HEPES-IVG Polymer: A Novel Biorelevant Media for use in Sperm migration/penetration Assays.
Description
Sperm migration distance in relation to time. Effect of capillary size on sperm penetration distance in simulated cervical mucus. Effect of freezing on sperm penetration distance in simulated cervical mucus samples. Pictures of simulated cervical mucus samples. Pictures of capillaries. Micrographs showing sperm motility
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Sperm motility assay This was conducted at the fertility clinic from where semen samples were acquired. To examine the sperm motility, 0.1 mL of the sample was incorporated into each preparation of synthetic cervical mucus and viewed on a microscope slide using a magnification of ×400. The observed motility level was then classified into four types identified as rapid progressive, slow progressive, non-progressive, and immotile. This served as a preliminary test to the penetration assay to confirm the quality of sperm sample and level of motility in each sample of cervical mucus (Ivic, 2002). Penetration assay The Kremer test (swim-up sperm count method) was used to conduct the penetration assay as described by Carlborg (Hwang & Lamb, 2013). The synthesized cervical mucus was introduced into long flattened capillary tubes (Dimension – 0.30 × 3.0 mm, 0.300 mm wall thickness) which were thereafter sealed at one end with Parafilm®. This filling process was carried out while ensuring that no trapped air bubbles were found within the samples in the tubes. The open end of each tube was placed in the semen sample in a sample bottle to allow for migration/penetration, after which the capillary tube was viewed under a microscope. A ×40 eyepiece was used to carry out the observation and the migration distance of the farthest sperm cell in the sample was recorded as the sperm penetration distance. The assay was conducted and reported in four steps: Spread of data in repeated penetration tests The swim-up penetration test was repeated to improve accuracy and reliability in taking the readings. One sperm sample was used for all preparations of cervical mucus, and the multiple tests were recorded in a table for which the means, standard deviations (SDs), and coefficients of variation were calculated. Sperm migration in relation to time Eight CM samples were used to conduct this experiment. Since the human cervical canal measures only up to 25 mm, the capillary tube was filled with the CM samples up to 40 mm and the migration rate of the sperms was recorded every 5 min. A line graph was used to represent the results and the correlation coefficients from the data were determined. Effect of capillary size Each sample of cervical mucus was tested with the sperm sample and repeated with another size of capillary tube (Dimension – 0.70 × 7.0 mm, 0.460 wall thickness). The means and standard deviations (SD) were calculated with all results presented as mean ± SD. Effect of freezing cervical mucus One sperm sample was used in this method with different cervical mucus samples. Here, the cervical mucus samples were initially used to conduct the penetration test. Following this, the samples were stored at -23°C and thawed afterwards, then the tests were repeated in triplicate.
Institutions
- University of Lagos Faculty of Pharmacy
Categories
Funders
- Grand Challenges AfricaGrant ID: GCA-R13-01