Fertility potential and safety assessment of residual ovarian cortex in young women diagnosed with epithelial borderline and early-stage malignant ovarian tumors.

OBJECTIVE: To establish the safety and quality of ovarian cortex surrounding epithelial ovarian tumors in women eligible for fertility-sparing surgery by identifying occult malignant lesions and characterizing the ovarian follicle pool. METHODS: Multicentric retrospective study of 48 subjects (15-45 years), diagnosed with borderline ovarian tumors (BOTs) or early-stage epithelial ovarian cancers (EOCs) and eligible for fertility-sparing surgery. Histological samples of ovarian cortex surrounding tumors were analyzed to characterize the follicle pool, find any occult malignant lesion using tumor-specific markers (cytokeratin 7 and mucin 1), and quantify tumor-infiltrating lymphocytes (TILs) by CD3 and tumor associated macrophages (TAMs) by CD68. RESULTS: Occult ovarian lesions were observed in 6 out of 45 cases investigated (14.6%), including one mucinous stage-I BOT (1/14), one serous stage-I BOT (1/13), 3 advanced-stage serous BOTs (3/11) and one early-stage serous EOC (1/7). Notably, follicle density was significantly lower in subjects diagnosed with ovarian tumors compared to controls (p < 0.001) and at a younger age. Significantly higher follicle atresia was encountered in the ovarian tumor group then in controls (20.1 ± 8.8% vs 9.2 ± 9.4%, p < 0.001) at all ages. Both TILs and TAMs were found in ovarian tumors irrespective of histotype, but no link was established with the status of the ovarian reserve. CONCLUSIONS: Personalized counseling for fertility preservation is required in the event of BOTs and early-stage EOCs. Fertility-sparing surgery and adjuvant gamete preservation should be considered, balancing the oncological risks according to tumor stage and histotype and fertility potential, especially at a younger age.

Use of mesenchymal stem cells to enhance or restore fertility potential: a systematic review of available experimental strategies.

STUDY QUESTION: To what extent does regenerative medicine with stem cell therapy help to address infertility issues for future clinical application? SUMMARY ANSWER: Regenerative medicine using different stem cell sources is yielding promising results in terms of protecting the ovarian reserve from damage and senescence, and improving fertility potential in various preclinical settings. WHAT IS KNOWN ALREADY: Regenerative medicine using stem cell therapy is emerging as a potential strategy to address a number of issues in the field of human reproduction. Indeed, different types of adult and fetal mesenchymal stem cells (MSCs) have been tested with promising results, owing to their ability to differentiate into different tissue lineages, move toward specific injured sites (homing), and generate a secretome with wound-healing, proangiogenic, and antioxidant capacities. STUDY DESIGN SIZE DURATION: Guided by the checklist for preferred reporting items for systematic reviews and meta-analyses, we retrieved relevant studies from PubMed, Medline, and Embase databases until June 2023 using the following keywords: 'mesenchymal stem cells' AND 'ovarian follicles' OR 'ovarian tissue culture' OR 'ovarian follicle culture' OR 'cumulus oocyte complex'. Only peer-reviewed published articles written in English were included. PARTICIPANTS/MATERIALS SETTING METHODS: The primary outcome for the experimental strategies was evaluation of the ovarian reserve, with a focus on follicle survival, number, and growth. Secondary outcomes involved analyses of other parameters associated with the follicle pool, such as hormones and growth factors, ovarian tissue viability markers including oxidative stress levels, oocyte growth and maturation rates, and of course pregnancy outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Preclinical studies exploring MSCs from different animal origins and tissue sources in specific conditions were selected (n = 112), including: in vitro culture of granulosa cells, ovarian tissue and isolated ovarian follicles; ovarian tissue transplantation; and systemic or intraovarian injection after gonadotoxic or age-related follicle pool decline. Protecting the ovarian reserve from aging and gonadotoxic damage has been widely tested in vitro and in vivo using murine models and is now yielding initial data in the first ever case series of patients with premature ovarian insufficiency. Use of MSCs as feeder cells in ovarian tissue culture was found to improve follicle outcomes and oocyte competence, bringing us one step closer to future clinical application. MSCs also have proved effective at boosting revascularization in the transplantation site when grafting ovarian tissue in experimental animal models. LIMITATIONS REASONS FOR CAUTION: While preclinical results look promising in terms of protecting the ovarian reserve in different experimental models (especially those in vitro using various mammal experimental models and in vivo using murine models), there is still a lot of work to do before this approach can be considered safe and successfully implemented in a clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: All gathered data on the one hand show that regenerative medicine techniques are quickly gaining ground among innovative techniques being developed for future clinical application in the field of reproductive medicine. After proving MSC effectiveness in preclinical settings, there is still a lot of work to do before MSCs can be safely and effectively used in different clinical applications. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0077.14, FNRS-CDR J.0063.20, and grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, and the Fondation St Luc. None of the authors have any competing interest to disclose. REGISTRATION NUMBER: N/A.

Importance of oxygen tension in human ovarian tissue in vitro culture.

STUDY QUESTION: Is there any difference between 20% and 5% oxygen (O2) tension in vitro culture (IVC) on the viability and quality of human follicles contained in cultured ovarian cortex? SUMMARY ANSWER: An O2 tension of 5% yields higher follicle viability and quality than does 20% O2 tension after 6 days of IVC. WHAT IS KNOWN ALREADY: The primordial follicle (PMF) pool resides within the ovarian cortex, where the in vivo O2 tension ranges between 2% and 8%. Some studies suggest that lowering O2 tension to physiological levels may improve in vitro follicle quality rates. STUDY DESIGN, SIZE, DURATION: This prospective experimental study included frozen-thawed ovarian cortex from six adult patients (mean age: 28.5 years; age range: 26-31 years) who were undergoing laparoscopic surgery for non-ovarian diseases. Ovarian cortical fragments were cultured for 6 days at (i) 20% O2 with 5% CO2 and (ii) 5% O2 with 5% CO2. Non-cultured fragments served as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cortical fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; Ki67 staining to evaluate PMF proliferation; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine and gamma-H2AX (γH2AX) immunolabeling to detect oxidative stress damage and DNA double-strand breaks (DSBs) in oocytes and granulosa cells (GCs); and ß-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore the gene expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase 4 (GPX4) from the antioxidant defense system and cyclin-dependent kinase inhibitors (p21 and p16) as tissue senescence-related genes. MAIN RESULTS AND THE ROLE OF CHANCE: Apoptosis (P = 0.002) and follicle senescence (P < 0.001) rates were significantly lower in the 5% O2 group than in the 20% O2 group. Moreover, GCs in follicles in the 20% O2 group exhibited significantly (P < 0.001) higher oxidative stress damage rates than those in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (P = 0.001) in the 20% O2 group than in the 5% O2 group. SOD2 expression was significantly greater in the 5% O2 group compared to the 20% O2 group (P = 0.04) and the non-cultured group (P = 0.002). Expression of p21 was significantly increased in both the 20% O2 (P = 0.03) and 5% O2 (P = 0.008) groups compared to the non-cultured group. Moreover, the 20% O2 group showed significantly greater p16 expression (P = 0.04) than the non-cultured group, while no significant variation was observed between the 5% O2 and no culture groups. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study focuses on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue. The impact of O2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that 5% O2 tension culture is a promising step toward potentially solving the problem of poor follicle viability after IVC. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0064.22, CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.). The authors have nothing to disclose.

Role of apoptosis and autophagy in ovarian follicle pool decline in children and women diagnosed with benign or malignant extra-ovarian conditions.

STUDY QUESTION: Which biological mechanisms are responsible for physiological ovarian reserve decline owing to aging, or pathological follicle depletion triggered by inflammation or a pro-oxidant environment throughout a woman's lifetime? SUMMARY ANSWER: Ovarian follicle pool size is modulated by both apoptosis and autophagy, the first responsible for its physiological decline over time and increasing in the event of prior chemotherapy in children, and the latter playing a major role in physiological ovarian follicle pool diminution before puberty. WHAT IS KNOWN ALREADY: Among the different pathways of controlled cell death, apoptosis and autophagy are implicated in follicle loss. Apoptosis participates in eliminating damaged follicles, such as those impaired by chemotherapy (CHT), but its involvement in physiological age-related follicle decline is less well understood. Autophagy has proved crucial in follicle quiescence maintenance in murine models, but its contribution to human follicle pool modulation is still unclear. STUDY DESIGN, SIZE, DURATION: This retrospective study included 84 patients with benign or malignant extra-ovarian conditions aged between 1 and 35 years, with ovarian tissue stored for histological analyses at the time of cryopreservation (between 2012 and 2021) at a tertiary care center. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; cleaved caspase-3 immunostaining to identify follicle apoptosis; and microtubule-associated proteins 1A/1B light chain 3B immunolabeling to detect follicle autophagy. Transmission electron microscopy was also carried out to investigate ultrastructural features of oocytes and granulosa cells. All analyses stratified patients by age, menarchal status (premenarchal = 32; postmenarchal = 52), potentially gonadotoxic CHT before cryopreservation (n = 14), presence of endometriosis and use of hormonal treatment. MAIN RESULTS AND THE ROLE OF CHANCE: Premenarchal patients had a larger follicle pool in terms of total follicle density [mean, range 4979.98 (342.2-21789) versus 918.8 (26.18-3983), P < 0.001], but higher rates of morphologically abnormal [8.52 (0-25.37)% versus 3.54 (0-17.5)%, P < 0.001] and atretic [15.8 (0‒31.85)% versus 10.6 (0-33.33)%, P < 0.01] follicles than postmenarchal subjects. Apoptosis rates did not change with increasing age [27.94 (0-93.2)% in prepubertal subjects and 29.5 (0-100)% in postpubertal subjects], but autophagic follicles were around 10 times more common in premenarchal than postmenarchal subjects [10.21 (0-62.3)% versus 1.34 (0-25)%, P < 0.001], playing a crucial role in age-related follicle decline and elimination of 'abnormal' follicles, that are rarely seen after menarche. The impact of diagnosis and previous CHT varied according to age. In premenarchal patients with previous CHT, significantly more apoptotic [40.22 (0-100)% versus 26.79 (0-87)%, P < 0.05] and fewer abnormal [3.84 (0-10-76)% versus 9.83 (0-25.37)%, P < 0.01] follicles were detected than in subjects with no CHT prior to ovarian tissue cryopreservation, suggesting a direct effect on follicle elimination, especially of those with abnormalities. In postmenarchal subjects with previous CHT, quiescent follicle rates were lower than in patients with no CHT before tissue freezing [71.57 (0-100)% versus 85.89 (50-100)%, P < 0.05], suggesting accelerated follicle activation and growth. Moreover, increased autophagic activity was observed in the event of a cancer diagnosis compared to benign conditions after puberty [26.27 (0-100)% versus 9.48 (0-29.41)%, respectively, P < 0.05]. LIMITATIONS, REASONS FOR CAUTION: The impact of specific CHT protocols could not be investigated since the group of patients with previous CHT was highly heterogeneous. WIDER IMPLICATIONS OF THE FINDINGS: This study yields a deeper understanding of regulation of the follicle pool decline, showing for the first time that both apoptosis and autophagy pathways are involved in physiological follicle depletion, the latter being crucial before puberty. Moreover, our data showed a different response to non-physiological damage according to age, with higher apoptosis rates only in premenarchal subjects with previous CHT, confirming that this pathway is activated by drugs known to induce DNA damage in oocytes, such as alkylating agents, but not by cancer itself. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (F.R.S.-FNRS/FRIA FC29657 awarded to L.C., CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.), grants from the Fondation contre le Cancer (grant 2018-042 awarded to A.Ca.), the Fondazione Comunitaria del Varesotto and Provincia di Varese ('Amalia Griffini' Fellowship in Gynecology and Obstetrics awarded to A.Ce.), Fonds Spéciaux de Recherche, Fondation St Luc and donations from the Ferrero family. The authors have no competing interests to declare. TRIAL REGISTRAION NUMBER: N/A.

A synopsis of global frontiers in fertility preservation.

Since 2007, the Oncofertility Consortium Annual Conference has brought together a diverse network of individuals from a wide range of backgrounds and professional levels to disseminate emerging basic and clinical research findings in fertility preservation. This network also developed enduring educational materials to accelerate the pace and quality of field-wide scientific communication. Between 2007 and 2019, the Oncofertility Consortium Annual Conference was held as an in-person event in Chicago, IL. The conference attracted approximately 250 attendees each year representing 20 countries around the world. In 2020, however, the COVID-19 pandemic disrupted this paradigm and precluded an in-person meeting. Nevertheless, there remained an undeniable demand for the oncofertility community to convene. To maintain the momentum of the field, the Oncofertility Consortium hosted a day-long virtual meeting on March 5, 2021, with the theme of "Oncofertility Around the Globe" to highlight the diversity of clinical care and translational research that is ongoing around the world in this discipline. This virtual meeting was hosted using the vFairs ® conference platform and allowed over 700 people to participate, many of whom were first-time conference attendees. The agenda featured concurrent sessions from presenters in six continents which provided attendees a complete overview of the field and furthered our mission to create a global community of oncofertility practice. This paper provides a synopsis of talks delivered at this event and highlights the new advances and frontiers in the fields of oncofertility and fertility preservation around the globe from clinical practice and patient-centered efforts to translational research.

First live birth after fertility preservation using vitrification of oocytes in a woman with mosaic Turner syndrome.

PURPOSE: To report the case of a young woman diagnosed with Turner syndrome (TS) who achieved a live birth using her own oocytes that had been vitrified for fertility preservation. METHODS: A 25-year-old woman with mosaic (45,X/46,XX) TS was referred for fertility preservation (FP) counseling. Serum anti-Müllerian hormone (AMH) level was normal (6.4 µg/L). In view of the unpredictable rate of follicle loss in TS individuals, she requested FP and underwent two cycles of ovarian stimulation (OS) for oocyte cryopreservation (OoC) using a GnRH antagonist protocol and recombinant follicle stimulating hormone (rFSH), 200-250 IU daily for 8 resp. 12 days. RESULTS: In total, 29 metaphase II oocytes (MII) were vitrified after OS. After conceiving spontaneously and achieving a live birth, she returned to the clinic five years after OoC with a desire for pregnancy using in vitro fertilization (IVF) of her cryopreserved oocytes and preimplantation genetic testing (PGT-A). All 29 MII oocytes were thawed; 23 oocytes survived (79.3%) and were inseminated with partner sperm using intracytoplasmic sperm injection (ICSI). Thirteen oocytes were fertilized resulting in three good quality blastocysts which were vitrified after trophectoderm biopsy for PGT-A using array-CGH. Two blastocysts were found to be euploid. One was thawed and transferred to the uterus using a HRT priming protocol. An uneventful pregnancy occurred. The patient delivered a healthy baby girl weighing 3490 g at 40 weeks of gestation. CONCLUSIONS: We report the first live birth achieved using cryopreserved oocytes in a woman diagnosed with mosaic TS. Cryopreservation of oocytes after ovarian stimulation is a realistic option for FP in selected post menarche individuals with mosaic TS. Whether PGT-A may reduce the risk of pregnancy loss in TS has to be confirmed by further studies.

Ovarian tissue and oocyte cryopreservation prior to iatrogenic premature ovarian insufficiency.

Gonadotoxic treatments like chemotherapy or radiotherapy and ovarian surgery may result in an accelerated depletion of the ovarian reserve and subsequent premature ovarian insufficiency. Important determinants of this severe risk that require fertility preservation strategies are patient age, ovarian reserve, type of treatment, and administered dose. Oocytes and ovarian tissue can both be cryopreserved, with encouraging results in terms of pregnancy and live birth rates according to recent publications. Moreover, since ovarian tissue transplantation also results in long-term endocrine resumption, it represents a potential future therapeutic option for complete ovarian function restoration in patients with premature ovarian insufficiency.

Disease-inducing potential of two leukemic cell lines in a xenografting model.

PURPOSE: Cryopreserved ovarian tissue transplant restores ovarian function in young cancer patients after gonadotoxic treatment. However, leukemia is associated with increased risk of malignant cell transmission. We aimed to assess the tumor-inducing potential of two different leukemic cell lines when xenografted to immunodeficient mice. METHODS: Fifty-four female immunodeficient mice were grafted with either 100, 200, 500, 1000, and 10,000 chronic myeloid leukemia in blast crisis (BV-173) cells or relapsed acute lymphoblastic leukemia (RCH-ACV) cells, embedded inside a fibrin scaffold along with 50,000 human ovarian stromal cells. Two mice per cell line received the fibrin matrix without leukemic cells as negative controls. Clinical signs of disease were monitored for 20 weeks. Grafts, liver tissue, and masses were collected for macroscopic analysis and gene expression of BCR-ABL1 and E2A-PBX fusion transcripts present in BV-173 and RCH-ACV respectively. RESULTS: BV-173 cells: Mice grafted with 100, 200, or 500 cells showed no sign of disease after and were negative for BCR-ABL1 expression. Three of the 5 animals grafted with 1000 cells and all mice with 10,000 cells developed disease and showed BCR-ABL1-positive expression. RCH-ACV cells: Two out of 4 mice grafted with 100 cells developed disease and were E2A-PBX1-positive. All the animals grafted with higher cell doses showed signs of disease and all but one were E2A-PBX1-positive. CONCLUSION: The present work proves that the disease-inducing potential of BV-173 and RCH-ACV leukemic cells xenografted to SCID mouse peritoneum differs between cell lines, depending on cell number, type, status, and cytogenetic disease profile when ovarian tissue is harvested.

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.

Fibroid management in premenopausal women.

Uterine fibroids are the most common tumors affecting premenopausal women, responsible for bleeding, pain, and reduced quality of life. When symptomatic, their management mainly involves surgery, which is all too often radical (hysterectomy). While surgical options sparing the uterus (hysteroscopic and laparoscopic myomectomy) and other non-surgical approaches do indeed exist, drug-based therapies are associated with lower costs and morbidity rates. Since progesterone is required for fibroid growth, gonadotropin agonists have been used to control bleeding and decrease fibroid volume, but they only represent a temporary remedy due to adverse events. Ulipristal acetate (UPA), a selective progesterone receptor modulator, is indicated for fibroid management. It is safe, provides fast control of bleeding, and causes sustained fibroid volume reduction in the vast majority of cases (80%). Indeed, UPA-treated fibroids shrink by a combination of inhibition of cell proliferation, stimulation of cell death, and fibrosis resorption. In the case of symptom recurrence, repeated intermittent 3-month courses of daily UPA considerably maximize the impact of treatment, sometimes resulting in complete disappearance of treated fibroids. Despite the therapeutic dose of UPA being very well tolerated, patients with liver anomalies or disorders should be excluded at screening according to European Medicines Agency-Pharmacovigilance Risk Assessment Committee (PRAC) recommendations. We therefore propose new algorithms for fibroid management in premenopausal women with symptomatic fibroids, depending on their localization, the patient's wishes, and clinical response, while monitoring liver enzymes and bilirubin, as recommended by the PRAC, in order to minimize the risks of possible liver toxicity.

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.

Efficacy of ovarian tissue cryopreservation for fertility preservation: lessons learned from 545 cases.

STUDY QUESTION: How effective is ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: In our cohort of patients who underwent OTC, premature ovarian failure (POF) rates, return rates and pregnancy rates after autotransplantation were 31.5, 4.4 and 33%, respectively. WHAT IS KNOWN ALREADY: OTC for fertility purposes has been performed for >20 years now. With over 86 live births reported worldwide and success rates of ~30% after autotransplantation of frozen-thawed ovarian cortex, the procedure should no longer be considered experimental. However, very few publications report the efficacy of this procedure. STUDY DESIGN, SIZE, DURATION: Cases of ovarian tissue cryobanking for fertility preservation performed between 1997 and 2013 in a single institution were reviewed by analysis of the cryobank database and a prospective questionnaire sent out in March 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: There were 545 patients who underwent OTC during this period. The analysis included indications for OTC, survival rates, ovarian function and spontaneous pregnancies after OTC, come-back rates for ovarian tissue transplantation, pregnancy rates after transplantation, and complication and satisfaction rates. MAIN RESULTS AND THE ROLE OF CHANCE: OTC was performed in this cohort at a mean age of 22.3 ± 8.8 years for oncological indications (79%), benign gynecological pathologies (17.5%) and genetic risks of POF (3.5%). Of the 545 patients, 29% were under 18 years of age at the time of OTC and 15% were prepubertal. While 10% of patients died from their disease, 21 patients (3.9%) underwent autotransplantation, 7 of whom delivered a healthy baby, yielding a post-transplantation live birth rate of 33%. Of 451 patients who were sent the questionnaire, 143 agreed to respond (32%). Nevertheless, ovarian function could not be evaluated in 36% of those who answered. Of 92 evaluable patients, 31.5% were menopausal and 68.5% showed persistent ovarian function. Of 52 women who attempted to conceive naturally, 37 were successful (71%). Among 140 patients who answered the questionnaire, 96% were satisfied with the procedure and only 1 major complication (intra-abdominal hemorrhage) was encountered. Among all the patients, 12% have donated their ovarian cortex for research purposes or have had it destroyed. LIMITATIONS, REASONS FOR CAUTION: The questionnaire participation rate (32%), limited follow-up (mean 7.6 ± 3.5 years) and use of only clinical criteria for evaluation of ovarian function made it difficult to accurately assess the risk of POF and efficiency of OTC. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm a 30% pregnancy rate after ovarian cortex autotransplantation but also stress the difficulties of evaluating the real efficacy of OTC. STUDY FUNDING/COMPETING INTEREST(S): No funding was sought for this study and none of the authors have any conflict of interest. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registration ID: CRYOFONOV01.

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.