Leuven Institute for Fertility and Embryology

Intrauterine human chorionic gonadotropin (hCG) administration prior to embryo transfer has been explored as a method to improve embryo–endometrial synchrony, but clinical evidence remains inconsistent due to variations in dosage, timing, embryo stage, and cycle type. This study evaluated the clinical impact of a pre-transfer intrauterine hCG flush performed 48–72 h before frozen embryo transfer (FET) in hormonally prepared cycles. This prospective non-randomized cohort study included 200 women with primary infertility undergoing FET with at least one good-quality blastocyst. Participants were assigned either to the hCG flush group (n = 100) or a control group (n = 100). In the control group, 20 participants discontinued before FET for non-medical reasons, and one was lost to follow-up, leaving 79 for analysis. Outcomes included implantation rate, clinical pregnancy rate, live birth rate, miscarriage rate, multiple pregnancy rate, and procedure-related adverse events. Logistic regression generated odds ratios (ORs) with 95% confidence intervals (CIs). A p-value < 0.05 was considered significant. Baseline demographics and cycle parameters were comparable between groups (hCG flush vs. control group, respectively). Implantation rate did not differ significantly between the hCG flush and control groups (58.2% vs. 54.6%, respectively; p = 0.39). Clinical pregnancy rates were similarly comparable between the groups (73.0% vs. 79.7%, respectively; p = 0.88). The live birth rate was higher in the hCG group (70.0%) than the control group (62.0%), although not statistically significant (OR 1.51; 95% CI 0.76–2.99; p = 0.21). Multiple pregnancy rates were also comparable between hCG group and control group (15.0% and 24.0%, respectively, p = 0.18). However, a reduction in miscarriage rate was observed in the hCG group (3.0%) on comparing with the control group (16.4%) (p < 0.001). Pre-transfer intrauterine hCG administration 48–72 h before blastocyst-stage FET did not significantly influence implantation, clinical pregnancy, or live birth rates but was associated with miscarriage rate. These findings from this preliminary study suggest a potential biological effect of localized hCG exposure on early gestational maintenance and warrant confirmation through larger, multicentre randomized controlled studies.

Rescue in vitro maturation (rescue-IVM) refers to the post-retrieval culture of immature oocytes - typically at the germinal vesicle (GV) or metaphase I (MI) stage - under laboratory conditions, allowing them to complete maturation to metaphase II (MII) and become suitable for fertilization.This case report presents a successful pregnancy following rescue-IVM and preimplantation genetic testing for aneuploidy (PGT-A) in a 35-year-old woman with unexplained infertility and a history of ectopic pregnancy.Immature GV-stage oocytes retrieved during in vitro fertilization (IVF) were cultured using standard protocols, and several progressed to MII without pharmacologic stimulation. Following delayed intracytoplasmic sperm injection (ICSI), two euploid blastocysts were obtained, one of which led to a viable singleton pregnancy.This case underscores the clinical relevance of rescue-IVM in IVF cycles with high proportions of immature oocytes, particularly in patients with diminished ovarian reserve or tubal factor infertility. It highlights the importance of individualized assisted reproductive technology (ART) strategies, and the potential of advanced embryology techniques - such as time-lapse monitoring and optimized ICSI timing - to improve outcomes in poor-prognosis cases.

Endocrine dysfunctions refer to alterations in hormone production, release, or regulation that can significantly impact health. In pregnant women or those planning pregnancy, these conditions may manifest as disorders such as polycystic ovary syndrome, hypothyroidism, endometriosis, gestational diabetes mellitus, and other metabolic issues, which could potentially cause infertility or pregnancy complications. Research and clinical experience indicate that hormones play a crucial role in basic physiology and are essential for overall health and well-being. At the same time, lifestyle—defined as daily habits related to nutrition, exercise, sleep, stress management, and other factors—directly influences microbial composition and hormonal regulation. The human microbiome, a diverse community of microorganisms residing within the human body, plays essential roles in supporting overall health. The increasing prevalence of hormonal disorders, especially in urban populations, has heightened interest in how modern lifestyles—characterised by sedentary habits, chronic stress, imbalanced diets, and inadequate sleep—may contribute to the development or aggravation of these conditions, leading to higher infertility rates or pregnancy complications if untreated. This review investigates the interaction between hormonal dysfunction, the human microbiome, and lifestyle factors, with a focus on their effects on pregnant women and those seeking to conceive. Its purpose is to provide a comprehensive overview of the underlying pathophysiological mechanisms and to examine preventative and therapeutic approaches that could alter these patterns.

Are we able to establish endometrium epithelial organoids (EEOs) from endometrial samples obtained from women with PCOS, and do they differ from non-PCOS EEOs? We were able to establish, for the first time, PCOS EEOs which capture endometrial abnormalities present in women with PCOS, including increased inflammation and decreased receptivity-related gene expression. Patient-derived EEOs could serve as a tool to study endometrial dysfunction, as diseased tissue-derived organoid models typically retain the disease-related traits. In PCOS, endometrial dysfunction likely contributes to subfertility and pregnancy complications, yet previous research on the endometrial epithelial compartment has been scarce and, so far, no PCOS-derived EEOs have been established. EEOs were established from endometrial biopsies from two cohorts of women with PCOS-including overweight/obese (O-PCOS, n = 4) and lean (L-PCOS, n = 4)-along with BMI-matched controls (overweight/obese control (O-Ctrl), n = 4; lean control (L-Ctrl), n = 4). EEOs were exposed to combinations of steroid hormones (β-estradiol (E2), progesterone, cAMP, and the Wnt/β-catenin signaling (WNT) inhibitor XAV-939)for 6days to simulate the proliferative or secretory phases of the menstrual cycle, with or without simultaneous androgen exposure with dihydrotestosterone (DHT). Bulk RNA-sequencing was conducted to identify variations in gene expression between PCOS and Ctrl EEOs, while reverse-transcription quantitative PCR RT-qPCR was employed to validate these results. Morphological assessment of EEOs was performed using hematoxylin and eosin staining and immunostaining. The size of EEOs was evaluated after 6days of hormonal exposure. PCOS EEOs from both BMI groups demonstrated increased inflammation-related gene expression (including increased expression of Oncostatin M Receptor (OSMR) and Intercellular Adhesion Molecule 1 (ICAM1)) and showed a reduced diameter compared to their respective control EEOs. The O-PCOS EEOs displayed an aberrant response to steroid exposure with E2 and progesterone (including reduced expression of receptivity-related genes progestagen-associated endometrial protein and leukemia inhibitory factor)as compared to control EEOs. Addition of DHT to the culture media did not affect EEO transcriptome, aligning with the minimal androgen receptor (AR) expression in the EEOs. Sequencing data are available from the corresponding author upon request. The study should be replicated with a larger number of samples and with other PCOS phenotypes apart from different weight categories. Furthermore, as this work is the first one to establish PCOS EEOs, future studies should focus on incorporating other endometrial cell types, including immune cells, in a co-culture system. This novel in vitro organoid model for PCOS captures the endometrial abnormalities present in the two weight categories of women with PCOS, thereby providing a valuable tool to gain insights into PCOS-related endometrial dysfunction. Our findings propose potential links to the increased risk of pregnancy complications in women with PCOS, such as the role of altered receptivity and implantation environment including increased inflammation, which may contribute to aberrant placentation and subsequent placental dysfunction. Jusélius Foundation, Novo Nordisk Foundation, Research Council of Finland, Horizon 2020 Marie-Curie MATER Innovative Training Network (all to T.T.P.), Fund for Scientific Research Flanders-Belgium (FWO, G0A6719N to J.V. and GO99023N to H.V.); KU Leuven Research Fund (C14/21/116 to H.V. and C14/24/152 to J.V.), University of Oulu Scholarship Foundation Grant (to L.L.), and PhD grant of China Scholarship Council (CSC, to M.W.). The authors have no conflicts of interest to declare.

Long-read whole-genome sequencing (lrWGS) enhances haplotyping by providing more phasing information per read compared to short-read sequencing. However, its use for single-cell haplotype phasing remains underexplored. This proof-of-concept study examines lrWGS data from single cells for small variant (SNV and indel) calling and haplotyping using the Genome in a Bottle (GIAB) Ashkenazi trio. lrWGS was performed on single-cell (1 cell) and multi-cell (10 cells) samples from the offspring. Chromosome-length haplotypes were obtained by leveraging both long reads and pedigree information. These haplotypes were further refined by replacing them with matched parental haplotypes. In single-cell and multi-cell samples, 92% and 98% of heterozygous SNVs, and 74% and 78% of heterozygous indels were accurately haplotyped. Applied to human embryos for preimplantation genetic testing (PGT), lrWGS demonstrated 100% consistency with array-based methods for detecting monogenic disorders, without requiring phasing references. Aneuploidies were accurately detected, with insights into the mechanistic origins of chromosomal abnormalities inferred from the parental unique allele fractions. We show that lrWGS-based concurrent haplotyping and aneuploidy profiling of single cells provides an alternative to current PGT methods, with applications potential in areas such as cell-based prenatal diagnosis and animal and plant breeding.

Abstract Study question Should a national embryo transfer (ET) policy designed in the era of cleavage stage transfers be adapted for blastocyst transfers to avoid twin pregnancies? Summary answer Double embryo transfer policy results in unacceptable high twin rates at all ages and treatment ranks which is significantly more pronounced after double blastocyst transfer. What is known already In 2003, authorities in Belgium implemented a law to reduce multiple pregnancies after ART by tailoring ET policies based on age, embryo quality, and cycle rank. A choice between SET or Double Embryo Transfer (DET) depending on embryo quality is allowed in the second cycle for patients under 36y with SET being mandatory with top quality embryos. DET is allowed as of the first cycle for patients aged 36y or more regardless of embryo quality. Blastocyst transfer results in significantly higher live birth rates, implying an increased risk of multiple pregnancies after multiple blastocyst transfers. Study design, size, duration A analysis of 35,523 double embryo fresh transfer cycles over a period of 10 years from 2012 to 2021 from the Belgian national BELRAP (Belgian Registry on Assisted Procreation) registry was performed. The study included 26,930 double cleavage stage transfers (DET-CL) and 8,593 double blastocyst transfers (DET-BL). Participants/materials, setting, methods The primary outcomes were delivery rate (DR) per transfer and twin delivery rate (TDR). DR includes all deliveries with a least one live birth per transfer; the TDR includes all twin deliveries as a proportion of all deliveries. For analysis, the data were categorized into the following age groups: 18-25 years, 26-29 years, 30-35 years, 36-39 years and 40-45 years. Cycle ranks were categorized as follows: rank 1, rank 2 and cycle 3- ≥ 6. Main results and the role of chance For all age groups and cycle ranks, delivery rate per transfer and twin delivery rate per delivery were significantly higher after DET-Bl compared to DET-Cl (26.0% versus 19.9%; p &amp;lt; 0.0001 and 28.0% versus 20.6%; p &amp;lt; 0.0001, respectively). In the age groups 26-29 years, 30-35 years and 36-39 years, a significantly higher TDR was observed in DET-Bl compared to DET-Cl, respectively (34.4% versus 24.2%, p = 0.0005; 33.5% versus 24.9%, p &amp;lt; 0.0001 and 24.3% versus 17.3%, p = 0.0002). TDR after DET-Bl was significantly lower in women aged 40-45 years compared to all other age groups, 18-25 years, 26-29 years, 30-35 years and 36-39 years, respectively (11.3% versus 32.8%, p &amp;lt; 0.0001; 11.3% versus 34.4%; p &amp;lt; 0.0001; 11.3% versus 33.5%; p &amp;lt; 0.0001 and 11.3% versus 24.3%; p &amp;lt; 0.0001), respectively). DR was significantly lower in cycle rank 1 compared to cycle rank 2 (22.5% versus 25.5%; p &amp;lt; 0.0001) and compared to cycle rank 3 - &amp;gt; 6 rank 1 and (22.5% versus 27.1%; p &amp;lt; 0.0001). After DET-Bl, a significantly lower TDR was observed in cycle rank 1 compared to cycle rank 2 (15.6% versus 30.0%; p &amp;lt; 0.0001) and compared to cycle rank 3 - &amp;gt; 6 (15.6% versus 29.7%; p &amp;lt; 0.0001). Limitations, reasons for caution The retrospective design is a limitation. Outcomes might be influenced by clinical and laboratory improvements over the years. The registry does not collect embryo quality data making it impossible to assess the effect of embryo quality on the ET policy and outcome. Wider implications of the findings This data indicates that a double blastocyst transfer should only be considered from the age of 40 onwards. In the first cycle, regardless of age, we observe a significantly lower twin delivery rate, suggesting that a double ET is performed when only poor-quality blastocysts are available. Trial registration number Not applicable

The development and use of Cobots in industry, institutions, and healthcare, as well as the strong penetration of chatBots, reinforce the interdisciplinary nature of technological processes, which are flooded with data and information, making it almost impossible for the younger generation to go in depth and understand the requirements of a particular process. In contrast older experts (e.g., engineers aged 50–75) have a high level of expertise in technological process as well as other skills such as management knowledge, which are essential for the implementation of technological projects. The paper presents a learning management system (LMS) developed as an interactive human -computer-human online digital platform Wisdom of Age (WoA) aiming to close the gap between generations by transferring knowledge from senior experts to developing companies and opening the gates for new certified technological development. The social dimension (healthy aging for seniors, better training for juniors) and the sustainable development for companies are combined with a suitable digital dimension. The paper presents and examines based on survey results, which requirements must be placed on such a platform to optimally support and satisfy mentors and learners.

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention.