Andrei Tchirkov

STUDY QUESTION: What is the optimal stage (immature vs mature) and most efficient vitrification technique (semi-automated vs manual) to ensure the safety of the rescue-IVM (r-IVM) procedure for oocyte cryopreservation? SUMMARY ANSWER: Human oocytes should be cryopreserved after r-IVM at the mature stage (r-MII oocytes) by semi-automated or manual vitrification. WHAT IS KNOWN ALREADY: r-IVM of immature oocytes may increase the number of oocytes cryopreserved for fertility preservation. However, the best stage and vitrification system (semi-automated or manual) for cryopreserving oocytes with the least possible impact on nuclear quality is unclear. STUDY DESIGN, SIZE, DURATION: From January 2020 to June 2024, a prospective study was conducted on patients undergoing ICSI, including cases with at least one immature germinal vesicle (GV) stage oocyte on the day of oocyte collection, resulting in a total of 414 oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 175 patients under 37 years old with no ovulatory disorder and undergoing ICSI. A total of 414 immature oocytes were collected and divided into five groups. The control group included fresh in vitro-matured oocytes (IVM, n = 81). After r-IVM, oocytes were vitrified by a semi-automated technique (IVM+VITg group, n = 63) or a closed manual procedure (IVM+VITm group, n = 66). Before r-IVM, oocytes were vitrified using both techniques (VITg+IVM group, n = 113 and VITm+IVM group, n = 91). The fresh IVM group combined IVM, IVM+VITg, and IVM+VITm groups. Survival rates of oocytes were evaluated 2-h post-warming. r-IVM was performed in a time-lapse incubator , allowing the assessment of r-IVM rates and maturation kinetics, including GV breakdown (GVBD) and first polar body extrusion timings. We assessed the quality of oocyte nuclear maturation through the evaluation of meiotic spindle polarity and chromosomes alignment by 3D analysis of confocal microscopy images and aneuploidy rate by array-CGH (a-CGH). MAIN RESULTS AND THE ROLE OF CHANCE: The oocyte post-warming survival rate was lower when semi-automated vitrification was performed before r-IVM (50% for VITg+IVM group) compared with the three other groups (88% for IVM+VITg, 93% for IVM+VITm, and 80% for VITm+IVM, adjusted P-value < 0.001). IVM rates were not different between the three groups (fresh IVM: 80%, VITg+IVM: 80% and VITm+IVM: 69%, Pa = 0.131). Longer GVBD timings were observed when semi-automated vitrification was performed before r-IVM (VITg+IVM: 10.4 h) compared with Fresh IVM (7.9 h, Pa = 0.003). The percentages of oocytes with bipolar spindles or/and aligned chromosomes were not different between the five groups. Similarly, there was no difference in aneuploidy rates (monosomy or trisomy) between the five study groups (Pa = 0.847). Shorter GVBD timings were observed for oocytes with a bipolar spindle or aligned chromosomes (7.8 h) than for oocytes with a non-bipolar spindle or misaligned chromosomes (10.1 h, Pa = 0.011). LIMITATIONS, REASONS FOR CAUTION: The results are limited to the type of biological samples (GV-stage immature oocytes from stimulated ovaries) and the defined study population (patients < 37 years old without ovulatory disorders) used in this study. Furthermore, additional research is required to evaluate the ability of oocytes to undergo successful fertilization and embryo development, as well as their capacity to result in a live birth. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide reassurance regarding the use of semi-automated technique after r-IVM in clinics to optimize and standardize oocyte vitrification. The emerging field of r-IVM combined with cryopreservation by vitrification represents a promising option for fertility preservation, particularly for patients with a poor response to ovarian stimulation. Moreover, our study has revealed a previously unidentified correlation between extended GVBD timing and aberrant spindle morphology and chromosome misalignment. This provides a promising new non-invasive marker of nuclear oocyte quality for use in clinical practice. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the French Biomedicine Agency (grant number: ABM 20AMP004) and the University Hospital of Clermont-Ferrand (France, Bourse Innovation). There are no conflicts of interest to be declared for any of the authors. There are no patents, products in development, or marketed products to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov ID NCT03680937.

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

Cytogenetic aberrations are found in 65% of adults and 75% of children with acute leukemia. Specific aberrations are used as markers for the prognostic stratification of patients. The current standard cytogenetic procedure for acute leukemias is karyotyping in combination with FISH and RT-PCR. Optical genome mapping (OGM) is a new technology providing a precise identification of chromosomal abnormalities in a single approach. In our prospective study, the results obtained using OGM and standard techniques were compared in 29 cases of acute myeloid (AML) or lymphoblastic leukemia (ALL). OGM detected 73% (53/73) of abnormalities identified by standard methods. In AML cases, two single clones and three subclones were missed by OGM, but the assignment of patients to cytogenetic risk groups was concordant in all patients. OGM identified additional abnormalities in six cases, including one cryptic structural variant of clinical interest and two subclones. In B-ALL cases, OGM correctly detected all relevant aberrations and revealed additional potentially targetable alterations. In T-ALL cases, OGM characterized a complex karyotype in one case and identified additional abnormalities in two others. In conclusion, OGM is an attractive alternative to current multiple cytogenetic testing in acute leukemia that simplifies the procedure and reduces costs.

Telomere length appears to correlate with survival in early non-small-cell lung cancer (NSCLC), but the prognostic impact of telomere status in advanced NSCLC remains undetermined. Our purpose was to evaluate telomere parameters as prognostic and predictive biomarkers in advanced NSCLC. In 79 biopsies obtained before treatment, we analyzed the telomere length and expression of TERT and shelterin complex genes (TRF1, TRF2, POT1, TPP1, RAP1, and TIN2), using quantitative PCR. Non-responders to first-line chemotherapy were characterized by shorter telomeres and low RAP1 expression (p = 0.0035 and p = 0.0069), and tended to show higher TERT levels (p = 0.058). In multivariate analysis, short telomeres were associated with reduced event-free (EFS, p = 0.0023) and overall survival (OS, p = 0.00041). TERT and TRF2 overexpression correlated with poor EFS (p = 0.0069 and p = 0.00041) and OS (p = 0.0051 and p = 0.007). Low RAP1 and TIN2 expression-levels were linked to reduced EFS (p = 0.00032 and p = 0.0069) and OS (p = 0.000051 and p = 0.02). Short telomeres were also associated with decreased survival after nivolumab therapy (p = 0.097). Evaluation of telomere status in advanced NSCLC emerges as a useful biomarker that allows for the selection of patient groups with different clinical evolutions, to establish personalized treatment.

RNA splicing factors are frequently altered in cancer and can act as both oncoproteins and tumour suppressors. They have been found mutated or deregulated, justifying the growing interest in the targeting of splicing catalysis, splicing regulatory proteins, and/or specific, key altered splicing events. We recently showed that the DNA methylation alterations of CD34⁺CD15⁻ chronic myeloid leukaemia (CML) cells affect, among others, alternative splicing genes, suggesting that spliceosome actors might be altered in chronic-phase (CP)-CML. We investigated the expression of 12 spliceosome genes known to be oncogenes or tumour suppressor genes in primary CP-CML CD34⁺ cells at diagnosis (n = 15). We found that CP-CML CD34⁺ cells had a distinct splicing signature profile as compared with healthy donor CD34⁺ cells or whole CP-CML cells, suggesting: (i) a spliceosome deregulation from the diagnosis time and (ii) an intraclonal heterogeneity. We could identify three profile types, but there was no relationship with a patient’s characteristics. By incubating cells with TKI and/or a spliceosome-targeted drug (TG003), we showed that CP-CML CD34⁺ cells are both BCR::ABL and spliceosome dependent, with the combination of the two drugs showing an additive effect while sparing healthy donors cells. Our results suggest that the spliceosome may be a new potential target for the treatment of CML.

Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age of 25.0 ± 5.4 weeks and by CV biopsy at 18.1 ± 6.3 weeks. In neither sample type was TL influenced by gestational age or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P < 10⁻⁶), single (29%, P < 10⁻⁶) and multiple (44%, P < 10⁻⁶) malformations. Similar TL shortening was also observed in CV from abnormal fetuses but to a lesser extent (25%, P = 0.0002; 18%, P = 0.016; 20%, P = 0.004, respectively). Telomere shortening was more pronounced in cases of multiple congenital anomalies than in fetuses with a single malformation, suggesting a correlation between TL and the severity of fetal phenotype. Thus, TL measurement in fetal samples during pregnancy could provide a novel predictive marker of pathological development.

Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or testicular tissue (TT) is recommended to preserve fertility. However, the risk of reintroducing residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (n = 12) and TT (n = 14) were contaminated with tumor RD-ES cells (10, 100, and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with a strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (r = 0.93) and TT (r = 0.96) (p < 0.001). No transcript was detected in uncontaminated control samples. The invasive potential of Ewing cells was evaluated using co-culture techniques. After co-culturing, tumor cells were detected in OT/TT with histology, FISH, and RT-qPCR. In addition, four OT and four TT samples from children with metastatic EWS were tested, and no MRD was found using RT-qPCR and histology. We demonstrated the high sensitivity and specificity of RT-qPCR to detect EWS MRD in OT/TT samples. Clinical trial: NCT 02400970.