Comparative analysis of pregnancy rates after the transfer of early dividing embryos versus slower dividing embryos.

BACKGROUND: We compared retrospectively the pregnancy outcome in two subgroups of ICSI patients, using early division (26 h post injection) to the 2-cell stage as a criterion for embryo quality and viability (ability to produce a pregnancy). METHODS AND RESULTS: In the early dividing embryo (EDE) group, at least one of the transferred embryos was early dividing. In the late dividing embryo (LDE) group, no early dividing embryo was transferred. Additionally, tubal and uterine transfer in the two groups was also evaluated. Clinical pregnancy rates in the EDE group were significantly increased when compared with that in the LDE group (41.3 versus 20.0%). This was also true for ongoing pregnancy rates (33.3 versus 16.3%). The tubal transfer route showed increased (but not significant) ongoing pregnancy rates when compared with uterine transfer in both EDE (38.5 versus 25.0%) and LDE (22.7 versus 8.3%) groups respectively. In uterine transfer cycles, however, clinical pregnancy rates for EDE were significantly increased compared to LDE (37.5 and 11.1% respectively). The baby rate (number of live babies/embryos transferred) was also significantly increased in the EDE group and the tubal transfer group. Statistical analysis of pregnancy outcome, adjusted for the total number of embryos transferred (expressed as percentage risk difference - %RD), resulted significantly in favour of EDE compared to LDE (RD = 18%, P = 0.02). When adjusted for the combined factors: total number of embryos transferred, EDE and LDE, the pregnancy outcome result was significantly in favour of tubal transfer compared to uterine transfer (RD = 15%, P = 0.05). Pregnancy results of the LDE group only were significantly better in the tube compared to the uterus (RD = 19%, P = 0.04) but not significantly so for the EDE group (RD = 10%, P = 0.4). CONCLUSION: Early division is associated with embryo quality and a very easy and successful embryo transfer selection method. Our results also suggest that when EDE are available, both tubal and uterine embryo transfer can be considered. When only LDE are available, however, tubal transfer should be the preferred transfer route.

Intracytoplasmic sperm injection with testicular spermatozoa in men with azoospermia.

PURPOSE: The aim of the study was to gain an insight into the optimal management of the infertile couple with the husband suffering from azoospermia. METHODS: One hundred and forty-two intracytoplasmic sperm injection (ICSI) cycles performed with testicular extracted spermatozoa were retrospectively analysed. The following factors were investigated for their possible influence on fertilization, cleavage, damage, pregnancy, and ongoing pregnancy rates: the use of fresh, cryopreserved, and preincubated (24 h) spermatozoa and the etiology of the husbands' azoospermia (obstructive and nonobstructive). All microinjections were performed with apparently normal spermatozoa--a head with a tail of normal length. In 116 cycles at least two embryos were available for transfer. RESULTS: The overall fertilization, clinical pregnancy, and ongoing pregnancy rates obtained for the 116 cycles were 65.0, 30.2, and 22.4% respectively. Similar outcomes were obtained for cycles using fresh testicular and cryopreserved testicular spermatozoa. Similarly, no significant differences were obtained between the cycles using spermatozoa from obstructive or nonobstructive azoospermic patients. An increase in motility after a 24-h preincubation was observed, and although this group was relatively small (n = 17), a significant improvement in fertilization (73.7%) and pregnancy (53.9%) rate was obtained when the testicular sample was preincubated for 24 h. This improvement prevailed in the obstructive azoospermic group, but was less pronounced in nonobstructive patients. CONCLUSIONS: This study shows that the outcome of fresh and frozen-thawed testicular spermatozoa in ICSI is comparable, obstructive and nonobstructive etiologies perform the same, and that preincubation of testicular spermatozoa results in increased fertilization and pregnancy rates. All testicular biopsies are therefore performed the day before oocyte retrieval, superfluous spermatozoa cryopreserved, and the remaining testicular homogenate preincubated for the 24 h prior to oocyte retrieval. With this regime, most azoospermic patients are treated successfully, irrespective of the use of fresh or frozen-thawed spermatozoa from obstructive or nonobstructive cases.

Ultrastructural evaluation of recurrent and in-vitro maturation resistant metaphase I arrested oocytes.

An infertile couple whose female partner showed recurrent retrieval of immature metaphase I (MI) oocytes that were resistant to in-vitro maturation, was studied. Four spermiograms revealed teratozoospermia. Consistent non-fertilization and negative pregnancy outcomes were obtained after intrauterine insemination, gamete intra-Fallopian transfer and IVF. Two intracytoplasmic sperm injection (ICSI) cycles were finally performed. All oocytes (n = 17) in both cycles were arrested at MI and failed to mature after 48 h culture. ICSI also resulted in total non-fertilization. In the last cycle, two oocytes were analysed by transmission electron microscopy and showed almost identical results. All organelles showed normal characteristics of an MI oocyte. The main abnormality found was related to the MI spindle, with absence of microtubules and dispersion of the female chromosomes. Minor abnormalities were observed (immature fibrous appearance of the zona pellucida; the presence of small vesicle aggregates which formed a foam-like body). The injected sperm nucleus was arrested in the middle of the chromatin decondensation process, with no visible nuclear envelope reformation. Normal disruption of sperm acrosomal and flagellar components were observed. Only a partial cortical reaction was observed. This represents the first documented case of developmental arrest due to complete absence of spindle formation in association with an otherwise mature ooplasm.

Ongoing pregnancies resulting from intracytoplasmic sperm injection (ICSI) of spermatozoa from frozen-thawed testicular biopsy specimens.

Two clinical pregnancies following intracytoplasmic sperm injection of spermatozoa from frozen-thawed testicular biopsies in two azoospermic men are reported. The use of spermatozoa from cryopreserved testicular tissue is therefore a viable option for azoospermic men, as our results indicate that pregnancies is achievable in these cases.

Effect of sperm preincubation time on GIFT pregnancy outcome.

This investigation was conducted to evaluate the effect of human sperm preincubation time on the pregnancy outcome in gamete intrafallopian tube transfers. This was determined in a retrospective study on gamete intrafallopian transfer patients (ideopathic infertility) using logistic regression with the covariates, preincubation time (< or = 60 and > 60 min) and age (< or = 35 and > 35 years). The study included 485 consecutive gamete intrafallopian transfer cycles in which 3 metaphase II oocytes were transferred. Pregnancy outcome was evaluated by beta hCG levels on days 12 and 16 and was confirmed by the presence of a fetal heart 8 weeks after the procedure by means of sonography. The wife's age and sperm preincubation time were significant covariates in the prediction of ongoing pregnancy. The odds ratio for age (< or = 35 years) was 2.2 in the prediction of ongoing pregnancy, while the age-adjusted odds ratios for preincubation time (< or = 60 min) was 1.9. There was a critical relationship between sperm preincubation time and GIFT success, which confirms the effect of the wife's age on pregnancy. Close coordination is of importance between the clinical scientist and the physician to restrict the preincubation time to 1 h. The sperm preincubation time is a significant factor that has to be considered in the prediction of ongoing pregnancy in GIFT patients.

Fertilization and pregnancy using metaphase I oocytes in an intracytoplasmic sperm injection program.

PURPOSE: In this study we investigated whether metaphase I oocytes collected in an intracytoplasmic sperm injection program could successfully be matured and fertilized by injecting aged (> 20-hr) spermatozoa. MATERIALS AND METHODS: Metaphase I oocytes aspirated were preincubated for 20 hr to allow the oocytes to reach meiotic maturity. Only metaphase II oocytes were injected. The original sperm sample processed on the day of aspiration was used in the microinjection process. RESULTS: One hundred eighty-three oocytes were collected, of which 42 (23%) were metaphase I oocytes. These were incubated for 20 hr and microinjected with the original sperm sample. Thirty-one (74%) of the metaphase I oocytes reached meiotic maturity (extruded polar body): 67.7% showed two pronuclei 18 hr after injection and 61.3% embryo development 40 hr postinjection. No difference in fertilization and embryo development rate was found in metaphase II oocytes injected 6 hr postaspiration versus 20 hr postaspiration. An ongoing pregnancy was also achieved using only embryos obtained from matured metaphase I oocytes. CONCLUSIONS: Metaphase I oocytes can be successfully matured in vitro and injected using aged (> 20-hr) sperm samples. Matured metaphase I oocytes, if successfully injected, produce embryos able to induce pregnancy.

In vitro fertilization failure: identification of gamete defects by investigation of sperm-zona pellucida binding capacity of unfertilized oocytes.

The objective of the study was to determine whether fertilization failure was due to spermatozoal or oocyte factors. Twenty-five unfertilized oocytes from 12 IVF/GIFT couples showing total or partial fertilization failure were evaluated for sperm zona binding potential under hemizonaeassay (HZA) conditions. Hemizonae were separately incubated with a sperm sample from the husband and that of a fertile control. Tight sperm binding to hemizonae was assessed. First, among the 12 patients, results showed a possible zona defect thought to be the cause of fertilization failure in five cases. Second, in two cases, fertilization failure was possibly caused by poor sperm binding potential of spermatozoa. Third, in two cases, fertilization failure was possibly caused by an oocyte defect, and fourth, three cases showed a mixture of possible causes. The results stress the need to develop a sequential analytic programme for those couples with repeated total or partial fertilization failure.

Sperm decondensation and semen parameters: utilization of a simple staining technique for the evaluation of human sperm decondensation.

The ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin on penetration of the oocyte. Knowledge of what percentage of sperm decondenses is useful, especially in patients where other functional tests and sperm quality fail to explain the reported poor in vitro fertilization (IVF) rates. The objective of this study was (1) to compare sperm decondensation induced by either SDS/EDTA or heparin with semen parameters (volume, concentration, motility and morphology), and (2) to evaluate the use of a simplified staining technique (Diff QuikR [DQ]) in comparison with the standard phase contrast method (Rose Bengal-[RB]). Randomly selected semen samples from 31 men attending an assisted reproductive programme were analysed for basic semen parameters and decondensation with SDS/EDTA and heparin. Two staining methods for the evaluation of decondensation were compared (phase contrast microscopy after Rose Bengal [RB] staining and light microscopy after Diff QuikR (DQ) staining). Moderate and grossly swollen sperm heads were recorded. Semen samples included both fertile and unfertile semen parameters. Sperm decondensation results showed poor to moderate correlations with semen parameters. The SDS/EDTA (DQ) (moderate forms) showed a significant negative correlation (r = -0.46) with seminal volume and a significant positive correlation (r = 0.41) with normal sperm morphology. The heparin (DQ) (moderate forms) decondensation showed a significant positive correlation with motility (r = 0.61) and sperm concentration (r = 0.43). The DQ method was preferred over the RB method due to its optical and storage advantage.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of human sperm membrane antigens reacting with sera from antisperm antibody positive and negative patients by western blotting.

Immunological infertility is thought to be caused by the binding of antibodies to 'fertility-related' antigen(s) on the sperm membrane. We compared antibody profiles in sera from 20 ASA(+) and ASA(-) men, using a sperm membrane extract as an antigen. Antigens were separated by SDS-PAGE under reducing conditions. The patients were classed as ASA(+) by the MAR (> 50%), d-IBT (> 20%) and TAT (> 1:64). The results showed that immunoreactive bands in both the ASA(+) and ASA(-) groups were heterogeneous and included bands covering the whole molecular weight range. Statistical analysis showed significantly more patients in the ASA(+) group having immunoreactive bands at molecular weights of 32 Kd (P = 0.006) and 79 Kd (P = 0.02) when compared to the ASA(-) group. In the ASA(-) group significantly more patients had reactive bands at 81 Kd (P = 0.01) when compared to the ASA(+) group. The 32 Kd antigen reacted only with sera from ASA(+) patients. We conclude that differences exist between the ASA(+) and ASA(-) groups when this extraction method is used and that the isolation and purification of the 32 Kd protein may justify further investigation.

The influence of solubilized porcine zona pellucida protein on the binding capacity of human spermatozoa.

The acrosome reaction, sperm-zona pellucida binding, sperm-oolemma binding/fusion and subsequent fertilization are known to be influenced by homologous as well as heterologous follicular fluid and zona pellucida protein. In this study, the effect was investigated of different concentrations of solubilized porcine zona pellucida protein on the zona binding potential of human spermatozoa under hemizona assay conditions. Human spermatozoa incubated with 617 and 142 micrograms/ml porcine zona pellucida protein showed a statistically significant increase in zona binding when compared with control spermatozoa (106.5 +/- 18.0 versus 60.9 +/- 29.0, P less than 0.02 and 111.0 +/- 26.6 versus 63.0 +/- 25.5, P less than 0.0001, respectively). Concentrations of 67 micrograms/ml porcine zona pellucida protein did not show a significant increase in zona binding (78.7 +/- 21.7 versus 66.7 +/- 25.4, P greater than 0.05). Control zona binding values for different experiments did not differ significantly (60.9 +/- 29.0; 63.0 +/- 25.5; and 66.7 +/- 25.4, P greater than 0.6). In conclusion, it seems likely that a factor(s) present in the porcine zona pellucida might play a beneficial role during human sperm-oocyte binding. The results of the study might be used in future investigations to manipulate gamete interaction to such an extent that improved fertilization rates can be accomplished.

Comparison of sperm binding potential of uninseminated, inseminated-unfertilized, and fertilized-noncleaved human oocytes under hemizona assay conditions.

In this study, human oocytes obtained after ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were utilized to evaluate sperm/zona pellucida binding potential. Three groups of oocytes were evaluated: 1) uninseminated; 2) inseminated-unfertilized; and 3) fertilized-uncleaved. All oocytes had undergone germinal vesicle breakdown at the time of retrieval and were salt-stored (pH 7.2) for not more than 30 days. Sperm binding was recorded under hemizona assay (HZA) conditions using spermatozoa from eight fertile men (HZA control) and from 1) four teratozoospermic (HZA test) and 2) four normozoospermic (HZA test) infertile men. First, the mean numbers (+/- SD) of sperm tightly bound for fertile controls and teratospermic men to hemizonae from uninseminated oocytes were 69.7 +/- 16 and 14.5 +/- 7, respectively (P = 0.02). Likewise, hemizonae from uninseminated oocytes bound 102.0 +/- 19 and 114.0 +/- 28, respectively, for fertile controls and normospermic men (P = 0.5). Second, hemizonae obtained from inseminated-unfertilized IVF oocytes bound 44.2 +/- 12 and 19.7 +/- 6 for fertile controls and teratospermic men, respectively (P = 0.02). This category of oocytes bound 100.5 +/- 7 and 108.5 +/- 11 sperm, respectively, for fertile controls and normospermic semen (P = 0.3). Third, HZA results of fertilized but uncleaved oocytes showed a mean number of tightly bound sperm of 6.0 +/- 4 compared with 65.0 +/- 1 in control, uninseminated oocytes using fertile sperm. These results demonstrate that uninseminated and inseminated-unfertilized human oocytes, salt-stored under controlled pH conditions, give reliable information regarding sperm binding potential under HZA conditions.

Assisted reproductive technologies may obviate apparent immunologic infertility.

Spermatozoal autoantibodies have been associated with reduced fertilization by natural coital methods. Nine subfertile men were evaluated who repeatedly tested positive for spermatozoal autoantibodies as characterized by direct immunobead test. Using the hemizona assay, we determined whether tight binding of spermatozoa to the zona pellucida was reduced in these test males as compared to a fertile male whose semen had been cryopreserved and thawed immediately prior to testing. The average number of spermatozoa tightly bound to the zona pellucida from the subfertile male was significantly reduced compared to the fertile male (mean +/- SD, P less than or equal to 0.01) 19.5 +/- 8 versus 77.1 +/- 49. Seven of the nine couples eventually had successful fertilization using intrauterine insemination or gamete intrafallopian tube transfer and one couple conceived with natural coital insemination. Our findings, albeit limited, suggest that greater caution should be used in implicating associations of spermatozoal autoantibodies with absolute infertility, because novel assisted reproductive technologies often may obviate conventional encumbrances on opportunities for pregnancy.

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein.

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.

Detection of sperm antibodies on unwashed spermatozoa with the immunobead test: a comparison of results with the routine method and seminal plasma TAT titers and SCMC test.

This study investigated whether the immunobead test (IBT) could, for the purposes of simplicity and saving time, be applied directly on an unwashed semen sample instead of washed spermatozoa. These two methods were performed simultaneously on the semen samples of 15 men with a positive MAR test and 10 men with a negative MAR test. A possible association was found between the unwashed samples, showing positive IB binding (greater than 20%) on the tail and/or head and the seminal plasma TAT titers (P less than .00001, Fisher's exact test). In all cases with IB binding of greater than or equal to 20% on unwashed spermatozoa, positive seminal plasma TAT titers (greater than or equal to 32) and SCMC tests (greater than or equal to 50%) were found. In all cases where the binding of the beads was mainly located on the tailtips of the washed or unwashed spermatozoa, negative seminal plasma TAT titers and SCMC tests were found. Coating of the head and/or upper tail regions in both methods was always associated with high TAT titers and a strong-positive SCMC test. It is concluded that the IBT for IgA, but not for IgG, can be performed directly on unwashed semen and that the position of IB binding on the spermatozoa is of prognostic importance with regard to the expected outcome of the SCMC test and seminal plasma TAT titers.

The in vitro fertilisation programme at Tygerberg Hospital and the University of Stellenbosch. Five years' experience, April 1983-January 1988.

The results of the in vitro fertilisation programme at Tygerberg Hospital for the period April 1983 to January 1988 are presented. Of the 1117 laparoscopies performed, 825 patients reached the transfer stage. A live-birth rate of 9.3% was achieved. The pregnancy rate after transfer of 4 embryos was 25.9% compared with 15.4% after 2 embryos and 10.8% after 3 embryos (P = less than 0.0001). The multiple pregnancy rate was 2.8% in the group receiving 2 embryos and 11.7% and 10.4% in those receiving 3 and 4 embryos, respectively. Of the 77 successful pregnancies (90 babies), 1 baby died at 34 weeks' gestation as the result of abruptio placentae due to preeclampsia and 1 cot death occurred. The only congenital abnormality encountered was a cleft palate.

Treatment of male sperm autoimmunity by using the gamete intrafallopian transfer procedure with washed spermatozoa.

Sixteen couples were diagnosed as having immunological infertility. To detect sperm-bound immunoglobulin (Ig), i.e., IgA, IgG, and IgM antibodies, the direct immunobead test (IBT) was used. In each individual patient, the direct IBT was greater than or equal to 70% positive for either IgA or IgG or both. The indirect IBT was positive for IgA and IgG antibodies in the serum of all the patients. Semen was collected in 15 mL medium (Ham's F10 [Gibco, Grand Island, NY] + 10% whole blood serum) and prepared with the wash and swim-up method. Patients in the study group were treated for their immunological infertility problem by performing the gamete intrafallopian transfer (GIFT) procedure. An ongoing pregnancy was achieved in 7 of the 16 (43%) couples treated with the GIFT procedure with an ongoing pregnancy rate of 24.1% (7 of 29) per cycle. The GIFT procedure appears to be an effective and safe way of treating male immunological infertility.