microRNA-based signatures obtained from endometrial fluid identify implantative endometrium.

STUDY QUESTION: 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? SUMMARY ANSWER: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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). PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Aspiration of excess follicles before intrauterine insemination in high response cycles.

Purpose: To assess the outcome of excess follicle aspiration before intrauterine insemination (EFABI) in intrauterine insemination (IUI) cycles with 4-6 follicles ≥14 mm. Methods: A retrospective case-control study with 1559 patients undergoing IUI (donor and husband's sperm), of whom 86 underwent EFABI. We studied also an historical series of 2213 patients before EFABI implementation. For 3.5 years, all women undergoing IUI developing 4-6 follicles ≥14 mm were offered EFABI on the day of hCG administration. Pregnancy rates (PRs), multiple PRs, and adverse effects were measured. Results: EFABI was associated with a similar multiple PR (17.8% vs 17.5% in non-EFABI cases), with no triplets in EFABI patients. Live birth rates were significantly higher in EFABI cycles in IUI overall (25.5% vs 15.2%). When considered separately, the performance of EFABI resulted in significantly increased live birth rates in IUI-donor cycles (32.5% vs 18.5%), whereas the differences in IUI-husband cycles (19.5% vs 12.9%) did not reach statistical significance. The PR was 21.2% during the EFABI implementation period and 19.4% in the pre-EFABI period. Conclusions: EFABI in cycles in which 4-6 follicles reach ≥14 mm is a simple option that reduces cycle cancellation rates, results in higher PRs than cycles with 1-3 follicles, and lowers the risk of multiple pregnancy.

Proteomic pattern of implantative human endometrial fluid in in vitro fertilization cycles.

PURPOSE: To assess whether there are proteins in endometrial fluid aspirate (EFA) that predict implantation. METHODS: The population under study consisted of 285 women undergoing embryo transfer (ET). Endometrial fluid aspiration was performed immediately before ET. Results of proteomic analysis of EFA were compared between 33 cases who achieved pregnancy and 33 who did not. Samples were analysed by 2D electrophoresis and mass spectrometry. Blood samples were studied by ELISA Pregnancy rates and maternal complications were compared to those in women refusing aspiration. RESULTS: We found 23 proteins differentially expressed in the EFA in conception cycles: 4 up-regulated proteins and 19 down-regulated (FC = 0.31 0.78) (among others, arginase-1, actin B, PARK-7, cofilin-1, stathmin, annexin-2 and CAPZB). Among the five studied proteins that were differentially expressed in EFA, none was differentially expressed in serum. The aspiration procedure had no impact on pregnancy rate. No maternal complications were reported. CONCLUSIONS: We found a very different protein profile in implantative cycles, the majority of proteins being down-regulated. This probably reflects a different endometrial functional status, more favourable to implantation. EFA proteomic analysis could be a useful tool in the planning ET strategies.

Assisted Reproductive Techniques in Multiple Sclerosis: Recommendations from an Expert Panel.

INTRODUCTION: Multiple sclerosis (MS) is mainly diagnosed in women of reproductive age. However, there is a paucity of guidelines jointly prepared by neurologists and gynaecologists on managing women with MS and the desire for motherhood. Therefore, in this review we propose recommendations for such cases, with an particular focus on those requiring assisted reproductive techniques (ART). METHODS: A group of seven MS experts (4 neurologists and 3 gynaecologists) came together for three discussion sessions to achieve consensus. RESULTS: The recommendations reported here focus on the importance of early preconception counselling, the management of disease-modifying therapies before and during ART procedures, important considerations for women with MS regarding ART (intrauterine insemination, in vitro fertilisation and oocyte cryopreservation) and the paramount relevance of multidisciplinary units to manage these patients. CONCLUSIONS: Early preconception consultations are essential to individualising pregnancy management in women with MS, and an early, well-planned, spontaneous pregnancy should be the aim whenever possible. The management of women with MS and the desire for motherhood by multidisciplinary units is warranted to ensure appropriate guidance through the entire pregnancy.

Proteomics based drug repositioning applied to improve in vitro fertilization implantation: an artificial intelligence model.

Embryo implantation is one of the most inefficient steps in assisted reproduction, so the identifying drugs with a potential clinical application to improve it has a strong interest. This work applies artificial intelligence and systems biology-based mathematical modeling strategies to unveil potential treatments by computationally analyzing and integrating available molecular and clinical data from patients. The mathematical models of embryo implantation computationally generated here simulate the molecular networks underneath this biological process. Once generated, these models were analyzed in order to identify potential repositioned drugs (drugs already used for other indications) able to improve embryo implantation by modulating the molecular pathways involved. Interestingly, the repositioning analysis has identified drugs considering two endpoints: (1) drugs able to modulate the activity of proteins whose role in embryo implantation is already bibliographically acknowledged, and (2) drugs that modulate key proteins in embryo implantation previously predicted through a mechanistic analysis of the mathematical models. This second approach increases the scope open for examination and potential novelty of the repositioning strategy. As a result, a list of 23 drug candidates to improve embryo implantation after IVF was identified by the mathematical models. This list includes many of the compounds already tested for this purpose, which reinforces the predictive capacity of our approach, together with novel repositioned candidates (e.g., Infliximab, Polaprezinc, and Amrinone). In conclusion, the present study exploits existing molecular and clinical information to offer new hypotheses regarding molecular mechanisms in embryo implantation and therapeutic candidates to improve it. This information will be very useful to guide future research.Abbreviations: IVF: in vitro fertilization; EI: Embryo implantation; TPMS: Therapeutic Performance Mapping System; MM: mathematical models; ANN: Artificial Neuronal Networks; TNFα: tumour necrosis factor factor-alpha; HSPs: heat shock proteins; VEGF: vascular endothelial growth factor; PPARA: peroxisome proliferator activated receptor-α PXR: pregnane X receptor; TTR: transthyretin; BED: Biological Effectors Database; MLP: multilayer perceptron.

In-depth proteomics and natural peptidomics analyses reveal antibacterial peptides in human endometrial fluid.

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.

Recurrent Miscarriage and Implantation Failure of Unknown Cause Studied by a Panel of Thrombophilia Conditions: Increased Frequency of FXIII Val34Leu Polymorphism.

BACKGROUND: The role of acquired thrombophilia has been accepted as an etiology of recurrent miscarriage (RM); however, the contribution of specific inherited thrombophilic genes to this disorder has remained controversial. An increased incidence of RM has been suggested in women with inherited thrombophilia. METHODS: In this prospective study, assisted women with RM or repeated implant failure (RIF) were subjected to Thromboincode analysis, in order to identify 12 genetic variants for Factor V Leiden, Factor V Hong Kong, Factor V Cambridge, FII, FXIII, FXII, and A1 carriers. Patients included in this study were separated in RM cases (n=43), RIF cases (n=36) and RIF+RM (n=76). As a control group, patients undergoing IVF treatment (n=34) were used and a previously described 249 cases population as a representative sample of Spanish population were selected. Level of statistical significance was p<0.05 and groups were compared by Fisher test, except for age that was compared by t-test. RESULTS: Regarding FXIII, higher values were observed in RM (69.76%), RIF (70%) and in RM+RIF (68.42%) group when compared with our control group (52.94%) and general Spanish population (56.5%), but these differences were statistically significant only in RIF group (p=0.043, p=0.01). CONCLUSION: Concerning our findings, both RM and RIF patients had a very similar panel of thrombophilia polymorphisms, suggesting that, in both, thrombophilia might have an important contribution. High frequency of Val34Leu polymorphism in RM/ RIF presumably speaks in favor of a multifactorial RM genesis, wherean altered thrombophilia status plays a role.

Hysteroscopic hydrosalpinx occlusion with Essure device in IVF patients when salpingectomy or laparoscopy is contraindicated.

OBJECTIVE: To evaluate, in patients with hydrosalpinges, the effect on in vitro fertilization (IVF) outcome of the insertion by hysteroscopy of an intratubal blocking device, in cases where laparoscopic salpingectomy or laparoscopy was contraindicated. STUDY DESIGN: A prospective interventional case series study was conducted in fifteen women with unilateral (N=6) or bilateral hydrosalpinges (N=9) submitted for IVF. In all of them, laparoscopic salpingectomy was contraindicated. Hysteroscopic insertion of the Essure intratubal device in a consultation room setting was performed. IVF results were compared with those of women where hydrosalpinx was treated by laparoscopic salpingectomy (48 women, 76 cycles). RESULTS: There were no complications during or immediately after the procedure in any of the patients. There were four pregnancies from 16 embryo-transfers with own oocytes, one spontaneous pregnancy after unilateral Essure insertion, and one pregnancy after oocyte donation. In one case the hydrosalpinx grew and pelvic inflammatory disease developed 6 months after the insertion, requiring bilateral adnexectomy. Although not of statistical significance, IVF pregnancy rates were somewhat lower than in the laparoscopic salpingectomy group, which was attributed to the lower ovarian reserve before Essure insertion. CONCLUSION: The hysteroscopic insertion of the Essure intratubal device prior to IVF is a reasonable option in cases where laparoscopic salpingectomy is contraindicated. Larger series are required to assess pregnancy outcome.

Ovarian endometrioma but not deep infiltrating endometriosis is associated with increased serum levels of interleukin-8 and interleukin-6.

Cytokines, and specifically interleukin 6 (IL-6) and interleukin 8 (IL-8), have been associated with the pathogenesis of endometriosis. We studied serum concentrations of IL-6 and IL-8 in patients with deep infiltrating endometriosis (DIE) or ovarian endometriomas (OE), but no other forms of associated endometriosis disease type. We carried out a case-control study including 19 patients with OE alone (OE group), 14 patients with DIE alone (DIE group) and 24 healthy patients without endometriosis (C group). Serum concentrations of IL-6 and IL-8 were measured in the three groups of patients. Serum levels of both IL-6 and IL-8 were significantly higher in the OE group. A high positive correlation was found between serum IL-6 and IL-8 levels in the OE group but not in the DIE and C groups. Serum IL-8 alone achieved the highest predictive value of the presence of OE (adjusted OR: 1.44; sensitivity: 78.2%; specificity: 76.2%). The combination of IL-6 and IL-8 levels did not significantly improve the discrimination between subjects with OE and those with DIE over that of IL-8. OE but not DIE are associated with increased serum levels of IL-6 and IL-8, and thus these may become useful tools for discriminating OE alone from DIE.

Ovarian cystectomy versus laser vaporization in the treatment of ovarian endometriomas: a randomized clinical trial with a five-year follow-up.

OBJECTIVE: To investigate the effect of two laparoscopic techniques for treatment of ovarian endometriomas on recurrence rate. DESIGN: Prospective randomized clinical trial. SETTING: University teaching hospital. PATIENT(S): Ninety women with ovarian endometriomas. INTERVENTION(S): Patients were randomly selected to undergo either laparoscopic cystectomy (group 1) or laser vaporization (group 2) of ovarian endometrioma. MAIN OUTCOME MEASURE(S): Recurrence, evaluated by ultrasound scan examination, was assessed at 12 and 60 months of follow-up. RESULT(S): Endometrioma recurrence rate was higher, though not significantly different, in group 2 at 60 months of follow-up. Nevertheless, at 12 months of follow-up recurrences were statistically higher in group 2. CONCLUSION(S): The comparison between laparoscopic laser ablation and laparoscopic cystectomy for ovarian endometriomas after long-term follow-up showed earlier recurrences and a higher recurrence rate in the laser group, although at 5 years of follow-up there were no statistically significant differences.

Mifepristone-misoprostol midtrimester abortion: impact of gestational age on the induction-to-abortion interval.

BACKGROUND: This study was conducted to explore the effect of gestational age (GA) on the induction-to-abortion interval of mifepristone-misoprostol midtrimester termination of pregnancy (TOP) regimen. STUDY DESIGN: This study involved a consecutive series of 270 pregnancies between 12.0 and 22.6 weeks that have undergone legal TOP from April 2006 to June 2009. All women received a single oral dose of 200 mg mifepristone and, 36-48 h later, a course of misoprostol (an initial vaginal dose of 800 mcg plus four oral doses of 400 mcg at 3-hourly intervals). Treatment was considered to be a failure if abortion did not occur within 24 h. The impact of GA, parity and maternal age on the induction-to-abortion interval was assessed by means of Cox regression. RESULTS: Overall, the mean GA at TOP was 18.0 weeks. The mean induction-to-abortion interval was 9.8 h (SD=8.2 h; range=1-50 h), and 246 women (91%) aborted successfully within 24 h. GA at TOP and parity were the only two variables independently associated with the induction-to-abortion interval. The mean induction-to-abortion interval was increased by about 50% in patients undergoing TOP between 20.0 and 22.6 weeks (12.9 h, SD=8.9), as compared with those at 16.0-19.6 weeks (7.8 h, SD=5.9) and 12.0-15.6 weeks (8.2 h, SD=8.3) (p<.001). The effect of parity on the induction-to-abortion interval was more modest, with a 20% increase in induction-to-abortion interval in nulliparous (10.1 h, SD=9.1), as compared with women with a previous live birth (8.1 h, SD=6.7). CONCLUSIONS: The mean induction-to-abortion interval increases by 4 h after 20 weeks GA. This information may be relevant for counseling and planning of the procedure.

Unintended pregnancy after gonadal failure chemoprevention with gonadotropin-releasing hormone agonist in women with hematologic malignancies.

Many patients who receive co-treatment with GnRH agonists (GnRH-a) during chemotherapy treatment preserve their ovarian function and are at risk of unintended pregnancies. Therefore, it is important to offer them effective contraception. Also, pregnancies occurring after cancer therapy in women who received GnRH-a are not associated with adverse neonatal outcomes.