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STUDY QUESTION Are cumulative pregnancy rates better if supernumerary embryos are vitrified on Day 5/6 instead of Day 3? SUMMARY ANSWER The results do not show a significant difference in cumulative pregnancy rates between the Day 3 and Day 5/6 vitrification groups. WHAT IS KNOWN ALREADY Pregnancy and live birth rates following IVF or ICSI treatment are higher after extended embryo culture and blastocyst transfer (Day 5/6) compared to cleavage-stage (Day 3) transfer. Cumulative pregnancy rates from one oocyte retrieval (OR) cycle show no significant difference after fresh and frozen embryo transfers, but only one study has used vitrification for the cryopreservation of supernumerary embryos while four studies have used a slow freezing protocol. STUDY DESIGN, SIZE, DURATION Our prospective randomized controlled trial was performed in an academic centre between January 2018 and August 2020. Patients were randomized into vitrification Day 3 (n = 80) or Day 5/6 (n = 81) groups. The primary outcome was the cumulative ongoing pregnancy rate (cOPR), considering only the first pregnancy for each couple. The power calculation revealed that 75 patients were required in each group, when assuming a 50% cOPR with four embryo transfers in the vitrification Day 3 group vs two transfers in the vitrification Day 5/6 group. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients <38 years undergoing their first or second OR cycles were randomized at the start of the first cycle. Up to two cycles were included in the analysis. A fresh embryo transfer was performed on Day 3. Supernumerary embryos (with ≥6 cells, <25% fragmentation, and equal blastomeres) or blastocysts (with expansion grade ≥2 with inner cell mass and trophectoderm score A/B) were vitrified on Day 3 or Day 5/6, respectively, and then transferred at a later date. A time-to-event analysis was performed with the patient's first ongoing pregnancy as the event of interest and the number of embryo transfers as the time component. The statistical comparison was performed by a Cox proportional hazards model. Cumulative costs of vitrification on Day 3 vs Day 5/6 were explored and compared using Mann–Whitney U tests. MAIN RESULTS AND THE ROLE OF CHANCE By December 2021, 233 transfers (96 fresh and 137 frozen) in 77 patients were performed in the vitrification Day 3 group and 201 transfers (88 fresh and 113 frozen) in 77 patients were performed in the vitrification Day 5/6 group. The time-to-event analysis did not show a difference between the two arms with regard to the patient's first ongoing pregnancy as the primary study outcome (hazard ratio [HR] 1.25, 95% CI 0.82; 1.92, P = 0.30). The cumulative ongoing pregnancy rate after eight transfers (from one or two ORs) was 57% in the vitrification Day 3 group vs 58% in the vitrification Day 5/6 group. The median number of embryo transfers until a pregnancy was achieved was five vs four, respectively, in the vitrification Day 3 group vs the Day 5/6 group. Similar results were found for the secondary study outcome, i.e. clinical pregnancy with foetal heart rate (HR 1.19, 95% CI 0.78; 1.80, P = 0.41). The cumulative clinical pregnancy rate (cCPR) after eight embryo transfers was 62% in the vitrification Day 3 group vs 59% in the vitrification Day 5/6 group. The median number of transfers until a pregnancy was achieved was four in both groups. The healthcare consumption pattern differed between the two groups and we observed higher costs for the vitrification Day 3 group compared to the vitrification Day 5/6 group, although these differences were not statistically significant. LIMITATIONS, REASONS FOR CAUTION Although our power calculation revealed that only 75 patients were needed in each study group (β = 0.87, α < 0.05), the numbers were low. Also, different numbers of single and double embryo transfers were performed between the two groups, which may have affected the results. The cost analysis was performed on a subset of the patients and is therefore exploratory. WIDER IMPLICATIONS OF THE FINDINGS Our study shows no difference in the cumulative pregnancy rate nor costs after fresh and frozen embryo transfers of at most two sequential OR cycles between the Day 3 and Day 5/6 vitrification groups; however, obstetric and perinatal outcomes should be taken into account to determine the best strategy. STUDY FUNDING/COMPETING INTEREST(S) This study was funded as an investigator-sponsored study of S.D. by Merck nv/sa Belgium, an affiliate of Merck KGaA, Darmstadt, Germany, and by Gedeon Richter Benelux (PA18-0162). The authors declare no conflict of interest related to this study. TRIAL REGISTRATION NUMBER NCT04196036. TRIAL REGISTRATION DATE 15 January 2018. DATE OF FIRST PATIENT'S ENROLMENT 15 January 2018. [ABSTRACT FROM AUTHOR]

The article discusses the reasons why the gynecology and obstetrics field need to use a noninvasive diagnostic test for minimal to mild endometriosis with a high sensitivity. The presence of endometrial-like tissue outside the uterus is known as endometriosis, and it is associated with a chronic inflammatory reaction in the pelvis and often results in subfertility and pain. The stages of endometriosis include minimal, mild, moderate and severe.

Research Question: What are the perspectives of preimplantation genetic testing (PGT) patients in Belgium on the ethics of PGT for polygenic risk scoring (PGT-P)?, Design: In-depth interviews (18 in total, 10 couples, 8 women, n = 28) were performed with patients who had undergone treatment with PGT for monogenic/single-gene defects (PGT-M) or chromosomal structural rearrangements (PGT-SR) between 2017 and 2019 in Belgium. Participants were asked about their own experiences with PGT-M/SR and about their viewpoints on PGT-P, including their own interest and their ideas on its desirability, scope and consequences. Inductive content analysis was used to analyse the interviews., Results: Participants stated that their experiences with PGT-M/SR had been physically, psychologically and practically difficult. Most participants stated that, partly because of these difficulties, they did not see the added value of knowing the risk scores of embryos via PGT-P. Many participants worried that PGT-P could lead to additional anxieties, responsibilities and complex choices in reproduction and parenthood. They argued that not everything should be controlled and felt that PGT-P, especially non-medical and broad screening, was going too far. With regards to the clinical implementation of PGT-P, participants in general preferred PGT-P to be limited to people with a serious polygenic family history and wanted embryo selection decisions to be made by healthcare professionals., Conclusions: This study shows that individuals with experience of PGT-M/SR saw PGT-P as different from PGT-M/SR. They had various ethical concerns with regards to PGT-P, especially regarding broadly offering PGT-P. These stakeholder viewpoints need to be considered regarding potential PGT-P implementation and guidelines., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: This study aimed to assess the attitudes and experiences of subfertile couples applying for medically assisted reproduction (MAR) using their own gametes towards reproductive genetic carrier screening (RGCS) for monogenic conditions., Methods: A prospective survey study was conducted where subfertile couples were recruited from the fertility centre of a university hospital in Flanders, Belgium. Participants were offered RGCS free of charge and completed self-administered questionnaires at three different time points., Results: The study sample consisted of 26 couples. Most participants had no children, did not consider themselves as religious, and had some form of higher education. Overall, attitudes towards RGCS were mostly positive and the intention to participate in RGCS was high. Anxiety scores were only elevated and clinically relevant for a limited number of participants. A large proportion of participants would consider preventive reproductive options like prenatal diagnosis or in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) combined with pre-implantation genetic testing for monogenic conditions (PGT-M) in the event of an increased likelihood of conceiving a child with a hereditary condition. Participants were satisfied with their decision to undergo RGCS, and the majority would recommend RGCS to other couples., Conclusion: Our study findings suggest that subfertile couples applying for MAR using their own gametes find RGCS acceptable and have a positive attitude towards it. This study provides valuable insights into the perspectives of these couples, highlighting the need for appropriate counseling and timely information provision., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Reproductive genetic carrier screening (RGCS) allows for the identification of couples who have an increased likelihood of conceiving a child with a particular autosomal recessive or X-linked condition. The aim of this study was to assess the level of satisfaction, anxiety, knowledge retention, psychosocial and counseling-related aspects among couples who chose to have RGCS. Participants were initially informed about their screening results by telephone. After obtaining a written report of test results, participants were asked to complete an individual self-administered questionnaire. All participants (n = 67) felt they had enough information to make an informed choice. None of the participants regretted their choice to have RGCS. Test results were most often shared with parents (61%) or siblings (37%). Our findings demonstrate that the information/counseling and reporting strategy that was used in the context of this study led to high participant satisfaction, an increase in knowledge over time and favorable psychosocial and counseling-related outcomes., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Background: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive., Methods: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression., Results: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP 3 R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells., Conclusions: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst., (© 2023. The Author(s).)

Study Question: Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents?, Summary Answer: Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs., What Is Known Already: Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype., Study Design, Size, Duration: This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles., Participants/materials, Setting, Methods: To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5-10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping., Main Results and the Role of Chance: The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT., Limitations, Reasons for Caution: The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs., Wider Implications of the Findings: Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available., Study Funding/competing Interest(s): This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. All other authors have no conflict of interest to declare., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: The objective of this study was to examine the prevalence of chronic endometritis (CE) in infertile women, its impact on reproductive outcomes, and the accuracy of hysteroscopy as a screening tool for CE., Design: This was a prospective observational study., Participants: Participants involved in this study were 514 asymptomatic patients with infertility., Setting: The review was conducted in a tertiary care center., Methods: The participants underwent a hysteroscopy and endometrial biopsy (EMB). Antibiotics were given for cases of CE. We investigated the prevalence of CE in patients starting assisted reproductive technologies (ART) as a primary outcome. Secondary outcomes were the clinical pregnancy rate (CPR) in the ART cycle after hysteroscopy, EMB, and antibiotic treatment in cases of CE; the cumulative CPR in the subsequent 2 years after hysteroscopy and EMB; the sensitivity and specificity of hysteroscopy as a screening tool compared to EMB as the "gold standard" for diagnosing CE., Results: CE was identified in 2.8% of patients starting ART (11/393). CPRs did not differ significantly between patients with CE and the entire cohort of patients without CE in the subsequent ART cycle (OR: 0.43; 95% CI: 0.09-2.02) or in the 2 years after EMB (OR: 0.56; 95% CI: 0.16-1.97). In a matched control comparison (with matching for age, basal FSH, and cause of infertility), CPR in patients with CE did not differ in the subsequent ART cycle (OR: 0.39; 95% CI: 0.09-1.61); however, their CPR in the 2 years after EMB was significantly lower (OR: 0.22; 95% CI: 0.13-0.38). The sensitivity and specificity of hysteroscopy as a screening tool for diagnosing CE were 8.3% and 90.1%, respectively., Limitations: Due to our cohort's low CE prevalence, we could not detect significant differences in CPRs., Conclusion: CE is rare in our studied population of asymptomatic patients starting ART. Hysteroscopy cannot replace EMB for diagnosing CE., (© 2023 S. Karger AG, Basel.)

Objectives: This study aimed to assess if the indication for oocyte reception (OR) or embryo reception (ER) impacts the reproductive and obstetric outcomes by evaluating our experience at a tertiary fertility centre and by performing a literature review on this subject. Several previous studies have reported that, in contrast to other types of fertility treatment, the indication for OR/ER seems to have little impact on the outcomes. However, the compared indication groups vary considerably between these studies, and some data indicates worse outcomes in patients who developed premature ovarian insufficiency (POI) due to Turner syndrome or treatment with chemotherapy/radiotherapy., Design: A retrospective analysis of all cases of OR/ER at a tertiary fertility centre from 2001 until 2020 was conducted. We analysed 584 cycles from 194 individual patients. A literature review on the impact of indication on reproductive or obstetric outcomes of OR/ER was performed using the following databases: PubMed/MEDLINE, Embase, and the Cochrane Library. A total of 27 studies were included and analysed., Participants, Setting, Methods: For the retrospective analysis, patients were divided into three major indication groups: failure of autologous assisted reproductive technology, POI, and genetic disease carrier. To assess reproductive outcomes, we determined pregnancy rate, implantation rate, miscarriage rate, and live birth rate. For comparing obstetric outcomes, we reviewed the term of birth, mode of delivery, and birthweight. Outcomes were compared using Fisher's exact test, χ2 test, and one-way ANOVA utilizing the GraphPad tool., Results: There were no significant differences in reproductive and obstetric outcomes between the three major indication groups in our population, in line with the findings reported by existing literature. Data on impaired reproductive outcomes in patients with POI after chemotherapy/radiotherapy are conflicting. Obstetrically, these patients are at higher risk of preterm birth and possibly also low birthweight, especially after abdomino-pelvic or total body irradiation. For patients with POI due to Turner syndrome, most data suggest similar pregnancy rates but a higher rate of pregnancy loss, and obstetrically an increased risk of hypertensive disorders and caesarean section., Limitations: The small number of patients in the retrospective analysis resulted in low statistical power when evaluating differences between smaller subgroups. There were some missing data on the occurrence of complications during pregnancy. Our analysis covers a period of 20 years, during which several technological innovations have also been made., Conclusions: Our study shows that the important heterogeneity in couples treated with OR/ER does not significantly impact their reproductive or obstetric outcomes, except for POI due to Turner syndrome or treatment with chemotherapy/radiotherapy, where there seems to be an important uterine/endometrial component that cannot be entirely overcome by providing a healthy oocyte., (© 2023 S. Karger AG, Basel.)

Objectives: To explore informed choice in reproductive genetic carrier screening (RGCS)., Methods: Women visiting a gynaecologist practice in Flanders (Belgium) were asked to consider participation in a study where RGCS was offered for free to them and their male partner. A modified Multidimensional Measure of Informed Choice was used to determine whether couples who opted for RGCS made an informed choice. In addition, we assessed risk perception, feelings towards RGCS, anxiety and decisional conflict., Results: Most participants (82 %, n = 63/77) made an informed choice with regard to RGCS according to our modified MMIC. Thirteen participants made an uninformed choice due to insufficient knowledge and one participant because of insufficient knowledge and value-inconsistency. Anxiety scores were elevated for three participants. Two participants presented with decisional conflict., Conclusion: Our results show high rates of informed choice among non-pregnant couples who were offered RGCS in a research study and received up to 30 min of pre-test counseling., Practice Implications: Limited resources outside a research context may impact informed choice. Pre-test counselling initiatives for RGCS should ideally be organized in such a way that information can be provided at multiple time points to avoid information overload and to allow for a reflection period., Competing Interests: Declaration of Competing Interest E.V.S., H.P., H.V., J.V., K.P., G.M., and P.B. declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Through carrier screening couples at-risk of conceiving a child with an autosomal recessive or X-linked condition can be identified prior to conception. The aim of this study was to assess knowledge, attitudes and preferences regarding reproductive genetic carrier screening (RGCS) among reproductive-aged men and women in Flanders (Belgium). Women and men of reproductive age visiting their pharmacist were invited to answer a self-administered questionnaire. Prior to filling in the questionnaire, participants were asked to read an information leaflet explaining some key concepts about RGCS. Our sample included 387 individuals of reproductive age, of which 68.5% were female and 31.5% were male. Most of the participants were below 34 years old (72.9%), didn't have children (68.6%) and were currently in a relationship (69.1%). Offering RGCS to couples that want to have children was found acceptable by 86% of participants. However, fewer participants would consider RGCS for themselves in the future (61%). We observed a positive correlation between attitude score/knowledge score and the intention to have RGCS. Half of the participants (50.9%) preferred the disclosure of individual test results. Most of participants indicated that RGCS should be offered through the gynecologist (81.1%), followed by the GP (71.5%) and the Centre for Human Genetics (64.8%). About 68.9% of participants were willing to pay out-of-pocket for an RGCS test. We recommend that RGCS should ideally be implemented through a tailored implementation strategy whereby individual needs and preferences can be taken into account., (© 2022. The Author(s), under exclusive licence to European Society of Human Genetics.)

Background: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres., Results: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest., Conclusions: Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF., (© 2022. The Author(s).)