A protocol for urine collection and storage prior to DNA methylation analysis.

BACKGROUND: Urine poses an attractive non-invasive means for obtaining liquid biopsies for oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of urinary DNA, and in particular hypermethylated DNA (hmDNA), are yet to be determined. AIM: To determine the most optimal and practical conditions for urine storage that result in adequate preservation of DNA for hmDNA analysis. METHODS: DNA yield for use in methylation analysis was determined by quantitative methylation specific PCR (qMSP) targeting the ACTB and RASSF1A genes on bisulfite modified DNA. First, DNA yield (ACTB qMSP) was determined in a pilot study on urine samples of healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) and Urine Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) for four time periods (1, 2, 7, 28 days). Next, hmDNA levels (RASSF1A qMSP) in stored urine samples of patients suffering from bladder cancer (n = 10) or non-small cell lung cancer (NSCLC; n = 10) were measured at day 0 and 7 upon storage with and without the addition of 40mM EDTA and/or 20 µl/ml Penicillin Streptomycin (PenStrep) at RT and 4°C. RESULTS: In the pilot study, DNA for methylation analysis was only maintained at RT upon addition of preserving agents. In urine stored at 4°C for a period of 7 days or more, the addition of either preserving agent yielded a slightly better preservation of DNA. When urine was stored at -20 °C or -80 °C for up to 28 days, DNA was retained irrespective of the addition of preserving agents. In bladder cancer and NSCLC samples stored at RT loss of DNA was significantly less if EDTA was added compared to no preserving agents (p<0.001). Addition of PenStrep did not affect DNA preservation (p>0.99). Upon storage at 4°C, no difference in DNA preservation was found after the addition of preserving agents (p = 0.18). The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA (ACTB) in most cases, except when PCR values became inaccurate. CONCLUSIONS: Addition of EDTA offers an inexpensive preserving agent for urine storage at RT up to seven days allowing for reliable hmDNA analysis. To avoid bacterial overgrowth PenStrep can be added without negatively affecting DNA preservation.

An assessment of framelapse videography of the effect of cryopreservation on human sperm motility.

Sixteen sperm specimens collected from regular healthy donors at Groote Schuur Hospital Sperm Bank were assessed subjectively by phase contrast light microscopy and the results were compared with assessments made by framelapse videography. The same specimens were frozen and thawed and re-assessed by framelapse videography and the results compared with the pre-freeze assessments. The objective cryosurvival was found to be 53%. The subjective assessment varied by an average of 20% from the videographic method. Framelapse videography was found to be a practical reproducible method for assessment of sperm motility where accuracy is essential.

Ovulation induction for in vitro fertilisation using clomiphene citrate and low-dose human menopausal gonadotrophin.

Ovulation induction using a low dose of human menopausal gonadotrophins and clomiphene citrate for in vitro fertilisation and embryo transfer is described. Sixty-two cycles of ovulation induction were initiated in 37 patients. Forty-six laparoscopies were performed, yielding 116 oocytes. Of these, 90 (77.6%) cleaving embryos developed, and in 36 transfers 10 pregnancies (27.8%) were established. The use of low-dose hMG in conjunction with a programme on an outpatient basis may prove to be optimal for the purpose of in vitro fertilisation and embryo transfer.