In and registered in Iran Clinical Trials registry

In thisrandomized control trial study, 60 men referred to the andrology lab at Yazd clinical research participatedprospectively. This investigation wasconducted from Jan 2015 to May 2017 andregistered in Iran Clinical Trials registry with ID number IRCT201507285261N2.

The infertile men whodefined as oligoasthenoteratozoospermia (OAT) (n=30) according to WHO 2010criteria (17) and fertile men with normal semen parameters as the controlgroup (n=30) were included.   These patientswere randomly divided into two groups 15 OAT infertile men undergoing Three-drug treatment (Vit E 400 mg, selenium200 ?g and folic acid 5 mg daily) were administered and 15 OAT infertile men undergoing two-drug treatment (taking Vitamin E 400 mg andselenium 200 ?g daily. The prescriptionwas done for three months by the urologist. Patients inclusioncriteria were age 25-40, no use of phenytoin andPhenobarbital, sperm concentration 7-14 million/ml, total motility <40% andnormal morphology <4%. Presenceof known genetic disorders like cryptorchidism, chromosome Y deletion, men with a history ofvaricocele, unexplained infertility were exclusion criteria. This study was approved by the ethics committee of theYazd Research and Clinical Center for Infertility and informed consent formswere signed by all participants.  2.

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2. Sperm parametersSemensamples were collected by masturbation after 2-5 days of sexual abstinence.Each semen sample was allowed to undergo liquefaction, and then was evaluatedfor sperm motility, concentration, and morphology according to World Health Organization(WHO, 2010) criteria. For evaluating  morphological abnormalities and at least 200sperm were examined per slide (10). Sperm concentrationwas assessed by Makler counting chamber. For evaluating the sperm viabilityeosin- nigrosin staining technique was carried out. The amount of 10 ?l ofprepared sperm was mixed with 10 ?l of eosin- nigrosin stain on a glass slideand assayed using a light microscope to determine the percentage of live sperm.

At least 200 spermatozoa were assessed for each case. White or unstained spermswere classified as live and pink or red sperms were considered dead. Allanalyses were done by one experienced laboratory technician blinded to thestudy2.3.Sperm DNA integrity tests:SpermDNA integrity assessments were applied using four tests including terminaldeoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay forsperm apoptosis detection.

Aniline blue staining for detection of excessive histones in sperm chromatin andToluidine blue for the sperm chromatin condensation status (11) .2.3.1.TUNEL assayThe percentage of apoptotic spermatozoa in eachsample was determined by TUNEL assay by In Situ Cell Death Detection Kit (RocheDiagnostics GmbH, Mannheim, Germany) using fluorescent microscopy that normalDNA was detected Light green and damaged DNA was seen Bright green (18).