miRNAs Associated With Undescended and Retractile Testes
Conditions: MIRAS, Testicular Abnominalies, Undescended Testes
Sponsor: Dr. Mevlüt Keleş

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Could miRNAs be used as markers for distinguishing 
undescended testicles from retractile testicles
No NCT number assigned yet
31.03.2022
1. Study protochol

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1.1.Sample size
This study is designed as a prospective controlled trial to compare miRNA
changes across UDT, RT, and normal control groups. The sample size was
determined via G*Power 3.1.9.6. A one-way ANOVA power analysis for
miR-34c, which compared three groups, utilized an effect size (f=1.2955357)
from a previous study [10]. To achieve 99% power and a 1% error rate, a
total sample size of 24 was needed. To increase confidence, the study
ultimately enrolled 30 patients, who were divided into three groups of 10.
1.2.Patient population
Initially, 10 boys with undescended testicles (UDTs), 10 with retractile
testicles (RTs), and 10 healthy volunteers (controls) from our urology clinic
(dates between March 2022 and March 2023) were enrolled. However, one
RT patient and one control patient were excluded because of parental refusal
for blood sampling. Only palpable (unilateral or bilateral) UDT cases were
included, excluding nonpalpable types. To ensure accurate RT diagnosis,
initial physician examinations were performed in three positions (supine,
semisupine, standing), followed by a 1-month parental examination (twice
daily). Only RT patients whose testicles spent >50% of their time in the
scrotum
were
included.
The
exclusion
criteria
also
included
prior
inguinal/scrotal surgery, defective datasets, or unsuitable serum samples.
1.3.Collection of patient data and control samples
Detailed patient histories, physical examinations, and routine biochemical
tests were performed. UDT blood samples were collected presurgery to
prevent misinterpretation. Blood from all the groups was collected in 5 mL
biochemistry tubes and centrifuged at 3000 rpm for 10 minutes. The resulting

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particle-free serum was transferred to 1.5 mL tubes and stored at -80°C until
analysis.
1.4.Validation of the expression levels of miR-210, miR-449a and miR-
34c via real-time PCR
Serum samples from the urology clinic were subjected to RNA isolation via
the Quick-cfRNA™Serum & Plasma Kit (Zymo Research). The extracted
miRNA was reverse transcribed into cDNA via the miRNA All-In-One
cDNA Synthesis Kit (ABMGood). cDNA quality and quantity were assessed
spectrophotometrically with a BioSpec-nano instrument (Shimadzu) before
real-time PCR. miRNA expression analysis was performed via a Rotor Gene
Q (Qiagen) instrument. The levels of miR-210, miR-449a, and miR-34c were
quantified with ready-to-use primers and BlasTaq™2X qPCR MasterMix
(ABMGood).
1.5.Statistical analysis
Data normality was assessed via the Shapiro‒Wilk test, and variance equality
was assessed via Levene's test. Descriptive statistics are presented as the
means ± SDs for parametric data and medians (IQRs) and means (ranks) for
nonparametric data. For group comparisons, one-way ANOVA was used for
parametric data, and the Kruskal‒Wallis test was used for nonparametric
data, followed by the Dunn‒Bonferroni correction for post hoc multiple
comparisons. Spearman's rank correlation coefficient was used to analyse
correlations. Statistical significance was set at p < 0.05.
miRNA expression was quantified via qRT‒PCR via Ct (crossing point)
values and normalized to RNU6B_13. Relative miRNA expression levels
were compared among all groups via the 2-ΔCt method and calculated as 2Ct

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(target gene) −Ct (reference gene). All the statistical analyses were performed via IBM
SPSS v28 (IBM Inc., Chicago, IL, USA).