In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women.

STUDY QUESTION: Can human theca cells (TCs) be differentiated in vitro? SUMMARY ANSWER: It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. WHAT IS KNOWN ALREADY: There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. STUDY DESIGN, SIZE, DURATION: After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53-74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. PARTICIPANTS/MATERIALS, SETTING, METHODS: To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells. WIDER IMPLICATIONS OF THE FINDINGS: This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.

Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries.

PURPOSE: Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. METHODS: RNA was isolated from 5 normal human ovaries (52-79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75 years of age), and 4 PCOS ovaries (18-35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. RESULTS: Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. CONCLUSIONS: This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

STUDY QUESTION: How do elastic matrisome components change during the lifetime of the human ovary? SUMMARY ANSWER: The deposition and remodeling of mechanical matrisome components (collagen, elastin, elastin microfibril interface-located protein 1 (EMILIN-1), fibrillin-1 and glycosaminoglycans (GAGs)) that play key roles in signaling pathways related to follicle activation and development evolve in an age- and follicle stage-related manner. WHAT IS KNOWN ALREADY: The mechanobiology of the human ovary and dynamic reciprocity that exists between ovarian cells and their microenvironment is of high importance. Indeed, while the localization of primordial follicles in the collagen-rich ovarian cortex offers a rigid physical environment that supports follicle architecture and probably plays a role in their survival, ovarian extracellular matrix (ECM) stiffness limits follicle expansion and hence oocyte maturation, maintaining follicles in their quiescent state. As growing follicles migrate to the medulla of the ovary, they encounter a softer, more pliant ECM, allowing expansion and development. Thus, changes in the rigidity of the ovarian ECM have a direct effect on follicle behavior. Evidence supporting a role for the physical environment in follicle activation was provided in clinical practice by ovarian tissue fragmentation, which promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth and generation of mature oocytes. STUDY DESIGN, SIZE, DURATION: We investigated quantitative spatiotemporal changes in collagen, elastin, EMILIN-1, fibrillin-1 and GAGs from prepuberty to menopause, before conducting a closer analysis of the ECM surrounding follicles, from primordial to secondary stages, in both prepubertal and tissue from women of reproductive age. The study included ovarian tissue (cortex) from 68 patients of different ages: prepubertal (n = 16; mean age [±SD]=8 ± 2 years); reproductive (n = 21; mean age [±SD]=27 ± 4 years); menopausal with estrogen-based HRT (n = 7; mean age [±SD]=58 ± 4 years); and menopausal without HRT (n = 24; mean age [±SD]=61 ± 5 years). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative investigations of collagen and GAG deposition in ovarian tissue throughout a woman's lifetime were conducted by analyzing brightfield images. Characteristic features of collagen fiber content were based on polarized light microscopy, since polarized light changes with fiber thickness. To evaluate the deposition and distribution of elastin, fibrillin-1 and EMILIN-1, multiplex immunofluorescence was used on at least three sections from each patient. Image processing and tailored bioinformatic analysis were applied to enable spatiotemporal quantitative evaluation of elastic system component deposition in the human ovary over its lifetime. MAIN RESULTS AND THE ROLE OF CHANCE: While collagen levels increased with age, fibrillin-1 and EMILIN-1 declined. Interestingly, collagen and elastin reached their peak in reproductive-age women compared to prepubertal (P < 0.01; P = 0.262) and menopausal subjects with (P = 0.706; P < 0.01) and without (P = 0.987; P = 0.610) HRT, indicating a positive impact of secreted estrogen and hormone treatment on collagen and elastin preservation. Interestingly, HRT appears to affect elastin presence in ovarian tissue, since a significantly higher (P < 0.05) proportion of elastin was detected in biopsies from menopausal women taking HRT compared to those not. Higher GAG levels were found in adult ovaries compared to prepubertal ovaries (P < 0.05), suggesting changes in tissue ultrastructure and elasticity with age. In this context, elevated GAG values are suspected to participate in hampering formation of the fibrillin-1 network (r = -0.2475; P = 0.04687), which explains its decline over time. This decline partially accounts for the decrease in EMILIN-1 (r = 0.4149; P = 0.00059). Closer examination of the ECM surrounding follicles from the primordial to the secondary stage, both before and after puberty, points to high levels of mechanical stress placed on prepubertal follicles compared to the more compliant ECM around reproductive-age follicles, as suggested by the higher collagen levels and lower elastin content detected mainly around primordial (P < 0.0001; P < 0.0001, respectively) and primary (P < 0.0001; P < 0.001, respectively) follicles. Such a stiff niche is nonpermissive to prepubertal follicle activation and growth, and is more inclined to quiescence. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The duration and form of administered HRT were not considered when studying the menopausal patient group undergoing treatment. Moreover, we cannot exclude interference from other nongynecological medications taken by the study patients on ovarian ECM properties since there is no information in the literature describing the impact of each medication on the ECM. Finally, since the ECM is by definition a very heterogeneous meshwork of proteins, the use of two-dimensional histology could be a limitation. Single time points on fixed tissues could also present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. WIDER IMPLICATIONS OF THE FINDINGS: From a biomechanical perspective, our study revealed important changes to ECM properties dictating the mechanical features of ovarian tissue, in line with the existing literature. Our findings pave the way for possible therapeutic targets at the ECM level in the context of female fertility and ovarian rejuvenation, such as mechanical stimulation, antifibrotic treatments, and prevention or reversion of elastic ECM degradation. Our study also sheds light on the follicle-specific ECM composition that is dependent on follicle stage and age. These data will prove very useful in designing biomimetic scaffolds and tissue-engineered models like the artificial ovary. Indeed, they emphasize the importance of encapsulating each type of isolated follicle in an appropriate biomaterial that must replicate the corresponding functional perifollicular ECM and respect ovarian tissue heterogeneity in order to guarantee its biomimicry. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS research associate; grant 5/4/150/5 awarded to M.M.D.) and the Université Catholique de Louvain (PhD grant 'Coopération au développement' awarded to E.O.). None of the authors have any competing interests to declare.

Immunodetection and quantification of enzymatic markers in theca cells: the early process of ovarian steroidogenesis†.

The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 2, play a role in the cascade after luteinizing hormone-choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2, and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (<3000 µm), and large (>3000 µm) antral follicles and corpora lutea at 100× magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2, and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 -> 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome.

Adipose tissue-derived stem cells boost vascularization in grafted ovarian tissue by growth factor secretion and differentiation into endothelial cell lineages.

Adipose tissue-derived stem cells (ASCs) have multilineage differentiation potential, proangiogenic properties, and the ability to enhance vascularization in xenografted human ovarian tissue. The aim of the present study was to identify the mechanisms behind the proangiogenic effects of ASCs. For this purpose, severe combined immunodeficient (SCID) mice were grafted with frozen-thawed human ovarian tissue. ASCs were labeled by lentiviral transfection for expression of enhanced green fluorescent protein (eGFP), and human ovarian tissue was grafted using a previously described two-step procedure. In the control group, ovarian tissue was transplanted using the standard one-step approach. Samples were collected and analyzed after 7 days. Detection of the eGFP antigen by immunofluorescence showed ASCs surrounding and infiltrating ovarian tissue grafts. Significantly higher vessel density was observed in the ASC group (P = 0.0182 versus control) on Day 7. Co-expression of eGFP, CD34 and CD31 was demonstrated in human vessels, confirming ASC differentiation into human endothelial cell lineages. Increased gene expression of vascular endothelial growth factor (VEGF) was also shown in the ASC group (P = 0.0182 versus control). Immunohistochemistry targeting anti-human VEGF revealed significantly higher expression levels in the ASC group (P = 0.033 versus control), while VEGF and eGFP immunofluorescence showed greater growth factor expression in areas surrounding ASCs. In conclusion, ASCs differentiate into human vessels and promote secretion of VEGF when transplanted together with human ovarian tissue to SCID mouse peritoneum using a two-step ovarian tissue grafting procedure. This is a promising step towards potentially improving ovarian tissue quality and lifespan. Long-term studies should be conducted to investigate ASC safety and efficacy in the context of ovarian tissue transplantation.

Association between missed canals and apical periodontitis.

AIM: To evaluate the frequency of post-treatment apical periodontitis associated with root filled teeth with at least one untreated root canal. METHODOLOGY: Eight hundred and seven cone beam computed tomography images containing at least one root filled tooth were selected from a collection of 1543 images from Brazilian individuals. Scans were taken using ICAT Classic devices (Imaging Sciences, Hatfield, PA, USA) in a private oral radiology clinic from January to April 2015. All root filled teeth were analysed for the presence of missed canals and apical periodontitis. The chi-square and odds ratio tests were used to verify if there were an association and risk relationship between the occurrence of untreated canals and apical periodontitis. RESULTS: A total of 2294 teeth with evidence of root fillings were identified. Two hundred and eighty-one teeth had at least one untreated missed canal (12%). The frequency of apical periodontitis in teeth with at least one untreated canal was significantly greater in comparison to teeth with all canals treated (274/281, 98% versus 1736/2013, 86%) (P < 0.01). The odds for apical periodontitis to be present was 6.25 times greater for teeth with an untreated canal. The mesiobuccal roots of maxillary first molars had the greatest frequency of untreated canals (114/154, 74%), with the second mesiobuccal canal being the most frequently missed (n = 106/114, 93%). CONCLUSION: Root filled teeth with at least one missed canal had a high prevalence of post-treatment apical periodontitis.

Assessment of serotonergic system in formation of memory and learning / Avaliação do sistema serotonérgico na formação de memória e aprendizagem

Abstract We evaluated the involvement of the serotonergic system on memory formation and learning processes in healthy adults Wistar rats. Fifty-seven rats of 5 groups had one serotonergic nuclei damaged by an electric current. Electrolytic lesion was carried out using a continuous current of 2mA during two seconds by stereotactic surgery. Animals were submitted to learning and memory tests. Rats presented different responses in the memory tests depending on the serotonergic nucleus involved. Both explicit and implicit memory may be affected after lesion although some groups showed significant difference and others did not. A damage in the serotonergic nucleus was able to cause impairment in the memory of Wistar. The formation of implicit and explicit memory is impaired after injury in some serotonergic nuclei.

Resumo Avaliar a participação do sistema serotoninérgico em processos de formação de memória e aprendizagem em ratos Wistar adultos saudáveis. Cinquenta e sete ratos de 5 grupos tinham um núcleo serotoninérgico danificado por uma corrente elétrica. A lesão eletrolítica foi realizada utilizando uma corrente contínua de 2 mA durante dois segundos por cirurgia estereotáxica. Os animais foram submetidos a testes de aprendizagem e memória. Os ratos apresentaram respostas diferentes nos testes de memória, dependendo do núcleo serotoninérgica envolvido. A memória explícita e implícita pode ser afetada após a lesão, embora alguns grupos apresentaram diferença significativa e outros não. A lesão no núcleo serotoninérgico foi capaz de causar danos na memória de Wistar. A formação da memória implícita e explícita é prejudicada após a lesão em alguns núcleos serotoninérgicos.

Assessment of serotonergic system in formation of memory and learning.

We evaluated the involvement of the serotonergic system on memory formation and learning processes in healthy adults Wistar rats. Fifty-seven rats of 5 groups had one serotonergic nuclei damaged by an electric current. Electrolytic lesion was carried out using a continuous current of 2mA during two seconds by stereotactic surgery. Animals were submitted to learning and memory tests. Rats presented different responses in the memory tests depending on the serotonergic nucleus involved. Both explicit and implicit memory may be affected after lesion although some groups showed significant difference and others did not. A damage in the serotonergic nucleus was able to cause impairment in the memory of Wistar. The formation of implicit and explicit memory is impaired after injury in some serotonergic nuclei.

Two-step transplantation with adipose tissue-derived stem cells increases follicle survival by enhancing vascularization in xenografted frozen-thawed human ovarian tissue.

STUDY QUESTION: Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? SUMMARY ANSWER: Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. STUDY DESIGN, SIZE, DURATION: A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01). LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.

Adipose tissue-derived stem cells in a fibrin implant enhance neovascularization in a peritoneal grafting site: a potential way to improve ovarian tissue transplantation.

STUDY QUESTION: Do two different concentrations of human adipose tissue-derived stem cells (ASCs) embedded inside a fibrin scaffold have the potential to differentiate into vessels and aid vascularization in a peritoneal grafting site intended for ovarian tissue transplantation? SUMMARY ANSWER: Human ASCs in low and high concentrations differentiated into vessels when transplanted to mouse peritoneum inside a fibrin matrix, but only high ASC concentrations significantly increased human vessel area 14 days after transplantation. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, including proangiogenic properties and have been used in tissue engineering to enhance vascularization in transplanted tissues. Fibrin has been studied and used as an ASC-compatible biomaterial. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 22 severe combined immunodeficient mice. In total, 16 mice (eight per group) were intraperitoneally grafted with a fibrin scaffold loaded with two different human ASC concentrations (either 150 000 [L-ASC] or 1 500 000 [H-ASC] cells) and lithium phthalocyanine (LiPc) crystals as oxygen-sensitive probes. Six mice were grafted with an empty fibrin (EF) implant containing only LiPc and served as controls. Levels of partial pressure of oxygen (pO2) in implants were monitored in vivo by electron paramagnetic resonance oximetry (EPR). ASC identification, proliferation, and host and human vascularization were analyzed by immunohistochemistry (IHC). All analyses were performed on post-grafting Days 3, 7 and 14. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), as well as histological (hematoxylin-eosin staining) and IHC (anti-human vimentin, anti-human Ki67, anti-mouse and human double CD34) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 in implants was observed in all groups between Days 3 and 7 (P < 0.001). ASC-loaded implants displayed a tendency towards increased pO2 levels from Days 7 to 14, not observed in EF implants. ASC-loaded implants showed differentiation into human CD34-positive vessels. Total CD34-positive endothelial area was correlated to pO2 values obtained by EPR oximetry (r = 0.6506, P = 0.0019). In the H-ASC group, a greater human CD34-positive vascular surface area was found compared to the L-ASC group 14 days after transplantation (P < 0.0049). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, ASCs transplanted inside a fibrin matrix can differentiate into CD34-positive human vessels. However, other possible mechanisms involved in ASC angiogenic behavior remain to be investigated. WIDER IMPLICATIONS OF THE FINDINGS: High concentrations of ASCs loaded inside a fibrin scaffold could serve as a substrate to prepare a peritoneal grafting site over 14 days, in order to enhance vascularization once human ovarian tissue is grafted. Our proposed preparation of the grafting site would not only benefit ovarian tissue transplantation, but also other experimental avascular grafting procedures. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, Télévie Grant no. 7.6515.16F awarded to DDM and Grant 5/4/150/5 awarded to M.M.D. [CAA is FRS-FNRS research associate]), Fonds Spéciaux de Recherche, and Fondation St Luc, Foundation Against Cancer, and donations from the Ferrero family. None of the authors have any competing interests to declare.

Fibrin in Reproductive Tissue Engineering: A Review on Its Application as a Biomaterial for Fertility Preservation.

In recent years, reproductive medicine has made good use of tissue engineering and regenerative medicine techniques to develop alternatives to restore fertility in cancer patients. For young female cancer patients who cannot undergo any of the currently applied strategies due to the possible presence of malignant cells in their ovaries, the challenge is creating an in vitro or in vivo artificial ovary using carefully selected biomaterials. Thanks to its numerous qualities, fibrin has been widely used as a scaffold material for fertility preservation applications. The goal of this review is to examine and discuss the applications and advantages of this biopolymer for fertility restoration in cancer patients, and consider the main results achieved so far.

Further insights into the impact of mouse follicle stage on graft outcome in an artificial ovary environment.

STUDY QUESTION: Are mouse preantral follicles differently affected by isolation, encapsulation and/or grafting procedures according to stage? SUMMARY ANSWER: Isolated secondary follicles showed superior ability to survive and grow after transplantation, which was not related to a particular effect of the isolation and/or grafting procedure, but rather to their own ability to induce neoangiogenesis. WHAT IS KNOWN ALREADY: Isolated and encapsulated mouse preantral follicles can survive (6-27%) and grow (80-100%) in a fibrin matrix with a low concentration of fibrinogen and thrombin (F12.5/T1) after short-term transplantation. STUDY DESIGN, SIZE, DURATION: An in vivo experimental model using 20 donor Naval Medical Research Institute (NMRI) mice (6-25 weeks of age) and 14 recipient severe combined immunodeficient (SCID) mice (11-39 weeks of age) was applied. Each NMRI mouse underwent mechanical disruption of both ovaries and isolation of primordial-primary and secondary follicles with ovarian stromal cells, in order to encapsulate them in an F12.5/T1 matrix. Twelve out of 40 fibrin clots were immediately fixed as controls (D0) (10 for histology and 2 for scanning electron microscopy [SEM]) and the others (n = 28) were grafted to the inner part of the peritoneum for 2 (16 fibrin clots) or 7 (12 fibrin clots) days (D2 and D7). PARTICIPANTS/MATERIALS, SETTING, METHODS: This study involved the participation of the Gynecology Research Unit (Universitè Catholique de Louvain) and the Physiological Sciences Department (University of Brasília). Specific techniques were used to analyze the follicle recovery rate (hematoxylin-eosin staining), vascularization (CD34) and follicle ultrastructure (transmission electron microscopy [TEM] and SEM). MAIN RESULTS AND THE ROLE OF CHANCE: After follicle isolation and encapsulation, a statistically higher percentage of normal follicles was observed in the secondary group (62%) than in the primordial-primary group (47%). Follicle recovery rates were 34% and 62% for primordial-primary and secondary follicles on D2, respectively, and 12% and 42% on D7, confirming that secondary follicles survive better than primordial-primary follicles after grafting. Concerning vascularization, both follicle stages exhibited similar vascularization to that seen in control mouse ovary on D7, but a significantly higher number of vessels and greater vessel surface area were detected in the secondary follicle group. Despite structural differences in fiber density between fibrin clots and ovarian tissue observed by SEM and TEM, preantral follicles appeared to be well encapsulated in the matrix, also showing a normal ultrastructure after grafting. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results during the isolation procedure, we encapsulated a significantly higher number of round structures in the primordial-primary group than in the secondary group, which could partially explain the lower recovery rate of early-stage follicles in our previous study. However, it is not excluded that the physical and mechanical properties of the fibrin matrix may also play a role in follicle survival and growth, so further investigations are needed. WIDER IMPLICATIONS OF THE FINDINGS: This research represents one more key step in the creation of the artificial ovary. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) to C.A. Amorim as a research associate at FRS-FNRS and (grant 5/4/150/5 awarded to M.M. Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) (grant #013/14 CAPES/WBI awarded to C.M. Lucci, with F. Paulini receiving a post-doctoral fellowship), and Wallonie-Bruxelles International, and donations from the Ferrero family. None of the authors have any competing interests to declare in relation to the topic.

Evaluation of minimal disseminated disease in cryopreserved ovarian tissue from bone and soft tissue sarcoma patients.

STUDY QUESTION: What is the risk of finding malignant cells in cryopreserved ovarian tissue from sarcoma patients? SUMMARY ANSWER: Minimal disseminated disease (MDD) was not detected in frozen-thawed ovarian tissue from 26 patients by any of the sensitive methods applied. WHAT IS KNOWN ALREADY: In case of leukemia, the risk of malignant cell transmission through the graft is well known and widely documented. However, for bone cancer, like Ewing sarcoma or osteosarcoma, only a small number of case reports, have been published. These cancers often affect prepubertal girls, in whom ovarian tissue cryopreservation and transplantation is the only option to preserve fertility. STUDY DESIGN, SIZE, DURATION: The presence of malignant cells in cryopreserved ovarian tissue from patients with bone/soft tissue sarcoma was investigated with disease-specific markers for each patient, using immunohistochemistry (IHC), FISH and real-time quantitative RT-PCR (qPCR), with the original tumor serving as a positive control. PARTICIPANTS/MATERIALS, SETTING, METHODS: Forty-eight sarcoma patients were enrolled in the study, 12 of whom subsequently died. In each case, tissue from the primary tumor was investigated in order to identify markers (immunohistochemical and/or molecular) to analyze the ovarian tissue case by case. Ovarian tissue from osteosarcoma (n = 15), liposarcoma (n = 1) and undifferentiated sarcoma (n = 5) patients could not be evaluated, as no specific markers were detected by FISH or sensitive IHC in any of their primary tumoral tissue. One patient with Li-Fraumeni syndrome was also excluded from the study. IHC analyses were therefore performed on ovarian tissue from 26 patients and qPCR on 19. The primary tumors involved were Ewing sarcoma family of tumors (n = 14), rhabdomyosarcoma (n = 7), synovial sarcoma (n = 2), clear cell sarcoma (n = 2) and a malignant peripheral nerve sheath tumor (n = 1). MAIN RESULTS AND THE ROLE OF CHANCE: MDD was not detected in any of the 26 analyzed samples using sensitive techniques in this largest reported series, even from patients who subsequently died and/or those who presented with metastasis (11/26), hence the most aggressive forms of bone cancer. Indeed, anti-CD99 IHC and PCR performed on patients presenting with Ewing sarcoma family of tumors (n = 14) was negative in all cases. In patients with soft tissue sarcoma (n = 12) primitive tumor markers were detected by IHC and were negative in ovarian tissue. PCR could only be performed in 6/12 of these patients, again proving negative. LIMITATIONS, REASONS FOR CAUTION: Cryopreserved ovarian fragments to be transplanted cannot be tested, so this analysis of malignant cells cannot guarantee that all cryopreserved fragments will not contain any disseminated disease. Moreover, molecular markers are not readily available for all types of tumors. WIDER IMPLICATIONS OF THE FINDINGS: These results are reassuring regarding the risk of malignant cells in the ovary for transplantation, as the study involves a large series including different types of sarcomas. We believe this will help clinicians in their patient counseling for fertility preservation and restoration. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Fonds National de la Recherche Scientifique de Belgique-FNRS under Grants Nos 7.4578.14 (Télévie to MS) and 5/4/150/5 to MMD. The authors declare no competing financial interests.

Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane.

In vitro culture and transplantation procedures are essential protocols employed in the evaluation of ovarian follicle survival and development. Culture in the chorioallantoic membrane (CAM) of chick embryos is an intermediate method that provides important follicle development information and has not been tested for cat ovaries to date. The aim of this study was to investigate if in vitro and CAM culture could be used as short-term systems to study cat ovarian tissue development. The ovaries of eight cats were dissected into 3-mm(3) cubes, cultured in vitro and in CAM for up to 5 days, and stained with hematoxylin-eosin and Gomori trichrome. Cell proliferation was analyzed using anti-Ki67. Possible differences among groups were investigated by analysis of variance or the Kruskal-Wallis test followed by Bonferroni correction. The T-test or Wilcoxon test was used to verify differences between the CAM and IVC. Results revealed that 87.5% of all follicles were primordial during culture. The percentage of primordial follicles in the morphologically normal follicles (MNF) pool was always higher than 80%, with the exception of Day 3 of CAM culture, but the number of MNF reduced significantly from Day 0 (600 out of 777 follicles) to Day 5 in the CAM (91 out of 171) and IVC (296 out of 686). The number of primordial follicles in 1 mm(3) in Days 2, 3, and 5 in the CAM was significantly lower than that in the control (Day 0). No cellular proliferation was observed in culture. Vascularization occurred in the CAM culture, but with no association to follicular viability. In addition, both methods showed an increase in connective tissue during culture. Although no significant differences were observed in the percentage of MNF, there was a reduction in the total number of follicles, both for IVC and CAM-cultured ovarian tissue. Furthermore, anti-Ki67 did not stain any follicle after Day 0 in IVC or in CAM culture. Neither system was capable of promoting follicle growth and/or development. The results show that the CAM is not a suitable system for feline ovarian tissue and highlight the necessity to improve IVC systems in cats.

Influence of follicle stage on artificial ovary outcome using fibrin as a matrix.

STUDY QUESTION: Do primordial-primary versus secondary follicles embedded inside a fibrin matrix have different capabilities to survive and grow after isolation and transplantation? SUMMARY ANSWER: Mouse primordial-primary follicles showed a lower recovery rate than secondary follicles, but both were able to grow. WHAT IS KNOWN ALREADY: Fresh isolated mouse follicles and ovarian stromal cells embedded in a fibrin matrix are capable of surviving and developing after short-term autografting. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 11 donor Naval Medical Research Institute (NMRI) mice and 11 recipient severe combined immunodeficiency (SCID) mice. Both ovaries from all NMRI mice were mechanically disrupted and primordial-primary and secondary follicles were isolated with ovarian stromal cells. They were then encapsulated in a fibrin matrix composed of 12.5 mg/ml of fibrinogen (F12.5) and 1 IU/ml of thrombin (T1) (F12.5/T1), and grafted to the inner part of the peritoneum of SCID mice for 2 and 7 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to conduct histological (H-E staining) and immunohistochemical (Ki67, TUNEL) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Although all grafted fibrin clots were recovered, the follicle recovery rate on day 2 was 16 and 40% for primordial-primary and secondary follicles respectively, while on day 7, it was 6 and 28%. The secondary group showed a significantly higher recovery rate than the primordial-primary group (23%, P-value <0.001). Follicles found in both groups were viable, as demonstrated by live/dead assays, and no difference was observed in the apoptosis rate between groups, as evidenced by TUNEL. Their growth to further stages was confirmed by Ki67 immunostaining. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results, secondary follicles appear to be more likely to survive and develop than primordial-primary follicles in a fibrin matrix after both periods of grafting. These findings may also be attributed to the specific features of the fibrin matrix, which could benefit larger follicles, but not smaller follicles. WIDER IMPLICATIONS OF THE FINDINGS: This study is essential to understanding possible impairment caused by factors such as the isolation procedure or fibrin matrix composition to the survival and development of different follicle stages. It therefore provides the basis for further investigations with longer periods of grafting. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4578.14 and 7.4627.13, grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, the Foundation Against Cancer, and the Region Wallone (Convention N°6519-OVART) and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any competing interests to declare.

Impact of the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus monkeys.

PURPOSE: The aim of this study was to determine the best combination in terms of cryopreservation techniques and vascular bed preparation before grafting in order to obtain functional ovarian tissue after transplantation. METHODS: Five cynomolgus monkeys were used. Strips from 10 ovaries were cryopreserved, 5 by vitrification (V), and 5 by slow-freezing (SF). Pieces of fresh ovarian tissue were used for controls. After 1 month, the strips were autografted to two different vascular beds, healed (HB) or freshly decorticated (FDB), constituting four study groups: SF-HB, SF-FDB, V-HB, and V-FDB. These were compared to fresh tissue. After 6 months, the ovaries were removed and several parameters analyzed: follicle quality, stage, density, proliferation, apoptosis, functionality, vascularization, and fibrosis. Mixed effect linear regression models were built to assess the impact of cryopreservation and vascular bed preparation on ovarian tissue viability and functionality. p values were adjusted for multiple testing using the Benjamini-Hochberg method, and q values < 0.20 were considered significant in order to achieve a 20% false discovery rate. RESULTS: Compared to fresh tissue, no difference was observed in the percentage of morphologically normal follicles, while a significant increase was noted in the follicle proliferation rate (41%, q = 0.19), percentage of antral follicles (12%, q = 0.14), and number of vessels per area (3.3 times, q = 0.07) in the V-FDB group. CONCLUSIONS: Vitrification associated with FDB vascular bed preparation is the best combination to obtain functional autografted ovarian tissue. Further studies are nevertheless required, with confirmed pregnancies and live births before introducing the procedure into clinical practice.

The best source of isolated stromal cells for the artificial ovary: medulla or cortex, cryopreserved or fresh?

STUDY QUESTION: What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh? SUMMARY ANSWER: Ovarian cells from fresh medullary tissue, which can be isolated in larger numbers, show higher viability and are able to improve graft vascularization. WHAT IS KNOWN ALREADY: In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells. STUDY DESIGN, SIZE, DURATION: Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay. MAIN RESULTS AND THE ROLE OF CHANCE: Cryopreservation decreased ovarian cell yield (-2804 cells/mg, P = 0.015) and viability (-9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2.47-fold increase in human and total vascular surface area, respectively. P-values were adjusted for multiple testing using the Benjamini-Hochberg method to achieve a 10% false discovery rate and adjusted P-values < 0.1 were therefore considered significant. LIMITATIONS, REASONS FOR CAUTION: Pilot study involving a limited number of experiments. WIDER IMPLICATIONS OF THE FINDINGS: Knowing that fresh medullary tissue is the best source of stromal cells is important for construction of the artificial ovary, as isolated follicles require structural support and a rich vascular network for their survival and development. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (5/4/150/5 and 7.4518.12F), Fonds Spéciaux de Recherche, Fondation Saint Luc and Foundation Against Cancer, and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any conflicting interests to declare.

Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi).

We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue.

Is transplantation of cryopreserved ovarian tissue from patients with advanced-stage breast cancer safe? A pilot study.

PURPOSE: To assess the safety of reimplantation of cryopreserved ovarian tissue from advanced-stage breast cancer patients. METHODS: Cryopreserved ovarian cortical fragments were obtained from 13 advanced-stage breast cancer patients aged 17-35 years. After thawing, part of the ovarian cortical tissue was grafted to severe combined immunodeficient mice for 6 months. The presence of malignant mammary cells in ovarian tissue was evaluated after thawing as well as after grafting by 1) histology and immunohistochemistry (epithelial membrane antigen, Her2/neu and gross cystic disease fluid protein 15 identification), and 2) detection of the MGB2 gene by qPCR. RESULTS: No malignant cells were evidenced by histology and immunohistochemistry. None of the mice died during the 6-month grafting period, nor developed macroscopically visible masses. MGB2 gene expression was detected by qPCR and confirmed by sequencing in frozen-thawed ovarian tissue in 4 cases and in grafts in 1 case. CONCLUSIONS: This pilot study is the first to evaluate the risk of contamination of cryopreserved ovarian tissue from advanced-stage breast cancer patients by xenotransplantation for 6 months to immunodeficient mice, associated with more conventional screening methods. Our xenografting results are reassuring, but caution needs to be exercised, as MGB2 gene expression was detected in some cases. Larger numbers of ovarian tissue samples from patients with advanced-stage breast cancer are required to confirm our findings before ovarian tissue transplantation can be contemplated in these patients.