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Male Reproductive System > Tests of Sperm Function > Flashcards

Tests of Sperm Function Flashcards

1
Q

What forms of male infertility can semen analysis tell us?

A

non obstructive azoospermia
obstructive azoospermia
Total immotility
globozoospermia

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2
Q

what does routine semen analysis NOT tell us about sperm functional capabilities?

A
  1. undergo capacitation
  2. bind to zona pellucida
  3. penetrate the egg
  4. undergo initial steps of nuclear decondensation
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3
Q

What DOES a routine semen analysis tell us?

A
  1. testicular function (sperm production)
  2. genital tract secretions- prostate, seminal vesicle, proximal genital tract

NOT a test of fertility!

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4
Q

What sperm functions can be tested with additional tests?

A
  1. cervical mucus penetration
  2. acrosome reaction
  3. capacitation
  4. zona pellucida binding
  5. zona pellucida penetration
  6. oocyte-sperm fusion, sperm decondensation
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5
Q

What is the role of cervix/cervical mucus in fertility?

A
  1. cervix protects sperm from hostile acidic environment of vagina
  2. provides energy sources for sperm
  3. filter abnormal sperm
  4. sperm reservoir gradually releasing sperm over time
  5. initiate sperm capacitation
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6
Q

What is the suspected frequency of abnormalities of cervix and cervical mucous in female infertility?

A

approximately 15-30% of infertility in women

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7
Q

How is cervical mucus regulated by ovarian hormones?

A
  1. regulate secretion, physical characteristics and sperm receptivity of cervical mucous
  2. Production of cervical mucus is cyclic
  3. E2 stimulates copious amounts of thin, clear, acellular and alkaline mucus receptive to sperm penetration and survival
  4. P4 stimualtes small amounts of thick, viscous cellular and unreceptive mucus
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8
Q

When should it be collected and What parameters of cervical mucus can be evaluated?

A

Collected as close to ovulation as possible and basal body temperature can be used to measure this

  1. amount - mucus volume
  2. viscosity
  3. spinnbarkeit (stretchability)
  4. ferning (dinstinctive structure)
  5. Cellularity

Scored 4 pt score 0-3

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9
Q

What is the postcoital test (Sims-Huhner or Huhner test) and why isn’t it used anymore?

A

not standardized and dificult to interpret

determines number of active sperm in mucus and to evaluate sperm survival and behavior hours after coitus

Depends on proper: timing, technique, evaluation, interpretation

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10
Q

What may cause a hostile environment in the cervical mucus test?

A
  1. immunological infertility
  2. endocrine disturbances
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11
Q

What are 4 ways to measure sperm-cervical mucus interaction?

A
  1. slide test
  2. contact test
  3. capillary tube test
  4. bovine cervical mucus penetration test
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12
Q

What is PENETRAK test?

A
  1. Thin glass tube filled with bovine cervical mucus
  2. placed with semen
  3. after incubation distance travelled by vanguard sperm is measured microscopically

Only assesses sperm function to penetrate cervical mucus

donor sperm from fertile male is positive control

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13
Q

What did the ASRM practice committee conclude about the postcoital test (PCT) cervical mucus tests ?

A

PCT was traditional method for diagnosis of cervical factor infertility

Abnormalities of sperm/cervical mucus interaction are rarely the sole cause of infertility

PCT is not a valid method for evaluation of cervical factors

However it is subjective, poor reproducibility, inconvenient, does not predict inability to conceive, PCT is no longer recommended for evaluation of infertile female

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14
Q

What is the sperm hypo-osmotic swelling test (HOS)?

A

measures sperm ability to transport fluid- viability
Incubates sperm under hypoosmotic conditions
water transport into the cell will occur if membrane is functional
tail will swell
if fluid transport does not occur and tail does not swell, it is assumed membrane is inactive and will not function in fertilization process

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15
Q

What is the composition of the solution used for HOS test?

A

sodium citrate and fructose

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16
Q

What is a normal/abnormal result in the HOS test?

A
  1. positive = >60% sperm tail swelling
  2. negative = <50% swelling
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17
Q

How is HOS different from sperm vital staining?

A

Vital staining measures membrane disruption
HOS measures chemically inactive/active membranes

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18
Q

What does the acrosome reaction test measure?

A

capacitation

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19
Q

What are the 3 hydrolytic enzymes in the acrosome?

A
  1. hyaluronidase
  2. acrosin
  3. neuraminidase
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20
Q

When does the acrosome reaction occur in vivo during normal fertilization?

A

after sperm bind to zona pellucida and is required for fertilization

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21
Q

What initiates the normal AR after binding to zona pellucida?

A

Calcium influx

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22
Q

What are the 3 acrosome staining patterns observed?

A
  1. CAP
  2. BAR
  3. BLANK
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23
Q

What is the ARIC test? How is it calculated? What is a normal/abnormal result?

A

acrosome reaction to ionophore challenge

ARIC scored by calculating %AR Test minus %AR of control

normal is 15%
Values <10% considered abnormal
values 10-15% suggest sperm function may be abnormal

Note: measuring difference in AR in test vs control! How much more AR was induced in the treated sperm vs control

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24
Q

What sperm functional test can also be performed with chlortetracycline?

A

acrosome staining

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25
Q

How is the hemizona assay performed?

A
  1. zonas from eggs that failed to fertilize are cut in half by micromanipulation
  2. Wash and swim-up of sperm
  3. assess sperm paramaters
  4. aliquot specific concentration of sperm
  5. co-incubate sperm with zonas for 4 hours, then rinse
    one half exposed to donor sperm and the other half with patient
    those that bound are counted
    results expressed as ratio of patient to donor sperm bound
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26
Q

What is the competitive sperm zona binding assay?

A

can be used prospectively to predict sperm binding to zona prior to IVF or post-IVF to determine sperm or ova cause of IVF fertilization failure

donor sperm and patient are labeleled with fluorescent markers then incubated with zona pellucida for 4 hours then calculate the ratio

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27
Q

What did ASRM practice committee conclude about ASAB testing in 2012?

A

ASAB are rare cause of male subfertility that do not require routine testing
the clinical utility of the test is uncertain
ASAB testing is unnecessary if ICSI is planned

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28
Q

What is the estimated frequency of ASAB in infertile couples?

A

10-10%

29
Q

Where can antisperm antibodies be found?

A

ASAB can be derived from transudates from blood or secreted locally by submucosal plasma cells

ASAB can be found in serum but not tract fluid
or
ASAB can be found in tract but not serum

30
Q

List conditions that can cause ASAB to occur

A
  1. congenital anomaly
  2. vasovasostomy (procedure designed to bypass obstruction)
  3. cystic fibrosis (CBAVD)
  4. vasectomy (50-70% of men with vasectomy)
31
Q

What can cause ASAB in women?

A

mechanical or chemical disruption of mucosa or local inflammation/genital infection

ASAB can be detected in serum (IgG) or reproductive tract fluids (IgA)

32
Q

What pathogenesis can ASAB cause in sperm?

A
  • they bind surface of sperm
  • ASAB causes decrease in sperm penetration of cervical mucus resulting from agglutination or loss of motility
  • may also inhibit capacitation, block sperm-oocyte recognition, inhibit penetration of zona pellucida by sperm
33
Q

What is the most common method for measuring ASAB?

A

Immunobead test (IBT)
detects ASAB using polyacrylamide spheres or latex beads with covalently bound rabbit antihuman immunoglobulins

IgA, IgG, IgM or combination

can detect presence of specific immunoglobulin binding to specific areas of the sperm

34
Q

What is the direct IBT test?

A

detect ASAB directly on sperm
sperm are washed free of seminal plasma, resuspended in buffer
mix small amount of sperm with suspension of immunobeads on microscope slide
wait 2-10 minutes and examine under microscope
determine % of motile sperm with >2 beads bound
note location of binding
ignore tail=tip binding (not clinically significant)

35
Q

What is the indirect IBT test?

A
  1. detects ASAB in serum, seminal plasma or cervical mucus
  2. incubate patient fluid with donor sperm to passively transfer ASAB to sperm surface
  3. pellet sperm, resuspend in buffer, mix with beads as with direct IBT
  4. use donor sperm proven free of ASAB
  5. cervical mucus can be solubilized with bromelain
36
Q

What is a clinically relevant amount of bead binding in IBT test?

A

at least 200 sperm counted
clinically relevant if >50% of motile sperm have beads bound to them
binding to tail tip only not significant

ASAB in semen belong exclusively to either IgA or IgG
IgA is more important
IgM rarely found in semen even though high concentrations can be found in blood

37
Q

What is the MAR test?

A

mixed antiglobulin reaction (MAR)
* mix untreated semen with latex beads or red blood cells coated with human IgA or IgG
* Add antiserum to IgA or IgG or IgM
* antiserum binds immunoglobulin on the surface of the beads and if present on the sperm surface
* direct/indirect testing performed as with IBT

38
Q

What is the interpretation of the MAR test?

A

same as IBT
clinically relevant if >50% of motile spoerm have beads bound to them

39
Q

What is the treatment of ASAB?

A
  • condom therapy theoretically reudces antigenic exposure of women to sperm - not proven successful
  • corticosteroid therapy: immunosuppressive therapy, mixed results, main drawbacks include hip necrosis and exacerbation of ulcers
  • Sperm washing - not proven succesfful
  • IUI - some success with cervical mucus ASAB
  • ICSI appears to be best option
40
Q

What is the sperm penetration assay?

A
  • in vitro assay to measure fertilization potential of sperm
  • hamster eggs are promiscuous and permit heterologous sperm to penetrate
41
Q

How is the sperm penetration assay performed?

A
  1. ejaculated spoerm placed in TEST yolk buffer 42 hr at 4 C
  2. rapid warming followed by swim-up to collect motile sperm
    syncrhonize sperm and enhance ability to penetrate the egg
  3. incubation with zona-free hamster eggs
  4. extensive polyspermy-penetrated and decondensed heads counted
42
Q

How can ICSI be used in combination with SPA (sperm penetration assay)?

A

hamster ICSI SPA : tool for training, proficiency and prediction of ICSI fertilization
prediction of male-mediated fertilization failure in IVF and technical proficiency

43
Q

List direct and indirect tests of sperm DNA damage?

A

Direct Methods direclty measure the number of DNA breaks
* * Comet Assay
* TUNEL assay

Indirect Methods measure the susceptibility of sperm DNA to break after acid treatment
*Sperm chromatin structure assay (SCSA)

Comet assay:

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

44
Q

What is the SCSA assay?

A

Sperm chromatin structure assay indrectly measures the susceptibility of sperm DNA to break after acid treatment

chromatin and or DNA intercalating dyes such as acridine orange are used to differentiate single-strand from double-stranded DNA

45
Q

What is the comet assay?

A

single cell electrophoresis on sperm embedded in agarose
DNA migration assessed

46
Q

What is the halosperm assay?

A

DNA breakage detection using fluorescent in situ hybridization
Fragmented DNA fail to produce halo of DNA after acid denaturation and removal of nuclear proteins

opposite appearance of comet assay

47
Q

What are therapies to reduce sperm DNA damage?

A
  1. avoidance of gonadotoxins and heat
  2. anti-oxidant therapy - inconclusive
  3. treatment of semen infection
  4. varicocelectomy
  5. testicular sperm extraction
48
Q

What are the causes of sperm DNA damage?

A
  1. abnormal chromatin packaging
  2. defective DNA repair
  3. defective cell death
  4. Free radicals - ROS
  5. environmental or industrial toxins
49
Q

What did ASRM practice committee conclude about sperm DNA integrity testing?

A

existing data on relationshiop bewteen abnormal DNA integrity and reproductive outcomes are limited

Sperm DNA damage is more common in infertile men especially those with RPL or idopathic infertility

Results of sperm DNA integrity testing alone dontr predict pregnancy rates achieved through intercourse, IUI, IVF or ICSI

no proven role for routine DNA integrity testing in evaluation of infertility

no evidence that treatments for abnormal DNA integrity have clinical value

this thinking has changed in the last few years because of improved testing and studies

50
Q

What is the alkalinity/acidity of accessory gland secretions?

A

Seminal vesicle- alkaline
Prostatic- acidic

51
Q

What is the WHO pH reference value for semen?

A

7.2 is the lower value

52
Q

What could a pH of less than 7.0 in semen sample with low volume and low sperm numbers mean?

A

ejaculatory duct obstruction or CBAVD (seminal vesicles are poorly developed)

53
Q

What do pH increases with time provide little clinically useful information?

A

semen pH increases with time as natural buffering decreases

54
Q

What is the formula to calculate wet mount depth? and what is the minimum chamber depth?

A

D= V/A

greater than 20um

55
Q

What are the 4 grades/types of agglutination?

A

grade 1: isolated <10 spermatozoa per agglutinate, many free
spermatozoa

grade 2: moderate 10–50 spermatozoa per agglutinate, free
spermatozoa

grade 3: large agglutinates of >50 spermatozoa, some spermatozoa
still free

grade 4: gross all spermatozoa agglutinated and agglutinates
interconnected

56
Q

How many sperm per replicate should be assessed for assesment of motility?

A

at least 200 spermatozoa in a total of at least 5 fields in each replicate

57
Q

What is the difference between progressive motility and nonprogressive motility according to WHO 5?

A

Progressive motility (PR): spermatozoa moving actively, either linearly or in a large circle, regardless of speed.

Non-progressive motility (NP): all other patterns of motility with an absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed.

Immotility (IM): no movement.

58
Q

How should you proceed if the first replicates show differences that are greater than acceptable?

A

DO NOT TAKE 3rd and average with first two replicates

discard the first two values and reassess new replicates

59
Q

What is the lower reference limit for motility in WHO 5?

A

total motility = 40%

Progressive = 32%

60
Q

What is indicated by high percentage of immotile and non-viable cells?

A

necrozoospermia
epididymal pathology

61
Q

How is eosin-nigrosin scored?

A

live spermatozoa have white heads and dead spermatozoa have dark pink or red

light/faint pink head are alive

stained neck region only is also alive

62
Q

v What is the lower limit of the vital stain test?

A

58% vitality

63
Q

How is HOS test done therapeutically vs diagnostically?

A

diagnostic - 30 minutes incubation in sodium citrate and fructose solution

Therapeutic - 5 minutes incubation in 1:1 culture medium with sterile purified water

64
Q

What is the volume of one HPF at 200 or 400x

A

One HPF at 200 = 16nl
One HPF at 400 = 4nl

65
Q

What is the minimum volume of semen to be used in dilutions prior to counting?

A

50ul

66
Q

What is the formula to calculate sperm concentration in hemocytometer?

A

C= (N/n) x (1/20) x dilution factor

N= number of sperm countecd
n= number of rows counted
there are 20 nl in each row in grids 4,5,6

NOTE: for 1/20 dilutions the total number of sperm counted divided by the total number of rows assessed equials sperm concentration in 10^6/ml of semen

67
Q

What is the lower reference limit for sperm concentration?

A

15 X 10^6 spermatozoa per ml

68
Q

What is the reference limit for the total number of sperm in ejaculate?

A

39 x 10^6

69
Q

What is the general conception about when the term azoospermia can be used?

A

only if no sperm are found in the sediment of a centrifuged sample

Male Reproductive System (2 decks)

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