It is well accepted fact that cryo-preservation impairs sperm quality & viable sperm in number. Even after 60 years of continuous practice of sperm freezing in different species there is hardly much improvement in cryo-survival. Even till date the survival rate of sperm after freezing is 30-60%; It is little in difference from those 50 years ago. In other words we can say we are still 50 years back in term of outcome of cryo-survival of sperm. On the other hand, oocyte and embryo cryo-survival has increased many folds (as high as 90%).
Main reason seems lesser research in the field of sperm cryo-survival. One reasonable logic may be number of sperm. In comparison to oocyte, sperm are produced in million folds. But now scenario is changing. Now more people are facing with different type of sperm abnormality. Life long well being of offspring is another concern. Many abnormality (morphology, DNA / mitochondrial, etc) are now evident in the sperm following conventional sperm freezing. One day we will also know in detail, how these abnormality can affect ART offspring? And what will be the way out?
Where we stand?
It is worth to give reference of queen honey bee. She has multiple sex in one to two days during second week of her adult life. It is worth to mention here that she mates only for this period in her whole life. A queen honey bee lives for two to four years. After mating she stores sperm in a special organ. She withdraws sperm almost daily to lay eggs for rest of her life.
On another hand, still we are struggling; how to preserve sperm with improved outcome?
Major hurdles with sperm preservation.
There are numerous hurdles with sperm preservation. Which lead to a poor outcome. We will cover only major factors which affects cryo-survival of sperm. As well as our aim is to suggest possible solution. Suggested solution may not be available in present circumstances. But it provides a lead for further research.
Intracellular & extra-cellular ice crystal formation.
Intracellular & extra-cellular ice crystal formation.
Presently we follow slow freezing and slow thawing protocol to preserve and thaw sperm. Both the process generate ice-crystals inside & outside of sperm. Ice-crystals harm the sperm or in mainly two ways:
Ice-crystals make pointed projections. Which are capable to damage cell membrane and intracellular organelles.
During the course of ice formation, intracellular & extracellular ingredients get more and more concentrated. Results in very high osmotic pressure; which has potential to damage sperm. Worth to mention that cryo-preservative added to freeze sperm already has high osmotic pressure.
One mode of action of cryo-preservative is drawing out intracellular water. Hence increases ingradients concentration. This property finally results in increasing the osmotic pressure of intra-cellular component. Moreover osmotic pressure of freezing medium also high. This high osmolarity is further augmented by increasing concentration due to ice-crystal formation.
Certain compounds seems good for sperm freezing. But their toxicity are strong enough to make sperm dead or non-motile. The permeable cryo-preservatives with different toxicity are : ethylene glycol, propylene glycol, dimethylsulfoxide, glycerol, formamide , methanol, and butanediol. On other hand some non-permeable cryo-preservatives (sucrose &
polyvinyl pyrrolidone) are also toxic at certain concentration. Glycerol has proven toxicity at higher concentration. But in routine it is in use at lower concentration. Another reason to use glycerol; we do not have better alternative. Sucrose concentration in freezing medium is on higher side. Our aim should be to reduce glycerol & sucrose concentration as much as possible. All are going to affect knowingly or un-knowingly the cryo-survival of sperm.
During the process of sperm freezing and thawing there is massive release of ROS (reactive oxygen species). Which can directly affect cryo-survival. morphology and functions of sperm. A short boost in ROS in cells may cause damage to DNA, RNA, and proteins, and even cell death.
Semen have enough quantity of anti-oxidants. Therefore while preserving whole semen, available antioxidants may provide some protection. Addition of antioxidants is one option. But amount of antioxidant added is a burning issue. Extra antioxidant or wrong selection of antioxidants may do harm by reductive stress.
In this discussion we are not focusing on all factors affecting cryo-survival. The factors pointed out are only major factors which influence cryo-survival of human sperm. Following may be the possible solution.
Minimization of ice crystal formation is possible by fastest mode of cooling and warming only. It seems possible by freezing by process of vitrification and quick thawing at higher temperature.
Reduction of osmotic shock over the sperm is possible by reducing the ingredient concentration. Or in other words making the osmolarity of freezing solution near to iso-osmolar with sperm and seminal fluid.
The best way is to not use toxic ingredient for sperm freezing. In case it is not possible, fix the concentration of toxic ingredient to minimum level.
Oxidative stress is still a matter of discussion and research. It is well known that steroidogenic tissue (gonads & adrenal glands) contain a large concentration of antioxidants. Therefore it seems logical that freezing of whole semen may not require additional antioxidants.
Huge number of sperm per ejaculate is having significant advantage. But making cryo-survival of sperm better remains always an appreciated factor. Now this is a proper time to shift from old age conventional sperm freezing to new method of sperm freezing. Our ultimate aim should be to get best possible cryo-survival of sperm.