Den - 2 - An Appropriate kit for Sperm Preparation

Sperm Preparation for ART

Sperm Preparation for ART

—-  Why  & How

Double Layer Density Gradient - Sperm Preparation For ART

DEN – 2

When first  Sperm Preparation for ART started?  It is not very  clear. According  to available data  first IUI started  about a century ago, initially in cattle.  First  Assisted reproductive technology (ART)  baby  “Louise Brown”  took birth on 25 July 1978.  Following his birth,  scope and future expectation with the ART  increased.  As well as  demand of Sperm Preparation for ART technique increased.  The aim behind Sperm Preparation for ART is to give best possible result.

Why Sperm Preparation before ART is required :–

  • The human spermatozoa after ejaculation remain in a non- productive  stage.   They are  incapable to fertilize  if they are kept in close contact with ova.

  • Capacitation  of  human sperm is essential  for fertilization.  Inside the human body we call it In- vivo and  outside the body  In-vitro.  Seminal fluid contain more than one de-capacitation agent.  Hence  fertilization can not  take place in presence of  seminal fluid or seminal plasma.

  • Seminal fluid also contain some factors.  Which prolong contact with sperms can damage the sperm permanently.  After prolong contact of sperm with seminal fluid,   sperm may loose their  capability to penetrate cervical mucous.   Also other function of sperms  is being affected adversely and sometime permanently.

  • Prolong exposure to seminal plasma can permanently reduce the fertilizing ability of human sperm.  Therefore  sperm preparation  for ART (IUI or IVF) should  be performed  within  a pre-fixed time schedule (usually within 45 minutes)  Also one should be careful to remove seminal fluid  as completely  as possible.

  • Separation of  sperm also aim to make it free from  reactive  oxygen species (ROS) producing  cells.  Particularly  when sperm is ready for ART.

  • Consequently, we can say,  in order to fertilize an ova the  sperm must be separated from seminal plasma.  In other word separation of human sperm from seminal plasma is an essential  pre-step  to achieve capacitation and  making the sperm capable  to  fertilize the  ova.

The  ideal method for sperm preparation for ART should be  :–

  1. Easy & cost effective.

  2. Fast.

  3. Able to separate maximum number of motile sperm.

  4. Safe for human sperm.

  5. Able to separate dead sperm, leukocytes, bacteria, debris etc.

  6. Able to eliminate or neutralize harmful substance like  reactive  oxygen species (ROS).

  7. Capable to process large quantity of semen.

There are four  basic method of Sperm Preparation  for ART  / Sperm Wash

1.    Dilution of semen and washing.

  • Dilution of semen with washing media  followed by centrifugation.  It results in  pelleting  of all dead, alive & abnormal  sperms, leukocytes, debris, etc.

  • Leukocytes & abnormal sperm (particularly with extra cytoplasm) generate free radicals  like reactive oxygen species (ROS).  In this method leukocytes & abnormal sperm reach  the pellet along with normal sperm.  It  affect the normal sperm adversely.

2.  Sperm Migration. 

  • We know this method also as  direct swim up method. Here we gently layer washing media  gently over  liquefied semen.  Sperm  swim  out from the semen and migrate in washing media.

  • In this method it is preferable to divide the semen in multiple tube.  And layer the washing media gently over each part of semen.

  • In this method harmful sperm & leukocytes do not swim up to the washing media.

  • This method is better than dilution and washing of semen.  Since  this method does not pull  leukocytes &  abnormal sperms  along with normal sperm.  Hence normal sperms are safe from damaging effect of  ROS producing  leukocytes and abnormal sperm.                                                       Enhance                                                                                                                     Enhance – F

3.      Selective washing  of  semen (Density Gradient method)

  • May be single layer or multiple layer

  • Here isopycnic separation methods works to collect the healthy sperm in pellet.

  • Leukocytes & abnormal sperm responsible to generate ROS are not reaching to the pellet.

  • Only healthy sperms migrate to pellet.

  • This method separate seminal plasma very effectively.                                                                                     Den – 2

4.       Adherence method  (glass wool, glass beads & sephadex                                                                columns)

  • We are not  using this method in our lab.

  • Not enough literature is available to comment about this method.

  • In case of glass wool filtration there is possibility of contamination of final product with glass particle.

Colloidal  Silica

Presently  use of colloidal silica is very  common & widely used for sperm preparation for ART.  There are many intrinsic factor  which make colloidal silica  ideal for preparing density  gradient for separation of  human sperm.

The factors  making  colloidal silica  ideal  for Sperm Preparation  are :–

  • First as a mineral substance , it exert no osmotic effect  on culture media

  • Secondly it allows high specific gravity (density) media preparation

  • Thirdly it has low viscosity & does not slow down the sperm sedimentation.

  • Presently used  Silane Coated Colloidal Silica  is least  toxic to sperm.

From 1st Jan 1997, Percoll as density gradient for human are not in use because of the risk of endotoxins. 

Discussion : —

There are two basic reason for Sperm Preparation for ART / Sperm Washing for ART.   Firstly  to  remove  seminal plasma.   Secondly  to keep away the ROS  generating  cells (Leukocutes &  abnormal  sperm).

Certainly ROS producing leukocytes and abnormal sperm (with excess retained  spermatid  cytoplasma)  does not affect every man, particularly  with normal sperm quality.  But majority of patient coming to ART clinic are not having normal  sperm.

The  sperms with excess retained  spermatid  cytoplasma generate  ROS.  It  indicates  the  very specific  selection of  sperms by  density gradients can effectively  separate the less dense sperms  from the pellet after centrifugation.

Here it is worth  to  discuss regarding the effect of  centrifugation on sperms.  In  theoretical means  centrifugation seems  to be harmful for human sperms.  But practically it is not a problem until  gravitational force  crosses  800 G.  In our lab we are using maximum 400 G of gravitational force , which is  not harmful  to human sperms either in practical or theoretical means.

Conclusion   :–

In the light  of above article  and our vast practical  experience of  sperm separation,  double layer density gradient separation of human sperm  seems to be most suitable.   Not  only in sperm preparation for ART but also it gives good result for sperm cryo-preservation.  Particularly   when you  decide  to preserve the sperm after washing.

 

Our limitations :–

  1. The article prepared for normal person, who is not expert for ART management. Therefore we simply avoid using technical or heavy – heavy words.

  2. Our lab is equipped with basic equipment , so we have mentioned the result in very simple & basic way.

Referance

  1. Aitken and Clarkson (1988)

  2. Jeulin et al (1982)

 

 

 

 

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