Wednesday, 23 October 2013

Sperm vitality tests



The percentage of live spermatozoa is assessed by identifying those with an intact cell membrane, by dye exclusion test or by hypo-osmotic swelling testThe lower reference limit for vitality (membrane-intact spermatozoa) is 58%.



When?

When the percentage of sperm motility is low or absent, vitality tests are used in order to determine whether spermatozoa have lost their flagellation because of metabolic dysfunction and/or axonemal defects, or are simply dead (necrozoospermia). When ART is considered, viable sperms are picked out for use. Vitality results should be assessed in conjunction with motility results from the same semen sample. Necrozoospermia may indicate epididymal pathology. Vitality tests are routinely performed on all samples by some. They also provide a check on the motility evaluation, since the percentage of dead cells should not exceed the percentage of immotile spermatozoa. Vitality should be assessed as soon as possible after liquefaction of the semen sample, preferably at 30 minutes, but in any case within 1 hour of ejaculation, to prevent observation of deleterious effects of dehydration or of changes in temperature on vitality.



How?


Test
Principle
Description
Suitability for ART
Dye exclusion test (eosin*-nigrosin test)
Dead sperms allow eosin to penetrate and thus stain the cell as damaged membranes in dead sperms, allow entry of eosin. Live sperms exclude eosin.
Nigrosin is used to increase the contrast between the background and the sperm heads, which makes them easier to discern. It also permits slides to be stored for re-evaluation and quality-control purposes.  Spermatozoa with red /dark pink heads are considered dead, whereas spermatozoa with white/light pink heads are considered alive. If the stain is limited to only a part of the neck region, and the rest of the head area is unstained, this is considered a “leaky neck membrane”, not a sign of cell death and the sperm is considered alive. If eosin is used alone, it might be difficult to discern the pale pink stained heads, in such circumstances nigrosin is used to increase the contrast of the background.
Sperms are killed in the vital staining process and cannot be used subsequently for ART.
Hypo-osmotic swelling (HOS) test
It is test of the integrity of the semi-permeable plasma membrane of the viable sperm tail. If live sperms are placed in hypotonic solution, water will enter the cell and cause the cell to increase in volume; this becomes evident in   the tail region where swelling of the sperm tail can be easily seen.
Semen is diluted with a hypotonic solution so that water enters sperm tail osmotically. Intact spermatozoa are revealed by swelling-related changes enforcing a coiling of the tail. In contrast, dead cells have damaged membranes that provide no osmotic gradient for swelling of the sperm tail to occur.
Sperms are not killed during the test and the test can be used in a therapeutic manner for ART including testicular sperm extraction (when only non-motile sperms are present, the test allows the technician to pick out viable non-motile sperm). It should be noted that the time and preparing the reagents differ between diagnostic use and therapeutic use. 


 *Eosin (Greek, Eos goddess of dawn) is a red dye resulting from the action of bromine on fluorescein. Structures that stain readily with eosin are termed eosinophilic. There are closely related interchangeable compounds commonly referred to as eosin. These are the one usually used eosin Y (with very faint yellowish cast), and eosin B (with very faint bluish cast).


What else to be considered?

1 1) The possibility of the semen specimen being exposed to contaminants within the container or to extremes of temperature, or prolonged abstinence periods. Also, consider the possibilities of genital tract infections and varicocele2) Defective transport through the genital system. 3) Antisperm antibody assay. It is preferable to use direct assays since these determine the presence of sperm-bound antisperm antibodies, as compared with indirect assays that determine the presence of antisperm antibodies in serum. 4) Electron microscopy studies for ultra-structural defects (commonest is immotile cilia syndrome).






 This page was last updated in November 2013

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Main Works of Reference List (The first eight are my top favourites)

  • British National Formulary
  • British National Formulary for Children
  • Guidelines (BAD - BASHH - BHIVA - Uroweb)
  • Oxford Handbook of Genitourinary Medicine, HIV, and Sexual Health
  • Oxford Handbook of Medical Dermatology
  • Rook's Textbook of Dermatology
  • Simple Skin Surgery
  • Weedon's Skin Pathology
  • A Concise Atlas of Dermatopathology (P Mckee)
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  • Andrews' Diseases of the Skin
  • Andrology (Nieschlag E FRCP, Behre M and Nieschlag S)
  • Bailey and Love's Short Practice of Surgery
  • Davidson's Essentials of Medicine
  • Davidson's Principles and Practice of Medicine
  • Fitzpatrick's Colour Atlas and Synopsis of Clinical Dermatology (Klaus Wolff FRCP and Richard Allen Johnson)
  • Fitzpatrick’s Dermatology in General Medicine
  • Ganong's Review of Medical Physiology
  • Gray's Anatomy
  • Hamilton Bailey's Demonstrations of Physical Signs in Clinical Surgery
  • Hutchison's Clinical Methods
  • Lever's Histopathology of the Skin
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  • Macleod's Clinical Examination
  • Martindale: The Complete Drug Reference
  • Oxford Handbook of Clinical Examination and Practical Skills
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  • Stockley's Drug Interactions
  • Treatment of Skin Disease: Comprehensive Therapeutic Strategies
  • Yen & Jaffe's Reproductive Endocrinology