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 :–
Easy & cost effective.
Fast.
Able to separate maximum number of motile sperm.
Safe for human sperm.
Able to separate dead sperm, leukocytes, bacteria, debris etc.
Able to eliminate or neutralize harmful substance like reactive oxygen species (ROS).
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
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 :–
The article prepared for normal person, who is not expert for ART management. Therefore we simply avoid using technical or heavy – heavy words.
Our lab is equipped with basic equipment , so we have mentioned the result in very simple & basic way.
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