OBJECTIVE:To study whether the method of donor sperm cryopreservation, vitrification or slow freezing, affects embryological and clinical outcomes in in vitro fertilization (IVF) cycles. DESIGN:Retrospective cohort study. SUBJECTS:A total of 604 IVF cycles conducted between January 2023 and November 2024 using donor sperm cryopreserved by vitrification (n = 396) or slow freezing (n = 208). EXPOSURE:Cryopreservation method of donor sperm (vitrification vs. slow freezing). MAIN OUTCOME MEASURES:Fertilization rate, usable blastocyst rate, and clinical pregnancy rate after the first embryo transfer. RESULTS:Overall fertilization rates were similar between groups; however, stratified analysis by insemination technique revealed a significantly higher fertilization rate with vitrified sperm in conventional IVF cycles (76.4% vs. 65.9%; adjusted odds ratio [aOR] 1.95 [1.11-3.40]), whereas no difference was observed in intracytoplasmic sperm injection (ICSI) cycles (76.8% vs. 79.7%; aOR 0.84 [0.65-1.09]). Usable blastocyst and clinical pregnancy rates after the first embryo transfer were comparable between vitrification and slow freezing groups. Multivariable analyses adjusting for confounders confirmed these findings. CONCLUSION:Donor sperm vitrification is a safe alternative to conventional slow freezing and yields comparable blastocyst and clinical outcomes. Notably, in conventional IVF cycles, vitrification was associated with a significantly higher fertilization rate. This advantage was not observed in ICSI cycles, suggesting that vitrification better preserves sperm membrane functionality required for natural gamete interaction. Although clinical outcomes remain similar between both methods, reduced fertilization with slow-frozen sperm may decrease the number of embryos available, with potential implications for overall treatment efficiency.

Is ejaculatory abstinence period (EAP) associated with embryo developmental ability and ploidy status? The duration of ejaculatory abstinence does not affect fertilization rate, embryo development, or ploidy status. Ejaculatory abstinence period contributes to intra-individual variability in semen parameters. Although the World Health Organization (WHO) Manual recommends 2–7 days of ejaculatory abstinence for diagnostic purposes, the optimal ejaculatory abstinence for ART treatments could be different. While recent reviews highlight the association of short abstinence duration and improved semen quality in terms of motility and DNA fragmentation, its impact on IVF outcomes has been scarcely studied. Preliminary evidence suggests a potential link between shorter abstinence and higher embryo euploidy rates. Further research is needed to provide deeper insights into these associations. We conducted a retrospective cross-sectional study involving 2,450 ICSI cycles with PGT-A indication at the Reproductive Medicine Department of a University-affiliated Hospital between November 2017 and December 2023. Cycles were divided in three groups according to EAP: Short (<2 days), Standard (2-7 days), and Long (>7 days). Embryological outcomes (fertilisation rate, good-quality embryo rate, and euploidy rate) were analysed in relation to EAP. Patients underwent ovarian stimulation following standard short GnRH antagonist or progestin-primed ovarian stimulation protocol (PPOS). Mature oocytes were fertilised using ICSI, and laser-assisted hatching was performed on day 3. Viable blastocysts (days 5–7) were biopsied, and ploidy status was assessed via next generation sequencing (NGS). Comparisons between EAP groups were established using ANOVA or Pearson’s chi-square. Additionally, a logistic regression model was used to adjust results for potential confounders. Mean female age was 39.9 years (standard deviation, SD 3), while male was 41.4 years (SD 5.1). Mean EAP was 3.3 days (SD 2). Normozoospermic and abnormal semen samples accounted for 66% and 34%, respectively. Sample distribution per groups was: Short 282 cycles (12%), 818 blastocysts; Standard 2086 cycles (85%), 5857 blastocysts; Long 82 cycles (3%), 224 blastocysts. No differences in fertilisation rates were observed according to EAP: Short 78% [95% confidence interval (CI):74–82]; Standard 76% [95% CI:75–78]; Long 74% [95% CI:68–81]) Similarly, no differences were found in the proportion of good-quality embryos: Short 48% [95% CI:44–53]; Standard 56% [95% CI:54–57]; Long 55% [95% CI:46–66]). Embryo euploidy rates were also not associated to EAP: Short 37% [95% CI:32–41]; Standard 36% [95% CI:34–38]; Long 33% [95% CI:26–42]). Additionally, no differences were observed in mosaicism rate across groups: Short 5.4% [95% CI:3.9–7.2]; Standard 6.3% [95% CI:5.6–6.9]; Long 4.9% [95% CI:2.5–8.8]). Adjustment for potential confounding factors, including male age and normal vs. altered semen analysis, revealed no significant differences. The adjusted odds ratios (aOR) for embryo euploidy were 0.98 [95% CI: 0.82–1.17] for the Standard group and 1.11 [95% CI: 0.76–1.58] for the Long EAP group. The main limitations of this study are its retrospective nature, low numeric representation of Short and Long EAP and the relatively small difference in abstinence duration between the Short and Standard groups, which may impact the ability to detect subtle associations. Specific patient groups may deserve independent assessment. Should these preliminary results be confirmed, no recommendations on ejaculatory abstinence would be needed in the context of ART cycles. No

Study question What is the impact of discontinuing the reporting of low-grade mosaic embryos on PGT-A cycles’ outcomes? Summary answer Disregarding low grade mosaics leads to a significant increase in cycles with transferrable embryos without compromising clinical outcomes. What is known already In the context of PGT-A, uncertainty regarding the clinical relevance of chromosomal mosaicism has made it challenging for both professionals and patients to decide whether to transfer mosaic embryos. Consequently, many have been discarded. Yet, research has shown that such embryos can give rise to healthy babies. Lately, although there is still controversy, low-grade mosaics have been reported to be equivalent to euploid embryos in terms of clinical outcomes and risks. Thus, ESHRE guidelines on mosaicism opened the possibility of not reporting low-grade mosaicism. However, the impact of implementing such option in a PGT-A setting has not yet been assessed. Study design, size, duration Retrospective cohort study analysing 1718 PGT-A cycles performed from January 2021 to October 2024 at a university-affiliated fertility unit. Two groups were established based on low-grade mosaicism reporting policy: REPORTING (n = 1380) and NON-REPORTING (n = 338). Clinical pregnancy rates of transfers performed up to November 2024 were assessed for REPORTING group (n = 949) and NON-REPORTING group (n = 151). Additionally, PGT-A results of transferred embryos from NON-REPORTING group were revisited to detect low-grade mosaics and their outcomes were analysed. Participants/materials, setting, methods Trophectoderm biopsies from 5382 embryos were analysed by NGS. From January 2021 to January 2024, the REPORTING policy was applied: low-grade mosaics (30-50%) and high-grade mosaics (50-70%). From February 2024, NON-REPORTING policy was applied: euploid (<50%) and high-grade mosaics (50-70%). Primary outcomes measured were: mosaicism rate, % of cycles with mosaic embryos, % of cycles with only mosaic embryos for transfer, % cycles with no euploid embryos for transfer, and clinical pregnancy rates. Main results and the role of chance Both groups were similar in terms of female and male age, female BMI, number of pick-ups per PGT-A cycle, total dose of gonadotropins, days of ovarian stimulation, number of oocytes retrieved, number of MII oocytes, and number of biopsied blastocysts. As expected, mosaicism rate was inferior in NON-REPORTING group (3.9% [95%CI (%): 2.5-5.2]) compared to REPORTING group (6.8% [95%CI (%): 6.0-7.5]), while euploidy rate was higher (41.1% [95%CI (%): 37.6-44.6] vs 35.9% [95%CI (%): 34.2-37.5]). No differences were observed in aneuploidy rates (47.5% vs 52.4%, NS). The percentage of cycles with at least one mosaic embryo was lower in NON-REPORTING group 9.8% (33/338) compared to REPORTING group 18.5% (255/1380) (p < 0.001). Most interestingly, the percentage of cycles with no euploid embryos for transfer was significantly reduced in the NON-REPORTING group (34.3% vs 41.5%, p = 0.009). The adoption of the NON-REPORTING policy did not negatively impact on clinical pregnancy: 53.6% (81/151) vs 57.4% (545/949) in REPORTING group (p = 0.2). A total of 22 transferred embryos from NON-REPORTING group, that would have been classified as low-grade mosaics according to the REPORTING policy, presented the following clinical outcomes: biochemical pregnancy 54.5% (12/22), clinical pregnancy 45.4% (10/22), miscarriage 10.0% (1/10), and ongoing pregnancy 40.9% (9/22). All ongoing pregnancies are normally developing. Limitations, reasons for caution Retrospective study. Data on clinical outcomes for the NON-REPORTING group is preliminary and should be further confirmed with extended pregnancy follow-up and increased sample size.. NON-REPORTING policy of low-grade mosaicism should not be adopted without providing patients with an adequate pre-test consultation with full disclosure of the chosen policy. Wider implications of the findings Results suggest that disregarding low-grade mosaicism avoids unnecessary uncertainty to patients without compromising clinical outcomes. As a result, an increase in cumulative live birth could be expected. Given all, it may be the time to consider of no value the reporting of a low-grade mosaicism. Trial registration number No

STUDY QUESTION:In oocytes of advanced maternal age (AMA) women, what are the mechanisms leading to aneuploidy and what is the association of aneuploidy with embryo development? SUMMARY ANSWER:Known chromosome segregation errors such as precocious separation of sister chromatids explained 90.4% of abnormal chromosome copy numbers in polar bodies (PBs), underlying impaired embryo development. WHAT IS KNOWN ALREADY:Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years) and can affect over half of oocytes in this age group. This underlies the rationale for PB biopsy as a form of early preimplantation genetic testing for aneuploidy (PGT-A), as performed in the 'ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis' (ESTEEM) randomized controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), Meiosis II (MII) non-disjunction (ND), and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. STUDY DESIGN, SIZE, DURATION:Data were sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridization (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs (PGT-A group), and on embryo morphology in a subset of embryos, up to Day 6 post-insemination, from both the intervention (PB biopsy and PGT-A) and control groups. PARTICIPANTS/MATERIALS, SETTING, METHODS:ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (two pronuclei) in the PB biopsy group and 1082 in the control group were used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE:Overall, 461/693 PB pairs showed abnormal segregation in 1162/10 810 chromosomes. The main observed abnormal segregations were compatible with PSSC in Meiosis I (MI) (n = 568/1162; 48.9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%), and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9.6%), we observed a chromosome copy number in the first PB (PB1) and second PB (PB2) that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 days post-insemination compared to the control group, indicating that PB biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day, aneuploidy was associated with a decrease in embryo quality on Day 3 (adjusted odds ratio (aOR) 0.62, 95% CI 0.43-0.90), Day 4 (aOR 0.15, 95% CI 0.06-0.39), and Day 5 (aOR 0.28, 95% CI 0.14-0.58). LIMITATIONS, REASON FOR CAUTION:RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. WIDER IMPLICATIONS OF THE FINDINGS:Our finding of frequent unexplained chromosome copy numbers in PBs indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by mis-segregation of chromosomes during MI and MII. STUDY FUNDING/COMPETING INTEREST(S):Data were mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of PBs. None of the authors have competing interests. TRIAL REGISTRATION NUMBER:Data were mined from the ESTEEM study (ClinicalTrials.gov Identifier NCT01532284).

Research question: Is partial compaction during morula formation associated with an embryo's developmental ability and Design: Retrospective analysis of data from 196 preimplantation genetic testing for aneuploidy (PGT-A) cycles. Embryos starting compaction were grouped according to the inclusion or not of all the blastomeres in the forming morula (full compaction or partial compaction). The possible effect of maternal age and ovarian response on compaction was analysed. Morphokinetic characteristics, blastocyst formation rate, morphology and cytogenetic constitution of the obtained blastocysts were compared. Comparisons of reproductive outcomes after the transfer of euploid blastocysts from both groups were established. Finally, in a subset of embryos, the chromosomal constitution concordance of the abandoned cells and the corresponding blastocyst through trophectoderm biopsies was assessed. Results: A total of 430 embryos failed to include at least one cell during compaction (partial compaction group [49.3%]), whereas the 442 remaining embryos formed a fully compacted morula (full compaction group [50.7%]). Neither female age nor the number of oocytes collected affected the prevalence of partial compaction morulae. Morphokinetic parameters were altered in embryos from partial compaction morulae compared with full compaction. Although an impairment in blastocyst formation rate was observed in partial compaction morulae (57.2% versus 70.8%, P < 0.001), both chromosomal constitution (euploidy rate: partial compaction [38.4%] versus full compaction [34.2%]) and reproductive outcomes (live birth rate: partial compaction [51.9%] versus full compaction [46.2%]) of the obtained blastocysts were equivalent between groups. A high ploidy Conclusions: Partial compaction morulae show a reduced developmental ability compared with full compaction morulae. be a consequence of a compromised embryo development regardless of the chromosomal constitution of the excluded cells.

Abstract Study question Is a fully-automated system a faster and reliable sperm counting method? Summary answer A fully-automated sperm counting system is a reliable tool that significantly reduces time spent for semen evaluation. What is known already The emergence of automated tools in our everyday life is a reality. Fully-automated systems for the evaluation of seminal samples are available for the andrology laboratories. The expected advantages of such analysers would be a higher reliability and reproducibility of results, with lower inter and intra-individual variability. Although there are few published validation studies, available data have evidenced that results obtained with these methods are, in general, consistent with those obtained with manual/conventional analysis. Moreover, automatic sperm assessment could be useful in reducing the time spent for analysis. However, no data have been published in this regard. Study design, size, duration Observational retrospective study of 220 semen samples analyzed in 2022. Every sample was analyzed using three different methodologies: manual analysis, semi-automated and fully-automated. Manual assessment was performed by senior embryologists undergoing periodical quality control evaluations and included counting with improved Neubauer chamber, motility assessment with slide with phase-contrast optics, and morphology analysis by Diff-Quick stain. The semi-automated analysis was performed by SCATM plus manual morphology assessment. The fully-automated analysis was exclusively performed by SQA-VisionTM. Participants/materials, setting, methods Male patients consulting for infertility and undergoing a spermiogram in a single centre. The sperm parameters evaluated for correlation study among methodologies were: total sperm, motile sperm and progressive sperm concentrations (106 per ml), and morphologically normal forms (%). Duration of analysis (sperm count + motility + morphology) using each method was recorded and compared. Intraclass correlation coefficient (ICC[95%CI]) was used to compare agreement between methodologies and Wilcoxon’s test was used for time (median (p25-p75)). Main results and the role of chance The observed correlation between the three methodologies was moderate (ICC=0.5-0.79) to high (ICC >0.8) for all parameters analysed. Concerning total sperm concentration, both fully and semi-automated systems showed a high correlation with manual assessment (ICC=0.93 (0.90-0.95) fully-automated vs manual; ICC=0.83 (0.73-0.89) semi-automated vs manual, respectively). Concerning motile sperm concentration and progressive motile sperm concentration, both fully and semi-automated systems showed a high or moderate correlation with manual assessment (ICC =0.86 (0.77-0.91) and ICC=0.87 (0.76-0.92) fully-automated vs manual; ICC=0.54 (0.27-0.70) and ICC=0.62 (0.39-0.76) semi-automated vs manual, respectively). In terms of normal forms, fully-automated showed a moderate correlation with manual assessment (ICC=0.69 (0.57-0.78)). Regarding time spent for analysis, both fully and semi-automated analysers showed to be faster than manual method (p < 0.001) (1343 (1199-1472) seconds faster fully-automated vs manual and 608 (429-769) seconds faster semi-automated vs manual). Comparing time spent for analysis between the fully and the semi-automated systems, fully-automated was 707 (610-784) seconds faster (p < 0.001) than the semi-automated. Limitations, reasons for caution Retrospective study. SCATM is a semi-automated operator-dependent system based on image analysis and its accuracy relies on the number of sperm analysed and the operators’ ability in reviewing images. SQA-VisionTM is fully-automated, based on electro-optical signal analysing millions of sperm. Therefore, conclusions may not apply to other software operators. Wider implications of the findings Fully-automated systems for sperm assessment can be reliably introduced in andrology laboratories offering a dramatic reduction in the time spent for analysis. However, such analysers may only be cost-efficient in centres with a very high activity. Trial registration number not applicable

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

To determine whether embryo mosaicism prevalence in preimplantation genetic testing for aneuploidy (PGT-A) cycles is associated with the trophectoderm biopsy technique used (a. number of laser pulses or b. the use of flicking or pulling) or the time to tubing. Prospective observational study performed in a single IVF-PGT-A setting from May 2019 to May 2021. Trophectoderm biopsies were analysed by next-generation sequencing. Mosaicism was analysed in relation to the biopsy methodology (number of laser pulses and pulling vs flicking), time elapsed from biopsy to tubing (min), and time of sample cryostorage from tubing to amplification (days). As a secondary objective, the number of laser pulses and biopsy methodology were studied in relation to clinical outcomes of transferred euploid blastocysts. None of the analysed variables were associated to mosaicism prevalence. Multivariable regression analysis demonstrated that mosaicism prevalence was comparable either when  > 3 laser pulses were used as compared to ≤ 3 (13.9% vs 13.8%, aOR = 0.8726 [0.60–1.28]) and pulling compared to flicking (13.1% vs 14.0%, aOR = 0.86 [0.60–1.23]). Moreover, neither the number of laser pulses during biopsy (> 3 vs ≤ 3) nor the technique used (pulling vs flicking) were associated with clinical pregnancy after the transfer of frozen-thawed euploid blastocysts (54.9% vs 55.2%, aOR = 1.05 [0.53–2.09]; 61.1% vs 52.9%, aOR = 1.11 [0.55–2.25], respectively). Our results suggest that, as long as the biopsy and tubing procedures are performed following standardized high quality procedures, no specific approach would increase the generation of artefactual mosaicism as a result of trophectoderm biopsy. Trophectoderm biopsies should be performed regardless of the methodology but always aiming on minimising blastocyst manipulation.

ObjectiveTo assess patients' and embryonic characteristics that may have an influence on the decision to transfer a mosaic embryo. MethodSingle centre retrospective cohort study including 1247 PGT-A cycles. Demographic and clinical factors associated with a decision to transfer a mosaic embryo were studied. Female age, number of previous cycles, previous availability of euploid embryos, history of miscarriages and parity as well as percentage of mosaicism, type of anomaly and chromosome risk were studied in relation to decision-making. Outcomes after mosaic embryo transfer were assessed. ResultsTo date, in 7.9% of cycles (99/1247), patients have had to make a decision on the fate of their mosaic embryos. In 23.2% of cycles (23/99), patients decided to transfer. In most cases (79.8%; 79/99), patients underwent genetic counselling before the decision. None of the variables analysed were associated with the patients' decision, although parity and the high-degree mosaicism (>50%) seemed to be negatively associated with the decision to transfer (18.2% vs. 29.8%, p = 0.294; 10% vs. 32.2%, p = 0.052). ConclusionsNeither reproductive history nor information on mosaic embryo characteristics through counselling seems to be determinative for patients when deciding to transfer a mosaic embryo. Promising and increasing data on clinical outcomes after mosaic embryo transfer will be of utmost importance to soften risk perception regarding mosaic embryos and give a better, simplified and more evidence-based counselling.

Research question: Are intrinsic or extrinsic factors associated with embryo mosaicism prevalence in IVF cycles? Design: Retrospective cohort study of preimplantation genetic testing for aneuploidy (PGT-A) cycles carried out at a university-affiliated IVF clinic between October 2017 and October 2019. Trophectoderm biopsies were analysed by next generation sequencing. Mosaicism prevalence, type of anomaly and the chromosomes involved were analysed. Intrinsic and extrinsic factors potentially inducing mosaicism were studied: maternal and paternal age, antral follicle count, cumulus-oocyte complexes retrieved, female body mass index, PGT-A indication, sperm concentration, total dosage of gonadotrophins, embryo quality and day of blastocyst formation, single-step commercial media used and biopsy operator. Results: Overall prevalence of mosaicism in our PGT-A setting was 13.9%. In segmental mosaicism, larger chromosomes tended to be more affected, which was not observed in whole-chromosome mosaicism. Additionally, segmental mosaicism was mostly observed in monosomy (69.6%; P < 0.01) compared with whole-chromosome mosaicism (49.7% monosomies versus 50.3% trisomies; P = 0.83). Although a high inter-patient variability was observed, only paternal age showed a positive association with mosaicism (adjusted OR 1.26, 95% CI 1.02 to 1.54) among the analysed variables. Conclusions: Our results suggest remarkable differences in the mechanisms generating segmental and whole-chromosome mosaicism, indicating that they may deserve different consideration when studying them and when prioritizing them for transfer. Male factor seems to be associated with mosaicism and may be worthy of specific assessment in future studies.

STUDY QUESTION Does an individualised luteal phase support (iLPS), according to serum progesterone (P4) level the day prior to euploid frozen embryo transfer (FET), improve pregnancy outcomes when started on the day previous to embryo transfer? SUMMARY ANSWER Patients with low serum P4 the day prior to euploid FET can benefit from the addition of daily subcutaneous P4 injections (Psc), when started the day prior to FET, and achieve similar reproductive outcomes compared to those with initial adequate P4 levels. WHAT IS KNOWN ALREADY The ratio between FET/IVF has spectacularly increased in the last years mainly thanks to the pursuit of an ovarian hyperstimulation syndrome free clinic and the development of preimplantation genetic testing (PGT). There is currently a big concern regarding the endometrial preparation for FET, especially in relation to serum P4 levels around the time of embryo transfer. Several studies have described impaired pregnancy outcomes in those patients with low P4 levels around the time of FET, considering 10 ng/ml as one of the most accepted reference values. To date, no prospective study has been designed to compare the reproductive outcomes between patients with adequate P4 the day previous to euploid FET and those with low, but restored P4 levels on the transfer day after iLPS through daily Psc started on the day previous to FET. STUDY DESIGN, SIZE, DURATION A prospective observational study was conducted at a university-affiliated fertility centre between November 2018 and January 2020 in patients undergoing PGT for aneuploidies (PGT-A) IVF cycles and a subsequent FET under hormone replacement treatment (HRT). A total of 574 cycles (453 patients) were analysed: 348 cycles (leading to 342 euploid FET) with adequate P4 on the day previous to FET, and 226 cycles (leading to 220 euploid FET) under iLPS after low P4 on the previous day to FET, but restored P4 levels on the transfer day. PARTICIPANTS/MATERIALS, SETTING, METHODS Overall we included 574 HRT FET cycles (453 patients). Standard HRT was used for endometrial preparation. P4 levels were measured the day previous to euploid FET. P4 > 10.6 ng/ml was considered as adequate and euploid FET was performed on the following day (FET Group 1). P4 < 10.6 ng/ml was considered as low, iLPS was added in the form of daily Psc injections, and a new P4 analysis was performed on the following day. FET was only performed on the same day when a restored P4 > 10.6 ng/ml was achieved (98.2% of cases) (FET Group 2). MAIN RESULTS AND THE ROLE OF CHANCE Patient's demographics and cycle parameters were comparable between both euploid FET groups (FET Group 1 and FET Group 2) in terms of age, weight, oestradiol and P4 levels and number of embryos transferred. No statistically significant differences were found in terms of clinical pregnancy rate (56.4% vs 59.1%: rate difference (RD) -2.7%, 95% CI [-11.4; 6.0]), ongoing pregnancy rate (49.4% vs 53.6%: RD -4.2%, 95% CI [-13.1; 4.7]) or live birth rate (49.1% vs 52.3%: RD -3.2%, 95% CI [-12; 5.7]). No significant differences were also found according to miscarriage rate (12.4% vs 9.2%: RD 3.2%, 95% CI [-4.3; 10.7]). LIMITATIONS, REASONS FOR CAUTION Only iLPS through daily Psc was evaluated. The time for Psc injection was not stated and no serum P4 determinations were performed once the pregnancy was achieved. WIDER IMPLICATIONS OF THE FINDINGS Our study provides information regarding an 'opportunity window' for improved ongoing pregnancy rates and miscarriage rates through a daily Psc injection in cases of inadequate P4 levels the day previous to FET (P4 < 10.6 ng/ml) and restored values the day of FET (P4 > 10.6 ng/ml). Only euploid FET under HRT were considered, avoiding one of the main reasons of miscarriage and implantation failure and overcoming confounding factors such as female age, embryo quality or ovarian stimulation protocols. STUDY FUNDING/COMPETING INTEREST(S) No external funding was received. B.C. reports personal fees from MSD, Merck Serono, Ferring Pharmaceuticals, IBSA and Gedeon Richter outside the submitted work. N.P. reports grants and personal fees from MSD, Merck Serono, Ferring Pharmaceuticals, Theramex and Besins International and personal fees from IBSA and Gedeon Richter outside the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER NCT03740568.

Introduction The development of new methodologies for PGT-A has led to the detection of a certain percentage of mosaic embryos. It has been postulated that this could be a normal feature in human embryos. To date, the real incidence of mosaicism in blastocysts is unknown as illustrated by the very variable incidences reported between groups. Certain factors could influence mosaicism occurrence such as patients’ characteristics, ovarian stimulation and in vitro culture conditions. Biopsy sample manipulation could also induce artefactual mosaicism. Moreover, different diagnostic platforms, algorithms and detection limits are used by different groups, thus making comparisons difficult. Material & methods The cytogenetic results of 1339 blastocysts obtained from PGT-A cycles performed in our centre from September 2017 to January 2019 were retrospectively analysed. Embryos were biopsied at the blastocyst stage between day 5 and 7 of development and genetic analysis was performed by next-generation sequencing (NGS) according to VeriSeq™-PGS protocols (Illumina). Embryos were diagnosed as euploid, aneuploid or mosaic. An embryo was classified as mosaic when the percentage of affected cells ranged from 30-70%. Both the incidence of mosaicism in this cohort and the specific chromosomes affected were studied. Mosaicism was studied versus patients’ factors (age, indication for PGT-A, ovarian reserve), embryonic factors (blastocyst development and quality), potential iatrogenic factors (ovarian stimulation treatment and the use of two different single-step culture media), and the biopsy operator as a proposed source of artefactual mosaicism. A logistic mixed multivariable model was applied to estimate the odds for every endpoint. Patient and biopsy procedure were treated as random factor to control the correlated observations effect. Results The overall incidence of mosaic embryos was 13.0% (7.5% of euploid-aneuploid mosaic embryos and 5.5% of aneuploid-aneuploid mosaic embryos). Most mosaic embryos showed just one chromosome affected (85.5%). No chromosomes were especially associated with mosaic aneuploidy. However, segmental mosaicism was more common in larger chromosomes. No association could be stablished between embryo mosaicism and the factors studied. Neither maternal nor paternal ages showed any association with mosaicism (ORa=0.96(0.88-1.04); ORa=1.04(0.99-1.09)). The antral follicle count did not show any correlation either (ORa=0.987(0.95-1.02)) nor the indication for PGT-A. In terms of embryo development, no associations were found either: ORa for blastocyst formation in day 5 vs day 7 was 0.72(0.31-1.68); ORa for blastocyst formation in day 6 vs day 7 was 0.53(0.23-1.22); ORa for excellent and good quality embryos vs fair and poor quality embryos was 0.86(0.54-1.36). Regarding the studied factors that were potentially associated with iatrogenic mosaicism, neither type of stimulation (agonists vs antagonists), nor the use of two different single-step media showed an association. No impact on the incidence on mosaicism has been related to a given operator. Conclusions There is no preferential distribution of mosaicism among the chromosomes although large chromosomes seem to be more sensitive to segmental mosaicism. None of the studied potential sources of intrinsic, iatrogenic, or artefactual mosaicism showed an association with such feature.

A retrospective cohort study was performed to examine whether, in artificial endometrial preparation for frozen embryo transfer (FET) cycles, progesterone (P) levels the day prior to embryo transfer of euploid embryos have an impact on pregnancy outcomes. In a private university clinic, 244 FETs between January 2016 and June 2017 were analyzed. Endometrial preparation was achieved with estradiol valerate and vaginal micronized progesterone. Serum P and estradiol levels the day prior to embryo transfer were measured. A multivariable analysis to assess the relationship between serum P level and pregnancy outcomes was performed, adjusted for confounding variables. Mean P value was 11.3 +/- 5.1 ng/ml. Progesterone levels were split in quartiles: Q1: <= 8.06 ng/ml; Q2: 8.07-10.64 ng/ml; Q3: 10.65-13.13 ng/ml; Q4: > 13.13 ng/ml. Patients included in the lower P quartile had a significantly higher miscarriage rate and significantly lower live birth rate (LBR) compared to the higher ones. A low serum P level (<= 10.64 ng/ml) one day before FET is associated with a lower pregnancy and LBR following FET of euploid embryos.

One of the main limiting factors in preimplantation genetic testing for aneuploidies (PGT-A) for advanced maternal age is not having enough available embryos for biopsy in order to find at least 1 euploid blastocyst. This study aims to analyse, for the first time, the potential benefit of blastocyst accumulation in PGT-A in women of advanced reproductive age. Retrospective study performed in a University-affiliated fertility clinic. 284 women aged ≥38 years undergoing PGT-A were included. According to our policy of blastocyst accumulation and to the expected age-related aneuploidy rate, patients with >4 blastocysts in the first cycle didn't undergo further stimulations (group ">4 no acc"), whereas women with ≤4 blastocysts in the first stimulation were recommended to accumulate: 45% accumulated (group "≤4 acc"), 55% didn't (group "≤4 no acc"). Group ">4 no acc": n=107. Group "≤4 acc": n=79. Group "≤4 no acc": n=98. Trophoectoderm biopsy was performed at blastocyst stage, blastocysts were vitrified and comprehensive chromosome testing was done with aCGH. Patients with >1 euploid blastocyt might have done >1 embryo transfer; therefore main outcome is expressed as CPR's/patient. Categorical variables were compared with Chi-Square test, continuous variables with ANOVA, in case of a significant test, Bonferroni multiple comparisons adjustment was performed. Age, antral follicle count (AFC) and AMH, did not differ significantly between groups "≤4 acc" and "≤4 no acc". Mean age of patients of group ">4 no acc" was significantly lower compared to "≤4 acc" and "≤4 no acc" (40 vs. 40.9 vs. 40.7, respectively). AFC and AMH were significantly higher in patients of group ">4 no acc" compared to the other groups (AFC: 15.9 vs. 12.2 vs. 12; AMH: 3 vs. 1.8 vs. 1.8, respectively). Mean number of biopsied embryos was similar in ">4 no acc" and "≤4 acc" (7.1 and 6.7, respectively) but significantly lower in "≤4 no acc" (2.4). Mean number of euploid embryos was 2.2 in group ">4 no acc", 1.5 in "≤4 acc" and 0.6 in "≤4 no acc" (p<0.001). The probability of having ≥1 euploid embryo was 84% for ">4 no acc", 71% for "≤4 acc" and 44% for "≤4 no acc". Out of the patients that accumulated, 29% didn't have any euploid embryo, 28% had it not in the first but in subsequent stimulations. CPR/patient was 68% in ">4 no acc", 48% in "≤4 acc" and 26% in "≤4 no acc" (OR between groups "≤4 acc" and "≤4 no acc"=2.5, 95% CI [1.3-4.8]). According to our results, PGT-A should be offered to advanced reproductive aged women with a good profile of ovarian reserve markers or willing to undergo several stimulation cycles. If not so, PGT-A should be proposed with caution as, if ≤4 blastocyts are obtained, CPR/patient do not reach 30%.

The transition in biopsy timing from blastomere to trophectoderm biopsy has led to a remarkable decrease in the percentage of undiagnosed blastocysts. However, patients with few or no euploid blastocysts can be affected by this residual percentage of diagnosis failure. The aim of this study is to assess whether blastocyst rebiopsy and revitrification is an efficient and safe procedure to be applied in cases of no results after analysis. Fifty-three patients agreed to the warming of 61 blastocysts to perform a second biopsy and PGT-A by aCGH. Only 75.4% of the blastocysts survived, reexpanded, and could be rebiopsied. After the second biopsy and analysis, 95.6% of the blastocysts were successfully diagnosed with an euploidy rate of 65.9%. Eighteen euploid blastocysts were warmed and transferred to 18 patients with a 100% survival and reexpansion rate. Seven clinical pregnancies have been achieved with 4 live births, 1 ongoing pregnancy, and 2 miscarriages. Thus, although few transfers of rebiopsied and revitrified blastocysts have been performed till date, our preliminary results show that this approach is efficient and safe to be applied for undiagnosed blastocysts, as it ultimately allows the transfer of euploid blastocysts and good clinical outcomes.