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.

1. Raw or Neat semen preservation
2. Washed sperm preservation.

sperm preservation

Purpose of this discussion is to find  a third method  which theoretically & practically sound better.

1. 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.

2. 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.

3. 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.