Should donor semen be washed before or after freezing?

Donor Semen Processing: Washing After Freezing is Standard Practice

Donor semen should be washed after thawing, not before freezing, as this approach yields significantly higher post-thaw total motile sperm counts, progressive motility, and viability compared to pre-freeze washing. 1

Evidence-Based Processing Algorithm

Standard Clinical Practice for Donor Semen

• Freeze unwashed semen directly in cryoprotective medium (typically glycerol-based), then wash after thawing using standard WHO protocols (simple wash, swim-up, or density gradient centrifugation) before intrauterine insemination 2, 1

• The World Health Organization recommends that frozen sperm be thawed and prepared using standard sperm washing techniques before IUI, following WHO manual protocols 2

Why Washing After Freezing is Superior

• Post-thaw washing of frozen-unprepared samples yields higher total motile sperm counts (P < 0.0001), higher progressive motility (P = 0.005), and higher viability (P < 0.0001) compared to samples washed before freezing 1

• Pre-freeze washing followed by refreezing causes a 50% reduction in motility compared to unwashed thawed specimens, with significant impairment of linearity and morphologically normal spermatozoa 3

• Post-thaw dilution and washing exerts a deleterious effect on sperm motility when performed on already-washed frozen samples, but this effect is minimized when washing fresh-thawed unwashed samples 3

Critical Safety Consideration: Quarantine Period

• Donor semen must be quarantined for at least 6 months after collection, with the donor retested for HIV antibody before release, to exclude donors in the HIV "window period" who test negative but are actually infected 4

• This quarantine requirement makes pre-freeze washing impractical, as semen must be frozen immediately and stored during the mandatory waiting period 4

Practical Clinical Protocol

At Time of Donation

• Collect semen sample and perform initial HIV antibody testing on donor 4
• Freeze unwashed semen directly in cryoprotective medium (glycerol-based) 2, 5
• Store at -80°C or in liquid nitrogen for minimum 6-month quarantine 4

After Quarantine Period

• Retest donor for HIV antibody at 6 months 4
• If negative, release frozen samples for clinical use 4

At Time of Use for IUI

• Thaw frozen unwashed semen using standard protocols (typically 37°C thawing for rapid vapor-frozen samples or 22°C for slow computer-controlled frozen samples) 3
• Wash thawed semen using density gradient centrifugation, swim-up, or simple wash per WHO manual 2
• Verify post-preparation total motility and concentration 2
• Perform single insemination 24-40 hours after hCG trigger or 1 day after spontaneous LH surge 2

Common Pitfalls to Avoid

• Never wash before freezing and then refreeze - this causes cumulative damage with each freeze-thaw cycle, reducing motility by 50% and viability by 23% despite cryoprotectant use 3, 1

• Avoid multiple freeze-thaw cycles - freeze-thaw cycles are more deleterious to bacteria and sperm than duration of cryoconservation, with significant drops in motile and vital sperm recovery between each thaw (P < 0.01) 6, 7

• Do not skip the quarantine period - transmission of HIV from screened HIV-antibody-negative donors has occurred when samples were used before the 6-month retest window 4

Supporting Evidence for Post-Freeze Washing

• Cryopreservation of unwashed semen has been the cornerstone of donor insemination since 1953, with the primary advantage being the ability to exclude infections through quarantine before use 5

• Sperm DNA integrity is preserved during cryopreservation regardless of washing timing, though concentration and progressive motility decrease significantly 4

• The inclusion of seminal plasma does not protect human sperm during freezing and thawing, eliminating any theoretical benefit of freezing unwashed samples 6