human fertilization
TRANSCRIPT
Maryam Borhani-HaghighiPhD student
of Tehran Medical University
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Oocyte maturation
Nuclear maturation
Cytoplasmic maturation
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Oocyte continues meiosis.
Nuclear membrane of oocyte disappears.
First polar body separates, and it enters the perivitelline space.
Second meiotic division takes place and stops in the metaphase II.
This process is known as the maturation of oocyte nucleus.
Nuclear maturation
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Nuclear maturation
Maturation Promoting Factor (MPF) activity during oocyte maturation. MPF activity appears just
before germinal vesicle breakdown (GVBD). It falls down at the end of Meiosis I and reappears at the
beginning of Meiosis II (MII). It remains high during MII-arrest of oocytes and decreases following
fertilization..
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Cytoplasmic maturation
Cytoplasmicmaturation
Organelle redistribution
MitochondriaEndoplasmic
reticulum(ER)
Cortical
granules lipid droplet
Protein synthesis
Cytoskeleton dynamics
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Mitochondria
Before the beginning of oocyte maturation. Homogenous distribution of
mitochondria in the cell cortex is observed
After nuclear maturation, mitochondria are dispersed throughout the cytoplasm
except the very central region of oocytes.
BB - Balbiani bodyM - mitochondrian - nucleus
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Endoplasmic reticulum (ER)
ER :protein and lipid synthesis organelle
store of Ca2+ oocyte activation in during fertilization.
In mature oocytes, an accumulation of ER in the oocyte cortex,
specifically in the region with cortical granule exocytosis and sperm-egg fusion
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Cortical Granule (CG)
Exocitosis of cortical granules plays an essential role in the block against
polyspermy.
In immature oocytes, CGs are found in the whole cytoplasm (the cortex
cytoplasm and the inner cytoplasm)
In mature oocytes, an asymmetric distribution of CGs in the cortex is observed.
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Lipid droplets (LD)
Energy supply necessary for meiotic, maturation, fertilization and early embryo development
In GV - stage oocytes, a homogenous distribution has been reported.
In mature oocytes, Lipid droplets contribute to the oocyte polarization by surrounding the MII
spindle.
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Cytoskeleton dynamics
During the GV stage, microfilaments are distributed throughout the cytoplasm,
and during GVBD they migrate toward the oocyte cortex.
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
The sperm has to pass several barriers to enter the egg.1.layer of cumulus cell.2. zona pellucida
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Maryam Borhani-Haghighi
Zona pellucida
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The zona pellucida (plural zonae pellucidae, is a glycoprotein membrane
surrounding the plasma membrane of an oocyte.
Zona pellucida
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ZP3 mediates Sperm-Specific Egg Binding
ZP2 mediates subsequent sperm binding
ZP1 cross-links ZP2 and ZP3 as protein meshwork
Zona pellucida
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As a receptor
Inducer of Acrosomal Reaction
Blockade of heterospecific fertilization. Species-specific barrier
to sperm binding and penetration;
Prevention of polyspermy
Protection of the embryo before implantation
Functions of the ZP include
• ZP keeps sperm of foreign species out .( Not 100% effective; some cross-species fertilization can occur (e.g., horse & donkey = mule; some speciesof monkeys can cross fertilize) )
• Remove the ZP and other species sperm can fertilize and egg (e.g., Hamster Test for Male Fertility; human sperm can fertilize the zona-less hamster egg)
Maryam Borhani-Haghighi
• The mutant mice which lacked ZP3 couldn't bind the sperm to the zona pellucida and thus did not get fertilized
• When their missing ZP3 gene was replaced with a human ZP3 gene, the mice made the human ZP3 and it was incorporated into their zona pellucida. With the human ZP3 in their zonas the eggs could be fertilized by mouse sperm revealing that human and mouse ZP3 can mediate the same critical events.
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Zona pellucida
Inner zone
Outer zone
Birefringence
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(a): Outer surface of the ZP of a human mature oocyte. Many fenestrations
are present in which the filaments form a large meshed network
(b): Outer surface of the ZP of a human atretic oocyte. The filaments form a tight meshed network.
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(b): The ZP appears completely compact and with a smooth surface.
(d): High magnification of (b) showing the compact and dense structure of the zona.
Human atretic oocyte
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This metaphase II oocyte displays an atypical aspect of the
ZP. Projections from the ZP give the oocyte a hairy, brush-
like appearance.
Abnormality of the zona pellucida
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Maryam Borhani-Haghighi
Capacitation of sperm
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Capacitation of sperm
Reduction in membrane cholesterol increase fludity
Hyperactivaty of sperm ;lateral movement of head
Removal of coating factors
Only capacitated sperm can bind to the ZPMaryam Borhani-Haghighi
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Maturation-Ejaculation-CapacitationLeaving testis-Epididymis-seminal plasma-Oviduct
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5 sets of molecular changes are considered important for capacitation:
1.Sperm membrane sheds cholesterol by albumin
allowing for zona pellucida recognition
2.Protein/carbohydrate loss opens sperm-egg
recognition sites
3.Membrane potential of the cell membrane become
more negative activating cAMP production
4.Protein phosphorylation occurs forming a receptor
5.Acrosomal membrane changes
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Reduction In Membrane Cholesterol
Cholesterol efflux during the early phases of capacitationincreases plasma membrane fluidity,
facilitating the entry of bicarbonate (HCO3-) and calcium ions (Ca2+) into the sperm cytosol through specific membrane channels.
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Model of mammalian sperm capacitation
soluble adenylyl
cyclase(SACY)
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which then simultaneously inhibits the activity of tyrosine phosphatases (PTP) and activates tyrosine kinases (PTK).
cAMP, activate protein kinase A (PKA),
leading to the production of the second messenger cyclic AMP (cAMP).
soluble adenylyl cyclase (SACY) is activated by increases in intracellular bicarbonate, calcium and pH
increase in protein tyrosine phosphorylation
capacitation
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• membrane remodeling events(Cholesterol ) redistribution of membrane rafts to the anterior region of the sperm head.
• This event may serve to reposition key zonapellucida receptor molecules
• membrane remodeling events may be augmented by the action of chaperones that are themselves activated during capacitation.
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EGF
Epidermal growth factor
(EGF)interacts with its receptor
tyrosine kinase (EGFR)
stimulates the tyrosine
phosphorylation of several proteins
enhances actin polymerization
during capacitation
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Hyperactivaty of sperm
CatSper, sperm cation channel
that is localized in a principal
piece of the mature sperm tail.
gene knockouts that are CatSper
have poorly motile sperm that
completely fail to fertilize eggs
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Role of actin cytoskeleton in mammalian sperm capacitation
• In human sperm the regions reported to contain actin include the acrosomal space,
the equatorial and post acrosomal regions, and the tail.
actin polymerization and depolymerization might be involved in sperm
function.
actin polymerization occurs during capacitation
F-actin breakdown should occur in order to achieve the acrosome reaction.
inhibition of actin depolymerization by phalloidin inhibits the reaction.
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Sperm penetration to corona radiata
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hyaluronic acid–rich layer of cumulus cells surrounds the ovulated
oocyte.
Sperm penetration to corona radiata
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Acrosomal reaction & penetration to zp
The Acrosome Reaction:
• When sperm contacts the egg cell layers, the it has an intact acrosome.
• stimulation of the sperm by agents from the corona radiata followed by binding to the zona pellucida, leads to the acrosome reaction
Maryam Borhani-Haghighi
The Acrosome Reaction: Galactosyltransferase binding
• Here we will look at only one of the classic mechanisms of sperm-egg binding that leads to the acrosome reaction:
Galactosyltransferase binding that occurs at the zona pellucida.
Maryam Borhani-Haghighi
Galactosyltransferase
• Each sperm has galactosyltransferase(GalTase) enzymes on its head
• Galactosyltransferase is an enzyme that transfers a sugar group from one molecule to another
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Unlike normal enzyme reactions in which the enzyme binds its substrate and produces products, this enzyme reaction cannot go to completion. Since it can’t complete the reaction the enzyme and the substrate remain attached to each other.
Maryam Borhani-Haghighi
• Each sperm binds N-acetylglucosamine, GlcNAc) residues in ZP3 via galactosyltransferase (GalTase) enzymes in the sperm cell membrane. Thus the GlcNAc is the substrate for the GalTase enzyme.
• so the sperm remain attached via the sugar-enzyme binding because the enzyme reaction cannot go to
completion .
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ZP3 and the Acrosome Reaction
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• As more GalTase enzymes bind more GlcNAc substrates the receptors on the sperm head cluster together
• This clustering alters the sperm cell membrane causing calcium levels to increase in the sperm cytoplasm
• The increase of intracellular calcium mediates the fusion of the acrosomal and sperm cell membranes
• This allows the contents of the acrosome to flow out
• The released Acrosomal enzymes now begin to digest a path for the sperm through the zona pellucida
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Calcium & the Acrosome Reaction
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
In freshly ovulated eggs, ZP3 is in very close physical association with ZP2.
Acrosome-reacted sperm bind to ZP2 via their exposed inner acrosomal membranes, penetrate the zona, fuse with the oocyte, and fertilize the egg.
Maryam Borhani-Haghighi
Immediately after fertilization, cortical granules release a ZP2-specific protease and other enzymes into the perivitellinespace.
This cortical granule protease clips ZP2 and converts it into the cleaved form (ZP2c) that no longer supports the binding of acrosome-reacted sperm.
ZP3 dissociates from ZP2c, and undergoes a subtle modification that converts it into a form (ZP3f) that lacks sperm receptor and acrosome-inducing activity.
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Hyaluronidase
neuraminidase
acrosin
protease
trypsin-like enzyme
Arylsulfatase
PLC
Esterase
Maryam Borhani-Haghighi
Ca2+-
Calcium is an important modulator for capacitation and AR
and is probably the key messenger in the information
exchange between sperm and egg .
Progestrone
Follicular fluid
Coffein
K+ and Na+
Acrosomal reaction
SOCStore-operated channels
Progesterone cause Ca2+influx::::::Elevation of Ca2+in the midpiece/proximal flagellum ::::::leading to regulation of flagellar activity
Maryam Borhani-Haghighi
What is progesterone?Progesterone is a steroid hormone made by both men and women.
In women, it is made the corpus luteum of the ovarythe adrenal glands.In men, is producedin adrenal testicular tissue.
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Result of Acrosomal Reaction
Acrosomal bulb
Ca / cAMP
Ph (7.1 7.5)
Zp Zp2f
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Sperm oolemma binding & fusion
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Sperm oolemma binding & fusion
• The observation that acrosome intact sperm can bind to zona-freeeggs, but do not fuse with them, suggests that membrane alterations occurring during the acrosome reaction are required for fusion.
Sperm Binding to the Zona & the Egg Cell Membrane
• several proteins that mediate these membrane interactions, fertilin b. Mouse mutants that lack fertilin b have a markedly reduced fertility.
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The sperm plasma membrane
• Fertilin α
• Fertilin β
• Cyritestin
• ADAM 4
• ADAM 5
• IZUMO
molecules on the sperm surface, such as fertilin and cyritestin3, involved in sperm–egg bindingIzumo is essential for membrane fusion
On the egg, CD9 is required for fusion and might collaborate with other proteins such as integrins or glycosylphosphatidylinositol (GPI)-anchored proteins
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• members of a new family of membrane proteins, the ADAM family (a disintegrin and metalloprotease disintegrin
• cysteine-rich
• EGF-like
Fertilin
• fertilin, a heterodimeric (α and β subunits) sperm membrane protein, is involved in the fusion process.
• This protein is located on the posterior head of acrosome-reacted sperm.
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• Localization of cyritestin in the equatorial region is consistent with itsparticipation in sperm–egg fusion.
• Although both the equatorial regions and posterior head of the spermmembrane fuse with the egg membrane,
cyritestin
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The Egg
.CD9
.CD81
Glycosylphosphatidyl
inositol (GPI)-anchored
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• immunofluorescence microcopy shows that CD9 is localized to the microvillar-
rich region of the egg
CD9
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CD81
• CD81, which fairly closely resembles CD9 in structure
and some functions, has a role in gamete fusion.
However, the results of deleting the CD81 gene are
less dramatic than those seen with the deletion of
CD9.
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Sperm oolema binding & fusion
The egg plasma membrane can be divided into two major regions.
1.The part of the membrane that directly overlies the metaphase
chromosomes has a smooth surface devoid of microvilli.
2. The remainder of the egg is rich in microvillar protrusions. This is the region
of the egg where sperm both bind and fuse.
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Fertilization cone
Block to Polyspermy
• Special blocks to polyspermy exist:
• 1. Fast Block: electrical change in egg membrane
2. Slow Block: modification of zona pellucida
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Maryam Borhani-Haghighi 70
Slow block to polyspermy
• Elavation of Ca2+
• Cortical granule reaction
Exocytosis Perivitteline space
Zp2
β-hexoaminidase
Tyrosin
Slow block of polyspermyThe Cortical Reaction
• Calcium not only plays a role in the acrosome reaction, it also mediates the subsequent event of corticalgranuleexocytosis in the egg.
• Ca2+ induces local exocytosisof cortical granules
• Granules release to stimulate adjacent cortical granules to undergo exocytosis
• Wave of exocytosis occurs around egg in 3 dimensions from original site of sperm entry Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi
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Calcium signals at fertilization
• A “Ca2+ wave” that starts from the site of sperm–egg fusion
and propagates across the egg cytoplasm has been
extensively analyzed
Fast block of polyspermy
• is the electrical charge across the surface of the egg, which is caused by the fusion of the first sperm with the egg.
Elevation of intracellular PH
Change the membrane potential
Resting potential: -75 mV
Fertilization potential: +20 mV
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Maryam Borhani-Haghighi
a) Glutathione
b) Heparin
c) Zn
Decondensation of sperm nucleus
From the following cell is seen the protamineto histone transition where maternally derived
histones replace protamines resulting in the decondensation of the sperm head
Maryam Borhani-Haghighi
Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione
(GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor
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A significant fraction of the zinc is lost from sperm chromatin when the cysteinethiols in protamine are oxidized into disulfide bonds.
Extraction of zinc from the freshly ejaculated spermatozoon allows immediate decondensation
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• Male PN : 22.1 µm
• Female PN : 24.4 µm
• Male PN has more Nucleoli
• Female PN is close to polar body
The male pronucleus is colorized in blue; female in pink.
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Role of tail
sperm-aster
A centrosome with astral rays in the cytoplasm of
an inseminated ovum;
it is brought in by the penetrating spermatozoon
and gives rise to the mitotic spindle of the first-
cleavage division.
Proximal centriol
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syngamy
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Syngamy
Union of gametes resulting
in formation of a zygote
syngamy
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Singamy
At gamete fusion the sperm tail is incorporated into the ooplasm,the centriolar region forms the sperm aster while the sperm head is decondensing; this aster acts to guide the female pronucleus towards the male pronucleus. The centriole duplicates during the pronuclear stage, and at syngamy centrioles are found at opposite poles of the first cleavage.
The centrosome has several implications for human infertility.It is possible that immotile or nonprogressively motile spermatozoa may possess centriolar abnormalities or an absence of centrioles.Similarly, antisperm antibodies against centrioles may be responsible for mitotic arrest. One way of solving this problem would be the use of donor centrosomesMaryam Borhani-Haghighi
Maryam Borhani-Haghighi
Pronuclear scoring system
• 16-18 hours after fertilization
• Grading is based on:
– Pronuclear size, symmetry & position
– Size, number, equality & distribution of nucleoli
– Appearance of cytoplasm
Van Blerkom (1990)
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Pronuclear size & symmetry
Embryos containing tree or More PNs are polyploid:
injection of more than one sperm
sperm chromatin disperse
second polar body extrusion failure
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What is cleavage?
Cleavage is a rapid series of mitotic divisions that occur just after
fertilization.
There are two critical reasons why cleavage is so important:
1. Generation of a large number of cells that can undergo
differentiation and gastrulation to form organs.
2. Increase in the nucleus / cytoplasmic ratio. Eggs need a lot of
cytoplasm to support embryogenesis. It is difficult or impossible
for one nucleus to support a huge cytoplasm, and oocytes are one
of the largest cells that exist. One small nucleus just cannot
transcribe enough RNA to meet the needs of the huge cytoplasm.
Maryam Borhani-Haghighi
Cleavage differs from normal mitoses in 2
respectsNormal eukaryotic cells divide slowly,
once every several hours or days. The
cell cycle has G1 and G2 periods. During
G1 the cell synthesizes RNA and other
components for cell growth.
Cleavage consists of very rapid
successive mitoses. Since the egg has
stored large amounts of RNA and other
material, it does not need G1 or G2.
However, as the number of cells
increases, the nucleus / cytoplasmic ratio
also increases. The rate of cell division
slows because the cell now needs to
synthesize its own RNA and grow
between divisions. Thus, G1 and G2 are
restored
Maryam Borhani-Haghighi
Asynchronous cleavage: mammalian embryos are unusual in that they have
asynchronous cleavage. Not all blastomeres divide at the same time.
The first cleavage is meridional, and the second cleavage is rotational.
The 2 blastomeres divide in different planes (one is equatorial and one is meridional.
Maryam Borhani-Haghighi
Cleaved embryo assessment
24-28 hours after insemination
2 cells
40-44 hours after insemination
4 cells
64-68 hours after insemination
8 cells
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•Morula stage in mammals begins when the embryo
consist of 16 blastomers , occurs 3 to 4 days after
fertilization, when embryo passes from oviduct into
the uterus
MORULA
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Compaction
During compaction each of the eight blastomeres undergo a polarization.
Polarization and the formation of tight junction allow the blastomers to
create an inner embryo environment that is different from the outside
environment
Tight junctions develop beneath ext. surfaces and gap junction form
between the internal surfaces
Maryam Borhani-Haghighi
Blastocyst Morphology
• Thin zona pellucida.
• Smooth trophoectoderm.
• Equality & close adhesion of blastomeres.
• Clearly visible blastocyst cavity.
• Well developed inner cell mass.
Blastocyst scoring is based on:
blastocyst, Inner cell mass, trophectoderm
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eomesodermin
Cdx2
Trophoblast
Oct4
Nanog
ICM
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The act of hatching involves the local digestion of the zona by an enzyme produced in
patch of trophoblast cells situated opposite the inner cell mass (this location
minimizes the risk of collateral enzymatic damage to the embryo
Having escape from the zona ,the blastocyst begins the process of implantation
Hatching
Maryam Borhani-Haghighi
Metabolic Shift
• Maturing oocytes use more pyruvate than immature and non-transitional oocytes.
• In the early stages of early development the embryo uses pyruvate switching to
glucose.
• The use of pyruvate, glucose and lactate production indicating embryo health.
Maryam Borhani-Haghighi
• Blastocyst with a glucose uptake >5µg/h develop
better in culture and give rise to more pregnancies.
• Morphologically normal blastocyst use more
glucose than degenerating blastocyst.
Thank you for your attention
Thank you for your attentionMaryam Borhani-Haghighi
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Thank you for your attention
Developing EmbryoThank you for your attention
Maryam Borhani-Haghighi
Maryam Borhani-Haghighi