8 research outputs found
Concentration of glutathione and expression of glutathione peroxidases 1 and 4 in fresh sperm provide a forecast of the outcome of cryopreservation of human spermatozoa
Oxidative stress imbalance potentially leads to damage of the structure of the cell and macromolecules such as plasma membrane components, proteins, and DNA. The plasma membrane of the sperm cell, which has high levels of polyunsaturated fatty acids, renders it particularly sensitive to free radical–mediated attacks. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction techniques, which results in a highly variable and unpredictable reduction in the number of motile sperm cells. Subsequently, oxidative status can positively or negatively affect the motility, viability, and fertilizing capacity of thawed sperm. These effects are counteracted by various oxidative defense enzymes and anti-oxidants such as glutathione peroxidase isoforms GPx1 and GPx4, glutathione reductase (GR), and cellular glutathione (reduced) (GSH). In this way, oxidative status could represent a predictive marker of sperm quality following the freeze-thaw process. This study was based on 56 human sperm samples. We observed direct positive and negative relationships between the postthaw motile sperm recovery rate and GPx1 and GPx4 expression and activity, on the one hand, and GSH concentrations, on the other. No correlation was found between this recovery rate and GR or basic semen parameters. Predictive values clearly demonstrate that, among the molecules analyzed, the most accurate diagnoses result when analyses are conducted for GPx1 and GPx1 messenger RNA expression, GPx1 and GPx4 enzymatic activity, and GSH concentration. In conclusion, a reserve of glutathione, together with GPx expression, is necessary to eliminate free radicals using GSH or a like structural protein and seems to be essential for a good postthaw recovery. These molecules can be employed as indicators of postthaw sperm quality.Meseguer Anastasio, Manuel Francisco, [email protected] ; Simon Valles, Carlos Antonio, [email protected] ; Remohi Gimenez, Jose Alejandro, [email protected]
False positive rate of an arrayCGH platform for single-cell preimplantation genetic screening and subsequent clinical application on day-3
In this work, false positive rate of an arrayCGH platform for its use in day-3 single-blastomere analysis was calculated. For this purpose, 38 embryos diagnosed as abnormal on day-3 by FISH were re-biopsied on day-4. Single-cell day-4 arrayCGH diagnosis was then performed. A successful amplification was obtained in 97.4 % (37/38) of the day-4 cells analysed by arrayCGH. Day-3 FISH and day-4 arrayCGH diagnosis were concordant in 35/37 cases. The two discordant embryos were spread and all the cells from each embryo were re-analysed by FISH on day 5. The same error rate (2.7 %) for day-3 FISH and day-4 arrayCGH was obtained when comparing day-5 FISH re-analysis. After this pre-clinical phase, the platform was used for day-3 arrayCGH clinical application in 320 patients (1,760 embryos). Day-3 amplification rate was 98.6 %. An optimal reproductive outcome was obtained when applying arrayCGH to a clinical program: clinical pregnancy rate per cycle of 38.4 % and 60.3 % per transference were obtained, with an implantation rate of 53.5 %. Overall miscarriage rate was 10.6 %. Additionally, day-5 FISH re-analysis was performed in 42 of the embryos from the clinical phase, obtaining a concordance rate of 97.6 % with day-3 arrayCGH.Electronic supplementary materialThe online version of this article (doi:10.1007/s10815-012-9918-4) contains supplementary material, which is available to authorized users
New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization
The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation
Endometrial microbiota composition is associated with reproductive outcome in infertile patients
Background: Previous evidence indicates associations between the female reproductive tract microbiome composition and reproductive outcome in infertile patients undergoing assisted reproduction. We aimed to determine whether the endometrial microbiota composition is associated with reproductive outcomes of live birth, biochemical pregnancy, clinical miscarriage or no pregnancy. Methods: Here, we present a multicentre prospective observational study using 16S rRNA gene sequencing to analyse endometrial fluid and biopsy samples before embryo transfer in a cohort of 342 infertile patients asymptomatic for infection undergoing assisted reproductive treatments. Results: A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was associated with unsuccessful outcomes. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. Conclusions: Our findings indicate that endometrial microbiota composition before embryo transfer is a useful biomarker to predict reproductive outcome, offering an opportunity to further improve diagnosis and treatment strategies
Human embryo live imaging reveals nuclear DNA shedding during blastocyst expansion and biopsy
Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding
Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle
In a human menstrual cycle the endometrium undergoes remodeling, shedding and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, our understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, resolving cellular heterogeneity in multiple dimensions. We profiled the behavior of seven endometrial cell types, including a previously uncharacterized ciliated cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that the human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with a widespread decidualization feature in the stromal fibroblasts. Our study provides a high-resolution molecular and cellular characterization of human endometrial transformation across the menstrual cycle, providing insights into this essential physiological process
The human periconceptional maternal-embryonic space in health and disease
Pregnancy is established during the periconceptional period as a continuum beginning with blastocyst attachment to the endometrial epithelial surface followed by embryo invasion and placenta formation. This period sets the foundation for the child and mother's health during pregnancy. Emerging evidence indicates that prevention of downstream pathologies in both the embryo/newborn and pregnant mother may be possible at this stage. In this review, we discuss current advances in the periconceptional space, including the preimplantation human embryo and maternal endometrium. We also discuss the role of the maternal decidua, the periconceptional maternal-embryonic interface, the dialogue between these elements, and the importance of the endometrial microbiome in the implantation process and pregnancy. Finally, we discuss the myometrium in the periconceptional space and review its role in determining pregnancy health
Decoding the endometrial niche of Asherman's Syndrome at single-cell resolution
Asherman's Syndrome is characterized by intrauterine adhesions or scarring, which cause infertility, menstrual abnormalities, and recurrent pregnancy loss. The pathophysiology of this syndrome remains unknown, with treatment restricted to recurrent surgical removal of intrauterine scarring, which has limited success. Here, we decode the Asherman's Syndrome endometrial cell niche by analyzing data from over 200,000 cells with single-cell RNA-sequencing in patients with this condition and through in vitro analyses of Asherman's Syndrome patient-derived endometrial organoids. Our endometrial atlas highlights the loss of the endometrial epithelium, alterations to epithelial differentiation signaling pathways such as Wnt and Notch, and the appearance of characteristic epithelium expressing secretory leukocyte protease inhibitor during the window of implantation. We describe syndrome-associated alterations in cell-to-cell communication and gene expression profiles that support a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment