DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction  Jung Jin Lim, Ph.D., Jin Il Lee, M.Sc., Dong Hwan Kim, M.Sc., Seung-Hun Song, M.D., Hyung Joon Kim, Ph.D., Woo Sik Lee, M.D., Ph.D., Dong Ryul Lee, Ph.D.  Fertility and Sterility  Volume 100, Issue 6, Pages 1564-1571.e5 (December 2013) DOI: 10.1016/j.fertnstert.2013.08.017 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Schematic diagram of ligation-mediated real-time polymerase chain reaction (LM-RT-PCR) and LM-RT-PCR detection of DNA ladders on agarose gel. (A) Genomic DNA was purified and 5′-phosphorylated fragments (blue) were ligated to linkers consisting of a 24-bp oligonucleotide (red) and a complementary 12-bp oligonucleotide (black), thereby forming a blunt end. Heating the ligated DNA to 72°C removed the 12-bp oligonucleotides, and subsequent treatment with Taq polymerase filled in the protruding 5′ ends. The 24-bp oligonucleotide could then be used as a PCR primer, resulting in the selective amplification of DNA fragments with linkers at both ends. (B) Human sperm genomic DNA (50 ng/sample) was subjected to 25 cycles of LM-RT-PCR and examined by agarose gel electrophoresis. Lane 1: 100-bp size marker. Lane 2: sperm treated with 5 IU DNase I for 10 minutes (positive control for DNA fragmentation). Lanes 3–4: samples from the high DF% group. Lanes 5–6: samples from the low DF% group. Lane 7: sperm with DF% <1–2 (negative control for DNA fragmentation). Lane 8: reactions run without T4 DNA ligase (negative control for PCR). Fertility and Sterility 2013 100, 1564-1571.e5DOI: (10.1016/j.fertnstert.2013.08.017) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Ligation-mediated real-time polymerase chain reaction (LM-RT-PCR) for sperm DNA fragments derived amplification and melting curve. (A) LM-RT-PCR graph representing amplification curves (blue line: amplification curve of negative control; red line: amplification curve of positive control). The Ct values of experimental samples (green lines) were located between the positive and negative control. (B) LM-RT-PCR graph representing melting curves (blue line: amplification curve of negative control; red line: amplification curve of positive control). Relative fluorescence unit (RFU) values of experimental samples (green lines) were located between the positive and negative control. Note: The axis in the amplification curve represents PCR cycles (x-axis) and RFU (y-axis). The axis in the melting curve represents temperature (x-axis) and RFU (y-axis). Fertility and Sterility 2013 100, 1564-1571.e5DOI: (10.1016/j.fertnstert.2013.08.017) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Comparative analysis of ligation-mediated real-time polymerase chain reaction (LM-RT-PCR) for sperm DNA fragments rate using threshold cycle (Ct) and the relative fluorescence unit (RFU) value. (A) LM-RT-PCR graph representing amplification curves (red: amplification curve of the high sperm DF% group; green: amplification curve of the low sperm DF% group). (B) LM-RT-PCR graph represents melting curves (red: amplification curve of the high sperm DF% group; green: amplification curve of the low sperm DF% group). Note: Basic criteria were used for the TUNEL assay (sperm with fragmented DNA ≤20%: low DF%; sperm with fragmented DNA >20%: high DF% group). The axis in the amplification curve represents PCR cycles (x-axis) and RFU (y-axis). The axis in the melting curve represents temperature (x-axis) and RFU (y-axis). Fertility and Sterility 2013 100, 1564-1571.e5DOI: (10.1016/j.fertnstert.2013.08.017) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Evaluation of sperm DNA fragmentation using TUNEL and SCD assays. (A) Photomicrograph of human sperm stained using the TUNEL assay kit. TUNEL-negative sperm (unfragmented DNA) stain positive for DAPI (blue); the TUNEL-positive sperm (fragmented DNA) stain positive for tetramethylrhodamine (TMR) red (red) and DAPI (blue, in the head region). (B) Images of sperm with various morphologic features, as observed by bright field and fluorescence (DAPI/TUNEL) microscopy. (a) Normal morphology without fragmented DNA. (b) Abnormal morphology (large head) with fragmented DNA. (c) Normal morphology with fragmented DNA. (d) Abnormal morphology (amorphous head) without fragmented DNA. (C) Photomicrograph of human sperm stained using the SCD assay kit showing sperm with fragmented (no halo: c, d) and unfragmented (halo: a, b) DNA. Note: TUNEL assay results (i.e., the median ratios of sperm with fragmented DNA identified by the TUNEL assays were 20.5%) were used to categorize samples into low DF% (sperm with ≤20% fragmented DNA) and high DF% (sperm with >20% fragmented DNA) groups. Fertility and Sterility 2013 100, 1564-1571.e5DOI: (10.1016/j.fertnstert.2013.08.017) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Evaluation of sperm using strict morphologic criteria. Photomicrographs of human sperm stained using the Diff-Quick staining kit. (A) Normal morphologic features from low DF% group. (B) Different abnormal morphologic features from high DF% group: b, c, and d show an elongated head, coiled tail, and amorphous head, respectively. Fertility and Sterility 2013 100, 1564-1571.e5DOI: (10.1016/j.fertnstert.2013.08.017) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions