In vitro maturation and fertilization of oocytes recovered from free-ranging Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae).

A noninvasive repeatable method to harvest oocytes for in vitro fertilization (IVF) could potentially be used to assist reproduction in endangered equid species. The objectives of this study were to evaluate a specific transvaginal ultrasound-guided oocyte recovery procedure for use in zebra mares and the general applicability of IVF procedures in zebra. Ovaries were collected from Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae) mares at routine culling for Expt. I. Of the 144 oocytes recovered from these ovaries, 70% were of excellent quality. No significant difference in oocyte quality was found between the two zebra species. Zona drilling was performed on in vitro-matured oocytes prior to IVF. Epididymal sperm from culled Burchell's zebra stallions were used for IVF. The sperm either were exposed to calcium ionophore or were not treated and served as a control. In vitro fertilized oocytes were then co-cultured with zebra granulosa cells (ZGC) or with bovine oviduct cells (BOC) for up to 8 days. Overall, a 38% cleavage rate was obtained with 16% of sperm-exposed oocytes developing to the morula or blastocyst stage. All of the embryos that developed to at least the morula stage were cultured on BOC; whereas, none of those cultured on ZGC reached the morula stage during the same interval. Cleavage rates of oocytes inseminated with ionophore-treated or with control sperm were not significantly different, suggesting that ionophore treatment of epididymal sperm for IVF in these zebra species may be redundant. In Expt. II, 10 transvaginal ultrasound-guided oocyte aspiration procedures on five captive Burchell's zebra mares recovered a total of 33 oocytes (6.6 oocytes/female) of which 94% were considered viable. This approach may be an attractive means of producing gametes for assisted reproduction in endangered species. Furthermore, results from this study indicate that IVF may become a means of producing offspring from zebra and other equid species in the future.

The detection of blood contamination in human follicular fluid.

PURPOSE: The aim of this study was to compare the reliability of the methods conventionally used to identify low levels of blood contamination in human follicular fluid (hFF) as applicable in the clinical environment. METHODS: Follicular fluid (n = 339) and plasma samples (n = 20) were collected from patients (n = 138) attending the Centre for Fertility Studies, HF Verwoerd Hospital, University of Pretoria, South Africa. hFF blood contamination was assessed by means of (a) visual inspection, (b) hematocrit (Hct), (c) spectrophotometric analysis, (d) spectrophotometric hemoglobin kit, and (e) Combur-9-test urine sticks. RESULTS: (1) Neither hematocrit nor spectrophotometry provided reliable detection at low levels of blood contamination. (2) Visual inspection presented with a better discriminatory ability than either Hct or spectrophotometry. (3) Combur-9-test sticks identified up to 50% of blood-contaminated fluids. (4) Spectrophotometrically determined hemoglobin levels presented with weak discriminatory abilities for detecting blood-contaminated fluids. CONCLUSIONS: Visual inspection as performed in this study provides a fast and relatively reliable method for the determination of blood-contaminated hFFs. In a laboratory environment, however, it would be recommended that a combination of visual inspection, Hct, and spectrophotometric evaluation be employed for the selection of blood-free fluids.

Chronobiological fluctuations in semen parameters with a constant abstinence period.

This study examined the seasonal variation in three semen parameters (total sperm count, % grade a progressive motility and sperm morphology according to strict criteria) with an identical abstinence period of 24 h. A total of 340 spermiograms of 107 different men enrolled in an intrauterine insemination (IUI) program were examined. To reduce variation due to interindividual disparities in semen quality, differences of each test result from the mean value obtained for that individual were analyzed. Mean values resulted from at least 3 observations during different IUI cycles. Using ANOVA and spectral analysis, no differences in semen parameter results in function of the month of the year were observed. The lack of any significant periodicity may be explained by the large biological variation for individual semen parameters and/or the lack of significant differences regarding light exposure and temperature between summer and winter months in moderate climates.

The predictive value of basal follicle stimulating and growth hormone levels as determined by immunofluorometry during assisted reproduction.

PURPOSE: Our purpose was (a) to investigate relationships of baseline endocrine serum levels with selected assisted reproduction (AR) parameters and (b) to evaluate the clinical applicability of an immunofluorometric assay (IFMA) as an alternative to a radioimmunoassay (RIA). METHODS: Basal endocrine values (analyzed by RIA-rFSH, rLH, and rE2; analyzed by IFMA-fFSH, fLH, and fGH) were determined for female patients (n = 142) preceding ovarian stimulation for AR. RESULTS: Specific AR parameters correlated significantly with RIA- and IFMA-determined FSH levels, although IFMA correlations consistently exceeds that of RIA. Cut-off values of fFSH > or = 11.68 IU/L or rFSH > or = 15.0 IU/L indicated a poor response. The high-basal fFSH group was older (34 vs 31 years; P = 0.0334) and yielded fewer oocytes (2.9 vs 4.6 oocytes; P = 0.0018) than the low-basal fFSH group (< 11.68 IU/L). Lower cumulative embryo scores and conception rates were also associated with the high-fFSH group, compared to the low-fFSH group. CONCLUSIONS: This study confirms the negative impact of elevated basal FSH levels on AR. Basal LH, GH, and E2 levels are, in comparison to baseline FSH levels, unsuitable indices for estimating ovarian responsiveness. IFMA can be regarded as an alternative to RIA in both clinical and research laboratories.

Teratozoospermia and in-vitro fertilization: a randomized prospective study.

A prospective randomized study was conducted to assess the prognostic value of sperm morphology in an in-vitro fertilization (IVF) programme, using strict criteria. The first group (T, teratozoospermic) included 32 couples with an isolated teratozoospermia in the male partner (morphology < 9% normal). The second group (C, control) contained 36 couples with normal semen parameters, including morphology (> 9% normal, strict criteria). In both groups, 50 IVF cycles were performed. Patients were matched for indication for IVF. There was no difference between the two groups regarding age, duration of infertility, stimulation protocol, catheter used for embryo transfer and different sperm parameters. A statistically significant difference between the T and C groups respectively was observed regarding the fertilization rate (69.2 and 79.4%, P < 0.05), pregnancy rate per cycle (12.0 and 42%, P < 0.001), the pregnancy rate per transfer (13.9 and 42.0%, P < 0.01) and per embryo transferred (6.1 and 14.8%, P < 0.05). No pregnancy occurred in the poor prognosis group (morphology < 5% normal). In cases of moderate teratozoospermia, the fertilization rate appeared normal (78.6%) but the conception rate remained low. We concluded that the use of strict criteria in the assessment of sperm morphology is useful in predicting fertilization and pregnancy rate in the human in-vitro model.

Interleukin-1 beta, interleukin-6, and growth hormone levels in human follicular fluid.

PURPOSE: To investigate possible relationships of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and growth hormone (GH) with biochemical variables in human follicular fluid (FF) and selected in vitro fertilization (IVF) parameters. METHODS: A total of 67 FF samples (n = 67 patients undergoing oocyte retrieval for IVF) was evaluated. IL-1 beta, IL-6, GH, hLH, FSH, PRL, hCG, testosterone, total protein, fibrinogen, sialic acid, alpha 1-antitrypsin, plasminogen levels, and spectrophotometric absorbance at 458 nm were analyzed for selected FF. IL-6 and GH levels of serum and FF samples were also compared (n = 23). RESULTS: Immunoreactive levels of IL-1 beta, IL-6, and GH were detected in all FF samples. A positive correlation existed for IL-6 (r = 0.5069, P = 0.0161) when serum-to-FF levels were compared (concentration ratio, 1:1.857). Smaller-volume follicles (< 4 ml) were associated with high IL-1 beta levels (P = 0.0229), and an additional tendency of IL-1 beta to decrease with increasing embryo cleavage and scoring was observed. With the exception of a weak positive correlation between follicular IL-1 beta and testosterone levels (r = 0.3128, P = 0.025), no other relationship with biochemical variables or IVF parameters (etiology, e.g., endometriosis) could be implicated. CONCLUSIONS: Substantially higher IL-6 levels occurred in FF compared to serum, thus supporting intrafollicular production. Interleukin-1 beta, IL-6, and GH levels in FF are, however, unsuitable markers for in vitro fertilization outcome.

Spectrophotometric analysis of human follicular fluid with regard to in vitro fertilization (IVF) parameters, follicular protein, and hormone content.

PURPOSE: Our purpose was to investigate possible relationships with spectrophotometric absorbance (458-nm region) and biochemical variables in follicular fluid (FF) as well as in vitro fertilization (IVF) outcome. METHODS: This study included 227 normal ovulatory women undergoing oocyte retrieval for IVF. Blood-uncontaminated fluid samples, identified by spectrophotometry, were investigated. Spectrophotometric absorbance of FF at 458 nm (n = 426), as well as hLH, FSH, PRL, hCG, testosterone, sialic acid, alpha 1-antitrypsin and plasminogen of selected fluids, was analyzed. RESULTS: Small-volume follicles (< or = 2 ml) were associated with higher absorbance profiles (P < 0.05), when compared to volumes greater than 2 ml. Our data suggest that the presence or absence of an oocyte, the potential of an oocyte to fertilize or cleave, failed to show any relationship with maximum FF absorbance at 458 nm. Maximum absorbances were significantly lower in FF from patients who subsequently became clinically pregnant (P = 0.039). No correlation between FF absorbances and biochemical parameters (P > 0.15) were established. CONCLUSIONS: Absorbance of clear FF at 458 nm should not be viewed as the single parameter to predict oocyte development in vitro.

Multiple attempts at embryo transfer: effect on pregnancy outcome in an in vitro fertilization and embryo transfer program.

PURPOSE: This study derives from the observation that a correlation exists between failed first attempts (FFA) at embryo transfer caused by one or more embryos remaining in the catheter and reduced pregnancy rates (20.3 vs 3.0%). The aim of this study was to examine the relationship between failed first attempts at transfer and contamination of the transfer set; the related aspects of cervix dilatation and late embryo transfer were also investigated. RESULTS: The following observations were made. Retention of embryos in the transfer sets significantly reduced the pregnancy rate (P = 0.015); catheters contaminated with blood and cervical mucus indirectly contributed to this effect by increasing the incidence of failed first transfer attempts. Even though cervical dilatations, if indicated by uterus sounding, were done 2 days before embryo transfer, no pregnancies were effected in these 18 cases (P = 0.0001). Late transfers of embryos, due to delayed fertilization or slow cleavage rates, yielded a pregnancy rate of 10.5%. CONCLUSION: The approach of immediately retransferring retained embryos does not solve the problem of reduced pregnancy rates in FFA cases. It is suggested that ET should be repeated 1 day later in FFA cases in an attempt to improve pregnancy rates.

Pregnancy outcome after in vitro fertilisation at the Pretoria unit.

This retrospective study was conducted to ascertain the success rate of a university-based in vitro fertilisation (IVF) programme. Over a 4-year period a total of 881 patients was aspirated with an 81.8% embryo transfer (ET) rate. This resulted in 150 biochemical pregnancies (20.8% per ET), and 92 births that produced 100 babies were recorded (12.8% per ET). Multiple pregnancies accounted for 8.7% of births and 6.5% of the 100 babies presented with minor congenital abnormalities. Delivery took place by caesarean section in 46% of cases. Birth mass of babies ranged from 1.06 to 4 kg with a mean of 2.944 +/- 0.629 kg. Mean gestational age was 260 +/- 18.2 days. Twenty-three percent of babies born were preterm, 13.8% of these were twins and presented with a low birth mass. It was concluded that the conception rate of patients did not differ with regard to the number of IVF attempts but that those who conceived during the first two attempts had a significantly better chance (P < 0.05) of carrying to term.

Spectrophotometric absorbance of follicular fluid: a selection criterion.

PURPOSE: The aim of this study was to ascertain the absorbance profiles of uncontaminated follicular fluids (FF) vs blood and medium contaminated FF, and to test the hypothesis that blood contamination alters the biochemical status of FF. METHODS: FF (n = 655) from 230 patients were scanned spectrophotometrically during an ongoing in vitro fertilization (IVF) program. Based on spectrophotometric evaluation, blood contaminated and uncontaminated FF (n = 39 patients/FF) were analyzed for electrolyte content, inorganic phosphate, glucose, creatine, urate, total protein, albumin, bilirubin, cholesterol, alkaline phosphatase, gamma-glutamyltransferase, alanine aminotransferase, aspartate transaminase, and lactate dehydrogenase by means of a SMAC analyzer. RESULTS: Laparoscopic aspiration resulted in the collection of significantly more clear FF compared to the transvaginal procedure (P = 0.001). Uncontaminated fluid depicted a single mean absorbance at 458.0 nm. Blood contaminated FF could be identified by spectrophotometry and revealed three peaks at 418.12, 540.13, and 575.32 nm, respectively, according to the degree of blood contamination. Follicular fluid diluted with Earles Balanced Salt Solution (EBSS) displayed an additional mean peak at 561.4 nm. Potassium, glucose, and all of the above mentioned enzymes revealed significantly higher levels in blood contaminated FF (P < 0.05). CONCLUSIONS: From this study, it is concluded that blood contamination and dilution with culture medium influence the biochemical composition as well as the absorbance spectrum of follicular fluids. This procedure is advocated as a prerequisite before quantifying FF content.

Microbial flora in semen during in vitro fertilization.

Semen samples from 183 consecutive unselected men participating in an in vitro fertilization program were retrospectively studied to determine the bacterial and fungal contamination rate before and after antibiotic treatment. To ascertain the influence of semen preparation (wash and swim-up method) on the incidence of microorganisms, semen from 102 male patients was studied before and after swimup. Antimicrobial treatment by prescription of antibiotics decreased the incidence of pathogens by 16.3% (P less than 0.0001). Semen processing was more effective by ridding 57.4% of semen samples of microbial contaminants (P less than 0.0001). When infection of culture media was observed during routine microscopy, all infected oocytes were degenerated, without evidence of fertilization or pronuclei.

In vitro fertilisation when normal sperm morphology is less than fifteen per cent. Results of in vitro fertilisation and embryo transfer at H.F. Verwoerd Hospital, Pretoria.

The outcome of in vitro fertilisation and embryo transfer in 90 couples where the husband's normal sperm morphology was less than 15% were analysed. Based on the percentage of morphologically normal spermatozoa the patients were divided into three groups: group A--normal morphological features 0-5%; group B--6-10%; and group C--11-14%. A control group had normal morphological features greater than or equal to 15%. The fertilisation rate and number of embryos transferred was not significantly different in these groups. However, the pregnancy rate per embryo transfer was significantly different for groups A and B compared with group C and the control group (0% and 5.9% for groups A and B v. 13.9% and 18.3% for group C and the control group, respectively). It is concluded that when the normal sperm morphology is less than 11% the prospect of a pregnancy is poor.

Successful pregnancy despite retrograde ejaculation. A case report.

In retrograde ejaculation sperm is passed into the bladder causing infertility. Among patients seen at the Reproductive Biology Research Unit of the University of Pretoria retrograde ejaculation is relatively uncommon (0.004%), but is a potentially treatable cause of infertility. The technique for collecting sperm, sperm processing and recovery described was successful and resulted in a pregnancy after transfer of 1 four-cell embryo.

In vitro culture of mouse embryos in human amniotic fluid.

Human amniotic fluid was compared with Ham's F-10 culture medium as a possible alternative for use in in vitro fertilisation. The cleavage success of mouse embryos in human amniotic fluid (experimental group) was 92% compared with 86% in Ham's F-10 medium. It is concluded that human amniotic fluid is a viable alternative culture medium for mouse embryos.

A preliminary study of the fine structure of in vitro fertilized human embryos.

The fine structure of rejected human in vitro fertilized embryos was studied by light and transmission electron microscopy. Apart from normal cytoplasmic features, changes suggestive of degeneration are discussed. Cytoplasmic blebs and cellular debris as well as primitive interblastomeric junctions are also described.

Cost analysis study of an in vitro fertilisation programme.

In vitro fertilisation (IVF) and embryo transfer has established itself as a modality in the treatment of infertile patients. Estimates of the cost of this relatively new procedure were investigated with specific assumptions. The maximum cost per (first) cycle totalled R1611.16. Laboratory expenses were the single most expensive entity, accounting for 39.2% of the total cost. Patient contribution was established at 32.6% of total cost and the approximate breakdown of cost for an IVF and embryo transfer programme for a university-based unit is given.

A preliminary report on the effects of parasitism by the leeches Theromyzon cooperi and Placobdella garoui on the physiology of the redbilled teal Anas erythrorhyncha.

The effects of parasitism by leeches Theromyzon cooperi and Placobdella garoui on the redbilled teal Anas erythrorhyncha were investigated. Leeches with initial body mass ranging from 5.2 to 28.4 mg were allowed to parasitize redbilled teal clamped in Perspex containers. Theromyzon cooperi increased its body mass after parasitism with a mean of 10.5-fold compared to 6.9-fold for P. garoui. Changes were also observed in values of various haematological and biochemical variables of the redbilled teal monitored during this investigation.

Effects of melatonin on heat production and enzymatic activity in diurnal and in nocturnal rodents.

1. Heat production by means of oxygen consumption (Vo2), and enzymatic activity of malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH) were studied in diurnal (Rhabdomys pumilio) as well as nocturnal (Praomys natalensis) murids, as a response to melatonin treatment. 2. A significant (P less than 0.001) increase in Vo2 was noticed in melatonin treated mice of both species at ambient temperatures of Ta = 6 degrees C, 25 degrees C, 30 degrees C and 34 degrees C, when compared to sham operated mice. A significant (P less than 0.001) increase was also recorded in body temperature (Tb) at Ta = 6 degrees C and 25 degrees C. 3. Enzymatic activity was significantly higher: P less than 0.001 for MDH and LDH in R. pumilio, P less than 0.05 for LDH, and P less tha 0.01 for MDH in P. natalensis, after melatonin administration when compared to sham operated animals.