Understanding how the first cell lineages in human development are specified and maintained has fundamental importance and clinical implications for regenerative medicine, infertility and pregnancy loss. Although mouse models have provided valuable insights into transcription factors regulating early development, translating these findings to human embryos has been limited by ethical, technical and biological constraints. Functional studies of transcription factors in human embryos have been hindered by nuclease-based genome editing approaches that induce genotoxicity1-3. Here, to overcome this, we applied ABE8e adenine base editing4,5 to precisely target an exon splice donor site, resulting in a splicing defect and functional knockout of the developmental regulator NANOG in human embryos. This approach did not trigger genotoxicity and showed limited off-target editing. Loss of NANOG disrupts pluripotent epiblast specification and instead cells differentiate towards a primitive endoderm (yolk sac) or trophectoderm (placental) transcriptional programme. Retention of primitive endoderm differentiation in NANOG-edited human embryos reveals a functional compensation that is distinct from mouse, underscoring the importance of directly investigating human development. Our findings demonstrate an essential role for NANOG in human pluripotency and epiblast specification and highlight the utility of base editing for functional interrogation of human development.

To assess narrative clinical documentation quality in assisted reproductive technology (ART) consultations, by applying a transparent, reproducible AI-assisted scoring framework. In this cross-sectional observational study, 90 consecutive initial ART consultation records from 9 clinicians were evaluated using a predefined, domain-based documentation scoring framework. Documentation quality was assessed across three domains: Regulatory Alignment, Clarity of Plan of Action, and Disclosure Readiness. A rule-constrained AI-assisted tool applied fixed scoring criteria. Domain scores were normalised, and mixed-effects models were used to examine domain differences, associations, and clinician-level effects. Human–AI concordance was assessed in a blinded random subset scored by an independent expert clinician. Repeatability was assessed through repeated rescoring under identical conditions. Mean normalised documentation scores differed significantly across domains (Regulatory Alignment 0.830, Clarity of Plan of Action 0.677, Disclosure Readiness 0.753, Wald χ2 = 284.43 p < 0.001). Domain ordering was consistent across documents, with lowest scoring for Plan Clarity (Kendall’s W = 0.53, p < 0.001), and was unchanged after adjustment for consultation type and documentation length. Score agreement between AI and the expert was substantial (weighted Kappa = 0.65), with high concordance in domain ordering (mean Kendall T = 0.84) and no significant differences overall (p = 0.10). A constrained, domain-based AI scoring framework enables transparent and reproducible quantification of narrative documentation quality in reproductive medicine. This approach may help generate formative feedback and hypotheses for targeted documentation improvement, which should be evaluated against downstream outcomes in future work.

The human blastocyst contains the pluripotent epiblast from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signaling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterized NODAL signaling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signaling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signaling activity. Genome-wide transcriptional analysis showed that NODAL signaling inhibition did not decrease NANOG transcription or impact the wider pluripotency-associated gene regulatory network. These data suggest differences in the signaling requirements regulating pluripotency in the pre-implantation human epiblast compared with existing hESC culture.

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and maintain molecular markers of the epiblast. Our functional study demonstrates the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.

Existing methods to image chromosome segregation errors are not suitable for studying human embryos at advanced preimplantation stages. As chromosomal errors are a leading cause of miscarriage and infertility, it remains unclear whether missegregation arises postfertilization. Here we optimize nuclear DNA labeling via messenger RNA electroporation and apply light-sheet live imaging to reveal chromosome segregation errors immediately before implantation. We show that embryos at advanced preimplantation stages display missegregation, including multipolar spindle formation, lagging chromosomes, misalignment and mitotic slippage. Most lagging chromosomes are passively inherited rather than reincorporated. To trace individual nuclei, we developed an open-source, semi-automated segmentation method using a customized deep learning model optimized for variability in embryo size, shape and signal. With this approach, we find most labeled cells remain externally positioned, consistent with placental rather than inner cell mass fate. Our findings raise questions about clinical uses of preimplantation genetic testing for aneuploidy, while providing broadly applicable imaging and segmentation methods for studying diverse cellular structures in human embryos. A method to label cultured human embryos finds chromosome segregation errors in placenta-fated cells.

The increasing number of women with underlying health conditions seeking in vitro fertilization (IVF) necessitates a comprehensive risk assessment framework tailored to their needs. This review aims to provide a practical framework for IVF specialists to systematically assess and manage the risks associated with medically complex patients. The GRASP mnemonic (Genetics, Retrieval, Anaesthetics, Stimulation, Pregnancy) is introduced to facilitate a thorough risk assessment process. The review emphasizes the importance of obtaining detailed medical histories, corroborating information from other healthcare providers, and involving maternal medicine specialists in pre-pregnancy counselling. Furthermore, it highlights the ethical considerations of informed consent, advocating for fertility specialists to take a central role in coordinating care and ensuring patient understanding of risks. Continuous health evaluation throughout the IVF process and clear communication of risks are crucial for improving patient outcomes.

Meiotic and mitotic chromosome segregation errors in human development have been studied mostly prior to and at the time of fertilisation. Despite chromosomal errors being a leading cause of miscarriage and infertility, chromosome missegregation has not been extensively studied at later stages of human development. Here we optimised labelling, light-sheet live imaging and semi-quantitative analysis of human embryos and reveal chromosome segregation errors just prior to implantation. We found that human embryos exhibited a number of chromosome missegregation events including multipolar spindle formation, lagging chromosomes, misalignment and chromosome slippage. We found that the majority of lagging chromosomes were passively inherited by one of the daughter cells, instead of reincorporating into the nuclei, suggesting a distinct pattern of micronuclei inheritance. By semi-automated segmentation, we tracked the position of labelled cells in human embryos and observed that while most labelled cells remained segregated to the outside, and therefore restricted to a placental-progenitor fate, there was evidence of a rare cell migration to cells positioned on the inside, which suggests that there may be plasticity. Altogether, we found that mitotic chromosome segregation errors arise just prior to implantation, which has implications for our understanding of biological events that contribute to aneuploidy mosaicism. ### Competing Interest Statement The authors have declared no competing interest.

Studies in the mouse demonstrate the importance of fibroblast growth factor (FGF) and extra-cellular receptor tyrosine kinase (ERK) in specification of embryo-fated epiblast and yolk-sac-fated hypoblast cells from uncommitted inner cell mass (ICM) cells prior to implantation. Molecular mechanisms regulating specification of early lineages in human development are comparatively unclear. Here we show that exogenous FGF stimulation leads to expanded hypoblast molecular marker expression, at the expense of the epiblast. Conversely, we show that specifically inhibiting ERK activity leads to expansion of epiblast cells functionally capable of giving rise to naïve human pluripotent stem cells. Single-cell transcriptomic analysis indicates that these epiblast cells downregulate FGF signalling and upregulate molecular markers associated with naïve pluripotency. Our functional study demonstrates for the first time the molecular mechanisms governing ICM specification in human development, whereby segregation of the epiblast and hypoblast lineages occurs during maturation of the mammalian embryo in an ERK signal-dependent manner.### Competing Interest StatementThe authors have declared no competing interest.

Various fertility scientific societies have published pathways and recommendations for COVID-19 screening during fertility treatments. As there is currently very limited research evidence on how to best deliver this screening, it is not surprising that there are noticeable differences between their recommendations. This paper compares the screening pathways recommended by these guidelines, in the light of the emerging evidence. It proposes the more liberal use of viral testing for improving detection of asymptomatic or mildly symptomatic fertility patients. It also argues that a negative test result on symptomatic individuals should not be over-relied upon for allowing the treatment to proceed. In these cases, a low threshold for cancellation may still need to be maintained.

RESEARCH QUESTION:What are the incidence and risk factors for poor ovarian response (POR) during repeat IVF? DESIGN:A retrospective analysis of 1224 consecutive patients who underwent at least two IVF stimulations in a single centre over a 6-year period. Risk factors from the initial treatment were assessed for association with POR during repeat IVF using logistic regression analysis. A simple, practical predictive model was constructed and evaluated for accuracy and calibration, based on the factors that demonstrated significant association with subsequent POR. POR during repeat IVF was defined as ≤3 retrieved oocytes or cancellation before retrieval following recruitment of ≤3 mature follicles. RESULTS:The risk of POR during repeat IVF was approximately 11.5%. A higher POR risk during repeat IVF is associated with a reduced oocyte yield during the initial treatment (≤3 oocytes: odds ratio [OR] 14, 95% confidence interval [CI] 6.42-30.24; 4-9 oocytes: OR 4.13, 95% CI 2.00-8.54; 10-15 oocytes: OR 1) and low ovarian reserve (anti-Müllerian hormone [AMH] <5.4 pmol/l: OR 3.54, 95% CI 2.24-5.59; AMH 5.4-25 pmol/l: OR 1). Women with low ovarian reserve who experience POR during the initial IVF have the highest risk of suffering POR again during repeat IVF (57% within 1 year). Other groups, such as women with unexpected POR or expected poor responders with suboptimal ovarian response during the initial IVF, are also at risk of exhibiting POR during a subsequent treatment (28% within 1 year). CONCLUSIONS:As there is a clear association between POR and lower live birth rates, this practical model may help manage patients' expectations during repeat IVF treatment.

Purpose Several trials have shown that in men with overactive bladder (OAB) and benign prostate enlargement (BPE), the combination of an α-blocker with an anticholinergic is superior to α-blocker monotherapy. The purpose of this study is to explore whether urodynamic detrusor overactivity (DO) affects clinical outcomes in men with BPE and OAB. Methods This is a post hoc analysis of a prospective, randomized trial, designed to evaluate the changes of morphometric parameters of the prostate following monotherapy or combination therapy in men with BPE-OAB. The initial study recruited men aged ≥50 years, with BPE and predominantly storage lower urinary tract symptoms (LUTS). Patients were randomized to receive tamsulosin monotherapy or a combination of tamsulosin and solifenacin for 26 weeks. All participants completed a 3-day bladder diary and the International Prostate Symptom Score (IPSS), and then underwent pressure-flow and ultrasonographic studies. Data were stratified and analysed based on the urodynamic observation of DO at baseline (DO vs. non-DO). The primary endpoint was the mean change in urgency episodes from baseline. Secondary outcomes were the changes in nocturia, total IPSS, and urodynamic parameters. Results Sixty-nine men were initially randomized, but only 60 men were included in this analysis. Urgency episodes significantly improved in men with DO who received combination therapy compared to the DO monotherapy subgroup (P=0.04) or to the non-DO combination treatment subgroup (P=0.038). Nocturia also improved in the DO combination treatment subgroup as compared to the non-DO combination subgroup (P=0.037). The 24-hour frequency and total IPSS significantly improved from baseline without significant differences among the subgroups. Conclusions The present study suggests that baseline DO could be a prognostic factor for a better response to combination therapy over monotherapy in men with BPE and storage LUTS.

Aims: Ultrasound-estimated bladder weight (UEBW), is an emerging diagnostic tool, which has been used in both males and females with lower urinary tract dysfunction. The currently acknowledged UEBW calculation methods rely on the accurate measurement of bladder wall thickness (BWT). We aim to identify if subtle errors in BWT measurement have a significant impact on UEBW calculations. Materials and methods: Twenty patients were randomly selected from an overactive bladder patient cohort. The primary endpoint was to identify the range of false BWT measurements outside which significant changes in UEBW calculation occur. We used the Kojima method and a semi-automatic 3-D model that is based on Chalana's principle. Measurements were performed using the correct BWT and a series of faulty calculations from +0.5 mm to -0.5 mm using steps of 0.05 mm from true BWT. The effect of a fixed 0.5 mm BWT error was checked in bladder volumes above and below 250 ml and in three UEBW groups (< 35 gr; 36-50 gr; > 51gr). Results: BWT measurement errors above 0.25 mm cause statistically significant changes in UEWB calculation when a 3-D model is used and errors above 0.15 mm when Kojima's method is used. At a fixed BWT error of 0.5 mm and bladder volume < 250 ml, there is a 23.76% deviation from true UEBW, while at volumes > 250 ml the deviation is 32.72%. The deviation is inversely proportional to the UEBW result, and heavier bladders deviate less. Conclusions: UEBW is a promising diagnostic tool, but small errors in BWT measurement might cause significant deviation from the true values. A 3-D calculation model appears to minimize such risks.

ObjectivesTo identify the potential influence of antimuscarinics on morphometric parameters of the prostate in patients with benign prostatic enlargement and overactive bladder.MethodsNon‐neurological patients with prostate volume >30 mL, predominately storage lower urinary tract symptoms, three or more urgency episodes per 24 h, maximum flow rate ≥10 mL/s and post‐void residual ≤100 mL were recruited for this study. They were randomized to receive either tamsulosin or tamsulosin + solifenacin. Patients were submitted to transrectal and transvesical ultrasonography, pressure‐flow study and prostate‐specific antigen test, and completed the International Prostate Symptom Score, bladder diary and overactive bladder questionnaire at induction and at 6 months. End‐study changes in morphometric prostate parameters (total prostate and adenoma volumes, prostate vascularity), as measured by transrectal ultrasound, were the principal outcomes.ResultsA reduction in total prostate volume (mean −9.5%) was noted in the combination group, as opposed to an increase in the monotherapy group (+9.2%; P < 0.001). Similar changes were reflected in adenoma volume (monotherapy +17.4% vs combination −12.5%, P = 0.001) and in prostate vascularity (monotherapy +149.3% vs combination −19.8%, P = 0.001). Both treatment regimens improved the International Prostate Symptom Score (P = 0.001); monotherapy improved the voiding subscale (P = 0.01) more, whereas combination therapy improved the storage subscale (P = 0.024). Cystometric capacity improved in the combination group (P < 0.001). Post‐void residual was increased in the combination group (+34.79%), as opposed to a decrease in the monotherapy group (−17.05%; P = 0.001).ConclusionsThe results of this pilot study suggest that solifenacin might affect morphometric properties of the prostate, decreasing total prostate and adenoma volume, as well as vascularity. A molecular effect of antimuscarinics on the prostate, in parallel with their expected bladder effect, warrants further investigation.

BACKGROUND A substantial minority of women undergoing IVF will under-respond to controlled ovarian hyperstimulation. These women-so-called 'poor responders'-suffer persistently reduced success rates after IVF. Currently, no single intervention is unanimously accepted as beneficial in overcoming poor ovarian response (POR). This has been supported by the available research on POR, which consists mainly of randomized controlled trials (RCTs ) with an inherent high-risk of bias. The aim of this review was to critically appraise the available experimental trials on POR and provide guidance towards more useful-less wasteful-future research. METHODS A comprehensive review was undertaken of RCTs on 'poor responders' published in the last 15 years. Data on various methodological traits as well as important clinical characteristics were extracted from the included studies and summarized, with a view to identifying deficiencies from which lessons can be learned. Based on this analysis, recommendations were provided for further research in this field of assisted conception. RESULTS We selected and analysed 75 RCTs. A valid, 'low-risk' randomization method was reported in three out of four RCTs. An improving trend in reporting concealment of patient allocation was also evident over the 15-year period. In contrast, <1 in 10 RCTs 'blinded' patients and <1 in 5 RCTs 'blinded' staff to the proposed intervention. Only 1 in 10 RCTs 'blinded' ultrasound practitioners to patient allocation, when assessing the outcome of early pregnancy. The majority of trials reported an intention-to-treat analysis for at least one of their outcomes, with an improving trend in the recent years. Substantial variation was noted in the definitions used for 'poor responders', the most popular being 'low ovarian response at previous stimulation'. The preferred cut-off value for defining previous low response has been 'less or equal to three retrieved oocytes'. The most popular tests used for diagnosing diminished ovarian reserve have been antral follicle count and FSH. Although the Bologna criteria for POR were only recently introduced, they are expected to become a popular definition in future 'poor responder' trials. Numerous interventions have been studied on 'poor responders'. Most of these have been applied before/during controlled ovarian hyperstimulation. The antagonist protocol, the microdose flare protocol and the long down-regulation protocol have been among the most popular interventions. The analysis of outcomes revealed a clear improving trend in reporting live birth. In contrast, only 10% of RCTs reported significant improvement in reproductive outcomes among tested interventions. Twelve 'significant' interventions were reported, each supported by a single 'positive' RCT. Finally, trials of higher methodological quality were more likely to have been published in a high-impact journal. CONCLUSIONS Overall, the majority of published trials on POR suffer from methodological flaws and are, thus, regarded as being high-risk for bias. The same trials have used a variety of definitions for their poor responders and a variety of interventions for their head-to-head comparisons. Not surprisingly, discrepancies are also evident in the findings of trials comparing similar interventions. Based on the identified deficiencies, this novel type of 'methodology and clinical' review has introduced custom recommendations on how to improve future experimental research in the 'poor responder' population.

assisted reproduction (Wong et al., 2014). An example of the lack of scientific rigour in this area has been the random introduction of Intralipid as a therapy (Clark, 1994). Intralipid was used as a control infusion inoneof theearlier, nowdiscredited, incarnationsof ‘immunetherapies’ where trials of trophoblast syncytial membranes were injected IV (Johnson et al, 1991). The control has now become the treatment. This situation contrasts with the tremendous success story of cancer immunotherapy where the use of checkpoint inhibitors became possible after the accumulation of evidence from basic scientists over many years (Hurst, 2015). Reproductive immunologists could be inspired and encouraged by this precedent and use their resources and energies to invest in better basic research to benefit tomorrow’s patients. As a provider of immune tests and steroid-based IVF protocols (IVF Australia), Sacks argues that we have a duty to our patients to demonstrate that we are considering their frustrations by offering NK cell testing, and where is the harm in this? The harm is in the financial and emotional cost of an unproven investigation and the perpetuation of an acceptance that a clinician should bend to a patient’s will and not seek to protect them (sometimes from themselves).