1. SEMEN ANALYSIS: What? When? How? Why?
Dr Sohrab Salehi

2. BACKGROUND What is semen, exactly?
A mixture of seminal plasma and cells
Seminal plasma contains:
Prostatic fluid (~30% of the volume)
Epididymal plasma (~5% of the volume)
Seminal vesicle fluid (the remainder of the ejaculate)
The cells are:
Spermatozoa
Germ line cells
Leukocytes of various types
Bacteria
Epithelial cells
Occasional red cells
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3. BACKGROUND Formation of the sperm cell (1)
Formed in the seminiferous tubules, develop in close association with the Sertoli cells
Start as spermatogonia (self-renewing stem cell of the male germ cell line) – located on the basement membrane
The transformation from the round germ cell to the sperm cell occurs during passage to the centre of the seminiferous tubule
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4. BACKGROUND Formation of the sperm cell (2)
Spermatogenesis is a cascade of cell divisions:
Mitosis: spermatogonia to primary spermatocytes
First meiotic division: secondary spermatocytes
Second meiotic division: haploid spermatids
This process takes 70 ± 4 days in the human – so errors will take about 3 months to show up
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5. BACKGROUND Formation of the sperm cell (3)
Spermiogenesis: differentiation of the round spermatid into a spermatozoon
This is the process in which sperm morphology is largely determined
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6. BACKGROUND Sperm transport and seminal plasma
“Testicular sperm” need to undergo more maturation steps before they are ready to fertilize
Transported from the testes to the epididymis, where they mature, and acquire the ability to swim
Then moved to the vas deferens, for storage
At ejaculation, the sperm are transported out of the vas and mix with accessory gland secretions:
prostatic fluid (pH slightly acidic to neutral; contains citric acid and zinc)
seminal vesicle fluid (pH strongly alkaline; contains fructose)
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7. BACKGROUND Sperm function
The ejaculated sperm pass through the cervix, then the uterus, and enter the oviduct
The fertilizing sperm swims through the layers of cells around the egg (cumulus and corona), and reaches the zona pellucida
The sperm then loses the front membranes of its head (the “acrosome reaction”), binds to the zona, then forces its way through the zona to the egg membrane
When the sperm head binds to the egg membrane, its tail stops beating, and the egg incorporates the whole sperm cell
The egg unpacks the sperm, then the male and female pronuclei form.
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8. WHAT IS A SEMEN ANALYSIS?
An evaluation of spermatogenesis and spermiogenesis.
Traditional descriptive analysis:
interpretation based on population distributions of characteristics,
therefore prone to misinterpretation at the individual level.
Modern approach is to interpret with regard to:
diagnosis of specific lesions; and
indicators of dysfunctional and/or functional potential.
Requires understanding of the relevance of sperm patho-physiology.
In any case, the results must be accurate and reliable.
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9. WHY PERFORM SEMEN ANALYSIS?
Diagnosis of sterility
Diagnosis of infertility
Prognosis for fertility
Identify treatment options:
surgical treatment
medical treatment
assisted conception treatment
Therefore = a screening test to help direct management.
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10. What is the “standard” approach to semen evaluation?
International minimum standards are, by consensus, the World Health Organization’s Lab Manual.
Focus is on standardization with expanded section on quality control.
Methods amenable for use in any (“third world”) country.
Basic infertility work-up.
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11. SEMEN ANALYSIS Sample Collection
For a meaningful result, semen samples must always be collected under standardized conditions:
the container has to be sterile and known NOT to be spermotoxic (i.e. provided by the lab)
the man must have had 3 – 5 days of abstinence
the man must have washed his hands before collection (particularly if microbiological analysis is requested)
the man must NOT have used lubricants (except for Pre~Seed or His~Seed, the only “sperm-friendly” ones)
the sample must be kept at 37°C until analysis, which begins ideally within 30 min, but absolutely within 60 min, of ejaculation
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12. SEMEN ANALYSIS Sample Handling
The semen sample should be mixed gently during the liquefaction period to promote liquefaction
The sample should NEVER be vortexed (the only exception is for the fixed prep for concentration assessment)
The sample should NEVER be “needled” – if it is too viscous to work with, a known volume of sperm buffer (not PBS) should be added and the sample mixed gently. The added volume must be included in the sperm concentration calculation
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13. SEMEN ANALYSIS Macroscopic Evaluation
There are several macroscopic evaluations which give useful diagnostic information about the sample:
Appearance
Odour
Liquefaction
Volume
Viscosity pH
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14. SEMEN ANALYSIS Macroscopic Evaluation - Odour
Different people have different abilities to smell semen, so this cannot be standardized
However, when the lid is taken off the collection jar, it should be noted if there is a strong smell of urine or of putrefaction
Samples collected after a prolonged abstinence period (i.e. several weeks) are likely to have a stronger odour
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15. SEMEN ANALYSIS Macroscopic Evaluation – Liquefaction & Viscosity
Liquefaction is the breakdown of the gel portion of the seminal plasma – the enzymes for this are in the prostatic fluid
A sample with incomplete liquefaction has a gelatinous material in a liquid base – this can be seen when the sample is swirled for the appearance assessment
Viscosity is related to the fluid nature of the whole sample
This is rated subjectively according to the length of the thread of semen produced when the sample is allowed to run back out of the volumetric pipette used to measure the ejaculate volume
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16. SEMEN ANALYSIS Macroscopic Evaluation - Volume
The volume of the sample should be measured to allow an accurate determination of the sperm number
This is most easily assessed using a warmed disposable volumetric pipette (which is sterile and known NOT to be spermotoxic)
After the sample is measured, allow it to run back into the collection jar, noting its viscosity (a normal sample will have some viscosity – i.e. not watery, but it will flow easily)
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17. SEMEN ANALYSIS Macroscopic Evaluation - pH
pH is important because sperm die at pH < 6.9
The pH of liquefied semen is normally determined using test strips (we use EM Science ColorpHast type, pH 6.5–10.0)
We usually measure pH after volume and viscosity – by touching the “emptied” volumetric pipette to the test strip
The normal pH range is 7.2–8.4
Inflammatory disorders of the accessory glands can take the pH outside of this range
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18. SEMEN ANALYSIS The wet preparation – set-up
Place 10µl of thoroughly mixed, liquefied semen on a microscope slide and cover with a 22x22mm No 1½ coverslip
There are several important points to keep in mind:
The quality of sperm motility is affected by temperature – the lower the temperature, the poorer the motility, and then cold shock starts to occur at around 15°C. So great care must be taken to ensure that the slides and coverslips, as well as the pipette tips are kept at 37°C
The assessment must start as soon as the flow stops – if this is >1 minute, then a new wet prep must be made
Microscope: phase contrast optics and a heated stage
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19. SEMEN ANALYSIS The wet preparation – assessments
The characteristics assessed are:
Motility (to be discussed later)
Sperm aggregation (random clumping) – “some” is normal, but large clumps (each with hundreds of sperm) is abnormal
Spermagglutination (between specific sites) – could suggest the presence of antisperm antibodies.
Round cells: should be <1 per 40× field (~ 1 million/ml). If more abundant, a leukocyte test should be run
Epithelial cells: usually present in small numbers
Erythrocytes: should not be present
Debris: particles smaller than sperm head, may be plentiful
Bacteria and protozoa: presence indicates infection
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20. SEMEN ANALYSIS Motility assessment – interpretation
The WHO’s Reference values for motility
Total motility(np+pr) (38-42)
Progresivemotility(pr %) 32(31-34)
MARtest(motilespermatozoawithbound particles,%) <50
Immunobead test(motilespermatozoawith bound beads,%) <50
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21. SEMEN ANALYSIS Sperm concentration – interpretation
The WHO Reference values for:
Sperm concentration is > 15 × 106 sperm/ml(12-16)
Sperm count is > 39 × 106 sperm per ejaculate(38-42)
A persistently low sperm concentration is associated with impaired fertility
If a man has a sperm concentration < 5 × 106 sperm/ml, the WHO recommends assessment for numerical and structural abnormalities of sex chromosomes
Azoospermia can indicate a failure of spermatogenesis or blockage(s) in the male tract
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22. SEMEN ANALYSIS Sperm morphology
Normal morphology% 4(3-4)%
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23. SEMEN ANALYSIS Semen biochemistry
Zinc: marker for prostatic function – colorimetric assay (WHO)>2.4
Fructose: marker for seminal vesicle function, and is a substrate for sperm metabolism – spectrophotometric assay (WHO)>13
-Glucosidase: secreted exclusively by the epididymis and so is a marker for epididymal function – spectrophotometric assay (WHO)<20
Peroxidase-positive leukocytes<1
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24. SEMEN ANALYSIS Sperm Vitality
vitality(live spermatozoa,%)58(55-63)
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25. SEMEN ANALYSIS Leukocyte tests
Cytochemical stain for peroxidase activity – benzidine-cyanosine is the easiest to use (and it is available in a kit). Only stains those cells with peroxidase activity so does not detect activated polymorphs or lymphocytes
Immunocytochemistry: double antibody assay (primary antibody is a mouse MAb to CD45), reaction is with alkaline phosphatase:anti-alkaline phosphatase complex (available in a kit)
The tests are run on whole semen
The normal range is < 1 × 106 / ml
Values above this are considered to be clinically significant
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26. SEMEN ANALYSIS Retrograde ejaculation
In some men, the semen passes back into the bladder at ejaculation - this is confirmed by examination of a sample of post-ejaculatory urine
The man must take sodium bicarbonate the day before, and the day of, his appointment – to alkalinize his urine
Before collection, he should pass urine and then wait until he feels there is some urine in his bladder before masturbating
He then collects a urine sample for analysis
Assess volume and pH of the urine
Centrifuge (600g for 10 min), resuspend pellets to a total of 20ml with sperm buffer + protein
Re-spin, resuspend pellets and combine – add buffer to a final volume of 1ml
Perform a standard semen analysis with this suspension
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27. SEMEN ANALYSIS Post-vasectomy analysis
To determine the sperm concentration when few/none expected:
centrifuge as much of the sample as possible (1000 g for 15 min) in a disposable conical centrifuge tube – note the exact volume used
Remove most of the supernatant - note volume removed
Make at least 5 wet preps (10µl of resuspended pellet under a 22 × 22mm coverslip)
Search through at least 5 wet preps
If anything that looks like a sperm is seen – the report is “not clear”
Final decision is the responsibility of the Pathologist
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28. SEMEN ANALYSIS What tests should always be done?
Semen volume
Sperm concentration
Differential motility
Morphology
If there is an indication:
White cells
Vitality
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29. ABNORMAL SEMEN ANALYSIS Treatments to correct abnormalities
There is some evidence accumulating that there are some treatments which can improve semen analysis results, including:
antioxidants
carnitine
reducing scrotal temperature
However, there is still no good evidence that these changes in semen analysis results are related to improved fertility
Therefore, two abnormal semen analyses will often lead to a couple being recommended fertility treatments
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30. FERTILITY WORK-UP
Fertility is a “couple” problem, and so both partners are assessed
For the woman, the investigations may include hormone assessments (such as a day 3 FSH), a hysterosalpingogram, and/or a laparoscopy
Both partners also undergo physical examinations
For these functional assessments, the investigation is based on finding where the problem(s) lie – i.e. predicting failure
The treatment chosen is the one most likely to circumvent the problem(s) – i.e. increasing the likelihood of success
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31. FERTILITY vs STERILITY
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32. FERTILITY TREATMENT OPTIONS
If a partner is sterile (i.e. no gametes), then the couple would need donor gametes to achieve a pregnancy
If one or both partners are sub-fertile, then the treatment options are:
no treatment, or ovulation induction
intra-uterine insemination (+ ovulation induction)
in vitro fertilization (includes ICSI)
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33. INTRA-UTERINE INSEMINATION (IUI)
IUI is the least invasive of the ARTs - involves the selective washing of semen to isolate the motile spermatozoa (can’t put whole semen into the uterus)
Up to 15 million motile spermatozoa are inseminated
Advantages:
relatively inexpensive – simple procedures
minimal use of FSH
can be used in consecutive cycles
can usually start treatment virtually immediately
Disadvantages:
lower success rate per cycle than other ARTs
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34. IN VITRO FERTILIZATION (IVF) Overview
There are many types of IVF
For virtually all types, the woman is treated with “fertility drugs” to stimulate the development of a group of eggs (the average is around 10 – but the range can be enormous)
Just prior to ovulation, the oocytes are retrieved
That afternoon, they are inseminated with prepared sperm
Inseminated eggs checked the next day for fertilization
The fertilized eggs are kept in culture for up to 5-6 days
Embryo transfer / possibly cryopreservation
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35. IN VITRO FERTILIZATION (IVF) Fertilization and embryo development
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36. IN VITRO FERTILIZATION (IVF) Intracytoplasmic sperm injection (ICSI)
ICSI and its variants - the highest-level ART
One sperm is injected directly into an egg
Only mature eggs injected
After the insemination, the rest of the lab procedures are the same as for “standard” IVF
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37. IN VITRO FERTILIZATION (IVF) Risks and rewards
IVF gives the highest chance of pregnancy per cycle – although this is still controlled by the woman’s age
Traditionally there was a high risk of multiple pregnancy with IVF – as several embryos were transferred at once
The new culture systems give embryos with a much higher chance of implanting – so fewer are transferred
The advent of ICSI has allowed men with very poor fertility prognoses (including azoospermia) to become biological fathers
This can bring the requirement for genetic counselling prior to treatment (Y-chromosome deletions, etc.)
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38. ART SUCCESS AND FEES Pregnancy rates (women < 37 years old):
No treatment / fertile couple: around 15-20% per cycle
IUI + COH: 20% per cycle
IVF (includes ICSI): % per cycle (was about 10%)
Cost in BC (excluding drugs, which can be $2000–$4000):
IUI + COH: $300 – $400
IVF: $4500 – $4700
ICSI: $5750 – $6250
ICSI with aspirated sperm: $6250 – $6500 (+ urologist fees)
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39. TREATMENT OPTIONS Normal semen analysis
The recommended treatment will depend on the woman’s test results, and could be:
No treatment: if her results were normal, and she is young, and they have been trying for only a short time (< 1 year)
IUI: if the couple has already been trying for some years and the woman has normal results
IVF: if the woman is in her mid to late thirties, or if she has blocked tubes
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40. TREATMENT OPTIONS Low concentration (oligozoospermia)
The recommended treatment will depend on the woman’s test results, and could be:
IUI: if enough motile sperm can be recovered from the ejaculate (and if the woman’s results are normal)
IVF: if enough sperm can be recovered from the ejaculate, and if the woman is older, or has blocked tubes.
ICSI: only need to be able to isolate as many motile sperm as there are eggs, so used in cases of extreme oligozoospermia
ICSI with aspirated sperm: in the case of ejaculatory azoospermia (i.e. there are sperm in the testes but not in the ejaculate)
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41. TREATMENT OPTIONS Low motility (asthenozoospermia)
The recommended treatment will depend on the woman’s test results, and could be:
IUI: less likely, and depends on whether enough sperm with good progressive motility can be recovered, and if the woman’s results are normal
IVF: if enough motile sperm can be recovered from the ejaculate, with a final total motility of >90% with good progression (and if hyperactivation is seen)
ICSI: quite likely, as the sperm may not be able to generate enough power to break through the outer layer of the egg (the zona pellucida)
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42. TREATMENT OPTIONS Poor morphology (teratozoospermia)
If the sperm morphology is very poor, then ICSI is the most likely option
This is due to the high likelihood of failure of fertilization by IUI or IVF, related to functional failure in sperm-egg interactions, including:
Geometric interference in sperm-egg binding (head too round, or too long)
Interference in penetration of the zona (low energy, poor transmission of force due to midpiece or tail defects)
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43. RESEARCH & NEW TECHNIQUES Sperm kinematics
The way sperm swim affects their fertility
To get through the cervix, they have to swim in a straight path
To get through the outer layer of the egg, they have to generate a lot of power – this is seen as hyperactivated motility
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44. RESEARCH & NEW TECHNIQUES Sperm kinematics
The sperm tracks are analyzed and a number of kinematic parameters are derived, including:
Velocity (VCL, VSL and VAP)
Velocity ratios (expression of the path shape and regularity)
Amplitude of lateral head displacement
Beat/cross frequency
The proportion of sperm in a sample which meet particular kinematic criteria is used to predict (failure) of:
Mucus-penetrating ability
Hyperactivation (a marker of sperm function)
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45. RESEARCH & NEW TECHNIQUES CASA
Computer-aided sperm analysis
Able to assess the kinematics of hundreds of sperm in a couple of minutes
Means that these tests can be part of the infertility work-up
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46. RESEARCH & NEW TECHNIQUES ROS and sperm
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47. RESEARCH & NEW TECHNIQUES Sperm DNA Damage
Sperm Chromatin Structure Assay: SCSA™
© SCSA Diagnostics Inc, Brookings, SD, USA
Evenson et al. (1980) Science 240:
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48. RESEARCH & NEW TECHNIQUES Structured management
Use the test results to predict where blocks to fertilization are occurring
A treatment plan to go around these blocks is created for each couple
It is designed to maximize the chance of achieving a successful pregnancy in the most practicable and economical way
The treatment plan is implemented after consultation with the couple
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49. The WHO and Structured Management
(2000)
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50. STANDARDIZATION & QUALITY CONTROL
Fundamental requirements of all laboratory tests :
appropriate and robust methodology
careful training
operator experience
suitable assay control limits
regular internal comparisons = IQC
regular external comparisons = EQA
cost effectiveness
College of Reproductive Biology Inaugural Meeting, Houston, May 1997 }
accuracy & precision
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51. ISO 15189:2003
Medical laboratories – Particular requirements for quality and competence.
5.5.1 Appropriate procedures that meet the needs of the users of the laboratory’s services shall be used.
5.5.2 Only validated procedures shall be used.
5.5.3 All procedures must be documented and available:
a) purpose j) sources of interference
b) principle k) calculation of results (includes
c) performance specifications uncertainty of measurement)
d) primary sample(s) l) reference intervals
e) sample container m) reportable interval for patients
f) equipment & reagents n) alert &/or critical values
g) calibration o) interpretation by laboratory
h) procedural steps p) safety precautions
i) QC procedures q) potential sources of variability
5.5.4 Performance specifications must related to intended use.
5.5.5 Periodic review of biological reference intervals
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52. QC IN SEMEN ANALYSIS
Mean ± SD% differences between 4 trained andrology scientists using WHO manual/visual semen analysis methods on 60 determinations. (Calgary Diagnostic Semen Lab, ca. 1990)
Concentration
-2.3 ± 7.4
-1.7 ± 4.9
+4.5 ± 7.3
-0.5 ± 7.0
Total motility
+0.3 ± 3.0
-0.8 ± 3.1
-1.0 ± 3.3
+1.6 ± 2.9
Prog motility
+0.4 ± 2.6
-0.8 ± 2.9
-0.6 ± 3.3
+1.0 ± 2.8 A B C D
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53. TRAINING IN SEMEN ANALYSIS
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54. GOAL-ORIENTED TRAINING
Originally elaborated in Calgary during the mid/late 1980s as a means to facilitate the training of new staff.
Subsequently applied in numerous locations where we were involved in training andrology lab staff:
e.g. Sydney (Australia), London (UK), Stockholm (Sweden),
Boston (USA), Bangkok (Thailand).
Adopted by the ESHRE Andrology SIG Education Sub-committee as the basis for their Basic Semen Analysis Courses (25 run by the end of 2003).
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55. ESHRE Basic Semen Analysis Courses
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56. CONCLUSION
Semen analysis is an important laboratory test and should be thought of in the same way as any other diagnostic assay
It is used in determining treatment plans for infertility
The results can therefore have a huge impact on the level of intervention, with the associated emotional and financial costs to the couple
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