Long-term cryostorage of sperm in a human sperm bank does not ...

4 Yogev et al.Table II Effect and distribution of storage length on PMC (mean + SD) and median (in parentheses) of specimenscryopreserved for women future treatments (n 5 1239).Storage (years) Subgroup size (% of total group) T0 PMC and (median) T1 PMC and (median) D(T12T0).............................................................................................................................................................................................0.5–3 127 (10) 11.6 + 4.60 (11.0) 12.1 + 5.58 (11.0) 0.5 + 4.80.3–5 458 (37) 10.6 + 3.70 (10.0) 12.0 + 4.21 (11.0) 1.5 + 4.56.5–7 287 (23) 10.1 + 5.04 (9.0) 11.9 + 4.14 (11.0) 1.8 + 4.54.7–10 293 (24) 10.6 + 4.18 (10.0) 11.8 + 4.05 (11.0) 1.3 + 4.40.10 74 (6) 11.7 + 7.87 (9.0) 12.1 + 4.17 (12.0) 0.4 + 6.08PMC, progressive motility concentration (10 6 /ml), T0, value of sample after freezing/thawing and before storage, T1, value after storage and thawing before insemination. There aresignificant differences between the five subgroups for T0 (P ¼ 0.009) and for D(T12T0) (P ¼ 0.041), while there is no significant difference for T1 (P ¼ 0.96).Table III Progressive motility concentration (10 6 /ml) in the same 19 donors after short-term and long-term storage.# Donor Short-term storage......................................................Long-term storage....................................................P-valueNo. of straws PMC No. of straws PMC.............................................................................................................................................................................................1 65 13.5 + 6.8 27 12.3 + 3.1 0.2542 61 9.3 + 4.3 14 10.9 + 3.1 0.1963 52 10.8 + 4.3 56 10.3 + 3.1 0.4664 61 11.4 + 3.9 45 13.3 + 4.8 0.0275 61 10.3 + 3.3 17 9.9 + 2.6 0.7176 64 11.5 + 3.8 31 13.0 + 6.5 0.2537 62 10.0 + 2.93 15 11.6 + 2.1 0.0488 87 13.2 + 8.7 14 9.9 + 2.3 0.0059 75 11.5 + 4.1 13 11.8 + 5.3 0.85210 67 11.3 + 5.0 12 13.3 + 2.4 0.03311 67 14.4 + 5.5 2 20.5 + 6.4 0.12912 65 10.8 + 3.0 41 12.6 + 3.6 0.00813 63 12.3 + 4.4 29 8.8 + 1.4 ,0.00114 66 14.3 + 4.8 15 13.9 + 2.9 0.66615 60 12.8 + 3.6 12 12.3 + 3.4 0.70116 63 13.7 + 5.7 6 11.3 + 1.6 0.02417 61 10.4 + 2.5 16 10.4 3.3 0.99518 64 8.7 + 2.1 24 9.7 + 3.6 0.18719 122 12.2 + 4.0 20 11.8 + 2.1 0.704Values are mean + SD. There was no difference within the same individual donor (P . 0.05) for most of them.Effect of storage length on individual donorsTesting the possible influence of length in cryostorage on individualdonors revealed a significant influence in 11 of 72 donors, but the findingswere not uniform; four sperm donors had a negative Pearson correlation(a damage effect), whereas the remaining seven showed apositive one. The influence of length of time in cryostorage on thePMC in 20 individual sperm donors who contributed a relativelylarge number of specimens (more than 50 each) is shown inTable IV. Only two of them showed a significant storage influence(Nos. 8 and 13), and both showed a positive correlation, i.e. thelength of time in cryostorage ‘increased’ the specimen quality.Similar results were also shown when several aliquots (at leastthree) from the same ejaculate were thawed after different lengthsof time in storage (n ¼ 483 aliquots, r ¼ 20.04, P ¼ 0.27, Fig. 1).An influence of the storage length on D(T12T0) could not bedemonstrated.Comparison between raw and washed spermspecimens storageTo evaluate whether raw specimens (cryopreserved with seminalfluid) can survive cryostorage differently to washed sperm samples(i.e. after seminal fluid has been replaced by a human tubal fluidmedium), specimens were divided into two groups by type ofsample preparation before cryostorage. Raw specimens were mixedwith the freezing medium in Group A (n ¼ 1157) and the specimenswere washed before cryostorage in Group B (n ¼ 1365). The mean

Long-term cryostorage in human sperm banking 5Table IV The influence of storage length (mean + SD) on PMC (mean + SD) of individual donors (n 5 20) whocontributed more than 50 specimens each.#Donor Specimens (n) Storage length (years) T0 PMC (10 6 /ml) T1 PMC (10 6 /ml) P-value r.............................................................................................................................................................................................1 92 3.50 + 4.36 12.90 + 5.20 13.10 + 5.94 0.39 20.092 75 2.70 + 3.06 8.70 + 2.59 9.64 + 4.11 0.30 0.123 108 4.20 + 3.62 9.46 + 3.80 10.54 + 3.72 0.56 0.064 53 7.25 + 3.09 7.13 + 3.33 9.19 + 2.82 0.35 20.135 106 3.10 + 2.82 9.87 + 4.34 12.24 + 4.37 0.83 0.026 78 2.14 + 2.32 10.36 + 4.70 10.18 + 3.12 0.73 20.047 95 2.94 + 2.06 12.00 + 6.12 11.99 + 4.8 0.24 0.128 77 3.00 + 3.36 8.70 + 2.54 10.30 + 2.85 0.04 0.239 101 1.60 + 0.77 11.16 + 6.55 12.74 + 8.20 0.22 20.1210 88 1.70 + 0.82 9.44 + 3.83 11.56 + 4.29 0.53 20.0711 79 2.15 + 1.88 10.20 + 4.21 11.58 + 4.76 0.25 0.1312 69 1.72 + 1.49 11.90 + 3.19 14.58 + 5.58 0.25 0.1413 106 3.35 + 2.28 9.36 + 2.35 11.53 + 3.34 0.05 0.1914 92 2.61 + 1.92 9.60 + 3.11 11.17 + 4.02 0.06 20.2015 81 2.51 + 1.56 10.70 + 2.41 14.27 + 4.47 0.62 20.0616 72 2.42 + 1.38 10.36 + 2.99 12.69 + 3.53 0.35 20.1117 69 1.78 + 1.26 11.70 + 4.37 13.52 + 5.55 0.37 20.1118 77 2.30 + 1.96 8.80 + 1.81 10.40 + 2.66 0.89 0.0219 88 3.11 + 2.98 8.60 + 2.83 8.90 + 2.63 0.15 0.1520 142 2.50 + 1.78 10.34 + 2.26 11.90 + 3.79 0.86 0.01Nos. 8 and 13 had a significant storage influence, both with a positive correlation. PMC, progressive motility concentration, T0, value of sample after freezing/thawing and before storage,T1, value after storage and thawing before insemination.storage lengths of the two groups were 4.2 + 0.09 and 3.2 + 0.05years, respectively. The mean change in PMC, between the thawedsamples that were used for treatment (T1) and those that werethawed immediately after freezing and before cryopreserving (T0),was similar (1.56 + 0.12 and 1.38 + 0.13 10 6 /ml, respectively).Observation of the PMC after storage revealed that the length oftime in cryostorage did not influence the PMC values in eitherGroup A or Group B (r ¼ 0.02, P ¼ 0.48 and r ¼ 0.042, P ¼ 0.12,respectively).DiscussionThis evaluation of the largest ever-reported number of cryopreservedsperm bank donor specimens revealed that up to 14 years instorage caused no demonstrable decrease in progressive motility.This finding is very encouraging, because progressive motility percentagesand concentrations are reportedly the best prognostic parametersfor pregnancy in IUI with donor semen (Larsen et al., 2000;Freour et al., 2009). Moreover, sperm motility after thawing providesthe most significant parameter for predicting donor sperm fertilitypotential (Marshburn et al., 1992).Whatever procedure of freezing is used, sperm cells are damagedby undergoing either structural or functional changes or both(O’Connell et al., 2002; Thomson et al., 2009). Damage to humanspermatozoa can be recognized immediately after freezing–thawingas a loss in motility, but there is a possibility that further harmcan be caused to the sperm cells during storage (Desrosier et al.,2006).Sperm frozen in seminal fluid has appeared to be more resistant todamage associated with freezing than washed sperm (Donnelly et al.,2001; Saritha and Bongso, 2001). When we assessed the influence ofthe length of time in cryostorage on the PMC of specimens cryopreservedwith and without seminal plasma, however, we found no differencebetween the two groups.Unexpectedly, there was a significant difference in PMC afterfreezing process survival of samples between partially filled andtotally filled straws. Consequently, instead of showing zero value ofD(T12T0) because of the absence of storage influence, an increasein PMC that was not influenced by the storage length could bedemonstrated. In order to establish the quality of each freezing(before storage), we therefore recommend using a full straw thathas a small volume (e.g. 0.25 ml) for saving the valuable specimen,instead of partially filling a 0.5-ml straw. Still, the change in PMCmay be due to ‘technical’ artifacts rather than to a biologicaleffect. A recent study demonstrated that with a decrease involume, and therefore an increase in surface area-to-volume ratio,the intra-container pressure during freezing increases and causesmore cryodamage to the cells than does large volume freezing (Saragustyet al., 2009). Further studies are required to identify thefactor(s) responsible for the decline in PMC in a partially filledstraw, such as free radicals that may be produced during the freezingprocess or the effect of high pressure in a small aliquot.

6 Yogev et al.Figure 1 Progressive motility concentration (PMC) values in ejaculates of nine donors (A–I). Each drawing represents a PMC of at least threealiquots from a single specimen that was thawed after different lengths of storage time (T1). The value of PMCs of thawed samples beforestorage (T0) is shown as a horizontal line. (E) Three different ejaculates of the same donor are shown by three indexes (empty circle, filled-incircle and square). A significant influence of the lengths of cryostorage time on D(T12T0) could not be demonstrated.There were varying degrees of influence of length of time in cryostorageon individual donors. There was a decrease of PMC in someand an increase in others. Interestingly, three ejaculates of the samedonor (Fig. 1E) showed greater, less and no influence of the lengthof time they were in cryostorage. These results support the positionthat there is no remarkable and lasting change in PMC after long-termcryostorage.In a previous report from our sperm bank, an overall pregnancy rateof 12.1% was achieved with frozen–thawed spermatozoa (Botchanet al., 2001). A comparison between the fertility rate of the cryopreservedsemen after short- and long-term storage would obviously contributeto the significance of the present study. However, the olderage of the women in the present study taken together with theextended storage time can be expected to dramatically reduce thevalue of such a comparison.We concluded that the length of time during which sperm is storedin liquid nitrogen has no injurious effect on PMC and consequently, onthe fertilizing potency of donated sperm. When the effect of freezing isexamined by the thawing of a small sample volume in partially filledstraws, it should be borne in mind that the PMC will be lower inthe sample than in the rest of the stored specimen.Authors’ rolesL.Y.: conception and design, collection, assembly of data and writingthe manuscript. S.E.K.: critical revision of the manuscript. E.S.: analyzingthe data and performing the statistical evaluation. A.B.: revisionof the manuscript. G.P.: contribution to the conception ofthe model; revision of the manuscript. R.H.: critical revision ofthe manuscript; approval of the final version. O.L.: design andcoordination of the research. H.Y.: conception and design of theresearch, correction of the manuscript. R.G.: concept and designof the research, data interpretation and critical revision of themanuscript.AcknowledgementThe authors thank the laboratory technicians of The Institute for theStudy of Fertility for skillful and excellent technical work, Ms EstherEshkol, Tel Aviv Medical Center, for expert editorial assistance andMs Sigalit Siso of The Graphics Unit, Tel Aviv Medical Center, forthe skilled graphic illustration.

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