Dependency of mitochondrial quantity on blastocyst timeline obscures its actual effect to pregnancy outcomes.

Objectives: To explore the correlation between mitochondrial quantity and the blastocyst development timeline as well as their respective contributions to early pregnancy. Methods: A retrospective study was conducted using a dataset comprising 2,633 embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2023. The study was divided into three subsets to address distinct aspects: the representativeness of a single trophectoderm (TE) biopsy for mitochondrial quantity (n=43), the correlation between morphokinetic features and mitochondrial quantity (n=307), and the association analysis among mitochondrial quantity, blastocyst timeline factor, and reproductive outcomes (n=2,283). Distribution assessment of mitochondrial quantity across an individual blastocyst involved the identification within multiple biopsies and spent culture media. Timeline evaluation included correlating mitochondrial quantity with time-lapse datasets. Finally, multivariate logistic regression models, incorporating potential effectors alongside mitochondrial quantity, were employed to analyze their respective contributions to early pregnancy endpoints. Results: Of distribution assessment, mitochondrial quantity exhibited an even distribution across the entire trophectoderm (Spearman's ρ=0.82), while no detectable mtDNAs in the corresponding spent culture media. Then the timeline correlation study revealed significant association between mitochondrial quantity and blastocyst features of both the day of expanded blastocyst formation (95% Confidence intervals, CIs: 0.27~4.89, p=0.03) and the timing of expanded blastocyst formation (tEB) (95% CIs: -0.24~-0.01, p=0.04) in the regression model, indicating a strong dependency between mitochondrial quantity and the blastocyst development timeline. For the contribution to early pregnancy, multivariate logistic regression models showed that the day of expanded blastocyst formation contributed to four endpoints persistently: positive for HCG (odd ratio, OR: 0.71, p=0.006), gestational sac (OR: 0.78, p=0.04), fetal heartbeat (OR: 0.71, p=0.004), and progression to 14 weeks (OR: 0.69, p=0.002). Contrastingly, no notable correlation was observed between the mitochondrial quantity and these endpoints. Conclusions: Strong interaction was observed between mitochondrial quantity and the blastocyst timeline, particularly the timing of expanded blastocyst formation. It suggests that the primary determinant influencing pregnancy outcomes lies in the time-dependent parameter of blastocyst rather than in the specific mitochondrial quantity.

Reduced mitochondrial DNA content correlate with poor clinical outcomes in cryotransfers with day 6 single euploid embryos.

Objective: To investigate whether the mitochondrial DNA (mtDNA) content of a single biopsy at trophoblast correlates with the developmental potential and reproductive outcomes of blastocyst. Methods: A retrospective analysis applied the dataset of 1,675 embryos with preimplantation genetic testing for aneuploidy (PGT-A) from 1,305 individuals, and 1,383 embryos involved cryotransfers of single euploid embryo between January 2015 and December 2019. The studied cohort was divided for algorithm establishment on the NGS platform (n=40), correlation of biological features (n=1,635), and correlation of reproductive outcomes (n=1,340). Of the algorithm derived from the NGS platform, the reliability and repeatability were validated via qPCR assay and inter-run controls, respectively. Of the correlation across biological features, stratification analyses were applied to evaluate the effect from a single contributor. Eventually, the correlation between the mtDNA ratios and reproductive outcomes was adjusted according to the significant effector(s). Results: The mtDNA ratios showed statistically different between embryos with different days of blastocyst formation ([Day 5]: 1.06 vs. [Day 6]: 0.66, p=0.021), and between embryos with different expansion stages ([Expansion 5]: 1.05 vs. [Expansion 6]: 0.49, p=0.012). None or weakly correlated with the maternal age, morphology, ploidy, and gender. Analyzed by the different days of blastocyst formation with fixed expansion score as 5 in the euploid single embryo transfers (eSET), the day 6 eSET showed significantly lower reduced mtDNA ratio (n=139) in failure groups of fetal heartbeat (p=0.004), ongoing pregnancy (p=0.007), and live birth (p=0.01); however, no correlation between mtDNA ratios and pregnancy outcomes was observed in the day 5 eSET (n=1,201). Conclusions: The study first demonstrated that mtDNA ratio was dependent on the days of blastocyst formation while expansion stage was fixed. Lower mtDNA ratios were observed in the day 6 eSET with adverse outcomes. The present stratification analyses reveal that the timeline of embryo is an important covariate to the mtDNA content.

Mycobacterial species and their contribution to cholesterol degradation in wastewater treatment plants.

Mycobacterium often presents as an abundant bacterial genus in activated sludge in many wastewater treatment plants (WWTPs), but the species-level taxonomy and functions remain poorly understood. In this study, we profiled the mycobacterial communities in eleven WWTPs from five countries by pyrosequencing the rpoB amplicons and searching against a customized database of mycobacterial rpoB sequences. Results indicated that major mycobacterial species were related to M. brumae, M. crocinum, M. sphagni, etc., most of which belong to poorly characterized rapidly-growing group. A few opportunistic pathogenic species were detected, suggesting the potential risk of mycobacteria in WWTPs. Genomic analysis of four isolates from activated sludge indicated these genomes contained genes of degradations of alkane, aromatics, steroids and a variety of cytochrome P450 families. Additionally, a few key genes responsible for cholesterol degradation were detected in a full-scale activated sludge metatranscriptomic dataset reported previously and taxonomically assigned to mycobacteria. Evidence showed that all isolates can degrade cholesterol, a major composition of sewage. Relative abundance of mycobacteria in activated sludge was enriched by 4.7 folds after adding cholesterol into the influent for one week. Our results provided the insights into mycobacterial species and functions in WWTPs.