Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.

STUDY QUESTION Are the LH levels at the start of ovarian stimulation predictive of suboptimal oocyte yield from GnRH agonist triggering in GnRH antagonist down-regulated cycles? SUMMARY ANSWER LH levels at the start of ovarian stimulation are an independent predictor of suboptimal oocyte yield following a GnRH agonist trigger. WHAT IS KNOWN ALREADY A GnRH agonist ovulation trigger may result in an inadequate oocyte yield in a small subset of patients. This failure can range from empty follicle syndrome to the retrieval of much fewer oocytes than expected. Suboptimal response to a GnRH agonist trigger has been defined as the presence of circulating LH levels <15 IU/l 12 h after triggering. It has been shown that patients with immeasurable LH levels on trigger day have an up to 25% risk of suboptimal response. STUDY DESIGN, SIZE, DURATION In this retrospective cohort study, all patients (n = 3334) who received GnRH agonist triggering (using Triptoreline 0.2 mg) for final oocyte maturation undergoing a GnRH antagonist cycle in our centre from 2011 to 2017 were included. The primary outcome of the study was oocyte yield, defined as the ratio between the total number of collected oocytes and the number of follicles with a mean diameter >10 mm prior to GnRH agonist trigger. PARTICIPANTS/MATERIALS, SETTING, METHODS The endocrine profile of all patients was studied at initiation as well as at the end of ovarian stimulation. In order to evaluate whether LH levels, not only at the end but also at the start, of ovarian stimulation predicted oocyte yield, we performed multivariable regression analysis adjusting for the following confounding factors: female age, body mass index, oral contraceptives before treatment, basal and trigger day estradiol levels, starting FSH levels, use of highly purified human menopausal gonadotrophin and total gonadotropin dose. Suboptimal response to GnRH agonist trigger was defined as <10th percentile of oocyte yield. MAIN RESULTS AND THE ROLE OF CHANCE The average age was 31.9 years, and the mean oocyte yield was 89%. The suboptimal response to GnRH agonist trigger cut-off (<10th percentile) was 45%, which was exhibited by 340 patients. Following confounder adjustment, multivariable regression analysis showed that LH levels at the initiation of ovarian stimulation remained an independent predictor of suboptimal response even in the multivariable model (adjusted OR 0.920, 95% CI 0.871–0.971). Patients with immeasurable LH levels at the start of stimulation (<0.1 IU/l) had a 45.2% risk of suboptimal response, while the risk decreased with increasing basal LH levels; baseline circulating LH <0.5 IU/L, <2 IU/L and <5 IU/L were associated with a 39.1%, 25.2% and 13.6% risk, respectively. LIMITATIONS, REASONS FOR CAUTION The main limitation of the study is its retrospective design. WIDER IMPLICATIONS OF THE FINDINGS This is the largest study of GnRH agonist trigger cycles only, since most of the previous research on the predictive value of basal LH levels was performed in dual trigger cycles. LH values should be measured prior to start of ovarian stimulation. In cases where they are immeasurable, suboptimal response to GnRH agonist trigger can be anticipated, and an individualized approach is warranted. STUDY FUNDING/COMPETING INTEREST(S) There was no funding and no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

37Rodríguez et al. Nueva metodología geométrica para evaluar la morfología del eritrocito normaL Nueva metodología geométrica para evaluar la morfología del eritrocito normalNew physical and mathematical methodology to evaluate the morphology of normal erythrocyte ResumenObjetivo. Desarrollar una nueva metodología para caracterizar la estructura del eritrocito normal mediante el espacio ocupado por el anillo del eritrocito normal caracterizado con el método de Box Counting. Método. se analizaron las imágenes de 20 extendidos de sangre periférica, cuyos eritrocitos fueron evaluados por un experto como normales. Se superpusieron dos rejillas Kp de 5 x 5 pixeles y Kg de 10 x 10 pixeles, para calcular el espacio ocupado por dos regiones del eritrocito estos son, el disco y centro de este, visto de manera frontal mediante el método de Box Counting.Resultados. Los espacios ocupados por la región del disco con la rejilla Kp variaron entre 47 y 56, la región del centro del eritrocito, varió entre 9 y 14. La dimensión fractal de estas dos regiones varió entre 0,941 y 1,115 para el disco, entre 0,652 y 1,222 para el centro. Conclusiones. La estructura del eritrocito normal puede ser caracterizada mediante el espacio ocupado por cada una de las regiones del eritrocito a partir de la geometría fractal.Palabras claves: matemáticas, fractales, hematología, eritrocito, morfofisiología, geometría. AbstractObjective. Develop a new methodology to characterize the structure of the normal erythrocyte through the space occupied by the ring of the normal erythrocyte characterized by the method of Box Counting. Method. Images of 10 peripheral blood smears were analysed, whose erythrocytes were evaluated by an expert as normal. There were superimposed two Kp grids of 5 x 5 pixels and Kg of 10 x 10 pixels, to calculate the space occupied by two regions of the erythrocyte which are, disc and centre of this, seen of way frontal by the method of Box Counting. Results. The spaces occupied by the disc region with grid Kp varied between 47 and 56, the central region of the erythrocyte, varied between 9 and 14. The fractal dimension of these two regions varied between 0,941 and 1,115 for the disc, between 0.652 and 1,222 for the centre. Conclusions. The normal erythrocyte structure can be characterized by the space occupied by the regions erythrocyte from fractal geometry.

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Gallbladder cancer is a rare, aggressive malignancy that has a poor overall prognosis. Effective treatment consists of early detection and surgical treatment. With the wide spread treatment of gallbladder disease with minimally invasive techniques, the rate of incidental gallbladder cancer has seen an equitable rise along with stage migration towards earlier disease. Although the treatment remains mostly surgical, newer modalities such as regional therapy as well as directed therapy based on molecular medicine has led to improved outcomes in patients with advanced disease. We aim to summarize the management of gallbladder cancer along with the newer developments in this formidable disease process.