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Standardizing RNA quality is key to interpreting RNA-seq data as a compromised sample can mask the underlying biology. The challenge remains when evaluating RNA quality in samples with high RNA fragmentation. For example, programmed fragmentation and cytoplasmic expulsion, integral to sperm maturation, is a prime example of the complexities of interpreting RNA-seq data, given that fragmentation can be random and\or targeted. To meet this challenge, we developed an algorithm that accurately measures RNA quality in samples with high fragmentation, such as spermatozoa. The integrity of 1,000 previously identified abundant sperm transcripts were independently visualized and evaluated using the Transcript Integrity Index (TII) algorithm to identify intact transcripts. Full-length transcripts from visual and the TII algorithm were evaluated for testis preference in humans using the GTEx tissues database. Samples were then filtered by the Interquartile Range (IQR), identifying those in which the greatest number of transcripts failed to pass the visual or TII thresholds. Transcript lists were overlapped, forming the set of intact transcripts used as TII standards. Each sample was re-evaluated as a function of this TII set of intact transcripts, with poor quality samples identified as those failing in the largest number of transcripts. While ontologically enriched in roles related to spermatogenesis and/or fertilization, samples did not segregate based on birth outcome. The TII algorithm proved an effective means to identify samples of similar quality from sperm, a cell type enriched in biologically fragmented RNAs. The algorithm should facilitate other studies using samples compromised by high levels of RNA fragmentation, such as Formalin-Fixed Paraffin-Embedded samples. Requisite to assessing male health, TII provides a solution to the long-sought-after standard that identifies samples of similar quality. [ABSTRACT FROM AUTHOR]

Abstract Male fertility and spermatogenesis are directly linked to the Sertoli cell's ability to produce factors associated with germ cell development. Sertoli cells express receptors for FSH and testosterone, and are the major regulators of spermatogenesis. Recent studies report that regulatory RNA molecules, such as microRNAs (miRNAs), are able to modulate testicular function during spermatogenesis and that their altered expression may be involved in male infertility. miRNAs may play a role in the response to xenobiotics that have an adverse consequences to health. An important group of xenobiotic organic compounds with toxic potential are dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experimental models of TCDD exposure in mice demonstrated that TCDD exposure causes low sperm count and delayed puberty. This study below examines the mechanism of TCDD's action in human Sertoli cells, through interrogating the expression profile of miRNAs and mRNAs, that enabled us to identify dysregulated molecular pathawys in Sertoli cell. 78 miRNAs presented altered expression, with positive regulation of 73 and negative regulation of 5 miRNAs when compared to the control group. Regarding gene expression profile, 51 genes were deregulated, of which 46 had positive regulation and 5 genes with negative regulation. Important pathways have been altered by the action of TCDD as AhR pathway, GPR68, FGF2 and LIF. This study has opened the door to new perspectives on the TCDD toxicity pathway as it affects Sertoli cells physiology that can ultimately lead to male infertility. [ABSTRACT FROM AUTHOR]