Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation.

STUDY QUESTION: Are uterine fluid-derived extracellular vesicles (UF-EVs) a 'liquid biopsy' reservoir of biomarkers for real-time monitoring of endometrial status? SUMMARY ANSWER: The transcriptomic cargo of UF-EVs reflects the RNA profile of the endometrial tissue as well as changes between the non-receptive and the receptive phase, possibly supporting its use for a novel endometrial receptivity test. WHAT IS KNOWN ALREADY: EVs have been previously isolated from uterine fluid, where they likely contribute to the embryo-endometrium crosstalk during implantation. Based on a meta-analysis of studies on endometrial tissue implantation-associated genes and the human exosomes database, 28 of the 57 transcripts considered as receptivity markers refer to proteins present in human exosomes. However, the specific transcriptomic content of receptive phase UF-EVs has yet to be defined. STUDY DESIGN, SIZE, DURATION: Two experimental series were set up. First, we simultaneously sequenced RNA species derived from paired UF-EVs and endometrial tissue samples collected from physiologically cycling women. Second, we analyzed RNA species of UF-EVs collected during the non-receptive (LH + 2) and receptive (LH + 7) phase of proven fertile women and from the receptive (LH + 7) phase of a population of women undergoing ART and transfer of euploid blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS: For paired UF-endometrial tissue sampling, endometrial tissue biopsies were obtained with the use of a Pipelle immediately after UF collection performed by lavage of the endometrial cavity. Overall, n = 87 UF samples were collected and fresh-processed for EV isolation and total RNA extraction, while western blotting was used to confirm the expression of EV protein markers of the isolated vesicles. Physical characterization of UF-EVs was performed by Nanoparticle Tracking Analysis. To define the transcriptomic cargo of UF-EV samples, RNA-seq libraries were successfully prepared from n = 83 UF-EVs samples and analyzed by RNA-seq analysis. Differential gene expression (DGE) analysis was used to compare RNA-seq results between different groups of samples. Functional enrichment analysis was performed by gene set enrichment analysis with g:Profiler. Pre-ranked gene set enrichment analysis (GSEA) with WebGestalt was used to compare RNA-seq results with the gene-set evaluated in a commercially available endometrial receptivity array. MAIN RESULTS AND THE ROLE OF CHANCE: A highly significant correlation was found between transcriptional profiles of endometrial biopsies and pairwise UF-EV samples (Pearson's r = 0.70 P < 0.0001; Spearman's ρ = 0.65 P < 0.0001). In UF-EVs from fertile controls, 942 gene transcripts were more abundant and 1305 transcripts less abundant in the LH + 7 receptive versus the LH + 2 non-receptive phase. GSEA performed to evaluate concordance in transcriptional profile between the n = 238 genes included in the commercially available endometrial receptivity array and the LH + 7 versus LH + 2 UF-EV comparison demonstrated an extremely significant and consistent enrichment, with a normalized enrichment score (NES)=9.38 (P < 0.001) for transcripts up-regulated in LH + 7 in the commercial array and enriched in LH + 7 UF-EVs, and a NES = -5.40 (P < 0.001) for transcripts down-regulated in LH + 7 in the commercial array and depleted in LH + 7 UF-EVs. When analyzing LH + 7 UF-EVs of patients with successful versus failed implantation after transfer of one euploid blastocyst in the following cycle, we found 97 genes whose transcript levels were increased and 64 genes whose transcript levels were decreased in the group of women who achieved a pregnancy. GSEA performed to evaluate concordance in transcriptional profile between the commercially available endometrial receptivity array genes and the comparison of LH + 7 UF-EVs of women with successful versus failed implantation, demonstrated a significant enrichment with a NES = 2.14 (P = 0.001) for transcripts up-regulated in the commercial array in the receptive phase and enriched in UF-EVs of women who conceived, and a not significant NES = -1.18 (P = 0.3) for transcripts down-regulated in the commercial array and depleted in UF-EVs. In terms of physical features, UF-EVs showed a homogeneity among the different groups analyzed except for a slight but significant difference in EV size, being smaller in women with a successful implantation compared to patients who failed to conceive after euploid blastocyst transfer (mean diameter ± SD 205.5± 22.97 nm vs 221.5 ± 20.57 nm, respectively, P = 0.014). LARGE SCALE DATA: Transcriptomic data were deposited in NCBI Gene Expression Omnibus (GEO) and can be retrieved using GEO series accession number: GSE158958. LIMITATIONS, REASONS FOR CAUTION: Separation of RNA species associated with EV membranes might have been incomplete, and membrane-bound RNA species-rather than the internal RNA content of EVs-might have contributed to our RNA-seq results. Also, we cannot definitely distinguish the relative contribution of exosomes, microvesicles and apoptotic bodies to our findings. When considering patients undergoing ART, we did not collect UFs in the same cycle of the euploid embryo transfer but in the one immediately preceding. We considered this approach as the most appropriate in relation to the novel, explorative nature of our study. Based on our results, a validation of UF-EV RNA-seq analyses in the same cycle in which embryo transfer is performed could be hypothesized. WIDER IMPLICATIONS OF THE FINDINGS: On the largest sample size of human EVs ever analyzed with RNA-seq, this study establishes a gene signature to use for less-invasive endometrial receptivity tests. This report is indeed the first to show that the transcriptome of UF-EVs correlates with the endometrial tissue transcriptome, that RNA signatures in UF-EVs change with endometrial status, and that UF-EVs could serve as a reservoir for potential less-invasive collection of receptivity markers. This article thus represents a step forward in the design of less-invasive approaches for real-time monitoring of endometrial status, necessary for advancing the field of reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by a competitive grant from European Society of Human Reproduction and Embryology (ESHRE Research Grant 2016-1). The authors have no financial or non-financial competing interests to disclose. TRIAL REGISTRATION NUMBER: NA.

Safety of fertility treatments in women with systemic autoimmune diseases (SADs).

Introduction: Systemic Autoimmune Diseases (SADs) include systemic lupus erythematosus, antiphospholipid antibody syndrome, rheumatoid arthritis, systemic sclerosis, Sjogren's syndrome, mixed connective tissue disease, idiopathic inflammatory myopathies and vasculitis. SADs often occur in women of childbearing age and can affect fertility. Both infertility treatments and fertility preservation techniques are thus often indicated. Areas covered: The literature regarding the safety of fertility-related drugs for both fertility preservation and infertility treatment in patients affected by SADs was reviewed. Based on current knowledge, all the options for fertility preservation should be contemplated in patients with SADs who are at risk for fertility loss, including GnRH analogue administration, oocyte/embryo vitrification and ovarian tissue cryopreservation. Similarly, if pregnancy is not contraindicated in a patient with a SAD, neither should be any fertility treatment. Expert opinion: Women with SADs should postpone conception until a stable disease has been achieved for at least 6 months. When infertility treatments are needed, women with antiphospholipid antibodies should receive concomitant anticoagulation. If in vitro fertilization/intra-cytoplasmic sperm injection and embryo transfer is required, ovarian hyperstimulation and the inherent risk of thrombosis should be eliminated by GnRH-agonist trigger and cycle segmentation. Counselling about adherence to anti-rheumatic therapy to prevent disease exacerbations is also critical.

Embryonic extracellular vesicles as informers to the immune cells at the maternal-fetal interface.

Extracellular vesicle (EV) exchange is emerging as a novel method of communication at the maternal-fetal interface. The presence of the EVs has been demonstrated in the preimplantation embryo culture medium from different species, such as bovines, porcines and humans. Preimplantation embryo-derived EVs have been shown to carry molecules potentially able to modulate the local endometrial immune system. The non-classical major histocompatibility complex (MHC) class I molecule human leucocyte antigen (HLA)-G, the immunomodulatory molecule progesterone-induced blocking factor and some regulatory miRNAs species are contained in embryo-derived EV cargo. The implanted syncytiotrophoblasts are also well known to secrete EVs, with microvesicles exerting a mainly proinflammatory effect while exosomes in general mediate local immunotolerance. This review focuses on the current knowledge on the potential role of EVs released by the embryo in the first weeks of pregnancy on the maternal immune cells. Collectively, the data warrant further exploration of the dialogue between the mother and the embryo via EVs.

Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study.

STUDY QUESTION: Does female obesity affect the dynamic parameters of embryo quality assessed by time-lapse analysis? SUMMARY ANSWER: Female obesity does not affect the dynamic embryo quality as determined by image acquisition and time-lapse analysis. WHAT IS KNOWN ALREADY: Female obesity impairs natural and assisted reproduction but there is no agreement on the specific contribution of gametes, embryos or endometrial receptivity. In this preliminary study the dynamic parameters of embryo quality are assessed for the first time by time-lapse analysis. STUDY DESIGN, SIZE, DURATION: Two-year cohort retrospective study comparing embryos from three groups of patients according to the presence of infertility and/or obesity. PARTICIPANTS AND SETTING: Participants attended a University-affiliated private clinic where ICSI was performed. Using an IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we monitored individual embryos from 89 patients: 71 embryos from 13 obese infertile women, 242 embryos from 45 normoweight infertile women and 111 embryos from 31 normoweight fertile oocyte donors. The chronological pattern of cell divisions (timings of cell cleavages) and other morphologic features (time-dependent cell size and nucleation) was recorded. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos from obese and normoweight infertile women showed similar cleavage patterns, but occurring more slowly, to those from fertile donors. These differences were statistically significant for t2 (time of cleavage to two-blastomere embryo) (P = 0.016), t3 (P = 0.014), t4 (P = 0.003) and t5 (P = 0.040). LIMITATIONS, REASONS FOR CAUTION: These are preliminary data from a retrospective analysis with a limited sample size. GENERALIZABILITY TO OTHER POPULATIONS: Not recommended until further studies using time-lapse analysis of a larger sample have been performed. STUDY FUNDING/COMPETING INTEREST(S): None.

[Vascular access for hemodialysis. Clinical experience]. / Accesso vascolare per emodialisi. Una esperienza clinica.

Seventy-nine arteriovenous fistulas for periodic hemodialysis were created in 62 uremic patients: 64 primary vascular accesses and 15 salvage operations for malfunctioning fistulas. Sixty-three distal and 1 proximal arteriovenous fistulas between the radial artery and the cephalic vein, were created as primary vascular access. The utilization time of the primary arteriovenous fistulas was longer than 5 years in 34%, 3-4 years in 25% and 0-2 years in 41% of cases. Eleven fistulas evidenced thrombosis: a salvage operation (resection-anastomosis) was possible for 9 (89%). A salvage operation was possible for 100% of poor flow fistulas. The utilization's time of the salvage fistulas is longer than 5 years in 15%, 3-4 years in 15% and 0-2 years in 70% of cases.

The use of dura mater allograft in the surgical repair of large defects of the abdominal wall.

The dura mater allograft is being used as an abdominal fascia substitute and major advantages are documented. It was used successfully in four patients at this institution, with a ten month follow-up period. Its strength, durability, tissue tolerance, flexibility, ease of handling, nonallergenic property, availability and stability in the presence of infection make it a good graft to be used in the repair of large abdominal hernias. We are quite convinced that long term follow-up results will verify the results of the animal experiments done by others and affirm the durability of the graft. In one patient, this graft has been shown to be adequate for placement in an infected bed and to have a definite resistance to infection. The results of incidental operative biopsy in another patient, have shown an excellent incorporation of the dura graft into the surrounding tissues. In our hands, the experience with the dura graft using monofilament sutures has been quite positive.

Preliminary studies of nonvirion antigens associated with herpes simplex virus 1 and 2 (HSV1 - HSV2).

Biochemical and radioimmunological studies have indicated the presence of a nonvirion antigen associated with herpes virus. Guinea pig kidney cells were grown in culture and infected (I-cells) for 3h with herpes virus. Uninfected cells (N-cells) served as control. The cells were collected and sonicated to obtain, after centrifugation, "soluble extract" (SI, SN). The pellets were extracted with KCl (ISP, NSP). SN, SI were submitted to gel-filtration on Sephadex G-200. Extracts from I and N cells gave similar patterns. After labelling with 125I, greater resolution was obtained in a second gel-filtration with a much smaller load. ISP contained protein species not present in NSP. Polyacrylamide gel electrophoresis of peaks from SN and SI showed that SI contained some radioactive bands not found in SN. Labelled materials from SI and SN were incubated with sera from cancer patients and normal subjects. No differences in binding were seen with SN, but SI showed 14-30% more 125I binding with sera of cancer patients than with sera from normal subjects. No such difference was seen between ISP and NSP.