SPERM FUNCTION TEST IN EMBRYOLOGY .pptx

SPERM FUNCTION TEST
DR. DEEPTHI REPALLE
IVF LAB DIRECTOR
MOHAK IVF, SAIMS

Normal functions of the sperm
Fertilization

SPERM OOCYTE INTERATION
Capacitation in the cervical mucus
Acrosome reaction
Sperm Zona pellucida interaction
Sperm-Oocyte membrane fusion
Decondensation of sperm nucleus and finally
fusion of male and female pronucleus

HOWEVER…
• The sperms should be sufficient in number and have normal structure
for fertilizing the eggs
• Sperms should have intact and functioning membrane to survive the
vaginal and uterine environment
• Normal functioning of the biochemical markers and proteins
• Motility and mitochondrial function to cross the reproductive barrier
• Sperm should able to survive from stress
• Proper Acrosome functioning to fertilize the oocyte
• Transfer the proper genetic material to the oocyte during the process
of fertilization

Sperm function tests?
What are sperm function tests
Sperm count
Total sperm number
Sperm motility
Progressive motility
These tests detect function of a certain part of spermatozoa and give insights on the events in
fertilization of the oocytes

Who needed these tests?
• Patients with normal semen analysis parameters may not need these
specialized test immediately but, may be later
• Certain patients need some specialized test to understand the
functioning of their sperm better like
• Borderline semen parameters
• Pathological semen parameters
• Previous fertilization failures/TFF
• Prolonged Unexplained infertility/unexplained male infertility

What these tests specify
• Understanding the human sperm and fertilization biochemistry
• Information about important measures like kinematics of sperm
capacitation, hyperactivation,
• The ability of sperm to bind zona-pellucida,
• The ability to undergo acrosome reaction,
• Decondensation of the chromatin to form male pronucleus

Biochemical tests
• Each secretion has a characteristic marker to detect presence, absence,
dysfunction or infection of specific glands of male reproductive system.
• Each specific substance is contributing to sperm function, but how is still unsure
S.no Secretions from Specific Markers Functions
1 Prostate gland Acid phosphatase, Citric
acid, Zinc and
Magnesium
Zinc –protecting and
stabilizing condensed
sperm chromatin
2 Seminal Vesicles Fructose and
Prostaglandins
Fructose-energy source
for sperm for anaerobic
metabolism
3 Epididymis L-Carnitine, alpha-
glucosidase and
glycerophosphocholine

Biochemical tests
Fructosetest
• Patients with azoospermia and in low volume samples (<1 ml)-
seminal obstruction or atresia or ejaculatory duct obstruction.
• Reduced fructose leads to reduced motility .
• Men with high sperm and low motility due to relative deficiency of
fructose.
• Concentration of fructose in semen ranges from 63-500 mg/dl(3.5-28
mmol/l).
• Seliwanoff’s test for ketose sugars or qualitative assessment of
fructose by colorimetric test.

Leucocyte detection tests
• There are different methods for assessing leukocytes in semen,
including identification of round cells, immunohistochemical staining
using monoclonal antibodies, the Endtz test, the peroxidase test, and
flow cytometry.
• The WHO also recommends the peroxidase test, although this test
distinguishes only granulocytes and no other WBC types.
• The gold standard for assessment of WBCs in semen is
immunohistochemical staining using monoclonal antibodies against
specific WBC subpopulations, but this method is expensive, time-
consuming, and not standardized.

• The World Health Organization (WHO) defines leukocytospermia as
>1 × 106 WBCs/mL in a semen sample.
• Leukocytospermia is considered an inflammatory disease. In most
cases, the inflammatory syndrome is secondary to a urogenital
bacterial disorder. However, other conditions may also lead to
leukocytospermia, including viral infections, varicocele, smoking, or
trauma such as spinal cord injury.
• Studies have evaluated the use of antibiotics, anti-inflammatory
agents, and antioxidants as treatment; however, results have been
varied, and controversy remains.

Peroxidase
test

• Endtz test

Reactive oxygen species(ROS)
• Spermatozoa require oxygen for metabolism but the deleterious
metabolites produced such as ROS can modify cell function/damage.
• Seminal plasma naturally contains different antioxidants that help to
protect spermatozoa against such oxidants.
• Seminal plasma oxidative stress develops as a result of an imbalance
between ROS generating and scavenging activities.
• Spermatozoa are more likely susceptible to ROS-induced damage
compared to other cells as they have larger quantities of
polyunsaturated fatty acids(DHA) in the membrane, and their
cytoplasm contains a low concentration of scavenging enzymes.

Reactive oxygen species (ROS)
Ros in semen produced by
- Immature sperms
- Leukocytes
- Epithelial cells
ROS can be estimated by using different methods
• Chemiluminescence using luminol as a substrate
-Normal range <0.2 × 10 6 cpm per 20 million spermatozoa
-Abnormal values >0.2 × 10 6 cpm per 20 million spermatozoa
• Semi-quantitative assays using oxidative indicators
ROS is inversely correlated with normal sperm function and directly
correlated to male infertility

Sperm morphology
• The classification system evolved by Kruger et al. and subsequently
accepted by WHO give very low threshold for accepting normal
morphology and very few samples show more than 25% normal
spermatozoa with most in the range of 4–30% in fertile population.
• According to strict accepted criteria considering normal spermatozoan are:
• Normal sperm head is considered to be 3–5 μ in length and 2–3 μ in width
with perfect oval shape
• Mid-piece is about 1 μ in diameter with straight and regular outline. It
must be aligned to the longitudinal axis of the head and should be 7–8 μ in
length
• The tail must be slender, uncoiled and at least 45 μ in length.

Sperm morphology
Staining methods
• The morphology is evaluated by using air dried smears prepared from
whole semen on glass slides and stained using Diff-Quick or PAP
method.
PAP
Diff
quick

Sperm vitality
• It represents the sperm membrane functionality when it enters the
female tract.
• There are different test for sperm vitality
HOST test (Hypo Osmotic Swelling Test)
Eosin nigrosin test
Eosin test

Sperm vitality
• Eosin-nigrosin test

Mitochondrial activity test
• Mitochondria is necessary for motility of the sperm
• The energy required for the flagellar activity is derived from ATP produced
in the mitochondria concentrated in the mid piece of spermatozoa.
• The long journey of spermatozoa through the female tract requires an
optimal quantity of ATP.
• The mitochondrial oxido-reductive enzyme apparatus can be tested using
indicators like nitro blue tetrazolium, which product blue insoluble pigment
in and around mid-piece.
• Evaluation can be carried out in smears showing stained mid-piece from
good sperms to poor staining of nonmotile or poor mitochondrial activity
sperms.

Acrosome reaction tests
CAPACITATION
• Capacitation can be defined as time dependent, reversible changes sperm
undergo especially in the female genital tract, which lead to the ability of
sperm to acrosome react.
• The changes occur with loss of extrinsic proteins, including acrosome
stabilizing factors along with the loss of membrane cholesterol.
• These intricate steps results in sperms having more fluid and pliable
membranes. During capacitation sperm motility changes to the
characteristic pattern, and induction of AR becomes possible.
• The rapid spiraling pattern of the sperm movement and subsequently
hyperactivated sperm motility is commonly associated with sperm
capacitation.

ACROSOME REACTION
• The integrity of the acrosome structure and the ability to undergo acrosomal
exocytosis are necessary for normal fertility.
• The acrosome reaction is a process that in vivo occurs in the proximity of the
oocyte, and which must take place before the spermatozoon can penetrate the
oocyte vestments and fuse with the oocyte.
• Several stimuli are known to induce acrosome reaction. Among these, zona
pellucida Proteins and progesterone are considered possible physiological
inducers of acrosome reaction in view of their elevated concentration in the
proximity of the oocyte.
• Other stimuli, such as calcium ionophores, will induce the acrosome reaction, but
the results are not or less related to those obtained from the zona pellucida-
induced acrosome reaction

Acrosome reactions can be detected by variety of methods like
1. Labeling with fluorescent lectins,
2. Monoclonal antibodies to specific proteins,
3. Histochemical staining or by binding with antibody-bound beads.
4. Flow cytometry also can be used to detect acrosome reacted sperm
using specific fluorochrome staining.

Sperm penetration assay
.To assess the capacitation and acrosome reaction
also known as “ZONA –FREE HAMSTER ASSAY”
• Zona-free hamster oocyte following the sperm penetration assay.
Positive penetration is demonstrated by completely decondensed
sperm heads; the tail is still visible (white arrows).
• All other sperm are seen attached to the surface
only and are considered negative.

HEMI-ZONA ASSAY
• The zona pellucida is isolated
and divided in half. one half is
incubated with fertile donor
sperm (positive control) and the
other half is incubated with
patient sperm. the ratio of fertile
to donor binding is measured.

Hyaluronan binding assay
To test the sperm capacitation in vitro, HBA is the best method

Sperm DNA fragmentation tests
• The integrity of paternal genome delivered by the spermatozoa is of
paramount importance in the initiation of viable pregnancy.
• The fragmented DNA delivered by the sperm is incompatible with
normal embryonic development.
• DNA damage can occur due to various reasons
• DNA damage can be tested by various tests

Anti sperm antibodies (ASAB)
• ASAB in females due to repeated exposure to sperms,
• ASAB can be present in the cervical mucus (IgA type) or can be
circulating in the blood and other body fluids (IgG type).
• ASAB effect the sperm oocyte interaction and fertilization
• To what extent the antibodies can affect fertility of sperms is still
questionable

Anti sperm antibodies (ASAB)
• There are Various tests to detect the antibodies
• 1. Immunobead test (IB)- no commercial IB tests are available
• 2. Mixed antiglobulin reaction (MAR)- commercially available
• 3. Elisa estimation of antibodies
Results of IB/MAR
• 10-39% of sperms reacted - suspected
• 40% of sperm reacted- high probability
• More than 50% reaction is significant cause of subfertility

Sperm function tests Do we need them in this era?
• Many feel and believe that sperm function testing is now irrelevant
due to advances in IVF, especially ICSI technique.
• ICSI has definitely given hope to couples who previously had virtually
no chance of conception with their own gametes.
• Whether these tests are really needed in the era of ICSI!!!
• Success of ICSI had led to the cessation of research in the area of
sperm function and defects.

Importance !!!
• Because sperm contribute 50% of the genetic material
• For better understanding of basic and applied biology of subfertility
• For couples without severe sperm defects we can provide better
sperm function tests to ensure normal fertilization without the use of
ICSI.
• It is essential to identify the pathology and treat the male partner
which may allow the couples to improve their fertility potential and
improve the ART results and also helps in conceiving naturally too.
• With the rapid growth in ART especially ICSI male partner is
overlooked which is responsible for the half of the infertility cases

Conclusion
• The complex nature of molecular interactions between oocyte and
sperm leading to fertilization needs further clarification
• Since there is more than one sperm function involved, no single test
of a single function will have perfect sensitivity. Therefore a batteries
of tests are required for diagnosis.
• Tests with high sensitivity, high predictive values and low false
positive rates are desirable.
• Sperm function test should not be routine investigations as they are
complex, expensive, not rigorously tested, do not always provide
clinically useful information and typically do not effect treatment

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