Normal Karyotype Research Articles

Generation of a homozygous (MCRIi031-A-3) WT1 knockout human iPSC line

The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.

Generation of iPSC lines from three Laing distal myopathy patients with a recurrent MYH7 p.Lys1617del variant

Variants in MYH7 cause cardiomyopathies as well as myosin storage myopathy and Laing early-onset distal myopathy (MPD1). MPD1 is characterized by muscle weakness and atrophy usually beginning in the lower legs. Here, we generated iPSC lines from lymphoblastoid cells of three unrelated individuals heterozygous for the most common MPD1-causing variant; p.Lys1617del. iPSC lines showed typical morphology, expressed pluripotency markers, demonstrated trilineage differentiation potential, and had a normal karyotype. These lines represent the first iPSCs derived from MPD1 patients and complement existing MPD1 animal models. They can provide in vitro platforms to better understand and model MPD1 pathomechanisms and test therapies.

Comprehensive management of Mayer-Rokitansky-Küster-Hauser syndrome management: A case report.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, also known as Müllerian aplasia, is a congenital condition characterized by uterine and upper vaginal aplasia. It affects females with a normal female karyotype and typical secondary sex characteristics. The aim of this case report was to highlight the multidisciplinary management approach for MRKH syndrome, focusing on tailored interventions to address physical and psychological challenges and improve reproductive prospects. A 26-year-old married female presented to Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi, Wardha, India, in January 2023 with primary amenorrhea. Physical examination revealed a blind vagina and a hypoplastic uterus, indicative of MRKH syndrome. Further evaluation, including pelvic magnetic resonance imaging (MRI), confirmed Müllerian duct abnormalities and bilateral ovarian anomalies. The absence of a functional vagina significantly impacted the patient's quality of life, leading to difficulties with sexual intercourse and emotional distress related to infertility. A collaborative approach involving a gynecologist and a psychiatrist at AVBRH was initiated to address these challenges. The patient underwent vaginoplasty to create a neovagina, enhancing the sexual function and intimate relationship with the spouse. However, due to the hypoplastic uterus, achieving motherhood through traditional means was not possible. Therefore, assisted reproductive techniques, in particular surrogacy, were explored. Normal, functional ovaries were harvested from the patient for use in surrogacy procedures. This comprehensive management strategy exemplifies the challenges associated with MRKH syndrome and underscores the importance of tailored interventions and long-term follow-up. The case highlights the significance of collaborative care in improving the quality of life and reproductive prospects for individuals with MRKH syndrome.

Generation of four induced pluripotent stem cell lines (KEIUi004-A, KEIUi005-A, KEIUi006-A, and KEIUi007-A) from patients with sensorineural hearing loss with mutation in EYA4 gene

Disease-related cells differentiated from patient-derived iPSCs are useful for elucidating the pathophysiological mechanisms underlying these diseases. In this study, four iPSC lines were established from independent patients with sensorineural hearing loss and a mutation in EYA4. These iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes, suggesting that these lines are useful for the pathological study of sensorineural hearing loss and drug screening for ear disorders.

P-570 A cell-free DNA test in semen of nonobstructive azoospermic men to predict spermatogenesis

Abstract Study question Can a genetic assessment of cell-free DNA, isolated from seminal plasma of nonobstructive azoospermic (NOA) men, predict successful sperm extraction at testicular biopsy? Summary answer Seminal cell-free DNA (cfDNA) isolated from the ejaculates of NOA men with failed testicular sperm retrieval outcomes concurrently and consistently displayed mutations on three genes. What is known already The most puzzling form of azoospermia is the testicular type, where the chances of successful microdissection testicular sperm extraction (micro-TESE) are unpredictable. Therefore, developing a method that could identify or predict individuals with absent spermatogenesis would be most useful. While histopathology is considered the most reliable method for predicting successful micro-TESE, it is invasive and often inconclusive. Recently, cfDNA testing has been gaining momentum as a noninvasive method for prenatal screening and even with the potential for eventually replacing PGT-A. However, the prognostic value of using cfDNA to predict the presence/absence of spermatozoa in men undergoing micro-TESE is relatively unknown. Study design, size, duration Over a period of 8 months, we recruited azoospermic men with normal peripheral karyotypes and for whom an extensive semen analysis did not identify spermatozoa. For consenting patients (n = 12), we performed genetic assessments on cfDNA from their ejaculates (IRB 1006101085). These patients subsequently underwent micro-TESE, and their cfDNA mutation profiles were compared according to whether spermatozoa were successfully retrieved ((+)Sperm, n = 5), or not ((-)Sperm, n = 7). Participants/materials, setting, methods Using a commercially available kit, circulating cfDNA was eluted from the ejaculates of consenting NOA men, as well as a normozoospermic control (n = 2). The isolated cfDNA specimens subsequently underwent whole exome sequencing (WES) on an Illumina HiSeq at 2x150bp. Gene mutations were detected by CLC Genomic Server 9.0 and compared within, as well as between, the +Sperm and -Sperm cohorts in comparison to the control group. Main results and the role of chance All 12 men (35.5±4yrs) had normal peripheral karyotypes and tested negative for Y-microdeletions. They had no history of genetic diseases and were off any supplements, medications, and/or hormones before or during the study. Genetic assessment on cfDNA from the +Sperm cohort (n = 5; 38.3±2yrs) identified 8 genes (ADAMTS7, ATP9B, CBFA2T3, FAM20C, FCGBP, PEBP4, PHF2, RPS9) that were concurrently mutated among all NOA men from this group. Although ADAMTS7 plays a potential role in sperm-egg fusion, the remaining genes are unrelated to sperm production. Whole exome sequencing of cfDNA from the -Sperm cohort (n = 7; 33.5±4yrs) consistently detected mutations on 3 genes, including one involved in spermatogenesis (CFAP54), one essential for oxidative regulation of spermatozoa (GSTT4), and one that encodes testis antigens that are normally expressed during spermatogenesis (BAGE2). Moreover, we concurrently and consistently identified pathogenic mutations on an additional gene in the -Sperm cohort. This gene (MUC2) is involved in the epithelial coating of mucus membrane-containing organs, and its expression has been correlated with spermatogenesis. Interestingly, these genes were all unaffected in men with successful micro-TESE outcomes. Limitations, reasons for caution Although we detected candidate gene mutations that correlate with micro-TESE outcome, these are preliminary findings that need further validation in a larger study cohort. Furthermore, while cfDNA analysis has shown great promise, it is not specific enough on its own and should be corroborated by epigenetic testing. Wider implications of the findings Genetic screening of seminal cfDNA provides a noninvasive alternative to histopathology and can be used to predict the absence of spermatogenesis, sparing these men from unnecessary surgery and cost. This novel biomarker approach lays the groundwork for precision medicine in male infertility testing. Trial registration number n/a

P-419 Relevance of intravenous immunoglobulins in patients with unexplained recurrent pregnancy loss that occurred at the same gestational weeks every time

Abstract Study question Are intravenous immunoglobulins (IVIG) effective for the selected patients who experienced unexplained recurrent pregnancy loss (RPL) that occurred at the same gestational weeks every time? Summary answer IVIG before and after the gestational week-limit (GWL), where miscarriage occurred at the same gestational weeks every time, may improve pregnancy outcomes in RPL patients. What is known already There is no established treatment for patients with unexplained RPL. Some investigators have indicated that IVIG may exhibit therapeutic efficacy in women with unexplained RPL. Since it has been reported that live birth rate significantly increased in women with four or more RPL of unexplained etiology, who received a high dose IVIG at 4-5 weeks of gestation in 2022, ESHRE Guideline Group on RPL (2022) has changed the recommendation to the following: the use of repeated and high doses of IVIG very early in pregnancy may improve live birth rate in women with four or more unexplained RPL. Study design, size, duration We performed a retrospective study between September 2013 to March 2023 in two fertility centers. We included 164 patients in the study who experienced 2 or more miscarriages between 5 and 21 weeks of gestation that occurred at the same gestational weeks every time, who had all negative results for our routine RPL work up and still unsuccessful result after treating these conditions, and whose products of conceptus (POC) revealed at least one normal karyotype. Participants/materials, setting, methods IVIG (Venoglobulin IH 5%, Japan Blood Products Organization, 0.4g/kg) injection was performed twice before and after 1 week of GWL. We divided 164 patients into IVIG group and non-IVIG group by their own choice. The primary outcome was live birth rate, and the secondary outcome was modified live birth rate excluding pregnancies with abnormal fetal karyotype, maternal and neonatal complications. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance IVIG group (n = 116) and non-IVIG group (n = 48) showed no significant differences in the background of patients on BMI (20.3±3.7 and 21.1±2.8), age (37.4±4.2 and 36.7±3.2) and Childbirth history (20.7% and 23.0%), respectively (average±SD), except for the history of past miscarriages. Average numbers of previous miscarriages on IVIG group and non-IVIG group were 3.2±1.3 and 2.5±0.8 (p < 0.001), respectively. After multivariate analysis for BMI, age, Childbirth history and previous miscarriages, live birth rate of IVIG group (70.7%, 82/116) was significantly higher than that of non-IVIG group (60.4%, 29/48; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Similarly, modified live birth rate of IVIG group (73.9%, 82/111) was significantly higher than that of non-IVIG group (63.0%, 29/46; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Treatment outcomes when IVIG was used for each number of previous miscarriages, the modified birth rate was 88.3% for two times miscarriages, 77.0% for three times, and 57.1% for four or more miscarriages. Maternal complications were observed in 2 cases in IVIG group (chest tightness, obstetric critical bleeding). There were no significant differences in the birth weight, gestational weeks, preterm birth, or fetal growth restriction of newborns between two groups. Limitations, reasons for caution Since retrospective nature and we did not perform POC tests for all patients whose pregnancies had ended in miscarriage, we could not assert the effectiveness of IVIG. We need to conduct randomized controlled trial using placebo group to demonstrate the IVIG efficacy before and after the GWL using euploid blastocysts. Wider implications of the findings IVIG before and after the GWL adjusted for each patient may improve pregnancy outcomes in RPL patients. Although IVIG is expensive, the current method requires only two IVIG injections, that may be acceptable for much more patients who want to avoid miscarriages and who didn’t use IVIG for economic reasons. Trial registration number NA

P-612 Euploidy rates between cycles triggered with gonadotrophin-releasing hormone agonist (GnRHa), human chorionic gonadotrophin (hCG) and dual trigger

Abstract Study question Is there any difference in euploidy rates between in vitro fertilization (IVF) cycles triggered with GnRHa, hCG or dual trigger? Summary answer There is no difference in the euploidy rate in preimplantation genetic testing for aneuploidy (PGT-A) cycles according to the trigger method used. What is known already The use of PGT-A has shown that the success of meiosis in the IVF laboratory is inversely correlated with maternal age. However, other uninvestigated factors involved in the IVF process may interfere with normal oocyte physiology. Since the final maturation obtained after ovarian stimulation depends on the use of drugs to promote the resumption of meiosis, it is worth investigating whether the triggering method could impact on oocyte and embryo euploidy. This study aims is to evaluate differences in embryo euploidy rates between PGT-A cycles triggered with GnRHa, hCG or dual trigger (GnRHa + hCG). Study design, size, duration This is a single-center retrospective study of 934 patients who underwent 1085 PGT-A cycles by Next-Generation Sequencing (NGS) from January 2017 to December 2023, using one of three possible trigger methods to induce final oocyte maturation: GnRHa, hCG, or dual trigger. The couples included in the study had normal karyotypes and no severe male factor. Participants/materials, setting, methods Data on the number of oocytes retrieved, ratio of metaphase II (MII) oocytes and fertilization rate, blastocyst development and embryo euploidy were assessed and compared between the GnRHa, hCG and dual trigger treatments. A secondary analysis was performed to evaluate data stratified by patient age. Main results and the role of chance A total of 1085 PGT-A cycles involving 934 patients (38.07 ± 3.01 years) and 2803 biopsied blastocysts were included in the analysis. Dual trigger was used in 726 (77.73%) cycles, GnRHa in 287 (26,4%) cycles and hCG in 72 (7.71%) cycles. The number of oocytes retrieved was significantly higher in the GnRHa group (GnRHa: 18.72 ± 8.59; hCG: 9.88 ± 5.64; dual trigger: 10.58 ± 5.65; p < 0.001) with similar maturation rate (GnRHa: 77.46%; hCG: 74.58%; dual trigger: 76.03%; p = 0.789), fertilization rate (GnRHa: 78.12%; hCG: 76.37%; dual trigger: 79.12%; p = 0.168), blastulation rate (GnRHa: 61.03%; hCG: 58.32%; dual trigger: 58.73%; p = 0,629), high-quality blastocyst rate (GnRHa: 27,88%; hCG: 33,67%; dual trigger: 27,88%; p = 0.543) and embryo euploidy per biopsied embryo (GnRHa: 42.48%; hCG:45.49%; dual trigger:46.98%; p = 0.112). Analysis of the data stratified by age showed similar rates of euploidy rates with the three trigger methods in women younger than 41 years (<35 years: GnRHa 66.50%; hCG 66.67%; dual trigger 56.97% p = 0.290; 35-37 years: GnRHa 49.45% hCG 51.04%; dual trigger 50.23%; p = 0.988; 38-40 years: GnRHa 40.93%; hCG 41.27%; dual trigger 39.13% p = 0.257). However, in patients ≥41 years old the euploidy rate was statistically higher in the GnRHa group (GnRH 31.51%; hCG 18.75%; dual trigger 16.89%; p = 0.032). Limitations, reasons for caution The main limitations of the study are: its retrospective design, the possibility that the trigger method was chosen based on the ovarian response and the limited number of patients in both the hCG trigger and women ≥ 41 years groups. Wider implications of the findings Our results suggest that triggering with GnRHa, hCG or dual trigger leads to comparable euploidy rates. Triggering with GnRHa should be recommended in older patients since it has been associated with a higher rate of embryonic euploidy. Trial registration number Not Applicable

P-537 Linking genetic variation to infertility and embryonic genomic instability. Lessons from a database of whole genomes from targeted populations: challenges, and future directions

Abstract Study question Can novel genomic analysis methods be developed to identify genetic variants that predispose to specific types of infertility and genomic instability in embryos? Summary answer Whole genome analysis and the creation of a genomic variation database of highly targeted populations yielded several genomic links to human infertility and genomic instability. What is known already Infertility is a phenotypic manifestation that originates from highly variable causes, like repeated miscarriage (RM), repeated implantation failure (RIF) or various male and female factors. Specific genetic variations can predispose carriers to distinct types of infertility however quantifiable genetic risks are difficult to be established clinically due to the high amount of variables that exist in any relevant infertility studies. In addition, the combination of the maternal and paternal genetics further complicates the interpretation of genomics results especially when genetic instability in embryos are studied. Several studies have indicated multi[le risk variants however few have managed to establish causal relationships. Study design, size, duration The causes of infertility are extremely variable, which presents a challenge in the design and analysis of genome sequencing studies that involve large populations of participants with similar characteristics. In this study a targeted approach highly selected populations allows more detailed examination of the whole genome of the participants and the different subgroups that exist among them. The participants were recruited using specific selection criteria and samples were collected over a period of 5 years. Participants/materials, setting, methods Couples presenting with infertility and went through preimplantation genetic testing for aneuploidy (PGT- A) were invited to participate. Selection criteria were applied: i) referred for PGT-A after >3 assisted reproduction failed cycles, ii) Normal karyotypes, iii) Female age less than 37, iv) Embryonic instability in their subsequent PGT-A cycles in more than 40% of the produced embryos. After whole genome sequencing (WGS) and variant calling, a bespoke genomic SQL database was constructed for genomic analysis. Main results and the role of chance From 118 individuals that consented to participate 28 individuals with repeated implantation failure (RIF) and 8 individuals RIF/RM passed the selection criteria and their DNA was processed with WGS using a short read platform. In addition, WGS Validation was performed using genomes with known mutations. After the WGS quality control and validation. In total 50 genomes were processed and produced variant results. A novel MySQL database was successfully constructed and validated. Variant z scores were calculated for each participant and the significantly flagged variants were pulled and analysed and comparison with other bespoke and publicly available databases was performed. Ethnicity bias in novel variations detected and corrected. Several smaller cohorts were identified with similar genomic characteristics and accumulation of specific sets of polymorphisms, that were associated with certain types of infertility and preimplantation embryo abnormalities. Of particular interest were variants in synaptonemal complex genes associated with gamete failure, meiotic errors and instability in embryos. Structural variations were significant for male factor infertility. The WGS approach also revealed novel intronic and extragenic variants that would not be usually picked up by other methods like exome sequencing but can play a vital role in gene expression. Limitations, reasons for caution As with all genomic studies relating to infertility, association does not mean causation. Further functional analysis would be required to establish definitive causal pathways based on significant risk variants. In addition, although our study is highly targeted more genomic data can enhance to validation of these results. Wider implications of the findings This highly targeted study using a personalised approach to genome analysis combined with WGS can lead to protocols that determine the cumulative clinical risk for infertility, by revelling the accumulation of specific sets of polymorphisms, intronic and exonic, can be associated with certain types of infertility and preimplantation embryo abnormalities. Trial registration number not applicable

P-056 NOA-SERA: combined autologous progenitor cells and platelet-derived growth factors for male factor infertility due to non-obstructive azoospermia and severe oligoasthenoteratozoospermia (OATS) – a case series

Abstract Study question Can NOA-SERA be utilizing autologous progenitor cells and platelet-derived growth factors,effectively address male factor infertility in non-obstructive azoospermia (NOA) and severeoligoasthenoteratozoospermia (OATS) Summary answer Intra-testicular NOA-SERA transplantation of autologous progenitor cells andgrowth factors shows promise in improving spermatogenesis and fertilization potential,presenting potential breakthrough for male factor infertility. What is known already Male infertility affects 10–15% of couples, with non-obstructive azoospermia (NOA)diagnosed in 60% of azoospermic men. Current interventions, including platelet-rich plasma and stem cells, show variable success. This study introduces a novel protocol, NOA-SERA, combining autologous mobilized progenitor cells and platelet-derived growth factors to restore spermatogenesis. Despite advancements in assisted reproduction, male factor infertility due to primary NOA remains challenging. This case series explores the safety and efficacy of NOA-SERA, avoiding in vitro breeding risks and emphasizing intratesticular transplantation’s safety and efficacy Study design, size, duration his case series (March 2019–November 2023) includes 7 male factor infertility patients,consisting of 4 non-obstructive azoospermic NOA, 1 cryptozoospermia and 2 severe oligoaesthenoteratospermic (OATS, strongly desirous of pregnancy with self-gamete. All patients received NOA-SERA following written consent. One-year follow-up included hormonal profiling, semen analysis, and testicular biopsy assessment Participants/materials, setting, methods Seven men, with normal karyotype, underwent NOA-SERA due to male factor infertility. This intervention involved intra-testicular transplantation of autologous CD34+/CD133+ enriched peripheral blood mononuclear stem cells (MPB-MNCs) combined with platelet derived growth factors. The procedure, avoiding in vitro breeding and manipulation, prioritized safety and efficacy. Follow-up for one year included hormonal profile, semen analysis, testicular biopsy assessments, clinical pregnancy (b-hCG), sustained implantation (>8 weeks), and live birth rates were followed. Main results and the role of chance Analysis of NOA-SERA treatment in this case series revealed positive outcomes in 70% of patients, showing increased testicular size, elevated testosterone, and reduced FSH levels. In our study, out of 4 NOA, 2 NOA remained NOA, 1 progressed to OATS, 1 progressed to normal semen analysis after 3 months. Couple conceived with intrauterine insemination (IUI) first pregnancy and they also had a subsequent natural conception. 1 Crypto progressed to OATS and 1 severe OATS remained the same and one progressed to OATS. Although no appearance of sperm in the ejaculate was observed, 2 patients displayed sperms in Testicular Sperm Aspiration (TESA) sufficient for ICSI. Couples undergoing IVF therapy post-biopsy achieved successful fertilization in two cases, leading to positive pregnancies and sustained implantation and livebirth of healthy full-term babies. Post-procedure FSH was slightly lower than pre-procedural levels, indicating hormonal improvement without observed therapy-related deterioration. The novelty lies in the intratesticular transplantation of mobilized purified autologous CD34+/CD133+ enriched MPBMNCs with platelet-derived growth factors, avoiding in vitro risks.” To summarise, 3 out of 7 achieved successful pregnancy, 2 NOA with ICSI, 1 NOA progressed to normal semen analysis and pregnancy with IUI Limitations, reasons for caution Despite interesting results, limitations include the self-controlled case series design and asmall patient cohort, lacking a comparison to a control group. Future studies should include more patients, stratified for age, aetiology, and other risk factors, to further elucidate this intervention’s mechanism of action Wider implications of the findings NOA-SERA demonstrates promise in enhancing spermatogenesis and fertilization potential, emphasizing necessity for larger randomized trials to ascertain clinical efficacy, particularly in subgroups of OATS&NOA patients. NOA-SERA emerges as safe, cost-effective adjuvant for optimizing spermatogenesis and improving IVF success and pregnancy outcomes in male factor infertility. Trial registration number Not applicable

P-518 Differentially gene expression identified by RNA seq in aneuploid human blastocysts is more influenced by the phenomenon of aneuploidy than the involved chromosome

Abstract Study question What insights do RNA-Seq profiles provide about gene expression in aneuploid blastocysts with small and large chromosomes compared to euploid blastocysts? Summary answer The difference in gene expression is affected by aneuploidy and is more related to the presence of aneuploidy than the size of the aneuploid chromosomes. What is known already Embryonic aneuploidy is one of the main common causes of implantation failure and miscarriage. RNA-Seq investigations of the embryo in the early stages of preimplantation indicate that with the gradual replacement of the embryonic profile and the reduction of the oocyte residues, the gene expression profile in the blastocyst is more dependent on the embryo’s genome. A recent study by comparing the gene expression profile of euploid blastocysts and specific aneuploidies such as trisomy 21, trisomy 18, XXY and XO syndromes using RNA-Seq, showed how the type of aneuploidy might affect gene expression. Study design, size, duration This study was conducted on surplus embryos of the patients with history of recurrent implantation failure or recurrent miscarriage following PGT by Day-5 trophectoderm biopsy. Inclusion criteria involved maternal age ≤37, normal parental karyotypes, and ovulation by long stimulation protocol. As advanced maternal age and ovarian stimulation method can affect the aneuploidy rate and expression profile, embryos belonging to mothers > 37 years and included only patients with the long stimulation protocol were excluded. Participants/materials, setting, methods The normal embryos and those with single aneuploidy were selected for comparison of gene expression profiles using RNA sequencing. The single aneuploid embryos were classified in two groups: with defined aneuploidy(DA) of small chromosomes (including chromosomes 16, 20, 21, 22), and with undefined aneuploidy(UA) as single aneuploids for other chromosomes. To neutralize the effect of genomic content on gene expression profile in DA group, embryos were pooled reciprocally (trisomy and monosomy for the same chromosome). Main results and the role of chance Principal Component Analysis (PCA) validated embryos classification considering their differentially expressed genes (DEG) clustering into three distinct groups. To minimize the role of chance, only genes with significant adjusted P-values in both STAR and Hisat data analyses were included in consequent interpretations. In addition to comparison between and among the DA, UA and normal groups, the entire set of aneuploid groups was compared with the normal group. The DEG in both aneuploid groups compared to the normal group represented eminent upregulation rather than downregulation. Gene Ontology analyses indicated an upregulation of transcription, ubiquitination, autophagy, mitochondrial, chromosome segregation and DNA repair pathways in both DA and UA compared to normal group. Overexpression of the DNA repair pathway and other validated genes of interest in aneuploid embryos was in agreement with qPCR findings. Translation and protein synthesis pathways also showed overexpression as part of the functional response to aneuploidy. Upregulation of apoptotic process pathway was evident in the UA group. While comparison of gene expression between DA and UA showed overexpression of 143 genes, but cellular lipid synthesis was the only pathway with a significant difference. The distribution of DEG on chromosomes in the DA group was not associated with selected aneuploidies. Limitations, reasons for caution The number of biological replicates in each category was limited in order to provide more confident data by minimizing the time interval between providing the samples and RNA sequencing. Larger studies might change some affected pathways due to differences in identified DEG. Wider implications of the findings This study shed light on the black box of human preimplantation aneuploidy. Considering that the transcriptome is affected by the genomic status and some debates on genomic based current PGT approaches, this study is a step for selection of healthy and viable embryos by revealing the molecular mechanisms. 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

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

Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity

BackgroundThe human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.MethodsFirst, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.ResultsWe found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.ConclusionThe HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

Establishment of a transgene-free iPS cell line (SDCHi007-A) from a young patient bearing a ATP1A2 mutation and suffering from Epilepsy

Epilepsy is a chronic neurological disease. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient diagnosed as epilepsy caused by ATP1A2 gene mutation. Induced pluripotent stem cells (iPSCs) were developed using non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and C-MYC. The established iPSC line (SDCHi007-A) displayed pluripotent cell morphology, high expression levels of pluripotency markers, differentiation potential in vitro, normal karyotype, and remaining the original ATP1A2 gene mutation.

Overview of prevalence, etiology, clinical features, diagnosis, and management of primary ovarian insufficiency

Primary ovarian insufficiency (POI) is the development of primary hypogonadism before the age of 40 years in women with normal karyotypes. Patients present with symptoms that are similar to those of menopause, including irregular periods, reduced fertility, and climacteric symptoms due to hypoestrogenism, such as hot flashes, vaginal dryness, dyspareunia, irritability, and night sweats. The incidence of spontaneous POI is approximately 1 in 250 by 35 years of age and 1 in 100 by 40 years of age. Genetic, inflammatory, metabolic, autoimmune, and idiopathic factors contribute to POI. POI pathophysiology is explained by the presence of fewer primordial follicles and their rapid degeneration. Detailed history and examination are important for diagnosis. Diagnosis requires biochemical evidence and laboratory testing, including measurements of follicle-stimulating hormone, thyroid-stimulating hormone, prolactin, and estradiol (E2) levels. In addition, transvaginal ultrasound is preferred to evaluate ovarian reserve. POI should be managed through a multidisciplinary approach involving gynecologists, endocrinologists, and psychologists. Hormonal replacement therapy is the mainstay treatment for all women with POI. Lifestyle modifications such as weight-bearing exercises, smoking cessation, and Vitamin D and calcium supplementation are highly recommended to improve bone density and reduce fractures. Psychological care has been suggested to improve self-esteem and social support, along with family planning guidance. Despite recent discoveries, additional screening techniques are needed.

Design of iPSC-based cell model to study the functions of the <i>UBE2A</i> gene

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. UBE2A mutations lead to congenital X-linked mental retardation syndrome-type Nascimento. How UBE2A participates in the central nervous system development is still unknown. AIM: To establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of UBE2A in neurogenesis. METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, a cell model based on iPSCs from two healthy donors was designed. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of UBE2A. In addition, iPSCs were obtained by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A locus. RESULTS: The obtained iPSCs demonstrate an ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA-1-81 and have normal karyotypes. iPSCs with UBE2A knockout or hyperexpression had significantly increased nuclei size compared with the isogenic control. CONCLUSION: The developed iPSC-based cell model can be used for fundamental studies of the functions of UBE2A in neurogenesis.

The likelihood of detecting abnormal karyotypes in fetuses with a single major anomaly or "soft" marker on ultrasonographic scanning.

Fetuses with abnormal karyotypes often exhibit distinctive ultrasonographic markers, including major anomalies and "soft" markers, indicating potential chromosomal issues. A crucial consideration arises when a single fetal anomaly is detected, raising the question of whether karyotyping is warranted, given the associated procedural risks. Our objective was to establish correlations between single fetal anomalies identified through ultrasound and chromosomal abnormalities. A cross-sectional study analyzed the karyotype of 1493 fetuses and detected a single ultrasonographic anomaly over a 16-year period. Karyotyping was performed using the standard karyotype technique. Moreover, data regarding the type of anomaly detected ultrasonographically, karyotype results, and outcomes following interventions were collected. Among other methods, the use of positive likelihood ratios (LR+) was used to evaluate the diagnostic accuracy of ultrasound compared to karyotyping. In total, an aberrant karyotype was identified in 99 fetuses (6.6%). This was most commonly observed in cases involving a "soft" marker, occurring in 27 out of 218 fetuses (12.4%). The most frequently detected aberrant karyotype resulted from aneuploidies (80.6% of cases), notably trisomy 21 (50.5%). "Soft" markers predicted chromosomal issues (LR+ = 1.9; OR = 2.4), and isolated polyhydramnios (LR+ = 1.54; OR = 1.6) showed significance in predicting fetal chromosomal aberrations. When assessing the necessity for karyotyping in fetuses with single major anomalies or "soft" markers, it is crucial to consider individual risks for chromosomopathies, including the LR+ of the detected marker. In cases where fetuses exhibit isolated anomalies with a normal karyotype, additional diagnostic measures, such as molecular cytogenetic and molecular genetics techniques, may become necessary.

Microcarrier-based clinical-grade manufacturing of therapeutic Wharton's jelly mesenchymal stromal cells

Due to their immunomodulatory and anti-inflammatory properties, tissue repair capabilities and regenerative potential, Wharton's jelly mesenchymal stem/stromal cells (WJMSCs) have been widely investigated as potential treatment for diverse clinical indications. WJMSCs have been found to be well-tolerated and safe, positioning them as a promising candidate for cellular therapy. To address the commercial need for manufacturing WJMSCs for clinical applications, the production scale should be capable of generating large quantities of cells that retain their expected identity, purity and potency. This study aimed to establish a current Good Manufacturing Practice (cGMP) compliant robust and scalable expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house cGMP-manufactured WJMSCs, which are currently being tested in a Phase Ib clinical trial (NCT03158896) using two-dimensional (2D) planar systems, we optimized various culture parameters including type of microcarrier, seeding density, agitation and culture feed regime in a 3D microcarrier-based culture system in spinner flasks. The results showed that cell adhesion was potentiated under intermittent stirring (3 min of agitation at 25 rpm followed by a period of non-agitation for 30 min), with reduced supplementation (0.05%) during the initial 8 h of cultivation with an initial cell concentration of 0.45 × 105 cells/mL. Microcarrier-based WJMSC expansion in spinner flasks achieved greater cell densities of 1.67 × 106 cells/mL with a maximum of 37-fold expansion, yielding ∼84 × 106 cells after 6 days of culture with a 95% harvest efficiency. Additionally, post 3D expansion, WJMSCs maintained their phenotypic characteristics, differentiation potential, normal karyotype, functional properties and sterility in the culture systems evaluated. This cGMP-compliant expansion process described herein demonstrates a successful transition of an established 2D planar culture process of clinical grade WJMSCs to 3D microcarrier-based suspension process generating higher cell yields, is cost-effective and represents an important step toward fulfilling the commercial demand of clinical grade mesenchymal stromal cells.

Generation of a gene-corrected human isogenic iPSC line from an Alzheimer’s disease iPSC line carrying the PSEN1 H163R mutation

We report the generation of a gene-edited human induced pluripotent stem cell (iPSC) line from an Alzheimer’s disease patient-derived iPSC line harbouring the PSEN1 H163R mutation. This line demonstrates pluripotent stem cell morphology, expression of pluripotency markers, and maintains a normal karyotype.

The significance of follow-up in patients with dysmorphic features: a case from clinical practice

Background. Kabuki syndrome is a rare disorder, that is characterized by typical facial dysmorphism, hypotonia, delay in intellectual and motor development. Case report. We present a case of a girl in whom polycystic left kidney was prenatally established. Born prematurely in 37 weeks by C section due to oligohydramnios. After birth, atresia of the anus with fistula, cysts in the left and reduced dimensions of the right kidney, were further established. A normal female karyotype was found and targeted sequencing analysis was conducted on a panel of 81 genes associated with renal abnormalities – no pathogenic variants were detected. The child was then followed up by its general practitioner. At the age of 2 years, she was again referred for genetic counseling, which revealed the following dysmorphic signs – long eye palpebral fissuras with ectropion of the lower eyelid, sparsed lateral eyebrows, depression of the nasal bridge, brachydactyly and others characteristic of Kabuki’s syndrome. The conducted molecular genetic analysis confirmed the clinical diagnosis – a likely pathogenic variant in the KMT2D gene was established. Conclusions. Certain pathognomonic facial may not present at birth and only appear after a few years. Therefore, monitoring of the evolution of dysmorphic traits is required.