INTRA UTERINE INSEMINATION

Dr. Suryakant Hayatnagarkar

Cryo Cell India Pvt. Ltd.

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Rationale for doing I.U.I

Optimization of gamete availability at the site of fertilization

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Reduction in Distance traveled by Sperm

Tackling of occult Seminal problems Optimization of ovulation & Endometrium

Indications for I.U.I.

Female Factors Cervical factors Immunological factors Sexual dysfunction Un-explained Infertility

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Indications for IUI

Male

Factors Anatomic defects Abnormal Semen parameters Retrograde ejaculation Sexual / Ejaculatory dysfunction

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Semen Analysis

Collection of samples
Testing of samples
Physical characters

Volume,Color,Viscosity,pH

Count
Motility
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W.H.O. CRITERIA

Volume pH Sperm Conc.

: : :

2.0ml or more 7.2 - 8.0 15 million/ml or more

Motility

50% or more with forward progression

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Ideal Goals of Sperm Processing

Select highest number of motile sperms Remove Seminal Plasma & Prostaglandins Remove dead , damaged & abnormal sperms

Remove white cells & round cells

Induce Capacitation & Hyperactivation

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Sperm Processing Methods

Simple washing
Wash and spin
Oldest method Aim is to remove seminal Plasma only Does not separate motile sperm from dead sperm and debris

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Sperm Processing Methods

Methods using sperm motility

Wash and swim-up

Earliest most widely used method Advantages are

Useful in normal samples to get good motile sperm Effectively removes bacteria and cell debris

Disadvantage is pelleting of good sperm with debris thereby exposing to ROS

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Sperm Processing Methods

Methods using sperm motility

Swim-up from neat semen (layering)

Centrifugation is avoided
Yield is good for IVF but poor for IUI

Multiple tube technique can be used to improve yield

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Sperm Processing Methods

Methods using sperm motility

Self-migration and sedimentation

Special devices used

Advantage of rapidity Potential of developing into a product

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Sperm Processing Methods

Methods using sperm motility

Swim-up from ejaculate into

hyaluronic acid column

Modification of overlay method using hyaluronic acid media

Hyaluronic acid is known to stimulate motility

Advantage of conversion into a new product.

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Sperm Processing Methods

Methods based on density differences

Percoll, PureSperm, Polysaccharide or Isolate gradients

Ficoll gradient
Nycodenz Very popular methods use Percoll or like gradients Very effective and safe technique

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Sperm Processing Methods

Methods depending on other physical characteristics

Glass wool filtration Glass bead separation Glass Beads and Glass wool will bind dead sperm and allow the normal sperm to pass through. Very simple to perform Damage to sperm is the reality Sephadex columns

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Media Used In Sperm Processing

Use media manufactured by reputed manufacturer, do not use from cottage manufacturer or fly-by-night operators Do not use media in open package, or bulk pckings as it is likely to be contaminated before finishing. Insist on quality certification / check for manufacturers instructions before using.

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Media Used In Sperm Processing

HAM F-10 EBSS HUMAN TUBAL FLUID MEDIUM Additives Pentoxyphyline Albumin

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SWIM UP TECHNIQUE

Open the ampoules by holding the blue dot on the ampoule neck in front and firmly flicking the tip backwards
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SWIM UP TECHNIQUE
Cryo HTF medium
Semen Sample

Add 3 ml Cryo HTF medium to the sample & mix completely, centrifuge for 10 minutes at 1500 RPM

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SWIM UP TECHNIQUE
Cryo HTF medium
Semen Sample

Add 3 ml Cryo HTF medium to the sample & mix completely, centrifuge for 10 minutes at 1500 RPM

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Remove

SWIM UP TECHNIQUE

Supernatant Pellet

Following centrifugation, remove the supernatant without disturbing the pellet.

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Remove

SWIM UP TECHNIQUE

Supernatant Pellet

Following centrifugation, remove the supernatant without disturbing the pellet.

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SWIM UP TECHNIQUE
Cryo HTF medium
Pellet

Carefully layer 1.5 to 2 ml of Cryo HTF medium over this pellet. Keep the tube inclined in the incubator for 45 50 minutes.

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Remove & shift to tube two Supernatant

SWIM UP TECHNIQUE

Pellet
Aspirate the supernatant without disturbing the pellet and transfer to second tube. Centrifuge the tube at 1000 RPM for 4 5 minutes. Remove the supernatant. Resuspend the pellet in 0.5 ml of Cryo HTF medium do post wash examination on a small aliquot & calculate the yield.

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Remove & shift to tube two Supernatant

SWIM UP TECHNIQUE

Pellet

After transferring supernatant fraction from

incubation tube to separate conical bottom tube, centrifuge briefly at 1500 RPM for 5-7 minutes. Remove supernatant and add 0.5 ml of CryoHTF. Mix the Pellet and examine a small drop in Cryocell Sperm Counting chamber for Count and Motility. Use for insemination immediately

Aspirate the supernatant without disturbing the pellet and transfer to second tube. Centrifuge the tube at 1000 RPM for 4 5 minutes. Remove the supernatant. Resuspend the pellet in 0.5 ml of Cryo HTF medium do post wash examination on a small aliquot & calculate the yield.

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Cryo HTF 1 ml
Semen 0.5 ml 1 ml

SWIM UP BY LAYERING TECHNIQUE

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Cryo HTF 1 ml
Semen 0.5 ml 1 ml

SWIM UP BY LAYERING TECHNIQUE

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SWIM UP BY LAYERING TECHNIQUE

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After pooling fractions from all the layered Remove supernatant and add 0.5 ml of
Cryo-HTF.

SWIM UP BY LAYERING TECHNIQUE

tubes in conical bottom tube centrifuge briefly at 1500 RPM for 5-7 minutes.

Mix the Pellet and examine a small drop in

Cryocell Sperm Counting chamber for Count and Motility.
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Use for insemination immediately

Double Layer Density Gradient Technique

Open the ampoules by holding the blue dot on the ampoule neck in front and firmly flicking the tip backwards
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Cryo 45 medium

Double Layer Density Gradient Technique

Cryo - 90

Transfer entire contents Cryo 90 medium reagent ampoule to a conical tube. Carefully layer contents of Cryo 45 medium ampoule over the Cryo 90, without mixing them.

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Semen sample

Double Layer Density Gradient Technique

Cryo 45 medium Cryo 90 medium

Transfer liquefied & well mixed semen sample on top of the media layers. Centrifuge for 15 20 minute at 1500 RPM

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Remove Supernatant

Double Layer Density Gradient Technique

Pellet

Following centrifugation remove the supernatant without disturbing the pellet. Layer 1 to 2 drops of Cryo HTF medium over the pellet & resuspend the pellet. Shift to second tube.

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Double Layer Density Gradient Technique

Cryo HTF medium
Remove Supernatant Pellet

Add 3 4 ml of Cryo HTF medium and mix well. Centrifuge the tube at 1500 RPM for 5 minutes

Pellet

Resuspend pelle Ready for IUI

Remove the supernatant

Resuspend the pellet in 0.5 ml of Cryo HTF medium do post wash examination on a small aliquot & calculate the yield

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Single Layer Density Gradient Technique

Open the ampoules by holding the blue dot on the ampoule neck in front and firmly flicking the tip backwards

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Single Layer Density Gradient Technique

Semen

Cryo - One
Transfer entire Cryo One 2 ml medium to a tube carefully layer semen sample over it. Centrifuge at 1500 RPM for 15 20 minutes.

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Single Layer Density Gradient Technique

Semen

Cryo - One
Transfer entire Cryo One 2 ml medium to a tube carefully layer semen sample over it. Centrifuge at 1500 RPM for 15 20 minutes.

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Remove Mix Remove

Single Layer Density Gradient Technique

Transfer

Remove carefully seminal plasma, interphase & Cryo One layer. Add One drop of Cryo HTF medium & transfer pellet to a new tube using a new pipette

Add 3 4 ml Cryo HTF medium and centrifuge at 1000 RPM for five minutes

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Single Layer Density Gradient Technique

Ready for IUI

Remove supernatant. Add 0.5 ml Cryo HTF medium & incubate at 37oC for 15 minutes

Centrifuge briefly at 1500 RPM for 5-7 minutes. Remove supernatant and add 0.5 ml of Cryo-HTF. Mix the Pellet and examine a small
drop in Cryocell Sperm Counting chamber for Count and Motility. Use for insemination immediately

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Advantages of Density Gradient Centrifugation

1. Can be used for both normal and oligospermic samples 2. Concentrates highly motile sperm in the pellet. 3. Effectively removes seminal plasma, round cells and dead sperm 4. Reduces oxidative damage to sperm. 5. Viscous semen samples can be processed. 6. Enhances percentage of spermatozoa with normal morphology.
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IUI is an effective,non invasive,

relatively simple & inexpensive

method of treatment. It can be provided easily in simple setups.

Careful selection & counseling of

couples is essential.
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SETTING UP IUI LAB

Place

Equipment
Personnel Facilities
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PLACE

One room preferably 100 sq feet Air conditioned Away from busy areas, dust Preferably in basement Near OT or insemination room
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EQUIPMENT
Binocular

Microscope Cryocell chamber, Makler chamber Centrifuge Incubator, Tube warmer

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EQUIPMENT
Refrigerator
Kits

and accessories Laminar Flow Bench

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PERSONNEL

Trained Gynecologist Trained Technician Other support services

Assistants Sonologist

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WHAT CRYOCELL CAN DO FOR YOU Supply Quality reagents and Kits Help in designing IUI lab and selecting equipments for the lab Help in procuring the equipment
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WHAT CRYOCELL CAN DO FOR YOU

Train

Gynecologists in procedures Train Technicians in processing techniques Supply tested Quarantined Frozen Donor Semen samples Troubleshooting
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