Abstract Study question Does Microfluidic Sperm Selection (MSS) improve the blastocysts’ development rate and the euploidy rate compared to Swim-up sperm selection? Summary answer MSS doesn’t improve the rate of blastocyst development and euploidy compared to Swim-up. Furthermore, in women age ≥ 37 years D6 aneuploid blastocyst rate increases What is known already Sperm defects have been shown to have a negative impact on embryo quality and development. MSS was introduced as an alternative to traditional centrifugation-based sperm preparation techniques to efficiently isolate highly motile and healthy spermatozoa. Centrifugation has been suggested to compromise membrane integrity, motility, mitochondrial energy production, and DNA structure. Less DNA fragmentation and ROS formation was observed in spermatozoa isolated with MSS compared to standard techniques. Our study aims to analyze whether MSS also improves biological outcomes. Study design, size, duration In a prospective study of 51 ICSI/PGT-A cycles performed between September 2022 and November 2023, freshly ejaculated semen sample was divided into two aliquots and processed by Swim-up and MSS. Split sperm sample was used to inject siblings’ mature oocytes (249 vs 274 oocytes in Swim-up vs MSS groups). Inclusion criteria: retrieved MII oocytes ≥8, oligoteratozoospermic and normozoospermic semen. Results were analyzed according to women’s age <37 years (21 cycles) and ≥37 years (30 cycles). Participants/materials, setting, methods Spermatozoa were selected using either conventional Swim-up or a microfluidics device (ZyMōtTM). Post treatment sperm parameters (sperm count; total sperm motility; progressive motility and hyperactivated motility) were examined for both. Embryo culture was performed in Irvine Continuous Culture-NX media until day5 or day6 and biopsy was performed at the blastocyst stage by next generation sequencing. Primary endpoint was fertilization rate, blastocyst development and usable blastocysts rate. Chi-square analysis was performed and p < 0.05 was considered significant Main results and the role of chance There is no significant differences overall between Swim-up and MSS in fertilization rate (73% vs 75%), blastocyst development rate (47% vs 54%) and ploidy rate: aneuploid (47% vs 50%), usable blastocyst (i.e. euploid+mosaics) (53% vs 50%). When considering female age, no significant difference is observed in the primary endpoints in the <37 years group. However, in women aged ≥37 years, the rate of blastocyst development is similar in the two sperm preparation groups, but a significant difference is observed in the timing of blastocyst development: The percentage of blastocysts formed on day 6 in the MSS group is significantly higher than in the Swim-up group (43% vs. 19% p < 0.05). In addition, the aneuploidy rate of blastocysts on day 6 is also significantly higher than in the Swim-up group (79% vs. 50% respectively, p < 0.05). The concentration of sperm recovered post-treatment was similar in Swim-up vs MSS (23 x106 vs 19 x106); total motility and progressive motility were similar in Swim-up compared to MSS (92.7% vs. 95.3% and 85% vs. 87% respectively). Only hyperactivated motility assessed with the SCASCOPE CASA System (Microptic) was significantly higher in MSS vs Swim-up 41% vs 4% respectively (p < 0.0005). Limitations, reasons for caution Our study included couples with different infertility diagnosis and semen parameters. Further studies with an increased number of patients may provide more comprehensive data to better evaluate biological and clinical outcomes to eventually suggest the use of MSS in laboratory routines Wider implications of the findings Recently, some studies reported that microfluidic-sorted sperm showed significantly less ROS formation and DNA fragmentation compared to those treated with conventional Swim-up method. Nevertheless, our data does not currently show the expected increase in embryo euploidy rate to justify the routine use of these expensive devices in male infertility treatment Trial registration number non applicabile

Abstract Study question Which are the clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism? Summary answer The level of mosaicism negatively influences implantation and ongoing pregnancy rate. 6/7 cases in which mosaicism persisted in the foetuses were from low-level mosaic. What is known already Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation to their reproductive potential has been defined. Study design, size, duration We collected the clinical outcomes of 3074 mosaic embryos transferred in women who underwent IVF between May 2019-May 2023. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow-growing embryos. The clinical outcome obtained after the transfer of mosaic embryos with the different chromosomal constitutions was compared with each other. Prenatal and post-natal outcomes were collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing (PGT) was performed using high-resolution next-generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis, mosaic embryos were divided into groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >5Mb, MS). Main results and the role of chance Embryos classified as ‘low-mosaic’ by NGS-based PGT-A have a higher likelihood of achieving implantation compared to ‘high-mosaic’ embryos (48% vs. 39.%; p < 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p < 0.05). Chromosomal composition of mosaicism abnormalities dictates the success rate of mosaic embryo transfers, with low and high segmental mosaics being preferable over low- and high-mosaics involving whole chromosomes. For 621/670 pregnancies, parental tests and post-natal data were available. The majority (99.75%) of the babies were largely healthy by routine physical inspection by neonatologists (no gross abnormalities in babies from mosaic embryos, n = 495). A combination of NIPT, CVS, and Amniocentesis prenatal testing results were collected for 552 pregnancies of mosaic embryo transfers, with predominantly normal findings. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 7 out of 552 pregnancies (0.9%), in which the mosaicism identified with PGT-A at the blastocyst stage was reflected in gestation by prenatal chromosomal testing as true fetal mosaicism. Limitations, reasons for caution Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer. Wider implications of the findings The international registry of mosaic embryo transfers continues to grow in sample size, in turn increasing the power of analysis. The findings of the mosaic embryo transfer registry can help educate the management and selection of embryos in the clinic. Trial registration number no

Abstract Study question How much of an impact has the maternal age on cumulative life births after euploid Single Embryo Transfer (SET) in autologous and donor oocyte cycles? Summary answer A maternal age ≥ 42 years old affects negatively the cumulative Life Birth Rates (cLBRs) even after euploid SET, but only in the autologous cycles. What is known already Despite the improvements in assisted reproduction technologies (ART) in the last decades, the LBRs remain suboptimal, especially when advanced maternal age is considered. Although several factors could explain these outcomes (i.e. infertility history; uterine pathologies/surgery; advanced paternal age), maternal age-related aneuploidies remain the most limiting factor for a favorable clinical outcome. Thus, the Genetic Testing for Aneuploidies (PGT-A) has been integrated into ART as a strategy to evaluate chromosomal status before transfer. Interestingly, recent studies show that, even after the transfer of euploid blastocyst, the maternal age negatively influences the LBRs. Study design, size, duration This study, carried out in Villa Mafalda Reproductive Medicine Department (Italy), included 568 euploid SET: 542 from autologous cycles (divided in four subgroups according to maternal age: ≤ 34 y.o. (n = 207); 35-38 y.o. (n = 193); 39-41 y.o. (n = 106); ≥ 42 y.o. (n = 36)) and 26 from oocyte donation (OD) cycles (average recipient age is 44.2). All cycles were performed from May 2020 to February 2023. PGT for Monogenic disorders (PGT-M) cycles were excluded. Participants/materials, setting, methods All blastocysts included in this study were analyzed by Next Generation Sequencing (NGS) after a day 5/6 trophectoderm biopsy. For each subgroup was annotated the embryo morphology- divided into good (AA-AB-BA); average (BB-BC-CB) and poor (CC) according to Gardner/Schoolcraft blastocyst grading system- and the day of expanded blastocyst formation (grade 4 of Gardner/Schoolcraft blastocyst grading system). Primary analysis included the cumulative LBRs. Secondarily, blastocyst morphology and blastocyst expansion were examined for all embryo categories considered. Main results and the role of chance The pairwise comparison between groups was performed by Chi-square analysis and p < 0.05 was considered statistically significant. The cLBRs for each subgroup were respectively: ≤ 34 (63.09%); 35-38 (58.47%); 39-41 (56.84%); ≥ 42 (48.57%); OD (69.23%). Data showed that cLBRs decreased with the increase of maternal age, although statistical significance was observed only among women ≤ 34 y.o. and ≥ 42 y.o. (P=0.029). Notably, there was no statistical significance in cLBRs comparing women ≤ 34 y.o. and OD patients (P=0.086). The good-quality blastocyst rate was respectively: ≤ 34 y.o. (54.58%); 35-38 y.o. (47.66%); 39-41 y.o. (60.37%); ≥ 42 y.o. (50%); OD (59.37%). The pairwise comparison showed no significant difference between groups. Furthermore, there was not found significant difference about the day of expanded blastocyst formation: ≤ 34 y.o. (D5: 78.92%; D6 21.07%); 35-38 y.o. (D5: 78.49%; D6: 21.50%); 39-41 y.o. (D5:75.70%;D6: 24.29%); ≥ 42 y.o. (D5:69.44%; D6: 30.55%); OD (D5: 87.5%; D6: 12.5%). Data showed that maternal age ≥ 42 y.o. affects negatively cLBRs even after euploid SET, suggesting that other factors age-related, rather than aneuploidies, influence implantation. Interestingly, these factors do not affect OD recipients, with advanced maternal age, who, instead, reach successful LBRs like patients ≤ 34. Limitations, reasons for caution These results should be evaluated carefully because of the small sample size of the OD patients. A greater number of OD cycles are needed to verify the results obtained. Wider implications of the findings Our findings suggest that embryonic age-related factors - changes in gene expression, metabolism or epigenetics - could influence the embryo capability to, successfully, support implantation other than the ploidy status. This thesis was supported by the OD recipient’s behavior, who had cLBRs comparable to young autologous patients. Trial registration number Not Applicable

Abstract Study question Does an increase in usable blastocyst rates with low lactate culture medium lead to increased live birth rates? Summary answer Increased overall usable blastocyst rate correlates with an increased live birth rate following low lactate embryo culture. What is known already Lactate is essential to the oocyte and early embryo and while it is produced naturally through the normal glycolytic pathways of the same, it is nonetheless added to available embryo culture media. In embryo culture using a continuous culture medium containing only 1mM lactate, an increase in day 5 and overall usable blastocyst rates has been documented as compared to higher lactate (6-10mM) in a sequential media system. There are now also studies showing an increase in normal fertilization with a decrease in no fertilization and abnormal fertilization with oocytes placed in low lactate at oocyte retrieval. Study design, size, duration In a prospective interventional study of IVF cases between October 2020 and April 2021, patient oocytes were divided randomly amongst a control (Vitrolife G1/G2) and treatment (Continuous Single Culture Medium-NX Complete) group. Oocytes were split following oocyte retrieval and embryo culture proceeded in one of the two groups through day 6. Total oocytes in the control group, 258, and in the treatment group, 273. Patients of all ages and diagnosis’ were included. Participants/materials, setting, methods All cases were inseminated via intra-cytoplasmic sperm injection, blastocyst biopsy performed for next generation sequencing, and blastocysts vitrified with Vit Kit or Vit Kit–Freeze NX for frozen embryo transfer. All blastocysts were warmed with Vit Kit–Warm NX and transferred using SG/G2-Plus. Primary analysis included initial beta-HCG, clinical pregnancy rate (fetal cardiac activity) and live birth. Secondarily, fertilization rates were examined for comparison of normal (2PN), abnormal (1PN and 3PN), and no fertilization (0PN). Main results and the role of chance Chi-square analysis was applied on all considerations to assess statistical significance and p < 0.05 considered significant. In the analysis of fertilization parameters for the control versus treatment group, normal fertilization (69.0% vs 74.0%), abnormal fertilization (6.2% vs 8.0%) and no fertilization (19.8% vs 15.8%) rates were not significant. The initial beta HCG rates following frozen embryo transfer in the control and treatment groups were significant at 65.3% and 90.9%, respectively (p = 0.036). The rate of clinical pregnancy in the control was 38.5% and in the treatment group was 68.2% (p = 0.040). Live birth rate in the control versus treatment group was 30.8% and 59.1%, respectively (p = 0.049). There was no difference in the implantation rate between groups (control = 56.7%, treatment = 80.0%) (p = 0.065). In patients where the first embryo transfer did not result in clinical pregnancy, a subsequent transfer with an embryo from the opposite culture medium yielded a live birth rate of 11.1% (control) and 33.3% (treatment). Limitations, reasons for caution The pregnancy data in this particular study all result from frozen embryo transfers, with no fresh embryo transfers, and the possible effect or interference of the vitrification media, recovery medium and/or transfer medium cannot be accounted for, therefore, more study is needed. Wider implications of the findings The pregnancy and live birth rates in this analysis support previous findings that low lactate culture medium increases the instance of usable blastocysts and demonstrates that those increases can ultimately correlate with, and result in, a higher live birth rate for patients. Trial registration number Not Applicable

Abstract Study question Which are the results obtained by the International Registry of Mosaic Embryo Transfers? Summary answer An update of clinical outcomes and post-natal results of mosaic embryos with low and high-level of mosaicism is provided. What is known already Chromosomal mosaic embryos are characterized by the presence of chromosomally different cell lines within the same embryo. While the transfer of these embryos is now offered as an option for women who undergo in vitro fertilization (IVF), several concerns remain. For instance, the limited data on pregnancy outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Therefore, some argue that mosaicism should be not reported until a clear classification of such embryos in relation to their reproductive potential has been defined. Study design, size, duration We collected the clinical outcomes of 2045 mosaic embryos transferred in women who undergoing IVF between May 2019-May 2022. All embryos were cultured to blastocyst stage; trophectoderm (TE) biopsy was performed on Day-5 of development or Day6/7 for slow-growing embryos. The clinical outcomes obtained after the transfer of mosaic embryos with the different chromosomal constitutions were compared. Prenatal and post-natal outcome was collected for available cases. Participants/materials, setting, methods Preimplantation genetic testing for aneuploidies (PGT-A) was performed using high-resolution next-generation sequencing (NGS) methodology. TE biopsies were classified as mosaic if they had 20%-80% abnormal cells. For statistical analysis, mosaic embryos were divided into groups based on mosaic levels and chromosomal constitution detected in TE: single mosaic aneuploidy (monosomy/trisomy; SM), double mosaic chromosomes (monosomy/trisomy or combination, DM), complex mosaic aneuploidy (>2 different aneuploidies; CM) and mosaic segmental aneuploidy (single and double deletion/insertion >5Mb, MS). Main results and the role of chance Embryos classified as ‘low-mosaic’ by NGS-based PGT-A have a higher likelihood of achieving implantation compared to ‘high-mosaic’ embryos (48% vs. 39.%; p < 0.05 ), as well as ongoing pregnancy/live birth (40% vs. 29%; p < 0.05). Chromosomal composition of mosaicism abnormalities dictates the success rate of mosaic embryo transfers, with low and high segmental mosaics being preferable over low- and high-mosaics involving whole chromosomes. For 550/670 pregnancies, parental tests and post-natal data were available. The majority (99.75%) of the babies were largely healthy by routine physical inspection by neonatologists (no gross abnormalities in babies from mosaic embryos, n = 495). Prenatal testing performed on pregnancies from mosaic embryo transfers were generally normal. The mosaicism detected at the embryonic stage by PGT-A was reflected in prenatal testing in only 5 out of 550 pregnancies (0.9%) in which the mosaicism identified with PGT-A at the blastocyst stage was reflected in gestation by prenatal chromosomal testing as true fetal mosaicism. Limitations, reasons for caution Additional clinical data must be obtained to evaluate the contribution of each different chromosome before this approach can be evaluated as an additional tool to choose mosaic embryos for transfer. Wider implications of the findings The International Registry of Mosaic Embryo transfers continues to grow in sample size, in turn increasing the power of analysis. The findings of the mosaic embryo transfer registry can help educate the management and selection of embryos in the clinic. Trial registration number Not applicable

Study question Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst? Summary answer Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology. What is known already Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome. Study design, size, duration This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test. Participants/materials, setting, methods 771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA. Main results and the role of chance In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p < 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( > = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p < 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient. Limitations, reasons for caution Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer. Wider implications of the findings While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible. Trial registration number Not Applicable

Abstract Study question Which chromosomal mosaic features do women under IVF treatment show? Summary answer Single aneuploidies were the most represented among mosaic embryos and their frequency as well as the level of mosaicism increased in older women. What is known already Chromosomal mosaic embryos are characterised by the presence of chromosomally different cell lines within the same embryo. We previously demonstrated that reproductive potential of mosaic embryos is affected by the complexity of and the number of aneuploid cells present in trophectoderm (TE) biopsy. Although with the introduction of next generation sequencing (NGS) chromosomal constitution of human embryos have been elucidated, only limited number of mosaic have been characterised. Therefore, we performed a larger-scale multicenter study on mosaic embryos to examine the patterns and prevalence of chromosome specific mosaicisms in TE samples. Study design, size, duration This is a retrospective cohort study from May 2019 to May 2021 of 20200 embryos obtained from 5770 women under IVF treatment. From this cohort, 2280 mosaic embryos were analysed. All embryos were cultured to blastocyst stage; TE biopsy was performed on Day-5 of development or on Day-6/7 for slow growing embryos. Participants/materials, setting, methods TE biopsies underwent comprehensive chromosome screening (CCS) utilizing validated NGS. TE biopsies were classified as mosaic if they had 20%-80% of abnormal cells. For statistical analysis, embryos were divided in: single whole-chromosome trisomy, double whole-chromosome trisomies, single whole-chromosome monosomy, double whole-chromosome monosomies and complex aneuploidy (more than two different aneuploidies) groups. In addition, the frequency of single, double and complex segmental aneuploidies was evaluated. For each group, the trend related to maternal age was examined. Main results and the role of chance Among 2282 mosaic embryos single (whole-chromosome and segmental) aneuploidies were the most represented(52%) followed by complex(36%) and double ones(14%). When embryos were divided based on different types of aneuploidies, complex aneuploidies were the most frequent(34%) followed by segmental(31%), single monosomies(14%), single trisomy(12%). The more affected chromosomes within trisomies were 16(7%), 19 (7%), 21 and 22(6%). Monosomy involved mainly chromosomes 7(8%), 21 and 22(7%). Chromosomes 1, 5(9%), and 9(8%) were the most targeted by segmental duplication while segmental deletion mostly involved chromosomes 1(11%), 2(13%) and X(8%). The frequency of single aneuploidies as well as the percentage of mosaicism increased in older women. Limitations, reasons for caution This study was retrospective and observational, demonstrating the relative frequency of mosaicisms but not offering any direct insight into the clinical relevance of the findings. More clinical data must be obtained before this approach can be evaluated as an additional tool to choose mosaic embryos for the transfer. Wider implications of the findings This study provides a large dataset of mosaic embryos and offers detailed information on the distribution of different types of mosaicism among a general population of women under IVF treatment. Trial registration number Not applicable

Abstract Study question Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst? Summary answer Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology. What is known already Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome. Study design, size, duration This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test. Participants/materials, setting, methods 771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA. Main results and the role of chance In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p < 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( > = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p < 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient. Limitations, reasons for caution Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer. Wider implications of the findings While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible. Trial registration number Not Applicable

Study question Which chromosomal mosaic features do women under IVF treatment show? Summary answer Single aneuploidies were the most represented among mosaic embryos and their frequency as well as the level of mosaicism increased in older women. What is known already Chromosomal mosaic embryos are characterised by the presence of chromosomally different cell lines within the same embryo. We previously demonstrated that reproductive potential of mosaic embryos is affected by the complexity of and the number of aneuploid cells present in trophectoderm (TE) biopsy. Although with the introduction of next generation sequencing (NGS) chromosomal constitution of human embryos have been elucidated, only limited number of mosaic have been characterised. Therefore, we performed a larger-scale multicenter study on mosaic embryos to examine the patterns and prevalence of chromosome specific mosaicisms in TE samples. Study design, size, duration This is a retrospective cohort study from May 2019 to May 2021 of 20200 embryos obtained from 5770 women under IVF treatment. From this cohort, 2280 mosaic embryos were analysed. All embryos were cultured to blastocyst stage; TE biopsy was performed on Day-5 of development or on Day-6/7 for slow growing embryos. Participants/materials, setting, methods TE biopsies underwent comprehensive chromosome screening (CCS) utilizing validated NGS. TE biopsies were classified as mosaic if they had 20%-80% of abnormal cells. For statistical analysis, embryos were divided in: single whole-chromosome trisomy, double whole-chromosome trisomies, single whole-chromosome monosomy, double whole-chromosome monosomies and complex aneuploidy (more than two different aneuploidies) groups. In addition, the frequency of single, double and complex segmental aneuploidies was evaluated. For each group, the trend related to maternal age was examined. Main results and the role of chance Among 2282 mosaic embryos single (whole-chromosome and segmental) aneuploidies were the most represented(52%) followed by complex(36%) and double ones(14%). When embryos were divided based on different types of aneuploidies, complex aneuploidies were the most frequent(34%) followed by segmental(31%), single monosomies(14%), single trisomy(12%). The more affected chromosomes within trisomies were 16(7%), 19 (7%), 21 and 22(6%). Monosomy involved mainly chromosomes 7(8%), 21 and 22(7%). Chromosomes 1, 5(9%), and 9(8%) were the most targeted by segmental duplication while segmental deletion mostly involved chromosomes 1(11%), 2(13%) and X(8%). The frequency of single aneuploidies as well as the percentage of mosaicism increased in older women. Limitations, reasons for caution This study was retrospective and observational, demonstrating the relative frequency of mosaicisms but not offering any direct insight into the clinical relevance of the findings. More clinical data must be obtained before this approach can be evaluated as an additional tool to choose mosaic embryos for the transfer. Wider implications of the findings This study provides a large dataset of mosaic embryos and offers detailed information on the distribution of different types of mosaicism among a general population of women under IVF treatment. Trial registration number Not applicable

STUDY QUESTION:Can anti-Müllerian hormone (AMH) help predict how many oocytes will be retrieved following double stimulation (DuoStim)?SUMMARY ANSWER:A simple clinical tool can use serum AMH values to predict ovarian response following DuoStim in IVF cycles.WHAT IS ALREADY KNOWN:The knowledge that multiple follicular waves arise during a single ovarian cycle has led to the introduction of unconventional ovarian stimulation protocols. The DuoStim protocol involves two successive ovarian stimulations performed during a single ovarian cycle and has been proposed as an approach for patients with poor ovarian response and for medical fertility preservation. As AMH has been used as a marker of ovarian reserve and stimulation response, the current study aimed to investigate the diagnostic performance of AMH in predicting the number of retrieved oocytes following DuoStim.STUDY DESIGN, SIZE, DURATION:This is a retrospective observational study involving 116 patients who received IVF treatment from January 2021 to September 2022.PARTICIPANTS/MATERIALS, SETTING, METHODS:The study was conducted at a private IVF centre. Only patients who had their AMH measured prior to treatment and had complete patient records regarding their clinical and IVF/ICSI cycle characteristics were included. The primary outcome was the correlation between AMH values and the number of oocytes retrieved following DuoStim. Parametric and non-parametric tests were used to compare baseline characteristics and outcomes. Spearman's R was used to analyse correlations between variables, while the C statistic was used to calculate the diagnostic performance of AMH.MAIN RESULTS AND THE ROLE OF CHANCE:AMH levels were significantly correlated with the total number of oocytes retrieved after the DuoStim (R 0.61; CI 0.44-0.70; P < 0.0001). The difference in the total number of oocytes retrieved between the first (median 4 oocytes, interquartile range (IQR) 2-6) and second (median 6 oocytes, IQR 3.2-8) stimulation was statistically significant (P < 0.0001). However, there was no significant difference in the number of mature oocytes that were retrieved (median of 3 and 4 in the first and second stimulations, respectively). After the first stimulation, 68% of patients had at least one blastocyst available, while after the second stimulation, 74% did (NS). Based on linear regression, each 0.25 ng/ml increase in basal AMH corresponds to one additional oocyte recovered at the end of both stimulations (R2: 0.32, P < 0.0001).LIMITATIONS, REASONS FOR CAUTION:The results are limited owing to the observational nature of the study and the number of participants.WIDER IMPLICATIONS OF THE FINDINGS:Counselling infertile couples regarding the intermediate outcome of IVF (i.e. number of retrieved oocytes) is one of the most demanding tasks that clinicians face. To our knowledge, this is the first study that provides an easy-to-use clinical tool that enables the quantitative prediction of ovarian response following DuoStim, based on serum AMH values.STUDY FUNDING/COMPETING INTEREST(S):No external funding was obtained for this study. The authors declare no conflicts of interest.TRIAL REGISTRATION NUMBER:N/A.

Abstract Study question Does Continuous Single Culture Medium NX, an embryo culture medium containing 1mM lactate, support increased blastocyst development over high lactate Vitrolife G1/G2 Series sequential culture? Summary answer There is a statistically significant increase in day 5 usable blastocysts in low lactate culture medium compared to the one with high lactate medium. What is known already Studies have shown that day 5 is the most desirable day to obtain blastocysts that are of an expansion, grade and quality to be utilized for transfer and/or vitrification procedures as those result in the highest success of clinical pregnancy,as compared to day 6/ 7 blastocysts, that do not meet criteria. Moreover, recent studies have indicated that there is an increase in chromosomal correctness of embryos cultured in a 1mM lactate environment as opposed to the higher 6-10mM lactate concentrations that have historically been believed necessary for successful blastocyst culture and resulting pregnancy. Study design, size, duration A prospective split sibling oocytes study was performed on 50 ICSI and IMSI cycles from October 2020 through April 2021. Oocytes were divided into the low lactate medium and high lactate gradient medium immediately following ICSI/IMSI and thereafter cultured in those medium until the final day of blastocyst culture. All patient ages were included in the sample population. Participants/materials, setting, methods This study was carried out in a private clinic. All patient stimulation protocol information and diagnosis’ were recorded; however, there was no restriction on participation. The endpoint was to analyse the resulting usable blastocyst rates on day 5 and day 6 in both arms of the study, using a denominator of normal 2PN fertilization. If a blastocyst was transferred or cryopreserved on day 5 or day 6, it was determined to be usable. Main results and the role of chance The resulting data was stratified not only by day 5 and day 6 usable blastocyst rates but also by patient age. It illustrates a statistically significant improvement in day 5 usable blastocysts for patients <35 in CSCM-NXC vs G1/G2 at 56% and 42%, respectively, a 14% increase (p < .05). The overall day 5 usable blastocyst rate was also statistically significant in CSCM-NXC (47%) as compared to G1/G2 (36%), (p < .05) with all ages considered. Additionally, on day 5, there was a higher percentage of usable blastocysts demonstrated in low lactate vs high in patients aged 35-37 (65% vs 42%, respectively) and 41-42 (41% vs 15%). Statistical significance was reversed in patients <35 on day 6, with G1/G2 having 24% usable blasts and CSCM-NXC 10% (p < .05). Interestingly, though not significant, G1/G2 had an increase in usable blastocyst percentage on day 5 in patients >42 (20% vs 11%), but overall, CSCM-NXC saw an increase in that same age group by 16%. Limitations, reasons for caution Though statistical significance was found in this study, a greater number will help to bolster the statistical power of the observations. Additionally, more studies are needed in order to ascertain if low lactate has an effect on the development prior to ICSI and resulting culture. Wider implications of the findings The mechanism of action that leads to the successful embryo development in low lactate embryo culture medium is vastly unknown, so further studies are required in order to understand the complexities and the impact of the observations provided. Trial registration number not applicable.

Abstract Study question Do kinetic parameters change among euploid, mosaic and aneuploid blastocysts? Is the KIDscoreTMDay5 version 3.0 (KS-5.3) correlated to preimplantation genetic testing for aneuploidies (PGT-A) results? Summary answer The KS-5.3 differs in embryo ploidy classes. The analysis of the kinetic variables showed that the aneuploid embryos were significantly slower than euploid and mosaic. What is known already Chromosomal abnormalities affect more than 50% of embryos in women with >35 years of age and PGT-A is the best way to predict embryo’s ploidy status decreasing implantation failure and miscarriage. However, this procedure is not always possible due to social or moral issues. So, the use of the non-invasive time lapse monitoring could be helpful to determine the morphokinetic characteristics in the different ploidy classes. KS-5.3 (vitrolife,Sweden) is a scoring model based on morphokinetic data, developed to predict the pregnancy rate of day-5 blastocysts. Recent publications showed differences in kinetic parameters between euploid and aneuploid embryos. Study design, size, duration This retrospective study analyzed 728 blastocysts with PGT-A results obtained at Villa Mafalda Clinic from May 2020 to June 2021. Embryos were cultured in EmbryoScope+ time-lapse system (Vitrolife) at 37 °C, 6%CO2, and 5% O2. The PGT-A was performed using next-generation sequence (NGS) technology on the trophectoderm biopsy sample on day 5/6/7. Automatic annotations for division times and quality gradings were performed by senior embryologists and all kinetic values were reported in hours post microinjection. Participants/materials, setting, methods 728 blastocysts were classified in: (E) euploid (n = 172), (M) mosaicism (n = 171) and (A) aneuploid (n = 385). In this study, they were considered KS-5.3 and the following kinetic variables: the time to reach 2 cells (t2), 3 cells (t3), 4 cells (t4), 5 cells (t5), and the blastocyst formation (TB). Continuous variables were reported as the median and interquartile range (IQR). For the statistical analysis, nonparametric tests were performed and p < 0.05 was considered statistically significant. Main results and the role of chance KS5.3 was significantly different between groups [E = 6.6(4.6-7.9) vs M = 5.3(2.9-7.2) vs A = 4.0(2.5-6.6), p < 0.0001]. It was significantly higher in euploid than in mosaic and aneuploid (EvsM p = 0.0007, EvsA p <0.0001, MvsA p = 0.0077). A significant delay in t2,t3,t4 and tb was showed in aneuploid embryos compared to euploid and mosaic, whereas there was no significant difference between euploid and mosaic: [t2: E = 25.80 (24.56-28.09), M = 25.99 (24.49-28.91), A = 27.02 (25.30-29.47), EvsA p <0.0001, AvsM p = 0.03, EvsM p = 0.32]; [t3: E = 37.08 (34.74-39.34), M = 36.69 (34.55-40.02), E = 38.45 (35.93-41.14), EvsA p = 0.0003, MvsA p = 0.002, EvsM p >0.99]; [t4: E = 38.28 (35.63-41.19), M = 38.49 (35.47-42.13), A 39.72 (37.25-43.31), EvsA p = 0.0001, MvsA p = 0.02, EvsM p = 0.65]; [tb: E = 107.70 (102.20-114.30), M = 110.10 (103.60-116.80), A = 113.7 (106.80-122.70), EvsA p <0.0001, MvsA p <0.0001, EvsM p = 0.42]. As for t5, there were no differences among the groups. Longer cell cycles in aneuploid embryos could be associated with activated DNA repair mechanism or during chromosome segregation. Instead, regarding the mosaics, there was a significant difference with euploid embryos only in KS5.3. The age was similar between euploid and mosaic [E = 36.29 (33.42-39.00) vs M = 36.71 (34.00-39.33) p = 0.99], whereas that was significantly higher in aneuploid embryos [A = 39.11(36.01-42.27), EvsA/EvsM p <0.0001]. Limitations, reasons for caution All these findings have to be validated in a larger sample size. Furthermore, for the retrospective nature of this study, there were some confounding factors, such as protocol of stimulation, female age, and malefactor. This research did not consider the importance of every single kinetic parameter. Wider implications of the findings A further study is needed to verify if there is a correlation between morphology and ploidy status. This could clarify the difference in KS-5.3 between euploid and mosaic. In order to decrease age bias, we should enlarge the sample size to analyze a subgroup of patients with higher maternal age. Trial registration number not applicable

Abstract Study question Can MACS increase euploid blastocyst rate in Pre-implantation Genetic Testing (PGT) cycles for AMA-APA (Advanced Maternal-Paternal Age) in patients with high sperm DNA fragmentation (SDF)? Summary answer A slight increase in euploid blastocyst rate was found using MACS in infertile patients with high SDF undergoing PGT cycles compared to the control group. What is known already Many authors have shown a close correlation between the presence of apoptotic markers on spermatozoa and the failure of assisted reproduction treatments. In normal physiological conditions, apoptotic spermatozoa with phosphatidylserine (PS) residues externalized on the plasma membrane, are eliminated along female genital tract, preventing oocyte fertilization. MACS eliminates apoptotic sperm whit PS residues using superparamagnetic microbeads conjugated with annexin V. This technique reduces the proportion of sperm with high rates of SDF and can be used to maximize ART procedures results. MACS application improves sperm quality, fertilization, cleavage and pregnancy rates reducing miscarriage rate. Study design, size, duration From June to November 2020, 10 couples in which MACS was applied to select non-apoptotic spermatozoa, were randomly enrolled in our study (MACS group) and 8 couples without MACS were considered as controls (No-MACS Group). All couples in both groups underwent a PGT cycle and had high sperm DNA Fragmentation (> 20%). A higher rate of euploid and diploid-euploid mosaic blastocysts were obtained in the MACS group compared to the control group. Participants/materials, setting, methods Patients with severe oligoastenoteratozoospermia were excluded. MACS protocol was performed as follows: semen sample was analyzed (WHO 2010) and washed with buffered medium; pellet was removed and a swim-up was performed. Retrieved spermatozoa were washed with a binding buffer (Miltenyi Biotec), centrifuged (400 g x 4 minutes) and supernatant discarded. Pellet was covered with Annexin-V and re-suspended. After 15 minutes incubation at room temperature, the sample was eluted through the column and collected for ICSI. Main results and the role of chance In MACS group, female and male mean age ± SD were 41.6 ± 2.1 and 43.5 ± 7.3, respectively. Female and male mean age ± SD were 41.7 ± 2.8 and 44.6 ± 8.1 in the No-MACS group, respectively. In MACS and No-MACS groups, injected oocytes were 44 and 35, fertilized oocytes were 32 (72.3%) and 27 (77.1%) (NS), blastocyst formation rates were 71.8% (23/32) and 48.1% (13/27) (NS), respectively. In No-MACS group, only 1 euploid and 1 diploid-euploid mosaic blastocysts were obtained (1/13 = 8%) (NS). In MACS group, 4 euploid blastocysts were formed (4/23 = 17.4%) whereas mosaic diploid-euploid blastocysts were 3/23 (13.0%) (NS). Aneuploid blastocysts were 16/23 (69.6%) in MACS group and 11/13 (84.6%) in No-MACS group (NS). Limitations, reasons for caution AMA and APA of couples enrolled should be considered as a limit of the study. A larger number of patients and biopsied blastocysts are needed to analyze clinical results and perform a robust statistical analysis establishing if MACS is useful to improve transferable blastocyst rate in patients with high SDF. Wider implications of the findings: MACS is useful to select non apoptotic sperms; although fertilization, cleavage and blastocyst rates are not improved, aneuploid blastocysts rate slightly decreases using MACS. It I possible that, selecting spermatozoa free from PS residues, MACS allows to choose spermatozoa with a better DNA packaging, thus affecting the embryo ploidy. Trial registration number non applicable

Abstract Study question We assessed the outcome and predictors of successful salvage microdissection testicular sperm extraction (mTESE) in non-obstructive azoospermia (NOA) men previously submitted to unfruitful classic (cTESE). Summary answer The sperm retrieval rate at salvage mTESE was almost 50%. Hypospermatogenesis and low FSH values were associated with positive outcomes at salvage mTESE What is known already In men with NOA testicular sperm can be retrieved using cTESE in approximately 50% of cases. mTESE has been proposed as a salvage treatment option for men with a previously failed TESE, but data are scarce. Study design, size, duration Multicenter, cross-sectional study. Complete data from 61 NOA men who underwent mTESE after a failed cTESE between 01/2014 and 10/2020, at 6 tertiary referral centers in Italy were analysed. Participants/materials, setting, methods All men underwent testicular ultrasound, hormonal and genetic blood testing. Histopathological diagnosis from TESE was collected in every man. Semen analyses were based on the 2010 WHO reference criteria. mTESE was performed according to the technique of Schlegel et al. (1999). Descriptive statistics and logistic regression models were used to investigate potential predictors of positive sperm retrieval (SR+) after salvage mTESE. Main results and the role of chance Overall, median (IQR) age and testicular volume were 35 (31–38) years and 10 (6–15) ml, respectively. Baseline serum FSH and total testosterone levels were 17.1 (8.6–30.4) mUI/mL and 4.7 (3.5–6.4) ng/mL, respectively. Sertoli-cell-only (SCO) syndrome, maturation arrest (MA) and hypospermatogenesis were found in 24 (39.3%), 21 (34.4%) and 16 (26.2%) men after cTESE, respectively. Spermatozoa were retrieved in 30 (49.2%) men at salvage mTESE. Patients with a diagnosis of hypospermatogenesis had a higher rate of SR + [12/16 (75%)] than those with MA [12/21 (57.1%)] and SCOS [6/24 (25%)] after salvage mTESE (p < 0.01), which was bilateral in 36 (59%) cases. FSH was higher [16.5 (8–22) vs. 8.9 (5–13) mUI/mL, p < 0.01] in SR- patients compared to SR+. No difference in clinical characteristics was found between patients with SR+ and SR- at salvage mTESE. There were no significant complications after mTESE. Multivariable logistic regression analysis showed that hypospermatogenesis (OR 9.7; p < 0.01) and low FSH levels (OR 0.9, p < 0.001) were independent predictors of SR+ after salvage mTESE, after accounting for age. Limitations, reasons for caution Despite we analysed one of the largest series of salvage mTESE, the samples size is too small to draw general conclusions. Because of the multicenter nature of the study we cannot rely on standardization of surgical techniques for TESE. Wider implications of the findings: This is one of the larger studies on salvage mTESE. The selection of patients for salvage mTESE is of critical importance. Trial registration number na

Abstract Study question To explore the effect of chromosomal mosaicism detected in preimplantation genetic testing (PGT-A) on prenatal and postnatal outcome of mosaic embryo pregnancies Summary answer No significant difference between euploid and mosaic embryos was observed in terms of weeks of gestation, average weight, and developmental defect of the babies born What is known already Mosaic embryos have the potential to implant and develop into healthy babies. Transfer of these embryos is now offered as an option for women who undergo IVF resulting in no euploid embryos. While, prenatal diagnosis has shown the depletion of chromosomal mosaicism in mosaic embryos, several concerns remain. For instance, the direct effects of different kind of mosaicism on prenatal/postnatal outcome and the possibility that intra-biopsy mosaicism in the TE is a poor predictor of the ploidy status of the ICM. Thus, there is certainly a need for comprehensive analyses of obstetrical and neonatal outcome data of transferred mosaic embryos. Study design, size, duration Compiled analysis from multicenter data on transfers of mosaic embryos (n = 1,000) and their outcome, with comparison to a euploid control group (n = 5,561). To explore the effect of embryonic mosaicism on newborns, we matched mosaic embryos resulting in a birth with a euploid embryo by a series of parameters (maternal age, embryo morphology, and indication for PGT-A). Prenatal tests and birth characteristics of > 200 neonates from mosaic embryo transfers were compared to > 200 euploid embryos. Participants/materials, setting, methods PGT-A was performed on blastocyst-stage embryos with 24-Chromosome whole genome amplification (WGA)-based Next Generation Sequencing (NGS). In accordance with established guidelines, embryos were categorized as mosaic when PGT-A results indicated 20-80% aneuploid content. Prenatal testing where performed in 30% of pregnancies with amniocentesis, 4% did an extra analysis for potential UPD for the suspected mosaic chromosome, and an additional 16% performed chorionic villus sampling (CVS) and 9.5% performed noninvasive prenatal testing (NIPT). Main results and the role of chance Of the 465 mosaic embryos that implanted, about 20% miscarried, and out of those, 75% were early spontaneous abortions. Of the pregnancies, 3 out of 368 were stillborn (2 out of them were twins that were extremely premature at 23 weeks, and the other died during pregnancy from a heart defect). The remaining 99% of those have been born or are late ongoing pregnancies at the time of analysis. Prenatal tests were performed in > 200 pregnancies and the vast majority tested normal. All 5 abnormal cases were amniocentesis tests showing microdeletions or insertions of sizes smaller than the resolution used during PGT-A, so they were unrelated to the mosaicism detected with PGT-A. In fact, in none of the cases did the prenatal test reflect the mosaicism detected at the embryonic stage. Matching each of the 162 mosaic embryos resulting in a birth with a euploid embryo, we found that the length of gestation was similar on average, and so was the average weight of the babies at birth. We also gathered information on the routine physical examination performed on babies at birth, and of those 162 babies from mosaic embryo transfers, none had obvious developmental defects or gross abnormalities. Limitations, reasons for caution Even though newborns resulting from mosaic embryo transfers in this study invariably appeared healthy by routine examination, concerns for long-term health cannot yet be entirely dispelled. The question must therefore be carefully considered by each clinic and patient situation. Wider implications of the findings Prenatal testing of > 200 pregnancies from mosaic embryo transfers showed no incidence of mosaicism that matched the PGT-A findings, indicating the involvement of self-corrective mechanisms. Pregnancy and obstetric data indicates that mosaic embryos prevailing through gestation and birth have similar chromosomal and physiological health compared to euploid embryos. Trial registration number none

Abstract Study question Could advanced paternal age influences the embryos aneuploidy rate in eggs donation cycles with poor sperm quality? Summary answer In case of severe male factors increased paternal age can affect embryos aneuploidy rate in egg donation cycles. What is known already While the impact of advanced maternal age on reproductive is well understood, the effect of paternal age on reproductive function is controversial. Many studies have shown that Advanced Paternal Age (APA) could impact on male fertility potential affecting testicular function and sperm quality. Moreover, APA also has been associated with increased epigenetics changes and DNA mutations. Increased paternal age could be associated with different types of disorders such as autism, schizopherenia and bipolar disorders. Egg donation cycles, controlling female variables, represent the ideal model for the study of the impact of paternal age on reproductive outcomes. Study design, size, duration We retrospectively analyzed 43 egg donation cycles (October 2014-January 2020) with ≥ 50% survival rate of vitrified/warmed oocyte. Only cycles with poor sperm quality were considered. Cycles were divided in two GROUPS: group–1 included male paternal age ≤ 45 while group–2 included male paternal age >45. Data, shown as avarage±SD, were analyzed with Chi square or Student-t test. Participants/materials, setting, methods Group–1 included 20 cycles and 219 oocytes, male age was 40,89 ±6.12; Group–2 included 17 cycles and 173 oocytes, male age was 51±6.06. Respectively, in Group 1 and in Group 2, donor age were 22.4±2.65 and 24.8±3.88 (NS). All oocytes were injected with abnormal sperm samples according to WHO 2010. Embryos were cultured in time-lapse system until blastocyst stage. Trophectoderm biopsy and PGT-A analysis were performed according to standardized laboratory protocols. Main results and the role of chance Oocytes survival rates in Group1 and 2 were 86% (188/219) and 90.7% (157/173) (NS), respectively. Fertilization rates in Group1 and –2 were 71.42 (135/189) and 73.45% (119/162) (NS), respectively. The total number of obtained embryos (transferred + frozen) were 81 and 801 in Group–1 and –2, respectively. The rates of obtained embryos per reiceved occytes were 37% (81/219) and 46.24% (80/173) in Group–1 and –2 (p < 0.7), respectively. The PGT-A analysis showed 38.7% (31/80) and 31.17% (24/77) of euploid (NS) and 25% (20/80)and 42.85% (33/77) of aneuploid embryos (P < 0.05) in Group–1 and –2, respectively. Mosaic embryos were 33.5% (26/80) and 27.27%(21/77), in Group–1 and –2, respectively. (NS). These results indicate that in presence of severe male factor, advanced paternal age could increase embryos aneuploidy rate raising incidence of chromosomal abnormalities. Limitations, reasons for caution Each donor was stimulated with different protocols according to her history and hormones levels. Nothing is known about which type of sperm parameters (semen amount, morphology or motility) have a major impact when focusing on the embryos genetic outcome. Wider implications of the findings: To better known the effect of APA, it could be necessary identify embryos chromosomal abnormalities and the correlation with specific sperm parameters. Further studies should be done to confirm the APA effect in patients with severe male factors and define a cut-off male age where PGT-A should be recommended. Trial registration number Not applicable