Abstract

PIP: An apparatus for in vitro fertilization and culture of mammalian eggs that permits close control over variations in temperature, lighting, and pH has been developed to imitate natural conditions as far as possible. All handling of the oocyte and zygote are performed inside a baby incubator which has a system of air saturation using water vapor. A binocular dissecting microscope and an inverted compound microscope are built into the enclosure to permit simultaneous observation of the egg at widely different magnifications. Lighting is restricted to the longer wavelengths, which do not harm mammalian eggs. The microscope is equipped with a camera system. A gas mixture of 5% CO2, 5% O2, and 90% N2 is delivered at a pressure of 1 bar by a double reduction valve arrangement. The gas passes into the incubator through a filter and is humidified and warmed inside the incubator by bubbling through a bottle of sterile distilled water. The gas is finally distributed by a series of culture tubes. The culture tubes are usually kept in darkness; their position is changed as the development of the egg progresses to take advantage of the slight temperature differentials within the apparatus. Less than 1 minute after puncture of the follicle, the fluid is transferred to the incubator and is examined in a petri dish using the stereomicroscope. The oocyte is immediately transferred to a culture tube containing medium and placed at 36.8 degrees Celsius. 16-20 hours after insemination the egg is transferred under the stereomicroscope by pipetting directly into a new tube, where the medium is supplemented by a small quantity of maternal serum. It is kept at 37.2 degrees Celsius. After 2-4 days of culture in vitro, if normal development is observed, the egg is transferred to the patient's uterus. The variation in temperature at any given point in the apparatus is less than .2 degrees Celsius. 114 human oocytes have been collected, of which 58% were fertilized. 82% of the fertilized eggs underwent normal cleavage, and 38 embryos were replaced in the uteri of 34 patients. 8 early pregnancies resulted, of which 2 lasted more than 3 months. The fertilization rate was closely related to the quality of male and female gametes; it was 94% when both were favorable.