Mitochondrial activity and cytoskeleton organization in three pronuclei oocytes after intracytoplasmic sperm injection.

SummaryDigyny, the presence of a third pronucleus due to the failure of second polar body extrusion, is problematic after intracytoplasmic sperm injection (ICSI) practices. Mitochondria have critical roles such as production of adenosine triphosphate (ATP) and regulation of Ca2+ homeostasis during oocyte maturation, fertilization and the following development, while the regulation of meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis is closely associated with the cytoskeleton. In this study, mitochondrial membrane potential, distribution of F-actin and γ-tubulin, and the ultrastructure of three pronuclear (3PN) oocytes were investigated. 3PN oocytes after ICSI procedure were taken from patients who were enrolled in assisted reproduction programmes. For mitochondrial membrane potential analysis, fresh oocytes stained with the mitochondrial membrane potential probe JC-1, were evaluated under fluorescence microscopy. The mitochondrial membrane potential of three pronuclear oocytes showed similar results to normal zygotes. γ-Tubulin was stained strongly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not the perinuclear, area. Cytoplasmic halos were moderately or not detected by electron microscopy; lipofuscin granules, degenerated mitochondria, and multilamellated bodies were seen in the ooplasm. Immunohistochemistry and electron microscopic findings suggested that mitochondrial membrane potential has no direct effect on second polar body extrusion. This abnormality can be associated with an altered cytoskeleton due to poor oocyte quality.

Antioxidant activity of CAPE (caffeic acid phenethyl ester) in vitro can protect human sperm deoxyribonucleic acid from oxidative damage.

PURPOSE: Sperm processing (e.g., centrifugation) used in preparation for assisted reproduction can result in excessive generation of reactive oxygen species (ROS) and potential sperm damage. The use of antioxidants during sperm processing has been shown to prevent iatrogenic sperm damage, including DNA damage. In this study, we evaluated the effect of caffeic acid phenethyl ester (CAPE) on oxidative stress mediated sperm dysfunction and DNA damage. METHODS: Semen samples were obtained to liquefy at room temperature. After centrifugation and washing protocols, spermatozoa were incubated in a single step supplemented medium with either of 10, 50 or 100 µmol/L CAPE for 2 hours at 36 °C. After incubation period, MDA levels of seminal plasma were measured. The fragmentation in sperm DNA was detected by light microscopy via use of an aniline blue assay, while ultrastructural morphology was analyzed by transmission electron microscopy. RESULTS: Significant increase has been observed in percent chromatin condensation (assessed by aniline blue staining) and Malondialdehyde (Mmol/L) in oligoasthenoteratozoospermia group before the centrifugation (0.57 ±â€¯0.15). Incubation of samples with 100 µmol/L CAPE after centrifugation resulted in a significantly lower percent chromatin condensation compared to samples incubated without CAPE (0.42 ±â€¯0.12) (P < 0.0033). Incubation of all samples with CAPE (10 µmol/L, 50 µmol/L, 100 µmol/L.) after centrifugation resulted in a significantly lower percentage of Malondialdehyde levels. CONCLUSIONS: The data suggests that preincubation of spermatozoa with the antioxidant CAPE offers protection against oxidative DNA damage in vitro.

Serum substance P concentrations to predict oocyte maturation index and clinical pregnancy.

AIM: The aim of this study was to assess the predictive value of serum substance P (SP) concentrations on oocyte maturation and clinical pregnancy. METHODS: Ninety-three women with unexplained infertility underwent intracytoplasmic sperm injection (ICSI) cycles. Antagonist protocol was started for each participant and at the day of oocyte pick up, serum samples were obtained from each participant to assess SP concentrations, and these concentrations were utilized to predict mature/total oocyte ratio and clinical pregnancy. RESULTS: SP concentration was a significant predictor for mature/total oocyte ratio > 0.75 and clinical pregnancy. In correlation analyses, maturation index was significantly correlated with FSH (r= -0.226, p = 0.03), estradiol (r = 0.239, p = 0.021), peak estradiol (r = 0.414, p < 0.001), and substance P (r = 0.796, p < 0.001). In multivariate analyses, number of immature (beta coefficient = -0.379, p < 0.001), mature oocyte (beta coefficient = 0.473, p < 0.001), SP concentration (beta coefficient = 0.723, p < 0.001) and maturation index (beta coefficient = -0.387, p = 0.003) were significantly associated with clinical pregnancy. CONCLUSION: SP concentrations at the day of oocyte pick up may be used to predict clinical pregnancy and may be an indirect indicator for cycle outcome in assisted reproductive technology (ART).

The assesment of follicular fluid presepsin levels in poor ovarian responder womenandits relationship with the reproductive outcomes.

A considerable proportion of all women undergoing IVFrespond poorly to gonadotropin stimulation. These women are reported to be associated with increased cancellation rates and lower pregnancy rates. It has been hypothesized that poor response to ovarian stimulation is a first sign of ovarian ageing or premature ovarian failure, which might be related to altered inflammatory response in the body. We aimed to compare follicular fluid presepsin levels between poor- and normo-responder patients to ovarian stimulation, to assess its relationship with reproductive outcomes. This study included infertility patients who underwent ovulation induction with either long GnRH agonist or GnRH antagonist protocols and who subsequently underwent IVF/ICSI. Included patients were assigned to two groups according to the Bologna criteria for poor ovarian response. Group 1 and 2 consisted of normo- and poor-responder patients, respectively.The 2 groups were compared in terms of FF presepsin levels. Also, any relationship between the FF presepsin levels and fertility outcomes was assessed within the groups. The groups were compared by using student's t-test, Mann-Whitney U test and X(2) test, where appropriate. Pregnancy rates were not significantly different between the groups (22.6% and 17.6%; P=0.650, respectively). FF presepsin levels were higher in Group 1, however, the difference was not statistically significant (298.0±797.4 and 149.2±422.3; P=0.190, respectively). FF presepsin levels did not significantly differ between pregnancy positive and the pregnancy negative patients in both Group 1 (243.6±531.1 and 314.3±866.5; P=0.055, respectively) and Group 2 (112.2±79.8 and 157.1±464.3; P=0.394, respectively). Consequently, FF presepsin seems not to be a reliable marker in predicting pregnancy in both normo-responder and poor-responder infertility groups.

Can blood or follicular fluid levels of presepsin predict reproductive outcomes in ART; a preliminary study.

Many stages of COH protocols are considered to potentiate a state of systemic inflammation. The limit beyond which inflammation has negative impacts on the formation of conception and the reproductive outcomes are compromised still remains unclear. Presepsin is a novel biomarker for diagnosing systemic inflammation and sepsis. We aimed to investigate whether plasma and follicular fluid presepsin values on oocyte pick-up (OPU) day, embryo transfer (ET) day and pregnancy test (PT) days could predict reproductive outcomes during IVF treatment in women with UEI. Patients were assigned to two groups according to pregnancy test results; pregnant (Group 1) and non-pregnant (Group 2). From all patients included in the study, 2 cc of venous blood was sampled on the three days and follicular fluid (FF) was collected during oocyte retrieval. Plasma presepsin, CRP and WBC values and FF presepsin values were measured and compared between the 2 groups. There was no significant difference between FF and plasma presepsin levels on the OPU day (298±797.4 ve 352.9±657.1; P=0.701, respectively). Plasma WBC, CRP and presepsin levels on the OPU, ET and PT days and FF presepsin levels on OPU day were not different between the 2 groups. Plasma presepsin course on the separate 3 days were different between the groups.

Effect of lower than expected number of oocyte on the IVF results after oocyte-pickup.

OBJECTIVES: To investigate whether a lower than expected number of oocyte after ≥14 mm follicle aspiration during OPU has any effect on pregnancy outcomes Methods: This is a retrospective study done between 2010 and 2013 at the IVF Unit of the Zeynep Kamil Women and Children Diseases Education and Research Hospital, dealing with the medical records of infertile patients who underwent IVF cycle and controlled ovarian stimulation with long agonist or fix antogonist protocol. The patients included into the study were those diagnosed with a primary infertility, aged between 23 and 39, at a BMI of 22-28 kg/m(2) and having received the first or second IVF treatment. Male factor, presence of uterine anomaly, patients with serious endometriosis and patients with low ovarian reserve were all excluded from the study. Typically, oocyte pick-up was performed in all the patients 35.5 hours after the hCG implementation. Single or double embryo transfer was performed, where available. Patients were classified into two groups. Group 1 consisted of those with no difference between ≥14 mm aspirated follicle number and expected number of oocyte or with 1 missing number of oocyte at the most. Group 2 consisted of those with at least ≥2 missing number of oocyte between aspirated follicle number and expected number of oocyte. Statistical analysis was performed using Student's t test for continuous variables and chi-square test for categorical variables. Additionally, a Linear regression analysis was conducted between the total number of oocyte and pregnancy. RESULTS: In total, 387 treatment cycles were included into the study. Group 1 consisted of 134 patients and Group 2 consisted of 252 patients. Antral follicle number (12.8 ± 4.3 and 14.5 ± 4.1, P = 0.0007), hCG day E2 value (1990.7 ± 1056.4 and 2515.2 ± 1332.7, P < 0.0001) and the the number of aspirated follicle during OPU (9.1 ± 4.4 and 13.7 ± 5.5, P < 0.0001) were significantly higher in Group 2; whereas on the other hand, daily gonadotropin dose (290.9 ± 79.9 and 273.4 ± 74.4, P = 0.034) and total gonadotropin doses (2545 ± 1031.8 and 2247.7 ± 901.9, P = 0.004) were significantly higher in Group 1. The pregnancy rate was significantly higher in Group 1 (29.1% and 19.4%, P = 0.041). No correlation was observed between the number of oocyte and pregnancy (r = 0.082, P = 0.107). CONCLUSIONS: The number of aspirated follicles during IVF treatment being higher than the collected number of oocyte leads to a statistically significant fall in the pregnancy rates. There is no correlation between the number of oocyte and pregnancy.

Detection of epithelial cell transfer in spinal areas by light microscopy and determining any tissue coring via cell culture during combined spinal-epidural interventions.

BACKGROUND AND OBJECTIVES: Epithelial tissue coring by spinal needles during subarachnoid injections may cause intraspinal epidermal tumors. Previous studies have investigated tissue transfer with different needle types during subarachnoid or epidural injection. This study deals with the transfer of epithelial tissue during combined spinal-epidural (CSE) anesthesia. METHODS: We studied 68 American Society of Anesthesiologists I to III adult patients. CSE anesthesia was induced under aseptic conditions at the L2-3 or L3-4 interspace with patients in the lateral decubitus position. Cerebral spinal fluid, spinal needle stylet, fluid used to flush the interior of the spinal needle, fluid used to wash the exterior of the spinal needle, fluid used to flush the interior of the epidural needle, and fluid used to wash the exterior tip of the epidural needle were examined under light microscopy (n = 30 patients) or incubated in a cell-culture medium (n = 38 patients). Samples were incubated in cell-culture medium alone (n = 13) or in a cell-culture medium for 3 weeks and then in a medium with epidermal growth factor (n = 25). As a positive control, skin tissue samples were taken by punch biopsy from 10 randomly chosen patients who underwent CSE interventions. These samples were incubated in an enriched medium serum. RESULTS: Light microscopy revealed that there was cell transfer in all phases in various rates: samples 1, 2, 3, 4, 5, and 6 contained epithelial cells and debris in ratios of 6.9%, 20.7%, 6.9%, 20.7%, 26.7%, and 33.3%, respectively. Epithelial cell colonization was detected in the cell-culture samples taken from the control group but not in the samples taken from the CSE group. CONCLUSIONS: We could not reproduce the cells or cell debris obtained during the CSE interventions in vivo, which can be explained by a possible structural deformation of cells or the inadequacy of the amount of cells that were transferred.