Acridine orange binding to RNA interferes DFI calculation in SCSA

18 03 2010

To the Editor:

In the era of assisted reproductive technique (ART), it is very important to assess DNA fragmentation index (DFI) and establish threshold levels beyond which there is poor ART outcome, pre- or post-implantation failure or recurrent spontaneous abortions. The study by Smit et al. (1) is very informative and of immense clinical significance.

Sperm chromatin is a highly organized, condensed and compact structure, which is considered to be an important factor for the normal fertilization and pregnancy outcome (2). Sperm chromatin structure assay (SCSA) is an excellent and most predictable and clinically correlated test widely used in estimation of DFI. Acridine orange (AO), a metachromic dye has the property of shifting fluorescence to red when it binds to single stranded DNA or RNA from green when it is bound to native double-stranded DNA. In our recent work on SCSA, we found decreased mean red fluorescence after treatment with RNAase compared to the mean value without RNAase treatment (unpublished data). This shows that the number of cells showing red fluorescence (indicating DNA damage) may be overestimated by SCSA due to presence of single stranded RNA which also binds to AO.

Until recently it was believed that sperm is a terminally differentiated and transcriptionally inert cell. However, 15% of sperm DNA is bound to histones and remains transcriptionally active and codes for a complex cohort of mRNA (3,4) which are of developmental importance (HOX gene cluster), miRNA cluster and imprinted gene clusters and genes involved in cell proliferation and signal transduction. These transcripts are critical for early post fertilization events and are also important for embryogenesis (5). Ostermeir et al., 2004 (6) reported that about 18,000 transcripts are delivered to the oocyte at the time of fertilization.

Though mRNA usually have a short half-life of about 5 hours, sperm mRNA lie in close proximity to the nucleus, in the nuclear region or embedded in the nucleus, and persist until after fertilization. The presence of these transcripts is indicative of fertility status and of diagnostic and prognostic importance. However, since AO binds to single stranded DNA and RNA, this actually may result in overestimation of cells showing red fluorescence. This was observed after RNAase treatment of semen ejaculate, which showed significant decline in red fluorescence. Thus we recommend that the semen sample used for SCSA should be pretreated by RNAase to actually quantify ssDNA and calculate accurate DFI.

Sundararajan Venkatesh
Rima Dada, M.D., Ph.D
Laboratory for Molecular Reproduction and Genetics
Department of Anatomy
All India Institute of Medical Sciences (AIIMS)
New Delhi, India

References
1. Smit M, Romijn JC, Wildhagen MF, Weber RF, Dohle GR. Sperm chromatin structure is associated with the quality of spermatogenesis in infertile patients. Fertil Ster [epub ahead of print].

2. Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl 2006; 8:11-29.

3. Barroso G, Valdespin C, Vega E, Kershenovich R, Avila R, Avendano C et al. Developmental sperm contributions: fertilization and beyond. Fertil Steril 2009; 92:835-48.

4. Miller D, Ostermeier GC. Towards a better understanding of RNA carriage by ejaculate spermatozoa. Hum Reprod Update 2006; 12:757-67.

5. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature 2009; 460:473-8.

6. Ostermeier GC, Miller D, Huntriss JD, Diamond MP, Krawetz SA. Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature 2004; 429:154.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.03.039

The Authors Respond:

We thank Venkatesh and Dada for their interest in our paper in which we investigate the prevalence of sperm DNA damage in 279 infertile men with a diversity of andrological diagnoses.

We used the sperm chromatin structure assay (SCSA) to assess sperm DNA damage. The assay is based on the susceptibility of sperm chromatin to DNA denaturation following low pH treatment, after which samples are stained with Acridine Orange. This metachromic dye emits green fluorescence when bound to native, intact DNA and red fluorescence when bound to single strand or damaged DNA. The extent of sperm DNA damage is expressed as the DNA fragmentation index (DFI), reflecting the ratio of red to total fluorescence.

SCSA is widely used in clinical studies and increased DFI levels are reported to be associated with decreased in vivo fertilizing potential (1,2) and intra uterine insemination (IUI) results (3). Whether or not DFI is prognostic for the outcome of assisted reproduction remains controversial (4).

Earlier studies that were the basis for the development of SCSA, concluded that although the combination of in situ denaturation followed by Acridine Orange staining measures DNA and RNA content simultaneously, RNA content in mature sperm is low and doesn’t interfere with SCSA data (5,6).

In their letter, Venkatesh and Dada consider this former belief and postulate that binding of mRNA present in the sperm nucleus to Acridine Orange may overestimate damaged sperm DNA levels. In unpublished data they observed decreased red fluorescence after treatment of samples with RNAse.

Our results indicate limited diagnostic use for SCSA in Andrology patients, because DFI reflects impaired spermatogenesis irrespective of its cause. We look forward to the publication of the recent observations by Venkatesh and Dada. If their results indicate a more accurate assessment sperm DNA integrity using SCSA, the clinical use may be optimized. Further studies will be needed to clarify if the proposed modification of the SCSA has clinical implications.

Marij Smit M.D.
Gert R. Dohle M.D., Ph.D
Andrology Unit of the Department of Urology
Erasmus MC
Rotterdam, The Netherlands

References
1. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14:1039-49.

2. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 2000;73:43-50.

3. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 2007;22:174-9.

4. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 2008;89:823-31.

5. Darzynkiewicz Z, Evenson D, Kapuscinski J, Melamed MR. Denaturation of RNA and DNA in situ induced by acridine orange. Exp Cell Res 1983;148:31-46.

6. Evenson D,Jost L. Sperm chromatin structure assay for fertility assessment. Curr Protoc Cytom 2001;Chapter 7:Unit 7 13.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.03.040

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