Cover -- Half-title -- Title -- Copyright -- Dedication -- Contents -- Preface -- Acknowledgments -- 1 Introduction -- Further reading -- 2 Producing gametes -- Oocyte growth -- Follicle development -- Storing information -- The regional organization of the oocyte: polarization -- Oogenesis in the human -- Meiotic arrest and resumption of meiosis -- Resumption of meiosis -- Spermatogenesis in mammals -- Further reading -- 3 Sperm-oocyte interaction -- The acrosome and the vitelline coat -- Sperm-oocyte fusion -- Activation of the spermatozoon -- Motility -- Chemotaxis -- Capacitation -- Acrosome reaction -- Sperm-oocyte interaction in mammals -- Capacitation in mammals -- Acrosome reaction -- Sperm-oocyte fusion -- Oocyte activation -- Releasing meiotic arrest -- 1.The G-protein hypothesis -- 2.The soluble sperm factor hypothesis -- Intracellular calcium release -- Calcium and the cell cycle proteins -- The cortical reaction -- The cortical reaction in mammals -- The role of cortical reorganization -- Fusion, centrosomes and pronuclei -- Centrosomes -- Formation of pronuclei -- Pronucleus formation in mammals -- Syngamy -- 'The sun in the egg' -- Further reading -- 4 First stages of development -- Activation of the zygote genome -- Imprinting -- Compaction -- Causes of embryo arrest -- Paternal factors -- Metabolic requirements of the early mammalian embryo in vitro Yves Ménézo -- Cleavage patterns -- Cytoplasmic segregation and the formation of cell lines -- Further reading -- 5 Endocrine control of reproduction -- Further reading -- 6 Assisted reproductive technology in farm animals -- Artificial insemination -- Bovine IVF -- (with thanks to R.Brittain) -- Maturation (IVM ) -- Fertilization (IVF ) -- Embryo culture (IVC ) -- IVF during pregnancy -- Results -- The large calf syndrome -- Pregnancy results.

Sex distribution of in vitro produced calves -- Gender selection -- Microsurgery in mammalian embryos -- Further reading -- 7 The clinical in vitro fertilization laboratory -- Introduction -- Setting up a laboratory: equipment and facilities -- Ambient air quality -- Laboratory space layout -- Incubators -- Water quality -- Supplies -- Daily cleaning routine -- Washing procedures -- Pipettes -- Non-disposable items (handle with non-powered gloves, rinsed in purified water) -- Tissue culture media -- Serum -- Growth factors -- Follicular flushing -- Quality control procedures -- Suggested useful routine QC procedures include the following: -- 1. Sperm survival test -- 2. Culture of surplus oocytes -- 3. Multipronucleate embryo culture -- 4. Culture of 'spare' embryos -- Tissue culture systems -- Oil preparation -- Serum supplements -- Preparation of maternal serum -- Basic equipment required for the IVF laboratory -- Embryology -- Details of IVF media can be obtained from the following manufacturing companies: -- Further reading -- 8 Semen analysis and preparation for assisted reproductive techniques -- Semen assessment -- Collection of semen samples -- Liquefaction -- Measure the volume using a graduated pipette or syringe -- Assessment of density and motility -- Progression -- Progression is assessed on a scale of 0-4 (The Macleod Scale) -- Morphology -- Chromatin staining -- 1. Chromomycin (CMA3) staining -- 2. Aniline Blue Staining (AAB) -- Antisperm antibodies -- Preparation of sperm for in vitro fertilization/GIFT/intrauterine insemination -- Sterile technique must be used throughout -- Standard swim-up or layering -- Pellet and swim-up -- Discontinuous buoyant density gradient centrifugation -- Methods -- Isotonic gradient so·lution -- Mini-gradient (95/70/50) -- Sedimentation method or layering under paraffin oil.

High-speed centrifugation and washing -- Chemical enhancement of sperm motility prior to insemination -- Sperm preparation for ICSI -- No motile sperm -- The hypo-osmotic swelling test (HOS) -- 100% abnormal heads -- Sticky sperm -- Excessive amounts of debris -- Retrograde ejaculation and electroejaculation: sperm preparation -- Obstructive and nonobstructive azoospermia: epididymal and testicular sperm -- Spermatid identification -- Sperm preparation: equipment and materials -- Further reading -- 9 Oocyte retrieval and embryo culture -- Programmed superovulation protocols -- Protocol -- Preparation for each case -- Microdroplets under oil -- Four-well plates -- Oocyte retrieval (OCR) and identification -- Insemination -- Microdroplets under oil -- Four-well dishes -- Scoring of fertilization on Day 1 -- Embryo dissection -- Dissection techniques -- Making narrow-gauge pipettes -- Scoring of pronuclei -- Reinsemination -- Selection of surplus pronucleate embryos for cryopreservation -- Embryo quality and selection for transfer -- Blastocyst transfer -- Selection of embryos for transfer -- Fragments -- Cytoplasmic donation -- Embryo transfer -- Grade 1 -- Grade 2 -- Grade 3 -- Grade 4 -- Grade 5 -- Grade 6 -- Remaining embryos -- Embryo transfer procedure -- Materials -- Preparation of embryos for transfer -- Gamete intrafallopian transfer (GIFT) -- GIFT procedure -- Transport IVF and transport ICSI -- Coculture systems -- Trypsinization -- Further reading -- 10 Cryopreservation -- Benefits and concerns of an embryo cryopreservation programme -- Principles of cryobiology -- John Morris -- Direct effects of reduced temperatures -- Physical changes associated with ice formation -- The effects of freezing on cells -- Supercooling and cell survival -- Ice nucleation -- Vitrification -- Storage of cryopreserved samples -- 1. Within the freezing apparatus.

2. During storage -- 3. From liquid nitrogen -- Embryo freezing and thawing -- Embryo selection for cryopreservation -- Media preparation -- MATERIA LS -- Freezing solutions -- Thawing solutions -- Method -- Loading into straws -- Ampoules -- Freezing programme -- Thawing -- Blastocyst freezing -- Planer Kryo 10 operation -- Clinical aspects of frozen embryo transfer -- Transfer in a natural menstrual cycle -- Transfer in an artificial cycle -- Results -- Oocyte cryopreservation -- Ovarian tissue cryopreservation -- Semen cryopreservation -- Sample preparation -- Manual freezing -- Kryo-10 semen freezing programme -- Insemination -- Cryoprotective medium (CPM) -- Freezing of testicular and epididymal samples -- Method -- Sample processing -- Cryopreservation of semen for cancer patients -- Further reading -- 11 Micromanipulation techniques -- Introduction -- Intracytoplasmic sperm injection -- Patient selection: indications for treatment -- Practical aspects -- Important considerations regarding the location of micromanipulation facilities -- Microtools -- Materials -- PVP preparation -- Oocyte preparation and handling -- Preparation for injection: cumulus-corona removal -- Preparation for injection -- Materials -- Micromanipulator -- Transfer the gametes to the injection dish -- Sperm selection and immobilization -- Injection procedure -- Injection procedure: important points!!! -- Assessment of fertilization and cleavage -- No fertilization after ICSI -- Transport ICSI -- Assisted hatching -- Protocol -- Step-by-step -- Equipment for ICSI -- For microinjection -- For making microtools -- Supplies -- Adjustment of Narishige manipulators for ICSI (with thanks to Terry Leonard) -- Micromanipulator parts -- Adjusting the coarse manipulators -- Attaching the fine manipulator -- Attaching the ball joint/tool holder attachment.

Finding the best position for the micromanipulators -- Forward/backward movement -- Left/right movement -- Up/down movement -- Alignment of the ICSI needle in the manipulator -- Ratio of movement and joysticks -- Microtool preparation -- Equipment -- Capillary tubing -- PCP7 Plug board (Research Instruments) -- IMMAC power cleaner -- Micropipette puller (MPP11, Research Instruments) -- Microforge (Research Instruments) -- Microbeveller or microgrinder (Research Instruments) -- Pipette storage containers -- Oven for heat sterilization -- Procedure -- Pulling -- Holding pipettes -- Injection pipettes -- Notes on pipette pulling -- First pull (injection pipette) or Only pull (holding pipette) -- Notes on the Microforge -- Setting up -- Setting a glass bead onto the filament -- Cleaning glass from the filament -- Loading the micropipette -- APPENDIX -- Causes of azoospermia -- Pathologies -- Chromosomal anomalies -- Klinefelter's syndrome -- 46 XX Klinefelter's -- 46 XX (Noonan's syndrome) -- Robertsonian translocation -- Further reading -- 12 Preimplantation genetic diagnosis -- The genetics of inherited disease -- Autosomal recessive disease -- Autosomal dominant disorders -- X-linked diseases -- Triplet repeat disorders -- Chromosomal abnormalities -- Age-related aneuploidy -- Serum screening -- Ultrasound -- Prenatal diagnosis of inherited disorders -- Chorionic villus sampling (CVS ) -- Amniocentesis -- Fetal blood sampling, cordocentesis or PUBS (percutaneous umbilical blood sampling) -- Fetal tissue sampling -- Diagnostic testing -- PCR -- Karyotyping -- FISH -- Preimplantation genetic diagnosis -- Embryo biopsy -- Zona drilling -- Polar body biopsy -- Cleavage stage biopsy -- Blastocyst biopsy -- Single cell diagnosis -- PCR diagnosis -- Contamination -- Allele dropout -- FISH diagnosis -- PGD of age-related aneuploidy -- Problems with PGD.

Chromosomal mosaicism.