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Submitted by Dr. Hesham Al-Inany, M.D. Lecturer, Gynaecology & Obstetrics dept. Kasr El-Aini hospital, Cairo University, Egypt.

During Gamete Micromanipulation sperms are injected into an ovum to assist in union of the gametes.     send to a friend   printer friendly version           Related   Gametogenesis: a basic review Anomalies of the female genital tract

Quality control of laboratory procedures

Basic quality control in the ART laboratory is essential in order to maintain high fertilization rate and embryo viability.

Incubator

Should be temperature-controlled using an independent thermocouple with a print out over 24 hours . The utilized gas is usually 5% carbon dioxide in room air. Some units use a mixture of 90% nitrogen, 5% CO2 and 5% oxygen. A high humidity of 98% is also of high importance (Brooks & Bobby,1991).

Microscope

The commonly used microscope is the directing phase with a high magnification power. Other items such as luminar flow blood, heated stages on the microscope and warming trays for the pipettes are advisable (Brooks & Bobby ,1991) .

Culture media

A wide range of culture media have been used in ART and can be classified as: simple and complex media. 

Complex media

Ham's F-10 was the first one that had been used but it contain hypoxanthine which has been shown to inhibit the development of embryo in vitro. Under these circumstances, it is difficult to recommend the use of Ham's F-10 for culture of human embryos in vitro.
Other complex media are Menezo B2 or B3 culture solutions. B3 contain human serum albumin and doesn't require the addition of human serum for use. . (Menezo et al, 1984).

Simple media

The majority of ART centers use relatively simple balances salt solutions for egg and insemination and culture of embryos. Earle's, Tyrode's and Hepes media have been successfully introduced. They are available commercially as single strength or concentrated solution. The osmolarity, ph of the culture medium are very important since the embryo development is strongly related to any variations in these parameters ( Brooks & Bobby,1991).
It appears that the media presently in use enable human embryo to develop the blastocyst stage in vitro but their development is probably retarded compared with that in vivo and their viability reduced with increasing time in culture (Cohen et al, 1985). In conclusion, the medium of choice depends on the acceptance of quality control and availability of media rather than any specific type (Wood and Trounson, 1989).

Human co-culture

Addition of any fluid or cells to the known medium-culture is termed co-culture. In the last few years, researchers have begun to develop methods to co-culture human zygotes obtained by ART.
Different methods of co-culture were described either using fluids as amniotic fluid or using helper cells as ampullary cells, Vero kidney cells, fibroblast or cumulus cells. (Seibel et al, 1993).
Co-culturing of human embryos help to develop them to more advanced morphologic stages in vitro prior to transfer to increase the chance of pregnancy. H uman tubal fluid (Quinn et al, 1989) or human ampullary cells reduce cleavage abnormalities of the embryo (Bongso et al, 1989).
Possibly the most meaningful use of culture system in the future will be better evaluation of embryos and to discard poor quality or questionable-quality embryo in vitro prior to replacement, thus increasing the overall pregnancy rate (Seibel et al , 1993).

The primary element of concern to the medical community, however, would be the potential for transmission of pathologic agent from human co-culture cells to the embryo during in vitro incubation. Studies with embryos from farm animals to date indicate that an intact Zona Pellucida during collection and transfer procedures protects the recipient female against extrazonal disease transmission (Seibel et al, 1993).

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Evaluation of eggs

Determination of egg quality is still difficult. Generally speaking, a loosely aggregated granulosa, an expanded cumulus and radiant corona indicate a mature oocyte, while compact and tightly aggregated granulosa cells, dense cumulus and a compact or occasionally absent corona characterize an immature oocyte (Veeck et al ,1989 )
Nuclear status at harvest gives more reliable prediction of oocyte maturity and capacity to fertilize. An oocyte with one polar body is mature and has a high chance of fertilization. A germinal vesicle can be observed in fully immature oocytes. Granular cytoplasm and vacuoles in the cytoplasm are poor prognostic signs (Veeck et al, 1989).
If immature eggs are identified, they may be cultured for 20-24hs before insemination but viability of such eggs is likely to be reduced (Wood & Trounson, 19 8 9 ). Eggs which do not have pronuclei by 17-20hs are likely to have increased incidence of chromosomal anomalies and should be discarded.

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