Identification of candidate microRNA markers of endometriosis with the use of next-generation sequencing and quantitative real-time polymerase chain reaction

Abstract

Objective: To identify novel candidate diagnostic microRNA (miRNA) markers of endometriosis by means of an unbiased search with confirmation by means of targeted polymerase chain reaction (PCR). Design: Retrospective cohort. Setting: University teaching hospitals. Patient(s): Women with endometriosis and control women, confirmed with the use of laparoscopy. Interventions(s): Diagnostic laparoscopy and blood sample. Main Outcome Measure(s): Next-generation sequencing (NGS) and quantitative real-time PCR (qRT-PCR).Result(s): Candidate miRNAs differentially expressed in women with endometriosis compared with control women were identified by means of NGS and selected for qRT-PCR. Plasma samples from another cohort of women with surgically confirmed endometriosis (n ¼ 53) and disease-free control women (n ¼ 53) were checked for hemolysis using spectrophotometry and the ratio of miR-23a and miR-451 by means of qRT-PCR. MicroRNA signatures were quantified by means of qRT-PCR in hemolysis-free plasma samples of case subjects (n ¼ 25) and control subjects (n ¼ 28) with the use of miRcury LNA miRNA. Circulating levels of eight miRNAs (miR-199a-3p, miR-143-3p, miR-340-5p, let-7b-5p, miR-21-5p, miR-17-5p, miR-20a-5p, and miR-103a-3p) were significantly lower in case subjects compared to control subjects. The sensitivity and specificity for individual miRNAs ranged from 0.36 to 1.00 and from 0.43 to 1.00, respectively, but when combined produced sensitivity and specificity of 0.92 and 0.86 with positive (PPV) and (NPV) predictive values of 0.85 and 0.92, respectively. However, combination of five miRNAs (miR-17-5p, miR-20a-5p, miR-199a-3p, miR-143-3p, and let-7b-5p) produced sensitivity and specificity of 0.96 and 0.79 with PPV and NPV of 0.80 and 0.96, respectively. Conclusion(s):We conclude that a panel of candidate miRNAs was comparable to laparoscopy in distinguishing between women with endometriosis and control women. (Fertil Steril Ò 2020;113:1232-41. Ó2020 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo.

Characterization of micro-RNA in women with different ovarian reserve

“…observed an up-regulation of miR-21-5p in endometrial cells 38 , while Papari et al . found a reduced expression level of miR-21-5p in the plasma of women with endometriosis 39 . A physiological reduction in miR-21-5p was observed in human endometrial stromal cells (hESC) in preparation for pregnancy (decidualization) 40 .…”

Биомаркёры Эндометриоза: Что Мы Знаем О Них Сегодня?

“…Однако данная процедура является инвазивной, что включает в себя определѐнное количество рисков, поэтому в наши дни продолжается активное изучение эндометриоза; его распространѐнность и наличие маркеров к нему. Существует мнение, что из-за отсутствия специфических диагностических малоинвазивных маркеров, дигностика эндометриоза в среднем затягивается до 7 лет [5].…”

Diagnosing endometriosis by measuring plasma micro ribonucleic acids: it may take a miRacle

“…Strengths of the current report include [1] an unbiased discovery phase using NGS, [2] rigorous phenotyping of cases and controls by laparoscopy, [3] careful selection of circulating reference miRNAs (''housekeeping miRNAs'') for which expression is high and remains stable between cases and controls, and [4] elimination of plasma samples with evidence of hemolysis (which is expected to alter the miRNA profile ordinarily present in cell-free plasma). Two experimental methods were used to identify hemolysis (or red blood cell contamination)-spectrophotometry and low expression of the red blood cell-expressed miR-451 relative to the plasma-expressed miR-23a (3).…”

“…Interstudy inconsistencies may be attributed to disparities between study populations (including variable disease severity and diverse criteria for selecting the control groups, as well as diverse genetic backgrounds), variable specimens (plasma vs. serum) and specimen handling, different stages of the menstrual cycle, and variation in plasma miRNA detection platforms (NGS, global qRT-PCR, microarrays). Papari et al (1) suggest that an additional source of experimental variation may relate to suboptimal choice of reference RNAs when normalizing miRNA expression levels by qRT-PCR (there is evidence that the popular reference RNAs RNU6 and miR-16-5p are not stable in human plasma). Although likely to be true for some studies, this reasoning ignores studies that used alternative reference RNAs and implies failure of multiple research groups to properly validate their methodological approaches.…”

“…Whether peripheral miRNAs correlate with severity of disease, or the degree of pelvic pain, similarly remains unknown. We must commend Papari et al (1) for their rigor in phenotyping cases and controls and for excluding samples with evidence of hemolysis. Practical implementation of a peripheral miRNA-based diagnostic test for endometriosis will depend on greater consistency in methods and analysis as well as rigorous replication in validation sets.…”