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Sperm preparation before cryopreservation improves cryo-survival and utilisation

Although the evidence-base is relatively thin, recent data suggests that preparing sperm on density gradients (before cooling) not only improves outcome but also, at least for donor sperm, confers clear advantages in: dose control, smarter utilisation of donations and simpler preparation for DIUI. Read below for more…..

Unlike the cryopreservation of many other cells, sperm survival post thaw is related less to intra-cellular ice formation and more to osmotic damage sustained during cryoprotectant (CPA) addition and subsequent removal (Gilmore et al, 1998; Morris et al, 2006). It seems that only a particular cohort within the highly heterogeneous sperm population is able to withstand the movement of cryoprotectant, water and other solutes across the plasma membranes, many not surviving and others becoming damaged in the process (see below). The video below shows a relatively common osmotic stress response to freeze-thaw, where sperm show morphological abnormalities induced by membrane damage, where motility might be retained but is clearly atypical.

Despite the many variables involved, recent published evidence demonstrates that sperm preparation using density gradient centrifugation (DGC) prior to cooling provides significantly improved cryo-survival and independently of the general enrichment-effect that DGC has on viability/motility (Androni et al, 2021).

One can only speculate at this stage as to why this is so but it could be related to (i) the removal of cells with increased DNA damage and associated free oxygen radicals and the effect they might have on surviving cells or perhaps (ii) by removing seminal plasma which contains many of the same substances found in typical cryoprotectants (sugars, trace elements, proteins), biochemistry is normalised (from sample to sample), viscosity reduced and the mixing and penetration of CPA is more consistent. In addition, sperm preparation confers a number of more practical advantages over simply freezing sperm in its raw state in that:

(i) Donations that don’t meet the laboratory’s normal threshold (based on total motile sperm number) for IUI, need not be wasted as they can simply be concentrated and fewer units/straws produced.

(ii) The final dose can be adjusted to provide IUI and IVF quality units (if required)

(iii) Space savings within the freezer can be made by storing fewer frozen units of higher quality

(iv) Although sperm processing takes longer, workforce savings are made at the time of insemination, since donor units simply need to be thawed, washed to remove cryoprotectant and loaded into the catheter

Data from a number of internal projects, published data (Androni et al, 2021) and solid clinical outcome data over the past 15 years or so (Tomlinson et al, 2010; NUH Life – have all served to validate this change to previous procedure

However, over the years, implementing this as routine procedure has led to the following repeatedly visited questions?

Should donor sperm intended for IVF (as opposed to IUI) be prepared prior to cryopreservation?

Possibly not – since many IVF centres often to prefer to DGC prepare their own samples (whether it has been prepared previously or not) in order to improve the removal of as many immotile sperm as possible. In this case, some donations could be cryopreserved without preparation, purely for IVF treatments

Should patients undergoing fertility preservation e.g. prior to chemotherapy have their sperm DG prepared prior to freezing?

In this case also probably not. The uptake and utilisation of sperm from this group is low at significantly less than 10% according to the literature. Therefore, the routine preparation would be an inefficient use of resources for the remaining 90%. Moreover, in the vast majority of cases, IVF or ICSI would be the treatment of choice, in which case as is outlined in the question above, IVF units would prefer to DG prepare their own samples


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