A randomized, non-inferiority trial on the DuoStim strategy in PGT-A cycles.

RESEARCH QUESTION: Is the DuoStim strategy an effective alternative to two conventional ovarian stimulation cycles in poor-prognosis patients undergoing preimplantation genetic testing for aneuploidies (PGT-A) to improve euploidy rates and obtain the first euploid embryo in less time? DESIGN: This randomized controlled trial was performed at IVI Madrid between June 2017 and December 2020 and included 80 patients with a suboptimal profile aged 38 or older undergoing PGT-A cycles. Patients were blindly randomized into two groups: 39 women underwent two ovarian stimulations in consecutive cycles (control group), whereas the double stimulation strategy was applied to 41 women (DuoStim group). The main outcome was the euploidy rate in each group. The secondary outcomes were the time it took to obtain a euploid embryo and the main cycle outcomes. RESULTS: The baseline characteristics of the patients were similar. No differences were found between the control group and the DuoStim group in the mean days of stimulation (21.3 ± 1.6 versus 23.0 ± 1.4, P = 0.10), total gonadotrophins (4005 ± 450 versus 4245 ± 430, P = 0.43), metaphase II oocytes (8.7 ± 1.8 versus 6.8 ± 1.7, P = 0.15) or euploid embryos obtained (0.8 ± 0.4 versus 0.6 ± 0.4, P = 0.45). The euploid rate per randomized patient (ITT) was 16.1% in the control group versus 22.7% in the DuoStim group, with P-values of 0.371, and the euploidy rate per patient treated was 39.0% versus 45.7% in the control versus DuoStim groups. However, there was a significant difference in the average number of days it took to obtain a euploid blastocyst, favouring the DuoStim group (44.1 ± 2.0 versus 23.3 ± 2.8, P < 0.001). CONCLUSIONS: The use of the DuoStim strategy in poor-prognosis patients undergoing PGT-A cycles maintains a similar euploidy rate while reducing the time required to obtain a euploid blastocyst.

A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs.

Accurate meiotic chromosome segregation critically depends on the formation of inter-homolog crossovers initiated by double-strand breaks (DSBs). Inaccuracies in this process can drive aneuploidy and developmental defects, but how meiotic cells are protected from unscheduled DNA breaks remains unexplored. Here we define a checkpoint response to persistent meiotic DSBs in C. elegans that phosphorylates the synaptonemal complex (SC) to switch repair partner from the homolog to the sister chromatid. A key target of this response is the core SC component SYP-1, which is phosphorylated in response to ionizing radiation (IR) or unrepaired meiotic DSBs. Failure to phosphorylate (syp-16A) or dephosphorylate (syp-16D) SYP-1 in response to DNA damage results in chromosome non-dysjunction, hyper-sensitivity to IR-induced DSBs, and synthetic lethality with loss of brc-1BRCA1. Since BRC-1 is required for inter-sister repair, these observations reveal that checkpoint-dependent SYP-1 phosphorylation safeguards the germline against persistent meiotic DSBs by channelling repair to the sister chromatid.

Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae. New insights into transcription-associated recombination.

Homologous recombination of a particular DNA sequence is strongly stimulated by transcription, a phenomenon observed from bacteria to mammals, which we refer to as transcription-associated recombination (TAR). TAR might be an accidental feature of DNA chemistry with important consequences for genetic stability. However, it is also essential for developmentally regulated processes such as class switching of immunoglobulin genes. Consequently, it is likely that TAR embraces more than one mechanism. In this study we tested the possibility that transcription induces recombination by making DNA more susceptible to recombinogenic DNA damage. Using different plasmid-chromosome and direct-repeat recombination constructs in which transcription is driven from either the P(GAL1)- or the P(tet)-regulated promoters, we have shown that either 4-nitroquinoline-N-oxide (4-NQO) or methyl methanesulfonate (MMS) produces a synergistic increase of recombination when combined with transcription. 4-NQO and MMS stimulated recombination of a transcriptionally active DNA sequence up to 12,800- and 130-fold above the spontaneous levels observed in the absence of transcription, whereas 4-NQO and MMS alone increased recombination 193- and 4.5-fold, respectively. Our results provide evidence that TAR is due, at least in part, to the ability of transcription to enhance the accessibility of DNA to exogenous chemicals and internal metabolites responsible for recombinogenic lesions. We discuss possible parallelisms between the mechanisms of induction of recombination and mutation by transcription.

Hpr1 is preferentially required for transcription of either long or G+C-rich DNA sequences in Saccharomyces cerevisiae.

Hpr1 forms, together with Tho2, Mft1, and Thp2, the THO complex, which controls transcription elongation and genome stability in Saccharomyces cerevisiae. Mutations in genes encoding the THO complex confer strong transcription-impairment and hyperrecombination phenotypes in the bacterial lacZ gene. In this work we demonstrate that Hpr1 is a factor required for transcription of long as well as G+C-rich DNA sequences. Using different lacZ segments fused to the GAL1 promoter, we show that the negative effect of lacZ sequences on transcription depends on their distance from the promoter. In parallel, we show that transcription of either a long LYS2 fragment or the S. cerevisiae YAT1 G+C-rich open reading frame fused to the GAL1 promoter is severely impaired in hpr1 mutants, whereas transcription of LAC4, the Kluyveromyces lactis ortholog of lacZ but with a lower G+C content, is only slightly affected. The hyperrecombination behavior of the DNA sequences studied is consistent with the transcriptional defects observed in hpr1 cells. These results indicate that both length and G+C content are important elements influencing transcription in vivo. We discuss their relevance for the understanding of the functional role of Hpr1 and, by extension, the THO complex.