IUI MADE EASY

SEMEN ANALYSIS, PROCESSING AND PRESERVATION

Dr Suryakant Hayatnagarkar
Cryo Cell India Pvt. Ltd New Delhi
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Cryo Cell Sperm Counting Chamber

Simple and Inexpensive Cryocell is easy to use and gives rapid and accurate counts from undiluted specimen
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CRYO CELL CHAMBER

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CRYO CELL CHAMBER

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CRYO CELL CHAMBER

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SPERM FUNCTION TESTS

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SPERM MEMBRANE FUNCTION

Sperm Vitality Test Eosin stain does not penetrate the intact cells Less than 50 % cells stain with eosin Hypo-osmotic Swelling Test Intact sperm Membranes allow the sperms to swell in Hypo-osmotic solutions with curling of tails More than 60 % Curling is normal associated with good fertility
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HYPO-OSMOTIC SWELLING TEST

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HYPO-OSMOTIC SWELLING TEST

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TESTS OF SPERM MEMBRANE BINDING, CAPACITATION AND PENETRATION

Hyper-activated Motility

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Hemi-Zona Assay
Zona free hamster oocyte test

ACROSOME DETECTION AND REACTION TESTS

Triple Staining
Fluorescent dyes binding
Various Lectins, Chlortetracycline, Antisera etc.

Acrosin Measurement
Gelatin Film Lysis test
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ACROSOME DETECTION AND REACTION TESTS

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BIOCHEMICAL ASSAYS OF SEMINAL PLASMA

PROSTATE
Zinc, Citric Acid, Acid Phosphatase

SEMINAL VESICLES
Fructose

EPIDIDYMIS
L-Carnitine, Neutral Glucosidase
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SEMEN FRUCTOSE TEST

Simple biochemical test

Denotes intact seminal vesicle function

When present the levels directly proportional to testosterone levels in blood

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ANTI-SPERM ANTIBODY TEST

ELISA TESTS

IMMUNOBEAD TEST
IgG and IgA types direct and indirect tests

MIXED ANTIGLOBULIN REACTION TEST

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COPMUTER ASSISTED SEMEN ANALYSIS

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DIFFICULTIES IN IUI

Imported bulk packing expensive media requiring CO2 incubators

Availability restricted to few people Technology unavailable. Training restricted

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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

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

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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In conclusion IUI is an
effective, non invasive, relatively simple &

inexpensive method of
treatment. It can be provided easily in simple setups.
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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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What is Cryopreservation? Cryopresrvation means preserving cells and tissues in live condition at such low temperature, that all the cell metabolism comes to a standstill.
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Mechanism of Damage to cells in Cryopreservation

Ice

crystals formation in the nucleus and cytoplasm results in disruption of membranes Rapid water loss due to osmotic gradient Damage due to reactive oxygen species release during cooling.
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CRYOPROTECTANTS

Glycerol Dimethyl Sulfoxide Propendiol

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EXTENDERS
Optimize

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Osmotic Pressure Optimize pH Provide Energy Source Prevent use of membrane phospholipids Prevent Bacterial Contamination

EXTENDERS

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Zwitteronic Buffers Egg Yolk Sodium Citrate Albumin Sucrose Dithiothritol

Factors affecting Sperm Cryosurvival

Maturation stage at Storage Initial Sperm Quality Sperm Processing Techniques Temperature Shock, Cooling Rates, Seeding etc. Duration of Storage Warming Rates
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INDICATIONS FOR CRYOPRESERVATION

HUSBANDS SEMEN
Malignancy Pre-Vasectomy Absentee

Husband Sexual Problems Neurological Diseases Pooling of samples in Oligo-spermia

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SPERM BANKING
Cryopreservation

of donor samples and donor sample service is the most important activity of sperm banks. ICMR has suggested setting up of Sperm Banks as an Independent Units free from administrative control of ART centres.
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SEMEN BANKING
Proper criteria for Selection of Donors Screening of Donors Quality control of procedures Quarantine Testing Storage should be followed
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Selection of Donors

Good Health Status Pleasant Personality Minimum College Education Age < 40 years Cooperative Spirit and Understanding of Program Requirements Commitment to the program and Cause
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Exclusion Criteria for Donors

Non-cooperative Attitude Hesitation in Commitment Detailed Medical and Genetic History and Exclusion for Possible Genetic Disease High Risk Sexual Behavior History of Sexually Transmitted Diseases
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SEMEN SCREENING
Probability of Pregnancy is directly related to the quality of semen Minimum acceptable parameters :

Volume Sperm Concentration Motility Efficiency of freezing

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>2 ml > 60 M/ml > 70 % > 60 %

LABORATORY SCREENING

General Lab tests CBC, Liver function tests etc Serological Tests Syphilis VDRL HIV HBsAg

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LABORATORY SCREENING
SEMEN EXAMINATION Gonorrhea Chlamydia tracomatis Genital Mycoplasma
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Completion of Questionnaire Semen Sample Quality and Freezability

Physical Examination Serological Tests HIV, VDRL, Hepatitis B Chlamydia
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FRESH V/S FROZEN ?

Strict Screening of Donors is not always possible One Time Testing at the time of Donation is not adequate as Antibodies to some Antigens take 3-6 months to appear in Blood after infection
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In most Developed Countries, use of Fresh Semen for Donor Insemination is Discontinued

Exclusive use of Cryopreserved Semen which has been Quarantined for 6 months is Recommended by CDC USA and American Fertility Society

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Advantages of Cryopreserved Semen

Quarantine of Donor Semen till Appropriate Testing can be Completed Ease of Scheduling Insemination when Ovulation is Optimal Availability of Consistant Quality of Samples

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Advantages of Cryopreserved Semen

Advanced Multiple

Matching of Donors Physical Characteristics data with Husband Inseminations in a given cycle or In same patient with the same Donors samples possible availability

Commercial
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SEMEN BANKING MANAGEMENT

PLACE

PERSONNEL EQUIPMENT
CONSUMABLES
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SPERM BANKING MANAGEMENT

SHOULD HAVE STANDARD OPERATING PROCECURES FOR SELECTION OF DONORS COLLECTION OF SEEN SAMPLES QUALITY CONTROLS MANDETORY TESTING STORAGE AND DISTRIBUTION POLICIES ADMINISTRATIVE CONTROLS
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Equipment

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Liquid nitrogen Containers Liquid Nitrogen Freezers Controlled Temperature Freezers Cryovials Canes and Canisters for holding Sample Vials Cryoprotectant and Processing Kits

Protocol for Freezing IUI Samples

Collection of Semen by Masturbation Semen Analysis Semen Processing Addition of Cryoprotectant Gradual Freezing in Liquid Nitrogen Vapors Storage in Liquid Nitrogen
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ASSEMENT OF CRYOSURVIVAL

Post-thaw Motility Morphology Longevity CASA

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CRITERIA FOR ACCEPTANCE OF CRYOPRESERVED SAMPLE

VOLUME MOTILE

: 0.4 0.6 ML : > 12 MILLION : > 35 %

SPERMS

MOTILITY
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SAMPLE STORAGE
It is necessary that every sample from every ejaculate is traceable Samples are labeled with donor ID # and Batch # with indelible ink. Record is maintained in register about every sample ID No., Date of collection, initial parameters, Processing parameters, No. of vials made and finally Quality of freeze-thaw.
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SAMPLE STORAGE

Once freeze-thaw QC is passed the samples are transferred to inventory container till the Quarantine is complete. Samples are segregated month-wise in different containers and only after retesting of quarantine donors is complete the samples are transferred to current inventory container for dispatch
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SAMPLE DISTRIBUTION

Current Inventory container is used to segregating samples for dispatch. Random quality check is performed on few samples to check the storage. As per the requirement from doctors and patients samples are randomly or specifically pulled out of current inventory container and separated to transport container.
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SAMPLE DISTRIBUTION

One master manifest is prepared of all the samples taken out for order with information about Donor ID #, Batch #, Blood Group, Height, Bone Structure, Color of Hair, Eye, Skin and No. of vials of each batch. Single sample record sheets are also prepared along with master manifest.
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SAMPLE DISTRIBUTION

Complete docket is prepared with master manifest, Single sample records for patient files, Invoice, Quality record sheet to accompany the container of samples ready for dispatch to doctors. All the records are maintained indefinitely.

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ETHICAL and LEGAL ASPECTS

There is a Need to evolved uniform legislation in issues arising from Artificial Insemination Must provide clear guidelines to Doctors in the use of Therapeutic Donor Insemination ICMR is making efforts to give comprehensive guidelines for issues in DI
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 Almost 30 % couples need donor

REGULATORY ISSUES IN DONOR INSEMINATION

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Insemination Increased awareness of donor Insemination Confidentially of DI is socially acceptable in comparison to Adoption The child is directly affected by the quality of service.

SAFETY ISSUES IN DONOR INSEMINATION

Child born out of DI Health issues of child Couple undergoing DI Communicable diseases Doctors performing DI Consumer relation issues involving restriction on using scientific judgment

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 Sovereign right of government to make legislations or give regulatory guidelines Efforts of FOGSI Efforts of ICMR
www.icmr.nic.in/art_clinic.htm
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ROLE OF REGULATORY BODIES

REGULATORY OPTIONS
PROFESSIONAL REGULATION Individual Practice Programme Guidelines Professional Associations Guidelines Commissions and Committees Government Guidelines
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PROFESSIONAL REGULATION

American Fertility Society Guidelines Very good guideline framework Provides good control

Lacks Legal Authority Problems of unscrupulous elements and flyby-night operators is great
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PUBLIC CONTROL

Sovereign right of government to make laws Always trail behind advances in technologies and changing social values Enforcement becomes mere interpretation of written word of law and real spirit is lost Attempts to interpret hinder medical judgments
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CURRENT REGULATIONS
USA U.K. AUSTRALIA SWEEDEN FRANCE DENMARK INDIA
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STATE REGULATION LAW REGULATIONS LAW COMMISSION LAW NONE

COUPLE

Assessment of need of DI and suitable treatment Should have written down procedures for selection treatment protocols selection of donor samples CENTRES SHOULD NEVER USE UNTESTED, UNQUARANTINED OR FRESH SAMPLES FOR DONOR INSEMINATION AT ALL COSTS
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DONOR

Collection, Preparation, Storage and Testing should be done in a specialized center Specific programs should be followed for selection of donor and health status assessment testing for genetic diseases testing for communicable diseases Counseling of Donors
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DOCTOR

PROPER COUNSELING PROPER RECORDS PROPER INFORMED CONSENTS QUARANTINE CONFIDENTIALITY

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