Time intervals between semen production, initiation of analysis, and IUI significantly influence clinical pregnancies and live births.

PURPOSE: Does IDEF mapping help monitor the technical process of IUI and explore the potential improvements which might contribute to increased pregnancy and live birth rates? METHOD: Retrospective analysis of 1729 homologous IUI cycles of couples attending a fertility clinic in a university hospital setting. Standardized conventional semen parameters were analyzed and the semen samples prepared via discontinuous density gradient centrifugation. RESULTS: There was no significant association between sperm concentration, motility and morphology (analysis phase), and pregnancy outcome. Only female and male ages were significantly associated with the pregnancy outcome. There was a significant difference in the odds on clinical pregnancies and live births when analysis was ≤ 21 min initiated, and < 107 min between sample production and IUI, adjusted for male and female age. CONCLUSIONS: Adjusting for the couple's age, we could show that time intervals between semen production and analysis and IUI when kept low significantly influenced clinical pregnancies and live births.

DNA fragmentation in concert with the simultaneous assessment of cell viability in a subfertile population: establishing thresholds of normality both before and after density gradient centrifugation.

PURPOSE: TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. METHOD: Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a non-significant increase in total fragmentation and in the vital fraction the trend was unclear. CONCLUSIONS: Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.

Sperm DNA fragmentation in the total and vital fractions before and after density gradient centrifugation: Significance in male fertility diagnosis.

BACKGROUND: Sperm DNA fragmentation measured by different techniques make comparisons impossible due to lack of standardization. Induction of DNA damage after sperm preparation in the entire fraction has been observed on independent occasions but findings are not consistent. METHODS: Men presenting at a University hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Sperm motility in neat semen inversely correlated with sperm DNA fragmentation in the total fraction, but, total count, leukocytes and immature germ cells significantly affected the vital fraction. Sperm DNA fragmentation was observed both in normal and subnormal semen samples, but was significantly different in the total fraction of astheno-, asthenoterato- and oligoteratozoospermic men. After density gradient centrifugation, sperm DNA fragmentation increased significantly in the total but decreased in the vital fraction. Advancing male age significantly influenced damage in the total but not in the vital population. CONCLUSIONS: These findings provide opportunities to investigate the significance of the total and the vital fractions both in natural conception and after different assisted reproductive technologies.

Validating semen processing for an intrauterine program should take into consideration the inputs, actions and the outputs of the process.

To validate semen preparation via density gradient centrifugation, we took into account the input via the semen sample, the action generated by technical and equipment characteristics and the output measured by the level of performance. Longer periods of abstinence reduced % yield, but increased viscosity and incomplete samples collected had no effect. Under controlled technical and equipment characteristics, precision and reproducibility were validated for density gradient. Additionally, as a good laboratory practice, internal and external quality control measures were implemented to guarantee the level of performance. Inseminating motile sperm count is an important predictive parameter for IUI success. In our group of patients, a yield of an absolute lower limit of 2 million motile spermatozoa was sufficient to contemplate IUI. Pregnancy rate of 13.8% where >2 million rapid progressive spermatozoa were inseminated was significantly higher than the pregnancies (4.4%) obtained with <2 million rapid progressive spermatozoa. This percentage was even higher than the national data registered for IUI (12.2%). To make IUI an attractive first-line treatment, standardization and proper validation of semen preparation procedure are mandatory.