N OCIETY' - the Society for Reproductive Biology

MICROMANIPULATION PROCEDURES FOR GAMETES AND EMBRYOSO. Lacham-Kaplan, G. M. Jones, A. TrounsonCentre For Early Human Development, Institute of Reproduction and Development, MonashUniversity Melbourne, Victoria, AustraliaMicromanipulation procedures have been used as an artificial reproductive technology (ART) for thetreatment of infertility in the human and other mammalian species. The zona pellucida, surroundingthe mammalian oocyte is the major barrier to fertilization. Sperm must pass through this layer inorder to reach and fuse with the oocyte membrane and initiate embryonic development. The zonapellucida is also believed to protect the embryo during its preimplantation development in-vivo.Upon implantation in the uterus embryos at the blastocyst stage expand and exit from the zonapellucida in a process known as 'hatching'. The zona pellucida may prevent fertilization of oocytesby subfertile sperm. Moreover, due to abnormal thickening and hardening of the zona pellucidaembryos may fail to hatch, hence reducing the chances of implantation and pregnancies. In thesecases, breaching or complete removal of the zona pellucida is necessary. Breaching of the zonapellucida is mainly done by micromanipulation procedures.To facilitate fertilization, a microneedle is used to penetrate the zona pellucida and leave the spermin close proximity to the plasma membrane or directly inside the oocyte cytoplasm in a processknown as intracytoplasmic sperm injection (lCS!). In this procedure, sharp glass capillaries areoperated manually to pierce the zona pellucida and the plasma membrane. Using lCSl, fertilizationwith very low numbers of normal sperm, immotile sperm, epididymal and testicular sperm, and germcells such as round spermatids and secondary spermatocytes have been achieved in humans and inother mammalian species. These include mice, horses, cows, domestic cats and others and may bethe optimal procedure to be used for conservation of endangered species.Intracytoplasmic injection is also used to inject somatic cells into enucleated oocytes for the purposeof cloning farm and laboratory animals. A recent use of non-sharp microinjection capillaries attachedto a Piezo system, which is operated by electromagnetic pulses, has been shown to be less invasiveand more effective when germ cells and somatic cells were injected directly into the oocytecytoplasm.Assisted hatching and embryo biopsy for preimplantation diagnosis in the human are also done usingmicromanipulation procedures to partially remove the zona pellucida. Partial removal of the zonapellucida can be done chemically using low p~_ solutions, mechanically by a sharp microneedle, orby laser. The laser system for assisted hatching has been found to be easier to handle and morereproducible than the other two assisted hatching procedures. Using a laser beam directed on acertain point of the zona pellucida, accurate and repeatable holes are created. The procedure is safeand does not interfere with embryo development. The laser system offers a significantmethodological improvement over the chemical and mechanical assisted hatching. The threeprocedures are found to enhance implantation and pregnancies in cases of failed IVF due to eitherthick or hardened zonae, suggesting that the techniques may be beneficial for all IVF producedembryos.3435