Le blastocyste en milieu séquentiel (avantages et inconvénients)

PARIS-BIUP (751062107) / SudocSudocFranceF

First Birth after Sperm Selection through Discontinuous Gradient Centrifugation and Artificial Insemination from a Chromosomal Translocation Carrier

Introduction. Balanced chromosomal carriers, though usually healthy, are confronted with recurrent spontaneous abortions and malformations in the offspring. Those are related to the transmission of an abnormal, chromosomally unbalanced genotype. We evidenced that the proportion of unbalanced spermatozoa can be significantly decreased through a sperm preparation process called discontinuous gradient centrifugation (DGC). We therefore started offering intrauterine inseminations with this procedure to couples with a male translocation carriers. Case Presentation. We report the case of a 37-year-old man carrying a t(3;10)(q25;p13) reciprocal translocation. He and his partner had had trouble conceiving for ten years and had four spontaneous abortions. DGC in this patient decreased the proportion of unbalanced spermatozoa from 63.6% to 52.3%. They were therefore offered intrauterine insemination with DGC, which eventually led to the birth of a healthy female child carrying the paternal translocation. Conclusion. We showed that translocation carriers could be offered intrauterine inseminations with DGC. Before this, the only two options were natural conception with prenatal diagnosis and termination of chromosomally unbalanced fetuses or preimplantation genetic diagnosis, which is a much heavier and costly procedure. We are currently offering this option through a multicentric program in France, and this is the first birth originating from it

Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM) at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p<10-6), OR = 2.4; and p<0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI

Chromosomal segregation analysis and HOST-based sperm selection in a complex reciprocal translocation carrier

International audienceIntroduction: Complex chromosomal rearrangements (CCRs) involve two or more chromosomes and at least three breakpoints. Due to their complexity, they are associated with a high number of unbalanced gametes, whose fertilization is often incompatible with viable fetal development. Preimplantation genetic diagnosis (PGD) is usually offered to those patients and typically shows modest results considering the high number of unbalanced embryos. We previously showed that a sperm selection process using the hypo-osmotic swelling test (HOST) allows for an 83% reduction in the proportion of unbalanced spermatozoa (US) in male rearrangements carriers. This is the first report of the use of this procedure in a CCR carrier.Case description: We report on the case of a 36-year-old male t(4;7;14)(q12;p21;q11.2) carrier who presented to our center for infertility. Sperm fluorescent in situ hybridization showed an 88% proportion of unbalanced spermatozoa. After hypo-osmotic incubation and selection of spermatozoa with a specific flagellar conformation, the proportion of unbalanced spermatozoa dropped to 15%.Discussion: In the present case, we show that it is possible to select chromosomally balanced prior to in vitro fertilization in male CCR carriers. This technique has the potential of increasing the proportion of euploid embryos and therefore the chances of healthy pregnancy and birth

Molecular Profiling of Spermatozoa Reveals Correlations between Morphology and Gene Expression: A Novel Biomarker Panel for Male Infertility

International audienceChoosing spermatozoa with an optimum fertilizing potential is one of the major challenges in assisted reproductive technologies (ART). This selection is mainly based on semen parameters, but the addition of molecular approaches could allow a more functional evaluation. To this aim, we used sixteen fresh sperm samples from patients undergoing ART for male infertility and classified them in the high-and poor-quality groups, on the basis of their morphology at high magnification. Then, using a DNA sequencing method, we analyzed the spermatozoa methylome to identify genes that were differentially methylated. By Gene Ontology and protein-protein interaction network analyses, we defined candidate genes mainly implicated in cell motility, calcium reabsorption, and signaling pathways as well as transmembrane transport. RT-qPCR of high-and poorquality sperm samples allowed showing that the expression of some genes, such as AURKA, HDAC4, CFAP46, SPATA18, CACNA1C, CACNA1H, CARHSP1, CCDC60, DNAH2, and CDC88B, have different expression levels according to sperm morphology. In conclusion, the present study shows a strong correlation between morphology and gene expression in the spermatozoa and provides a biomarker panel for sperm analysis during ART and a new tool to explore male infertility

Simple FISH-based evaluation of spermatic nuclear architecture shows an abnormal chromosomal organization in balanced chromosomal rearrangement carriers

International audienceIntroduction: Interphasic DNA has a constant three-dimensional conformation, which is particularly striking for spermatic DNA, with distinct chromosomal territories and a constant chromosomal conformation. We hypothesized that this organization is fragile, and that an excess or a lack of chromosomal segments could hinder the genomic structure as a whole.Methods: Five human male chromosomal translocation carriers and five controls were included. Spermatic DNA spatial organization was studied, in both balanced and unbalanced spermatozoa, with two-dimensional fluorescent in situ hybridization (FISH) via analysis of chromosomes not implicated in the cases' translocations, compared to that of normal controls. Two parameters were studied: the distance between the two telomeric ends of chromosome 1, and the area of the chromosomal territories of chromosomes 1 and 17.Results: Sperm FISH analysis of rearrangement carriers revealed changes in the nuclear architecture compared to that of controls. Inter-telomeric distance and chromosomal territories areas were both significantly increased.Discussion: We show that an excess or lack of chromosomal segments can hinder the normal spatial nuclear architecture in sperm. These results show that nuclear architecture is a fragile assembly, and that local chromosomal abnormalities may impact the nucleus as a whole. This suggests a potential avenue for selection of spermatozoa prior to in vitro fertilization, not only in rearrangement carriers but also in the infertile population at large. Furthermore, we suggest that 2D-FISH could possibly be a useful tool in assessing spermatic nuclear organization in a way to evaluate male fertility

Double chromosomal translocation in an infertile man: one-step FISH meiotic segregation analysis and reproductive prognosis

International audienceBackground: The prevalence of chromosomal translocations is 1/500 in the general population. While in the vast majority of cases, carriers have a normal phenotype; they can present with difficulty conceiving due to the presence of a proportion of unbalanced gametes as a consequence of abnormal chromosomal segregation during meiosis. Since complex translocations involve three or more chromosomes, meiotic segregation leads to a greater number of possible combinations which effectively complicate both their study and therapeutic care.Case presentation: We report on the case of a male carrier of a complex homogeneous double Robertsonian translocation: 44, XY, der(13;14)(q10;q10),der(21;22)(q10;q10). We studied his meiotic segregation by FISH on spermatozoa from the initial sample, as well as following discontinuous gradient centrifugation and after incubation in an hypo-osmotic solution.Conclusion: We report a method to study in a simple single-step manner the meiotic segregation of double Robertsonian translocations in spermatozoa. Further, our results suggest that reproductive prognosis of affected individuals may be markedly improved by HOST-based sperm selection (HBSS)

Potential selection of genetically balanced spermatozoa based on the hypo-osmotic swelling test in chromosomal rearrangement carriers

International audienceChromosomal translocations and other balanced rearrangements, although usually associated with a normal phenotype, can lead to the transmission of an abnormal unbalanced genome to the offspring. Balanced and unbalanced spermatozoa, being indistinguishable, cannot be selected or deselected for prior to IVF and pre-implantation genetic diagnosis. Spermatozoa from 16 chromosomal rearrangement carriers were studied. After incubation in a hypo-osmotic solution (hypo-osmotic swelling test, or HOST), spermatozoa were fixed on microscope slides. The chromosomally balanced or unbalanced status corresponding to each observed class of flagellar conformation was evaluated through fluorescent in-situ hybridization (FISH). We show here a specific type of spermatozoa, with a distinct flagellar conformation that was associated with a balanced genetic content. HOST is a simple, low-cost and time-honoured procedure initially developed to distinguish immotile viable from non-viable spermatozoa. We demonstrate that it can also be used to identify genetically balanced spermatozoa in chromosomal rearrangement carriers, with a 96% decrease in the proportion of unbalanced spermatozoa after selection. This may potentially improve reproductive prognosis in affected couples if used prior to pre-implantation genetic diagnosis (PGD), and clinical utility and efficacy should be evaluated in further studies