Intrauterine insemination (IUI) is a fertility treatment that involves placing sperm inside a woman’s uterus to facilitate fertilisation. The goal of IUI is to increase the number of sperm that reach the fallopian tubes and subsequently increase the chance of fertilisation.

1. Basics of Semen Analysis/ Preparation/
Cryopreservation
Vishnu Priya Subramanian
Masters of Clinical Embryology
FEMELIFE FERTILITY
www.femelife.com
www.wikiHealthNews.com

2. SEMEN
ANALYSIS
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3. Human Semen
Semen can be divided into 4 fractions:
1. Pre-ejaculatory –secretion from urethral and bulbourethral gland (no sperms)
2. Dilute fluid from prostate gland (no sperms)
3. Major portion of sperms from vas deferens and distal epididymis (sperms)
4. Seminal vesicle secretion (few sperms)
Fraction 1, 2, 3 made up of the first portion of the ejaculate (30% of total
volume). Therefore important to ensure the first portion is collected.
IMPORTANT – Mix semen well before analysis

4. PRE-REQUISITES FOR SEMEN ANALYSIS
 Previous SA reports
 VIRAL MARKERS – HIV, HBV, HCV , VDRL
 ABSTINENCE – 3 to 7 days
 HOME COLLECTION – pre-cautions
 COLLECTION DIFFICULTIES
o Coitus interruptus OR use of special latex free condoms
o Sidenafil support
o Vibratory therapy
o Electro ejaculation
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5. CARE OF THE SPECIMEN
The sample should be collected only in sterile disposable cups
If in the condoms,tie and knot the open end and place it in A container
If collected outside – should reach the lab within 30-60 min
Should be carried in shirt or pant pocket
Container should be labelled with patient id, partners name, date and time of collection

6. PRECAUTIONS
First drop of sample should not be spilled as it is rich in sperms
No lubricants or gel should not be used as it is spermicidal
Container should not be washed before collection as water is spermicidal

7. INITIAL MACROSCOPIC
• Liquefaction
• Semen viscosity
• Appearance of the ejaculate
• Semen volume
• Semen ph

8. HYPOSPERMIA HYPERSPERMIA ASPERMIA
Volume Less Than 1ml More Than 5 Ml Absence Of Ejaculate
Reason
Bilateral Ejaculatory Duct
Obstruction
Bilateral Congenital
Vasal Aplasia
Inadequate Erection
Improper Situation
Incomplete Collection
Infection
Long Abstinence
Dilutional
Oligozoospermia
Retrograde Ejaculation
Anejaculation

9. INITIAL MICROSCOPIC
• Mucus strand formation
• Sperm agglutination
• Presence of other cells
• Sperm count
• Assessment of sperm motility
• Sperm Morphology

10. ROUND CELLS
• Round Shape Cells- Epithelial Cells,immature Germ Cells; Spermatid,
Spermatocytes, Spermatogona

11. LEUKOCYTOSPERMIA
• Sample Has To Be Sent To Microbiology Lab For Culture
• Patient Has To Be Appropriatelty Treated
• Repeat The Analysis After 2-3 Months

12. W
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PARAMETER LOWER REFERENCE LIMIT
Total Sperm number (per ejaculate) 39millions (33-46millions)
Sperm Concentration (per ml)
15 millions/ml (12-16 millions/ml)
Total Motility (PR+NP, %) 40 (38-42)%
Progressive Motility – Sum of Rapid + Slow (PR, %)
32 (31-34)%
Vitality (live spermatozoa, %) 58 (55-63)%
Sperm Morphology (Normal forms, %)
>4 (3-4)%
White Blood Cells (peroxidase positive cells/ ml) < 1million/ml (OR) 1-2 WBC / HPF 12

13. Makler Chamber
 A 10 Ul Of Semen
 Allow 1 Minute To Settle And Spread
Evenly.
 Use X20 Objective.
 A Row Of 10 Squares Is Counted
 Usually 3 To 5 Randomly Selected Rows
Are Read.
 If < 5 Sperms Per Row Of 10 Squares Is
Seen, Then The Entire Grid Is Counted.
A chamber specially designed for semen
analysis

14. SPERM MOTILITY SPERM MORPHOLOGY
 REFERENCE VALUE
 Progressive motility – 32%
 Total motility – 40%
 ASTHENOSPERMIA
 MANAGEMENT
 ≥32 % – wait & watch /
OI+TI
 30 % – IUI
 <30 % – Probably IVF /
ICSI; + / - intervention
 REFERENCE VALUE
 4 % or MORE (KRUGER’s)
 TERATOZOOSPERMIA
 MANAGEMENT
 ≥4% – – wait & watch / OI+TI
 2-4 % – IUI
 <2 % – Probably IVF / ICSI; + / -
intervention
 Abnormal morphology - ICSI
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15. NORMAL SPERM

17. Retrograde Ejaculation
 Retrograde ejaculation occur whereby the semen is ejaculated into the bladder.
 The acidity of the urine will kill sperms quickly.
 Alkalination of the urine is very important in order to recover live motile sperms.
 The patient is instructed to alkalinize his urine by intake of sodium bicarbonate, 3g (two table spoons) dissolved in a glass
of water in the evening before bed.
 In the morning, the patient must empty his bladder completely and drink another glass of sodium bicarbonate before
coming directly to the laboratory.
 Ask the patient to empty his bladder before semen collection. Provide two containers for collection, a small one for semen
and a larger one for urine. Instruct the patient to collect the semen by masturbation and to urinate immediately after
masturbation.
 The urine is divided into tubes and centrifuged for 10 min at 1500 rpm.
 The supernatant is removed leaving behind the pellet. The contents of all tubes are pooled.
 Analyzed as SA
 Add 2-3 ml of culture medium before proceeding with a gradient column.

18. TERMINOLOGY

19. SPERM FUNCTIONAL TESTS
4hr/24hr sperm survival – Good/ Avg / poor
Sperm vitality testing - <58% go for IVF/ICSI
Hyperactivation challenge test (Mol Hum Reprod. 2011 Aug;17(8):500-10. Epub 2011
May 23.)
 ROS levels & DFI (Fertil Steril. 2011 Oct;96(4):843-7. Epub 2011 Aug 11.)
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20. SPERM VIABILITY TESTS
DYES TESTS – EOSIN AND NIGROSIN
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HYPO OSMOTIC SWELLING TEST

21. DNA FRAGMENTATION INDEX (DFI)
• Raised DFI negatively affects the outcome
• Helpful in deciding the management line
• Testing modalities – SCSA, Staining (aniline Blue)
• SCSA – Sperm Chromatin Structural Assay
• Gold standard
• Cut off values - <15% / 15-25% / >25%
• Management of Raised DFI
• Frequent ejaculation
• Managing oxidative stress
• Anti-oxidants for 3 months
• Testicular sperm aspiration 21

22. Transient Poor Semen Analysis
Poor SA can result from factors such as:-
• Incorrect semen collection technique – spillage, dirty container, long delay in
delivering sample
• Poor technical expertise
• History of recent illness – flu or high fever may depress sperm counts
• Long period of abstinence – increased abnormal sperm morphology and
decrease motility
• Short abstinence period – lower semen volume and sperm count
As it take 10 weeks (64-70 days) for a new batch of sperm to be generated by the
testes, it is best to repeat SA after a period

23. Improving semen analysis
 Quality Assurance Program
 Standard Operating Procedures
 Laboratory Manual
 Documentation
 Sample ID and Tracking
 External QC
 Comparison of tests with an external source
 Internal QC
 Minimized variation by training
 Purchased QC samples with known values

24. POINTS TO CONSIDER
• Never start any treatment with out a basic Semen Analysis
• Is your semen analysis method and reporting format standardized
• Repeat semen analysis every 2-3 months is a must
• Interpreting the report
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25. SEMEN
PREPARATION
Vishnu Priya Subramanian
Masters of Clinical Embryology
FEMELIFE FERTILITY

26. SEMEN PREPARATION
• Semen sample must be washed prior to being used for IUI, IVF and ICSI
techniques.
• Washing of sperm removes the seminal plasma, dead sperm and other cells.
• In addition, washing selects for motile sperm.
• If the sperm count and motility are adequate, swim up and washing is suitable.
• If the semen quality is poor and includes large number of other cells, gradient
centrifugation is preferred.

27. METHODS OF SEMEN PREPARATION
 Direct swim up
 Swim - up (migration)
 Density Gradient (swim-down)
 Direct layer techniques
 Multilayer direct layer technique
 Direct wash

28. DIRECTSWIM UP METHOD
 Ideal for normozoospermic samples.
 It allows self selection of motile sperms.
PROCEDURE :
 Pipette 0.5ml of neat liquefied semen into the round bottom test tube
 Overlay 2ml of IVF medium. Others use IUI media, sperm preparation media or flushing media.
 Place swim up tubes in the incubator at +37oC and 5% CO2 for 30 - 60 minutes.
 Aspirate the top 1ml of medium and place in a clean conical tube.
 Add 5ml of IVF media and mix thoroughly.
 Centrifuge at 1500rpm for 10 minutes.
 Aspirate and discard the supernatant.
 Resuspend the pellet with 0.8 to 1ml of IVF media.
 Assess the count and motility and Sample is ready for IUI.

29. Density Gradient Centrifugation
• 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

30. DENSITY GRADIENT CENTRIFUGATION METHOD
 Pipette 1ml of 80% density gradient medium into a conical tube.
 Slowly layer 1ml of 40% density gradient medium on top of it.
 Gently layer 2ml of semen sample on top of the 40% layer.
 Avoid adding too much of semen as it will results poor separation.
 Centrifuge at 1500rpm for 15 minutes.
 Discard the top two layers.
 Loose the pellet and transfer into a sterile conical tube.
 Resuspend the pellet with 3 - 5 ml of IVF media.
 Centrifuge again at 1500rpm for 5 minutes.
 Discard the supernatant and resuspend the pellet with 0.5 - 1ml of IVF media.
 Assess count and motility and use for IUI.
 Once the sample is ready, the sample should be inseminate within 30 min.

32. INSTRUCTIONS TO BE FOLLOWED WHILE HANDLING SEMEN SAMPLE
 The sperm preparation should be performed in a clean sterile work area (Laminar air flow)
 Non-toxic, non-powdered gloves should be worn while handling semen sample.
 Sample should be collected in sterile non-toxic wide mouthed collection container.
 Semen sample should be collected not more than 1 hour before preparation.
 The longer semen reside in seminal plasma the less fertile it become relative to certain procedures.
 Damage to the sperm from dilution, temperature change.centrifugation,and exposure to potentially toxic
material must be minimised
 Dilution should be perform slowly
 Temperature changes should be gradual
 Preparation should be perform at 37`c
 A thorough identification protocol of the patient should be performed.

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34. HUMAN SPERM CRYOPRESERVATION
Principle, Technique & Implications for ART
Vishnu Priya
Subramanian
Masters of Clinical Embryology
FEMELIFE FERTILITY

35. What is cryopreservation?
The procedure that stabilizes the living cells at
cryogenic temperatures (-196°C) is called
cryopreservation.

36. Cryoprotectants
 Cryoprotectants are low-molecular-weight and highly permeable
chemicals used to protect spermatozoa from freeze damage by ice
crystallization.
 Glycerol, ethylene glycol, sucrose, dimethyl sulphoxide, and 1,2-
propanediol- Commonly used cryoprotectants

37. Function of Cryoprotectants
Cryoprotectants act by decreasing the freezing point of a substance
Reduces the amount of salts and solutes present in the liquid phase of
the sample
Decreases ice formation within the spermatozoa

38. Cryoprotectants
Criteria for choosing Cryoprotectant:
• Least toxic to cells
• Should be permeable to cells
• Should be soluble in water during freezing

39. Cryostorage- Devices used
Straws Cryovials Cryo sleeves
Aluminium Canes
Liquid Nitrogen
Dewars
Canisters

40. Techniques of Cryopreservation
MANUAL – SLOW FREEZING
Add equal volumes of semen and cryoprotectant media
Keep in room temperature (37°C ) for 10-15 mins
Step wise decrease in temperature to be done manually
Sample is brought to 5°C from 37°C @ 0.5–1°C/min
Sample is then frozen from 5°C to −80°C @ 1–10°C/min
Sample is then plunged into liquid nitrogen at −196°C

41. Techniques of Cryopreservation
The sample is initially mixed in drop wise manner with equal volume of cold cryoprotectant
The mixture is loaded into the straws/vials and left to incubate at 4°C for 10 minutes.
The vials are then placed at a distance of 15–20 cm above the level of liquid nitrogen (−80°C) for 15 min
The vials are then immersed in liquid nitrogen (-196°C).
RAPID FREEZING

42. Equipment for semen Freezing
• Kryo10 series programmable freezer
• The freeze control-Cryologic
• Biotronics
• Biofreezer

43. Planner K-10 Semen freezing programme
• Start temperature 24oC (Room temperature)
• Cooling: 2oc per minute to 0oc
• 10o per minute to –100oc
• Hold :10 minute
• Total programme time 37min
• Transfer to LN2 flask for temporary storage before placing in LN2 of cryo storage
dewar.
• Record all storage details appropriately.

44. Cryopreservation
APPROPRIATE LABELLING ESSENTIAL

45. Liquid Nitrogen (LN2)- Medical grade
 Refilling of LN2 to be
done at regular intervals
LN2 evaporates at the
rate of 300ml per day
Air-conditioned storage
area prevents evaporation

46. Thawing Procedure
 The thawing procedure is an equally important step
 The cell must be allowed to recover its normal biological activities trying to avoid abrupt thermal
changes as far as possible.
Several thawing techniques are used, among which are
1. Thawing at room temperature for 10 min and subsequent thermostat pass at 37°C for another
10 min
2. Thawing in a thermostat and water-bath at 37°C for 10 min
3. Thawing at room temperature for 15 min.

47. Thawing Procedure
Post- Thaw- Semen used for ART’s
Once the semen is thawed, it is separated from the
cryopreservation medium by washing in culture medium and
centrifuging for 5-10 mins at 1500rpm

48. IMPLICATIONS IN ART
• Preservation of male fertility before radiotherapy or chemotherapy which may lead to
testicular failure or ejaculatory dysfunction.
• In Azoospermic patients, who have undergone TESE/PESA, sperm cryostorage avoids
repeated biopsies or aspirations.
• For Donor insemination programs, after routine screening and quarantining the samples for 3
months.
• Routinely performed in patients who plan to start an ART- freeze semen sample to avoid
inconveniences due to failed ejaculation often associated with “semen collection stress,”
certain emotional states, or other commitments at the time of oocyte retrieval.

49. Cryopreservation and Reproductive Outcome
IUI or in vitro fertilization (IVF) with frozen-thawed spermatozoa result in lower
pregnancy rates compared with insemination with fresh sperm
Large scales studies not yet done to justify these results

50. SEMEN BANK
Vishnu Priya
Subramanian
Masters of Clinical Embryology
FEMELIFE FERTILITY

51. COUNSELLING OF DONORS
• He should understand that if treatment is successful, some where there will
be a child born that he will never know, have no connection, but
nevertheless be the biological father.
• Not more than 10 pregnancies by a single donor is allowed by HFEA.
• All prospective donors must be counselled that if their sample is found sub
optimal or if there is a positive screening for a disease, their sample will not
be used.

52. SCREENING & SELECTION OF DONORS
• History taking : Medical and family history up to grand parents (including
history of heritable disorders), personal history including hobbies.
• Physical exam - Height, weight, skin colour, eye and hair colour
• Tests - Blood group and Rh factor
HBsAg, HCV, HIV 1&2. VDRL
• Semen should be collected near the andrology lab.

53. • The donors must be registered by name and the code number allocated to
them by the
sperm bank
• Relevant information sheets should be given to prospective donors so that
they understand fully the legal as well as moral obligations of becoming a
sperm donor.
SCREENING & SELECTION OF DONORS

54. THANK YOU