Comparison of Two Different Sperm Processing Methods and Their Effects on Sperm DNA Fragmentation and Embryo Development

The method is based on the Sperm Chromatin Dispersion (SCD) test (Fernández et al., J. Androl 24:59-66, 2003; Fertil Steril 84:833-842, 2005). Intact unfixed spermatozoa (fresh, frozen/unthawed, diluted or neat samples) are immersed in an inert agarose microgel on a pretreated slide. An initial acid treatment denatures DNA in those sperm cells with fragmented DNA. Following this, the lysing solution removes most of the nuclear proteins, and in the absence of massive DNA breakage produces nucleoids with large halos of spreading DNA loops, emerging from a central core. However, the nucleoids from spermatozoa with fragmented DNA either do not show a dispersion halo or the halo is minimal. Slides are stained with Wright's stain. A minimum of 300 sperm per sample will be counted. The percentage of sperm with fragmented DNA will be calculated by dividing the sperm with small and no halos (fragmented sperm) by the total number of sperm counted.

Assessed using a sperm class analyzer - SCA (CASA system) for sperm count. Semen samples will be loaded into 20 microliter depth fixed volume slides. Slides will be placed on the Microoptics SCA machine. Videos will be captured of fields until at least 200 motile sperm are captured. Concentration will be analyzed by counting the number of sperm within each field, corrected for the depth of the chamber, and multipled by the volume of the sample. Concentrations between fields will not vary by more than 15%.

Assessed using a sperm class analyzer - SCA (CASA system) for sperm motility. Semen samples will be loaded into 20 microliter depth fixed volume slides. Slides will be placed on the Microoptics SCA machine. Videos will be captured of fields until at least 200 motile sperm are captured. Motility will be calculated as the number of motile sperm counted divided by the total number of sperm counted. Types of motility will be noted according to WHO semen analysis groupings, hyperactivation, and kinetic motion parameters.

10 uL volumes of sperm samples will be placed on pre-stained morphology slides using methylene blue/cresyl violet stain. A minimum of 200 sperm will be counted and assessed for normal vs abnormal sperm morphology using Kruger strict criteria. Percentage of normal sperm will be assessed my dividing the number of normal sperm by the total number of sperm counted.

The number of fertilized eggs (zygotes) will be divided by the number of eggs inseminated

Embryo development will be monitored using a time-lapse incubator. A score will be generated based on when the embryos meets developmental time points. The embryo will receive 2 points for each time point it successfully completes with expected parameters. Embryos will receive 1 point for each time point it completes but outside the expected time parameters. i.e. if an embryo has the first cleavage between 23-25 hours it will receive 2 points. If it cleaves earlier than 23 hours or later than 25 hours it will receive 1 point. The total score an embryo receives will be tallied from the time of insemination until the embryo is frozen.

All embryos will be graded according to a 3-part grading scale at the time of cryopreservation between days 5 and 7 of culture. The embryo will be given a number score of 1-6 based on the embryo's size, size of the blastocoel fluid cavity, and whether or not it is inside, hatching out, or completely hatched out the zona. Each embryo will get a letter grade for the quality of the inner cell mass either A, B, C, or D. Each embryo will get a second letter grade for the quality of the trophectoderm A, B, C, or D.

Calculated by the number of patients that become pregnant (assessed by the presence of a fetal heartbeat 5-6 weeks following embryo transfer) divided by the total number of embryos transferred. Live birth will be calculated by the number of patients that give birth divided by the number of embryos transferred.