精蟲DNA碎片過多(>27.3%)會對高齡女姓ICSI懷孕結果產生不利影響

Fertil Steril. 2015 Apr;103(4):910-6. doi: 10.1016/j.fertnstert.2015.01.014. Epub 2015 Mar 4.

Effect of sperm DNA fragmentation on the clinical outcomes for in vitro fertilization and intracytoplasmic sperm injection in women with different ovarian reserves.

Jin J1, Pan C1, Fei Q1, Ni W1, Yang X1, Zhang L1, Huang X2.

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Abstract

OBJECTIVE:

To investigate effect of sperm DNA fragmentation (SDF) on clinical outcomes of assisted reproductive technology in women with normal ovarian reserve (NOR) versus reduced ovarian reserve (ROR).

DESIGN:

Retrospective clinical study.

SETTING:

University-affiliated tertiary teaching hospital.

PATIENT(S):

A total of 2,865 consecutive couples undergoing their first in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycle.

INTERVENTION(S):

SDF assessed using sperm chromatin dispersion in sperm samples 1-2 months before treatment.

MAIN OUTCOME MEASURE(S):

SDF, IVF, and ICSI outcomes.

RESULT(S):

The grouping criteria were [1] basal follicle stimulating hormone >10 IU/L, [2] antral follicle count <6, and [3] female age ≥38 years. Women fulfilling two of the three criteria were considered to have ROR, and those not meeting any criteria were considered to have NOR. The area under the receiver operating characteristic curve was 0.594 (0.539-0.648) for the ROR group and 0.510 (0.491-0.530) for the NOR group. A cutoff value for SDF to predict the clinical pregnancy rate (CPR) in the ROR group was 27.3%. When the SDF exceeded 27.3%, the live-birth and implantation rates in the ROR group were statistically significantly decreased, but the clinical pregnancy, live-birth, and implantation rates were not affected in the NOR group. The risk of early abortion increased significantly in the NOR group when the SDF exceeded 27.3%.

CONCLUSION(S):

Sperm DNA fragmentation has a greater impact on IVF and ICSI outcomes among women with ROR, so SDF testing may be of particular clinical significance for these couples.

Each SCD assay was performed using the Halosperm kit (INDAS Laboratories) with a slight modification. In brief, the sperm concentration was diluted to 5–10 million per milliliter. The total volume was split into 30-μL aliquots on agarose gel in tubes that were placed in a water bath at 90–100°C for 5 minutes to fuse. The tubes were then placed in a water bath at 37°C for temperature equilibration. Next, 18 μL of the semen sample was added to each tube and mixed with the 30 μL of fused agarose, then 18 μL of the semen-agarose mixture was pipetted onto a precoated slide and covered with an 18 × 18-mm coverslip. The slide was placed in a refrigerator at 4°C for 5 minutes to allow the agarose to produce a microgel embedded with sperm cells. The coverslip was gently removed, and the slide was immediately immersed in an acid solution for 7 minutes. Next, the slide was immersed in lysing solution for 25 minutes. After 5 minutes of washing in a tray with abundant distilled water, the slide was dehydrated in increasing concentrations of ethanol (70%, 90%, and 100%) for 2 minutes each, and was then air dried.

For bright-field microscopy, each slide was covered with a mixture of Wright's staining solution and phosphate buffer solution (1:1) for 5–10 minutes. The slide was briefly washed in running water for 10 seconds and allowed to dry. Strong staining is preferred to achieve easy visualization of the periphery of the dispersed DNA loop halos. A minimum of 400 spermatozoa for each patient was scored according to the patterns established by Fernandez et al. (17). Sperm nuclei with fragmented DNA produce very small or no halos of dispersed DNA, whereas nuclei without DNA fragmentation release their DNA loops to form large halos. The percentage of sperm with very small or no halos was defined as the SDF level.