Evaluation Study Between Density Gradient and Swimming Down for Semen Processing
Condition: Infertility
MeSH terms: Infertility
Study type: Observational

Summary: 
      This study evaluates the quality of sperm produced after semen processing using Density
      Gradient (DG) and swimming down (SD).
    

Detailed description: 
      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.
    

Intervention name(s): Diff-Quik staining for morphology evaluation; Motility Evaluation for grade A and B; Concentration (mil/ml); 24 H half life; 48 H half life; HBA Testing; Bacterial contamination
Intervention type(s): Procedure; Procedure; Procedure; Procedure; Procedure; Procedure; Biological
Intervention description(s): 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.; 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.
Other intervention name(s): Abnormal forms %; Motility %; Concentration for final output; Motility after 24 hours incubation at 37 Dc.; Motility evaluation after 48hours incubation at 37 Dc.; Sperm physioogical activity evaluation HA binding; Bacterial contamination presence of post processinf samples
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Cases arranged for study were submitted for routine semen analysis. Patient agreements
        collected as consent form signed during sample submission.
      
Biospecimen description: 
      Semen samples
    
Keywords: IUI/AIH; Semen preparation; Semen processing; ICSI/IVF; ART; Density Gradient; Swimming down; hyaluronan affinity testing; Sperm vitality testin