Follicle activation and burn-out contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness

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1 Human Reproduction, Vol.29, No.5 pp , 2014 Advanced Access publication on February 11, 2014 doi: /humrep/deu015 ORIGINAL ARTICLE Reproductive biology Follicle activation and burn-out contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness Zohar Gavish 1, Gil Peer 2, Roness Hadassa 1, Cohen Yoram 1,3 and Dror Meirow 1,3, * 1 Fertility Preservation Center, Department of Obstetrics and Gynecology, Sheba Medical Center, Tel-Hashomer, Ramat-Gan 52621, Israel 2 IVF Division, Carmel Medical Center, The Ruth & Bruce Rappaport Faculty of Medicine, Technion Institute of Technology, Haifa, Israel 3 Sackler School of Medicine Tel-Aviv University, Tel Aviv, Israel *Correspondence address. Tel: ; Fax: ; meirow@post.tau.ac.il Submitted on September 27, 2013; resubmitted on January 13, 2014; accepted on January 15, 2014 studyquestion: What are the effects of thin ovarian grafts compared with grafts of the standard thickness on follicle loss post-transplantation? summary answer: Transplantation of reduced-thickness ovarian grafts led to intense activation and burn-out a short time after transplantation resulting in significant folllicle loss. what is known already: Transplantation of fresh and frozen thawed ovarian tissue has been proved successful, but techniques vary and are not optimised, often resulting in significant follicular loss. Follicle loss is mostly related to the freezing thawing process and to post-transplantation hypoxia. study design, size, duration: Bovine ovarian tissue strips (n ¼ 55) were prepared in two groups of conventional-thickness strips (1 2 mm) or thin strips ( mm). Fresh or frozen thawed samples were xenotransplanted into sterilized immune-deficient mice (n ¼ 49). Non-transplanted conventional size fresh samples were used as controls(n ¼ 6). Grafts from all study groups were recovered after 7 days foranalysis. participants/materials, setting, methods: Morphometric differential counting of follicle classes was performed by two observers. Immunohistochemistry was conducted for proliferation (Ki67), cortical fibrosis (Masson tri-chrome) and blood-vessel density (CD31). Results were expressed as the mean number of dormant or growing follicle (GF) type per section or total follicle counts per graft. Blood-vessel density was calculated per mm 2. P-values,0.05 were considered statistically significant. main results and the role of chance: The loss of all follicle types, and most noteably of primordial follicles (PMFs), was observed 7 days post-transplantation (P, 0.05). The relatively high number of GFs and the positive Ki67 staining in all recovered grafts indicated that follicle activation was depleting the resting follicle pool. The reduced graft thickness had an adverse effect on the number of recovered follicles, especially on the resting non-gfs in the fresh, and more so in the frozen-thawed, samples (P, 0.05). Extensive stromal fibrosis and high bloodvessel density were observed in all grafts with no advantage in the thin prepared grafts. limitations, reasons for caution: This study used only one species of ovaries (bovine) for xenotransplantation. The immediate post-transplantation events were not visualized directly nor were the molecules involved in follicle activation studied. wider implications of the findings: Follicle activation and burn-out appear to be important in follicle loss after transplantation. Reducing graft thickness in an attempt to improve freezing conditions and reduce post-transplantation ischemia has adverse effects on the graft follicle pool due to increased activation and loss. Agents which prevent burn-out will potentially improve follicle pool survival. study funding/competing interest(s): This study was funded by research grants from the Israeli Science Foundation (No. 1675/10), the Israeli Jack Craps foundation and the Israel Cancer Research Fund (ICRF No ). The authors have no competing interest to declare. Key words: fertility preservation / ovarian tissue transplantation / ovarian reserve / graft thickness / follicle activation & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 990 Gavish et al. Introduction Transplantation of cryopreserved and fresh ovarian tissue has been successfully used to restore fertility in animal models (Gosden et al., 1994) and sterile patients (Meirow et al., 2005; Silber et al., 2005; Donnez et al., 2011). Nowadays, ovarian tissue cryopreservation (OTCP) and transplantation procedures are commonly used to preserve fertility and overcome the iatrogenic sterility caused by cancer treatments (Meirow et al., 2007a,b; Meirow, 2008; Donnez et al., 2010; Andersen et al., 2012; Chung et al., 2013) and premature menopause (Silber and Gosden, 2007). Although there is a constantly increasing number of live births as a result of OTCP and transplantation procedures and a few reports indicating long-term graft survival (Baird et al., 2004; Kim et al., 2009; Arav et al., 2010; Andersen et al., 2012), a standard protocol for OTCP does not yet exist. As a result, there is large heterogeneity in OTCP results. One of the major limitations of OTCP is the rapid follicle loss observed after transplantation (Gosden et al., 1994; Nugent et al., 1998; Baird et al., 1999; Dolmans et al., 2007; Silber et al., 2010; Silber, 2012; von Schonfeldt et al., 2012). OTCP and transplantation is a multi-step procedure where each step has its effect on the survival of the follicle pool in the ovarian cortical strip. Disconnection of the ovarian tissue from blood and oxygen supply and its neighboring tissue, preparation of the tissue for immediate transplantation or more commonly for freezing, the specific freezing thawing procedure, and the choice of the transplantation site and grafting procedures all contribute to follicle loss (Meirow and Wallace, 2009). Thinning the cortical strip prior to freezing is critical to enable penetration of the cryoprotectants and better graft survival. Reperfusion ischemia and hypoxia play a major role in follicle depletion during the first days after transplantation and this continues for about a week (Newton et al., 1996; Aubard et al., 1999; Yang et al., 2008; Soleimani et al., 2011). During this phase, oxygen and nutrients from the surrounding tissue diffuse into the graft, salvaging it from ischemia and hypoxia until new blood vessels are formed within both the graft and surrounding tissue and the revascularization process is completed (van Eyck et al., 2010). Thus, an important variable influencing the success of cryopreservation and transplantation is the physical dimensions of the graft. The surface-to-volume ratio of the graft has to be high in order to ensure good penetration and evacuation of cryoprotective agents, to reduce the probability of damage caused by ice crystals during freezing and thawing, and to quickly overcome the ischemia and hypoxia that occurs during the initial steps of graft acceptance (Gavish et al., 2008). Currently, ovarian cortical tissue is cryopreserved in strips of 1 2 mm thickness either by slow freezing or vitrification protocols (Kagawa et al., 2009). However, in terms of diffusion of oxygen in living tissue, 1 2 mm thickness is a great distance. Diffusion distances.0.2 mm are generally poorly tolerated (Loffredo and Lee, 2008), and for larger molecules, such as growth factors and nutrients, diffusion distances are even shorter. Therefore, it has been suggested (Gavish et al., 2008; Revel et al., 2011) that reducing graft dimensions might enhance diffusion of oxygen, growth factors and nutrients into the ischemic graft and improve follicle survival. In order to clarify the possible role of graft dimension, we investigated the effects of ovarian tissue graft thickness on the follicle reserve, ovarian stroma and vascular bed a short time after transplantation of frozen thawed ovarian tissue. Materials and Methods Ethical approval The use of animals and xenotransplantation procedures in this study were approved by the Sheba medical center ethical committee (No. 599/10). Animals Fresh bovine ovaries (milking cows, ages years old, n ¼ 12) were collected from the local abattoir and transferred to the lab in sterile 378C saline supplemented with 75 mg/ml penicillin and 50 mg/ml streptomycin (Telfer et al., 2008). The total time from harvest to the lab was,1 h. For transplantations, 4-week-old male immunodeficient mice (Athymic nude, Harlan Israel, n ¼ 57) were anesthetized by intraperitoneal (i.p.) injection of 100 mg/kg Ketamine (Vitamed, Bat-Yam, Israel) and 10 mg/kg Xylazine (UDIM Pharmacy, Israel) solution following castration by bi-lateral orchiectomy through abdominal incisions. Xenotransplantation experiments commenced at least 2-weeks post-orchiectomy. Tissue preparation Ovarian cortical tissue was obtained by removal of the underlying stromal tissue and any visible growing follicles (GFs) using a scalpel and forceps under a stereomicroscope. Slices were initially cut into strips with identical cortical surface areas of 10 2mm (L W ) using a scalpel and 21G needles in Leibovitz medium at 378C (Beit Ha emek, Israel) supplemented with sodium pyruvate (2 mm), glutamine (2 mm), penicillin G (75 mg/ml) and 50 mg/ml streptomycin (all from Sigma-Aldrich, Israel). By using 21G needles and forceps under a stereomicroscope equipped with 1000 mm reticule, the strips were further processed and prepared in two groups of strips of conventional thickness of 1 2 mm, as routinely used in OTCP procedures (Meirow et al., 2005; Andersen et al., 2008), or thin strips with thickness reduced by 50% (Figure 1). The total preparation time was,1 h. Cryopreservation Slow freezing and thawing were performed according to a clinically used protocol (Meirow et al., 2007a,b). Briefly, tissue fragments were introduced into cryopreservation solution, consisting of Sydney IVF gamete wash buffer (Cook-medical, USA) supplemented with serum substitute supplement (Irvine Scientific, USA), 0.1 M Sucrose and 1.5 M dimethylsulphoxide (DMSO) (both from Sigma-Aldrich, USA), and incubated with agitation for 30 min on a shaker. Samples were transferred to cryovials (Nunc. USA) and introduced into the cryopreservation device (Planer, UK) using the following program. The starting temperature was 208C followed by cooling to seeding temperature (278C) at a rate of 218C/min. Next was slow cooling at a rate of 20.38C/min to 2368C, followed by cooling at a rate of 258C/min to 21008C and then the vials were placed into liquid N 2 for storage. Thawing was performed by taking the vials out of the liquid N 2 container and keeping them at RT for 30 s followed by 1 min in a 378C water bath until the tissues were fully thawed. Samples were then transferred into thawing solution, consisting of Sydney IVF gamete wash buffer supplemented with serum substitute supplement, 0.1 M Sucrose and 1 M DMSO, and incubated for 5 min. Samples were then incubated in decreasing DMSO gradient solutions and transferred for transplantation. Surgical procedures Mice were anesthetized by i.p. injection of 100 mg/kg Ketamine (Vitamed) and 10 mg/kg Xylazine (UDIM Pharmacy) solution. In all transplantation procedures, a longitudal incision was made in the dorsum using a scalpel, the skin was lifted and the ovarian cortical strips were inserted subcutaneously. Each graft was attached in place with a 6 0 non-absorbable prolene suture.

3 Follicle loss post-ovarian tissue grafting 991 Figure 1 Schematic representation of experiment design. The in vitro phase involved preparing ovarian cortical tissue strips as follows; slices were initially cut into strips with identical cortical surface area (in red) of 10 2 mm (a), the strips were further prepared with either a conventional thickness of 1 2 mm (b) or as thin strips of mm (c). The impact of the difference in thickness was tested in vivo by xenotransplantation of either fresh (boxes in solid lines) or frozen thawed (boxes in dashed lines) samples (d) for 7 days in immunodeficient mice (e). Recovered grafts (f) were compared with untransplanted conventionally prepared fresh tissue. The incision was sutured using 4 0 sutures with emphasis on using the suture to press the underlying graft. Transplanted tissue was recovered after 7 days in order to pass the wound healing period. Fresh non-grafted conventionally prepared (1 2 mm) tissue strips were used as controls. Histology and immunohistochemistry Grafts and control samples were fixed in 4% paraformaldehyde, embedded in paraffin, serially sectioned (5 mm) and stained with hematoxylin and eosin. For the classification of follicles as primordial or growing, every fifth section was counted by two independed observers under a light-microscope (Kalich- Philosoph, 2013). In the bovine ovary, PMF size is mm while antral follicles reach 130 mm(van Wezel and Rodgers, 1996; Braw-Tal and Yossefi, 1997). Hence, in order to verify that GFs were not counted more than once (which might create a counting bias), we used two additional counting methods: (i) GFs were counted every 10th section (i.e. in 50 mm intervals) and (ii) only the largest mid-section from each graft was counted in order to present the growing to PMF ratio as previously published (Da Silva-Buttkus et al., 2009). Follicles were classified as either PMFs or GFs according to their morphology; only follicles with a clearly visible oocyte encircled by a granulosa cell layer were counted. PMF were defined as an oocyte surrounded by a single layer of flattened pregranulosa cells. GF include follicles in all developing stages, including follicles with a single layer of flattened and at least one layer of cuboidal granulosa cells. Proliferation of granulosa cells in developing follicles was demonstrated by immunohistochemistry for Ki-67 (1:100, Novocastra, clone MM1, Leica Biosystems, UK). The Vectastain Elite Kit (Vector Laboratories, CA, USA. PK-6102) and diaminobenzidine were used as a secondary antibody detection system and the sections were counterstained with hematoxylin. To evaluate fibrotic depositions in the ovarian stroma following transplantation, Masson tri-chrome staining was performed (Sigma Chemical, Israel). Newly formed blood vessels were detected by immunostaining with polyclonal antibovine CD31 (1:250, Abbiotec, San Diego, USA) and the Histostain-Plus kit (Invitrogen, USA). Blood-vessel density analysis was performed by two independent observers under a light-microscope. CD31-positive endothelial cells were counted and the mean number per section was divided by the section s surface area, calculated by measuring the longitudal and horizontal axis of each section using the microscope 1000 mm reticule. Statistical analysis Results are expressed as the mean number of primordial or growing follicles per section + standard error of the mean (SEM), or total follicle counts per graft + SEM. Blood-vessel density was calculated per mm 2 + SEM. Analysis of variance was performed and followed by non-paired student t-test and F-test. P-values,0.05 were considered statistically significant. Results All grafts were successfully recovered 1-week post-transplantation (n ¼ 49). The number of all follicle types in each section of the recovered graft showed significant follicle loss compared with non-grafted controls (Table I). Thin prepared samples (mean thickness 0.72 mm ) transplanted as either fresh or frozen thawed tissues demonstrated significantly (P, 0.05) lower follicle counts in recovered grafts compared with conventionally prepared samples (mean thickness 1.52 mm ). Follicle loss was more extensive in the frozen thawed groups compared with fresh grafts. Moreover, the PMF pool, which represents the future cortical strip reproductive reservoir, was better preserved in the conventionally prepared samples compared with the thin samples, both in the fresh and cryopreserved-thawed groups (Figure 2). In the conventionally prepared grafts, GF numbers were increased or remained unchanged after transplantation (Table I), while in thin recovered grafts, GF numbers were significantly reduced. To express the dynamics of the different follicle subgroups posttransplantation, the ratio of GF to total follicles was calculated in the various treatments groups. Recovered grafts of all groups exhibited a high ratio of growing follicles to total follicles. The increase in ratio was

4 992 Gavish et al. Table I Follicle counts in ovarian tissue samples recovered 1-week post-transplantation. Follicles per section Primordial Growing Total Mean (range) SEM P < 0.05 Mean (range) SEM P < 0.05 Mean (range) SEM P < Non-grafted control 6.47 (0 41) (0 21) 0.54 # (0 52) 1.33 N/A Transplanted Fresh Conventional 3.59 (0 29) (0 28) 0.42 # 9.62 (0 54) Thin 2.29 (0 23) (0 13) 0.20 * 4.70 (0 27) Frozen Conventional 1.58 (0 5) (0 15) 0.61 # 6.58 (0 15) Thin 0.28 (0 3) (0 10) 0.49 * 2.33 (0 13) P, 0.05 compared with all treatment group, # P, 0.05 compared with thin (fresh and frozen) samples, * P, 0.05 compared with conventional (fresh and frozen) samples. most prominent in frozen thawed grafts, with a maximum of 0.88 in thin frozen thawed recovered grafts compared with 0.43 in control grafts (Figure 3). The use of the two additional counting methods validated the results presented. Counting GFs in every 10th section and counting the largest mid-section from each graft resulted in similar results to those achieved using the standard counting protocol: thin recovered grafts exhibited greater follicle loss than convenionally prepared grafts (both total and PMF numbers) and frozen thawed tissues showed greater loss than fresh tissues. Ratios of growing/total follicle counts calculated using these different counting techniques were likewise increased after transplantation and as above, and the highest ratio was observed in thin frozen thawed samples (data not shown). The crucial value of each graft rests on its post-transplantation fertility potential, as represented by the total number of residual follicles within each graft and, more importantly, the total residual number of PMFs. As described, all grafts were prepared having identical a cortical surface area but with different thickness. Total follicle counting of the different graft groups clearly indicated (Figure 2) that thin prepared grafts had significantly smaller reservoirs of follicles of all types in general and specifically smaller PMF contents. This decrease in follicle numbers was clearly shown in fresh groups, but was significantly more marked in frozen thawed grafts. Histological analysis demonstrated the short-term changes occurring in grafted tissue 1-week post-transplantation (Figure 4). Granulosa cells of early stage follicles as well as developing follicles stained positive with the proliferation marker Ki67, indicating follicle activation. Positive Ki67 follicle staining was clearly observed in grafted tissues (Figure 4e and f). Evaluation of stromal fibrosis using Masson tri-chrome staining as soon as 7 days post-transplantation revealed extensive early fibrosis in all grafts (Figure 4g). There was no reduction in fibrosis in thin samples compared with conventionally prepared tissue. More positive CD31 immunostaining was observed in grafted tissue compared with nontransplanted controls demonstrating increased neovascularization (Figure 4h). The density of newly formed blood vessels was significantly higher (P, 0.03) in grafted tissue, however, we could not demonstrate significant differences between thin grafts and conventionally prepared samples (Figure 5). Discussion Ovarian tissue transplantation has been proved to restore hormone secretion and fertility, using both fresh and frozen thawed grafts. All stages including tissue collection, cortex preparation, freezing thawing and transplantation influence graft survival and procedure success (Meirow, 2010). Extensive follicle loss following ovarian tissue transplantation is a major factor that may negatively impact transplantation results and long-term graft survival (Gosden et al., 1994; Nugent et al., 1998; Dolmans et al., 2007). A number of surgical modifications and pharmacological interventions have been tried in attempts to diminish follicle loss (Nugent et al., 1998; Kim et al., 2004; Silber et al., 2008; Hancke et al., 2009; Dath et al., 2010; Smitz et al., 2010; Donnez et al., 2011; Shikanov et al., 2011; Friedman et al., 2012; Wang et al., 2012). Studies have demonstrated that reperfusion ischemia and hypoxia play a major role in follicle depletion during the first days after transplantation and continues for about a week (Newton et al., 1996; Aubard et al., 1999; Soleimani et al., 2011). During this phase, oxygen and nutrients from

5 Follicle loss post-ovarian tissue grafting 993 Figure 2 Comparison of follicles count per graft. Total follicle (black) or primordial follicle (PMF) (grey) counts in fresh or frozen thawed thin or conventionally prepared ovarian tissues recovered 1-week post-transplantation. Values represent the mean per graft + SE. P, 0.05: a ¼ compared with control, b ¼ compared with fresh conventional, c ¼ compared with frozen conventional. Figure 3 Ratio of growing to total follicles counts (per section) in fresh or frozen thawed thin or conventionally prepared ovarian tissues recovered 1-week post-transplantation. The horizontal dashed line represents normal values as indicated by the non-transplanted controls. the surrounding tissue diffuse into the graft until newly formed blood vessels emerge from both graft and surrounding tissue and the revascularization process is completed (Van Eyck et al., 2010). It has been theorized that reducing graft dimensions and thickness would reduce ischemia and enhance graft vascularization, therefore improving transplantation results (Gavish et al., 2008; Kagawa et al., 2009; Ferreira et al., 2010; Revel et al., 2011). It has also been suggested that increasing the surface-to-volume ratio of cryopreserved grafts would have a beneficial effect on follicle survival after transplantation by minimizing the damage caused by ice crystals during freezing and thawing (Ferreira et al., 2010; Revel et al., 2011; Donnez et al., 2013). This study investigated the short-term effects of reducing graft thickness and clearly demonstrated, to the contrary, that significant more extensive follicle loss occurred in thin ovarian tissue grafts, whether fresh or frozen thawed. Post-transplantation ischemia is one mechanism that is known to be directly responsible for follicle loss in OTCP (Liu et al., 2002; Oktay and Oktem, 2010; Soleimani et al., 2011) preferentially affecting granulosa cells of developing follicles that are more metabolically active than those of dormant PMFs (Bols et al., 2010). In addition, the cryopreservation process has a differential deleterious effects on different follicle populations; PMFs have been shown to be more tolerant of cryopreservation than developing follicles (Newton et al., 1996; Baird et al., 1999; Gosden et al., 2002). Analysis of the different follicle subpopulations a short-time post-transplantation in this study not only provides evidence for the disadvantage of thin grafts, but also proposes an important mechanism of follicle loss. Significant differences between follicle populations were observed; PMF loss in recovered grafts was more extensive than loss of GFs, when compared with ungrafted controls. The differences in follicle populations were clearly shown as a higher ratio of GF/total number. Thin grafts demonstrated a greater loss of PMF and higher GF/total follicle ratio compared with conventionally prepared recovered samples. In frozen thawed recovered grafts, the differences between

6 994 Gavish et al. Figure 4 Histological examination of fresh conventionally prepared tissues; untransplanted controls (a d) and ovarian grafts recovered 1-week posttransplantation (e h). Immunostaining with Ki-67 antibody indicates cell proliferation (a, b, e, f). Arrows points to a proliferating granulosa cells. Cortical fibrosis was demonstrated by Masson tri-chrome staining (c, g). Newly formed blood vessels were localized by CD31 immunostaining (d, h and arrows). Counterstaining was by hematoxylin. Figure 5 Density of blood vessels in thin or conventionally prepared fresh bovine ovarian tissue transplanted for 1-week. There is a significant increase in blood-vessel density in recovered grafts compared with non-transplanted controls (*P, 0.05), however, there was no significant difference in blood-vessel density between thin and conventionally prepared tissues. PMF and GF populations were considerably more significant than in fresh recovered grafts. Although there is a large variance within each treatment group, resulting from the natural physiologic characteristics of the bovine ovarian cortex, all calculated results were significant using 1SEM, and 55 70% (for PMF and GF, respectively) of the results were significant using 2SEM. Immunohistochemical staining for Ki-67 clearly demonstrated granulosa cell proliferation and post-transplantation follicle activation in all treatment groups. The unexpected increase in GF numbers, together with the greater loss of PMF compared with GF, and the granulosa cell proliferation all suggest that follicle activation is occurring in the short-term post-transplantation. Under normal physiological conditions, PMF activation and dormancy are regulated by the PI3K/Akt follicle activation pathway as well as inhibitory factors such as AMH that are secreted by growing follicles (Visser et al., 2006). The balance between these signals is crucial for preservation of the long-term reproductive functioning of the ovary (Kim, 2012). Mechanical preparation of ovarian tissue strips, the freezing thawing procedure and posttransplantation ischemia cause immediate and extensive GF loss, which results in vastly reduced levels of inhibitory signaling factors. This may lead to PMF activation and follicle growth, and could explain the larger than expected number of GFs we observed 1-week after transplantation. Regulatory system imbalances have also been described after chemotherapy, where cyclophosphamide induced activation of the PI3K/Akt pathway leading to extensive activation and burn-out of dormant follicles and follicle-depleted ovaries (Kalich-Philosoph, 2013). This study shows that follicle activation and burn-out is also likely to be an important mechanism of follicle loss post-transplantation of ovarian tissue grafts. Our study results indicate that follicle burn-out is more intense in thinly prepared tissues and even more in frozen thawed grafts than in fresh grafts. This mechanism of follicle loss significantly contributes to a reduced PMF pool, resulting in a diminished graft reproductive potential and shortened graft survival. Targeting the regulatory pathways of PMF dormancy with agents that prevent or attenuate follicle activation was previously shown to reduce follicle loss postchemotherapy. Given the possibility that similar mechanisms appear to be responsible for follicle loss in OTCP and transplantation, the use of such agents might also improve the graft outcomes and fertility potential of ovarian tissue grafts.

7 Follicle loss post-ovarian tissue grafting 995 Extensive fibrosis post-transplantation was evident as previously reported (Nisolle et al., 2000; Dath et al., 2010) in all recovered grafts, and we could not demonstrate any advantage of thin grafts as the amount of fibrosis was similar across all groups. A possible association between stromal fibrosis and follicle activation might exist, as stromal rigidity has previously been suggested to contribute to dormant follicle activation (Woodruff and Shea, 2011) and extensive fibrosis is also observed post-chemotherapy (Meirow et al., 2007a,b). The follicle reservoir after transplantation mainly depends on rapid tissue salvage from ischemia with growth of newly formed blood vessels providing oxygen and nutrients. We observed extensive neovascularization 1-week posttransplantation, however, reducing the graft thickness had no positive effect on the revascularization of the graft, with no difference in vascular density observed between the thin and conventional grafts. In conclusion, PMF activation and burn-out play an important role in follicle loss post-transplantation and is more significant in frozen thawed grafts. There does not appear to be any benefit in reducing graft thickness beyond 1 mm; on the contrary, thinning the grafts results in more extensive PMF loss and generates increased follicle burn-out without improving neovascularization or fibrosis. Future use of attenuating agents to maintain the balance between activation and suppression signals should be studied and might increase OTCP graft survival and function. 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