Routine cryopreservation of spermatozoa is safe--evidence from the DNA methylation pattern of nine spermatozoa genes.
OBJECTIVE
Assess short- and mid-term impact of cryopreservation on DNA methylation status of different genes in spermatozoa.


METHODS
Semen samples from 10 healthy normozoospermic men were collected at the Department of Clinical Andrology of the Centre of Reproductive Medicine and Andrology (Muenster, Germany). Each was divided into four equal aliquots: 1) untreated, 2) diluted in cryoprotectant, 3) short term (2 days) cryopreserved and 4) mid term (4 weeks) cryopreserved. Samples were "swim-up" purified prior to analysis. DNA fragmentation was measured using comet assay and Flow cytometric evaluation with Acridine Orange (FCEAO). The degree of methylation of nine genes was determined by bisulfite pyrosequencing of genomic DNA.


RESULTS
Analysis of three maternally imprinted genes (LIT1, SNRPN, MEST), two paternally imprinted genes (MEG3, H19), two repetitive elements (ALU, LINE1), one spermatogenesis-specific gene (VASA) and one gene associated with male infertility (MTHFR) in semen samples demonstrated no alteration in methylation pattern regardless of duration of cryopreservation.


CONCLUSIONS
The lack of any changes in the sub-fraction of the genome examined in our study, implies that sperm DNA methylation is unaffected by cryopreservation and suggests that this daily clinical routine is safe in terms of DNA methylation.
Centre of Reproductive Medicine and Andrology, University Clinics Muenster, Albert-Schweitzer-Campus 1, Muenster, Germany.
Ruth
R
Kläver
Andreas
A
Bleiziffer
Klaus
K
Redmann
Con
C
Mallidis
Sabine
S
Kliesch
Jörg
J
Gromoll
D000165:Acridine Orange / Q000737:chemistry; D020087:Alu Elements; D020552:Comet Assay; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D003451:Cryoprotective Agents / Q000737:chemistry; D053487:DEAD-box RNA Helicases / Q000235:genetics; D053938:DNA Fragmentation; D019175:DNA Methylation; D005434:Flow Cytometry; D015894:Genome, Human; D018392:Genomic Imprinting; D006801:Humans; D007248:Infertility, Male / Q000235:genetics; D008297:Male; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics; D015203:Reproducibility of Results; D055101:Semen Analysis / Q000379:methods; D012680:Sensitivity and Specificity; D013076:Sperm Count; D013094:Spermatozoa / Q000166:cytology*; D013997:Time Factors
D003451:Cryoprotective Agents; C485621:KCNQ1OT1 long non-coding RNA, human; D024642:Potassium Channels, Voltage-Gated; C573423:MTHFR protein, human; D042965:Methylenetetrahydrofolate Reductase (NADPH2); C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases; D000165:Acridine Orange