Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT05383599 |
| Other study ID # |
22114CROSP_IM |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
July 16, 2022 |
| Est. completion date |
August 1, 2023 |
Study information
| Verified date |
February 2023 |
| Source |
Universitair Ziekenhuis Brussel |
| Contact |
ileana mateizel |
| Phone |
003224776690 |
| Email |
ileana.mateizel[@]uzbrussel.be |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
The present study aims to determine which of the sperm selection techniques used in IVF (In
Vitro Fertilization) laboratory leads to selection of the best/healthiest sperm population
based on their morpho-functional parameters. Different procedures are currently used in our
laboratory to select the sperm population that will be used to inseminate the oocytes:
Density Gradient Centrifugation (DGC), DGC in combination with Magnetic Activated Sperm
Sorting (MACS), and Microfluidic Sperm Sorting (MSS). The sperm cells selected by these
techniques will be evaluated for specific morpho-functional features such as, motility,
morphological abnormalities, and apoptotic status [phosphatidylserine translocation (early
stages of apoptosis)] and DNA (Deoxyribonucleic Acid) fragmentation as an indicator of stages
of apoptosis.
Description:
Different procedures are currently available to select the sperm population that will be used
to inseminate the oocytes during Medical Assisted Reproduction (MAR). The selected population
should contain fully functional spermatozoa that possess the ability to reach the
fertilization site and allow optimal fertilization and embryo development. These abilities
are strictly dependent on specific sperm morpho-functional features such as, but not limited
to, motility, morphological abnormalities, apoptotic status [phosphatidylserine translocation
(early stages of apoptosis)] and DNA fragmentation (later stages of apoptosis), The present
study aims to determine which of the sperm selection techniques used in our IVF laboratory
lead to selection of the best/healthy sperm population based on their morpho-functional
parameters. For this purpose, unused raw semen after routine diagnostic analysis will be
split in 3 fractions and will be processed immediately by different sperm preparation
methods: 1) DGC, 2) DGC and MACS, and 3) MSS.
The sperm cells selected by each method will be analyzed for morphological and functional
parameters.
Initial sperm concentration (×106/ml) will be assessed using a counting chamber.
Sperm motility will be evaluated on 200 spermatozoa under 40× magnifications and the results
will be expressed as percentages.
After each selection procedure, concentration and motility will be determined with the use of
a Neubauer counting chamber.
Spermatozoa with normal morphology will be checked on smears stained using Diff Quick
Staining on dried smears, 200 spermatozoa will be classified as normal/abnormal according to
Kruger strict criteria.
Phosphatidylserine (PS) translocation (early apoptotic marker): will be analyzed using
fluorescence microscopy following sperm cell suspension incubation with Annexin V-FITC
(Fluorescein Isothiocyanate) and propidium iodide (PI). The spermatozoa will be classified
as: viable spermatozoa without PS translocation (PI negative/Annexin negative); viable with
translocated PS (PI negative/Annexin positive); dead (PI positive/Annexin positive or PI
positive/Annexin negative).
DNA fragmentation index (percentage of sperm cells showing DNA fragmentation per number of
cells analyzed; DFI) will be analyzed using terminal deoxynucleotidyl-transferase-mediated
deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL) assay protocol (commercially
available kit "In Situ Cell Death Detection"; Roche Diagnostic). The cleavage of genomic DNA
during apoptosis leads to both single-strand breaks (nicks) and double-stranded,
low-molecular-weight DNA fragments. These DNA strand breaks can be identified in an enzymatic
reaction in two stages: (1) labeling of DNA strand breaks with TdT (Terminal Deoxynucleotidyl
Transferase), and (2) incorporation of Fluorescein isothiocyanate (FITC)-dUTP (deoxyuridine
triphosphate) into nucleotide polymers. TUNEL-positive sperm stain green and TUNEL-negative
samples stain red and it can be directly detected and quantified by fluorescence microscopy.
This kit is designed to be a precise, fast, and simple nonradioactive technique to detect and
quantify the number apoptotic cells. It is specific as it labels DNA strand breaks generated
during apoptosis, which enables the test to discriminate between apoptotic and necrotic
cells.
Descriptive statistics will be used to report mean, median and minimum and maxi-mum values
for each variable. Exact test for Friedman's test will be used to investigate the presence of
differences across the 3 techniques. Exact test for Wilcoxon signed rank test will be used
for pairwise comparisons. A Bonferroni correction will be applied to adjust for multiple
comparisons. Level of significance is set at p<0.05, 2-tailed. Statistical analysis will be
performed by means of STATA 15.1 and SPSS 26.0 (Statistical Package for the Social Sciences).
A number of 50 patients will be included in the study.
The results obtained will help to improve the sperm selection process in the IVF laboratory
