Barcelona IVI Clinic

IntroductionThe uterus exhibits dynamic peristaltic activity across the menstrual cycle, playing a critical role in reproductive processes such as sperm transport and embryo implantation. However, current imaging methods to assess this activity provide little information and are limited by subjectivity and lack of sensitivity. A recent pilot study has shown the potential of intracavitary electrohysterography (IC‐EHG) to study peristalsis in the uterine fundus. This study aimed to generalize previous results, compare peristalsis in other uterine regions, and study more peristaltic features.Material and MethodsThis prospective multicenter cohort study included 40 healthy women with proven fertility. IC‐EHG signals were recorded from different uterine sites for 30 min during three menstrual phases: mid‐follicular (MF), early luteal (EL), and late luteal (LL) using a custom‐designed multipolar catheter. Primary outcomes: contraction frequency (CT/min) and amplitude (μV); secondary outcomes: basal amplitude, contraction time percentage, contractility index and local organization index. Statistical comparisons between phases and regions were performed using Wilcoxon signed‐rank tests.ResultsA total of 95 fundal and 90 lower‐segment IC‐EHG recordings were analyzed. Contraction frequency peaked during the EL phase (fundus: 3.91 CT/min; lower segment: 4.01 CT/min) and was lowest during MF (fundus: 3.28 CT/min, p = 0.042; lower segment: 3.65 CT/min, p = 0.024). Fundal contraction amplitude decreased progressively from MF (16.27 μV) to LL phase (10.56 μV, p < 0.001). Basal amplitude, contraction time percentage and contractility index were also lowest in the LL phase for both uterine regions. Except for frequency, fundus peristaltic activity features were smaller than those of the lower segment, significantly during the MF phase. Local coordination index revealed lower local cell organization in the fundus across all phases, with maximum coordination during EL in both regions (p < 0.01).ConclusionsIC‐EHG technique enables objective, reproducible, and quantitative assessment of multiple aspects of uterine peristalsis, revealing distinct regional and cycle‐phase–dependent patterns. The decline in most contractile features during the LL phase supports the physiological uterine quiescence required for embryo implantation. The uterine fundus is more active during the MF phase. This study provided reference values for healthy, fertile conditions and could inform further investigation of alterations due to disorders or intervention strategies.

STUDY QUESTION:Which mechanisms of action and candidate drugs can be used to treat endometrial failure caused by molecular alterations rather than endometrial timing?SUMMARY ANSWER:Genistein, pioglitazone, alprostadil, flunisolide, and tenoxicam emerged as potential therapies to treat two molecular causes of endometrial failure not originating in endometrial timing.WHAT IS KNOWN ALREADY:Several studies have described molecular profiles of endometrial failure unrelated to endometrial timing and proposed diagnostic tools based on clinical and transcriptomic data, but effective therapeutic options remain lacking. Hence, there is a pressing need for tailored treatments to enable personalised medicine in endometrial-factor infertility.STUDY DESIGN, SIZE, DURATION:This multicentre prospective study, conducted at 5 fertility clinics in Spain between January 2019 and August 2022, included 192 patients undergoing in vitro fertilisation with hormone replacement therapy, whose endometrial biopsies were collected during the mid-secretory phase.PARTICIPANTS/MATERIALS, SETTING, METHODS:Of 291 endometrial biopsies, 192 met the quality criteria and 161 were classified according to clinical and transcriptomic data using a semi-supervised learning model for prognosis. Before classification, transcriptomic variation related to endometrial timing was corrected using our validated transcriptomic endometrial-dating model. Profiles were analysed using systems pharmacology approaches combining network analysis and reversal signature matching to identify therapeutic drugs capable of reversing molecular disruption. Candidate drugs were grouped by mechanism of action and prioritised by side-effect profile. Selected drugs were validated in endometrial cells through RT-PCR, F-actin staining, and enzyme-linked immunosorbent assays.MAIN RESULTS AND THE ROLE OF CHANCE:Four transcriptomic profiles were identified using artificial intelligence models, each with distinct clinical implications. Two profiles were associated with poor prognosis: clinical miscarriage-associated (CMA, n = 27) and biochemical miscarriage-associated (BMA, n = 16). CMA was characterised by upregulation of differentiation-related genes and BMA by upregulation of immune-related genes. The 4 profiles were homogeneous in demographic and embryological parameters (age, BMI, and embryo quality), reinforcing their biological relevance. Approved drugs capable of reversing these disrupted expression patterns were identified. Both BMA and CMA were linked to abnormal decidualisation, while BMA also showed immune dysregulation. Genistein and pioglitazone promoted decidualisation in vitro, whereas alprostadil, flunisolide, and tenoxicam inhibited immune responses in endometrial cell cultures, supporting their potential therapeutic role in endometrial failure not originating in endometrial timing.LIMITATIONS, REASONS FOR CAUTION:Although our artificial intelligence-based stratification model revealed clinical and functional differences among profiles, it was designed for drug repurposing rather than predictive diagnosis. A larger, specifically designed study would be required to validate predictive performance and generalisability. Further clinical trials are needed to evaluate the proposed drugs as personalised treatments for this condition.WIDER IMPLICATIONS OF THE FINDINGS:This is the first application of a systems-based drug repurposing strategy in IVF to develop tailored therapeutic interventions. We propose genistein, pioglitazone, alprostadil, flunisolide, and tenoxicam as approved, safe drugs identified through an evidence-based approach that could prevent loss of good-quality embryos and miscarriage due to maternal endometrial factors. These findings, supported by functional in vitro validation, pave the way for future clinical trials advancing personalised medicine in endometrial-factor infertility.TRIAL REGISTRATION NUMBER:Not applicable

Abstract Study question What are the number of collapses and the percentage of embryo surface reduction during blastocyst collapse from which clinical outcomes are impaired? Summary answer Results showed that a single embryo collapse can reduce clinical outcomes, but the threshold of embryo surface reduction varies with the patient’s age. What is known already Embryo collapse before hatching is frequently observed in blastocysts obtained from Assisted Reproductive Technologies (ART). During the collapse, the blastocyst surface reduces transiently and further expands once or more times. Weak contractions seem to be beneficial for blastocyst hatching, but intense or vigorous collapses impair both hatching and embryo development. The incidence of embryo collapse varies between studies, from 15 to 55%, mainly because the embryos with weak collapses may be considered as non-collapsed. Yet, a clear shrinkage threshold for substantial collapses has not been consistently established, the cut-off values ranging from 20 to 50% of blastocyst surface reduction. Study design, size, duration This retrospective study included 1,548 blastocysts obtained by ICSI fertilization of the patient’s own oocytes between January 2016 and December 2022. Patients were admitted to four sanitary centres, but oocyte fertilization and embryo culture were conducted at the same IVF lab. Patients (n = 461) were split into three groups: ≤34 years old (y/o), 35-39 y/o, and ≥40 y/o. All patients consented to participate. Participants/materials, setting, methods Fertilized oocytes were incubated in single-step medium until day+6 in an Embryoscope™ TL incubator. Blastocyst collapse was assessed by counting the number and intensity of collapses (% reduction of embryo surface in each collapse). Surface measures were conducted with Elypse-EmbryoviewerTM using the trophectoderm area in contact with the inner surface of the zona pellucida as a reference. Blastocysts were distributed into three groups according to the maternal age. Results are expressed as mean ± SD. Main results and the role of chance The number and intensity of blastocyst collapses impaired clinical outputs, but differences existed based on the maternal age. In the group of patients ≤ 34 y/o, the pregnancy and live birth rates did not differ between non-collapsed and single-collapse blastocysts (P &amp;gt; 0.05), but clinical outcomes decreased significantly if a second collapse occurs (P &amp;lt; 0.05). When patient’s age was equal to or higher than 35 y/o, a single collapse significantly affected clinical outcomes (P &amp;lt; 0.05). Regarding their intensity, embryo collapses associated with a surface reduction of 10-15% did not affect implantation rates in any of the maternal groups, but significantly impaired pregnancy and live birth rates in patients ≥ 40 y/o (P &amp;lt; 0.05). When embryo collapses resulted in a surface reduction ≥ 20%, all clinical outcomes were significantly lower in patients ≥ 35 y/o than in those ≤ 34 y/o. It should be noted that, in the youngest group, the threshold of surface reduction during the collapse significantly affecting clinical outcomes was higher (40%). Limitations, reasons for caution This preliminary study examined embryos from patients admitted to four different IVF clinics that were fertilized and incubated in a single IVF lab. To strengthen the robustness of our results, the study should be extended to other centres and increase the sample size. Wider implications of the findings Blastocyst collapse should be used as a criterion for embryo selection in ART when using patient own’s oocytes. A single collapse impairs clinical outcomes when its intensity is above the threshold 10% for patients ≥ 40 y/o, 20% for patients of 35-39 y/o, and 40% for patients ≤ 34 y/o. Trial registration number No

Abstract Preimplantation Genetic Testing (PGT) enables the genetic analysis of embryos generated through in vitro fertilization (IVF). Traditional applications of PGT include PGT for aneuploides (PGT-A - to identify whole-chromosome aneuploidies), PGT for structural rearrangements (PGT-SR - to detect chromosomal imbalances resulting from a balanced structural rearrangement, such as chromosomal translocations or inversions, in one of the patients), and PGT for monogenic disorders (PGT-M - to minimize the risk of transmitting a known inherited monogenic disorder). Recent advancements in Next Generation Sequencing (NGS) technologies used in PGT, combined with a substantial reduction in associated costs, have made Whole Genome Sequencing (WGS) a feasible approach at the embryonic level. This methodology provides genome-wide data, albeit typically at low coverage depth.These technological developments have enabled novel applications of PGT, such as screening embryos for monogenic variants (including de novo mutations) and polygenic embryo screening (PES). However, these approaches raise significant limitations and challenges, particularly from ethical and genetic counseling perspectives.This session will offer an overview of the key ethical and counseling dilemmas associated with the use of these emerging technologies, in order to facilitate a broader discussion on the scope and boundaries of PGT.

Abstract Study question Is there evidence of cell cycle checkpoint activation in preimplantation embryos with double stranded breaks (DSBs) in their DNA, induced using CRISPR? Summary answer Cells with persistent double strand DNA damage exhibit longer timeframes to reach key morphological milestones than those with successful repair, consistent with checkpoint activation. What is known already In most cell types, DNA damage leads to activation of cell cycle checkpoints that halt the cycle while DNA repair is attempted. It has been hypothesised that early embryos lack (or have relaxed) checkpoints, and that this may be necessary to allow the embryo to divide rapidly and synchronously during the first few mitoses. It is thought that maternal (oocyte derived) inhibitors may be responsible for suppressing checkpoints until activation of the embryonic genome. Inadequate checkpoint control may be responsible for the genetic instability and sensitivity to DNA damage seen during early preimplantation stages, which is of clinical importance. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods Precise induction of DNA damage involved injection of CRISPR-Cas9 ribonuclear complex (RNP), along with the sperm at the time of fertilization (ICSI). All embryos were disaggregated on day-3 and their cells subjected to whole genome amplification. Amplified products underwent low-pass next generation sequencing to detect segmental aneuploidy related to failure of DNA repair, while the site targeted using CRISPR-Cas9 was PCR amplified and sequenced to confirm whether it had successfully undergone repair. Main results and the role of chance Embryos that successfully repaired the induced DNA damage showed timings of pronuclei appearance/disappearance and cell divisions that were indistinguishable from control embryos. Rates of embryo arrest prior to completion of the second mitotic division were also similar between the two groups (25% versus 24%). In contrast, embryos with unresolved DNA damage, as evidenced by the detection of chromosomal fragments involving a breakpoint at the site targeted using CRISPR, were delayed in reaching the same developmental milestones (p &amp;lt; 0.0001 for time to first cleavage division) and displayed a much higher incidence of arrest (63% before the second mitotic division; p = 0.0002). The delay in mitotic progression in blastomeres with DNA damage represents strong evidence that a checkpoint is active in the cells of cleavage stage embryos, sensing double strand DNA breaks and slowing the cell cycle. However, the fact that 36% of embryos with unresolved DNA damage continued to progress, albeit at a slower rate, is consistent with the notion that checkpoint control is less stringent in early human embryos. This relaxation of control is likely to contribute to the genetic instability seen at the cleavage stage, including a risk of unresolved DNA damage and a high frequency of chromosomal malsegregation (causing mosaicism). Limitations, reasons for caution While altered cell cycle timings are consistent with checkpoint activation, other factors could conceivably influence the duration of key morphokinetic events. Complimentary experiments to test the functionality of specific checkpoints and/or the activity of their individual components will be required for definitive proof that embryonic checkpoints are active but weakened. Wider implications of the findings Segmental aneuploidy, micronucleation and mosaicism are frequently seen in human preimplantation embryos and are associated with reduced likelihood of ongoing pregnancy. Such abnormalities are symptomatic of genetic instability associated with excessively permissive checkpoints. These results highlight the need for culture systems that protect embryos from cellular stressors, especially DNA damage. Trial registration number N.A.