A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro.

To determine predictive values of routine semen analysis, sperm morphology evaluation using strict criteria and DNA status for in-vitro fertilization (IVF), 66 consecutive couples undergoing IVF in a university hospital IVF programme were prospectively investigated. Semen samples from 66 men were evaluated by routine semen analysis, morphology evaluation using strict criteria and acridine orange staining for determination of DNA status. A new technique is described for acridine orange scoring which consisted of evaluation of two smears per case, with and without heat treatment. Resistance to heat-provoked denaturation was determined by the difference between two evaluations. A logistic regression model was built and receiver operating characteristic curves were constructed to determine the threshold values and to compare diagnostic properties. Morphology evaluation using strict criteria and concentration of progressively motile spermatozoa were found to be the principal parameters determining the sperm fertilizing capacity in vitro. The logistic regression model composed of morphology evaluation using strict criteria and acridine orange score had a powerful diagnostic capability for prediction of fertilization in vitro.

Micromanipulation of mouse gametes with laser microbeam and optical tweezers.

Micromanipulation of mouse gametes with a commercially available compact laser microbeam system was studied. Both the normal in-vitro fertilization (IVF) group and the laser zona dissection (LZD) group were tested under normal (2 x 10(6) motile spermatozoa/ml) and low (500,000 motile spermatozoa/ml) insemination conditions. Subzonal insemination (SUZI) was also tried in a small group of gametes and the results were compared with those of the low insemination groups. Fertilization rates and blastocyst formation rates for the IVF and the LZD-treated groups were respectively 53 and 60% and 60 and 78%, which were not significantly different. However, under low insemination conditions, the results were significantly better in the LZD-treated group (58% fertilization rate and 83% blastocyst formation rate) compared to the results of the IVF group (33 and 48%) (P < 0.05). The SUZI-treated group showed the lowest fertilization rate (18%). No significant difference between the LZD and the IVF group was observed with respect to parthenogenetic activation. LZD has a beneficial effect on fertilization rates in cases of reduced sperm quality.

The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos.

OBJECTIVES: To evaluate the occurrence of injury due to physical factors in embryo cryopreservation and the effect of the polymers dextran, polyvinylpyrrolidone (PVP), and Ficoll on this mechanical damage. DESIGN: Damage to the zona pellucida (ZP) observed after cryopreservation was taken as indication of cryoinjury caused exclusively by physical factors. Human and mouse ZPs from oocytes remaining unfertilized after previous IVF attempts and mouse two-cell embryos were frozen in the presence of different polymers. After thawing, they were checked carefully for signs of physical damage (cracks). A possible toxicity of the use of the polymers in cryoprotection was evaluated by development to the blastocyst stage of mouse two-cell embryos that survived the freezing and thawing process. RESULTS: Incidences of damaged ZPs in groups of human and mouse ZPs and two-cell embryos frozen without polymers were found to vary between 20% and 29%. The use of any of the tested polymers resulted in significantly lower incidences of damaged ZPs (0% to 15%). Damage to the ZP after freezing and thawing in mouse embryos was accompanied by low survival rates of the embryo itself. Of mouse embryos that survived the cryopreservation process, blastocyst formation was not significantly different in groups frozen without polymer (80%) or in the presence of either dextran (90%) or Ficoll (82%); however, embryos frozen in the presence of PVP showed low blastocyst formation (12%). CONCLUSIONS: Polymers can protect embryos against cryoinjury by avoiding mechanical strain occurring during cryopreservation. Polyvinylpyrrolidine is toxic to mouse two-cell embryos when present during freezing and thawing.

Predictive value of morphologically normal sperm concentration in the medium for in-vitro fertilization.

Morphological evaluation of spermatozoa using strict criteria (MEUSC) and conventional sperm parameters were studied with respect to in-vitro fertilization and pregnancy outcome before and after a swim-up selection procedure. Recovered oocytes were inseminated with 50,000 progressively motile spermatozoa, and this study assess the influence of the total number of spermatozoa and of the percentage with strictly normal morphology in the insemination sample on the outcome of IVF. The results showed that the percentages of spermatozoa with normal morphology using strict criteria, both in native and in post-swim-up samples, were the best predictors of IVF outcome. Their respective cut-off points were 5% and 8%. The number of morphologically normal spermatozoa inseminated also showed a good correlation with fertilization. However, it was not possible to find a proper cut-off point for this parameter. The patients were categorized on the basis of their native and post-swim-up scores. Category 1, in which both parameters were below their respective cut-off points, showed a 7% fertilization rate and a 0% pregnancy rate. Category 3, in which both parameters were above their cut-off points, showed a 70% fertilization rate and a 23% pregnancy rate. This suggests that sperm morphology can be used as a criterion for patient selection for IVF as an aid to identification of possibly subfertile males.

Male factor as determinant of in-vitro fertilization outcome.

The effect of different semen parameters was evaluated in 200 consecutive couples in an in-vitro fertilization (IVF) programme. All semen analyses were performed on the native aliquot of semen which was subsequently prepared and used for in-vitro insemination. Morphology evaluation using strict criteria (kappa 0.46 and r = 0.565) was compared with progressive motile sperm density (kappa 0.37 and r = 0.333) and the conventional World Health Organisation (WHO) evaluation of morphology (kappa 0.31 and r = 0.378). Results show that morphology evaluation using strict criteria is the best predictor of IVF and density of progressively motile spermatozoa can be an optional method. The combined results of strict morphology and motile concentration progressively showed that if both parameters were below the cut-off points of 5% and 3 x 10(6)/ml respectively, the fertilization rate per oocyte was very low (18%). No pregnancies were achieved in this group. When both parameters were above the cut-off points, the fertilization rate per oocyte was high (72%) (P less than 0.005) and the pregnancy rate per embryo transfer was 27%. Predictive values indicate that morphology evaluation using strict criteria and the number of progressive motile spermatozoa can be used as patient selection criteria for infertility clinics.

Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro.

It has been shown, in both human and mouse in-vitro fertilization (IVF), that an excess number of spermatozoa in the insemination medium leads to reduced fertilization rates. In this study, we evaluated human embryonic development after dividing the oocytes of each of 62 IVF attempts into two groups on the basis of insemination with two widely used concentrations (50,000 and 100,000 spermatozoa/ml). The embryonic growth was retarded in the group inseminated with 100,000 spermatozoa/ml: significantly fewer fast developing embryos (4-cell and 5- to 8-cell stages) were found (53.4% in the 100,000/ml group and 65.5% in the 50,000 group; P less than 0.05). In two experimental series, mouse embryonic development was evaluated in the presence of 0, 50,000, 100,000 and 500,000 spermatozoa per ml. In the first series, the spermatozoa were present during 5-20 h after insemination, while in the second series, the spermatozoa were present during the whole culture period of 120 h. The development of mouse embryos was impaired when 500,000/ml spermatozoa were present during the whole culture period. In contrast with human IVF results, the presence of up to 500,000 spermatozoa during the first 20 h after insemination did not have any significant detrimental effect on blastocyst formation in the mouse.

Comparison between the hypoosmotic swelling test and morphology evaluation using strict criteria in predicting in vitro fertilization (IVF).

BACKGROUND: The role of male factor with respect to sperm morphology, progressive motility, and density is studied under in vitro conditions. METHODS: The semen samples of 67 males participating in an in vitro fertilization program were evaluated by the conventional WHO criteria of spermatogram, by morphology evaluation using strict criteria (MEUSC), and by the hypoosmotic swelling test (HOST). All sperm tests were performed in the original semen sample as delivered on the day of IVF, before further sperm treatment. The correlations between these parameters and the fertilization outcome were evaluated and their predictive values were calculated. RESULTS: When the patients were divided into two groups, namely, fertile (fertilization rate per oocyte greater than 0%) and infertile (fertilization rate per oocyte = 0%), only mean sperm density and morphology were significantly different between the groups (P less than 0.05). The correlation with fertilization rate in vitro was in favor of MEUSC. CONCLUSIONS: Our results show that the HOST is inferior to MEUSC and conventional WHO sperm analysis in predicting fertilization in vitro.

Evaluation of human sperm morphology using strict criteria after Diff-Quik staining: correlation of morphology with fertilization in vitro.

New, very strict criteria were used after Diff-Quik staining for evaluating sperm morphology. The results of morphology scoring were correlated with the fertilization rate in vitro. Semen samples from 64 men participating in an in-vitro fertilization programme were used for this study. All men had to have a sperm concentration of greater than or equal to 20 million/ml and a progressive motility of greater than 30%. The morphology evaluation using strict criteria was performed on the same aliquot of semen as that used for in-vitro insemination. If strict criteria showed that normal morphology was less than or equal to 4%, the fertilization rate per oocyte was 23%. If normal morphology was greater than or equal to 11%, 77% fertilization occurred. For proportions of normal morphology between 4 and 11%, the fertilization rate per oocyte was 59% (P less than 0.000001). Among all these morphology groups, classical semen parameters, such as the mean volume, the mean concentration and the mean motility, did not differ significantly, except for the morphology evaluation using WHO criteria. The correlation with fertilization was better for morphology evaluation using strict criteria than for WHO. In conclusion, the method of evaluating sperm morphology based on very strict criteria allows a more accurate prediction of the chance of fertilization in vitro. Further studies should be done to establish the most appropriate cut-off points for severely impaired, intermediate and high fertilization rates.