. Flower buds of appropriate size (equal sepal to petal ratio or slightly longer petal) were collected from the greenhouse and acetocarmine solution was used to determine the growth and development stage of microspores. The results showed that the most suitable stage for embryogenesis induction was the late mononuclear stage and the early binuclear stage. In order to sterilize the flower buds, 70% ethanol for 30 seconds and 5% sodium hypochlorite for 20 minutes were used and after each stage they were washed three times with sterile distilled water. Then, the anthers were separated from the flower bud and placed in C medium containing 2 mg/L naphthalene acetic acid (NAA), different concentrations of BAP (0, 0.1, 0.5, and 1 mg/L) and different concentrations of iron oxide nanoparticles (0, 1, 10, and 20 mg/L). After that, the explant cultured in C medium were kept at 35°C in the dark for 8 days in order to apply heat treatment. Then, they were transferred to 25°C in the light for 4 days. After this period, in order to induce embryogenesis, the explants were transferred from C medium to R medium and were subcultured every three weeks until embryos emerged. For further growth and root development, the embryos were transferred to V medium. Results: The results of the analysis of variance of the data showed that using different concentrations of iron oxide nanoparticles had a significant effect on the percentage of embryogenesis, regeneration and rooting of the plant, but did not have a significant effect on the percentage of callus formation. The results of comparing the average data showed that among the different concentrations of iron oxide nanoparticles, the 1 mg/L treatment had the highest percentage of embryogenesis (11.11). Also, the results of comparing the average effect of the nanoparticles on the percentage of regeneration showed that the 1 mg/L treatment had the highest percentage of regeneration (16.66). The results of the interaction effects showed that among the different concentrations of iron oxide nanoparticles and BAP, the highest percentage of embryogenesis was observed in the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP. Also, the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP had the highest percentage of regeneration (33.33%). After sufficient growth and root formation, the obtained plants were removed from the glass culture containers and transferred to pots containing sterilized culture medium and watered for adaptation. The tops of the pots were covered with plastic cups, and after three days, the cups were pierced and the plastic was gradually removed from the plant for further adaptation. Ploidy levels were determined by chromosome counting by staining the root tip cells. The results showed that out of the 23 obtained plants, 21 were diploid and had 2n=2x=24 chromosomes and 2 were haploid and had n=x=12 chromosomes. Conclusion: In bell pepper anther culture, different concentrations of iron oxide nanoparticles along with plant growth regulators at different concentrations showed a great effect on embryogenesis, regeneration, and rooting.

Abstract During the past 30 years, researchers have made a remarkable progress in identifying the biological (bacteria, viruses), biochemical (chemical compounds) and biophysical (ionizing radiation) cause of human cancer. The term ˝Cancer˝ refers to 277 forms of cancer diseases.
Scientists have determined the process of cancer formation from a consequence of accumulating multiple mutations in human genome. These genetic disruptions would eventually change the normal process of cellular proliferations and differentiation.
The genetic alterations are frequently indicative of poor prognosis for most human cancers.
Both nonhereditary and hereditary cancers are caused by genetic disorders that change the cellular growth control system.
 Genes associated with human cancer formation include four classes of genes: 1. Oncogenes (which increasing their activities end to uncontrollable growth of cells), 2. Tumor suppressor genes, 3. DNA repairing genes, 4. Apoptotic genes.
Over activated oncogenes which cause cellular proliferation. In contrast, inactivated tumor suppressor genes lose their inhibitory effect which is crucial to prevent inappropriate growth. DNA repairing proteins fix the damage and apoptotic proteins cause the pre-cancer cell to commit suicide.
We have over millions of genes in each somatic cell of our body. After sequencing all human genome in 2003, we noticed that Only 23,500 genes are active which encode over 400,000 proteins needed for physiological functions.
99.9% of genome is identical in all humans worldwide. Only 0.1% of the whole genome differ which cause the genetic variations.
Up to 93% of all human cancers are non-hereditary and the remaining 7% are hereditary. A wealth of information has been indicated by the potential use of bioinformatics and molecular techniques for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies. In recent years, molecular genetics have greatly increased our understanding of the basic mechanisms in cancer development. The essential outcome of these molecular studies is that the cancer can be considered as the genetic disease.
 

Abstract Aim: Pentoxifylline (PTX) is a methylxanthine derivative medicine used to improve motility of human spermatozoa in-vitro. It is commonly used in treatment of male-factor infertility, including asthenozoospermia. This study aimed to evaluate the effect of PTX on human sperm parameters and DNA integrity from asthenozoospermic problem.
Material and methods: A total of 38 infertile men with asthenozoospermia were allocated in this experimental study.  Specimens were randomly divided into experimental group treated with 3.6 mM PTX, and control group. All samples were incubated at 37˚ C for 45 min. Semen parameters and sperm DNA fragmentation were measured using sperm chromatin dispersion (SCD) test.
Results: PTX improved sperm motility, significantly, compared to the control (85.76±5.96 Vs 79.44±9.37, respectively, p < .01). There was also a significant decrease in sperm viability in the PTX- treated group in comparison to controls (87.7±8.3 Vs 83.5±9, respectively, p < .01). In addition, sperm DNA fragmentation was higher in PTX-treated group compared to control (23.36±10.25 and 18.5±8.74, respectively, p < .0001).
Conclusion: PTX might have some negative impact(s) on sperm DNA quality, although it has improved the sperm motility. Further studies are needed to elucidate the safety of PTX treatments in ART clinics.

Abstract Aim: The aim of this study was to investigate the effect of vitamin E on sperm parameters in adult rat treated with para-nonylphenol.
Material and methods: Adult male rats were divided into four groups: Control, para-nonylphenol, vitamin E and para-nonylphenl+vitamin E. Oral treatments were performed till 56 days. At the end of treatments, body and left testis weight were recorded and left caudal epididymis was cut in a medium. Released spermatozoa were used to analyze sperm parameters such as sperm number, viability, morphology and motility. Sperm chromatin quality was assessed by nuclear staining using acridine orange and aniline blue.
Results: Body and testis weight as well as normal sperm morphology showed no significant change in four groups. A significant decrease in the number, viability and motility of the sperm was found in rats treated by para-nonylphenol compared to the control. This decrease was significantly compensated by vitamin E in para-nonylphenol+vitamin E group compared to para-nonylphenol group. The application of vitamin E alone could significantly increase sperm viability and motility as comared with the control. Para-nonylphenol had no effect on sperm DNA integrity and histon-protamine replacement compared to the control.
Conclusion: Vitamin E, as a potent antioxidant, could protect the adverse effect of para-nonylphenol on certain sperm parameters in adult rats.
 

Abstract Plants produce a big group of secondary metabolites which are used as medicinal compounds. According to recent estimates, global market value of herbal medicines, including medicinal plants and their products, significantly has been increasing. Considering to the fact that most of the world market for medicinal plants, production and supply of secondary metabolites derived from these plants are concerned and the plant secondary metabolites are of high economic value. Chemical synthesis of these metabolites is an expensive process. So production of metabolites by different biotechnological methods such as cell culture is a useful alternative. Molecular recognition and elicitor-plant receptors interaction is a complex process requiring for signal transduction. Biotic elicitors induce secondary metabolites and hypersensitive responses by activation of defense mechanisms. Manipulation of cell culture media by elicitors is an important strategy for inducing secondary metabolism and production of valuable metabolites. Molecular recognition and elicitor-plant receptors interaction is a complex process requiring for signal transduction. Following perception of elicitor signals, rapid defense responses can be organized as follows: increase of ionic currents across the plasma membrane, reactive oxygen species (ROS) production, activation of defense gene expression, structural changes in the cell wall and phytoalexin production. In this study, different aspects of increasing the production of secondary metabolites in cell culture of plants by biotic elicitors is investigated.  

Abstract Cryopreservation is the most effective method for long-term maintenance of sperm. There are several procedures for freeze-thawing of semen and each may impose damage on sperm function, viability and finally decreases semen quality and fertility potential. In addition to decreased percentage of sperm viability and motility after freeze-thawing, percentage of DNA damage is also increases due to high level of oxidative stress. To minimize these damages, we need to increase our insights regarding different cryopreservation procedures, cryoprotectant and antioxidant supplements, which can protect sperm membrane during cryopreservation. Therefore, by using these experiments, we can improve the efficiency of these procedures. In this review, we discuss about principles of cryopreservation, types of freeze-thawing methods, advantages and disadvantages of each of these methods, effects of freezing on sperm parameters and clinical outcomes, and finally role of antioxidants in preservation of sperm integrity during freeze-thawing. For this review, all relevant information was collected via databases such as PubMed and Google Scholar during the period of 1966-2017.
 

Abstract Aim: This study was done to evaluate the therapeutic effects of Curcuma longa L. rhizome powder and bovine ghee mixture on experimental stomach ulcers in rat.
Material and methods: Stomach ulcer induction was done in 48 food deprived male rats (250-300 gr) using orally Indomethacin (50 mg/kg suspended in 1% carboxymethyl cellulose. Animals divided randomly into five groups. The normal and untreated (Indomethacin) group just received normal saline. The treated groups received different doses of C. longa and bovine ghee (500 and 750 mg C. longa in 10 ml ghee/per kg) for three successive days. On the third day, the rats were killed and their stomachs were removed for histological studies. Stomach ulcers number and length were measured and also ulcer index was calculated.
Results: Results showed that the mixture of C. longa and bovine ghee is significantly caused decreasing gastric ulcer index, inflammatory cells, blood capillaries densities (p < 0.001) and increasing mucosal layer thickness (p < 0.001) and secreted mucus (p < 0.05) in treated groups in comparison with untreated group.
Conclusion: According to the results, the mixture of C. longa and bovine ghee is significantly accelerated healing of experimental stomach ulcers.

Abstract Aim: The main goal was to evaluate the impact of different incubation time intervals on human sperm DNA status using terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) test.
Material and methods: This prospective study involved 21 normozoospermic specimens. After direct swim-up, sperm cells were incubated at 37^°C and DNA damage was evaluated at different time intervals (0, 1, 2 and 3 h).  After slide fixation with methanol 100% for 4 minutes and rinse in phosphate-buffered solution (PBS), TUNEL was added to each slide and incubated for 60 minute in dark. Eosin-Nigrosin and Papanicolaou staining protocols were applied in order to assess sperm viability and morphology, respectively.
Results: Sperm viability and normal morphology was improved after sperm processing (100%, 72.3% respectively, p < 0.0001). The rate of DNA damage was significantly higher after 2h compared to 0h (9.19±0.8% Vs 4.9±0.9%, respectively, p=0.008). Also there was significant difference in abnormal sperm DNA between 3h and 1h (10.95±0.7% Vs 7.1±1%, respectively, p=0.020).
Conclusion: Incubation of prepared normozoospermic samples at 37^°C more than 2 h may be associated with sperm DNA fragmentation. Therefore, it seems that incubation of human spermatozoa at 37^°C should be limited up to 2h prior to use in ART clinics.