Background: The aim of this study was to examine the effectiveness of Tumor Dissociation Enzyme (TDE) on the viability of follicles after digestion of fresh and cryopreserved ovarian cortex fragments (OCFs). Methods: Fresh and thawed OCF from 14 patients (29 ± 6 years), sized 20 to 210 mm were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1, frozen OCF digested with TDE; Group 2, frozen OCF digested with LiberaseTM; Group 3, fresh OCF digested with TDE; and Group 4, fresh OCF digested with Liberase TM. Evaluation of follicle viability was performed under light microscope after staining with Neutral red. For visualization of viable and dead cells under a confocal laser scanning microscope, the follicles were stained with Calcein AM and ethidium homodimer-1. Results: The results showed that the number of retrieved follicles was significantly higher (990 vs 487; P < 0.01) in the TDE-treatment group compared to the Liberase TM-group. The presence of intense neutral red stained follicles was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (70.3% ± +/− 6.22 vs 53,1% ± 2.03 and 94.2% ± 6.6 vs 79.1% ± 2.1; P < 0.01). The percentage of Calcein AM stained follicles of class V1 was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (95.97% ± 7.8 vs 87.87% ± 2.4; 97.1% ± 6.8 vs 91.3% ± 2.3; P <0.01). Conclusion: The enzymatic digestion of ovarian cortex with TDE provides recovery of a higher number of healthy preantral follicles in contrast to earlier described Liberase TM procedure. Keywords: Artificial ovary, Follicle, Ovarian tissue, Enzymatic isolation, Cryopreservation, Liberase TM, Tumor dissociation enzyme * Correspondence: email@example.com Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Gynaecology, University Maternal Hospital, Cologne University, Kerpener Str. 34, 50931 Cologne, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 2 of 14 Background factors that are secreted by activated primary follicles. Over the last decades, research in medicine has made a The growth and development of secondary follicles is af- great progress in cancer therapy. Long-time survival of fected by steroid hormones that are produced by interac- cancer patients has increased significantly [1, 2] and tions between granulosa and thecal cells [21, 22]. after convalescence many patients can lead normal lives. Ovarian medulla is composed by dense fibrotic stroma However, highly aggressive and gonadotoxic chemother- consisted from spindle-shaped cells (fibroblasts) and fol- apy and radiotherapy have strong side effects such as licles of different stages of maturity . Development premature ovarian failure and infertility [3, 4]. At of follicles, their ovulation and luteinisation are finely present, the only possibility to maintain fertility as well controlled by physiologic hormonal milieu. Therefore, as hormonal cyclicity and endocrinological function is stromal cells of adult ovary actively contribute to the the cryopreservation and autologous re-transplantation synthesis and phase remodelling both extracellular of ovarian cortical fragments taken before chemotherapy matrix and blood vessels . Ovarian stromal and or radiotherapy. This method has resulted in large num- endothelial cells are essential to ensure graft survival ber of live births worldwide [5, 6]. and quality . However, this procedure runs the risk of re-transplant- In fact, two arteries (arteria ovarica and the branch of ing of malignant cells, which makes it unsuitable for pa- the arteria uterina) penetrate the ovarian hilus and form tients suffering from neoplastic diseases whose network of blood vessels . They are an opening port malignant cells are likely to metastasize into the ovary. for malignant cells  which could preferentially gener- Cancers that are considered to have a high risk for ovar- ate special kind of stroma cells for aberrant proliferation ian metastases are leukaemia, lymphoma, carcinoma coli and invasion . In this case the including of stromal and cervical as well as uterine cancers [7–10]. In the cells by creating of an artificial ovary plays important same time, the re-transplantation of isolated primordial role only for healthy patients, because of the risk of follicles could be an alternative to safe fertility restor- transfer of malignant cells included in ovarian stroma ation in women with premature ovarian failure [11–13]. [7, 8]. The fibroblasts and some other cell types (for It is possible because the basal lamina surrounding the example, pre-vascular cells and mesenchymal stem cells) follicles separates them from the surrounding ovarian potentially include cancer-associated fibroblasts . stroma, blood vessels and nerves, which prevents the in- At present, for isolation of preantral follicles from vasion of metastatic cells . Therefore, primordial fol- human and animal ovarian cortex, a number of mechan- licles can be isolated and used for the generation of a ical, enzymatic and a combination of both methods have transplantable artificial human ovary [15, 16]. Con- been described [26–40]. struction of an artificial ovary could be the optimal way of The human ovarian tissue has a relatively high density preserving fertility as well as protecting female cancer pa- and the current mechanical isolation of follicles tients, against the re-transplantation of malignant cells, in- demonstrates unsatisfactory performance. However, the dependent from type of neoplastic diseases . combination of mechanical isolation technique with This possibility was shown by Laronda et al. . It enzymatic digestion significantly improved the viability was demonstrated the real possibility for successful of isolated follicles [36, 41, 42]. re-transplantation of follicles seeded with scaffolds to Our own experience supports the effectiveness of the use SCID-mice with subsequent pups born through natural of a commercial enzyme -cocktail “Tumor Dissociation mating. Successful isolation of preantral follicles from Enzyme Reagent” (TDE, Innovative Diagnostic Systems cryopreserved tissue is especially important in global Dr. Christian Sartori, Hamburg, Germany) for enzymatic practice: a large amount of ovarian tissues have been digestion of gynaecological solid tumors with good viability already conserved from patients with different types of and developmental rate of cancer -and non -cancer cells cancers. According to data, preantral follicles can be (non-published data). successfully cryopreserved before or after their isolation This especially designed commercial enzyme - cocktail without impairing their ability to survive and grow in was developed for gentle enzymatic digestion of solid tu- vitro [18, 19]. mors [43–45] and allows to isolate tumor cells without The important step of the artificial ovary creation from appearance of apoptosis or necrosis. Despite the highly preantral follicles is the isolation of these follicles from secured composition of TDE due to patent , it was fresh or cryopreserved (frozen and thawed) ovarian tissues. discerned that the contents of this drug are various types In fact, ovarian stromal cells play an important role in of highly purified collagenase (personal communication the process of development of preantral follicles. Ac- with Dr. Christian Sartori Labor, Hamburg, Germany) cording to Young and McNeilly , the thecal cells that allowed the digested tissue suspensions to be not around the secondary preantral follicles appear to be re- sticky and not viscous, and easy to handle and thus, can cruited from the surrounding stromal tissue layer by obtain a good number of viable cells. Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 3 of 14 The aim of our experiments was to study the Tumor sectioned at 5 mm, stained with haematoxylin/eosin, and Dissociation Enzyme effects on the integrity and viability analyzed under a light inverted stereomicroscope Nikon of cell -complexes (follicles) after digesting of these cells SMZ1270 (Nikon, Düsseldorf, Germany) under 400× from cryopreserved ovarian cortex. magnification. The following types of preantral follicles were evaluated: (1) primordial follicles composed of an Methods oocyte surrounded by a layer of flattened follicular cells, Except where otherwise stated, all chemicals were (2) primary and secondary follicles that are similar to obtained from Sigma (Sigma-Aldrich Chemie, GmbH, primordial follicles, but in which the oocyte is sur- Schnelldorf, Germany). rounded by one to two layers of cuboidal granulosa cells. Morphology of the follicles was evaluated on the basis of Tissue collection, dissection, and distribution into groups parameters previously described . The number of This study was approved by the Ethics Boards of viable and damaged follicles was counted. To avoid Cologne University (applications 99,184 and 13–147). overcounting of the same follicles, only the section with Written informed consents were obtained from all a visible oocyte nucleus was counted. Normality of folli- study participants aged 18 and over. cles was evaluated based on the parameters previously Tissues were obtained from 14 patients aged between 22 described by Paynter et al. . Three types of follicles and 39 (29.1± 5.9) years. According to our approved proto- were distinguished: Type I follicle is spherical with ran- col, 10% of ovarian tissues (ovarian tissue biopsies, OTBs) domly distributed granulosa cells around the oocytes. collected from patients were used for ‘patient-oriented’ re- The cytoplasm is homogenous with slightly granulated search. This refers to research done in order to assess the nucleus, in the center of which condensed chromatin is viability of the tissues for re-transplantation. detected in the form of dense spherical structure. Type The patients with following diseases were indicated: II follicle is spherical; however, granulosa cells do not breast cancer (3 patients), Hodgkin lymphoma (2 patients), cover the oocytes regularly. The oocytes can be flat, and non-Hodgkin lymphoma (2 patients), acute lymphoblastic condensed chromatin is not detected in the cytoplasm. leukemia (3 patient), uterine cancer (1 patient), naso- Type III follicle has partly or fully disrupted cytoplasm pharyngeal cancer (1 patient) and soft tissue sarcoma and pyknotic nucleus. The cytoplasm of granulosa cells (2 patient). For our research, the OTBs from three pa- has damages similar to oocytes. Follicles of Type I and tients were used after surgery without cryopreservation, Type II were denoted as normal, and those of Type III and OTBs for patient-oriented research from eight pa- were denoted as degenerated. tients were cryopreserved long before these experiments. The medium used for OTBs transport and dissection Tissue cryopreservation (freezing and thawing) (the basal medium) was composed by Leibovitz L-15 This procedure was performed as published previously with 5% Dextran Serum Substitute (Irvine Scientific, [48–58]. In our protocol, we used DMSO and ethylene Santa Ana, USA). After collection, fresh ovarian tissue glycol as cryoprotective cocktail to support a multi-cellular fragments were transported at 32 °C to 34 °C to the la- structure of ovarian tissues to protect all types of cells . boratory within 20 min of surgery. On the day of freezing, OCFs were placed for 30 min at Using sterile surgical tweezers and no. 22 scalpels, room temperature in a 20-ml freezing medium composed the medullary part of the fragment was removed to of basal medium supplemented with 6% dimethyl sulfox- achieve ~ 1 mm thickness of the cortical part. A small ide, 6% ethylene glycol and 0.15-M sucrose. Then pieces piece of fragment (~ 1 mm ) was fixed in Bouin solution were put into a standard 5-ml cryo-vials (Thermo Fisher for histological evaluation and served as a fresh control. Scientific, Rochester, NY, USA) previously filled by 4.5 ml Depending on the size of ovarian fragment intended for freezing medium and frozen in an IceCube 14S freezer this research, the size of OCFs for patient-oriented re- (Sy-Lab, Neupurkersdorf, Austria). The slow cooling pro- search was ranged from 20 to 210 mm (Table 1). Each file started at − 6 °C, and the samples were then cooled OCF from each patient was cut into two equal parts and from −6°Cto − 34 °C at a rate of 0.3 °C/min. At − 34 °C, cooled to 5 °C for 24 h. After this, the cooled OCF was en- the cryo-vials were finally plunged into liquid nitro- zymatically digested or frozen (one OCF per cryo-vial) gen and stored until thawing. The freezing protocol with subsequent storage in liquid nitrogen until thawing for cryopreservation of this ovarian tissue included an and use. Before each enzymatic treatment, the OCF was auto-seeding step at −6°C. weighted using analytical balance. To thaw the samples, cryo-vials were removed from li- quid nitrogen and held for 30 s at room temperature; Histological examination of ovarian tissue cortex they were then immersed in a 100 °C (boiling) water For histological investigation, the OCFs were fixed in bath for 60 s. The exposure time in the boiling water Bouin’ solution, imbedded in paraffin wax, serially was visually controlled by the presence of ice in the Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 4 of 14 Table 1 Total number of follicles isolated from frozen/thawed ovarian tissues after enzymatic digestion with different kinds of enzymes in 14 experiments Number Patient age Treatment Enzymatic digestion (follicles number) Volume of Byopsy weight (g) Follicle of patient (years) of ovarian piece (mm ) density/ TDE** Liberase TM*** Whole byopsy Byopsy for TDE Byopsy for cortex (mm ) weight digestion Liberase TM digestion 1 26 frozen 10 5 20 0.0192 0.0090 0.0102 8 2 29 frozen 15 7 30 0.0738 0.0378 0.0360 7 3 33 frozen 23 6 45 0.0941 0.0460 0.0482 7 4 22 frozen 43 20 40 0.0973 0.0479 0.0494 12 5 31 frozen 12 9 22 0.0257 0.0125 0.0132 7 6 39 frozen 14 7 36 0.0854 0.0440 0.0414 5 7 23 frozen 55 13 180 0.2805 0.1396 0.1409 5 8 29 frozen 34 6 55 0.1342 0.0665 0.0677 8 9 34 frozen 125 84 165 0.2348 0.116 0.1188 10 10 20 frozen 321 168 144 0.2220 0.116 0.106 24 11 34 frozen 216 115 157 0.2253 0.1114 0.1139 17 12 32 fresh 44 16 200 0.4592 0.2310 0.2286 4 13 34 fresh 30 12 210 0.5775 0.2905 0.2870 2 14 28 fresh 48 19 55 0.1401 0.0691 0.0710 11 Total –– 990 487 –– Mean ± SD 70.7 ± 87.7* 34.8 ± 48.7 81.2 ± 71.8 0.18 ± 0.17 0.096 ± 0.1 0.095 ± 0.1 9.1 ± 5.5 *asterisk corresponding the statistical (P < 0.01) **TDE (Dr. Christian Sartori, Labour, Hamburg, Germany) is a commercial Enzyme-cocktail (Patent Nr. WO 2006031867 A2, 2004) ***Liberase TM Research grade belongs to the group of Liberase Research Grade Purified Enzyme Blends with reduced endotoxine levels and are mixtures of highly purified Collagenase I and Collagenase II, and with a medium concentration of Thermolysin medium; as soon as the ice was 2 to 1 mm apex, the Mannheim, Germany) with the Tumor Dissociation cryo-vial was removed from the boiling water, at which Enzyme (TDE) from the DCS ATP-Chemo-sensitivity Assay point the final temperature of the medium was between Kit (DCS Innovative Diagnostik-Systeme, Dr. Christian 4 °C and 10 °C. Within 5–10 s after thawing, the pieces Sartori Labor, Hamburg, Germany) on the survival of dif- from the cryo-vials were transferred to a 10 ml thawing ferent stages of isolated follicles after enzymatic digestion solution (basal medium containing 0.5-M sucrose) in a of fresh and cryopreserved human ovarian cortex. 100 ml specimen container (Sarstedt, Nümbrecht, The TDE (commercially purchased from the Firma Germany). For stepwise dilution of cryoprotectants, the Dr. Christian Sartori Labor, Hamburg, Germany) is a container was placed on a shaker and continuously agi- commercial enzyme -cocktail for enzymatic digestion of tated with 200 osc/min for 15 min at room temperature. any solid tumors [43–45]. Rehydration of the tissue by stepwise rehydration All enzyme solutions were prepared on Leibovitz L-15 followed. This was also performed using the same, previ- medium of appropriate concentrations in combination ously published [5, 48–58] ‘dropping’ methodology: slow with 50 μg/ml neutral red dye, a vitality dye that shows a addition of basal medium to the solution of sucrose with deep red colour in acid cell structures for the express- ovarian pieces. For ‘dropping’, we used 50-ml of basal visualization of viable follicles (Fig. 1a, b). medium in a 50-ml tube (Greiner Bio-One GmbH, Twenty-two frozen/thawed and six fresh OCFs ob- Frickenhausen, Germany). The final sucrose concentra- tained from 11 patients were divided into four treatment tion was 0.083-M, resulting in almost isotonic conditions groups: Group 1: 11 frozen OCFs for enzymatic diges- [5, 48–58]. The last step involved three washes in a basal tion with TDE; Group 2: 11 frozen OCFs for enzymatic medium for 10 min immediately prior to preparation. digestion with Liberase TM: and two fresh control groups: Group 3: 3 fresh OCFs for enzymatic digestion Preparation of fresh and thawed ovarian tissue and with TDE; Group 4: 3 fresh OCFs for enzymatic diges- isolation of follicles tion with Liberase TM (Fig. 2). We compared the influence of the enzyme Liberase TM The treatment procedure was performed as previously Research Grade (Thermolysin Medium Concentration, described  with some modification. OCFs were Roche Diagnostics GmbH, Roche Applied Science, mechanically cut into ~ 0.5 × 1 to 1 × 1 mm pieces onto Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 5 of 14 Fig. 1 Example of follicles treated with neutral red dye and live/dead fluorochrome assay. a neutral red positive follicle; b B neutral red negative follicle. Neutral red dye is a viability stain which shows a deep red colour in acid cell structures like lysosomes. c V1, fully viable isolated follicle (granulosa cells as well as oocyte show bright green fluorescence) positive to Calcein AM, negative to ethidium homodimer-1; d V2, minimally damaged follicle with two cells positive (bright red colour) to ethidium homodimer-1; e negative control: dead follicle showing bright red fluorescence (positive to ethidium homodimer-1, negative to Calcein AM); f group of follicles, mostly V1, arrow indicates a follicle that was mechanically disrupted during the manipulation process. Bar = 50 μm a sterile 100 mm Falcon Petri dish (Falcon, Corning (Group 2 and Group 4). The optimal concentration for Incorporated-Life Science, Corning, USA) pre-cooled on digestion with TDE was titrated in previous experiments ice to + 4 °C and put onto a cold plate to stabilize the (unpublished data). Incubation with enzymes was per- temperature. The cutting of OCFs was performed with formed in CO -incubator at 37 °C on KS 260 basic quick simultaneous movements of two scalpels no. 22. shaker (IKA, Staufen, Germany) with 100 rotations/min The ovarian tissue pieces were then suspended in basal for 75 min. Enzymatic digestion was inhibited by the medium containing 16% TDE (Group 1 and Group 3) or addition of an equal volume of 4 °C cold Leibovitz L-15 in basal medium containing 0.05 mg/ml Liberase TM medium containing 20% fetal calf serum (FCS). The cell Fig. 2 Experimental design Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 6 of 14 suspension was immediately put onto ice, gently aspi- oocytes and all granulose cells are viable; minimally dam- rated up and down with a 1-ml pipette, filtered through aged follicles of class V2: the presence of less than 10% 100-μm cell strainers (Falcon, Corning Incorporated-Life dead follicular cells; moderately damaged follicles of class Science, NY, USA) and examined by two experienced V3: 10–50% dead follicular cells; and follicles of class V4: co-workers under a light inverted stereomicroscope both the oocytes and all follicular cells are dead. Nikon SMZ1270 (Nikon, Düsseldorf, Germany) in the Confocal images were processed and analyzed using presence of follicles stained and non-stained with neutral the program Fiji, an open-source platform for scientific red follicles. image processing, which is an advanced version of the commonly used Image J. Collection and morphological evaluation of follicles Isolated follicles were collected with 135 μm V-denuded Statistical analysis capillaries (Vitromed GmbH, Jena, Germany) and The enzymatic digestion of ovarian cortex with two washed four times in pre-cooled to 4 °C basal medium types of enzymes was repeated at least three times on to discard stromal cells. The number of stained and different days. Results were expressed as mean ± stand- unstained follicles was calculated for both groups. To ard deviation (SD). Statistical evaluation of flow cytome- identify the isolated primordial follicles, the classification trical results was performed with the GraphPad Prism 5 described by Gougeon and Fortune [61, 62] was used: software package (GraphPad, La Jolla, USA), applying primordial follicle (< 60 μm), oocytes surrounded by a sin- D’Agostino’s K2 test to assess Gaussian distribution. To gle layer of flattened pre-granulosa cells; primary follicle evaluate the effect of two different enzymatic treatments (> 60 μm- ≤75 μm), oocytes with single layer of cuboidal on the cellular viability of ovarian cortex (stromal cells granulosa cells; secondary follicle (> 75 μm-<200 μm). and follicles) immediately after digestion, a T-test for Immediately after isolation of follicles, they were ob- equal variances was performed. P-values less than 0.05 served under Nikon microscope SMZ25. The follicle were considered statistically significant. diameter was measured by Program Zen (Nikon, The correlation test (EXCEL – 2010) was performed Düsseldorf, Germany). for multi-variables (recovered follicle number, patient’s age, size and volume of biopsies) to investigate the rela- Confocal laser scanning microscopy tionship between these parameters. The value of The neutral red staining method provides a fairly rough correlation coefficient (r) was always between + 1 and − 1 estimation of viability. However, dead granulose cells are inclusive, where 1 is total positive linear correlation, 0 is difficult to identify  and we have chosen a live/dead no linear correlation, and − 1 is total negative linear cor- fluorochrome assay for exact analysis of viability using relation. A commonly used scale for the interpretation of confocal laser scanning microscopy. To check the viabil- correlation coefficient is the following: no correlation: r ity of cellular structures of isolated ovarian follicles (oo- from 0 to 0.1 or from 0 to − 0.1; weak correlation exists: r cytes surrounded by flat or cubical follicular cells) from 0.1 to 0.3 or from − 0.1 to − 0.3; moderate correlation collected follicles were stained with Calcein AM for exists: r from 0.3 to 0.5 or from − 0.3 to − 0.5; and strong visualization of viable cells and ethidium homodimer-1 correlation exists: r from 0.5 to 1 or from − 0.5 to − 1. for visualization of dead cells as previously described by Cortvrindt and Smitz . The follicles were exposed to Results 2 μM of Calcein AM and 5 μM of ethidium Retrieval rate of isolated follicles homodimer-1 in Dulbecco’s phosphate buffer saline In experiments total of 1477 follicles were isolated from (DPBS) for 15 min at 37 °C in the dark. During this time, fresh and frozen OCFs (169 follicles were isolated from living cells for intracellular esterase activity converted 6 fresh and 1308 follicles were isolated from 22 cryopre- the non -fluorescent cell-permeable Calcein AM in served OCFs). From fresh biopsies, 122 follicles were re- fluorescent Calcein producing an intense bright uniform covered using TDE and 46 follicles were recovered using green fluorescence (ex/em, 495 nm/515 nm). In contrast Liberase TM. From frozen biopsies, 868 follicles were re- to Calcein AM, ethidium homodimer-1 shows a red covered using TDE and 440 follicles using Liberase TM fluorescence when bound to the DNA of dead cells (P < 0.05) (Table 1). It was established that the retrieval (ex/em, 528 nm/617 nm) (Fig. 1c-f). After exposure to rate of follicles from the biopsies of patients 22–39 years these dyes, the follicles were washed in PBS and visualized old has a strong negative (r = − 0.6) correlation with age. using a confocal laser scanning microscope Olympus However, the relationship between the patient age and Fluoview FV 1000 (Olympus, Hamburg, Germany) with a number of follicles per 1 mm had no correlation (r = 0.06). multi-line argon laser (458, 488, 515 nm) to record the The correlation between the volume of biopsies and num- fluorescent images. Follicle viability was done as described ber of retrieved follicles is a moderate positive (r =0.3). by Paulini , as follows. Viable follicles of class V1: The same moderate correlation (r = 0.4) was found Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 7 of 14 between the volume of biopsies and number of follicles live follicles with the application of neutral red dye and per 1 mm . The correlation between the weight of bi- subsequent evaluation of follicles under a light inverted opsies and number of retrieved follicles was very stereomicroscope and (2) by fluorescence-staining tech- weak (r = 0.1). However, between the weight of biopsies nique with Calcein AM and ethidium homodimer-1 for and number of follicles per 1 mm , medium correlation visualization of viable and dead cells under a confocal (r = 0.) was found. Between the age of patient and volume laser microscope. of biopsies or weight of biopsies, the same very weak correlation (r = 0.1 and r = 0.2, respectively) was observed. Express technique for visualization of follicle vitality However, the relationship between the weight of biopsies Immediately after enzymatic treatment and simultaneous and its volume has a strong positive correlation (r =1) staining with Neutral red dye, the suspension of ovarian (Table 1). stromal cells and follicles were examined under an inverted microscope. Follicles were collected and neutral Quantity and morphology of isolated follicles red -uptake was investigated based on the optical pres- As shown in Fig. 3a the most of follicles in Group 1 are ence of their red staining. fully isolated (P < 0.01). The data on Fig. 5a show that the presence of intense Compared to digestion with TDE, the digestion with red -stained follicles in the suspension from fresh ovar- Liberase TM (Fig. 3b) has resulted in incomplete tissue ian tissues digested with TDE was significantly higher digestion (black arrows). Extruded oocytes were found than in the suspension of tissues digested with Liberase in both treatment groups, but it was extremely rare (< TM (94.2 ± 6.6% in Group 1 vs 79.1 ± 2.1% in Group 2, 3%). Note that apart from the good three-dimensional respectively). structure, the general morphology of isolated follicles The percent of light red- stained follicles was signifi- was well maintained independently from the type of en- cantly higher (P < 0.05) in the suspension of tissues zymatic treatment. After evaluation of isolated follicles digested with Liberase TM than after digestion with under an inverted microscope, it was noted their normal TDE (Group 2 vs Group 1: 16.6 ± 0.6% vs 4.3 ± 0.4%, re- spherical form with mostly one layer of granulosa cells spectively), while the presence of non -stained follicles around the oocyte. The data for the quantity of retrieved was not significantly different in these groups (Group 2 follicles of different maturity inside each treatment are vs Group 1: 4.2 ± 0.3% vs 1.4 ± 0.3%, respectively). The shown (Fig. 4a). It was also demonstrated that the num- presence of intense red -stained follicles in the suspen- ber of retrieved follicles independently from their matur- sion of cryopreserved ovarian tissues digested with TDE ity was significantly (P < 0.01) higher in the TDE was significantly higher (P < 0.05) in frozen ovarian tis- -treatment group in comparison with Liberase TM sues (70.3 ± 6.2% in Group 3 vs 53.1 ± 2.0% in Group 4, groups (Group 1[n = 122] vs Group 2 [n =46], Group 3 respectively) than in the suspension of tissues digested [n =868] vs Group 4 [n = 440]. However, these differ- with Liberase TM. ences in all treatment groups were no significant (P > The amount of light red -stained follicles was not sig- 0.1) in the distribution of follicles according to their nificantly different (P > 0.1) between Group 3 and Group maturity (Fig. 4b). 4 (17.7 ± 1.5% vs 20.2 ± 1.0%, respectively), while the presence of non-stained follicles was significantly higher Viability of follicles (P < 0.05) (12.0 ± 1.3% in Group 3 vs 26.8 ± 0.9% in The viability assessment of follicles was performed using Group 4, respectively) in the suspension of tissues two techniques: (1) express technique for visualization of digested with Liberase TM. The data on Fig. 5b (digested Fig. 3 Typical view of follicle suspension after enzymatic digestion. a follicles isolated from frozen ovarian cortex with TDE-enzyme cocktail; b follicles isolated from frozen ovarian cortex with Liberase TM. The black arrows show the clustered and partially isolated from incompletely digested stroma follicles. Bar = 50 μm Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 8 of 14 Fig. 4 Distribution of follicles according to their maturity in each treatment group. a Percent of retrieved follicles of different maturity inside of each treatment group. b Number of retrieved follicles of different maturity inside of each treatment group. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences (P < 0.05) fresh tissue) and Fig. 5c (digested frozen tissue) No significant difference (P > 0.1) between class V1 characterize the vitality of follicles according to their primordial follicles was found in ovarian tissues of Group maturity. No significant difference (P > 0.1) according to 1 (fresh, TDE-digested ovarian tissues) (58.5 ± 4.2%), the vitality of different maturity stages of follicles be- Group 2 (fresh, Liberase TM-digested ovarian tissues) tween treatment groups was found. (55.8 ± 1.3%), Group 3 (frozen, TDE-digested ovarian tissues) (53.9 ± 4.2%), and Group 4 (frozen, Liberase TM-digested ovarian tissues) (52.1 ± 1.3%). Vitality visualisation with confocal laser scanning The presence of class V2 and V3 follicles was not signifi- microscopy cantly different between all treatment groups, independent All isolated follicles after assessment of vitality using the of the stage of preantral follicles, (P > 0.1). Class V4 pre- Neutral red dye were stained with fluorescent dye to antral follicles in all treatment groups were absent. identify the presence of living and dead cells in each fol- licle. The data on Fig. 6a show that the percent of recov- ered class V1 follicles in the suspension from fresh Histological evaluation of fresh and frozen ovarian tissue ovarian tissues are not significantly different between Histological evaluation of non-treated pieces of ovarian Group 1 and Group 2 (97.1 ± 6.8% vs 91.3 ± 2.1%, cortex (fresh control) shows that the most of exanimated respectively) (P > 0.1). However, a significantly higher follicles (96.8 ± 2.5%) were morphologically normal. rate (96.0 ± 7.8% vs 87.9 ± 2.4%, respectively) (P < 0.05) Histologic analysis of haematoxylin-eosin stained of recovered follicles of class V1 in Group 3 (cortical tissue ovarian cortical tissues showed morphologically normal suspension digested with TDE) compared to Group 4 was preantral follicles. The follicles were surrounded by found. The amount of class V2 and V3 follicles in all treat- non-disrupted intact basement membrane. The oocytes ment groups was not significantly different (P > 0.1). Class were slightly stained, without signs of degeneration or V4 follicles were absent in all treatment groups. retraction. The percentage of morphologically normal The data on Fig. 6b (digested fresh tissue) and Fig. 6c follicles was not significantly different (P > 0.1) between (digested frozen tissue) characterize the vitality of folli- the fresh and frozen ovarian tissues samples (96.8 ± 2.5% cles according to their maturity stage. vs 97.1 ± 5.1%, respectively). Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 9 of 14 Fig. 5 Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested applying of Neutral Red dye. a Vitality of follicles in different treatment groups independent of their maturity stage; b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences (P < 0.05) Discussion automation, and is low cost with the possibility to obtain The latest work in the field of follicle isolation from hu- more cells of interest [66–68]. The feasibility of enzymatic man and animal ovarian cortical tissue indicates their digestion is a good alternative tissue sample preparation excellent survival after thawing and the ability to further method to the technique of mechanical tissue dissection. development both in culture and after xenotransplant- It is well known that the human ovarian cortex pos- ation [18, 19, 64, 65]. An important task is isolation of sesses a highly dense and fibrous structure. Therefore, the follicles from ovarian cortex to recover a maximal using of special enzymatic digestion technique is neces- number of high quality viable follicles. sary for effective isolation of the follicles from the In this work, we applied our original routine cryo- surrounding tissue. preservation protocol for human ovarian tissue, which The widely used enzyme collagenase is known to de- presupposes the long-time tissue pre-cooling step before grade connective tissues to allow tissue dissolution and freezing. The reason is our previous establishment that to get the single-celled suspensions. However, enzymatic the 24 h cooling to 5 °C before cryopreservation is bene- digestion with the use of collagenase also has serious ficial for the cryopreservation of human ovarian tissues, disadvantages. These disadvantages are well described especially of follicles [5, 48–58].  on the example of hepatocytes. Enzymatic digestion is commonly used for tissue Thus, it is known that most isolation protocols result in dissociation and cell harvesting and offers the advantages damage of cell junctions, cell membranes, to cell surface of unattended quick sample preparation, potential receptors and antigens, and cytosolic contents [70, 71]. Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 10 of 14 Fig. 6 Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested using of Calcein AM for visualization of viable cells and ethidium homodimer-1 for visualization of dead cells. a Viability of follicles in different treatment groups. b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences (P < 0.05) By enzymatic follicle isolation, the basal membrane of isolated follicles (collagenase IX and deoxyribonuclease disruption occurs often . Collagenase digestion also IV, ) and about high amounts of premature oocyte induces oxidative stress observed 4-8 h after isolation of extrusions from the enzymatically isolated follicles hepatocytes, leading to a loss of cytochrome enzyme (collagenase Type II)  that shows that the enzyme colla- activity . genase has a batch-to-batch variation in effectiveness . This could be why many isolated preantral follicles According to the latest data [36, 40, 42, 80], the degenerate within the first 24 h of in vitro culture viability of enzymatically isolated follicles depends on and only a few of them could reach the early antral the level of purity, the type of collagenase and on stage [34, 35, 38, 41]. combination with other enzymes, which could reduce its In attempts to standardize the protocol of enzymatic toxicity. Such products are Liberase Research Grade Puri- digestion and to improve the quality of isolated follicles, fied Enzyme Blends and are mixtures of highly purified the various types of collagenase (Ia, II, IX, XI) alone collagenase and neutral protease enzymes, formulated for [36, 37, 39, 74–76] or in combinations with DNA-se efficient, gentle, and reproducible dissociation of tissues [35, 38, 41, 76–78] were used. However, it was reported from a wide variety of sources (Roche Diagnostics GmbH, about increased amount of lipid droplets in granulosa cells Mannheim, Germany). Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 11 of 14 The works of Dolmans et al. , Vanacker and We have decided to apply the neutral red dye in our Kristensen  have shown the beneficial role of different experiments, because this vital dye has no deleterious types of Liberase for follicle isolation during enzymatic effects on enzymatic activity within cellular organelles digestion. In the work of Lierman  both Liberase TM  and proves to be nontoxic with no long-term nega- combined with collagenase IV and Liberase DH were tive effects on the follicles [39, 82]. Neutral red (toluy- shown to be better for isolating high-quality primordial lene red, Basic Red 5, or C.I. 50,040) is a eurhodin dye follicles, compared with mono-enzyme collagenase IV. used for many staining methods in histology. Neutral The Liberase TM (Thermolysin Medium, 0.04 mg/ml, red is used also as a vital stain because the live cells Roche Diagnostics GmbH, Mannheim, Germany), we used incorporate neutral red into their lysosomes . As in our experiments belongs to the group of Liberase cells begin to die, their ability to incorporate neutral red Research Grade Purified Enzyme Blends with reduced diminishes [84, 85]. This vital dye allows also easy endotoxin levels and is a mixture of highly purified visualization of the follicles in cell suspension. This Collagenase I and Collagenase II with a medium concen- technique was first applied by Kristensen  for follicle tration of Thermolysin (a non-clostridial neutral extremely visualization and their vitality assessment, and it allows stable Zn-metalloendopeptidase (www.Sigma-Aldrich.de) accelerating the process of follicle collection. Our results formulated for efficient, gentle, and reproducible show that the presence of intense red stained follicles in dissociation of tissues from a wide variety of sources the ovarian cortical tissue suspension digested with TDE (http://www.roche-applied-science.com; Worthington En- for both fresh and frozen ovarian tissues was signifi- zyme Manual, V Worthington Biochemical Corporation). cantly higher than in the suspension of tissues digested Their activity is directed to break the peptide bonds in with Liberase TM. collagen (Collagenase), fibronectin, collagen IV, and to a Visualization of viable and dead cells under a confocal lower extent collagen I, however, it does not cleave colla- laser scanning microscopy with Calcein AM and eth- gen V or laminin of neutral protease (V Worthington Bio- idium homodimer-1 showed that digestion ovarian tissue chemical Corporation). with TDE for both fresh and frozen samples provides a Compared to Liberase TM, TDE is a commercial en- significantly higher integrity rate of recovered follicles zyme -cocktail  for enzymatic digestion of any solid (class V1) compared to the Liberase TM -groups. The ef- tumors [43–45]. Due to highly secured patenting , fectiveness of the TDE technique is similar to the data we could not obtain the full composition of this enzyme for the Liberase follicles isolated from fresh cortical tis- cocktail and we can only assume, according to the qual- sues  and with the data for follicles isolated from fro- ity of the digested material (not sticky, not viscous, and zen tissues [18, 86–88]. easy to handle), that this cocktail, together with highly According to the latest research data, the best survival purified types of collagenase, may also include enzymes of the cells of an oocyte-granulosa-cells complex immedi- such as protease, dispase or DNA-se. ately after enzymatic isolation of preantral follicles from The comparison of two digestion protocols according to fresh ovarian tissue with subsequent one-week in vitro the quality of digestion of stromal tissues showed that culture provides the Liberase DH enzymatic cocktail . TDE allows complete digestion of the stromal tissue with Our data presented here with use of TDE commercial good preservation of follicle integrity. The digested tissue enzyme-cocktail for isolation of preantral follicles are not suspension is easy to handle, not sticky and able to obtain principally different from the results achieved with use of a good number of viable follicles. In contrast, the tissue Liberase DH for digestion of fresh ovarian tissue (95% digested with Liberase TM remained poorly dissolved, was TDE vs 95% Liberase DH, P >0.5) . The use of highly sticky and stretchy, and many follicles were still tightly purified commercial enzyme-cocktails allows eliminating embedded in the tissue and was very difficult to isolate, the possibility of obtaining undesirable results through the and this support the finding described . The compari- fault of the drug manufacturer [35, 37, 41, 79]. In fact, son of the two treatment groups also showed that the there are not principal differences between these two en- number of fully isolated follicles in the TDE groups was zymatic cocktails: they content seemingly different types significantly higher than in the Liberase TM groups both of collagenase. for fresh and frozen tissues, considering that the size of In the future work we suppose to perform a compara- digested ovarian cortex was similar in both groups. tive evaluation of effectiveness of these two commercial For evaluation of the quality of recovered follicles from enzymatic cocktails (TDE and Liberase DH) with de- cryopreserved ovarian cortex, we used two techniques: (1) tailed investigation of all apoptotic degenerations (early vital staining with neutral red dye for visualization of live apoptotic, late apoptotic and necrotic cells) which could follicles and (2) fluorescence -staining of follicles with take place in enzymatically isolated follicles using spe- Calcein AM and ethidium homodimer-1 for visualization cific surface markers for follicular cells by applying flow of viable and dead cells under a confocal laser microscope. cytometry and confocal microscopy. Schmidt et al. Reproductive Biology and Endocrinology (2018) 16:57 Page 12 of 14 Conclusion phosphatidylserine (Ptd-L-Ser) in human ovarian tissue. PLoS One. 2015;10:e0129108. New method for isolation of preantral follicles with 6. Donnez J, Dolmans MM. Fertility preservation in women. N Engl J Med. tumor dissociation enzyme (TDE) that can be used for 2017;377:1657–65. the construction of artificial ovary, provides a higher 7. Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra'anani H, et al. Searching for evidence of disease and malignant cell contamination in ovarian number of healthy preantral follicles from cryopreserved tissue store from hematologic cancer patients. Hum Reprod Oxf Engl. ovarian cortex in contrast with the earlier described 2008;23:1007–13. Liberase Thermolysin Medium method. 8. Dolmans MM, Marinescu C, Saussoy P, Van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients Abbreviations with acute lymphoblastic leukemia is potentially unsafe. Blood. DNA: Deoxyribonucleic acid; Liberase TM: Liberase Thermolysin Medium; 2010;116:2908–14. OCF: Ovarian tissue cortical fragment; OTB: 24 ovarian tissue biopsies; 9. Dolmans MM, Jadoul P, Gilliaux S, Amorim CA, Luyckx V, Squifflet J, et al. PBS: Phosphate buffer saline Dulbecco; SD: Standard deviation; TDE: Tumor A review of 15 years of ovarian tissue bank activities. J Assist Reprod Genet. dissociation enzyme 2013;30:305–14. 10. Rosendahl M, Andersen MT, Ralfkiaer E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female Acknowledgements patients with leukemia. Fertil Steril. 2010;94:2186–90. Supported by Alexander von Humboldt Foundation. 11. Amorim CA, Van Langendonckt A, David A, Dolmans MM, Donnez J. Survival of human pre-antral follicles after cryopreservation of ovarian tissue, Funding follicular isolation and in vitro culture in a calcium alginate matrix. The authors did not receive any specific funding for this study. Hum Reprod. 2009;24:92–9. 12. Soares M, Saussoy P, Maskens M, Reul H, Amorim CA, Donnez J, et al. Availability of data and materials Eliminating malignant cells from cryopreserved ovarian tissue is possible in The data sets supporting the conclusions of this article are included within leukaemia patients. Br J Haematol. 2017;178:231–9. the article. 13. Amorim CA, Shikanov A. The artificial ovary: current status and future perspectives. Future Oncol. 2016;12:2323–32. Authors’ contributions 14. Rodgers RJ, Irving-Rodgers HF, Russell DL. Extracellular matrix of the Conceived and designed the experiments: EI, VS, GR, VI, PM; performed the developing ovarian follicle. Reproduction. 2003;126:415–24. experiments: VS, EI, GR; analyzed the data: GR, GR, BH, BM; contributed 15. Luyckx V, Dolmans MM, Vanacker J, Scalercio SR, Donnez J, Amorim CA. materials/analysis tools: GR, VS, PM, wrote the manuscript: EI, VI, VS; manuscript First step in developing a 3D biodegradable fibrin scaffold for an artificial drafting and critical discussion: EI, VI, VS, GR; finally approved of the version to ovary. J Ovar Res. 2013;6:83–92. be published: EI, VI. All authors read and approved the final manuscript. 16. Camboni A, Van Langendonckt A, Donnez J, Vanacker J, Dolmans MM, Amorim CA. Alginate beads as a tool to handle, cryopreserve and culture Ethics approval and consent to participate isolated human primordial/ primary follicles. Cryobiology. 2013;67:64–9. This study was approved by the Ethics Boards of University Cologne 17. Laronda MM, Rutz AL, Xiao S, Whelan KA, Duncan FE, Roth EW, et al. A (applications 9 99,184 and 13–147). Written informed consent was obtained bioprosthetic ovary created using 3D printed microporous scaffolds restores from all the participating couples. ovarian function in sterilized mice. Nat Commun. 2017;8:15261. 18. Vanacker J, Luyckx V, Amorim C, Dolmans MM, Van Langendonckt A, Competing interests Donnez J, et al. Should we isolate human preantral follicles before or after The authors declare that they have no competing interests. cryopreservation of ovarian tissue? Fertil Steril. 2013;99:1363–8. 19. Lunardi FO, de Aguiar FL, Duarte AB, Araújo VR, de Lima LF, Ribeiro de Sá NA, et al. Ovine secondary follicles vitrified out the ovarian tissue grow and Publisher’sNote develop in vitro better than those vitrified into the ovarian fragments. Springer Nature remains neutral with regard to jurisdictional claims in Theriogenology. 2016;85:1203–10. published maps and institutional affiliations. 20. Young JM, McNeilly AS. Theca: the forgotten cell of the ovarian follicle. 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Reproductive Biology and Endocrinology – Springer Journals
Published: Jun 2, 2018
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