The influence of sera, follicular fluids and seminal plasma on human sperm-zona pellucida binding.

The purpose of this study was to assess the influence of male, female and fetal cord sera, follicular fluid, and seminal plasma on human sperm-zona pellucida binding, using the hemizona assay. Steroids, gonadotrophins, growth hormone and prolactin concentrations in follicular fluid and sera were also analysed. The influence of follicular fluid (10 or 50%, v/v) and sera (10%) on sperm-zona pellucida binding was investigated by supplementing the sperm processing medium as well as the sperm-hemizona incubation medium. Different seminal plasma concentrations (1 or 10%) were added to the sperm-hemizona incubation medium. Supplementation with 10% day 3 donor serum was used as a control throughout experimentation. Although supplementation with male sera and fetal cord serum exerted a stimulatory effect (36 and 90% respectively; P < 0.029) on sperm-zona pellucida binding, hemizona indices obtained with addition of male sera, fetal cord serum and sera obtained from sub-fertile in-vitro fertilization (IVF) patients on day 12 of their menstrual cycle did not differ significantly (P > 0.05). Final progesterone concentrations in sperm-zona pellucida incubation media (10% follicular fluid supplementation), which ranged from 0.788 to 3.85 microg/ml, enhanced sperm binding to the zonae by >100% (P < 0.02). The utilization of follicular fluid (10%) as a natural physiological stimulus to enhance sperm-zona pellucida binding in an IVF setting is recommended. The presence of seminal plasma in the spermzona pellucida incubation media showed no beneficial effect on the binding ability of sperm, and can be viewed as an unfavourable substance in the proximity of the oocyte.

The effect of seminal plasma on human sperm-zona pellucida binding.

The aim of this study was to determine the effect of different concentrations of seminal plasma (SP) in insemination medium on human sperm-zona pellucida (ZP) binding. The effects of fresh SP [50% (n = 10) and 1% (n = 9)], frozen-thawed versus fresh SP [5% (n = 10)] and frozen-thawed SP [5% (n = 10) and 1% (n = 9)] on sperm-ZP binding were examined using hemizona assays. The validity of the hemizona assay as performed in this study was also examined. Relative sperm binding percentages were determined for respective hemizonae, and the hemizona index was calculated for each experimental group and compared statistically. The 50% native SP inhibited sperm-ZP binding by approximately 70% (P = 0.0051), while 1% native SP enhanced sperm-ZP binding by approximately 350% (P = 0.0051). Significantly more spermatozoa (mean +/- SD, 172.00 +/- 54.12) bound to zonae in the presence of 5% frozen-thawed SP than with 5% SP that had not been frozen (mean +/- SD, 127.00 +/- 69.18; P = 0.0431). The 1 and 5% frozen-thawed SP stimulated sperm-ZP binding by approximately 400 and approximately 250% respectively (P = 0.0077 and 0.0051 respectively). It is concluded that SP concentrations found in insemination media during assisted reproductive techniques do not inhibit but in fact enhance sperm-ZP binding.

Effectiveness of various sperm processing methods in removing seminal plasma from insemination media.

The effectiveness of the wash and swim-up (1 and 2 wash cycle), two layer Percoll gradient, SpermPrep and underlay sperm preparation methods in removing seminal plasma from insemination suspensions was investigated. The number of wash cycles needed to rid sperm suspensions of seminal plasma (n = 15) was also determined. All sperm preparation methods were compared to the control wash and swim-up method, performed using Earle's buffered salt solution (EBSS), without serum supplementation so as not to mask seminal plasma concentrations. Control processing comprised 1 ml semen subjected to two wash cycles in EBSS followed by a swim-up period of 60 min under 5% CO2 in air. The Percoll method comprised 1 ml semen layered on a discontinuous 36 and 81% Percoll gradient (n = 14). In the SpermPrep method, 1 ml semen was run through a SpermPrep II column (n = 10), and underlay samples (n = 12) were processed by two wash cycles, after which sperm pellets were resuspended in the remaining medium, layered under 1.2 ml EBSS and allowed to swim up under 5% CO2 in air for 1 h. Seminal plasma and supernatant fractions obtained after each processing phase were stored at -20 degrees C and protein concentrations were determined by spectrophotometry. The wash and swim-up method was the most effective in removing seminal plasma from sperm suspensions, followed by the two-layer Percoll gradient, underlay and finally the SpermPrep II processing methods.

Electron microscopic study of human sperm membrane isolation.

OBJECT: Our purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants. METHODS: Donor semen samples collected after masturbation were stored at -70 degrees C and eventually pooled. Each attempt at sperm membrane isolation required 800 x 10(6) spermatozoa which were sonicated by ultrasound (40% output; Vibra Cell). The effect of sonication time (3 x 5, 3 x 15, and 180 sec) on membrane isolation was investigated. Sonicated samples were centrifuged (500 g, 5 min) and the supernatant was pipetted off. The supernatant of the centrifuged sample was layered on either a sucrose cushion (supernatant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifuged (100,000 g, 1 hr). Contents of supernatants of sonicated samples and fractions (sucrose interfaces) were then fixed in 1.0% tannic acid and 2.5% buffered glutaraldehyde and examined electron microscopically using standard procedures. RESULTS: (1) The optimal sonification time was found to be 3 x 15 sec. (2) Membrane isolation using a sucrose cushion was found to be inadequate, showing significant cellular contamination. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective. CONCLUSION: The advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.

The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme.

The cumulative embryo score system involves three aspects of relevance in pregnancy achievement during in-vitro fertilization (IVF) and embryo transfer: cleavage rates, morphological qualities and the number of embryos transferred. The scores of 602 IVF/embryo transfer trials were calculated and analysed to determine the system's relationship to pregnancy rate, pregnancy outcome and the incidence of twin and triplet pregnancies. The system was also applied to cycles where endotoxins were either present in or absent from culture medium, in order to evaluate its validity in quality control analyses. Pregnancy rates were found to increase from 4%, with scores between 1 and 10, to 35% in the 41-50 group. The score of 20 was the criterion for separating patients into poor and good pregnancy prognosis groups (P = 0.00001). Biochemical abortions occurred more frequently with scores < 20 (P = 0.00978), but a similar relationship was not found in clinical abortion rates (P = 0.62206). Birth rates below and above a score of 20 (2.8 and 19.2%, respectively) differed significantly (P = 0.0005). The scores of twins overlapped extensively with those of singleton births, but those of all triplets were > 40. The system did not reflect a correlation between embryo quality and the presence of endotoxins in culture medium.

Primate in vitro fertilization research: preliminary results on the folliculogenic effects of three different ovulatory induction agents on the chacma baboon, Papio ursinus.

1. A study was conducted on the chacma baboon, Papio ursinus with three ovulation induction agents in an effort to define a preferential stimulatory protocol with regards to the number and quality of oocytes obtained. 2. Three folliculogenic agents applied in four stimulatory protocol regimens comprised clomiphene citrate in a high (100 mg/day) and low (50 mg/day) dosage, a combination of clomiphene citrate and pregnant mare serum, and human menopausal gonadotropin. 3. A total of 159 oocytes were aspirated by laparotomy from 10 baboon females in 20 induced cycles with an average of 8.0 +/- 5.4 oocytes per aspiration. 4. The highest mean number of oocytes (11.3 +/- 6.7) were obtained with the clomiphene/pregnant mare serum gonadotropin combination. 5. The best fertilization rate was obtained with clomiphene 50 mg. 6. The highest incidence of oocytic cleavage and embryo transfer were achieved with human menopausal gonadotropin (14.8%).

Supplementation of Ham's F10 culture medium with three different sera in the culturing of baboon oocytes.

1. Ten female baboons (Papio ursinus) were stimulated for a total of 20 cycles with 3 ovulation induction agents. 2. Oocytes obtained were randomly allocated to Ham's F10 culture medium supplemented with human fetal cord serum, primate serum or commercial fetal bovine serum respectively. 3. Fertilization occurred (38.1-45.5%) in all 3 supplements, but cleavage and embryo development was more successful in culture medium supplemented with fetal bovine serum. 4. Eight embryos were cultured and 6 (75%) of these were cultured in fetal bovine serum supplemented medium.

DDT administration: haematological effects observed in the crowned guinea-fowl (Numida meleagris).

The continued use of chlorinated hydrocarbons as pesticides prompted an investigation into the effects of DDT (dichlorodiphenyltrichloroethane) on the blood of the crowned guinea-fowl (Numida meleagris). Birds were acclimated for 10 weeks, and haematological data obtained after this period served as baseline values. DDT was then administered per os at 75 ppm/kg body mass. This treatment was continued daily for 5 days. Subsequent blood analyses illustrated significant effects on several of the test parameters.