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Risk management: being proactive
Table 9.2 (cont.) Contributory factor
Classification/Explanation
Solution
6 Wash medium not kept under CO2 during egg search procedure
Contributory: As per factor 5, the wash mediuma would not have maintained its pH and oocytes already recovered from the follicular aspirates would have been exposed to an alkaline medium.
7 Cooling of cumulus-oocyte complexes during recovery from follicular aspirates
Contributory: r A very large Petri dish (15 cm diameter) was used for the aspirated material, taking longer to search than the typical 60 or 100 mm dishes; and r When the first oocyte was found it was aspirated into the Pasteur pipette (Fig. 9.7D, black arrow) and held there, exposed to the cooling effect of the air in the laminar flow cabinet, until all oocytes had been found and recovered, taking a minute or more.
A properly-formulated HEPES-buffered wash medium was to be used to hold all oocytes until the completion of the egg search procedure, at which time all the oocytes were washed through a dish of CO2 -equilibrated culture medium (held until that time in the small incubator in the work station) before transferring into the IVF culture dishes. Using a smaller Petri dish to search the aspirates was recommended, along with transferring each oocyte to the wash dish as soon as it was recovered. The wash dish was to be kept on the recalibrated warm stage (see factor 5, above) until that time.
a
The wash medium being used was a standard-buffered culture medium (25 mM bicarbonate) with 7.5 mM HEPES added. This amount of HEPES would not have held the pH while under an air atmosphere due to the “stress� of the 25 mM bicarbonate buffer being left without a CO2 -enriched atmosphere. A normal HEPES-buffered medium has 20 mM HEPES with the bicarbonate (which is an essential cofactor) reduced to 10 mM (see D. Mortimer et al., 2002).
medical and legal perspectives (Mortimer, 2004b; using technical and biological information reviewed in Mortimer, 2004a). Table 9.3 illustrates a formal risk analysis, in the format of an FMEA, performed on the four most common packaging devices used for cryopreserving human gametes and embryos and leads to the clear conclusion