Francesca Vidal

The purpose of this study is to provide novel information through Next Generation Sequencing (NGS) for the characterization of viral and bacterial RNA cargo of human sperm cells from healthy fertile donors. For this, RNA-seq raw data of poly(A) RNA from 12 sperm samples from fertile donors were aligned to microbiome databases using the GAIA software. Species of viruses and bacteria were quantified in Operational Taxonomic Units (OTU) and filtered by minimal expression level (>1% OTU in at least one sample). Mean expression values (and their standard deviation) of each species were estimated. A Hierarchical Cluster Analysis (HCA) and a Principal Component Analysis (PCA) were performed to detect common microbiome patterns among samples. Sixteen microbiome species, families, domains, and orders surpassed the established expression threshold. Of the 16 categories, nine corresponded to viruses (23.07% OTU) and seven to bacteria (2.77% OTU), among which the Herperviriales order and Escherichia coli were the most abundant, respectively. HCA and PCA displayed four clusters of samples with a differentiated microbiome fingerprint. This work represents a pilot study into the viruses and bacteria that make up the human sperm microbiome. Despite the high variability observed, some patterns of similarity among individuals were identified. Further NGS studies under standardized methodological procedures are necessary to achieve a deep knowledge of the semen microbiome and its implications in male fertility.

In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recombination and segregation. However, when the pairing process begins, it is still under investigation. Contrasting data exists in Mus musculus, since both leptotene DSB-dependent and preleptotene DSB-independent mechanisms have been described. To unravel this contention, we examined homologous pairing in pre-meiotic and meiotic Mus musculus cells using a three-dimensional fluorescence in situ hybridization-based protocol, which enables the analysis of the entire karyotype using DNA painting probes. Our data establishes in an unambiguously manner that 73.83% of homologous chromosomes are already paired at premeiotic stages (spermatogonia-early preleptotene spermatocytes). The percentage of paired homologous chromosomes increases to 84.60% at mid-preleptotene-zygotene stage, reaching 100% at pachytene stage. Importantly, our results demonstrate a high percentage of homologous pairing observed before the onset of meiosis; this pairing does not occur randomly, as the percentage was higher than that observed in somatic cells (19.47%) and between nonhomologous chromosomes (41.1%). Finally, we have also observed that premeiotic homologous pairing is asynchronous and independent of the chromosome size, GC content, or presence of NOR regions.

Study question: Can we accurately assess sex-chromosomes number in testicular cells (germ and somatic) of Klinefelter Syndrome (KS) prepuberal patients by DNA-FISH in paraffin embedded sections? Summary answer: We have evaluated the sex-chromosomes content of testicular cells in KS prepuberal patients and the presence of XY lines evidenced a gonadal mosaicism status. What is known already: Prepuberal boys with Klinefelter Syndrome usually have fertility problems in adulthood, mainly azoospermia. Due to the symptoms cause by the disease, Spermatogonial Stem Cells (SSCs) are less abundant and spermatogenesis does not occur. These patients do not have any alternative to restore the fertility in the future. When a boy has both XY and XXY cell lines, this patient is mosaic (46,XY/47,XXY). Meaning some cells could undergo spermatogenesis and generate gametes with normal sex-chromosomes number. The aim of this work is to test if KS patients diagnosed as pure can have testicular cell lines with XY chromosome number (mosaic). Study design, size, duration: We obtained samples from human prepuberal patients diagnosed with pure Klinefelter Syndrome (47,XXY) for the last 5 years. Testicular biopsy fragments are fixed for histological studies and other fragments are cryopreserved. Some fragments are used for immunofluorescence and subsequently processed by DNA-FISH to determine the sex-chromosomes content of testicular cells. Participants/materials, setting, methods: In this study, we used 10 prepuberal patients with KS and 5 prepuberal patients with other fertility problems with normal chromosome set as controls. We performed immunofluorescence to determine expression of germ cells (VASA) and SSCs (MAGEA4) markers and somatic cells markers such as Leydig cells (StAR) and Sertoli cells (SOX9). Afterwards, we perform DNA-FISH, with probes specific for chromosomes X and Y and chromosome 18 as a control. Main results and the role of chance: The methodology used allows cytogenetic characterization of testicular tissue in paraffin embedded sections. Testicular mosaicism has been observed in all patients diagnosed as pure KS. We have observed a degree of mosaicism of 66-80% in SSCs, of 20-50% in Sertoli cells and of 30-50% in Leydig cells. We pursued a protocol with a good FISH efficiency that allows colocalization of previous immunocharacterized testicular cells. Limitations, reasons for caution: The DNA-FISH technique is made just in a single paraffin section of each KS patient testicular sample. Due to the limited and difficulty to obtain KS testicular tissue, we use a limited number of samples. Wider implications of the findings: We succeed to demonstrate the mosaicism of testicular cells in prepuberal patients that are diagnosed with KS. Thus, the better understanding of the SSCs with normal chromosome set (XY) could be useful for future in vitro expansion and stem cell therapies. Trial registration number: not applicable

Purpose To determine whether embryo mosaicism prevalence in preimplantation genetic testing for aneuploidy (PGT-A) cycles is associated with the trophectoderm biopsy technique used (a. number of laser pulses or b. the use of flicking or pulling) or the time to tubing. Methods Prospective observational study performed in a single IVF-PGT-A setting from May 2019 to May 2021. Trophectoderm biopsies were analysed by next-generation sequencing. Mosaicism was analysed in relation to the biopsy methodology (number of laser pulses and pulling vs flicking), time elapsed from biopsy to tubing (min), and time of sample cryostorage from tubing to amplification (days). As a secondary objective, the number of laser pulses and biopsy methodology were studied in relation to clinical outcomes of transferred euploid blastocysts. Results None of the analysed variables were associated to mosaicism prevalence. Multivariable regression analysis demonstrated that mosaicism prevalence was comparable either when > 3 laser pulses were used as compared to ≤ 3 (13.9% vs 13.8%, aOR = 0.8726 [0.60–1.28]) and pulling compared to flicking (13.1% vs 14.0%, aOR = 0.86 [0.60–1.23]). Moreover, neither the number of laser pulses during biopsy (> 3 vs ≤ 3) nor the technique used (pulling vs flicking) were associated with clinical pregnancy after the transfer of frozen-thawed euploid blastocysts (54.9% vs 55.2%, aOR = 1.05 [0.53–2.09]; 61.1% vs 52.9%, aOR = 1.11 [0.55–2.25], respectively). Conclusion Our results suggest that, as long as the biopsy and tubing procedures are performed following standardized high quality procedures, no specific approach would increase the generation of artefactual mosaicism as a result of trophectoderm biopsy. Trophectoderm biopsies should be performed regardless of the methodology but always aiming on minimising blastocyst manipulation.

Chromosome territoriality is not random along the cell cycle and it is mainly governed by intrinsic chromosome factors and gene expression patterns. Conversely, very few studies have explored the factors that determine chromosome territoriality and its influencing factors during meiosis. In this study, we analysed chromosome positioning in murine spermatogenic cells using three-dimensionally fluorescence in situ hybridization-based methodology, which allows the analysis of the entire karyotype. The main objective of the study was to decipher chromosome positioning in a radial axis (all analysed germ-cell nuclei) and longitudinal axis (only spermatozoa) and to identify the chromosomal factors that regulate such an arrangement. Results demonstrated that the radial positioning of chromosomes during spermatogenesis was cell-type specific and influenced by chromosomal factors associated to gene activity. Chromosomes with specific features that enhance transcription (high GC content, high gene density and high numbers of predicted expressed genes) were preferentially observed in the inner part of the nucleus in virtually all cell types. Moreover, the position of the sex chromosomes was influenced by their transcriptional status, from the periphery of the nucleus when its activity was repressed (pachytene) to a more internal position when it is partially activated (spermatid). At pachytene, chromosome positioning was also influenced by chromosome size due to the bouquet formation. Longitudinal chromosome positioning in the sperm nucleus was not random either, suggesting the importance of ordered longitudinal positioning for the release and activation of the paternal genome after fertilisation.

“Apenas um ateu pode ser um bom cristão [...]”, declara o filósofo Ernst Bloch em seu livro Ateísmo no cristianismo. Como ele chega a uma tal tese? Que representação de messianismo marca a sua filosofia da esperança? O texto mostra como Bloch quer liberar as motivações rebeldes do religioso a fim de que possam ser explicitados os seus conteúdos de esperança. Também deixa claro por que o autor presume que o ateísmo já está ativo no próprio cristianismo.

The production of functional spermatozoa through spermatogenesis requires a spatially and temporally highly regulated gene expression pattern, which in case of alterations, leads to male infertility. Changes of gene expression by chromosome anomalies, gene variants, and epigenetic alterations have been described as the main genetic causes of male infertility. Recent molecular and cytogenetic approaches have revealed that higher order chromosome positioning is essential for basic genome functions, including gene expression. This review addresses this issue by exposing well-founded evidences which support that alterations on the chromosome topology in spermatogenetic cells leads to defective sperm function and could be considered as an additional genetic cause of male infertility.

The transition in biopsy timing from blastomere to trophectoderm biopsy has led to a remarkable decrease in the percentage of undiagnosed blastocysts. However, patients with few or no euploid blastocysts can be affected by this residual percentage of diagnosis failure. The aim of this study is to assess whether blastocyst rebiopsy and revitrification is an efficient and safe procedure to be applied in cases of no results after analysis. Fifty-three patients agreed to the warming of 61 blastocysts to perform a second biopsy and PGT-A by aCGH. Only 75.4% of the blastocysts survived, reexpanded, and could be rebiopsied. After the second biopsy and analysis, 95.6% of the blastocysts were successfully diagnosed with an euploidy rate of 65.9%. Eighteen euploid blastocysts were warmed and transferred to 18 patients with a 100% survival and reexpansion rate. Seven clinical pregnancies have been achieved with 4 live births, 1 ongoing pregnancy, and 2 miscarriages. Thus, although few transfers of rebiopsied and revitrified blastocysts have been performed till date, our preliminary results show that this approach is efficient and safe to be applied for undiagnosed blastocysts, as it ultimately allows the transfer of euploid blastocysts and good clinical outcomes.

Summary: Shortly after the implementation of comprehensive chromosome screening (CCS) techniques for preimplantation genetic testing for aneuploidies (PGT-A), the discussion about the transition from day 3 to blastocyst stage biopsy was initiated. Trophectoderm biopsy with CCS is meant to overcome the limitations of cleavage-stage biopsy and single-cell analysis. The aim of this study was to assess the results obtained in our PGT-A programme after the implementation of this new strategy. Comparisons between the results obtained in 179 PGT-A cycles with day 3 biopsy (D+3) and fresh embryo transfer, and 204 cycles with trophectoderm biopsy and deferred (frozen–thawed) embryo transfer were established. Fewer embryos were biopsied and a higher euploidy rate was observed in the trophectoderm biopsy group. No differences in implantation (50.3% vs. 61.4%) and clinical pregnancy rate per transfer (56.1% vs. 65.3%) were found. Although the mean number of euploid embryos per cycle did not differ between groups (1.5 ± 1.7 vs. 1.7 ± 1.8), the final number of euploid blastocysts available for transfer per cycle was significantly higher in the trophectoderm biopsy group (1.1 ± 1.3 vs. 1.7 ± 1.8). This factor led to an increased cumulative live birth rate in this last group (34.1% vs. 44.6%). Although both strategies can offer good results, trophectoderm biopsy offers a more robust diagnosis and the intervention is less harmful for the embryos so more euploid blastocysts are finally available for transfer and/or vitrification.