Sperm preparation by Dr.Renukadevi

12 Apr 2017

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Sperm preparation by Dr.Renukadevi

  1. IUI-Sperm Preparation Methods DR.RENUKADEVI SENIOR EMBRYOLOGIST ARC International Fertility & Research Centre
  2. Steps of IUI  Pretreatment workup  Counselling for IUI  Ovarian Stimulation  Monitoring of Response  Ovulation Trigger & Timing of IUI  Semen Collection  Sperm Preparation  Insemination  Post Insemination follow up
  3. What is IUI  IUI is deliberate introduction of sperms in the female uterus in order to achieve pregnancy  One of the simplest techniques of ARTs  Minimally invasive and uncomplicated procedure  First therapeutic step in treatment of infertility  Cost –effective, simple, can be learned easily  Husband or Donor sperm (frozen and quarantined) can be used.
  4. Sperm Preparation Methods • Semen is a mixture of motile, non-motile and dead spermatozoa with cells, cellular debris and sometimes micro- organisms. • There are various sperm separation and isolation methods. • These methods try to replicate in vitro the natural process in which viable sperm are separated from other constituents of the ejaculate as they actively migrate through the cervical mucus.
  5. • The World Health Organization recommendation is to process sperm sample within one hour after ejaculation in order to prevent damage from leukocytes and other cells present in the semen. • The sperm suspension obtained using these methods is used for IUI, IVF, ICSI and different sperm tests.
  6. Intrauterine Insemination (IUI) Goal is to Maximize the Chance of Fertilization • Increase Number of Eggs • Position Sperm Closer to Eggs
  7. The most commonly used sperm preparation methods in ART are : Simple wash • This method is used if the semen sample is very poor and it mainly removes seminal plasma from the sperms. This procedure provides the highest yield of spermatozoa. • Therefore it is often used for IUI. • In this procedure the culture medium is added to the ejaculate after liquefaction and the mixture is centrifuged twice using lower centrifugation forces and fewer centrifugation steps in order to minimize the damage caused by ROS.
  8.  Mix the semen sample well  Dilute the entire semen sample 1 + 1 (1:2) with supplemented medium to promote removal of seminal plasma.  Transfer the diluted suspension into multiple centrifuge tubes, with preferably not more than 3 ml per tube.  Centrifuge at 1200g for 5–10 minutes.  Carefully aspirate and discard the supernatants.  Resuspend the combined sperm pellets in 1 ml of supplemented medium by gentle pipetting.  Centrifuge again at 1200g for 3–5 minutes.  Carefully aspirate and discard the supernatant.  Resuspend the sperm pellet, by gentle pipetting, in a volume of supplemented medium appropriate for final disposition, e.g. insemination, so that concentration and motility can be determined (Who laboratory manual for the examination and processing of human semen 2010) The Common Steps of this Method are:
  9. SWIM-UP  This method is used for semen samples with normal count and motility.  Allow the semen sample for liquefaction  Microscopic assessment of the sample should be done to confirm count and motility.  Label the conical tubes with the patients name & ID.
  10. Swim Up Method This technique relies on the ability of spermatozoa to swim out of seminal plasma into culture medium and is suitable for semen with high to moderate motility. This procedure selects spermatozoa for their motility. The common steps of this method are:  Evaluate the percentage of motile and total number of sperm on a Makler Chamber  Fill the round-bottom test-tubes with 2 ml Sperm Preparation Medium (medium)  layer 1ml of the liquefied semen underneath the medium
  11.  Incubate the sample in the incubator for 60 min. Placement of tube at 45° angle creates a larger surface area for sperms to swim-up.  Aspirate the medium of the upper part of the test-tubes into new prewar med round-bottom test-tubes without disturbing the lower layer  Add 5 ml of the medium to the sample and centrifuge 5 min/1200 rpm  Aspirate the supernatant but save 0.5 ml of the medium to resuspend the pellet.  Evaluate the percentage of motile and total number of sperm on a Makler Chamber
  12. Swim Up Method wash media (2ml) Semen sample ***Semen sample, wash media vol must be equal Centrifuge normal sample 10 mins 1200 rpm. Viscous Sample 15mins 1500 rpm Discard Sup. Add wash media (2ml) & mix gently sample Centrifuge Sample 5 mins 1200 rpm Discard Supernatant Add 1 ml of Wash Media w/o Directly Pouring it on the Pellet (Around the Sides of the Tube) Incubation: Tube Slanted for 30 mins at 45o. All Motile Sperms Swim up to the Surface. After Incubation Sup. Taken out and given to Doctor, pellet Discarded
  13. DENSITY GRADIENT This method is used to wash poor quality of semen samples.  Low motility  Poor forward progression  Large amount of debris  High number of cells  Antisperm Antibodies  Highly viscous
  14. Density-Gradient Systems  Density gradient centrifugation separates cells based on their density.  Morphologically normal and abnormal spermatozoa have different densities and at the end of centrifugation they are located accordingly.  The solutions like colloidal silica, poly-sucrose and others have higher density than semen. Because of that this system can separate the debris, cells, microorganisms and non-motile sperms from the motile ones.  Centrifugal force enables the motile sperms to swim from a less dense seminal fluid into a denser solution. Cellular debris and non-motile microorganisms are trapped at the interphase between the two solutions.
  15. High Density Gradient (80%) Low Density Gradient (40%) Semen Sample Centrifuge 2000 rpm 10 – 15 mins; 15 – 20 mins for high viscous 2 ml wash media poured into new tube, then pellet mixed gently into new tube Sup discarded Centrifuge 1200 rpm 5 mins Remove sup., add 0.5 ml wash media to the pellet, then mix well Load the sample for analysis DENSITY GRADIENT TECHNIQUE *** Semen Sample, gradient volumes must be equal
  16.  Evaluate the precentage of motile and total number of sperm on a Makler Chamber  Layer 2 ml of the lower density medium (40% ) into conical testtube  Put the layer of the high density medium (80%) underneath the upper layer (lower layer will raise and a sharp interface is formed)  Layer 2 ml of the liquefied semen on the top of the gradient  Centrifuge the sample 15 min/1200 rpm  Use a new sterile Pasteur pipette to aspirate, in a circular movement from the surface, everything except the pellet and 4-6 mm of lower phase. The common steps of this method are:
  17.  If no pellet is seen after centrifugation, remove all fluid except the lowest 0.5ml.  Aspirate the pellet or the lowest 0.5ml liquid. Transfer sperm pellet to new tube and re-suspend pellet in 5ml sperm wash.
  18.  Centrifuge the sample at 1200 RPM for 5 minutes. Do not use the brake.  Aspirate sperm wash supernatant leaving as little (0.25-0.50ml according to the sperm count and motility) liquid as possible above pellet for swim-up.  Add 1.5ml of media without disturbing the pellet.  Incubate the tubes at a 45° angle for 20 – 30 minutes for swim-up in vertical rack in a 37°C incubator.  Aspirate sperm wash supernatant above pellet and mixed with suitable volume of culture medium to obtain the required sperm concentration.  Evaluate the precentage of motile and total number of sperm on a Makler Chamber
  19. Preparing Retrograde Ejaculation Samples  In some men, semen passes into the bladder at ejaculation, resulting in aspermia, or no apparent ejaculate.  Confirmation of this situation is obtained by examining a sample of post-ejaculatory urine for the presence of spermatozoa.  If pharmacological treatment is not possible or not successful, spermatozoa may be retrieved from the urine.
  20.  Alkalinization of the urine by ingestion of sodium bicarbonate, for example, will increase the chance that any spermatozoa passing into the urine will retain their motility characteristics.
  21.  At the laboratory, the man should be asked to:  Urinate without completely emptying the bladder;  Produce an ejaculate by masturbation into a specimen container.  Urinate again into a second specimen vessel containing culture medium (to alkalinize the urine further).  Both the ejaculate, if any, and urine samples should be analysed.  Because a large volume of urine may be produced, it is often necessary to concentrate the specimen by centrifugation (1200g for 5-10 minutes).  The retrograde specimen, once concentrated, and the antegrade specimen, if produced, can be most effectively processed using the density-gradient preparation method.
  22. Preparing HIV-infected semen samples  If the human immunodefciency virus (HIV) is present in semen, viral RNA and proviral DNA can be found free in seminal plasma and in non-sperm cells.  As HIV receptors (CD4, CCR5, CXCR4) are expressed only by non-sperm cells, a combination of density-gradient centrifugation followed by swim-up has been proposed as a way of preventing infection of uninfected female partners.
  23. References  WHO Laboratory manual for the examination and processing of human semen. Fifth edition, 287 pages, ISBN 9789241547789, prepublication version, 2010  A. Aribarg, N. Sukcharoen, 1995 intrauterine insemination of washed spermatozoa for treatment of oligozoospermia. Int J Androl, 18 1 (1995), 62 66
  24.  Textbook of Assisted Reproductive Techniques- Laboratory and Clinical Perspectives by David K Gardner, Ariel Weissman Colin M Howels, Zeev Shoham, 2nd Edition  Textbook of In Vitro Fertilization and Assisted Reproduction- The Bourn Hall Guide to Clinical and Laboratory Practice, 3rd Edition, Edited by Peter R Brinsden