A fully equipped laboratory with state-of-the-art air control; meticulously calibrated and maintained incubators and microscopes; culture media and medium supplements scrupulously selected after stringent after-market assays; validated, efficient and effective laboratory protocols and techniques; skilled, experienced, knowledgeable embryologists. What could go wrong? Why and how could there ever be a poor outcome, such as failure of fertilization, oocyte degeneration, arrested development of embryos or low blastocyst formation rate, poor survival of embryos after cryopreservation or low embryo implantation rates? Every laboratory and clinic must be prepared to encounter one, some or all of these undesired events and set out on a systematic analysis of the entire process to identify and address ‘the problem’. In this chapter, some suggested strategies and tools for an effective investigation and focussed troubleshooting are presented. These are designed to assist the embryologist, as well as the entire clinical team, to embark on a structured evaluation of each factor that may underlie, cause and/or contribute to a failure or a suboptimal outcome. Indeed, the engagement of the team is encouraged, providing a valuable perspective in a time when one must resist the temptation to ascribe poor performance in in vitro fertilization (IVF) solely to the laboratory.

The purpose of this chapter is to review some of the factors that influence laboratory and clinical outcomes, broadly under the heading of optimal handling techniques. Two principal environments are encountered – inside and outside the incubator. The chapter will address media buffers, gas atmosphere, timing and setting up culture or holding vessels, protection of medium performance, temperature relative to handling gametes and embryos, lighting, pH, incubation choices, and workflow. The importance of the interplay between these variables cannot be overlooked.