The effect of delayed processing on ovarian tissue stored for fertility preservation

Background Ovarian tissue cryopreservation (OTC) is important for fertility preservation and conservation. Delay in OTC may be required for transport or workflow management, however little is understood about the effect of processing delay on the tissue. Objective To determine whether a delay of 24-48 hours to OTC affects primordial follicle (PF) health. Methods Ovaries (n=6 sheep) were processed immediately or after storage at 4°C (24h, 48h). Tissue was fixed fresh, after cryopreservation or 10-day xenotransplantation. Morphological assessment of follicle health and development was performed. Findings A total of 1541 follicles were analysed. A 24h processing delay did not impact PF health in fresh or cryopreserved tissue. In fresh tissue a 48h delay had an adverse effect on follicle health (OR=2.47, 95% CI 1.29-4.71). Interestingly, a 48h delay resulted in cryopreserved tissue being less likely to be graded as unhealthy compared to control (OR=0.56, 95% CI 0.36-0.87). There was no difference in PF health or development across groups following xenotransplantation. Conclusion Ovarian tissue can be stored for up to 48 hours prior to cryopreservation with no net impact on PF health.


44
At present, over 80% of children and adolescents diagnosed with cancer will survive the disease and 45 reach adulthood (1). Furthermore, as of 2011 there are an estimated 388,000 childhood cancer 46 survivors in the US (2) and over 30,000 in the UK (3). Childhood cancer treatment can, however, 47

97
Tissue collection 98 Pairs of ovaries from six female lambs (Ovis aries, breed unknown) were collected from a local 99 abattoir and transported on ice in 100 mL of transport medium [Leibovitz's L-15 medium (Thermo 100 Fisher, UK) supplemented with 100 U/mL penicillin and 100 g/mL streptomycin (Sigma, UK) and 101 2.5 g/mL amphotericin B (Sigma)]. Upon arrival at the laboratory, one ovary from each pair was 102 immediately processed (no delay), while the other ovary was stored at 4°C in transport medium for 103 24 h or 48 h. 104 105 Ovarian tissue processing and cryopreservation 106 Ovaries were processed for cryopreservation using procedures aligned with the relevant standard 107 operating procedures (SOPs) applied at the Oxford Cell and Tissue Biobank (Oxford, UK), with some 108 modification. Each ovary was bivalved and the ovarian medulla was gently dissected away using 109 curved scissors under aseptic conditions. The outermost 1 mm of the ovarian cortex was cut to strips 110 of approximately 5 x 2 x 1 mm using a surgical scalpel blade. All tissue handling was carried out in 111 transport media on a Medicool ice block. Cortical strips were either fixed directly after processing 112 (fresh samples) or cryopreserved. For cryopreservation, individual cortical strips were transferred to 113 Nunc cryotubes and equilibrated in 1 mL of cryoprotectant medium containing L-15 supplemented 114 with 1.5 M ethylene glycol (Sigma), 0.1 M sucrose (Sigma) and 3 mg/mL bovine serum albumin 115 (BSA, Fisher Scientific, UK) for 1 h on ice. Cryopreservation was carried out in a controlled-rate 116 freezer (IceCube 14S, SY-LAB, Sweden) using the following cooling programme: start temperature: 117 4°C; cooling rate I: -2°C/min to -9°C; cooling rate II: -0. 3°C Each graft's position was secured before the incision was closed. Mice were housed individually for 145 the first 24 h after surgery. Recovery was monitored by observation and measuring body weight post-146 transplant; mice were weighed twice daily in the first 48 h and once daily thereafter. After a period 147 of 10 days, the animals were sacrificed by cervical dislocation and all grafts retrieved. 148

Histological analysis 150
Cortical strips were fixed in Bouin's solution overnight at room temperature and embedded in paraffin 151 wax. Serial sections were obtained at 5 µm thickness and slides were stained with haematoxylin and 152 eosin (H&E). Samples were imaged at x400 magnification using a Leica DM2500 microscope. 153 Images were captured using QCapture Pro 7 software. Follicles were graded in a blinded manner by 154 two independent researchers, with a concordance rate of over 90%. is indicated for each analysis. Statistical significance was defined as p<0.05. Data are presented as 172 mean ± SEM or as odds ratios (ORs) with 95% confidence intervals (CI).

The effect of processing delay on fresh ovarian tissue 175
Pairs of sheep ovaries were collected and either processed immediately (n=6) or stored at 4°C for 24 176 h (n=3) or 48 h (n=3) before processing. Primordial follicles were graded based on oocyte 177 morphology as healthy (grade 0) or affected, degenerating or atretic (grades 1-3, hereafter referred to 178 as 'unhealthy') based on the presence of nuclear condensation or pyknosis and shrinkage or 179 eosinophilia of the ooplasm (Figure 1). The vast majority of follicles in all fresh tissues were healthy; 180 89.0±3.5% for the immediately processed (no delay) group, 90.6±1.7% for the 24 h-delay group and 181 79.2±8.2% for the 48 h-delay group ( Table 1). Conversely, follicles were observed in two out of 207 three grafts for the 24h-delay group and all three grafts for the 48h-delay group. Many primordial 208 follicles were healthy after 10-day xenografting, however considerable variation was observed 209 between grafts, particularly for the 48h-delay group. The proportion of healthy primordial follicles 210 was 55.7±9.8% for the immediately processed group, 81.5±3.5% for the 24h-delay group and 211 43±28.8% for the 48h-delay group ( Figure 5A  Of the eight grafts containing follicles, six contained both primordial and growing follicles indicating 217 that follicle activation occurred during the xenografting period (Table 1; Figure 6). The other two 218 grafts that contained follicles contained only primordial follicles and in very low numbers; 2 and 6 219 follicles ( Table 1). The proportion of growing follicles was 17.3±30.0% for tissue processed 220 immediately after collection, 46.0±2.8% for tissue processed after 24 h and 45.3±10.8% for tissue 221 processed after 48 h ( Figure 6A), with the highest variability between animals in the no delay group 222 ( Figure 6B). There was no difference in the likelihood of a follicle being classified as 'growing' 0.55-3.96, p>0.05; Figure 6C Although cryopreservation of ovarian tissue with as little delay as possible seems obvious, and is 292 considered the gold standard in most countries (29), it is possible that transient cold ischaemia during 293 delayed processing induces a metabolic adjustment which enables quiescent primordial follicles to 294 better withstand ischaemia prior to revascularisation. Energetic adjustment to ischaemia is well-295 documented in muscle tissue (30) and it is possible that the oocyte is also equipped with the capacity 296 to adjust to metabolism in avascular conditions. Furthermore, there is some evidence to suggest that 297 ischaemic preconditioning can improve the outcomes of organ transplantation (31) and in this context, 298 our findings might suggest that the benefits of ischaemic preconditioning are not lost after 299 cryopreservation and thawing of the tissue. This, taken together with studies using human tissue, 300 which demonstrated that follicles can survive a processing delay of delay of up to 20 h at 4°C and 301 lead to a live birth (8,12,13), indicates that ovarian tissue responds to delayed cryopreservation 302 favourably without a negative impact on the primordial pool. 303

304
The results we present here have provided strong evidence to support the idea that ovarian tissue can 305 be chilled for 24 or even 48 h prior to cryopreservation with no net negative impact on the health of 306 the primordial pool of follicles after transplantation and in vivo development of the tissue. This lends 307 strength to an emerging body of evidence suggesting the delayed processing is safe, or even 308 beneficial, for cryopreserved ovarian tissue. Validation of these findings using human ovarian tissue 309 or other endangered species of interest would thereby present a new, feasible approach to increasing 310 access to ovarian tissue freezing for fertility preservation. It should be noted that in the present study 311 ovaries were left intact until the time of processing, therefore, it cannot be concluded that ovarian 312 biopsies would have a similar response. Nevertheless, these findings are of high relevance to fertility 313 preservation and, in particular, the way ovarian tissue cryopreservation is organised on a local and 314 national level for humans and endangered species. An update of the current guidelines to reflect an 315 extended window for tissue processing will ensure that the modality is more widely available to 316 patients without the need to create new facilities. Fundamentally, such an advancement is a great step 317 towards ensuring that every cancer patient who has a need, has an option.   (1), degenerating (2) or atretic (≥3). C) Same data as in B, showing the distribution of health grades 434 between animals (mean ± SEM). D) Proportional ORs and 95% CIs of follicles being graded as ≥ 1 435 when processed after 24h or 48h compared to the no delay group. 436  Proportional ORs and 95% CIs of follicles being classified as growing when processed after 24h or 486 48h compared to the no delay group. 487