Chromosomal Translocations Research Articles

P-249 Morphokinetic parameters comparison between balanced and unbalanced embryos from patients with structural chromosome abnormalities

Abstract Study question Are there morphokinetic differences between balanced and unbalanced embryos after PGT-SR? Summary answer The total time embryos remain at the morula stage (TiCAV- TM) could be a morphokinetic marker to consider in PGT-SR cycles. What is known already Due to the preimplantation genetic testing for structural rearrangements (PGT-SR) it is possible to identify embryos with an excess or defect of genetic material associated with a parental structural rearrangement. This technique is suitable for patients with a normal phenotype but a balanced chromosomal rearrangement. These patients have a higher risk of spontaneous abortion, as well as higher aneuploidy rates. Within structural chromosomal alterations, translocations are the most common presentation of chromosomal rearrangement. Study design, size, duration Within this multicentre retrospective study, we evaluated 133 embryos, from 39 cycles in which one of the partners had a karyotype with balanced reciprocal or Robertsonian translocations, from January 2021 to September 2023. After PGT-SR was carried out, we obtained 40 balanced embryos and 93 unbalanced embryos. Participants/materials, setting, methods We compared the conventional morphokinetic parameters and the duration of each state between two groups after PGT-SR samples were analysed. All embryos were cultured in Geri® time-lapse incubators, and underwent embryo biopsy on day 5-7. Statistical analysis was conducted using R software (v.4.3.1), including the following confounding variables: origin of karyotype alteration (maternal or paternal), type of translocation (Robertsonian or reciprocal), female and male gamete age, type of oocyte (fresh or frozen) and oocyte origin. Main results and the role of chance We observed that the mean age of the oocyte was significantly higher in the unbalanced embryos compared to the balanced ones (32.85±5.32 years vs.30.45±5.01 years). In terms of the altered karyotype, the paternal origin is more prevalent than the maternal one (60.9% vs. 39.1%). Regarding the type of translocation, reciprocal translocation is more predominant than Robertsonian translocation (83.5% vs. 16.5%) in our study population. When comparing morphokinetic parameters between both groups, we observed that unbalanced embryos tend to reach an expanded blastocyst stage later than balanced embryos (107.62±10.02h vs. 102.48±8.87h, respectively; p = 0.008, corrected p-value=0.186). We observed the same tendency with hatching initiation time (110.64±10.49h vs. 105.91±9.38h; p = 0.013, corrected p-value=0.296). We observed that unbalanced embryos have a slower development than balanced embryos (TiCAV-TFB 10.10±4.46h vs. 8.35±3.79h; p = 0.006, corrected p-value=0.437; TiH-TiCAV 13.03±5.98h vs. 11.79±5.50h; p = 0.017, corrected p-value=0.816). We did observe significant differences in total time at morula stage (TiCAV – TM) when comparing unbalanced and balanced embryos (12.60±6.94 vs. 8.88±5.65; p = 0.003, corrected p-value=0.001). Limitations, reasons for caution A higher number of embryos should be evaluated in order to enhance the statistical power of this study, as there is an important difference in sample size between balanced and unbalanced embryos in the PGT-SR cycles included in this study. Wider implications of the findings Further morphokinetic study of embryos from patients with structural chromosomal abnormalities can open up new lines of research and can help us to predict the final outcome of the cycle, accompanied by a genetic study. Trial registration number Not applicable

P-525 Is there an impact of chromosomal structural rearrangements on IVF laboratory outcomes in PGT-SR cycles? a propensity score matching-based study

Abstract Study question Are IVF laboratory outcomes altered in PGT-SR cycles, and how are these outcomes affected by the type of rearrangement and the carrier’s gender? Summary answer PGT-SR cycles showed a decrease in overall blastulation and top-quality blastocyst rates compared to PGT-A cycles. Transferable blastocyst rate correlates with chromosomal structural rearrangement type. What is known already Chromosomal structural rearrangements (SR) are associated with altered gametogenesis, resulting in the formation of gametes with chromosomal imbalances. For affected couples, undergoing IVF cycles with PGT-SR is recommended to select embryos with a normal or balanced karyotype. Scarce data exist on IVF laboratory outcomes in couples undergoing PGT-SR cycles. Findings regarding blastocyst rate, mainly focused on high-quality blastocysts, remain unclear. In addition, structural rearrangement carriers yield fewer transferable blastocysts than patients undergoing PGT-A. Conclusive evidence regarding the influence of carrier gender and type of chromosomal SR on the development of transferable blastocysts is lacking, with existing studies yielding contradictory results. Study design, size, duration This retrospective cohort study was conducted at a single university-affiliated obstetrics and fertility center between January 2019 and November 2023. The analysis involved 558 patients undergoing IVF cycles, including 136 in PGT-SR and 422 in PGT-A cycles. Participants/materials, setting, methods Trophectoderm biopsies from day 5/6/7 blastocysts underwent comprehensive chromosome screening utilizing validated NGS. Propensity Score Matching with a 1:1 match ratio was performed to balance baseline characteristics between the PGT-SR and PGT-A groups. The impact of chromosomal structural rearrangements on laboratory outcomes was assessed using a logistic regression model. Subsequently, within the PGT-SR population, the association between the carrier’s gender and the transferable blastocyst rate was evaluated through logistic regression, adjusting for potential confounders. Main results and the role of chance There was no significant difference in the fertilization rate between the PGT-A and PGT-SR groups (OR = 1.26, 95% CI 1.01-1.60, p-value=0.14). However, the PGT-SR group exhibited a significantly lower overall blastulation rate (36.7%) compared to the PGT-A group (42.8%) (OR = 0.76, 95% CI 0.63-0.93, p-value=0.03). Notably, a significant difference was observed in the top-quality blastocyst rate, with PGT-A patients showing a rate of 21.7% versus 9.57% in PGT-SR patients (OR = 0.45, 95% CI 0.29-0.69, p-value<0.01). In the PGT-SR cohort, female carriers constituted 44.1%, while male carriers were 56.6%. Reciprocal translocations represented 56.5% of the structural rearrangements, with the remaining 43.5% being other types. When comparing female to male carriers, no significant association was observed between the transferable blastocyst rate and the carrier’s gender (OR = 0.90, 95% CI = 0.35-2.31, p-value=0.83). However, a notable association was identified between the transferable blastocyst rate and the type of structural chromosomal rearrangements (OR = 0.16, 95% CI = 0.09-0.29, p-value<0.01). Specifically, in cases where the patient carried a chromosomal SR other than reciprocal translocation, the transferable blastocyst rate was 83.2%, in contrast to 44.9% in patients with reciprocal translocations. Limitations, reasons for caution The retrospective design of this study represents a limitation, and the limited sample size of patients with structural chromosomal rearrangements restricts the statistical power. Wider implications of the findings These findings reveal that patients with SR yield fewer embryos for biopsy than those undergoing PGT-A, showing a reduced rate of transferable blastocysts in cases of reciprocal translocations. Consequently, providing comprehensive counseling and managing expectations for couples undergoing PGT-SR is essential. Trial registration number not applicable

O-262 Karyotyping of single sperm from non-obstructive azoospermia patients and obstructive azoospermia patients using a combination of micromanipulation and next-generation sequencing

Abstract Study question Is there a difference in the frequency of aberrant karyotype in sperm when the cause of infertility is non-obstructive azoospermia (NOA) versus obstructive azoospermia (OA)? Summary answer Aberrant karyotype was found in 7 of 46 sperm with NOA, none of 26 sperm with OA and none of 28 sperm with control. What is known already It is known that aberrant karyotype occurs more frequently in sperm from infertile men than sperm from general population, and that the severity of infertility is proportional to the frequency of sperm chromosomal abnormality. In fact, miscarriage rates are high in cases where TESE-ICSI is performed for azoospermia, and embryonic chromosomal aneuploidy derived from sperm is thought to be one of the causes of miscarriage. Azoospermia is classified into OA and NOA. However, it is unclear whether there is a difference in the frequency of sperm chromosomal abnormality between NOA and OA. Study design, size, duration Sperm were picked up individually by micromanipulation for the OA and NOA groups who underwent TESE in clinical practice. For comparison, ejaculated sperm from males who underwent IVF or ICSI for female infertility were picked up. As a quality control, ejaculated sperm from balanced reciprocal translocation (RcT) carriers were also picked up. Each sperm was karyotyped by next-generation sequencing (NGS) to compare the number of normal haploid sperm and aberrant sperm. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients, we collected human sperm which were discarded after clinical use. Sperm samples were picked up individually by micromanipulation. Following whole-genome amplification (WGA), karyotyping was performed by NGS. For each sperm, if even one of all chromosomes had abnormality, that sperm was counted as aberrant sperm, and if all chromosomes were haploid, it was counted as haploid sperm. Main results and the role of chance We analyzed 10 sperm per patient. Karyotyping analysis was performed on sperm from 2 patients in the RcT group,4 patients in the control group, 4 patients in the OA group, and 5 patients in the NOA group. WGA was successful in 90% (18/20) of RcT carriers group, 95% (38/40) of the control group, 72.5% (29/40) of the OA group, and 92% (46/50) of the NOA group. Acquisition rate of karyotyping was 90% (18/20) of RcT carriers group, 70% (28/40) of the Control group, 65% (26/40) of the OA group, and 95% (46/50) of the NOA group. The results of karyotype analysis showed 28 haploid sperm and no aberrant sperm in the control group, 26 haploid sperm and no aberrant sperm in the OA group, and 39 haploid sperm and 7 aberrant sperm in the NOA group. Karyotyping of sperm from RcT carriers group showed sperm with unbalanced chromosomes derived from translocations of the carriers. The frequency of aberrant karyotype was significantly higher in NOA than control (P = 0.0297). It was also higher in NOA than OA(P = 0.0363). Limitations, reasons for caution In our research, micromanipulation was performed manually to isolate sperm, and the number of sperm that could be analyzed was small. Sperm with good morphology are selected using micromanipulation and analyzed by NGS, so the results do not reflect the results of all sperm actually collected by TESE. Wider implications of the findings In this study, sperm with good morphology were selected by micromanipulation and analyzed, so it is thought that the karyotype of sperm actually reflects the karyotype used in clinical practice. These results suggest that PGT-A is particularly effective in preventing miscarriages among patients whose azoospermia was caused by NOA. Trial registration number not applicable

P-714 Prevalence of chromosomal abnormalities in oocyte donor candidates: a French survey of over 8200 karyotypes

Abstract Study question Is karyotyping worth considering for fertile and childless oocyte donor candidates? Summary answer Karyotyping reduces the genetic risk of utilizing oocytes with balanced structural chromosomal rearrangements for recipient couples. What is known already While some countries including France consistently advocate for karyotype to be routinely performed, others suggest it as optional, specifically advising it for donors with a medical history indicating chromosomal rearrangements. Ravel et al. have demonstrated a higher incidence of balanced structural chromosomal rearrangements among 1196 french fertile oocyte donor candidates (FODC) compared to unselected newborns, raising the question whether oocyte donor recruitment could be biased towards chromosomally at-risk women. In 2016, France authorized childless oocyte donor candidates (CODC) to enhance recruitment, yet as of now, there is no reported study on their genetic status. Study design, size, duration A retrospective observational multicentric study conducted from January 2005 to October 2021. Participants/materials, setting, methods ODC were recruited within the French CECOS (Centre d’Etude et de Conservation des Œufs et du Sperme humain) network. Both FODC and CODC were compared to a control group composed of female newborns from literature. Quantitative and qualitative data were expressed as the median [25%–75%] or n (‰), respectively. Non-parametric Mann-Whitney tests were employed for quantitative parameters, while Chi-square tests or Fisher’s exact tests were used, as appropriate, to compare frequencies between groups. Main results and the role of chance A total of 8229 ODC from 22 CECOS centers were included in this study. FODC (n = 6339) and CODC (n = 1890) were compared to 8102 control female newborns. In total, 65 chromosomal abnormalities (7.9‰) were identified, leading to the exclusion of the ODC. This outcome was comparable to the 53 abnormalities observed in the control group (6.54‰, p = 0.3059). The occurrence of balanced structural chromosomal rearrangements was increased in ODC population (4.8‰) compared to controls (2.34‰, p = 0.0086). The occurance of reciprocal translocations was increased 5-fold in CODC (3.70‰) compared to the control group (0.74‰, p = 0.013). The number of gonosomal mosaics was notably increased in FOCD, with 17 cases (2.68‰) compared to the control group with six cases (0.74‰, p = 0.0052). Among chromosomal aberration carriers only two childless women (one reciprocal translocation and one gonosomal mosaïc) had fertility issues in their medical history with a diagnosis of premature ovarian failure. Limitations, reasons for caution This retrospective study was designed to examine ODC within the framework of French bioethics laws. ODC should adhered to specific criteria: being volunteers, anonymous, aged 18 to 38, and without any financial compensation. Since 2016, donors were permitted to retain around 50% of their gametes for personal oocyte cryopreservation. Wider implications of the findings Karyotyping is a straightforward and cost-effective approach to identify structurally balanced chromosomal abnormalities. Thus, chromosomal analysis limits genetic risks whithout resorting to the ethically debated expanded genetic screening in the ODC population. Nevertheless, it is crucial to communicate to gamete recipients the inherent genetic risks associated with any conception. Trial registration number not applicable

P-561 Clinical outcomes following PGT-M and PGT-SR cycles

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

P-534 Toward enhanced genetic counseling with PGT-SR: exploring the impact of chromosomal rearrangement type and carrier gender on the acquisition of transferable embryos

Abstract Study question Do chromosomal rearrangement type and gender of carriers influence the ability to obtain transferable embryos? Summary answer Carriers of a reciprocal translocation, particularly female carriers, could have a higher risk of having non-transferable embryos. What is known already Chromosomal translocations are common structural rearrangements in the general population, occurring even 10 times more frequently in the infertile population, as carriers may produce unbalanced chromosomal gametes. Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) is a crucial tool for selecting embryos to transfer. There are numerous factors that can affect the availability of transferable embryos, and some of the less studied ones are the gender of the carrier and the type of chromosomal rearrangement. Study design, size, duration This is a single-center retrospective study performed between 2016 and 2023, that included 265 biopsies. Embryos were classified as transferable (n = 80, 30%) if they were euploid/balanced or exhibited low-grade mosaicism (< 50%) without the involvement of chromosomes 13, 14, 16, 18, 21, and X. Non-transferable embryos (n = 185, 70%) included those that were unbalanced, aneuploid, or displayed high-grade mosaicism. Differences between groups were assessed using Chi-square and Fisher’s exact tests. Values with p < 0.05* were deemed significant. Participants/materials, setting, methods NGS-based PGT-SR was performed on 265 embryos from 71 cycles of 47 couples. Couples were divided into Robertsonian translocation carrier group (ROB-T, 105 embryos) and Reciprocal translocation carrier group (REC-T, 160 embryos). Subsequently, data was categorized by maternal age: ≤37 (ROB-T=91, REC-T=122) and >37 years (ROB-T=14, REC-T=38), as well as by the gender of the carrier: male carrier group (MC) (ROB-T=77, REC-T=97) and female carrier group (FC) (ROB-T=29, REC-T=74). Main results and the role of chance Of the embryos belonging to ROB-T group, 45/105 (43%) were transferable, while in the REC-T group only 35/160 (22%) were transferable. This difference was statistically significant (p = 0.0004612). It was assessed whether these differences were attributable to maternal age or gender of carriers. In the ≤37, 43% (39/91) of the ROB-T embryos were transferable, but only 25% (30/122) were transferable in the REC-T group (p = 0.007583). In the >37, 43% (6/14) of the ROB-T embryos were transferable versus 13% (5/38) in the REC-T group (p = 0.04951). In both age groups, the obtaining transferable embryos were influenced by the type of translocation. In the MC group, 47% (36/76) of the ROB-T embryos were transferable and only 24% (22/90) were transferable in the REC-T group (p = 0.003467). In the FC group, 31% (9/29) of the ROB-T embryos and 19% (13/70) of the REC-T embryos were transferable (p = 0.2749). In the MC group, the obtaining transferable embryos were influenced by the type of translocation. However, within the FC group, no significant differences were observed. When evaluating non-transferable embryos (185), 71% were unbalanced and 29% were balanced ones with aneuploidies. Unbalanced embryos were more frequent in REC-T carriers in all analyzed groups. Limitations, reasons for caution This study is limited by its retrospective nature and its small sample size. Additionally, it is worth noting the difference in sample size between groups when segmenting the data by maternal age. Wider implications of the findings Our results indicate that the type of rearrangement is relevant in obtaining transferable embryos. Carriers of REC-T, especially female carriers, could have a higher risk of having non-transferable embryos compared to carriers of ROB-T. These findings could be significant for genetic counseling of individuals carrying chromosomal translocations. Trial registration number Not applicable

P-090 Is preimplantation genetic testing for aneuploidy (PGT-A) necessary in the treatment strategy for ICSI with testicular sperm?

Abstract Study question Does the combination of testicular sperm extraction (TESE) and ICSI increase the risk of embryo aneuploidy? Summary answer Embryo aneuploidy occurrence is not increased when using testicular sperm and is not affected by the etiology of azoospermia. What is known already TESE-ICSI, especially in patients with non-obstructive azoospermia (NOA), has been reported to result in poor outcomes and difficulty in obtaining good blastocysts, which may be due to immature sperm retrieved by TESE. In addition, patients who require TESE have been shown to be at particularly high risk of chromosomally abnormal sperm. These factors may increase the risk of embryo aneuploidy in TESE-ICSI and contribute to poor outcomes. However, few reports have investigated the results of PGT-A following TESE-ICSI. This study was therefore designed to investigate whether using testicular sperm is more likely to result in embryo aneuploidy. Study design, size, duration This retrospective study included 81 couples and 197 TESE-ICSI cycles undergoing PGT-A (average maternal age: 38.1±5.4 years) between September 2016 and November 2023. Of the total cycles, 94 cycles were case of obstructive azoospermia (OA) and 53 cycles were cases of NOA. The etiologies of NOA included were unexplained (22 cycles), Klinefelter’s syndrome (KS) (10 cycles), post-chemotherapy (3 cycles), post-orchiopexy (11 cycles), and microdeletion of azoospermia factor (AZF) c on the Y chromosome (7 cycles). Participants/materials, setting, methods ICSI outcomes were compared between different etiologies (OA vs. NOA). A total of 356 blastocysts acquired were analyzed using PGT-A with next-generation sequencing (NGS). The PGT-A results were classified according to maternal age (<38, 38-41, >41 years) and paternal age (<40, ≥40 years). In addition, TESE-ICSI cycles were limited to the maternal age of < 38 years to remove the effect of maternal factors, the euploidy rates were compared between different etiologies or morphological grade. Main results and the role of chance The rates of fertilization and blastocyst formation for TESE-ICSI were significantly lower in NOA than OA (47.4% vs. 61.6%, P < 0.001 and 37.3% vs. 50.7%, P < 0.01, respectively). The rate of good-quality blastocyst (16.6% vs. 21.2%) was also lower, but not statistically significant. The euploidy rate of all evaluated TESE-ICSI embryos decreased with increasing maternal age (52.9% in < 38, 29.3% in 38-41, 10.0% in > 41 years), but was not affected by the paternal age (40.6% in < 40, 42.5% in ≥ 40 years). For cycles with the maternal age of < 38 years, the euploidy rate was not significantly different between OA and NOA patients (55.3% vs. 49.3%). In addition, the association between embryo euploidy and morphological grade (using Gardner’s classification, ≥BB for good-quality vs. <BB for poor-quality embryos) was evaluated, and the euploidy rate was significantly higher in good-quality embryos compared to poor-quality embryos (60.3% vs. 33.3%, P < 0.01). These results indicate the frequency of embryo aneuploidy in TESE-ICSI is comparable to commonly used ejaculated sperm. Furthermore, it suggests that embryo aneuploidy is dependent on maternal age or morphological grade of embryo, but not on paternal age or the etiology of azoospermia. Limitations, reasons for caution Patients with chromosomal abnormalities, i.e. KS, could not be evaluated due to the small number of cases and patients with chromosomal translocation were not included in this study. Therefore, the effects of male factors in NOA on embryo aneuploidy require extended investigation. Wider implications of the findings In TESE-ICSI, especially in the case of NOA, it is difficult to obtain good quality embryos due to poor ICSI outcomes. However, if good quality embryos are obtained, the euploidy rate is high and there is no need to perform PGT-A on the sole basis of using testicular sperm. Trial registration number not applicable

P-529 Optical Genomic Mapping (OGM) enable cryptic chromosomal rearrangements in a couple with recurrent miscarriages undergoing preimplantation genetic diagnosis

Abstract Study question Could Optical Genomic Mapping (OGM) detect chromosomal structural variations (SVs) below the detection limit of conventional cytogenetic diagnostics in couples with recurrent miscarriages? Summary answer The Next-generation cytogenetics of Optical Genomic Mapping confirmed a cryptic chromosomal translocation in a couple with recurrent miscarriages undergoing preimplantation genetic testing for aneuploidies (PGT-A). What is known already Chromosomal structural variants (SVs) significantly contribute to infertility, recurrent pregnancy losses and congenital defects... Approximately 2-4% of couples facing recurrent miscarriages carry chromosomal abnormalities. Currently, their detection is based on convencional cytogenetic methods (karyotyping, FISH or array-CGH), however cryptic chromosomal rearrangements are difficult to identify. Next-generation cytogenetics such as OGM is able to detect SVs with a resolution of < 500 kb, allowing a better understanding of the cause of patient’s infertility. In our study, we utilized OGM to confirm a cryptic translocation in a couple experiencing recurrent miscarriages, aiming to enhance our understanding of the underlying causes of their infertility. Study design, size, duration Our study focuses on a non-consanguineous couple diagnosed with recurrent pregnancy loss that attended to our private fertility clinic to undergo assisted reproductive treatment (ART) with their autologous semen, oocytes and PGT-A. Conventional cytogenetic technique was performed on both partners by G-banding karyotyping. PGT-A was performed by NGS (Veriseq, Illumina). Optical Genomic Mapping (OGM) served as a diagnostic tool, complemented by lymphocyte FISH analysis to confirm chromosomal structural variations in these infertile patients. Participants/materials, setting, methods Ultra High Molecular Weight genomic DNA (UHMW gDNA) was extracted from frozen human peripheral blood of both partners. UHMW gDNA was quantified using Qubit dsDNA assay BR kit and Qubit 4 Fluorometer (Thermo Fisher Scientific). We used Saphyr system (Bionano Genomics) for cytogenetic diagnosis. The consensus genomic map was constructed by de novo assembly in the Bionano Solve data analysis software. SVs were identified by aligning Optical Genomic Map with the reference genome GRCh38/hg38. Main results and the role of chance The couple had suffered four prior miscarriages and undergoing three PGT-A cycles. Both partners provided a normal karyotype. Among 13 biopsied embryos, four showed CNVs on chromosomes 2(p24.3è25.3, 12Mb) and 6(p24.1è25.3, 11Mb). Following this result we suspected that the couple might has a cryptic chromosomal structural abnormalities. To test our hypothesis, we performed OGM. We analysed UHMW gDNA samples from both partners. OGM generated an average of 823.67Gb. The average N50 molecule length (≥150kb) was 318kb. The average mapping rate was 90.75% and the average labeling rate was 15.30 labels per 100kb. The average effective coverage depth was 242X. OGM successfully identified a cryptic translocation in the male partner ogm[GRch38]t(2;6)(p24.3;24.1), while the female partner showed no abnormalities. Translocation breakpoints identified by OGM were Chr2:12.560.508 and Chr6:11.622.593. The translocated segments were near to the detection limit of conventional cytogenetic analysis and with similar pattern that could be the reason for missed detection by karyotyping. Importantly, we identified one gene disrupted by OGM rearrangement breakpoint: MIR3681HG. A previous study considered that CNV loci of 2p24.3, including MIR3681HG gene, could be associated with spermatogenesis. The lymphocyte FISH analysis finding corroborated the OGM result. Limitations, reasons for caution OGM is a very powerful method for detecting chromosomal structural variants in human genomes and is able to detect cryptic structural variants. However, this is a very new diagnostic application, so there is limited experience with OGM for correlating structural variations results to infertility diagnosis in couples with recurrent miscarriages. Wider implications of the findings A balanced cryptic translocation has been identified in a couple with recurrent miscarriages. Our case report demonstrates the high diagnostic capability of Optical Genomic Mapping for cryptic SVs. OGM technology allows a more accurate cytogenetic diagnosis by detecting cryptic structural variants with high resolution in infertile patients with recurrent miscarriages. Trial registration number not applicable

O-032 Intravenous immunoglobulin and prednisolone to women with unexplained recurrent pregnancy loss undergoing assisted reproductive technology treatment: a randomized, double-blinded, placebo-controlled trial

Abstract Study question Does intravenous immunoglobulin (IVIG) and prednisolone in combination affect ongoing pregnancy rate among patients with recurrent pregnancy loss (RPL) after assisted reproductive technology (ART) treatment? Summary answer The results of this trial will provide important information about whether immunomodulatory treatment to RPL patients undergoing ART treatment can increase clinical pregnancy rate. What is known already Two retrospective cohort studies suggest that IVIG and prednisolone in combination result in a 34-37% live birth rate in RPL patients undergoing ART which is higher than the expected 12% for patients with RPL after ART. A randomized controlled trial (RCT) testing IVIG in RPL patients conceiving naturally indicated that very early administration of IVIG caused more positive effects than late administration, and prospective cohort studies suggest that treatment efficacy depends on presence of immune disturbances. No RCT has tested the IVIG/prednisolone combination starting before implantation and continued in very early pregnancy for RPL patients undergoing ART. Study design, size, duration In a randomized, double-blinded, placebo-controlled trial, patients undergoing IVF/ICSI/FET were treated with 400mg/kg IVIG and 5-10mg/day oral prednisolone or IV human albumin and oral placebo between January 2021 and March 2024. Oral treatment was administered from day one of menstrual cycle to GW 8 + 4, while IV treatment was given once in a short time interval before or after embryo transfer and repeated three times before GW 8 + 0. Primary outcome was ongoing pregnancy in GW 12 Participants/materials, setting, methods Seventy-four women <41 years old with ≥2 consecutive biochemical or clinical early pregnancy losses after ART were randomized 1:1 and underwent trial treatment along with their next IVF/ICSI/FET treatment. Patients were followed with weekly ultrasound scans in first trimester, however, if no implantation occurred, trial participation ended, and no re-entry was allowed. Exclusion criteria included antiphospholipid syndrome, BMI >35, uterine malformations, parental chromosomal translocations, AMH <4 pmol/l, drug contraindications, thyroid disease, and prior IVIG treatment. Main results and the role of chance Seventy-four patients have been randomized, and currently we are awaiting the last two patients to complete the treatment protocol before the code can be disclosed. Blinded interim analysis after inclusion of 38 patients showed no serious adverse events and an ongoing pregnancy rate of 30.0% and 9.5% (RR 1.8, 95% CI 1.0-3.1, Fisher’s exact test: p = 0.13), in the two allocation groups, respectively. Final, unblinded data on ongoing pregnancy rate, miscarriage rate and adverse events will be presented at the Congress if selected for presentation. Limitations, reasons for caution No published study has tested the same treatments in comparable patient groups which could have been the basis for the sample-size calculation, and the study is therefore at risk for being underpowered to evaluate the treatment effect in patients suffering from a combination of infertility, recurrent implantation failure, and RPL. Wider implications of the findings Blood samples were collected before first IV treatment and approximately 4 weeks later, which allow analysis for immunological disturbances and searching for biomarkers identifying those who will benefit from the treatment. This treatment could potentially increase success rate in patients undergoing ART after multiple unsuccessful treatment attempts. Trial registration number NCT04701034

Molecular Modeling of Single- and Double-Hydrocarbon-Stapled Coiled-Coil Inhibitors against Bcr-Abl: Toward a Treatment Strategy for CML.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias and a subset of acute lymphoblastic leukemias. As a result of the so-called Philadelphia chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase, which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown that conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation relative to traditional small-molecule therapeutics. Here, we iterate a new generation of CCmut3 inhibitors against Bcr-CC-mediated Bcr-Abl assembly designed to address these constraints through incorporation of all-hydrocarbon staples spanning i and i + 7 positions in α-helix 2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to evaluate single- and double-stapled CCmut3 candidates in silico for dynamics and binding energetics. We further model a truncated system characterized by the deletion of α-helix 1 and the flexible loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems devoid of the CPP, with a cyclized CPP, and with an open-configuration CPP, for a total of six systems that comprise our library. From this library, we present lead-stapled peptide candidates to be synthesized and evaluated experimentally as our next iteration of inhibitors against Bcr-Abl.

Analysis and Identification of Rare and Prevalent Breakpoints in Chromosomal rearrangements in Adult and Pediatric with B-Acute Lymphoblastic Leukemia (B-ALL): A Systematic Review

Background: B-cell acute lymphoblastic leukemia (B-ALL) is a complex disorder that includes multiple genetic changes, one of the main causes of which is rare and common chromosomal translocations that lead to abnormal gene fusions. This abnormal fusion produces a new protein that causes the leukemia cells. These types of rearrangements usually occur in lymphoma and result in the movement of genetic material between different chromosomes or within chromosomes. This systematic review aims to evaluate published studies and investigate the role and importance of common and rare chromosomal translocations in the occurrence of B-ALL. Material and Methods: This systematic review investigated and evaluated the evidence regarding the effect of chromosomal translocations in adults and pediatrics with B-ALL. This review was based on the preferred reporting items for systematic reviews and meta-analysis checklists. A literature search was conducted using international databases (such as PubMed, Web of Science, Scopus, Research Gate, Google Scholar, and Cochrane Systematic Reviews database). Only English-language articles published between January 2010 and January 2023 including MESH terms such as ALL, B-ALL, and chromosomal translocations were selected. Seventy-five related studies had the necessary criteria to be examined in the present study. Results: A total of 237 articles were retrieved in the online search. The excluded articles included research on other types of leukemia in adults and children, descriptive studies, case studies, studies related to animal models of leukemogenesis, and comparisons of common treatment methods of leukemia grouping cancers. Finally, seventy-five studies were identified as eligible for inclusion in this systematic review. Conclusion: This review provides a comprehensive assessment of common and rare chromosomal translocations that can lead to the development of cancer cells. Chromosomal translocations play a role as diagnostic markers and important prognostic indicators and help specialists adjust treatment approaches according to their occurrence.

Recent Advances in The Definition of the Molecular Alterations Occurring in Multiple Myeloma.

Multiple myeloma (MM) is a disorder of the monoclonal plasma cells and is the second most common hematologic malignancy. MM initiation and progression are dependent upon complex genomic abnormalities. The current pathogenic model of MM includes two types of primary events, represented by chromosome translocations or chromosome number alterations resulting in hyperdiploidy. These primary molecular events are observed both in MM and in monoclonal gammopathy, its premalignant precursor. Subsequent genetic events allow the progression of monoclonal gammopathy to MM and, together with primary events, contribute to the genetic complexity and heterogeneity of MM. Newer therapies have considerably improved patient outcomes; however, MM remains an incurable disease and most patients experience multiple relapses. The dramatic progresses achieved in the analysis of the heterogeneous molecular features of different MM patients allowed a comprehensive molecular classification of MM and the definition of an individualized prognostic model to predict an individual MM patient's response to different therapeutic options. Despite these progresses, prognostic models fail to identify a significant proportion of patients destined to early relapse. Treatment strategies are increasingly. Based on disease biology, trials are enriched for high-risk MMs, whose careful definition and categorization requires DNA sequencing studies.

Radiation Research Society Journal-based Historical Review of the Use of Biomarkers for Radiation Dose and Injury Assessment: Acute Health Effects Predictions.

A multiple-parameter based approach using radiation-induced clinical signs and symptoms, hematology changes, cytogenetic chromosomal aberrations, and molecular biomarkers changes after radiation exposure is used for biodosimetry-based dose assessment. In the current article, relevant milestones from Radiation Research are documented that forms the basis of the current consensus approach for diagnostics after radiation exposure. For example, in 1962 the use of cytogenetic chromosomal aberration using the lymphocyte metaphase spread dicentric assay for biodosimetry applications was first published in Radiation Research. This assay is now complimented using other cytogenetic chromosomal aberration assays (i.e., chromosomal translocations, cytokinesis-blocked micronuclei, premature chromosome condensation, γ-H2AX foci, etc.). Changes in blood cell counts represent an early-phase biomarker for radiation exposures. Molecular biomarker changes have evolved to include panels of organ-specific plasma proteomic and blood-based gene expression biomarkers for radiation dose assessment. Maturation of these assays are shown by efforts for automated processing and scoring, development of point-of-care diagnostics devices, service laboratories inter-comparison exercises, and applications for dose and injury assessments in radiation accidents. An alternative and complementary approach has been advocated with the focus to de-emphasize "dose" and instead focus on predicting acute or delayed health effects. The same biomarkers used for dose estimation (e.g., lymphocyte counts) can be used to directly predict the later developing severity degree of acute health effects without performing dose estimation as an additional or intermediate step. This review illustrates contributing steps toward these developments published in Radiation Research.

Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eμ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Cumulative Dose from Recurrent CT Scans: Exploring the DNA Damage Response in Human Non-Transformed Cells.

Recurrent computed tomography (CT) examination has become a common diagnostic procedure for several diseases and injuries. Though each singular CT scan exposes individuals at low doses of low linear energy transfer (LET) radiation, the cumulative dose received from recurrent CT scans poses an increasing concern for potential health risks. Here, we evaluated the biological effects of recurrent CT scans on the DNA damage response (DDR) in human fibroblasts and retinal pigment epithelial cells maintained in culture for five months and subjected to four CT scans, one every four weeks. DDR kinetics and eventual accumulation of persistent-radiation-induced foci (P-RIF) were assessed by combined immunofluorescence for γH2AX and 53BP1, i.e., γH2AX/53BP1 foci. We found that CT scan repetitions significantly increased both the number and size of γH2AX/53BP1 foci. In particular, after the third CT scan, we observed the appearance of giant foci that might result from the overlapping of individual small foci and that do not associate with irreversible growth arrest, as shown by DNA replication in the foci-carrying cells. Whether these giant foci represent coalescence of unrepaired DNA damage as reported following single exposition to high doses of high LET radiation is still unclear. However, morphologically, these giant foci resemble the recently described compartmentalization of damaged DNA that should facilitate the repair of DNA double-strand breaks but also increase the risk of chromosomal translocations. Overall, these results indicate that for a correct evaluation of the damage following recurrent CT examinations, it is necessary to consider the size and composition of the foci in addition to their number.

Analysis of the pharmacoeconomic effectiveness of the tyrosine kinase inhibitors therapy in patients with chronic myeloid leukemia in a Single Hematology Center in Plovdiv, Bulgaria

Summary: Chronic myelogenous leukemia (CML) is a pluripotent hemopoietic stem cell malignancy characterized by the presence of a BCR-ABL1 fusion gene derived from a balanced translocation between the long arms of chromosomes 9 and 22 [t(9;22) (q34; q11)] known as the Philadelphia (Ph) chromosome. CML is an acquired hematopoietic stem cell disease common to myelo- and lymphopoiesis, characterized by uncontrolled proliferation of granulopoiesis (Shuvaev et al. 2015; Yordanov and Varbanova 2019). Relevance and goals: Second-generation tyrosine kinase inhibitors (TKIs) (nilotinib, dasatinib, and bosutinib) have an advantage over imatinib (first-generation) in the frequency and speed of achieving cytogenetic and molecular responses in the treatment of chronic myelogenous leukemia (CML), but they cause severe adverse effects and are much more expensive than imatinib, especially if we compare them to the prices of the registered generic products of Imatinib and Dasatinib. “Novartis Tasigna® trial shows superior results to Glivec® in patients with early-stage chronic myeloid leukemia”, reported on 10/20/2021 by Pierre Perrin-Montlouis. In the first direct comparison of these two oral therapies back in 2009 (Тasigna (nilotinib) 2009), as first-line treatment for CML, the results of Tasigna showed statistically significant improvement over Glivec in every measure of efficacy, including major molecular response (MMR), complete cytogenetic response (CCyR) and prevention of progression to accelerated or blast phase, with responses achieved faster in the Tasigna group than in the Glivec group. Furthermore, in the last ten years, CML patients who have achieved a stable deep molecular response for at least 2 years have been included in clinical trials for the management of treatment-free remission (TFR) (Kim et al. 2013; Saußele et al. 2016). On the other hand, the ever-increasing costs of diagnosis, treatment and monitoring of the response of CML to the various therapeutic strategies require conducting pharmacoeconomic analyses of the cost-effectiveness and cost-utility types in order to evaluate which are the cost-effective strategies with a view to introducing them into therapeutic practice. The present study aims to analyze the pharmacoeconomic efficiency of the TKI inhibitors used by the patients with CML-CP in the first and second lines, treated in the hematology clinic at UMHAT “St. George”, MU- Plovdiv during the period 2018–2022. Methods: An economic analysis of the medicinal use of TKIs for a 5-year period (2018–2022) was performed at the national level according to data from the National Health Insurance Fund and the availability, accessibility, and usability of original and generic TKIs in Bulgaria were evaluated. The direct medical costs for the therapy of all patients were calculated, including the costs of the TKI therapy, laboratory tests, and monitoring of the molecular response for the entire treatment period from the appointment of the TKI therapy until the end of 2022. A comparative analysis was conducted to assess the cost-effectiveness of the different therapeutic strategies with TKIs on the first and second lines of treatment of patients with CML-CP in the hematology clinic at UMHAT “St. George” Medical University, Plovdiv, using the decision analysis method and conducting one-way and probalistic sensitivity analyses. Results: The sensitivity analyzes of all pharmacoeconomic models showed the robustness and reliability of the obtained results. The threshold limits of medical costs and the frequency of achieving a deep molecular response determining the choice of first- and second-generation tyrosine kinase inhibitors as first- and second-line therapy for patients with chronic myeloid leukemia in the chronic phase have been determined. Prescribing doctors prefer the original MPs to generic analogues, which is also assumed by the current regulations, according to which even for expensive MPs dispensing by protocols, the prescription is by trade and not by international non-proprietary names (INN), which is why the use of the much cheaper generic MPs is negligibly low compared to original MPs. A personalized approach to the patient’s therapy and monitoring the patient’s molecular response to it, as well as stopping therapy in 25–30% of patients suitable to stop it safely when in TFR phase with a probability of more than 50% of not having a relapse will save additional costs that, by improving the cost-effectiveness of therapy for patients with CML, will be directed towards the treatment of new patients with this or other diseases. Conclusion: These pharmacoeconomic models can be applied to improve diagnostic and therapeutic standards in clinical practice and for the efficient use of the very limited resources for health care in countries like Bulgaria. The conducted cost-effectiveness analyses confirmed that the hematologists at the University Center in Plovdiv adhere to the recommendations of Leukemia Net and the Bulgarian Medical Society of Hematology and achieve not only good therapeutic but also pharmacoeconomic efficiency in the treatment of CML-СР patients in first- and second- line therapy.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Pattern of Translocations in Acute Leukaemia: A Single Centre Experience

A multifaceted approach is the current modality to the diagnoses of acute leukaemia. Given the importance of knowing chromosomal aberrations for rapid diagnosis and better treatment selection, this study assessed the frequency of common chromosomal translocations among acute leukaemia patients. In this retrospective study, both conventional karyotype and RT-PCR detecting translocations (HemaVision®-28N) were utilised in 400 patients with acute leukaemia of all subtypes in King Abdulaziz University, Jeddah (2015-2020). The researchers identified 38 acute leukaemia cases associated with translocations out of 400 new cases (9.5%), 326 samples were negative (81.5%) and 36 resisted PCR (9%). The most frequent observed translocation is t(9;22) (q34;q11.2) P190 (representing 28.9% of the total positive calls), followed by t(15;17) (q24;q21) (PML-RARA) and t(8;21) AML-ETO, each of which represents 13 percent. The resulting acute leukaemia translocation frequencies are similar to that published in the literature both locally and globally.

Pattern of Translocations in Acute Leukaemia: A Single Centre Experience

A multifaceted approach is the current modality to the diagnoses of acute leukaemia. Given the importance of knowing chromosomal aberrations for rapid diagnosis and better treatment selection, this study assessed the frequency of common chromosomal translocations among acute leukaemia patients. In this retrospective study, both conventional karyotype and RT-PCR detecting translocations (HemaVision®-28N) were utilised in 400 patients with acute leukaemia of all subtypes in King Abdulaziz University, Jeddah (2015-2020). The researchers identified 38 acute leukaemia cases associated with translocations out of 400 new cases (9.5%), 326 samples were negative (81.5%) and 36 resisted PCR (9%). The most frequent observed translocation is t(9;22) (q34;q11.2) P190 (representing 28.9% of the total positive calls), followed by t(15;17) (q24;q21) (PML-RARA) and t(8;21) AML-ETO, each of which represents 13 percent. The resulting acute leukaemia translocation frequencies are similar to that published in the literature both locally and globally

Combination of menin and kinase inhibitors as an effective treatment for leukemia with NUP98 translocations.

Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.