In vitro evaluation of exocytosis-associated SNARE molecules in human granulosa cells in polycystic ovary syndrome.

PURPOSE: Patients with polycystic ovarian morphology (PCOM) make up 20% cases for assisted reproductive technology (ART). Folliculogenesis is impaired in PCOS. Signaling molecules are involved in follicle development. Dysregulations of intrafollicular environment and signaling molecules are observed in PCOS. Granulosa cells (GCs) and oocytes secrete molecules into follicular fluid by exocytosis of SNAREs. The aim of this study is to evaluate vesicle transport and vesicle fusion proteins (SNAREs) in GCs from PCOS patients who have undergone IVF treatment. METHODS: Follicular fluids were collected from patients who undergo IVF/ICSI with the diagnosis of male factor (n = 10) and PCOS (n = 10) patients. GCs were separated and cultured. Each group of GCs was stimulated with FSH-hCG. The cells were examined under electron microscope. Immunofluorescent labeling was performed on cells for Stx6, SNAP25, StxBP1, FSHr, and KITL. Integrated density was analyzed from images of Stx6, SNAP25, StxBP1, FSHr, and KITL. RESULTS: Intercellular communication occurs by signal molecules; Stx6, SNAP25, and StxBP1 fusion proteins involved in exocytosis were decreased in the GCs of PCOS. There was no increase in in vitro stimulation with FSH-hCG either. In the electron microscope, it was observed that exocytosis of the vesicles was disrupted. CONCLUSIONS: Exocytosis and vesicular dynamics are among the basic physiological functions of human steroidogenic granulosa cells. Follicle development is necessary for production of competent oocytes and ovulation. Understanding the pathophysiology of PCOS at follicular level is important for disease management. According to our findings, deficits in vesicular dynamics of human granulosa cells in may be central to the treatment strategy for PCOS patients.

Vitrified-warmed blastocyst transfer timing related to LH surge in true natural cycle and its impact on ongoing pregnancy rates.

RESEARCH QUESTION: Does the timing of warmed blastocyst transfer in true natural cycle (tNC) differ according to six different commonly used definitions of LH surge, and do differences in timing have any impact on ongoing pregnancy rate (OPR)? DESIGN: Prospective monitoring, including repeated blood sampling and ultrasound analyses of 115 warmed blastocyst transfer cycles performed using tNC between January 2017 and October 2021. RESULTS: The reference timing of follicular collapse +5 days would be equivalent to LH surge +6 days in only 5.2-41.2% of the cycles employing the six different definitions of the LH surge. In contrast, the reference timing was equivalent to LH surge +7 days in the majority of cycles (46.1-69.5%) and less commonly to LH surge +8 days (1.8-38.3%) and +9 days (0-10.4%). For each definition of the LH surge, the OPR were comparable among the different warmed blastocyst transfer timings related to the LH surge (LH surge +6/+7/+8/+9 days). When logistic regression analysis was performed to evaluate the independent effect of variation of warmed blastocyst transfer timing (LH surge +6/+7/+8/+9 days) on OPR and taking LH surge +6 days as the reference, change in timing was not an independent predictor of OPR for any of the definitions of the LH surge. CONCLUSIONS: Employing a policy of performing warmed blastocyst transfer on follicular collapse +5 days and using six different definitions of the LH surge, vitrified-warmed embryo transfer timing is indeed equivalent to LH surge +7/+8 and even +9 days in a significant proportion of tNC with comparable reproductive outcomes.

A new isolation and culture method for granulosa cells.

Granulosa cells are the cell population who have an increasing importance in the female genital system and reproduction. Thus, nowadays in vitro studies to address these cells are also gaining importance and attracts researcher's attention. The aim of our study is to develop a more feasible, low-cost granulosa cell isolation and culture method compared to methods defined so far. Granulosa cells were isolated from follicular fluids obtained from both healthy women donors (n = 19) and polycystic ovary syndrome (n = 15) applied to in vitro fertilization treatment process. Granulosa cells were isolated by using Lymphosep® separation fluid that was not used for this purpose before. The isolated cells were cultured in suitable culture dishes with a mixture of BIO-AMFTM-1 and DMEM/F12 in the first seeding and only complete DMEM/F12 in the following feeds. Complete medium contains only 5% fetal calf serum, 4% L-glutamine and 1% penicillin-streptomycin-amphotericin. The new methods we have developed in granulosa cell isolation and in vitro culture have been successful. Reduction in supplement types and amount; improved the proliferation rate of the granulosa cells in culture. Our new methods of isolation and cell culture for granulosa cells from healthy women, have been also successful in samples of polycystic ovarian patients. With these developed methods, granulosa cells, which belong to humans and have an important role in the ovary, could be isolated and subsequently be maintained to reproduce (proliferate) more easily and cheaper.

Comparison of single euploid blastocyst transfer cycle outcome derived from embryos with normal or abnormal cleavage patterns.

RESEARCH QUESTION: To assess incidence of abnormal cleavage among biopsied blastocysts; to compare euploidy rates of the blastocysts with abnormal and normal cleavage; and to compare single euploid blastocyst transfer (SEBT) outcome derived from embryos with normal or abnormal cleavage. DESIGN: Retrospective analysis of prospectively collected data in a private IVF clinic. Consecutive 554 patients (749 cycles) undergoing preimplantation genetic testing for aneuploidy (n = 497; 671 cycles) or monogenic diseases (n = 57; 78 cycles) were included. All assessments for abnormal cleavage were carried out retrospectively; presence of abnormal cleavage was not a factor in deciding which euploid embryo to transfer. A total of 1015 blastocysts were biopsied and 295 SEBT procedures were carried out. Main outcome measure was live birth rate (LBR). RESULTS: Incidence of reverse cleavage, direct cleavage, and reverse plus direct cleavage, were 7.7%, 6.4% and 2.3%, respectively. Of the 1015 biopsied blastocysts, 35.0% were euploid. Blastocysts with abnormal cleavage, in total, had a significantly higher euploidy rate compared with blastocysts with normal cleavage (44.6% [74/166] versus 33.1% [281/849]; P = 0.017). The LBR after SEBT with normal, reverse and direct cleavage, and direct cleavage plus reverse cleavage, was 133/238 (55.9%), 6/26 (23.1%), 8/24 (33.3%) and 0/3 (0.0%) (P < 0.001). Generalized estimating equation analysis showed that the presence of abnormal cleavage pattern was the only independent predictor of LBR (OR 0.316; 95% CI 0.115 to 0.867; P = 0.013). CONCLUSIONS: Blastocysts with direct or reverse cleavage should be biopsied in preimplantation genetic testing cycles if they are morphologically eligible. Euploid blastocysts with abnormal cleavage, however, have approximately half the LBR of those euploid blastocyst with normal cleavage, hence, blastocysts with abnormal cleavage should have lower priority for transfer.

PGT for aneuploidy does not affect three-cycle cumulative IVF discontinuation rate in women of advanced maternal age.

RESEARCH QUESTION: Does preimplantation genetic testing for aneuploidy (PGT-A) influence the discontinuation rate in women with advanced maternal age (AMA) undergoing IVF? DESIGN: Retrospective longitudinal cohort study carried out at a single IVF clinic in Turkey. In total, 401 consecutive AMA cases were included. Discontinuation rates of pre-intervention (conventional IVF; June 2013 to October 2014; 203 couples; 270 cycles) and post-intervention (PGT-A; April 2015 to June 2016; 198 couples; 285 cycles) periods were compared. To delineate the reason for discontinuation, a telephone survey was conducted. Primary outcome measure was cumulative discontinuation rate before completing three cycles of IVF treatment without achieving an ongoing pregnancy. RESULTS: The discontinuation rates after the first and second failed cycles were comparable between the two arms as were the cumulative discontinuation rates before completing three cycles. The cumulative ongoing pregnancy rate per embryo transfer was significantly higher in the PGT-A arm (43.2% versus 16.8%; P < 0.001). The cumulative ongoing pregnancy rate per patient was comparable between the two arms (20.7% versus 16.3%, respectively). Female age was the only significant contributor to treatment discontinuation (hazard ratio [HR] 1.07; 95% CI 1.09 to 1.13). Of the 296 couples discontinuing treatment in both arms, 179 (179/296 [60.5%]) participated in the survey; overall, psychological burden was the main reason for treatment discontinuation (37/179 [20.7%]). CONCLUSIONS: About 90% of AMA cases not achieving an ongoing pregnancy discontinue IVF treatment before completing three cycles. Discontinuation rate is not reduced by carrying out PGT-A. Female ageing is the only significant contributor, with a hazard of discontinuing further IVF treatment of 7% with female ageing of 1-year.

The Impact of a Long-Acting Oral Sphingosine-1-Phosphate Analogue on Ovarian Aging in a Rat Model.

In animal studies, intravenous continuous infusion or peritoneal injection of sphingosine-1-phosphate (S1P) has been shown to decrease chemotherapy- and radiotherapy-induced apoptosis on primordial follicles. Although a long-acting oral form of an S1P analogue (FTY720, fingolimod) has been recently developed and utilized in women with multiple sclerosis, there are no data exploring its ability to avoid spontaneous follicle apoptosis. Thirty 10-month-old female rats were randomly assigned to 3 groups to investigate whether fingolimod would be able to decrease the spontaneous ovarian follicle apoptosis ratio. An oral analogue form of S1P was administered for 60 days at a dose of 0.1 mg/kg (n = 10) or dose of 1 mg/kg (n = 10) per day. The control group (n = 10) received physiological serum via an orogastric feeding tube. The main outcome measures were anti-Müllerian hormone (AMH) level and nonapoptotic follicle ratio. While low-dose S1P group had comparable AMH levels to high-dose S1P group and controls, high-dose S1P group had higher mean levels of AMH, reaching marginal significance with controls (5.72 ± 0.61 vs 4.81 ± 0.85 ng/mL, P = .050). For the nonapoptotic primordial follicle ratio, both low-dose S1P group (67.0% ± 16.4% vs 29.9% ± 19.5%, P < .001) and high-dose S1P group (51.1% ± 11.5% vs 29.9% ± 19.5%, P = .023) had superior rates when compared with controls. Interestingly, low-dose S1P groups also had a statistically higher nonapoptotic primordial follicle ratio than high-dose S1P group ( P = .047). Our findings suggest that a long-acting oral analogue of S1P might decrease spontaneous follicular apoptosis based on the nonapoptotic primordial follicle ratio and AMH levels when compared with placebo.

Time-lapse morphokinetic assessment has low to moderate ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account with the use of clustered data analysis.

OBJECTIVE: To study whether time-lapse morphokinetic (TLM) assessment predicts ploidy status when patient- and ovarian stimulation-related factors are taken into account. DESIGN: Retrospective cohort study. SETTING: Private IVF clinic. PATIENT(S): In total, 103 consecutive patients (415 blastocysts) were included. All embryos were individually cultured in a time-lapse incubator from intracytoplasmic sperm injection up to trophectoderm biopsy. Following trophectoderm biopsy on day 5 or 6, blastocysts were vitrified and 23 TLM parameters were analyzed. INTERVENTION(S): Correlations between patient- and ovarian stimulation-related factors and TLM parameters were tested in a multilevel mixed-effects linear regression model and assessed by means of intraclass correlation coefficient (ICC). MAIN OUTCOME MEASURE(S): Predictive ability of TLM parameters for euploidy. RESULT(S): The majority of TLM parameters had ICCs of 16%-47%. None of the patient- or ovarian stimulation-related factor had any systematic effect on any TLM parameter; however, body mass, total FSH dose, duration of infertility, number of previous cycles, antral follicle count, ovarian stimulation protocol, and E2 on the trigger day had a significant impact on some TLM parameters. With the use of multilevel mixed-effects logistic regression analysis, of the ten TLM parameters that were initially noted to be significantly different among euploid and aneuploid blastocysts in the univariate analysis, only five remained significant. However, the areas under the receiver operating characteristic curves at regression analysis were low, ranging from 0.55 to 0.63. CONCLUSION(S): Five TLM parameters, all related to timing of blastocyst development, have limited ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account.

Is Intracytoplasmic Sperm Injection Success Affected by the Testicular Histopathology in Nonobstructive Azoospermic Patients?

OBJECTIVE: To investigate whether the histological changes observed in testicular tissue might be correlated with pregnancy outcome in intracytoplasmic sperm injection-testicular sperm extraction (TESE) cycles in nonobstructive azoospermic patients. Embryo quality was also tested in various types of histology. STUDY DESIGN: We retrospectively analyzed the pathology results of 209 testicular biopsies from nonobstructive azoospermic patients in our assisted reproductive technologies clinic. According to the final pathology reports, the study group was divided into the following categories: Group A (Sertoli-cell-only), Group B (maturation arrest), Group C (hypospermatogenesis), Group D (normal spermatogenesis), and Group E (mixed pattern). RESULTS: Spermatozoa were identified in 114 of 209 cases (54.5%). Sperm retrieval and presence of motile spermatozoa after TESE was highest in normal spermatogenesis and lowest in Sertoli-cell-only groups. Fertilization rate was highest in the normal spermatogenesis and hypospermatogenesis groups. Embryo grades on day 3 were comparable between groups. Clinical pregnancy per embryo transfer was also similar. Both fertilization and clinical pregnancy rates were statistically higher in favor of motile spermatozoa group. CONCLUSION: Testicular histopathology not only gives information regarding the probability of retrieving sperm at TESE, but also is a good predictor of fertilization. Once a motile spermatozoon is identified, the chance of clinical pregnancy might improve probably due to relatively good optimal microenvironment within the testis.

The effects of iloprost, a prostacyclin analogue, in experimental ischaemia/reperfusion injury in rat ovaries.

Ovarian torsion is a surgical emergency affecting not only the ipsilateral ovary but also contralateral ovary. Although the conventional treatment is salpingo-oophorectomy, recent studies advocate detorsion. We hypothesized that iloprost, an analogue of prostacyclin with cytoprotective properties, may prevent the harmful effects of ischaemia-reperfusion injury in bilateral ovaries after unilateral ovarian torsion-detorsion in rat. In this study, 24 female Wistar-albino female rats were divided into four groups. Ovarian torsion was produced by applying vascular clamps to right ovaries. In Group I, bilateral oophorectomy was performed. In group II, bilateral oophorectomy was performed after a unilateral torsion period of 4h. In group III, bilateral ovaries were removed, following unilateral torsion-detorsion periods each lasted for 4h. Saline was injected i.p. 30 min before detorsion. In group IV, same experimental protocol, which was conducted in group III, was repeated. Iloprost was injected i.p. 30 min before detorsion instead of saline in group IV. Tissue levels of malondialdehyde (MDA) and nitric oxide (NO), which are the indicators for oxidative stress were determined and histopathological evaluation was performed in bilateral ovaries in all groups. The MDA and NO levels for ipsilateral ovaries of four groups were compared and no significant difference was found (p>0.05). The same comparison were done for the contralateral sides and no difference was seen either (p>0.05). In histological examination, iloprost produced improvement in I/R-induced alterations in ipsilateral and contralateral ovaries. In conclusion, these results showed that iloprost has beneficial effect on the histological appearances in both the ipsilateral and contralateral rat ovaries after unilateral torsion-detorsion.

Significance of intraepithelial lymphocytes in appendix.

The aim of this study was to investigate the importance of the increase in intraepithelial lymphocytes (IELs) in the mucosa of the appendix. One hundred and four retrospective appendectomy specimens were examined to evaluate the IELs. Intraepithelial lymphocytosis was identified in 11.5% (12 cases) of the specimens. Of these 12 cases, 6 cases with intraepithelial lymphocytosis were associated with parasitic infection. No increase in IELs was found in the 36 appendices that were removed in other primary operations. A wide range of immunologic stimuli can raise IELs in the gastrointestinal system. However, in appendectomies with clinical signs of acute appendicitis, an increase in IELs is more likely to be related to parasitic infection. This increase should be considered for the diagnosis of parasitic infections.

Nestin expression in neoplastic, fetal, and adult pancreatic endocrine cells.

BACKGROUND/AIMS: Pancreatic endocrine cells are multifunctional, differentiated cells originating from the differentiation of foregut endodermal precursors. Identification and transplantation of endocrine precursor cells appear to be the ultimate solution to diabetes mellitus, which requires lifelong treatment. Knowledge about specific markers that are used in the determination of precursor cells is limited. METHODOLOGY: In this study, the expression of nestin in well-differentiated pancreatic neuroendocrine tumors (n = 17) was investigated. In addition, developmental characteristics of nestin expression in the fetal (n = 5) and adult pancreas (n = 5) were examined. RESULTS: Findings indicated that nestin is expressed in proliferating and metabolically active cells, such as endothelium, independent of developmental and neoplastic processes, rather than endocrine precursor cells of the pancreas. CONCLUSIONS: In this study, it was concluded that nestin cannot be used as the only marker to identify pancreatic endocrine cell precursors for transplantation.

Prevention of tendon adhesions by the reconstruction of the tendon sheath with solvent dehydrated bovine pericard: An experimental study.

BACKGROUND: The achievement of the kinetic functions of a finger in full range of motion after the injury and the repair of the flexor tendon has been a challenging issue in hand surgery. Several current studies emphasize the importance of the tendon sheath for the healing of the tendon and for the prevention of the adhesive bands and advise primary repair or reconstruction of them. Various biological and synthetic materials have been used for this purpose with different rates of success. METHODS: In this experimental study, the sheaths of flexor tendons of chicken toe were excised and three groups were observed: group 1, primary sheath not repaired; group 2, sheath repair with autogenous fascia; and group 3, sheath reconstruction with solvent dehydrated bovine pericardium (SDBP). RESULTS: Histopathologic evaluations were performed on the 3rd and 12th weeks to determine the outcome of the repair methods regarding the gliding surfaces of the tendons, adhesion rates, and inflammatory reactions, which are the main issues on the healing of the tendon. The results indicated less formation of adhesions in group 3 compared with groups 1 and 2 (p = 0.001). CONCLUSION: The results showed that SDBP can be used for the reconstruction of the sheaths successively for the prevention of the adhesive bands in flexor tendon surgery.

Combined use of hair and fibrin glue for soft tissue augmentation: experimental study.

A vast variety of alloplastic materials together with autogenous grafts have been used for the correction of contour deformities related to aesthetic and reconstructive purposes. Despite a number of well-tolerated materials, the ideal has not yet been reached, although well-tolerated autogenous grafts have higher rates of resorption and distortion. The limited volume of autogenous grafts and concerns about donor-site morbidity may obligate the surgeon to use implantation materials. Covering almost every property of an ideal implantation material, hair, an organic autogenous nonvital tissue, was used for soft tissue augmentation in this experimental study. Hair pellets of 10 New Zealand rabbits were prepared and shaped as 1 x 1 x 1-cm cubes with the help of fibrin sealant, then inserted subcutaneously. The materials were evaluated 4 months later. No complications such as infection or extrusion were seen. The materials were intact, with no signs of resorption, but the shapes were distorted due to the pressure of the surrounding tissues. Histopathologic findings also demonstrated that the hair was well tolerated by the adjacent tissues. Different processes may enable hair to be used as a filler material in clinical practice. Well-tolerated, nonresorbable injectable materials or shaped implants may be obtained at low cost using hair.

Immunohistochemical study of cyclosporin-induced gingival overgrowth in renal transplant recipients.

BACKGROUND: Cyclosporin A (CsA) is an immunosuppressant widely used to treat transplant patients and various systemic diseases with immunological components. Gingival overgrowth (GO) is a common side effect of CsA administration; however, the pathogenesis of drug-induced GO is poorly understood. The aim of this study was to evaluate the expression of Ki-67, activation molecules (CD71, CD98), leukocytes activation antigens (CD45, CD45RA, CD50, CD11a, CD162, CD227, CD231), neurothelin (CD147), and novel endothelial cell antigens (B-F45, SCF87, B-D46, B-C44, VJ1/6) in gingival tissue in renal transplant recipients treated with CsA. METHODS: Tissues from 15 renal transplant patients with significant GO and 10 systemically healthy control subjects with gingivitis were studied. Frozen-section biopsies were stained with monoclonal antibodies specific for the above-mentioned antigens using an indirect immunoperoxidase technique. RESULTS: Comparison of the CsA-treated and control groups revealed no significant differences with respect to expression of Ki-67; CD50; activation molecules; neurothelin; or novel endothelial cell antigens B-D46, B-C44, and VJ1/6. However, expression patterns of CD45, CD45RA, CD11a, CD162, CD227, CD231, B-F45, and SCF87 were significantly different in CsA and control groups. CONCLUSION: Leukocyte activation antigens play an important role in CsA-induced gingival overgrowth.