Cryo-Preserved Sperm- Freezing to Insemination
Human sperm are being preserved with certain indications. Donor sperm are preserved to keep it viable for longer period, quarantine to rule out some disease, etc. Husband serum is also preserved in certain medical condition. In usual way sperm is first cryo – preserved , quarantined followed by Intrauterine insemination in either one of the two way.
- Raw or Neat semen preservation
- Washed sperm preservation.
Purpose of this discussion is to find a third method which theoretically & practically sound better.
- Wash — preserve — wash method.
Raw or Neat semen preservation
Raw or neat sperm is being cryo-preserved with a cryo-preservative media, which contain very high glycerol concentration (15% v/v).
- Washing of preserved sample is required after thawing by density gradient method — dead sperm, abnormal sperm, leucocytes, seminal plasma , debris, foreign body , cryo-preservative media etc. are removed & sperm is being concentrated in a pellet.
- Pellet is dispersed in washing media & centrifuged. Supernatant is removed. Pellet is again dispersed in appropriate quantity of washing media to make ready for The purpose of this step is to remove the silica particle which is used as density gradient.
- PROS —– Good quality sperm is inseminated .
–—- Dead sperms are almost removed .
—– Media used for insemination — HTF media
- CONS —- 1) Sperms are being preserved along with dead sperms & leucocytes which generate ROS (Reactive Oxygen Species) during freeze-thaw process and centrifugation. 2) In infected semen all the microorganism are preserved along with semen.
Washed sperm preservation
Semen is first washed by density gradient followed by washing by washing media.
After wash pellet is mixed with cryo-preservative media, which contain glycerol (5 to 8% v/v).
- During the process of semen wash with density gradient dead sperm, abnormal sperm, leucocytes, seminal plasma , debris, microorganism, foreign body , etc are removed and healthy sperms are concentrated in a pellet.
- The pellet is dispersed washing media and centrifuged. Supernatant is removed. Pellet is mixed with appropriate volume of cryo-preservative media. In this process silica particles are removed and robust sperm are selected for preservation.
- This type of preserved sperm is inseminated directly after
- PROS —- Easy to inseminate.
—- No processing is required after thawing.
- CONS —- Insemination is done along with cryo-preservative media which is not normal for uterus.
—- Dead sperms are also being inseminated.
Wash — preserve — wash method
Semen is mixed with equal quantity of washing media , incubated for 30 minutes at 340 C. This semen + washing media mixture is centrifuged over density gradient. Pellet is directly dispersed in appropriate quantity of cryo-preservative media, where glycerol concentration is 5 to 8%, followed by freezing.
- After thawing mix the content with equal quantity of washing media.
- Centrifuge at 250 G for 5 minutes, remove the supernatant. Mix the pellet with required quantity washing media to make ready for IUI.
- PROS —- Simple procedure is required after
—- Final content don’t have cryo-preservative media.
—- Final content are free from silica particle.
—- Dead sperms & leucocytes are separated before freezing, therefore possibility
of ROS generation is minimum during freezing and centrifugation.
—- Most of the microorganism are separated prior to preservation.
—- Practically we got less dead cell by this method.
- CONS —- Further study & opinion required.