Biomedical subjects
Publications and source records attributed to T F Kruger.
In order to maximise the chances of pregnancy, most successful in vitro fertilisation programmes use a combination of ovulation induction agents. This treatment can lead to the hyperstimulation syndrome. Aspiration of the follicles is believed to avoid this syndrome. Despite this approach, hyperstimulation syndrome may still develop. The clinical picture and treatment of a patient with severe hyperstimulation is discussed.
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In patients with acceptable sperm count and motility, two patterns of abnormal morphology, judged with strict criteria, were identified and described. Patients with less than 4% normal forms and less than 30% morphology index (summation of normal and slightly amorphous forms) had a fertilization rate of 7.6% of the oocytes (P pattern, poor prognosis). Patients with normal morphology between 4 and 14% had a significantly better fertilization rate of 63.9% of the oocytes (P less than 0.0001). Cases with greater than 14% normal forms fertilized within the normal range for the laboratory. By evaluating sperm morphology with the proposed strict criteria, its predictive value in in vitro fertilization is enhanced.
A new method for evaluation of sperm morphology using strict criteria is currently used in the andrology laboratory at the Eastern Virginia Medical School. A prospective study was designed to evaluate the following semen parameters in samples of all patients over a set period of time: sperm concentration and motility, and normal sperm morphology. These factors were correlated with results of the hamster zona-free oocyte/human sperm penetration assay (SPA). One hundred patients with a sperm concentration ranging from 2 to 219 X 10(6)/ml, a motile sperm fraction ranging from 6.9 to 87%, and normal sperm morphology ranging from 1 to 39%, were evaluated. The statistical analysis system general linear model was used to judge the influence of the different variables. There was a statistically significant relationship between the per cent of sperm with normal morphology and penetration rate in the SPA (P = 0.001). Outcome of the SPA was also correlated with in vitro fertilization, retrospectively, in 84 patients. Thirty-eight patients had an SPA less than 10%, with no fertilization in vitro in 13 patients (33.3%) and fertilization in 25 (66.7%). Forty-five had an SPA greater than 10% with fertilization in 37 (82.2%) and no fertilization in eight (17.8%) patients.
Acrosin, a neutral proteinase, is located within the acrosome. The aim of this study was to evaluate acrosin concentrations in patients with severe damage of the sperm head and to determine whether acrosin concentration could predict the chances of fertilization in an IVF program. Sixty patients were accepted into this study, prospectively. The patients were divided into two groups, those with a normal morphology of less than 14% (group I, n = 33) and those with normal morphology less than 14% (group II, n = 27). All patients had a sperm concentration of less than 20 million sperm/ml and less than 30% progressively motile sperm. The acrosin assays were performed on the semen sample obtained on the day of IVF. Routine IVF insemination procedures were used, and only mature oocytes were considered. The only factor that showed a significant correlation of fertilization was normal morphology (p less that 0.01). The mean acrosin level was 73.4 /+- 38.6 mED/10 million sperm in group I and 70.9 /+- 42.7 mIU/10 million sperm in group II (no significant difference). The fertilization rate in group I was 45.4% and in group II, 77.7% p less than 0.002). Acrosin levels were not significantly different in patients with and without fertilization (72.0 /+- 42.1 and 73.6 /+- mIU/10 million sperm, respectively).
Intra-uterine insemination with washed spermatozoa of the husband was evaluated in 42 infertile couples during 103 treatment cycles. A pregnancy rate of 47.3% was obtained after an average of 2 treatment cycles in a group of patients in whom hostile cervical mucus was the only known cause of infertility. A pregnancy rate of 21.7% was obtained after an average of 2.6 treatment cycles in a group of patients in whom low sperm morphology was the only known cause of infertility. The overall conception rate of 33.3% compares favourably with that in other published series.
The protocol for in vitro fertilisation (IVF) at Tygerberg Hospital is presented and the results are analysed. Indications for ovulation induction for IVF included the following: (a) irreversible tubal damage; (b) infertility due to immunological factors; (c) male factor-infertility; and (d) endometriosis. A combination of human menopausal gonadotrophin (HMG) and clomiphene citrate (Clomid; Mer-National) and human chorionic gonadotrophin was used. Clomid is given in dosages of 100 mg for 5 days depending on the cycle length. Three doses of HMG (150 IU) are given on alternate days, starting on the second day of clomiphene treatment. If the leading follicle has not reached a mean diameter of 14 mm the day after the last HMG dose, another dose is given. This dosage is continued until the leading follicle reaches a diameter of 14 mm. A total of 109 cycles in 100 patients was analysed. Ova were considered to be mature as soon as the dominant follicle had reached a sonographic average diameter of 18 mm, another two follicles of 16 mm average diameter being present. Serum luteinising hormone levels were determined 4-hourly from the stage when the leading follicles exceeded an average diameter of 14 mm. In this study the pregnancy rate was 21,3% per laparoscopy and 24,4% per embryo transfer. The oestradiol levels on the 5th day of treatment have a predictive value of the length of stimulation.
There are conflicting reports in the literature about the effect of the water source for growth and insemination media in an in vitro fertilisation (IVF) programme. A controlled trial was carried out in which two-cell mouse embryos were cultured in Ham F10 medium containing either grade 1 water (test group) or grade 2 water (control group). Of the two-cell embryos, 92.0% (69 of 75) in the test group and 91.8% (67 of 73) in the control group cleaved to the blastocyst stage (no statistically significant difference). On the basis of this experiment grade 2 water was used in the Tygerberg Hospital human IVF programme with good results. A continuing pregnancy rate of 23% per embryo transfer over a 1-year period is reported.
The effect of serum supplementation was evaluated on 108 oocytes obtained from 29 patients. The oocytes were all mature and randomly distributed in two groups. Each ovum was paired with another ovum within the same patient. The experimental group contained no serum in the insemination and growth medium, but in the control group 10% serum was added to the insemination and growth medium. In the experimental group the cleavage rate was 66.7%, compared to 83.3% in the control group. The embryos from each patient were individually evaluated. The sign test was used for statistical analysis. A significant difference between the two groups (P = 0.01) indicated that in this study the addition of serum to the medium gives better cleavage results. The effect of serum on embryos is discussed, as well as the practical implications of this finding.
A prospective study was planned to evaluate sperm morphology as a parameter to predict the fertilization outcome in an in vitro fertilization program. Couples applying to in vitro fertilization were admitted into this project when the sperm concentration was greater than 20 million per mL and motility greater than 30 per cent. Based on new strict criteria for evaluating normal sperm morphology, patients were divided prospectively into 2 groups. In group I (25 patients) normal sperm morphology was less than 14 per cent, and in group II (71 patients) normal sperm morphology was greater than 14 per cent, using a threshold established previously. Multiple regression analysis was used to evaluate different parameters: concentration, motility, and morphology against the dependent variables, fertilization, and cleavage. The only factor which was significantly correlated with fertilization and cleavage was normal sperm morphology (P less than 0.0001). The fertilization rate (per oocyte) and the cleavage rate were 49.4 per cent and 47.6 per cent in group I and 88.3 per cent and 87 per cent in group II (P less than 0.0001). The ongoing pregnancy rate per laparoscopy and per embryo transfer was 4 per cent and 5.5 per cent, respectively, in group I and 18.3 per cent and 18.5 per cent, respectively, in group II (no significant difference). This study demonstrates the value of analyzing sperm morphology using the criteria recommended in terms of predicting fertilization and perhaps pregnancy outcome. Patients can be better counseled and the probability of fertilization or no fertilization can be more accurately established. Furthermore a trend is shown in the pregnancy rate that may indicate the importance of the male genome in establishing a pregnancy.
The evaluation of sperm morphology is still an important parameter in the diagnosis of the infertile male. Most techniques used for staining human sperm are very time-consuming. A routine stain used for determining differential count of leucocytes (Diff-Quik stain) was evaluated against the standard Papanicolaou stain. Morphology results from 20 duplicate semen smears using both staining methods were determined separately by 2 technicians using a blind protocol. No significant differences were observed when comparing the two staining methods (paired Student's t-test). The advantages of the Diff-Quik stain are: a) complete staining-to-reading time under 7 min, b) commercially prepared reagents, and c) case of staining procedure.
One- and two-cell embryos were obtained from F1 hybrid female mice stimulated with human menopausal gonadotrophin and randomly distributed into two groups--group 1 (no serum) and group 2 (10% patient's serum). Fifty of 53 (94.3%) one-cell embryos in group 1 had cleaved to the blastocyst stage and 44 of 49 (89.79%) in group 2 after 96 hours (no significant difference--chi-square test). In the two-cell embryos in group 1, 78 of 89 (87.6%) reached the blastocyst stage after 72 hours and 80 of 86 (93.02%) in group 2 (no significant difference--chi-square test). According to microscopic evaluation cleavage to the blastocyst stage without serum supplementation is possible. The value of serum is discussed.
Phases II and III of the human in vitro fertilization programme at Tygerberg Hospital are presented. In phase II, 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 III. 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 II and III are discussed.
To determine whether there is a prognostic value in the percentage normal sperm morphologic features in a human in vitro fertilization (IVF) program, the authors conducted a prospective study in women with bilateral tubal damage. Based on the percentage of morphologically normal spermatozoa, the patients were divided into four groups: group I, normal morphologic features between 0% and 14%; group II, 15% to 30%; group III, 31% to 45%; and group IV, 46% to 60%. One hundred ninety successful laparoscopic cycles were evaluated. In group I, 104 oocytes were obtained, of which 37% fertilized, but no pregnancy resulted; in group II, 81% of 324 oocytes were fertilized, with a pregnancy rate per embryo transfer (ET) of 22%; in group III, 82% of 309 oocytes were fertilized, with a 31% pregnancy rate; and in group IV, 91% of 69 oocytes were fertilized, with a pregnancy rate of 12%. Probability models indicated that there was a clear threshold in normal sperm morphologic features at 14%, with high fertilization and pregnancy rate in the groups with normal sperm morphologic features greater than 14%.
Two-cell mouse embryos are used for quality control in a human in vitro fertilization programme. A controlled experiment was designed to evaluate the effect on cleavage of two-cell mouse embryos after a delay in embryo retrieval. In the test group, two fallopian tubes were incubated in Whittingham's T6 medium for 2 1/2 hours per experiment before the embryos were removed for culture. In the control group embryos were removed from the fallopian tubes immediately after the mice were sacrificed. Five experiments were performed. Eight of 141 two-cell embryos (5,7%) reached the blastocyst stage in the test group, and in the control group 143 of 151 two-cell embryos (94,7%) reached the blastocyst stage after 72 hours. Embryos must be removed immediately after the mice are sacrificed to obtain constant results. If not, poor cleavage can lead to unnecessary confusion in the laboratory.
Two-cell mouse embryos were subjected to fluorescent light, 2900 lux, for 30 minutes, and the cleavage compared with that in a control group. There was no statistically significant difference in the results. In both groups 90% of two-cell embryos reached the expected level of cleavage. The possible effect of fluorescent light on the oocyte is discussed.
In performing in vitro fertilization, a stable osmolarity in the medium surrounding the egg or embryo is of the utmost importance if a good fertilization and pregnancy rate is to be achieved. This study evaluated osmolarity changes in different volumes of fluid in the Falcon 3001 and 3037 Petri dishes and the Falcon 2058 tissue culture tube over a 24-hour period. It was found that the osmolarity was more stable in the Falcon 3037 Petri dish and in the tissue culture tube. The 3037 Petri dish was chosen for culturing human embryos.
Phase I (3 June - 31 October 1983) of the in vitro fertilization (IVF) programme at Tygerberg Hospital is outlined in this article. This programme led to the birth of the first IVF baby in South Africa after fertilization and embryo transfer (ET) took place at this institution. The baby was born on 29 April 1984; a second baby was born on 20 June 1984. During phase I 24 laparoscopies were performed; 32 oocytes were obtained from 56 follicles, of which 62,5% were fertilized. Eleven ETs were performed, which led to 3 pregnancies. An ongoing pregnancy rate of 18% per ET and a pregnancy rate of 12,5% per laparoscopy were achieved.