Therapeutic Effects of Mesenchymal Stem Cell Conditioned Medium in Rat Varicocele Model.

PURPOSE: This study aimed to examine the therapeutic effects of injection of conditioned medium of adipose-derived mesenchymal stem cells (ADMSC-CM) in a surgically created varicocele model in comparison with varicocelectomy. MATERIALS AND METHODS: Twenty-eight male Wistar Albino rats were randomly divided into four groups: sham group, varicocele group, varicocelectomy group, and ADMSC-CM injection group. Sperm parameters were analyzed in samples taken from the epididymis after treatment. Malondialdehyde and superoxide dismutase (SOD) levels in blood samples were examined by biochemical analysis. The testicular tissues were stained with hematoxylin-eosin for histological examination (Johnsen's Score). Additionally, Western Blot analyzes were performed to detect Claudin-11 levels, the functional protein of the blood-testis barrier, in testicular tissues. RESULTS: Varicocelectomy and ADMSC-CM treatments significantly improved mean sperm parameters (concentration, progressive motility, motility, normal sperm morphology) (p≤0.05 for all). Both treatment groups had increased SOD levels along with a decrease in malondialdehyde levels (p≤0.05 for all). No significant difference was observed between the ADMSC-CM group and the varicocelectomy group in preserving normal testicular histology according to Johnsen's Score (p=0.114). Levels of Claudin-11 were significantly higher in the varicocelectomy and ADMSC-CM groups compared to the varicocele group (p≤0.05 for all). CONCLUSIONS: The therapeutic effects of ADMSC-CM in varicocele model may involve secretion of anti-inflammatory and regenerative factors from ADMSC. ADMSC-CM injection appears to be a promising new strategy in the treatment of varicocele.

Splicing variants of versican in CD133+/CD44+ prostate cancer stem cells.

A cancer mass is composed of a heterogeneous group of cells, a small part of which constitutes the cancer stem cells since they are less differentiated and have a high capacity to develop cancer. Versican is an extracellular matrix protein located in many human tissues. The mRNA of versican has been shown to have "splicing patterns" as detected by RT-PCR, northern blot analysis, and cDNA sequencing. Based on this knowledge this study aims to reveal the splice variants of versican molecules, which are thought to be involved in the pathogenesis of the DU-145 human prostatic carcinoma cell line and prostatic cancer stem cells isolated from this cell line. In this study, RWPE-1 normal prostatic and DU-145 human prostate cancer cell lines have been used. Prostatic cancer stem cells and the remaining group of non-prostatic-cancer stem cells (bulk population) were isolated according to their CD133+/CD44+. RNA was isolated in all groups, and sequence analysis was accomplished for splicing variants by Illumina NextSeq 500 sequencing system. The results were analyzed by bioinformatic evaluation. As five isoforms of the versican gene in the differential transcript expression are analyzed, it was observed that a significant change was only found in the isoforms Versican 0 and Versican 1. In this study, we explored the function of this molecule which we think to be effective in cancer progression, and suggested that more valuable results can be obtained after the accomplishment of in vivo experiments.

Unravelling the impact of COVID-19 on pregnancy: In aspect of placental histopathology and umbilical cord macrophage immunoactivity with neonatal outcomes.

COVID-19 has turned into a global pandemic since it was first detected in 2019, causing serious public health problems. Our objective was to investigate the impact of COVID-19 on pregnant women and newborns, who belong to the vulnerable segments of society. Our study involved the histopathological examination of placentas and umbilical cords from two groups of pregnant women. Group I consisted of pregnant women who had never tested positive for COVID-19 during their pregnancy (n: 20). Group II consisted of pregnant women who had contracted COVID-19, exhibited moderate and mild symptoms, and recovered from the disease before giving birth (n: 23). Furthermore, we employed immunofluorescence techniques to detect macrophage activity in the umbilical cord. Prenatal assessments were based on maternal complete blood counts and coagulation assays (n:40 in both groups). Newborn conditions were evaluated using birth weight, height, head circumference, and APGAR (n:40 in both groups). Our analyses reveal that COVID-19 causes placental and umbilical cord inflammation and maternal and foetal vascular malperfussion. Our immunofluorescence investigations demonstrate a notable increase in macrophage numbers and the macrophage-to-total cell ratio within the COVID-19 group. In this aspect, this study provides the initial report incorporating macrophage activity results from Warton's jelly in pregnants who have recovered from COVID-19. We have also ascertained that COVID-19 abbreviates gestation periods and concurrently diminishes maternal haemoglobin concentrations. Consequently, COVID-19 with mild and moderate symptoms during pregnancy, causes significant changes to the placenta and umbilical cord, but propitiously does not cause a significant difference in the neonatal outcomes.

Efficacy of one-hour negative pressure wound therapy and magnetic field energy in wound healing.

OBJECTIVE: Wound healing is an important aspect of health but needs further research to identify the effects and interactions of different treatment approaches on healing. The aims of this study were to investigate the effectiveness of one-hour negative pressure wound therapy (NPWT) and compare histological differences between one-hour NPWT and magnetic field energy (MFE) in rats on early-stage wound healing, wound size and angiogenesis. METHOD: Standardised wounds were created on Wistar rats that were allocated and divided into NPWT, MFE and control groups. Both treatments were applied for 1 hour/day for 10 days. Wound size, histological changes and wound area blood flow were assessed. RESULTS: The wound size of all groups was similar on days 0, 2 and 10. The MFE group's wound size was smaller than the NPWT group on days 4, 6 and 8 (p<0.05). Development of the granulation tissue in both the one-hour NPWT and MFE groups was greater than in the control group. Additionally, the inflammatory phase was shorter, and wounds entered the proliferative stage faster in the MFE group than both of the other groups. CONCLUSION: Treatment with MFE may be more effective in terms of early stage wound closure and angiogenesis. On the other hand, the NPWT group's wound area blood flow was significantly greater than the other two groups. MFE is superior to one-hour NPWT in terms of wound area and angiogenesis. Furthermore, it is worthwhile to note that one-hour NPWT increases bloodflow in the wound area, which stimulates healing.

Tissue Factor and Vascular Endothelial Growth Factor in Detecting Thromboembolic Complications in Diabetic Atherosclerotic Patients.

Background: Atherosclerosis, which is one of the leading causes of death all over the world, can create major or minor thromboembolic complications with the exponentially increasing diabetic status. Despite all the studies, the mechanism by which endothelial damage in atherosclerosis is triggered in diabetic setting is still not fully understood. Methods: In this study, tissue factor (TF), which is thought to act together in the formation of vasular endothelial growth factor (VEGF-A) and coagulopathy in diabetic atherosclerotic patients, may be an important indicator in this regard, a total of 100 cases who were undergone off-pump coronary artery bypass (OPCAB) which were at same risk group examined by dividing into diabetic status. Early postoperative process and biochemical parameters analyzed in terms of TF and VEGF-A levels measured before and after the operation. Results: TF and VEGF-A expression of the T1DM group were statistically high compared to non-diabetics. Significantly longer hospital stays with changes in TF and VEGF-A were found in patients in the diabetic group compared to pre- and postoperatively, respectively; TF (95% CI: 0.879-0.992; p = 0.025), VEGF-A (95% CI: 0.964-0.991; p = 0.001) and hospital stay (95% CI: 1.96-7.49; p = 0.0001). Preoperatively measured carotid intima-media thickness (CT) was higher in diabetics and was significantly associated with atrial fibrillation (AF), (r = 0.873). Surgical team and protocols were same and OPCAB procedures were routinely applied to all patients in our clinic. No minor or major events were observed in any of the cases. Conclusion: TF and VEGF-A values in patients with diabetic atherosclerosis may be important in the early detection of thromboembolic complications.

Are ICSI results affected by months and seasons? A retrospective cohort study of fresh and frozen cycles.

In present study, we aimed to investigate the effect of seasonal variation on ICSI results both in fresh and frozen embryo transfer cycles. Between January 2007 and December 2019, a total of 4039 ICSI cycles (3227 fresh and 812 frozen ET) which resulted in embryo transfer were included in the study. We found no significant difference in the pregnancy outcome among different seasons and months. The best results were obtained for spring (41.0%) and the least for winter (37.1%) in fresh cycles and the best for spring (42.5%) and the least for winter (29.7%) in FET cycles. In monthly distribution, the best results were obtained for September (57.0%) and the least for November (24.1%) in fresh cycles and the best for October (49.3%) and the least for December (20.2%) in FET cycles. Our study did not show any significant influence of the months or seasons on clinical pregnancy rates in either fresh and frozen embryo transfers. However, the difference among months in frozen cycles was notable although it did not reach statistical significance. It can be suggested that the strict control of reproductive hormones especially in fresh cycles as well as the standardized laboratory temperature minimize the influence of seasonal effect on ICSI outcome.

Sperm selection with Annexin-V coated polystrene bead technique (APB-Tech): A novel and reliable method for the microscopic selection of viable and non-apoptotic sperm to be used for intracytoplasmic sperm injection.

Infertility is a growing health problem that affects 20% of couples who want to conceive. 'Intracytoplasmic sperm injection' (ICSI) is a commonly used assisted reproduction treatment technique that offers the couples to overcome a wide variety of infertility reasons, including female, male and unexplained infertility. A crucial step in ICSI is choosing the correct sperm with the highest potential to form a healthy embryo and thus a healthy offspring. The sperm selection strategies aim to obtain a sperm population with better motility and morphology, which are insufficient to predict a sperm's reproductive potential. The elimination of apoptotic sperms, which is shown to be higher in infertile males and which is shown to affect embryo development and reproductive cohort significantly should also be included in the selection strategies. Among all sperm selection methods, there are only a few which can eliminate apoptotic sperm, but because they need extra types of equipments, a long training period, and high costs, they couldn't find place in the most commonly used techniques in an IVF lab. Selecting the non-apoptotic sperm cells will help us choose a sperm that is more likely to be chosen by the natural selection mechanisms and thus will help to mimic the natural conception more. The study aimed to develop a novel, easy and a harmless individual sperm selection technique to enable choosing non-apoptotic viable sperm cells via light microscopy without any need for extra equipment, education and cost to be used for ''ICSI'. The technique is based on the binding ability of Annexin-V covered polystrene beads to the externalized phosphatidylserine at the outer leaflet of an apoptotic sperm's plasma membrane. After Annexin-V covered polystyrene beads were prepared, beads obtained were attached to BALB-c mice sperm, and the technique is optimized to obtain the most efficient attachment conditions. The results are then compared with the results of four well-known reliable apoptosis detection techniques to test the validity and sperm survival test to test the toxicity of the technique. The method is proven to be '''reliable' by comparing it with the results of well-known techniques, including TUNEL and SCSA (sperm chromatin structure assay), and '''safe' by showing its non-toxicity via sperm survival test (SST). In addition, the method enables the selection of sperm cells more closer to naturally-chosen ones from a pool of sperm that should not be allowed to be randomly chosen during microinjection. To date, it was impossible to distinguish a non-apoptotic sperm without harming it or without needing additional equipment other than a routine IVF lab and extra training other than routine andrology work. The technique is named as 'Annexin-V coated polystyrene bead technique (APB-Tech)'. Based on our results, further studies on APB-Tech should be focused on the possible improvement of ICSI outcomes and, thus, success rates.

Seminal trace elements and their relationship with sperm parameters.

Male reproductive problems may derive from many reasons including the environmental toxicants which may either intaken by occupational exposure, nutrition or bad air quality. The increased exposure to these substances due to rapid industrial development and technology has raised the questions: Is there a relationship between sperm parameters and these substances, and if so, in what extent? Results of studies on the subject reported conflicting results, many of which were not investigated in the seminal plasma. The aim of the current study was to evaluate the relationship between 23 metals and trace elements in human seminal plasma and semen parameters many of which were not investigated before. Levels of 23 metals in human seminal plasma were assessed by inductively coupled plasma mass spectrometry (ICP-MS). We examined the differences between subjects with normal ejaculate (normozoospermia) and pathologic ejaculate (with at least one abnormal semen parameter) according to the WHO criteria. The only significant difference was detected for Se while the other element's difference was not statistically significant. Se was statistically significantly increased in normal semen group suggesting the positive effect of this element on semen parameters.

The Role of TNF-α and Its Target HSP-70 in Triggering Apoptosis in Normozoospermic and Non-Normozoospermic Samples.

Objective: Semen analysis is performed as one of the screening tests for infertility, including motility, morphology, and concentration observation. We aimed to investigate the expression rates of tumor necrosis factor-α (TNF-α) and heat shock protein (HSP)-70 as two opposite affectors of apoptosis in men with normal semen parameters and abnormal parameters to find the possible effect of this pathway on sperm parameters. We also aimed to investigate the apoptotic markers (DNA fragmentation and Caspase-3 expression) to observe the correlation of this pathway with apoptosis. Materials and Methods: A total of 32 men who applied for infertility evaluation were included in the study. Semen analysis was performed according to WHO criteria. Liquefaction time, appearance, volume, pH, viscosity, sperm concentration, total motility rate, sperm motility, and percentage of spermatozoa with normal morphology were determined. TNF-α, HSP-70, and Caspase-3 immunolocalization were scored histologically. A sperm chromatin dispersion test was used to observe DNA fragmentation. Results: There was no significant difference in TNF-α protein expression rate (mild level). The HSP-70 expression rate was lower, especially in the head region of normo. Caspase-3 was higher totally in non-normo. DNA fragmentation levels were similar in both the groups. Conclusion: From TNF-α protein expression at the mild level in both the groups, it may be hypothesized that the apoptotic pathway might not be triggered by the extrinsic pathway. We found a negative correlation between HSP-70 and Caspase-3 expressions, providing further evidence that HSP-70 works as an inhibitor to apoptosis. This, particularly on specific points, made us think the communication might begin in the anterior chamber, then flow through the cell body to the tail. HSP-70 expression was lower in normo than in non-normo, indicating the possible role of HSP-70 as an answer to any type of stressor in non-normozoospermic patients. Correspondingly, it may be concluded that HSP has an antiapoptotic effect, causing inhibition in the elimination of abnormal sperm cells impairing sperm parameters.

Expression of pro-apoptotic and anti-apoptotic proteins in granulosa cells of women with diminished ovarian reserve.

PURPOSE: To evaluate the expressions of caspase-3 and cytochrome c and heat shock protein 70 (Hsp70) in granulosa cells (GCs) from women with normal ovarian reserve (NOR) and diminished ovarian reserve (DOR) undergoing intracytoplasmic sperm injection (ICSI). METHODS: GCs were collected from 117 infertile women during oocyte retrieval. Patients were classified into four groups as follows: DOR-COC score of 0, DOR-COC score of I, NOR-COC score of 0, and NOR-COC score of I. The caspase-3, cytochrome c, and Hsp70 analyses were performed immunohistochemically in GCs. The ICSI outcomes were evaluated prospectively. RESULTS: The clinical pregnancy and live birth rates were higher in DOR-COC score of I (15, 30.6%; 14, 38.9%) and NOR-COC score of I (19, 38.77%; 19, 52.7%) groups, compared with DOR-COC score of 0 (12, 24.4%; 3, 6.1%) and NOR-COC score of 0 (3, 6.1%; 0%) groups (p = 0.0001; 0.00002), respectively. Caspase-3 and cytochrome c expression levels were higher in DOR-COC score of 0 (23, 65.7%; 25, 71.4%) and NOR-COC score of 0 groups (19, 61.3%; 20, 64.5%), compared with DOR-COC score of I (8, 32%; 9, 36%) and NOR-COC score of I groups (7, 26.9%; 8, 30.8%) (p = 0.00297; p = 0.002), respectively. Lower expression levels of Hsp70 were found in DOR-COC score of 0 (11, 31.4%) and NOR-COC score of 0 groups (10, 32.3%), compared with DOR-COC score of I (16, 64%) and NOR-COC score of I groups (20, 76.9%) (p = 0.001), respectively. Hsp70 expression levels were positively correlated with the number of day 3 good-quality embryo and negatively correlated with estradiol levels in the DOR group. CONCLUSION: Our data suggest that COC score of 0 is associated with increased expression levels of apoptotic proteins, decreased expression levels of anti-apoptotic protein, and poor ICSI clinical outcomes in women with and without DOR.

In 2D and 3D Cell Culture Models, Effects of Endothelial Cells on E-cadherin / ß-catenin Expression Levels and Spheroid Sizes in Ishikawa Cells.

OBJECTIVE: Increasing evidence shows that three dimensional cell culture models better reflect the in vivo tumor microenvironment than two dimensional cell culture models. Co-culture models are ideal cell culture models for understanding the communication between cells and the in vivo microenvironment. Changes in expression levels of E-cadherin are closely related to cancer metastasis and progression. ß-catenin mediates cell adhesion of E-cadherin. Endothelial cells are stromal cell components in the tumor microenvironment. It is known that there is little or no expression of E-cadherin in endothelial cells. METHODS: In our study, both two-dimensional and three dimensional mono-culture and co-culture models were created using Huvec and Ishikawa cells (endometrial cancer cell lines) to better reflect cell interactions. Spheroids were followed for three days in the three-dimensional cell culture model. E-cadherin and ß-catenin expression levels of two-dimensional and three dimensional mono-culture and co-culture models were measured by immunofluorescence staining. Spheroid images were recorded using a Z-stack. Intensity measurements in both two-dimensional and three-dimensional mono-culture and co-culture models were made using the Image J software. Study groups were evaluated by one-way analysis of variance (One-Way ANOVA). Values of p <0.05 were considered statistically significant. RESULTS: The size of the co-culture spheroids was recorded significantly larger than the mono-culture spheroids (p <0.0001). In two (p = 0.0175) and three dimensional models (p <0.0001), expression levels of E-cadherin in the mono-culture of Ishikawa cells were recorded significantly higher than in Huvec and co-culture cells. Likewise, while the expression levels of ß-catenin were higher in the mono-culture of Ishikawa cells in two-dimensional models (p <0.05), no significant difference was observed in three dimensional models (p> 0.05). CONCLUSION: In summary, it has been noted that the expression levels of E-cadherin are significantly reduced in co-cultures of Ishikawa cells with Huvec cells in both two and three dimensions . These results support the idea that endothelial cells may cause changes in endometrial cancer progression by suppressing E-cadherin expression in Ishikawa cells.

Effects of Vincamine on Testicular Dysfunction in Alloxan-induced Diabetic Male Rats.

Background: Diabetes mellitus (DM) is frequently linked with problems of several organ systems, including retinopathy, neuropathy, and nephropathy. Additionally, patients have changes in sexual functioning, such as decreased libido and fertility. Vincamine, a monoterpenoid indole alkaloid, has hypoglycemic and antioxidant effects. Objectives: This research assessed the impact of vincamine on testicular dysfunction in alloxan-induced male rats by measuring fasting blood glucose, oxidative stress, seminal analysis, and histological examination of the testis. Methods: Wister-albino male rats were randomized into the following groups at random: Untreated-healthy, untreated-DM, vincamine-treated (20 mg/kg) DM, vincamine-treated (40 mg/kg) DM, and clomiphene-treated DM (5 mg/kg). On day 14, rats were sacrificed, and semen/blood samples were collected. Sperm count, motility, and morphological abnormalities were noted by microscopic examination. The testis was examined histopathologically and assessed using Johnsen's score. Results: Compared with the untreated diabetic group, a dosage of 40 mg/kg vincamine generate a significant reduction in fasting blood sugar (FBG). Compared with the untreated diabetic group, the vincamine-treated rats produced greater plasma testosterone levels and Johnsen scores. In the vincamine 20 mg/kg group, sperm concentration was higher than in the vincamine 40 mg/kg group. Conclusions: It is possible that vincamine has a potential preventive effect against diabetes-related reproductive problems attributable to its antioxidant activity and capacity to restore testicular steroidogenesis.

The creation of unilateral intermittent and unintermittent renal ischemia-reperfusion models in rats.

BACKGROUND AND AIM: This study aims to establish unilateral intermittent and unintermittent partial nephrectomy-like renal ischemia-reperfusion (I-R) model in rats and to compare the results with biochemical findings. MATERIAL AND METHODS: The study was conducted on 24 adult 8-week-old male Wistar-Albino rats, each weighing s200-250 g. The rats were divided into three groups. In the Sham group (n = 8), the kidney was surgically exposed and closed. We designed experimental I-R models in the second group (n = 8, a total of 30-min ischemia model in the manner of 3 intermittent sets 8 minutes clamping and 2 min unclamping) and in the third group (n = 8, one session of 30-min unintermittent ischemia). In postoperative day 1, the rats were sacrificed, and the effects of I-R models on the renal tissue were comparatively assessed by evaluating serum Neutrophil Gelatinase-Associated Lipocalin (NGAL), serum kidney injury molecule-1 (KIM-1), urinary NGAL, urinary KIM-1, and serum creatinine levels. RESULTS: Urinary NGAL and KIM-1 levels were significantly higher in the continuous ischemia group when compared to those in the sham and intermittent ischemia groups (P < 0.05). In the intermittent ischemia group, urinary NGAL and urinary KIM-1 levels were significantly higher than those in the sham group (P < 0.05). Although the results of serum NGAL, serum KIM-1, and serum creatinine levels seemed to be in parallel to the results of urinary markers, no statistically significant difference was found. CONCLUSION: Renal injury was significantly less in the intermittent I-R model when compared to that in the unintermittent I-R model in our experimental rat study.

A Histopathological Evaluation of Ovarian Hyperstimulation Syndrome on Reproductive and Vital Organs and the Role of the VEGF-PKA Pathway in a Mouse Model.

Ovarian hyperstimulation syndrome (OHSS) is one of the most common and iatrogenic complications of in vitro fertilization therapy, which is an exaggerated response to excess hormones resulting in the development of a large number of maturing follicles. Although the complications of and reasons for the condition are well known, the overall histopathological effects on systemic organs and the extent of the damage have not been fully elucidated. Besides, the mechanism that underlies the situation is not very well known. The aim of the present work was to analyse the histopathological effects of OHSS on reproductive (uterus and ovary) and vital organs (liver and kidney) and the possible role of the VEGF-PKA pathway in triggering the condition. Balb/c mice were used to establish an OHSS model. The OHSS group were injected with overdose PMSG while the normal responder group were injected with an optimal dose. Histopathological evaluation was utilised in the liver, kidney, ovary, and uterus stained with hematoxylin and eosin, Masson's trichrome, and periodic acid-Schiff stain. The expression profiles of VEGF (vascular endothelial growth factor), PKA (protein kinase A), and p-PKA (an activated form of PKA) were detected with immunohistochemistry and Western blotting. OHSS was demonstrated to have a negative histopathological effect on all of the organs analysed. These effects were associated with an overall increase in the expression levels of VEGF, PKA, and p-PKA. OHSS has a serious histopathological negative effect on the systemic and reproductive organs and is proven to affect overall health, and thus should be considered a dangerous complication during ART techniques. The activation of the VEGF-PKA pathway, which is indicated by the expression levels of VEGF, PKA, and p-PKA, is demonstrated to accompany this complication, which should be further elucidated to understand the mechanisms underlying the condition.

Effects of Gallic Acid on Endometrial Cancer Cells in Two and Three Dimensional Cell Culture Models.

BACKGROUND AND AIM: Cell culture studies are an indispensable tools used to understand basic physiological, biophysical and biomolecular mechanisms. Although traditional two-dimensional (2D) cell culture models are more preferred in experimental studies, three-dimensional (3D) cell culture models, attract more attention due to several advantages including mimicking tumor physiology, biochemistry and biomechanics. We aimed to investigate the effects of Gallic Acid, an antimutagenic, antioxidant and anticarcinogenic agent, on both 2D and 3D cultured endometrial cancer cells for the first time. METHODS: IC50 values were determined in 2D and 3D cultured endometrial cancer cells exposed to different doses of GA. In the 2D culture model exposed to GA, Caspase 3 expression levels were analyzed. In addition, the effect of GA on the migration of 2D cultured endometrium cancer cells was investigated. RESULTS: IC50 value in the 3D model was found significantly higher than the 2D model. In 2D culture model, Caspase 3 expression and apoptosis was increased significantly in cells of GA exposed group compared to the control group. GA did not have a significant effect on the migration profile of cells. CONCLUSION: Gallic Acid is shown to induce apoptosis in Ishikawa cells via Caspase 3 activation. We demonstrated a significantly higher  IC50 level in 3D model which provide evidence to prefer 3D models in drug-test trials. The data obtained in the current study will provide a basis for further experiments to analyze the effects of GA on endometrial cancer and to develop strategies for clinical treatment.

Chronic unpredictable stress disturbs the blood-testis barrier affecting sperm parameters in mice.

RESEARCH QUESTION: Does chronic stress affect the key proteins and sperm parameters of the blood-testis barrier (BTB)? DESIGN: C57Bl/6 mice were divided into two groups: a non-treated control group and a chronic unpredictable stress (CUS) applied group. The stress status of the animals was confirmed with behavioural tests. Histopathologic evaluation was conducted by haematoxylin and eosin staining and electron microscope. Malondialdehyde, corticosterone and testosterone levels were evaluated in peripheral blood. Expression levels of BTB proteins, namely zonula occludens-1 (ZO-1), claudin-11 (CLDN11) and clathrin in Sertoli cells, were assessed by Western blotting and immunofluorescence techniques. Sperm samples were collected from cauda epididymis, and sperm parameters analysed. RESULTS: The stress model was confirmed by behavioural tests. Histopathological evaluation of the testes demonstrated a mild degeneration in seminiferous tubules. Malondialdehyde (P = 0.008) and corticosterone levels increased (P = 0.004) and testosterone levels decreased (P = 0.005) in the CUS group. Electron microscopic evaluation confirmed the damage in BTB integrity in the CUS group. Western blot analysis showed that ZO-1 and CLDN11 levels were significantly decreased, although clathrin levels were unchanged. Although sperm concentration and total motility rate were not significantly different between the groups, progressive motility (P = 0.03), normal sperm morphology (P = 0.04), chromatin integrity (toluidine blue) (P = 0.002) and the acrosomal reaction rate (P = 0.002) were significantly decreased, and acrosomal abnormality rate was dramatically increased (P = 0.04) in the CUS group. CONCLUSIONS: In mice, CUS disrupted BTB integrity and impaired sperm parameters. A decrease in ZO-1 and CLDN11 expression levels may be proposed as the causative factor.

Factors related to follicular oxidative stress in intracytoplasmic sperm injection cycles and its effects on granulosa cells.

The aim of the present study was to investigate several common conditions that may potentially be correlated with follicular oxidative status during an intracytoplasmic sperm injection (ICSI) cycle and that include the serum oestrogen level on the day of oocyte pick-up, maternal age and pregnancy outcome. Patients that were enrolled in the study were classified randomly into three groups using their numerical order. The first group were classified based on maternal age (<35 and ≥35 years) (n = 398), the second group on the serum oestradiol (E2) level on the day of human chorionic gonadotropin (hCG) administration (levels >90th percentile and ≤ 90th percentile) (n = 491) and the third group on pregnancy outcome (positive/negative) (n = 376). The groups were matched for the other variables (stimulation protocol, dose of gonadotropin, duration of stimulation, antral follicle count, body mass index, basal follicle stimulating hormone (FSH), and E2 levels and day of hCG trigger) to prevent the possible contribution of those parameters to the results. Each group was matched for other variables (stimulation protocol, dose of gonadotrophin, duration of stimulation, antral follicle count, body mass index, basal FSH and E2 levels and day of hCG trigger) that may have affected the outcome, except for the parameter under investigation. Maternal age (P = 0.044,168 r = 0.418), oestrogen level on day of hCG administration (P = 0.001, r = 0.436) and pregnancy outcome (AUC = 0.65, P = 0.071) were found to be correlated with follicular oxidative status. The results obtained will help us to shield patients from possible situations that may cause oxidative stress and therefore adverse outcomes of an ICSI cycle.

Evaluation of Apoptosis Pathway of Geraniol on Ishikawa Cells.

Endometrial cancer is the most common type of cancer in the female reproductive system. Geraniol is acyclic monoterpene alcohol derived from essential oils of aromatic plants. This study aimed to investigate the apoptosis pathway of geraniol on Ishikawa cells. The cytotoxic effects of Geraniol on Ishikawa cells were determined by an MTT test. Ishikawa cells were seeded on cover slips, the IC50 dose was applied, and the cells were incubated with antibodies against Bax, Bcl-2, and TUNEL Assay. mRNA expression analysis of apoptosis-related genes was determined by RT-qPCR with an IC50 dose of Geraniol. The IC50 dose of Geraniol decreased Bcl-2 staining significantly, but it significantly increased Bax staining and TUNEL positive cells. A significant increase in the Bax, caspase3, caspase-8, cytochrome C and Fas genes and a significant decrease in the Bcl-2 gene was observed when the IC50 dose group was compared to the cells in the control group based on their mRNA expression levels.Analysis of expression of genes whose products are involved in apoptosis suggests the involvement of the mitochondrial pathway.

Seminal oxidation-reduction potential as a possible indicator of impaired sperm parameters in Turkish population.

Oxidative stress (OS) has been shown to have a key role in male infertility. Recently, a new measurement method has been developed to measure the overall oxidation-reduction potential (ORP) in a semen sample known as the MiOXSYS system. The aim of this study was to investigate the correlation of sperm parameters with oxidative stress levels determined by ORP and to evaluate whether the current limit is able to distinguish abnormal sperm parameters from normal ones in Turkish population. Semen samples of 121 patients who applied for infertility investigation were divided into two groups as (OS +; n:39) and (OS -; n:82). Semen parameters were compared between groups. Sperm concentration, total motility and progressive motility were found to be significantly lower in OS (+) patients compared to those OS (-), while immotile sperm count was significantly higher in OS (+) patients. Oxidative stress determined by MiOXSYS system was found to be related to reduced sperm parameters in Turkish population, which may be used as an indicator of poor sperm parameters and a support to routine semen analysis. In addition, recommended reference value was found to be reliable in distinguishing normal from impaired semen parameters.

Effects o follicular fluid oxidative status on human mural granulosa cells, oocyte competency and ICSI parameters.

PURPOSE: The aim of the present study was to understand the molecular and genetic alterations involved in follicular fluid oxidative process by investigating human mural granulosa cells and to find possible biomarkers for oocyte competency and ICSI outcome measures. METHODS: A total of 166 patients were included in the study. Total antioxidant and oxidant levels of follicular fluids were measured on the day of oocyte pick-up and oxidative status were calculated. Expression profiles of three potential target proteins in cases of oxidative stress (Hsp70, Tgf-ß, Notch1), DNA status and chromatin integrity of mural granulosa cells were analyzed. RESULTS: TAS levels were positively correlated with the Hsp70 and Tgf-ß expression patterns of mural granulosa cells. Mature oocyte rate and fertilization rates were affected negatively by the presence of oxidative stress and a significant positive correlation was found with the oxidative status and the fertilization rate, whereas no correlation with the remaining ICSI parameters in the overall group. CONCLUSIONS: Oxidative stress detected in follicular fluid adversely affects fertilization rates post-ICSI however no effect on the remaining parameters including embryo quality, pregnancy, and implantation rates. DNA damage, chromatin integrity were increased, whereas Hsp70 and Tgf-ß were decreased in mural granulosa cells in cases of oxidative stress which may indirectly reflect the oocyte competency and may be used as biomarkers for ICSI outcome measures.