Chapter
1.2 THE LEARNING CURVE AND ICSI TRAINING
1.3 PATIENT GROUPS AMENABLE TO TREATMENT WITH ICSI
1.3.1 Oligo-, astheno- and teratozoospermia
1.3.2 Anti-sperm antibodies
1.3.3 Non-ejaculated spermatozoa
1.3.4 Poor or failed fertilization
1.3.5 Pre-implantation genetic diagnosis
1.4 PATIENT GROUPS NOT AMENABLE TO TREATMENT WITH ICSI
2 Media and other consumables for micromanipulation
2.1 MATERIALS REQUIRED FOR THE PREPARATION OF SPERMATOZOA
2.1.1 Plasticware and glass consumables
2.1.3 Density gradient centrifugation media
2.1.4 Media for stimulating motility and assessing sperm viability
2.2 MATERIALS REQUIRED FOR THE PREPARATION OF OOCYTES
2.2.1 Plasticware and glass consumables
2.3 MATERIALS REQUIRED FOR MICROMANIPULATION PROCEDURES
2.3.1 Plasticware and glass consumables
3 Narishige micromanipulation workstation systems
3.1 INSTALLING THE NARISHIGE WORKSTATION
3.1.1 Mounting adaptor – installation
3.1.2 Coarse manipulators – installation
3.1.2.1 Fitting the MMN-1
3.1.2.2 Fitting the MM-89
3.1.3 Micromanipulators – installation
3.1.3.1 Hydraulic manipulators: a brief history
3.1.3.2 The z-axis confusion
3.1.3.3 Installing the MMO-202D
3.1.3.4 Installing the MMO-202
3.1.5.1 Narishige microinjectors: a brief history
3.1.6 Accessories and spare parts
3.1.6.1 Microinjector syringes
3.1.6.3 Dual tool holder HD-21
3.2 SETTING UP THE MANIPULATORS
3.3 FILLING THE MICROINJECTORS
3.4 ALIGNING THE PIPETTES
3.5.1 Manual coarse manipulators
3.5.1.1 Problem – the z-axis of the mechanical coarse manipulator drifts/runs down
3.5.1.2 Problem – the control knob is too loose or too tight
3.5.2 Motorized coarse manipulators
3.5.2.1 Problem – one axis of the motorized manipulator vibrates when activated, or moves intermittently, despite…
3.5.2.2 Problem – one of the motorized manipulators does not work in one axis
3.5.2.3 Problem – one of the motorized manipulators does not work in any axis
3.5.2.4 Problem – the pipettes vibrate when the motorized manipulator is activated
3.5.3 Hydraulic joystick-operated micromanipulators
3.5.3.1 Problem – the pipette moves in the opposite direction to the movement of the hydraulic joystick control…
3.5.3.2 Problem – the pipette moves in the opposite direction to the movement of the hydraulic joystick control…
3.5.3.3 Problem – the pipette moves in an axis at 90 degrees to the one commanded by the hydraulic joystick
3.5.3.4 Problem – the pipette hardly moves/does not move in response to commands from the hydraulic joystick
3.5.3.5 Problem – the pipette hardly moves/fails to move in one axis
3.5.3.6 Problem – there is a purple-coloured precipitate in the hydraulic lines of the micromanipulator
3.5.4.1 Problem – oil leaks from the syringe plunger – barrel interface
3.5.4.2 Problem – oil leaks from the syringe tip – tube connector interface
3.5.4.3 Problem – oil leaks from the pipette – holder interface
3.5.4.4 Problem – the glass syringe breaks when the control knob is turned
3.5.4.5 Problem – there is a delay between movement of the injector control and movement of the sperm in the pipette
3.5.4.6 Problem – there is generally poor control of the oil in the pipette
3.5.5 Micromanipulation workstation in general
3.5.5.1 Problem – the micromanipulator headstages are too far away to allow the pipette tips to be in the microscope field…
4 Eppendorf micromanipulation workstation systems
4.4 TRANSFERMAN NK FEATURES
4.5 TRANSFERMAN NK2 FEATURES
4.5.1 Mechanics, set-up and adjustment
4.5.2 Resolution, range and proportionality of the kinetics
4.5.3 Handling and software
4.8.1 Set-up and optimization of the CellTram Oil
4.8.2 Setting up the manipulators for ICSI
4.9.1.1 Problem – the tip of the pipette appears to vibrate excessively when moving
4.9.1.2 Problem – the x-motor stops moving inboard before it reaches the end of its travel
4.9.1.3 Problem – the z-motor stops moving upwards before it reaches the end of its travel
4.9.1.4 Problem – the z-motor stops moving downwards before it reaches the end of its travel
4.9.1.5 Problem – the pipette appears to jump slightly at a single point in its range of travel
4.9.1.6 Problem – when the home key is pressed, the pipette moves too short a distance to clear the sides of the dish
4.9.1.7 Problem – the motor moves the pipette in the opposite direction to the joystick
4.9.1.8 Problem – the joystick button will not depress
4.9.1.9 Problem – the micromanipulator bleeps continually and will not respond to commands from the joystick
4.9.1.10 Problem – the home setting of the NK2 micromanipulator automatically cancels itself and has to be reset
4.9.2.1 Problem – the griphead will not grip the pipette, no matter how hard it is tightened
4.9.2.2 Problem – the CellTram Air is sucking medium out of the dish, and it is hard to find the equilibrium point
4.9.2.3 Problem – the CellTram Oil has a black deposit on the inside of the cylinder
4.9.2.4 Problem – there is poor control of the sperm in the injection pipette (see also section 8.2.3)
5 Research Instruments micromanipulation workstation systems
5.1.2 Quadruple heated stage
5.1.3 Pipette angle adjustment
5.1.4 Damage-free visible pipette set-up
5.1.5 Screw-actuated syringe injectors
5.1.6 Dual variable-reduction lever stage
5.2.1.1 Problem – cannot focus on to the pipette tip
5.2.1.2 Problem – the pipette does not move smoothly in the xy planes
5.2.1.3 Problem – no fine movement (up and down) on the micromanipulator
5.2.1.4 Problem – joysticks and levers are too stiff/too loose
5.2.1.5 Problem – S16 yellow lever does not stay in the up position (pre-Integra systems only)
5.2.1.6 Problem – micropipette holder axial drive is not driven axially (pre-Integra systems only)
5.2.2.1 Problem – the display shows a ‘?’message
5.2.3.1 Problem – Poor control of the sperm
5.2.3.2 Problem – the sperm drift inside the injection needle after equilibration
6.1 SELECTING THE MICROSCOPE
6.2 SELECTING THE MICROMANIPULATORS
6.2.1 Micromanipulation Workstation Features
6.2.1.5 Pipette set-up and exchange
6.2.1.6 Double tool holders
6.2.1.8 Ease of installation
7 Preparation of gametes for micromanipulation
7.2 PREPARATION OF SPERMATOZO FROM THE EJACULATE
7.2.1 Preparation and use of density gradient centrifugation media
7.3 PREPARATION OF SURGICALLY RECOVERED SPERMATOZO AND SPERMATIDS
7.4 PREPARATION AND SELECTION OF VIABLE IMMOTILE SPERMATOZOA
7.5 PREPARATION OF OOCYTES
7.5.2 Assessing oocyte maturation and quality
7.5.3 In vitro maturation of oocytes
7.6.1.1 Problem – no spermatozoa are recovered following density gradient purification
7.6.1.2 Problem – no spermatozoa are recovered following preparation and washing
7.6.1.3 Problem – all of the spermatozoa are immotile or dead following preparation
7.6.1.4 Problem – the sperm preparation from a surgically recovered sample forms a sticky and viscous interface between the…
7.6.2.1 Problem – using hyaluronidase, it proves difficult to remove the oocyte from the cumulus mass
7.6.2.2 Problem – it proves difficult to pull a glass Pasteur pipette to a diameter appropriate for denuding oocytes
7.6.2.3 Problem – during denudation, an oocyte becomes stuck to the end of a pulled Pasteur pipette
7.6.2.4 Problem – despite using a pulled pipette of the correct diameter, it proves impossible to denude an oocyte adequately
8 Intracytoplasmic sperm injection
8.1 MEDIA AND MICROINJECTION DISH PREPARATION
8.2 FITTING, ALIGNMENT AND EQUILIBRATION OF MICROPIPETTES
8.2.1 Fitting micropipettes to their tool holders
8.2.2 Aligning the micropipettes
8.2.3 Equilibrating the micropipettes
8.3 MANIPULATION OF SPERMATOZOA
8.4 MANIPULATION OF OOCYTES
8.5.1 Microinjection of oocytes with mature spermatozoa
8.5.2 Microinjection of oocytes with immature spermatozoa
8.6 ASSESSMENT OF FERTILIZATION AND QUALITY CONTROL/ASSURANCE
8.7.1 Micropipette fitting, alignment and equilibration
8.7.1.1 Problem – oil fails to exit the tool holder when the injector screw is turned clockwise
8.7.1.2 Problem – the micropipette cannot be inserted past the sealing ring of the tool holder
8.7.1.3 Problem – the tool-holder cannot be attached to the retaining clip of the universal joint
8.7.1.4 Problem – the tool holder cannot be rotated about its long axis whilst held by the retaining clip of the universal…
8.7.1.5 Problem – the range of the coarse manipulator is insufficient to position the micropipette directly over the…
8.7.1.6 Problem – the holding pipette cannot be seen down the microscope after having been positioned directly above the…
8.7.1.7 Problem – the injection pipette cannot be seen down the microscope after having been positioned close to the…
8.7.1.8 Problem – the tip of the injection pipette cannot be brought into focus on the bottom of the microinjection dish
8.7.2 Gamete manipulation
8.7.2.1 Problem – no spermatozoa can be seen after loading the PVP drop with an aliquot of the sperm preparation
8.7.2.2 Problem – it is not possible to make contact with the sperm tail using the tip of the injection pipette.
8.7.2.3 Problem – spermatozoa stick to the tip of the injection pipette while attempting to immobilize them
8.7.2.4 Problem – the heads of the spermatozoa become detached from their tails during attempted immobilization
8.7.2.5 Problem – spermatozoa stick to the bottom of the microinjection dish by their tails following immobilization…
8.7.2.6 Problem – it proves difficult to aspirate immobilized spermatozoa by their tails from the bottom of the PVP drop
8.7.2.7 Problem – spermatozoa rush into the injection pipette uncontrollably while attempting to aspirate them
8.7.2.8 Problem – the spermatozoon does not fit into the injection pipette
8.7.2.9 Problem – spermatozoa drift within the injection pipette despite repeated attempts at stabilization
8.7.2.10 Problem – the tip of the holding pipette cannot be brought into the same focal plane as the oocyte
8.7.2.11 Problem – the oocyte either keeps falling off the holding pipette or becomes increasingly and excessively…
8.7.2.12 Problem – the oocyte becomes stuck to the holding pipette
8.7.3.1 Problem – the spermatozoon cannot be seen in the injection pipette following orientation of the PB of the oocyte
8.7.3.2 Problem – the spermatozoon falls out of the injection pipette tip before injection of the oocyte
8.7.3.3 Problem – the ZP proves difficult to penetrate with the injection pipette
8.7.3.4 Problem – the oolemma fails to rupture following its aspiration into the injection pipette
8.7.3.5 Problem – there is excessive aspiration of ooplasm following rupture of the oolemma and/or excessive outflow…
8.7.3.6 Problem – the oocyte and/or ooplasm sticks to the injection pipette when withdrawing it following sperm deposition
8.7.3.7 Problem – the oocytes appear shrunken following injection
9 Zona manipulation and embryo biopsy
9.1.2 Zona drilling with acid Tyrode’s
9.1.3 Zona dissection with micropipettes
9.1.4 Zona drilling by laser
9.3.1.1 Problem – backfilling the drilling pipette with acid Tyrode’s is extremely slow or impossible
9.3.1.2 Problem – the acid Tyrode’s appears to have no effect on the ZP
9.3.1.3 Problem – the embryo rolls off the holding pipette while attempting to pierce the ZP with an injection pipette
9.3.1.4 Problem – rubbing the ZP between the injection and holding pipettes fails to tear it
9.3.2.1 Problem – it is not possible to pass the biopsy pipette through the ZP following ZD
9.3.2.2 Problem – when the drilling pipette is replaced with the biopsy pipette on the double tool holder, the biopsy…
9.3.2.3 Problem – blastomeres remain attached to that being aspirated, and will exit the hole in the ZP if the procedure…
9.3.2.4 Problem – the embryo becomes dislodged from the holding pipette while attempting to aspirate a blastomere, and the…
9.3.2.5 Problem – the biopsied blastomere lyses
10.2 HISTORICAL PERSPECTIVE
10.3 CURRENT PIPETTE-PULLING TECHNOLOGY
10.5.2 The holding pipette
10.5.3 The injection pipette
10.6 PIPETTE STERILIZATION AND STORAGE
10.7.1 Micropipette puller
10.7.1.1 Problem – it is difficult to program parameters that produce the desired pipette shape
10.7.1.2 Problem – pipette shape is inconsistent from one pull to the next
10.7.2 Micropipette grinder
10.7.2.1 Problem – once grinding is complete, small particles of dust can be seen inside the pipette
10.7.2.2 Problem – there is inconsistency in the size of the tip opening between pipettes
10.7.3 Microforge (Narishige MF-90/900)
10.7.3.1 Problem – the heating element/glass bead is out of focus
10.7.3.2 Problem – it is difficult to focus clearly on the pipette and/or the heating element
10.7.3.3 Problem – the pipette holder will not rotate around the light source
10.7.3.4 Problem – the heater element will not glow, no matter how much power is supplied
10.7.3.5 Problem – when adjusting the x- or y-axis movement of the microscope, the pipette seems to wobble off-axis
11 Transgenesis and the generation of knock-out mice
11.1.4 Source of fertilized one-cell eggs/zygotes
11.1.6 Pronuclear microinjection
11.1.7 Transfer of injected embryos into recipient females
11.1.7.1 Oviduct transfer
11.1.7.2 Uterine transfer
11.2 GENERATION OF KNOCK-OUT AND TRANSGENIC MICE USING ESCs
11.2.2 Design of the targeting construct
11.2.4 Source of blastocysts
11.2.5 Preparation of ESCs for blastocyst injection
11.2.6 Blastocyst injection
11.3 GENERATION OF KNOCK-OUT MICE THROUGH MORULA CO-CULTURE
11.3.1 Preparation of ESCs for co-culture
11.3.1.1 For the preparation of a single cell suspension
11.3.1.2 For the preparation of small clumps of cells
11.4.1 Problem – neither pronucleus is visible during microinjection
11.4.2 Problem – pronuclei do not enlarge during microinjection of DNA
11.4.3 Problem – microinjection DNA becomes diluted with M2
11.4.4 Problem – the embryo rotates when sucked on to the holding pipette, causing the ICM to change position
11.4.5 Problem – the embryo moves around when it is being injected and is not held sufficiently steady by the holding pipette
11.4.6 Problem – it is hard to control the flow of ESCs into or out of the injection pipette
11.4.7 Problem – it is hard to penetrate the blastocyst with the injection pipette – the embryos collapse first
11.4.8 Problem – the ESCs rush out of the embryo when the injection pipette is withdrawn
11.4.9 Problem – it is not possible to keep the injected separate from non-injected embryos
11.4.10 Problem – the oviduct wall is punctured during transfer
11.4.11 Problem – backflow of media/bursal fluid/blood into the transfer pipette
11.4.12 Problem – bleeding in the infundibulum/oviduct area
11.4.13 Problem – unable to determine whether DNA is toxic to the embryo without waiting until recipient females give birth
11.4.14 Problem – unable to assess the pregnancy status of recipient females
11.4.15 Problem – lack of pups born to recipient females (uninjected blastocysts/unmanipulated morulae give plenty of pups)
11.4.16 Problem – chimaeras have low ESC-derived coat colour/are unable to transmit targeted allele to F1 pups
12 New and advanced techniques
12.1 CRYOPRESERVTION OF SPERMATOZOA WITHIN THE ZP
12.3 VISUALIZATION OF THE MEIOTIC SPINDLE FOR ICSI
12.4 OOPLASMIC TRANSFER AND GV TRANSFER
12.5 SOMATIC CELL NUCLEAR TRANSFER
Appendix Suppliers and manufacturers of equipment and consumables
Micromanipulation in Assisted Conception
Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.