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ICSI Lab Procedures
For Gynecologist
Aboubakr Elnashar
Benha University, Egypt
ABOUBAKR ELNASHAR
Lab Procedures
1. Semen Preparation.2. Oocyte Identification.3. Oocyte Denudation.4. Oocyte Assessment5. Oocyte Injection.6. Embryo Selection7. Embryo Transfer.8. Cryopservation.
ABOUBAKR ELNASHAR
qProcedures ú Day 0 Sperm collection, and preparation Oocyte retrieval Insemination in vitroú Day 1 Check for fertilization (presence of 2 pronuclei or PN's)ú Day 2 Embryos at the 4-cell or more stage of developmentú Day 3 Embryos at the 8-cell or more stage of developmentú Day 4 Embryos at the compacted morula (16-32 cell) stageú Day 5 Embryos at the blastocyst stage of development
ABOUBAKR ELNASHAR
1. Semen Preparation.
q Obtaining the semen sample 1. An abstinence interval:
3-5 days. 2. Collection of the semen:
In the clinic is better than in the patient’s home.3. Timing of processing of the semen:
With in 30 mins from ejaculation.4. A frozen back-up sample should be requested
if sperm collection difficulty on the day of OR is anticipated.
ABOUBAKR ELNASHAR
qAim of Sperm preparation: 1. Eliminate:
seminal plasma, debris and contaminants2. Concentrate:
progressively motile sperm3. Select:
against morphologically abnormal sperm.
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q Methods of Sperm preparation:
1. Discontinuous density gradient.2. Double wash.3. Swim up.The swim-up technique and discontinuous density-
gradient centrifugation are most frequently used and widely accepted.
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qSelection of Sperm preparation method:•Characteristics•Origin of individual samples.
qIn absence of motile sperm. -Phosphodiesterase inhibitors (pentoxifylline, theophylline) or-Hypo-osmotic swelling (HOS) test
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qAzoospermia on the day of OR and in the absence of a back-up sample:
•Sperm retrieval procedures or •Oocyte cryopreservation
qIf no sperm are observed. Enzymatic digestion of testicular tissue by collagenase
ABOUBAKR ELNASHAR
2. Oocyte identification
§ Aim:Identify the oocytes from the aspirated follicular fluid.oocytes are washed from the surrounding follicular
fluid (mimic physiology)§ The time between OR and culture of washed
oocytesshould be minimal. {Prolonged oocyte exposure to follicular fluid is not
recommended} § Exposure of oocytes to light
should be minimized.ABOUBAKR ELNASHAR
3. Oocyte Denudation
§ Chemical: repeated pipetting in hylaundase enzyme.
§ Mechanical: using thin pipettes of varying diameters (175-125 µ)
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A and B: GVC : MI D : MII oocyte.
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4. Oocyte Assessment
q Importance of oocyte quality:§ has a great influence on: § Fertilization§ early development§ implantation of embryos.§ In order that the oocyte would be successfully
fertilized and cleaved, the nucleus and the cytoplasm of the oocyte must be mature at the same time.
ABOUBAKR ELNASHAR
qNuclear Maturation1. Germinal Vesicle.
2. Metaphase 1 oocytes.
3. Metaphase 2 oocytes.
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Oocyte maturity after cumulus and corona cell removal. (A)Germinal vesicle (GV) stage oocyte is recognized by the
presence of a typical GV. (B)Oocytes that have undergone GV breakdown but not yet
extruded the first polar body are called metaphase I oocytes. (C)Metaphase II oocyte displays the presence of a first polar
body, which indicates that the oocyte is mature and has reached the haploid state. Only metaphase II oocytes are submitted to intracytoplasmic sperm injection.
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1. Germinal Vesicle GV oocyte.
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2. Metaphase 1 oocyte
§ After GV breakdown oocytes reach metaphase I, characterized by the absence of the first polar body extrusion.
§ Though some of these intermediate eggs may even be fertilizable (De Vos et al., 1999),
ABOUBAKR ELNASHAR
qRetrieved immature oocytes after COH ú Usually a variable percentage of the retrieved
oocytes from mature follicles after COH are GV or M I.ú These oocytes may change to M II oocytes in vitro,
and hence can be injected.ú However, the reproductive outcome of these
oocytes is very poor.ú This effect may not be evident until pre and early
postimplantation period.ú This poor outcome can be explained by the
asynchronous nuclear and cytoplasmic maturity.
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qFertilization and pregnancy potential of immature oocytes from stimulated ICSI cycles.(Shin et el , 2013)
§ The aim was to compare the outcome of retrieved mature oocytes and retrieved immature oocytes that were injected after maturation.
§ The fertilization rate of retrieved immature oocyteswas half that of retrieved mature oocytes (37% Vs 73%).
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3. Metaphase II oocyte
§ Large uniform cytoplasm§ Discrete intact first polar body
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Oocytes in first row represent different degrees of vacuoles in cytoplasm. Each oocytes in second row has increased central granularity.
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qCytoplasmic maturation
§ The only method: oocyte fertilizationembryo development in vitro.
§ If the cytoplasm of oocytes is immature: embryo cannot develop normally after
fertilization.
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qDefects in Cytoplasmic maturation lead to:
1. Retarded embryos2. Cleavage arrest3. Failure of implantation
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5. Oocyte Injection
1. Sperm selection.2. Sperm immobilization.3. Oocyte adjustment:
polar body is at 12 or 6 o'clock.4. Penetration of the oolemma5. Aspiration of the cytoplasm.6. Injection of the sperm.
ABOUBAKR ELNASHAR
(C) The injection pipette is introduced at the 3 o0clock position and rupture of the oolemma is ascertained by slight suction. Then the sperm cell is delivered into the oocyte with a minimal volume of medium; afterwards, the pipette can be carefully withdrawn.(D) A single sperm cell can be appreciated in the center of the ooplasma
A.single motile sperm isselectedand immobilized by pressing its tail between the microneedleand the bottom of the dish. The sperm cell is thenaspirated tail-first into theinjection pipette. B.Usingthe holding pipette, the mature oocyte is fixed with the polar body at the 6 o0clock position. The sperm cell is brought to the tip of the injection pipette.
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6. Embryo selection
qEmbryos are graded according to:1. Rate of cleavage: (retarded embryos).2. Anucleated fragments.3. Equal sized cells. §Class A embryos§Embryos of variable grades.
ABOUBAKR ELNASHAR
qScoring for fertilization §All inseminated or injected oocytes should be examined for the presence of pronuclei (PN) and polar bodies at 16 to 18 hours post insemination. §A normally fertilized oocyte contains 2PN and 2 polar bodies. §For conventional IVF, cumulus cells must be removed and 2PN oocytes transferred into new dishes containing pre-equilibrated culture medium.
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§Abnormal fertilizations. A : egg with1 pronucleus (PN) B with 3 C with 4 PN.
§Normal fertilizationOocyte in D, E or F, each has 2 PN.
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Fertilization outcome after intracytoplasmic sperm injection. (A) Oocytes are considered normally fertilized when two individualized or fragmented polar bodies are present together with two clearly visible pronuclei (2-PN) that contain nucleoli. (B) Abnormal fertilization may occur as one pronuclear (1-PN) oocyte, probably due to parthenogenic activation. (C) The occasional finding of three pronuclear (3-PN) oocytesafter injection of a single spermatozoon into the ooplasm isprobably caused by non-extrusion of the second polar body at the time of fertilization.
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§Embryos derived from ≥ 3PN oocytes should never be transferred or cryopreserved. §Under exceptional circumstances, if no transferable embryos derived from 2PN oocytes are available, embryos derived from 1PN oocytes or oocytes showing no PN but going through normal cleavage may be used for transfer.
§Even if no transferable embryos derived from 2PN oocytes are available, the use of embryos derived from 1PN oocytes or oocytes showing no PN is not recommended.
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The blastomere number is recorded and the embryos are scored accordingly to equality of size of the blastomeres and the presence of anucleate cytoplasmicfragments. On day 4 (sometimes already on day 3), a certain degree of compaction can be observed (E). For blastocyst (F) scoring, the classification system introduced by Gardner and Schoolcraft is used. Embryo transfer is usually done on day 3 (eight-cell stage) or day 5 (blastocyststage).
Embryo cleavage after intracytoplasmic sperm injection. Only embryosresulting from normally fertilized oocytes (A) will be transferred to patients. Embryo cleavage is evaluated daily. Two-cell embryos (B), four-cell embryos (C), and eightcellembryos (D) are usually obtained on day 1 (late afternoon), on day 2, and in the morning of day 3, respectively.
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Timeline for optimal blastocystdevelopment.
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7. Embryo transferqEmbryo culture and transfer §In order to optimise embryo development, fluctuations of culture conditions should be minimised. §Precautions must be taken to maintain adequate conditions of pH and temperature to protect embryo homeostasis during culture and handling.
ABOUBAKR ELNASHAR
qEmbryo scoring §should be performed at high magnification (at least 200x, preferably 400x) using an inverted microscope with Hoffman or equivalent optics.qEvaluation of cleavage stage embryos should include
1. cell number2. size and symmetry3. percentage of fragmentation4. Granulation5. Vacuoles6. nuclear status (e.g. multinucleation).
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qBlastocyst scoring should include 1. expansion grade, 2. blastocoel cavity size3. morphology of the inner cell mass (ICM) 4. trophectoderm (TE).
qAssessment should be performed at standardisedtimes post-insemination.
ABOUBAKR ELNASHAR
Scoring system for human blastocysts. Initially blastocysts are given a numerical score from 1 to 6 based upon their degree of expansion and hatching status: (1) early blastocyst; the blastocoel being less than half the volume of the embryo; (2) blastocyst; the blastocoel being greater than or equal to half of the volume of the embryo; (3) full blastocyst; the blastocoel completely fills the embryo; (4) expanded blastocyst; the blastocoel volume is now larger than that of the early embryo and the zona is thinning; (5) hatching blastocyst; the trophectoderm has started to herniate through the zona; (6) hatched blastocyst; the blastocyst has completely escaped from the zona. ABOUBAKR ELNASHAR
qTime-lapse imaging §Assess embryo development §more precise evaluation of the timing of consecutive events§not interfering with the embryo culture environment.
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qEmbryo selection for transfer is primarily based on 1. developmental stage2. morphological aspects. 3. Other selection parameters, such as time-
lapse kinetics, may be considered.
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qNumber of embryos to be transferred:§should be based on:
1. embryo quality2. stage of development3. female age4. ovarian response5. rank of treatment.
§Single embryo transfer is recommended to avoid multiple pregnancies. qIt is advisable not to transfer more than two embryos. qSupernumerary embryos may be cryopreserved, donated to research or discarded, according to their quality, patient wishes and national legislation.
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qEmbryo loading:Air bubbles to bracket the embryo-containing medium
Insufficient evidence to suggest that the fluid-only method is superior to the use of air brackets during embryo loading. (Abou-Setta, 2007)
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qTime interval between embryo catheter loading and discharging the embryos in the uterine cavity
§ The longer the duration, the lower the pregnancy and implantation rates.
§ The decrease in pregnancy and implantation rates is gradual until the duration of 120 s, and decreases sharply afterwards.
(Hum Reprod. 2004)
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9. Cryopreservation
qMethods:1. Slow freezing2. Vitrification
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qVitrification§ solidification of a solution (water is rapidly cooled
and formed into a glassy, vitrified state from the liquid phase) at low temperature§ Not by ice crystallization but
by extreme elevation in viscosity during cooling.§ During vitrification the § entire solution remains unchanged §water does not precipitate: no ice crystals are
formed.
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ABOUBAKR ELNASHAR
qCryopreservation
Steps:
1.1. Equilibration in the cryoprotectantEquilibration in the cryoprotectant2.2. Freezing processFreezing process3.3. Storage in LNStorage in LN22
4.4. Thawing (warming) processThawing (warming) process5.5. Removal of the cryoprotectantsRemoval of the cryoprotectants6.6. Culture in the physiological milieuCulture in the physiological milieu
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qAction of the cryoprotectant
Penetration of cryoprotectant in the cell andPenetration of cryoprotectant in the cell andpartially replacing thepartially replacing the↓ ↓ intracellular waterintracellular water↓↓dehydration of the celldehydration of the cell
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qCryopreservation can be performed for Gametesembryostissues.
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qSelection of mehod:according to the type of biological material. §For sperm:
slow freezing is still the method of choice, but rapid cooling is a possible alternative.
§For oocytesvitrification has been reported to be highly successful and is recommended.
ABOUBAKR ELNASHAR
qFor cleavage stage embryos and blastocystshigh success rates have been reported when using vitrification. However, for cleavage stage embryos good results can also be obtained using slow-freezing methods.
qFor tissuesthe method of choice is slow freezing, but vitrification of ovarian tissue is an option.
ABOUBAKR ELNASHAR
ABOUBAKR ELNASHAR
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