This study evaluates the quality of sperm produced after semen processing using Density Gradient (DG) and swimming down (SD).
Semen processing to be used for different ART procedures is essential step to isolate dead sperms as well as those abnormally structural or morphology. Semen preparation will maintain to remove the seminal fluids and present cells and contamination pus cells or bacterial cells. Among different protocols, Density Gradient and swim-up are the most common. For poor motility and number sample Density Gradient is indicated. While final evaluation for produced or separated sperm form processing procedures was not evaluated yet. Preparation for evaluation study to compare output of main used protocol Density Gradient (DG) with other protocol as swimming down using different study criteria can make more clear evaluation for better processing procedures. The cases arranged for evaluation were semen samples are within WHO 2010 normal values for motility and count to be more than 5 million/ml. All samples to be divided to two portions for processing by the two preparation techniques. Post preparation evaluation to be done for the two outputs using: Motility monitoring for 48 hours at 37.0 Dc, abnormal forms, hyaluronan affinity testing and contaminating bacterial growth. In this study we will show drawback effect of physical and time stress interface applied on sperms during processing, and compare the out put results with that results produced from sperm processed without centrifugation interface or more processing time. As the sperm will separate it self by its natural movement and normal behavior. Evaluation will be on three categories: 1. The final output quality: as net highly progressive sperms and their count number ( sperm morphology, count and motility): as our results showed more phonologically normal sperm retrieved from SD method with higher count and more percentage of progressively motile sperms. 2. The quality of sperm produced: through monitoring sperm vitality testing or sperm motility power among 2 days at 37.0 Ds. Motility will be evaluated after 24 hours and 48 hours. Both readings showed more decrease in sperm motility and vitality with time consuming with portions processed using density gradient. While portions processed using SD showed significant better readings. Another functional activity of sperm post processing is using HBA testing slide, as we evaluate the sperm affinity to hyaluronan. This Testing or assay indicate the physiological activity of sperm still maintained. While it showed HBA higher significant results with swimming down group study. 3. Final evaluation, that are used to present the quality of separation: is monitored using the contamination and carry over effect during semen manipulation and processing. Bacterial contamination using routine culture media (Blood agar) to evaluate 5 microns from each portions for presence and absence of any bacterial cells or during processing and manipulation.
Study Type
OBSERVATIONAL
Enrollment
100
Papanicolaou stain (haematoxylin, orange G6, and EA50) gives a clear difference between basophilic and acidophilic cell constituents and thereby enables a detailed examination of chromatin pattern, which is useful in the evaluation of sperm morphology and assessment of presence of immature spermatozoa. Cytoplasmic staining can vary between red and green dependent on ionic strength, pH, and composition of the cell department and the stain (OG6 and EA50). Morphological assessment for processed portions of semen. As separation process has to minimize the morphological abnormal sperm.
The better separation method will insure more progressive sperms after processing. Grading using A and B scale scored by (WHO 2010) references.
Al Baraka Fertility Hospital
Manama, Adliyah, Bahrain
Ibn Sina IVF Center- Ibn Sina Hospital
Sohag, Sohag Governorate, Egypt
HBA
Affinity is measured according to percentage of non motile sperm produced from 10 µL of processed sample.
Time frame: 1 day
Abnormal forms
Percentage normally stained sperms after processing
Time frame: 1 day
Final concentration
Concentration was fluctuation according to original motility and concentration, DG
Time frame: 1 day
24 Hour motility
Motility evaluation after 37.0 Dc incubation
Time frame: 1 day
48 Hour motility
Motility evaluation after 37.0 Dc incubation
Time frame: 2 days
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count of isolated sperms Using Makler chamber for count of sperm for each portion post processing.
percentage of active motile to non motile sperms. Microscopic examination for each sample after 24 hours incubation, to evaluate the progressively motile sperm and those non progressive or non motile.
percentage of active motile to non motile sperms. Microscopic examination for each sample after 48 hours incubation, to evaluate the progressively motile sperm and those non progressive or non motile.
The Hyaluronic Binding Assay (HBA®) is an important diagnostic tool for suspected male infertility in the analysis of semen. In a matter of minutes the HBA® slide provides an answer to the proportion of mature binding spermatozoa in the sample (The HBA® score %). By using HBA slide we can count the physiologically active sperms at each sample and calculate percentage of active sperms with reference to post processing count from 3rd investigating arm.
Routine bacterial culture using Blood Agar culture plates for 10 micron finally washed samples, to confirm presence of any bacteria contamination caused by carry over during processing.