MicroRNAs (miRNAs) are gene expression regulators. Altered miRNA levels are associated with diabetes, insulin resistance, and inflammation. Insulin resistance and inflammation are both features of ...Polycystic ovary syndrome (PCOS). The aim of this study was first to assess differences in selected miRNAs (miR-146a, miR-155, miR-320, miR-370, miR-486), involved in insulin sensitivity regulation and inflammation, in women with or without PCOS. Second, to investigate relationships among these miRNAs, insulin, High mobility group box 1 (HMGB1), and IL-6 in follicular fluid (FF), serum 17-beta estradiol (E2), and the number of dominant follicles.
Thirty PCOS and thirty-six non-PCOS women undergoing
fertilization were enrolled. RNA from granulosa cells (GC) and FF was extracted and the specific miRNAs were evaluated using qRT-PCR. HMGB1, insulin, and IL-6 in FF, and serum E2 were assayed using specific kits.
MiR-146a, miR-155, miR-486 were upregulated and miR-320 and miR-370 were downregulated in GC from the PCOS patients. In FF, miR-146a, miR-155, and miR-486 showed lower levels in PCOS, whereas miR-320 and miR-370 showed an opposite trend but no significant changes were observed. These miRNAs showed relationships with Body Mass Index (BMI), age, E2, number of dominant follicles, insulin, and HMGB1.
In conclusion, the miRNAs analyzed showed changes in PCOS ovaries and had relationships with indices of inflammation and insulin sensitivity within the ovary, providing evidence for new regulatory mechanisms.
Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzoapyrene (BaP), induce reproductive toxicity. Several existing ...studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.
Purpose
Several mathematical models have been developed to estimate individualized chances of assisted reproduction techniques (ART) success, although with limited clinical application. Our study ...aimed to develop a decisional algorithm able to predict pregnancy and live birth rates after controlled ovarian stimulation (COS) phase, helping the physician to decide whether to perform oocytes pick-up continuing the ongoing ART path.
Methods
A single-center retrospective analysis of real-world data was carried out including all fresh ART cycles performed in 1998–2020. Baseline characteristics, ART parameters and biochemical/clinical pregnancies and live birth rates were collected. A seven-steps systematic approach for model development, combining linear regression analyses and decision trees (DT), was applied for biochemical, clinical pregnancy, and live birth rates.
Results
Of fresh ART cycles, 12,275 were included. Linear regression analyses highlighted a relationship between number of ovarian follicles > 17 mm detected at ultrasound before pick-up (OF17), embryos number and fertilization rate, and biochemical and clinical pregnancy rates (
p
< 0.001), but not live birth rate. DT were created for biochemical pregnancy (statistical power–SP:80.8%), clinical pregnancy (SP:85.4%), and live birth (SP:87.2%). Thresholds for OF17 entered in all DT, while sperm motility entered the biochemical pregnancy’s model, and female age entered the clinical pregnancy and live birth DT. In case of OF17 < 3, the chance of conceiving was < 6% for all DT.
Conclusion
A systematic approach allows to identify OF17, female age, and sperm motility as pre-retrieval predictors of ART outcome, possibly reducing the socio-economic burden of ART failure, allowing the clinician to perform or not the oocytes pick-up.
In the absence of international guidelines indicating the usage of vitrification rather than slow-freezing, the study aim was to analyze a large cohort of slow-frozen/thawed embryos to produce a ...rationale supporting the standardization of IVF cryopreservation policy.
This retrospective analysis included 4779 cleavage stage embryos cryopreserved by slow-freezing/thawing from September 2009 to April 2017 at a single Center. Biological and clinical outcomes of three different commercial kits adopted sequentially, i.e. Vitrolife Cleave Kit® from Vitrolife (kit 1) vs. K-SICS-5000 Kit® and K-SITS-5000 Kit® from Cook Medical (kit 2) and Freeze/Thaw 1™ Kit® from Vitrolife (kit 3) were collected and compared in the light of cryoprotectants composition.
Kit 3 compared to kit 1 and kit 2 showed significantly (P < 0.001) higher embryo survival (79.9% vs. 75.6 and 68.1%, respectively) and frozen embryo replacement (91.5% vs. 86.5 and 83.3%, respectively) rates, and significantly (P < 0.001) lower blastomere degeneration rate (41.5% vs. 43.6 and 52.4%, respectively). No significant difference for clinical outcomes was observed among kits. Only a slight positive trend was observed for kit 3 vs. kit 1 and kit 2 on delivery rate per thawing cycle (7.12% vs. 4.19 and 4.51%, respectively; P < 0.058) and live birth rate (3.07% vs. 2.59 and 1.93%, respectively, P < 0.069). Thawing solutions of kit 3 were similar to those of any warming protocol.
A defined concentration of extracellular cryoprotectants in thawing/warming solutions had a beneficial effect on the embryo cryosurvival rate. Results could provide the rationale for the adoption of a single standardized warming protocol.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile ...dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses
. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test;
< 0.05,
= 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10
-1 × 10
ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells
. Intracellular cAMP production, Ca
increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test;
< 0.001;
= 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC
range = 12 ± 0.9-24 ± 1.7 ng/ml; β-arrestin 2 EC
range = 140 ± 14.1-313 ± 18.7 ng/ml; Kruskal-Wallis test;
≥ 0.05;
= 4). Kinetics analysis revealed that intracellular Ca
increased upon cell treatment by 4 μg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test;
< 0.05;
= 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC
s were demonstrated (Kruskal-Wallis test;
> 0.05;
= 5). Apart from preparation-specific intracellular Ca
increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.
Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female ...steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact
,
and
gene expression in hGLC, nor
and
expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.
The assessment of the embryo quality is crucial to maintain an high pregnancy rate and to reduce the risk of multiple pregnancy. The evaluation of the pronuclear and nucleolar characteristics of ...human zygote have been proposed as an indicator of embryo development and chromosomal complement. The aim of the current study was to assess the role of pronuclear morphology evaluation in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) cycles.
Retrospective clinical analysis on 755 non-elective transfers of only one embryo (ET). Embryo assessment was performed in days 1 and 2. Clinical and biological data were recorded and analyzed according to embryo and/or pronuclear morphology.
Both pronuclear and embryo morphology were significantly related to clinical pregnancy and live-birth rates. No significant difference in clinical pregnancy and live-birth rates was detected when the pronuclear and embryo morphology assessments were combined. Embryo morphology and maternal age were the only independent predictors of favorable outcome by logistic regression analysis.
Pronuclear evaluation is effective to select the best zygotes if ET is performed at day 1, whereas it did not improve the clinical outcomes when combined with embryo morphology evaluation in day 2.
Objective To investigate the hypothesis that Z-score criteria represent a reliable predictor of implantation rate and pregnancy outcome in in vitro fertilization (IVF) and intracytoplasmic sperm ...injection (ICSI) cycles, excluding the possibility of embryo selection before the embryo transfer. Design Retrospective clinical study. Setting Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova (ASMN), Reggio Emilia, Italy. Patient(s) We analyzed 393 pregnancies obtained by IVF or ICSI cycles. Intervention(s) Morphologic evaluations of Z-score in pre-embryos obtained from IVF or ICSI cycles. Main Outcome Measure(s) Evaluations of Z-scores, implantation rate, and clinical pregnancy outcome. Result(s) We did not find any statistically significant correlation between the Z-score of 1032 embryos transferred in 393 embryo transfers and the implantation rate or the pregnancy outcome. In particular, the best Z-score identified (Z1, 7.2%) did not seem to correlate with embryo implantation rate or pregnancy outcomes any better than those with worse scores (Z2, 6.9% and Z3, 85.9%). Conclusion(s) Our results seem to confirm that Z-score alone cannot be considered a better tool than standard morphologic criteria for identifying, controlling, or selecting embryos with a better chance of successful ongoing pregnancy.
Objective To evaluate the effect of the 2004 Italian regulations (insemination of ≤3 oocytes/cycle, transfer of all embryos, prohibition of embryo cryopreservation) on outcomes of assisted ...reproduction treatment (ART). Design Case-control study. Setting The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. Patient(s) Women undergoing ART for the first time. Intervention(s) Comparing outcomes of ART between 2 years before (n = 900) and after (n = 936) the law's implementation (March 10, 2004). Main Outcome Measure(s) Rates of fertilization, pregnancy, “take-home baby,” and multiple pregnancies. Result(s) During the pre-law period, statistically significantly more patients reached embryo transfer (odds ratio 1.9; 95% CI, 1.5, 2.5), and embryo transfer rate per cycle was statistically significantly higher (3.1 ± 1.7 vs. 2.2 ± 0.7), but the overall transfer of good embryos was lower (OR 0.6; 95% CI, 0.5, 0.8). The pregnancy rates per aspiration cycle were similar between the periods, but the pregnancy rate per embryo transfer and birth rate with at least one liveborn baby per embryo transfer were statistically significantly lower in the pre-law period (OR 0.7; 95% CI, 0.5, 0.9). The multiple births rate was not different between the two periods. Conclusion(s) In contrast to prior pessimistic expectations, the obligation to transfer all available embryos produced from ≤3 inseminated oocytes neither reduced success rates of ART nor increased the multiple births rate.
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