A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites (TFBSs) in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32%. Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http://bfgl.anri.barc.usda.gov/tfbsConsSites.

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites (TFBSs) in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32%. Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http://bfgl.anri.barc.usda.gov/tfbsConsSites.
Algorithms ; Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; genetics ; Cell Line, Tumor ; Computational Biology ; methods ; Conserved Sequence ; Electrophoretic Mobility Shift Assay ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Luciferases ; genetics ; metabolism ; Mice ; Normal Distribution ; Oligonucleotides ; genetics ; metabolism ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Rats ; Recombinant Fusion Proteins ; genetics ; metabolism ; Regulatory Sequences, Nucleic Acid ; genetics ; Reproducibility of Results ; Sp1 Transcription Factor ; genetics ; metabolism ; Transcription Factors ; metabolism ; Transfection

Many azoospermic men do not possess mature spermatozoa at the time of surgical sperm extraction. This study is a systematic review and meta-analysis evaluating outcomes following round spermatid injection (ROSI), a technique which utilizes immature precursors of spermatozoa for fertilization. An electronic search was performed to identify relevant articles published through October 2018. Human cohort studies in English involving male patients who had round spermatids identified and used for fertilization with human oocytes were included. Fertilization rate, pregnancy rate, and resultant delivery rate were assessed following ROSI. Meta-analysis outcomes were analyzed using a random-effects model. Data were extracted from 22 studies involving 1099 couples and 4218 embryo transfers. The fertilization rate after ROSI was 38.7% (95% confidence interval [CI]: 31.5%-46.3%), while the pregnancy rate was 3.7% (95% CI: 3.2%-4.4%). The resultant delivery rate was low, with 4.3% of embryo transfers resulting in a delivery (95% CI: 2.3%-7.7%). The pregnancy rate per couple was 13.4% (95% CI: 6.8%-19.1%) and the resultant delivery rate per couple was 8.1% (95% CI: 6.1%-14.4%). ROSI has resulted in clinical pregnancies and live births, but success rates are considerably lower than those achieved with mature spermatozoa. While this technique may be a feasible alternative for men with azoospermia who decline other options, couples should be aware that the odds of a successful delivery are greatly diminished and the prognosis is relatively poor.