What is good practice in ultrasound (US), and more specifically during the different stages of transvaginal oocyte retrieval, based on evidence in the literature and expert opinion on US practice in ART? This document provides good practice recommendations covering technical aspects of US-guided transvaginal oocyte retrieval (oocyte pick up: OPU) formulated by a group of experts after considering the published data, and including the preparatory stage of OPU, the actual procedure and post-procedure care. US-guided transvaginal OPU is a widely performed procedure, but standards for best practice are not available. A working group (WG) collaborated on writing recommendations on the practical aspects of transvaginal OPU. A literature search for evidence of the key aspects of the procedure was carried out. Selected papers (n = 190) relevant to the topic were analyzed by the WG. The WG members considered the following key points in the papers: whether US practice standards were explained; to what extent the OPU technique was described and whether complications or incidents and how to prevent such events were reported. In the end, only 108 papers could be used to support the recommendations in this document, which focused on transvaginal OPU. Laparoscopic OPU, transabdominal OPU and OPU for IVM were outside the scope of the study. There was a scarcity of studies on the actual procedural OPU technique. The document presents general recommendations for transvaginal OPU, and specific recommendations for its different stages, including prior to, during and after the procedure. Most evidence focussed on comparing different equipment (needles) and on complications and risks, including the risk of infection. For these topics, the recommendations were largely based on the results of the studies. Recommendations are provided on equipment and materials, possible risks and complications, audit and training. One of the major research gaps was training and competence. This paper has also outlined a list of research priorities (including clarification on the value or full blood count, antibiotic prophylaxis and flushing, and the need for training and proficiency). The recommendations of this paper were mostly based on clinical expertise, as at present, only a few clinical trials have focused on the oocyte retrieval techniques, and almost all available data are observational. In addition, studies focusing on OPU were heterogeneous with significant difference in techniques used, which made drafting conclusions and recommendations based on these studies even more challenging. These recommendations complement previous guidelines on the management of good laboratory practice in ART. Some useful troubleshooting/checklist recommendations are given for easy implementation in clinical practice. These recommendations aim to contribute to the standardization of a rather common procedure that is still performed with great heterogeneity. The meetings of the WG were funded by ESHRE. The other authors declare that they have no conflict of interest. NA. ESHRE Pages content is not externally peer reviewed. The manuscript has been approved by the Executive Committee of ESHRE.

STUDY QUESTION: Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? SUMMARY ANSWER: Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. WHAT IS KNOWN ALREADY: EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. STUDY DESIGN, SIZE, DURATION: We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. LIMITATIONS, REASONS FOR CAUTION: Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. WIDER IMPLICATIONS OF THE FINDINGS: This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates.

Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo–endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16–21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. The FASTQ data are available in the GEO database (access number GSE178917). One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.

The composition of endometrial fluid reflects the status of the endometrium; it is a good atraumatic source of information on embryo implantation processes and possible pathological conditions. Although some attempts have been made to characterise its proteome, the catalogue of its proteins remains incomplete and little has been done to analyse the natural peptides it contains. Here, we present a comprehensive analysis of the proteins and natural peptides of the endometrial fluid. The protein content of samples from 11 individuals was analysed using the novel timsTOF Pro mass spectrometer. We identified 4694 proteins with at least one peptide with FDR < 1%, of which 2261 were found in >50% of the samples. A pooled endometrial fluid sample was used for isolation and analysis of the natural peptides. Mass spectrometry analysis identified 3899 naturally occurring peptides from 238 different proteins. Among these, there were some putative natural antibacterial peptides. Antimicrobial activity of peptides derived from elafin and Cu/Zn superoxide dismutase was confirmed using microbiological assays. Our results substantially expand the catalogue of known endometrial fluid proteins and provide extensive new information on the natural peptide content of this fluid. SIGNIFICANCE: The endometrial fluid contains many proteins whose clinical relevance is still unknown. Some might be merely markers of endometrial function, but others might play a role in embryo nutrition and/or implantation. Human endometrial fluid analysis might open the door to new developments in embryo transfer strategies in in-vitro fertilisation programmes and lead to improvements in the composition of embryo culture media. Here, we report, for the first time, antimicrobial activity of endometrial fluid peptides. Such peptides could play an important role in the balance of the recently described uterine microbiota.

To investigate the influence of vitamin D status on in vitro fertilisation (IVF) results, a meta-analysis of 15 cohort studies of 3711 women undergoing IVF was performed. Women were classified into three groups according their vitamin D levels (≥30 ng/mL considered replete/sufficient; 21–29 ng/mL insufficient and <20 ng/mL deficient). Three different meta-analyses were performed: (i) sufficient vs deficient; (ii) sufficient vs ‘insufficient + deficient’; (iii) ‘sufficient + insufficient’ vs deficient. Comparing IVF outcomes in sufficient versus deficient groups (considering autologous and donor oocyte cycles together), we found women with sufficient vitamin D had significantly higher biochemical pregnancy (OR = 1.43 [1.06–1.95]), ongoing pregnancy (OR = 1.29 [1.02–1.64]), and live birth (OR = 1.74 [1.31–2.31]) rates, with a non-significant trend to a higher clinical pregnancy rate (OR = 1.31 [0.94–1.82]), whereas implantation and miscarriage rates were similar. When the meta-analysis was restricted to autologous oocytes, the parameters which had been significant in the joint analysis remained significant, and differences in implantation (OR = 1.64, [1.17–2.29]) and clinical pregnancy (OR = 1.47 [1.2–1.69]) rates became significant. No significant differences were found when considering only cycles with donor oocytes. The sufficient + insufficient vs deficient and sufficient vs ‘insufficient + deficient’ comparisons identified significant differences in live birth rate. The meta-analysis shows that sufficient vitamin D status is associated with better outcomes in IVF. Nonetheless, there are many demographic, geographic and clinical parameters that may be related to vitamin D status that need to be ascertained before concluding that the better results are due to the higher levels of vitamin D.