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.

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.

Implantation of a human embryo into the endometrium is a crucial event in gestation, as it marks the initiation of a pregnancy and is prone to high failure rates. We have limited understanding of these stages because of the inaccessibility of implanting embryos and the lack of suitable model systems. Here, we establish an in vitro model that recapitulates the luminal, glandular, and stromal compartments of the superficial layer of receptive human endometrium. Human embryos and blastoids implant into the endometrial model, achieving post-implantation hallmarks including advanced trophoblast structures that underlie early events in placental development. Single-cell RNA sequencing of the embryo-endometrial interface at day 14 uncovers predicted molecular interactions between conceptus and endometrium. Disrupting signaling interactions between extravillous trophoblast and endometrial stromal cells caused defects in trophoblast outgrowth, demonstrating the importance of crosstalk processes to sustain embryogenesis. This platform opens the opportunity to investigate early stages of human embryo implantation.

This retrospective cohort study was conducted in a single center to investigate the influence of paternal age on oocyte recipient cycles. 320 IVF/ICSI oocyte recipient cycles resulting in embryo transfers between 01/2015- 06/2022 and meeting the inclusion criteria: donors ≤35 years, endometrial thickness 6mm and normal endometrial cavity were analyzed. Donor or surgically retrieved sperm or PGT-A cycles were excluded. Paternal and recipient ages were grouped according to previous literature for comparability. Primary outcome was live birth rate, and secondary outcomes were clinical pregnancy and miscarriage rates. The median recipient, paternal and donor ages were 42 (IQR: 19-50), 43 (IQR: 38-46) and 26 years (IQR: 23-29). 55.6% [N=178] of the cycles were fresh and 44.3% [N=142] were frozen embryo transfers. Univariate and multivariate logistic regression, adjusted for semen WHO criteria, recipient age, IVF/ICSI and fresh/frozen cycles highlighted reduced odds of live birth by 79%, OR 0.21 [95%CI 0.11 to 0.38]; P<0.0001, clinical pregnancy by 58%, OR 0.42 [95%CI 0.31 to 0.89]; P<0.0001 and increased odds of miscarriage by 108%, OR 2.08 [95%CI 1.91 to 3.01]; P<0.0001 in those of paternal age 51 years compared to fathers ≤35 years. Our study identifies the negative impact of advanced paternal age.

Objectives: To study the effect of double trophectoderm biopsy on clinical outcomes following single euploid blastocyst transfer. Study design: Retrospective cohort study of 2046 single euploid frozen -thawed blastocyst transfers from January 2015 to June 2022 in a single centre. All patients undergoing a frozen -thawed embryo transfer (FTET) cycle with euploid blastocysts, biopsied for any indication, were included. The outcomes were compared for blastocysts which were biopsied and vitrified once (Group 1, n = 1684), biopsied once but vitrified twice (Group 2, n = 312) and biopsied and vitrified twice (Group 3n = 50). We adjusted for confounders and performed subgroup analysis for PGT-A, PGT-M and PGT-SR cycles. The primary outcome was live birth rate. Secondary outcomes included pregnancy, clinical pregnancy, birthweight and sex ratio. Results: After adjusting for confounders (previous failed euploid implantations, embryo quality and day of biopsy), embryos which were biopsied twice had lower OR for clinical pregnancy (0.48, CI 0.26-0.88, p = 0.019) and for live birth (0.50 CI 0.27-0.92, p = 0.025) compared to controls. Embryos which were biopsied once but vitrified twice had no different ORs for all reproductive outcomes compared to controls. No significant difference was observed for neonatal birthweight or sex ratio amongst the three groups. This is a retrospective single centre study with inherent bias and results may not be transferable to all settings. Conclusion: This study is the largest to date assessing the outcomes of FTET cycles following double trophectoderm biopsy. The results are in keeping with the existing literature and can be incorporated into patient counselling. Whilst double biopsy seems to adversely impact LBR, it is only one of the many factors that can affect success rates. The subfertility background and embryo characteristics should not be overlooked. This study provides reassuring evidence since double biopsied embryos still result in live births with no difference in sex ratio or birthweight. However, long term follow up of the off -springs is lacking and should be reported in future studies.

Abstract Purpose of the study Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant condition that leads to significant disability and morbidity, characterised by the formation of heterotopic hard tissues within connective tissues. The condition has an incidence of approximately one per two million people worldwide. There is no known single effective treatment available for FOP. We report the world’s first case of a healthy infant born following in vitro fertilisation (IVF) and preimplantation genetic testing for monogenic disorder (PGT-M) using Karyomapping for FOP. Case presentation A 30-year-old Caucasian female with FOP presented with her partner seeking IVF with PGT-M to achieve a healthy pregnancy with an embryo unaffected by FOP. Methods The couple underwent IVF and PGT-M using Karyomapping as the testing method. A multi-disciplinary team approach was utilised in planning this case, considering the additional risks of oocyte retrieval, pregnancy and childbirth in women with FOP. Main findings The oocyte retrieval was covered with a 5-day course of prednisolone to reduce the risk of a localised inflammatory reaction, which could result in subsequent heterotopic ossification. This was subsequently weaned down with reducing doses every two days. The patient underwent uncomplicated oocyte retrieval, yielding 12 mature oocytes. Following intracytoplasmic sperm injection (ICSI), ten zygotes having two pro-nuclei were cultured, and six underwent trophoectoderm biopsy and vitrification 5–6 days after retrieval. PGT-M via Karyomapping revealed four out of six (66.7%) of blastocysts were not carriers of the maternal high-risk FOP allele. In total, the patient had three separate embryo transfers. Pregnancy was achieved following the third frozen embryo transfer, which went to 37 weeks’ gestation, and delivered by Caesarean section. The baby was born in excellent condition and is unaffected by FOP. Conclusion IVF/ICSI and PGT-M using Karyomapping was successfully implemented to identify embryos carrying the high-risk FOP allele resulting in a healthy livebirth.

Abstract Study question Are the clinical outcomes following PGT-M Superior to PGT-SR and do female carriers of SR have more favourable outcomes than male carriers? Summary answer PGT-SR group was associated with improved clinical outcomes compared to PGT-M group. A higher PR and LBR observed amongst PGT-SR female carriers compared to males. What is known already Pre-implantation genetic testing for monogenic disorders (PGT-M) involves the genetic screening of embryos prior to transfer, where two individuals are carriers or affected by a specific genetic condition. Pre-implantation genetic testing for structural rearrangement (PGT-SR), however, tests for embryos in individuals who are carriers of Robertsonian or reciprocal translocations. The aim of both screening tests is to avoid passing on a known genetic condition to an offspring, resulting in fatal outcomes including physical or mental disabilities. Some studies have demonstrated improved clinical outcomes following PGT-M compared to PGT-SR. However, there is limited understanding of why this may be the case. Study design, size, duration Retrospective study of 916 patients who underwent PGD (M/SR) cycles carried out between 2015 to 2022 at a single fertility centre. There were (n = 759) who underwent PGT-M and (n = 157) PGT-SR. Clinical outcomes: pregnancy (PR), clinical pregnancy (CPR) and live birth (LBR) were determined following transfer of either an unaffected/euploid, or carrier/euploid embryo after PGT-M and transfer of a balanced/euploid embryo following PGT-SR. Subgroup analysis compared cycles including PGT-SR female carriers (n = 55) with male carriers (n = 102). Participants/materials, setting, methods Data was collected from the medical database (IDEAS). The overall mean age at oocyte retrieval was 32.9 (SD 3.7) years. SPSS software was used to determine whether PR, CPR and LBR were statistically significant when the two groups were compared. student t-test where (p < 0.05) is statistically significant was applied. Main results and the role of chance Clinical outcomes for PGT-M vs PGT-SR were comparable and not statistically significant: PR (66% vs 69%; p = 0.310) CPR (60% vs 61%; p = 0.930), LBR (55% vs 59%; p = 0.359) respectively. PR (80% vs 63%; p = 0.02), CPR (69% vs 56%; p = 0.10) and LBR (69% vs 54%; p = 0.06) were superior following PGT-SR amongst female carriers (n = 44/55), compared to male carriers (n = 64/102) respectively. However, only the pregnancy rate was statistically significant when the female was the carrier. Quality of the blastocyst(s) transferred was also higher amongst PGT-SR cycles where the female was the carrier. Limitations, reasons for caution The sample size of the PGT-SR group for both male and female carriers were significantly lower compared to the PGT-M group. A larger and equal sample size is required in order to generalise findings. Wider implications of the findings The quality of the blastocyst transferred has a significant effect on clinical outcomes, despite the euploid status. Such findings can be used to counsel patients or couples who consider undergoing IVF for the purpose of PGT-M/SR genetic screening prior to embryo transfer. Trial registration number IRB-0001CRGH-CITY-FL-2024

Abstract Study question Does embryo diameter and surface area at tsB and tB as determined by AI predict live birth (LB) potential amongst euploid blastocysts? Summary answer Euploid blastocysts with optimal diameter (tsB:120-129; tB:136-140μm) and surface area (tsB:11771-13577; tB:15000-15643μm2) are more likely to lead to LB than larger or smaller blastocysts. What is known already Current embryology consensus favours larger blastocysts based on Gardner grading, associating expansion with higher quality. Human assessment of embryos is subjective, and it is unknown whether this dogma holds when assessing pre-expansion blastocyst stages. The impact of blastocyst surface area and diameter at tSB and tB; and pace of development on LB remains unclear. This may help clarify whether large blastocysts are associated with accelerated embryo growth and abnormal metabolic processes. The study’s objective was to use AI to quantitatively determine the optimal embryo surface area and diameter of euploid blastocysts that lead to live birth. Study design, size, duration Retrospective study including 97 time-lapse videos of euploid embryos with known live birth outcomes from January to December 2021 Participants/materials, setting, methods Embryo diameter and surface area were measured at start of blastulation (tSB) and at time of blastulation (tB) by an AI model (CHLOE-EQ, Fairtility Ltd). Live birth (LB) outcomes were manually annotated by embryologists. An Optimal embryo surface area and diameter were determined by the highest live birth rate and the suboptimal range was determine by a decreased Live birth rate. A comparison between the optimal and suboptimal live birth rate was measured using chi-square. Main results and the role of chance Moderately sized embryos were found to have higher LB outcomes. At tSB, optimal surface area was 11771-13577 um2, while optimal embryo diameter was 120-129μm. LB rate for optimal embryo surface area was higher than for suboptimal [Optimal 80% (20/25) vs Suboptimal 55% (40/72), p < 0.001]. In addition, optimal embryo diameter had higher LB rates than embryos with suboptimal diameter [80% (16/20) vs 52% (31/60), p < 0.05). At tB, optimal embryo area was 15000-15643μm2 and optimal embryo diameter 136-140μm. LB rate for optimal embryo surface area was higher than for suboptimal 82% (14/17) vs 49% (36/73), p < 0.05]. Optimal embryo diameter had higher LB rates than embryos with suboptimal diameter (77% 20/26 vs 47% 30/64, p < 0.05). Embryos with an optimal surface area and diameter were slower than bigger suboptimal embryos: t2 (26±2 vs 25±2), t8 (59±8 vs 53±6), t9 + (71±6 vs 67±6), tM (82±7 vs 77±8), tSB (99±6 vs 93±6), tB (107±7 vs 100±6), tEB (137±2 vs 122±12); t8-t5 (12±10 vs 6±5), t8-t2 (33±8 vs 28±5), tEB-tSB (34±2 vs 23±9), p < 0.05. Optimal vs small suboptimal embryos were faster in t9 + (71±6 vs 76±4), tEB (121±11 vs 137±2, tEB-tSB (20±10 vs 34±1). But were slower in t8-t5 (12±10 vs 5±6), t8-t2 (33±8 vs 28±5), p < 0.05. Limitations, reasons for caution This is the first time a quantitative embryo surface area and diameter has been studied in its correlation with live birth. Generalisation requires the replication across diverse centers with varying demographic profiles to validate findings and establish broader applicability within the context of assisted reproductive technologies. Wider implications of the findings This data-driven approach can support the embryo selection process, correlating quantitative parameters with live birth outcomes, thereby refining the precision of the decision-making in IVF. This level of quantitative data of embryo surface area and diameter is only accessible through AI tools. Trial registration number IRB-007CITY-FL21.08.2023

Can an AI oocyte quality model improve blastulation prediction accuracy? An AI oocyte quality model presented an overall accuracy of 61-74%. Exhibiting an improvement of 26-48% for prediction of blastulation over random. Nowadays, the assessment of whether sufficient oocytes have been cryopreserved is purely based on patient age and number of oocytes collected. No morphological abnormalities provide quantitative guidance about the reproductive potential of the oocyte. Additionally, interobserver agreement and prediction of blastulation by experienced embryologists has been reported to be worse than chance (Behr et al., 2018). Therefore, there is a need to implement oocyte quality assessment tools to predict blastulation in oocyte donation, cryopreservation programs and IVF treatments. A retrospective study using an AI oocyte quality score (CHLOE-OQ, Fairtility Ltd) was performed in three private clinics evaluating 1749 oocytes that were inseminated with ICSI and cultured between October 2019 and December 2021. Overall and per clinic analysis were conducted (Clinic 1 n = 1131; Clinic 2 n = 576; Clinic 3: n = 42). An AI model provided an oocyte quality (OQ) score to assess prediction of blastulation, measured by binary logistic regression (AUC) per clinic and overall. The correlation between the OQ score and blastulation was measured using t-test. OQ score was classified into groups, Group A: 0.9-1.0, Group B: 0.7-0.9, Group C: 0.4-0.7 and Group D: 0-0.4. Blastulation rate was assessed in each OQ score subgroup using chi-square. OQ score prediction of blastulation presented an overall accuracy of 63% (1107/1749). Accuracy per clinic ranged from 61-74%, Clinic 1: 61% (690/1131), Clinic 2: 67% (386/576) and Clinic 3: 74% (31/42). Exhibiting an improvement of 24-48% for prediction of blastulation over random. Oocyte quality score was predictive of blastulation per clinic and overall Clinic 1: AUC 0.62, n = 1130; Clinic 2: AUC 0.62, n = 575; Clinic 3: AUC 0.78, n = 41; Overall: AUC=0.62, p < 0.001, n = 1749). Overall blastocyst development rate was 53% (929/1749). OQ score was higher in oocytes that blastulated than those that did not blastulate (0.85 ± 0.14 vs 0.74 ± 0.2, p < 0.001). There was a significant direct association between OQ score subgroups and blastulation rate. Group A with the highest score subgroup had the highest blastulation rate [61% (554/903), p < 0.05]. The blastulation rates for Group B [53% (283/539) p < 0.001], Group C [38%, (71/188), p < 0.001] and Group D [18% (21/119), p < 0.001] were decreasingly lower. This study was done in 3 centers in the same country. Therefore, further studies should focus on diversifying in terms of geographical location to address generalization of this AI assessment. Further studies should focus on comparing the efficacy of blastulation prediction by humans. Using AI to do an objective oocyte assessment aids clinicians in determining the optimal time to transition to oocyte donor programs. It assists the decision-making for oocyte banking, minimizing unnecessary cycles and guiding timely additional oocyte collections. This approach at the point of cryopreservation, mitigates risks associated with delayed decisions. N/A

Abstract Study question To determine if the introduction of concurrent testing with PGT-A has improved clinical outcomes for PGT-M/SR patients. Summary answer This study demonstrates that the introduction of concurrent testing may have improved clinical outcomes for this patient demographic irrespective of age. What is known already Preimplantation Genetic Testing (PGT) is utilised to investigate the genetic constitution of embryos. PGT-M (monogenic) and PGT-SR (structural rearrangements) are screening techniques considered in treatment for couples at risk of conceiving offspring with known genetic or structural chromosomal abnormalities respectively. Preimplantation Genetic testing for Aneuploidy (PGT-A) was developed in order to screen embryos for numerical chromosomal abnormalities, a leading cause of failed implantation. Nevertheless, routine use of PGT-A is discouraged due to lack of evidence regarding its efficacy. However, PGT-A is routinely used alongside PGT-M and PGT-SR despite the lack of evidence of its effectiveness in improving patient outcomes. Study design, size, duration The retrospective analysis of 496 PGT-M/SR single embryo transfer patient cycles between December 2013 and December 2021. Participants/materials, setting, methods The retrospective analysis of 496 PGT-M/SR single embryo transfer cycles at CRGH Portland was undertaken (single testing n = 77; concurrent testing n = 419). The clinical outcomes (positive urine pregnancy test, clinical pregnancy, live birth and miscarriage rates) were compared between groups using IBM SPSS Statistics (premium 27). Binary logistic regression was performed for statistical analysis, results with p < 0.05 were determined statistically significant. Main results and the role of chance Overall, concurrent testing was associated with statistically significant improved clinical outcomes compared to the reference group. Statistical significance was seen in positive urine pregnancy test (49% vs 68%, p = 0.012), clinical pregnancy (32.5% vs 63%; p = 0.000013), live birth rate (32.5% vs 59%; p = <0.00016) and miscarriage rates (34.2% vs 13%; p = 0.001). The results imply that the introduction of concurrent testing has improved the probability of obtaining a positive clinical outcome and has reduced the chance of miscarriage in this patient demographic. Further analysis showed concurrent testing improved the chances of obtaining a positive clinical outcome irrespective of the patients age or quality of the embryo. Limitations, reasons for caution This study has produced promising results. However, there are several limitations such as the small sample size, embryo biopsy techniques used, and the different molecular diagnostic techniques and providers utilised. Wider implications of the findings The results imply a justification for the use of PGT-A alongside PGT-M/SR screening techniques to improve embryo selection and clinical outcomes for this cohort of patients. Additionally, they demonstrate the efficacy of PGT-A in a previously underrepresented demographic. However, further research is required to confirm the clinical value of PGT-A. Trial registration number not applicable

ABSTRACT During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

To establish if preimplantation genetic testing for aneuploidy (PGT-A) at the blastocyst stage improves the composite outcome of live birth rate and ongoing pregnancy rate per embryo transfer compared to conventional morphological assessment. A systematic literature search was conducted using PubMed, EMBASE and Cochrane database from 1st March 2000 until 1st March 2022. Studies comparing reproductive outcomes following in vitro fertilisation using comprehensive chromosome screening (CCS) at the blastocyst stage with traditional morphological methods were evaluated. Of the 1307 citations identified, six randomised control trials (RCTs) and ten cohort studies fulfilled the inclusion criteria. The pooled data identified a benefit between PGT-A and control groups in the composite outcome of live birth rate and ongoing pregnancy per embryo transfer in both the RCT (RR 1.09, 95

Abstract Study question Does the outcome of the first, euploid transfer affect the success rate of the second PGT-A transfer cycle? Summary answer Live birth rate is higher in the second euploid transfer if the first euploid transfer results in at least a biochemical pregnancy versus no implantation. What is known already There is limited evidence on counselling patients for a second euploid embryo transfer based on previous outcomes. The existing literature is reassuring showing very high implantation rates even following two or more successive failed euploid transfers. Achieving implantation is a major step towards success. The association between a PGT-A embryo transfer cycle where implantation was achieved (at least biochemical pregnancy, miscarriage or live birth) with the outcome of the subsequent embryo transfer is interesting to explore and incorporate in patient counselling. Study design, size, duration This is a retrospective analysis of patients who underwent PGT-A treatment in a single centre from 2015-2021. Live birth rate was the main outcome of interest. Pregnancy rate and miscarriage rate were secondary outcomes. Means and standard deviations were used for demographic parameters. Embryos were biopsied at blastocyst stage for PGT-A and next generation sequencing was used. Embryo transfers were performed with uniform protocols by different, experienced clinicians. Participants/materials, setting, methods Patients who had their second euploid embryo transfer were included in the analysis. Outcomes were assessed in relation to the outcome of the first euploid cycle; whether at least a biochemical pregnancy was achieved versus a negative pregnancy test. Patients with uterine anomalies / suboptimal endometrial thickness were excluded. Protocols for endometrial preparation and add-ons were individualised and were not uniform for the two cycles. Protocols included both medicated cycles and ovulatory cycles. Main results and the role of chance 331 women were included in the analysis undergoing their second euploid embryo transfer cycle. 152 had one previous euploid implantation (the first cycle resulted in a biochemical pregnancy, miscarriage, or live birth) (group 1) and 179 had one previous failed implantation of a euploid embryo (the first cycle resulted in a negative pregnancy test) (group 2). There was no significant difference between the two groups in patient demographic characteristics including age, body mass index and age at egg collection. No significant difference was documented for embryo quality, day 5 versus day 6 embryos or endometrial thickness. More cycles were medicated in group 1 compared to group 2. All patients had an elective single embryo transfer. Live birth rate was significantly higher for group 1 versus group 2, 58% vs 44%, p = 0.013, OR 1.75 (1.12-2.70). No significant difference was observed for pregnancy rate or miscarriage rate. Limitations, reasons for caution This is a retrospective analysis in a single fertility centre, therefore prone to bias and results cannot be generalised unless validated by larger prospective multi-centre studies. There are multiple possible cofounders such as background and duration of subfertility, endometrial preparation protocols and add-on interventions which cannot be accounted for. Wider implications of the findings These findings suggest that the first euploid transfer outcome is important for future euploid transfer cycles. Women who achieved at least a positive pregnancy test in the first cycle seem to have higher success rates in subsequent PGT-A transfer. Further research is warranted to explore this association for patient counselling. Trial registration number not applicable

PURPOSE:Utilising non-invasive imaging parameters to assess human oocyte fertilisation, development and implantation; and their influence on transcriptomic profiles.METHODS:A ranking tool was designed using imaging data from 957 metaphase II stage oocytes retrieved from 102 patients undergoing ART. Hoffman modulation contrast microscopy was conducted with an Olympus IX53 microscope. Images were acquired prior to ICSI and processed using ImageJ for optical density and grey-level co-occurrence matrices texture analysis. Single-cell RNA sequencing of twenty-three mature oocytes classified according to their competence was performed.RESULT(S):Overall fertilisation, blastulation and implantation rates were 73.0%, 62.6% and 50.8%, respectively. Three different algorithms were produced using binary logistic regression methods based on "optimal" quartiles, resulting in an accuracy of prediction of 76.6%, 67% and 80.7% for fertilisation, blastulation and implantation. Optical density, gradient, inverse difference moment (homogeneity) and entropy (structural complexity) were the parameters with highest predictive properties. The ranking tool showed high sensitivity (68.9-90.8%) but with limited specificity (26.5-62.5%) for outcome prediction. Furthermore, five differentially expressed genes were identified when comparing "good" versus "poor" competent oocytes.CONCLUSION(S):Imaging properties can be used as a tool to assess differences in the ooplasm and predict laboratory and clinical outcomes. Transcriptomic analysis suggested that oocytes with lower competence may have compromised cell cycle either by non-reparable DNA damage or insufficient ooplasmic maturation. Further development of algorithms based on image parameters is encouraged, with an increased balanced cohort and validated prospectively in multicentric studies.

Abstract Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p < 0.0001), extent of blastocyst expansion (p < 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p < 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

To determine if oocyte yield in women undergoing cryopreservation for social (SOC), medical (MOC) and oocyte donation (OD) cycles is comparable when matched for age. 315 oocyte retrievals were performed for SOC, 116 for MOC and 392 for OD. Non-parametric Kruskal-Wallis tests and Poisson regression were used to assess the impact of age stratification. The median ages of women undergoing SOC, MOC, and OD were 38, 31 and 26 years respectively. The median (IQR) number of oocytes in the three categories was 7, 10, and 12. The oocyte yield was significantly higher in women aged 30-34 years undergoing SOC, compared to the MOC group. For the SOC group, age in years, oestradiol levels per 1000 pmol/and follicle count >12mm on the day of trigger were significant predictors of oocyte yield. Women embarking on SOC are significantly older than those undergoing MOC and OD, and thus oocyte yield is reduced when stratified for age. This study highlights the significant predictors of oocyte yield amongst women undergoing oocyte cryopreservation for specific indications. The findings can be used to optimise the yield and overall chance of successful livebirth.

To assess the relationship between the number of oocytes retrieved during elective oocyte cryopreservation (EOC) cycles with various clinical, biochemical, and radiological markers, including age, body mass index (BMI), baseline anti-Müllerian hormone (AMH), antral follicle count (AFC), Oestradiol level (E2) and total number of follicles ≥ 12 mm on the day of trigger. To also report the reproductive outcomes from women who underwent EOC. A retrospective cohort of 373 women embarking on EOC and autologous oocyte thaw cycles between 2008 and 2018 from a single London clinic in the United Kingdom. 483 stimulation cycles were undertaken amongst 373 women. The median (range) age at cryopreservation was 38 (26–47) years old. The median numbers of oocytes retrieved per cycle was 8 (0–37) and the median total oocytes cryopreserved per woman was 8 (0–45). BMI, E2 level and number of follicles ≥ 12 mm at trigger were all significant predictors of oocyte yield. Multivariate analysis confirmed there was no significant relationship between AFC or AMH, whilst on univariate analysis statistical significance was proven. Thirty six women returned to use their cryopreserved oocytes, of which there were 41 autologous oocyte thaw cycles undertaken. There were 12 successful livebirths achieved by 11 women. The overall livebirth rate was 26.8% per cycle. No livebirths were achieved in women who underwent EOC ≥ 40 years old, and 82% of all livebirths were achieved in women who had done so between 36 and 39 years old. Clinical, biochemical and radiological markers can predict oocyte yield in EOC cycles. Reproductive outcomes are more favourable when cryopreservation is performed before the age of 36, with lower success rates of livebirth observed in women aged 40 years and above.

Background:The purpose of the current study was to assess whether embryonic culture conditions has an impact on embryo ploidy in a preimplantation genetic testing for aneuploidy (PGT-A) cycle.Methods:In this retrospective single center cohort study, a total of 1099 blastocysts from 278 PGT-A cycles were analyzed. The generated blastocysts were biopsied on days 5 and 6. Inseminated oocytes were allocated in different incubators (benchtop and time lapse) and assisted zona hatching was performed on day 3 of embryo development to facilitate the biopsy process which was performed on days 5 and 6 (blastocyst stage).Results:The average age across the groups was 38.7±3.6 years and the total number of mature eggs was 2912 which were randomly distributed across both incubators. The euploidy rate obtained from both groups showed a higher proportion of euploid embryos in the TLM incubator (37.03%, 95% CI 31.9-42.1) compared to those cultured in the BT incubator (30.4%, 95% CI 23.1-37.7). Regression analysis showed that female age remains to be the key variable driving euploidy rates (0.85, 95% CI 0.82-0.88) although incubator type could be an important covariable (0.54, 95% CI 0.45-0.59). A subgroup analysis of 74 single euploid embryo transfers showed comparable pregnancy and live birth rates.Conclusion:This large cohort study demonstrates that uninterrupted controlled culture environment provides increased probability to develop euploid embryo in a PGT-A cycle. However, further evaluation is required to assess how environmental culture conditions at a cellular level could affect epigenetic mechanisms in embryo development and higher aneuploidy rate.

Abstract Study question Is raised paternal body mass index (BMI) important for the miscarriage rate following assisted reproductive technology (ART)? Summary answer Based on the available evidence, raised male BMI is not associated with higher risk of miscarriage following ART. What is known already More than half women and men of reproductive age worldwide are overweight or obese.There is extensive literature exploring the importance of normal female BMI for reproductive outcomes. However, little attention has been given to male BMI for couples seeking fertility treatment [1] .The adverse effect of male obesity on sperm parameters including DNA damage is well documented and there is evidence suggesting that raised male BMI results in significant decrease in live birth rate following ART [2] .Furthermore, emerging evidence from human and animal studies demonstrates that paternal obesity can affect the future health of the offspring through epigenetic pathways[3]. Study design, size, duration A computerized literature search was performed using EMBASE, MEDLINE, CINAHL and the Cochrane Central register of trials from database inception to November 2021. The aim was to explore the association between male BMI on miscarriage rate following ART. Reference lists of relevant studies were cross-checked. Only articles with full manuscripts available and published in English were included. Papers not relating to human subjects were excluded. All eligible studies were included (observational, prospective and retrospective studies). Participants/materials, setting, methods Included studies reported on couples undergoing ART for any indication using partner’s fresh sperm. Outcomes of interest were miscarriage rate and clinical pregnancy rate. Outcome data from each study were pooled and expressed as odds ratio (OR) with 95% confidence interval (CI) by using a random-effect model due to statistical heterogeneity in the outcome data[4]. Heterogeneity of treatment effects was evaluated using the I2 statistic to quantify the variation across studies caused by heterogeneity. Main results and the role of chance Abstract screening identified 197 relevant studies. After excluding duplicates, reviews and studies which did not fulfill the inclusion criteria, full manuscripts were accessed for 13 studies. Six studies were identified exploring the effect of male BMI on miscarriage following ART, two prospective and four retrospective. The quality of evidence was low using the GRADE framework. Meta-analysis was possible for three studies including 6793 couples undergoing ART. Outcomes were compared for male BMI < 25 kg/m2 versus BMI >/=25 kg/m2 . The pooled results did not show a statistically significant increase in miscarriage rate when the male partner was overweight or obese compare to normoweight (OR 1.32, 95% CI 0.82–2.1, P = 0.249). There was significant heterogeneity between the included studies (I 2 = 48.7%). There was no significant effect of male BMI on clinical pregnancy rate (OR 0.90, 95% CI 0.59–1.38, P = 0.637). For two of the remaining studies which could not be included in the meta-analysis due to missing data, the authors concluded that male BMI >25 was not associated with increased miscarriage risk whilst the most recent prospective study showed that high male BMI was associated with increased risk of chromosomal aberration-related miscarriages. Limitations, reasons for caution The number of the included studies and significant heterogeneity are the main limitations. It was not possible to account for important confounders such as age, subfertility diagnosis, type of stimulation and laboratory parameters including embryo grade. We grouped participants in two BMI categories therefore did not distinguish between overweight/obesity/morbid obesity. Wider implications of the findings Despite increasing evidence suggestive of adverse effect of raised male BMI on reproductive outcomes, there is limited literature exploring the impact on miscarriage rate following ART. More well-designed studies are needed for sound conclusions. Paternal characteristics, general health and preconception lifestyle should not be overlooked in the fertility consultation. Trial registration number not applicable