SAMJ VOLUME 69 1 MARCH 1986 297 fertilization Hospital, Results of the in vitro programme at Tygerberg phases 11 and III T. F. KRUGER, J. P. VAN DER MERWE, A. D. VAN DEN HEEVER, K. KOPPER, J. N. DE VILLlERS F. S. H. STANDER, R. MENKVELD, H. J. ODENDAAL, J. A. VAN ZYL, Patients and methods Summary Phases 11 and III of the human in vitro fertilization programme at Tygerberg Hospital are presented. In phase 11, 42 laparoscopies were performed and oocytes were obtained from 76% of the follicles aspirated, but with a fertilization rate of only 37%. The viable pregnancy rate per embryo transfer was 4%. Important changes took place in the programme. which led to a fertilization rate of 77% in phase Ill. Of the 78 patients subjected to laparoscopy. 65 (83%) reached the embryo transfer stage, resulting in a clinical pregnancy rate per embryo transfer of 23%. and with a 19% pregnancy rate per laparoscopy. The changes, methods and results of phases 11 and III are discussed. S Air Med J 1986; 19: 297-300. The methods used in theIn Vitro Fertilization Unit at Tygerberg Hospital and the results of the first 24 laparoscopies and II embryo transfers, which resulted in 3 pregnancies, were recently published. l In phase Il of the programme (1 January - 30 April 1984) almost the same laboratory methods were used, except for a few small changes. The ovulation-induction drug programme was changed to a combination of clomiphene and human menopausal gonadotrophin (HMG), as outlined by Kerin et al. 2 The results were not satisfactory. A reason for the poor results was sought and changes were instituted as from -1 May 1984. Phase III covered the period 1 May - 31 August 1984. These changes are discussed, as well as the results in phases Il and Ill. Department of Obstetrics and Gynaecology, University of Stellenbosch and Tygerberg Hospital, Parowvallei, CP T. F. KRUGER, M.PHARM.MED., M.MED. (0 & G). F.C.O.G. (S.A.). M.R.e.O.G. J. P. VAN DER MERWE, M.B.CH.B.,M.MED.(O&G),F.e.O.G.(S.A.) F. S. H. STANDER, Cycolechnician R. MENKVELD, M.Se. A. D. VAN DEN HEEVER, M.MED. (0 & G), F.e.O.G. (S.A.) K. KOPPER, Technician H. J. ODENDAAL, M.MED. (0 & G), F.e.O.G. (S.A.), F.R.e.O.G., M.D. J. A. VAN ZYL, M.B. CH.B., M.MED. (0 & G), M.D. J. N. DE VILLIERS, M.B. CH. B., M.O. &G.• F.R.e.O.G. Patients accepted into the programme had tubal damage and their male partners had to be fertile, as outlined in our previous article. 1.3 The ovulation induction programme in phase II was changed to a combination of clomiphene 100 mg and HMG 2 ampoules on alternate days, starting on the second day of clomiphene therapy. The HMG therapy was given 3 times, as discussed in a recent paper by Kerin ec al. 2 We used 50 mg less clomiphene per day than Kerin's group. Ultrasound examinations were performed on day I of the menstrual cycle and the cycle was cancelled if a cyst was detected. Thereafter ultrasound examinations were performed daily after the oestradiol (E 2 ) values reached a level of 500 pmoVI. When a diameter of 18 mm was reached by one ofthe follicles with at least one other follicle more than 16 mm in diameter (measured in two planes with a Phillips ST 7000), the HCG 10 000 U was given intramuscularly. The laparoscopy was performed 36 hours later. This was a change from our previous method of giving low doses of HMG or clomiphene 100 mg as the only method of ovulation induction. The laparoscopic technique also changed. The double-barrel Bourn Hall needle was replaced by the Monash needle, which is 14 cm in length, 17 mm in diameter and lined with Teflon. Flushing of the follicle took place only if the oocyte was not discovered in the follicular fluid or needle rinse. 0 heparin was used in phase III but the blood-stained fluid was transferred immediately to the laboratory. The needle was rinsed with 4-times distilled water after the procedure and every needle was relined after 4 aspirations. Dry heat was used to sterilize the needles. A 3033 Falcon tissue culture tube was used to obtain the follicular fluid and these bottles were prewarmed in an incubator to 37°C in phase III of the programme. This did not take place in phase 11. The follicular fluid was screened in the laboratory in 3003 Falcon Petri dishes. Care was taken to prewarm these dishes before use. The ovum was transported to a 3037 Falcon Petri dish as soon as pos- sible after it was rinsed in T6 medium with no serum added. The rest of the methods followed were outlined in a previous article. I Laboratory methods in phase Il were as in phase 1. Filtered T6 medium was used, made up once a week (filter 0,22 Gelman). The pH was 7,4 after 24 hours in a 5% carbon dioxide in air incubator (Forma Scientific 3157). The semen specimen was prepared by standard methods and described in a previous article;' 100000 motile spermatozoa were used per ml of insemination medium. Transfer to the growth medium took place after 20 hours. Oocytes were dissected and pronuclei documented (Table I). In phase III the medium was changed to Ham FIO because the poor fertilization rate in phase II could possibly have been caused by deterioration of the purivate quality in the medium. Fresh purivate could not be obtained immediately. The osmolarity was 280 mOsm/kg and the pH after 24 hours 7,4. The serum in the growth medium was 20%. No filters were used because wetting-agent free filters could not be obtained immediately. Because very satisfactory results were achieved, with no infection to date, reversion to the use of filters did not occur. All glassware was prepared as described by Whittingham.< The Pasteur pipettes were rinsed 3 times before use and handling of oocytes. In phase Ill, dissection of the oocyte was seldom performed after 20 hours but cleavage was observed 40 hours after insemination. In summary, the following changes took place: (c) T6 to Ham FIO; (ic) osmolarity 285 mOsm/kg to 280 mOsm/kg; (iiz) tubes and Petri