Overview of crosstalk between stromal and epithelial cells in the pathogenesis of adenomyosis and shared features with deep endometriotic nodules.

Since the first description of adenomyosis more than 150 years ago, multiple hypotheses have attempted to explain its pathogenesis. Indeed, research over recent years has greatly enhanced our knowledge of the underlying causes. This has opened up avenues for the development of strategies for both disease prevention and treatment of its main symptoms, such as pelvic pain, heavy menstrual bleeding, and infertility. However, the current means are still largely ineffective, so it is vital that we shed light on the pathways involved. Dysregulated mechanisms and aberrant protein expression have been identified as contributing factors in interactions between endometrial epithelial and stromal cells, ultimately leading to the growth of adenomyotic lesions. These include collective cell migration, epithelial-to-mesenchymal transition, hormonal influence, and signaling from non-coding RNAs and extracellular vesicles. We provide a concise summary of the latest insights into the crosstalk between glands and stroma in ectopic adenomyotic lesion formation. While there is an abundance of literature on similarities between adenomyosis and deep endometriosis, there are insufficient data on the cytochemical, molecular, and pathogenetic mechanisms of these two disorders. However, various shared features, including alterations of cell adhesion molecules, abnormal hormone regulation, and the presence of cancer-driving mutations and epigenetic modifications, have been identified. Nevertheless, the pathogenic mechanisms that contribute to the cause and development of these enigmatic diseases have not been fully elucidated yet.

Linzagolix therapy versus a placebo in patients with endometriosis-associated pain: a prospective, randomized, double-blind, Phase 3 study (EDELWEISS 3).

STUDY QUESTION: Does linzagolix administered orally once daily for up to 3 months at a dose of 75 mg alone or 200 mg in combination with add-back therapy (ABT) (1.0 mg estradiol; 0.5 mg norethindrone acetate, also known as norethisterone acetate [NETA]) demonstrate better efficacy than placebo in the management of endometriosis-related dysmenorrhea and non-menstrual pelvic pain? SUMMARY ANSWER: Combining 200 mg linzagolix with ABT was found to significantly reduce dysmenorrhea and non-menstrual pelvic pain at 3 months of therapy, while a daily dose of 75 mg linzagolix yielded a significant decrease only in dysmenorrhea at 3 months. WHAT IS KNOWN ALREADY?: A previously published Phase 2, dose-finding study reported that at a dose of 200 mg daily, linzagolix promotes full suppression of estradiol secretion to serum levels below 20 pg/ml and noted that the addition of ABT may be needed to manage hypoestrogenic side effects. At lower doses (75 mg and 100 mg/day), linzagolix maintains estradiol values within the target range of 20-60 pg/ml, which could be ideal to alleviate symptoms linked to endometriosis. STUDY DESIGN, SIZE, DURATION: EDELWEISS 3 was a multicenter, prospective, randomized, placebo-controlled, double-blind, double-dummy Phase 3 study to evaluate the safety and efficacy of linzagolix for the treatment of moderate-to-severe endometriosis-associated pain. Treatment was administered orally once daily for up to 6 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the EDELWEISS 3 trial, 486 subjects with moderate-to-severe endometriosis-associated pain were randomized at a 1:1:1 ratio to one of the three study groups: placebo, 75 mg linzagolix alone or 200 mg linzagolix in association with ABT. Pain was measured daily on a verbal rating scale and recorded in an electronic diary. MAIN RESULTS AND THE ROLE OF CHANCE: At 3 months, the daily 200 mg linzagolix dose with ABT met the primary efficacy objective, showing clinically meaningful and statistically significant reductions in dysmenorrhea and non-menstrual pelvic pain, with stable or decreased use of analgesics. The proportion of responders for dysmenorrhea in the 200 mg linzagolix with ABT group was 72.9% compared with 23.5% in the placebo group (P < 0.001), while the rates of responders for non-menstrual pelvic pain were 47.3% and 30.9% (P = 0.007), respectively. The 75 mg linzagolix daily dose demonstrated a clinically meaningful and statistically significant reduction in dysmenorrhea versus placebo at 3 months. The proportion of responders for dysmenorrhea in the 75 mg linzagolix group was 44.0% compared with 23.5% in the placebo group (P < 0.001). Although the 75 mg dose showed a trend toward reduction in non-menstrual pelvic pain at 3 months relative to the placebo, it was not statistically significant (P = 0.279). Significant improvements in dyschezia and overall pelvic pain were observed in both linzagolix groups when compared to placebo. Small improvements in dyspareunia scores were observed in both linzagolix groups but they were not significant. In both groups, hypoestrogenic effects were mild, with low rates of hot flushes and bone density loss of <1%. A daily dose of 200 mg linzagolix with ABT or 75 mg linzagolix alone was found to significantly reduce dysmenorrhea and non-menstrual pelvic pain also at 6 months of therapy. LIMITATIONS, REASONS FOR CAUTION: Efficacy was compared between linzagolix groups and placebo; however, it would be useful to have results from comparative studies with estro-progestogens or progestogens. It will be important to ascertain whether gonadotropin-releasing hormone antagonists have significant benefits over traditional first-line medications. WIDER IMPLICATIONS OF THE FINDINGS: Linzagolix administered orally once daily at a dose of 200 mg in combination with add-back therapy (ABT) demonstrated better efficacy and safety than placebo in the management of moderate-to-severe endometriosis-associated pain. The quality of life was improved and the risks of bone loss and vasomotor symptoms were minimized due to the ABT. The 75 mg dose alone could be suitable for chronic treatment of endometriosis-associated pain without the need for concomitant hormonal ABT, but further research is needed to confirm this. If confirmed, it would offer a viable option for women who do not want to wish to have ABT or for whom it is contraindicated. STUDY FUNDING/COMPETING INTEREST(S): Funding for the EDELWEISS 3 study was provided by ObsEva (Geneva, Switzerland). Analysis of data and manuscript writing were partially supported by ObsEva (Geneva, Switzerland), Theramex (London, UK) and Kissei (Japan) and grant 5/4/150/5 was awarded to M.-M.D. by FNRS. J.D. was a member of the scientific advisory board of ObsEva until August 2022, a member of the scientific advisory board of PregLem, and received personal fees from Gedeon Richter, ObsEva and Theramex. J.D. received consulting fees, speakers' fees, and travel support from Gedeon Richter, Obseva and Theramex, which was paid to their institution. C.B. has received fees from Theramex, Gedeon Richter, and Myovant, and travel support from Gedeon Richter-all funds went to the University of Oxford. He was a member of the data monitoring board supervising the current study, and served at an advisory board for endometriosis studies of Myovant. H.T. has received grants from Abbvie and was past president of ASRM. F.C.H. has received fees from Gedeon Richter and Theramex. O.D. received fees for lectures from Gedeon Richter and ObsEva and research grants for clinical studies from Preglem and ObsEva independent from the current study. A.H. has received grants from NIHR, UKRI, CSO, Wellbeing of Women, and Roche Diagnostics; he has received fees from Theramex. A.H.'s institution has received honoraria for consultancy from Roche Diagnostics, Gesynta, and Joii. M.P. has nothing to declare. F.P. has received fees from Theramex. S.P.R. has been a member of the scientific advisory board of Gedeon Richter and received fees from Gedeon Richter. A.P. and M.B. are employees of Theramex. E.B. was an employee of ObsEva, sponsor chair of the data monitoring board supervising the current study, and has been working as a consultant for Theramex since December 2022; she owns stock options in ObsEva. M.-M.D. has received fees and travel support from Gedeon Richter and Theramex. TRIAL REGISTRATION NUMBER: NCT03992846. TRIAL REGISTRATION DATE: 20 June 2019. DATE OF FIRST PATIENT'S ENROLLMENT: 13 June 2019.

Assessing the effect of adipose-tissue-derived stem cell conditioned medium on follicles and stromal cells in bovine ovarian tissue culture.

RESEARCH QUESTION: Does adipose-tissue-derived stem cell conditioned medium (ASC-CM) supplementation enhance follicle and stromal cell outcomes in vitro? DESIGN: Bovine ovaries (n = 8) were sectioned and cultured in vitro for 8 days in two different groups: (i) standard culture (OT Ctrl D8); and (ii) culture with ASC-CM supplementation (OT + CM D8). Half of the culture medium was replaced every other day, and stored to measure the production of oestradiol. Follicle classification was established using haematoxylin and eosin staining. Follicle and stromal cell DNA fragmentation was assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) staining served as a marker of follicle quality. Additionally, three factors, namely vascular endothelial growth factor (VEGF), interleukin 6 (IL-6) and transforming growth factor beta 1 (TGF-ß1), were evaluated in ASC-CM in order to appraise the potential underlying mechanisms of action of ASC. RESULTS: The OT + CM D8 group showed a significantly higher proportion of secondary follicles (P = 0.02) compared with the OT Ctrl D8 group. The OT + CM D8 group also demonstrated significantly lower percentages of TUNEL-positive follicles (P = 0.014) and stromal cells (P = 0.001) compared with the OT Ctrl D8 group. Furthermore, follicles in the OT + CM D8 group exhibited a significant increase (P = 0.002) in expression of GDF-9 compared with those in the OT Ctrl D8 group, and oestradiol production was significantly higher (P = 0.04) in the OT + CM D8 group. All studied factors were found to be present in ASC-CM. VEGF and IL-6 were the most widely expressed factors, while TGF-ß1 showed the lowest expression. CONCLUSIONS: Addition of ASC-CM to culture medium enhances follicle survival, development and oestradiol production, and promotes the viability of stromal cells. VEGF, IL-6 and TGF-ß1 could be paracrine mediators underlying the beneficial effects.

Impact of first chemotherapy exposure on follicle activation and survival in human cryopreserved ovarian tissue.

STUDY QUESTION: Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients? SUMMARY ANSWER: Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex. WHAT IS KNOWN ALREADY: OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status. STUDY DESIGN, SIZE, DURATION: Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining. MAIN RESULTS AND THE ROLE OF CHANCE: We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Could IVF replace reproductive surgery? No, reproductive surgery is still very much alive.

Could IVF replace reproductive surgery? The answer is no. Reproductive surgery still has a place, at least in some indications that will be explored in this contribution. While IVF can offer infertile couples the chance to have a healthy baby, it should be acknowledged that reproductive surgery can heal or harm the organs where reproduction takes place. This paper reviews different diseases and conditions with an impact on fertility, which may benefit from the technological innovations of recent decades, novel applications and the skill of reproductive surgeons. Reproductive surgery is certainly not dead. It lives on with the promise of restoring the functional anatomy to enhance the chances of pregnancy. It is our responsibility to train young residents adequately in this field to provide the right treatment at the right time.

M2 macrophages enhance endometrial cell invasiveness by promoting collective cell migration in uterine adenomyosis.

RESEARCH QUESTION: Are M2 macrophages implicated in endometrial invasiveness in adenomyosis? DESIGN: Seventeen formalin-fixed paraffin-embedded uterine samples and 16 fresh endometrial biopsies were collected from women with or without adenomyosis. Double immunofluorescence was performed to determine the predominant macrophage population in adenomyosis between M1 and M2 phenotypes. The invasion capacity of endometrial cells was assessed by invasion assays and quantitative polymerase chain reaction for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Specific mechanisms of invasion were investigated by immunohistochemistry for E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). RESULTS: Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both eutopic endometrium (P = 0.0109) and lesions (P = 0.0267) than healthy tissue. Co-culture with M2 macrophages significantly boosted invasion capacity in endometrial epithelial (P = 0.0002; P = 0.002) and stromal cells (P = 0.0469; P = 0.0047) from both adenomyosis patients and healthy controls. No gene expression differences indicating EMT were noted, either between co-cultured and control cells, or between healthy and adenomyotic cells. E- and N-cadherin protein expression did not differ significantly between endometrium from adenomyosis subjects and healthy tissue but MMP9 expression was increased in eutopic stroma from adenomyosis patients (P = 0.0492). In adenomyosis, both E-cadherin (P = 0.0379) and N-cadherin (P = 0.0196) were more extensively expressed in basal glands than functional glands. CONCLUSIONS: M2 macrophages accumulate in adenomyosis and enhance invasion capacity of adenomyotic and even healthy endometrial cells, implying that macrophage infiltration alone may be sufficient to promote the disease. This study failed to detect any changes pointing to EMT, suggesting an alternative mode of invasion. Strong E- and N-cadherin-positive intercellular junctions in basal (invasive) glands suggest the involvement of collective cell migration in the invasion process of endometrium.

Are lower levels of apoptosis and autophagy behind adenomyotic lesion survival?

RESEARCH QUESTION: How are markers of cell death, invasiveness and progesterone signalling expressed in endometrium and ectopic lesions from adenomyosis patients? DESIGN: Formalin-fixed paraffin-embedded tissue was collected from 15 control and 15 adenomyosis participants . To assess cell survival capacity, caspase 3 and microtubule-associated proteins 1A/1B light chain 3B (LC3B) were immunolabelled as markers of apoptosis and autophagy respectively. Matrix metalloproteinase 9 (MMP9) expression served as a marker of extracellular matrix degradation and invasion activity. Progesterone receptors were immunostained to detect evidence of progesterone resistance. RESULTS: Caspase 3 expression was significantly lower in the stromal (P = 0.0013) and epithelial (P = 0.0157) compartments of adenomyotic lesions than in healthy endometrial tissue. In the stroma, caspase 3 expression was significantly weaker in lesions than in corresponding eutopic endometrium (P = 0.0006). LC3B immunostaining was significantly decreased in adenomyotic stroma compared with corresponding eutopic endometrium (P = 0.0349). A significantly higher expression of MMP9 was detected in eutopic stroma from adenomyosis patients than in healthy tissue (P = 0.0295). Progesterone receptor immunostaining was found to be significantly weaker in the stroma of endometrium and ectopic lesions from adenomyosis patients than disease-free women (P = 0.0001; P = 0.0021). CONCLUSIONS: Adenomyotic lesions show lower levels of apoptosis and autophagy, suggesting that aberrant cell survival may be involved in disease pathogenesis. MMP9 appears to contribute to endometrial invasiveness in adenomyosis, as its expression is more pronounced in endometrium from these women than women without the disease. Evidence of progesterone resistance can be found in endometrium and ectopic lesions from adenomyosis patients, and may drive disease development and account for the failure of certain patients to respond to progestogens.

Summary of the ISFP congress, Brussels, 10-12 November, 2022.

The 7th International Congress of the ISFP was held in Brussels in November 2022. Hundreds of attendees from all over the world had the rare opportunity to hear the most distinguished leaders discuss and debate the latest advances in the field. Participants were also able to attend workshops under the guidance of skilled practitioners. Numerous topics were considered, including a recap on fertility preservation approaches in cancer and benign pathologies and a section on male factor infertility. Other aspects covered were in vitro maturation and poor responders, the impact of chemotherapy on the ovary, and future perspectives. Participants had the chance to listen to a symposium on fertility preservation techniques, and finally, a keynote lecture on fertility preservation in gynecological cancers brought this prominent and highly influential event to a close.

Impact of human ovarian tissue manipulation on follicles: evidence of a potential first wave of follicle activation during fertility preservation procedures.

PURPOSE: The aim of this study was to investigate the impact of processing human ovarian tissue on follicle activation dynamics. METHODS: Fresh ovarian tissue was retrieved from 9 women undergoing laparoscopic surgery for benign conditions. Biopsies from each patient were divided into 3 fragments, the first of which was immediately fixed in the operating room (T0) and the second and third just after processing at 25 (T25) and (T90) 90 min. To evaluate follicle activation, markers of the PI3K and Hippo signaling pathways were immunolabeled at each time point, targeting phospho-Akt (p-Akt) by immunohistochemistry and yes-associated protein (YAP) cellular localization in the granulosa cell layer by immunofluorescence. RESULTS: Four hundred forty primordial follicles were evaluated for p-Akt and 420 for YAP. Significantly stronger p-Akt expression was observed at T25 (23.01 ± 13.45%; p=0.04) and T90 (38.99 ± 25.21%; p<0.001) than at T0 (2.72 ± 3.35%). A significant nucleus-to-cytoplasm shift in YAP was detected at T25 (1.21 ± 0.25; p=0.015 compared to T0 (0.95 ± 0.09), while T90 (1.10 ± 0.16) values were similar to T25. CONCLUSION: Our data prove that ovarian tissue manipulation significantly impacts follicle dynamics by stimulating the PI3K and Hippo signaling pathways involved in primordial follicle activation. Further experimental evidence must nevertheless be gathered to understand and gain control of follicle activation mechanisms in non-physiological conditions (like ovarian tissue manipulation), in order to optimize fertility preservation and restoration strategies.

A synopsis of the 2021 International Society of Fertility Preservation bi-annual meeting.

On November 19, 2021, the first virtual meeting of the International Society for Fertility Preservation (ISFP) took place. Eight experts in the field of reproductive medicine presented important updates on their research in the field of fertility preservation and reproductive surgery for absolute uterine factor infertility. Presentations included talks on ovarian stem cell therapy for premature ovarian insufficiency, practical aspects of oocyte vitrification, ovarian stimulation for patients with breast cancer, in vitro maturation of oocytes at the time of ovarian tissue harvesting, male fertility preservation, and uterine transplantation. These presentations are summarized below and can be viewed in their entirety at www.isfp-fertility.org.

Ovarian tissue cryopreservation and transplantation in patients with central nervous system tumours.

STUDY QUESTION: Is there a possibility of reseeding cancer cells potentially present in frozen ovarian tissue from patients with central nervous system (CNS) tumours? SUMMARY ANSWER: Malignancy reseeding in cryopreserved ovarian tissue from 20 patients with CNS tumours was not detected by histology, immunohistochemistry (IHC), molecular biology or xenotransplantation. WHAT IS KNOWN ALREADY: Ovarian metastasis potential has been documented in patients with leukaemia, borderline ovarian tumours, advanced breast cancer and Ewing sarcoma. However, data on the safety of transplanting frozen-thawed ovarian tissue from cancer patients with CNS tumours are still lacking. STUDY DESIGN, SIZE, DURATION: This prospective experimental study was conducted in an academic gynaecology research laboratory using cryopreserved ovarian cortex from 20 patients suffering from CNS tumours. Long-term (5 months) xenografting was performed in immunodeficient mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects enrolled in the study were suffering from one of six types of CNS tumours including medulloblastoma, ependymoma, primitive neuroectodermal tumours, astrocytoma, glioblastoma and germinoma. The presence of malignant cells was investigated with disease-specific markers for each patient in cryopreserved and xenografted ovarian tissue by histology, IHC via expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP and ENO2 gene amplification. MAIN RESULTS AND THE ROLE OF CHANCE: Serial sections of cryopreserved and xenografted ovarian tissue from 20 patients showed no malignant cells by histology. All samples were negative for NSE and GFAP, although these neural markers were expressed extensively in the patients' primary tumours. Analysis by RT-ddPCR revealed no cancer cells detected in cryopreserved and xenografted ovarian fragments from subjects with astrocytoma, ependymoma, glioblastoma or medulloblastoma. Taken together, the study found no evidence of malignancy seeding in frozen-thawed and xenotransplanted ovarian tissue from patients affected by CNS cancers. LIMITATIONS, REASONS FOR CAUTION: This analysis cannot guarantee complete elimination of disseminated disease from all cryopreserved ovarian cortex, since we are unable to examine the fragments used for transplantation. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to be conducted in patients with CNS cancers undergoing ovarian tissue cryopreservation and transplantation, and clearly demonstrates no tumour seeding in their frozen-thawed and xenografted tissue. This information is vital for doctors to provide patients with meaningful and accurate advice on the possibilities and risks of ovarian tissue reimplantation. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique-the Excellence of Science (FNRS-EOS), number 30443682 awarded to M.-M.D. and T.Y.T.N., FNRS grant number 5/4/150/5 and FNRS-PDR Convention grant number T.0077.14 awarded to M.-M.D., grant 2018-042 from the Foundation Against Cancer awarded to A.C., and private donations (Ferrero, de Spoelberch). The authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

Ovarian tissue damage after grafting: systematic review of strategies to improve follicle outcomes.

Frozen-thawed human ovarian tissue endures large-scale follicle loss in the early post-grafting period, characterized by hypoxia lasting around 7 days. Tissue revascularization occurs progressively through new vessel invasion from the host and neoangiogenesis from the graft. Such reoxygenation kinetics lead to further potential damage caused by oxidative stress. The aim of the present manuscript is to provide a systematic review of proangiogenic growth factors, hormones and various antioxidants administered in the event of ovarian tissue transplantation to protect the follicle pool from depletion by boosting revascularization or decreasing oxidative stress. Although almost all investigated studies revealed an advantage in terms of revascularization and reduction in oxidative stress, far fewer demonstrated a positive impact on follicle survival. As the cascade of events driven by ischaemia after transplantation is a complex process involving numerous players, it appears that acting on specific molecular mechanisms, such as concentrations of proangiogenic growth factors, is not enough to significantly mitigate tissue damage. Strategies exploiting the activated tissue response to ischaemia for tissue healing and remodelling purposes, such as the use of antiapoptotic drugs and adult stem cells, are also discussed in the present review, since they yielded promising results in terms of follicle pool protection.

Investigation of the role of platelets in the aetiopathogenesis of adenomyosis.

RESEARCH QUESTION: Do platelets aggregate in adenomyotic lesions and participate in adenomyosis pathogenesis and related fibrosis? DESIGN: Eutopic and ectopic endometrium from 17 patients with adenomyosis and endometrium from 23 healthy controls were collected. Immunohistochemical analyses of platelet marker CD41, transforming growth factor beta 1 (TGF-ß1) and vascular endothelial growth factor (VEGF) were performed to investigate aggregation and activation of platelets in the stroma. Picrosirius staining was carried out to evaluate the extent of fibrotic tissue. RESULTS: Stroma in the control group showed higher CD41 staining levels than ectopic stroma from patients with adenomyosis (P < 0.001). In patients with adenomyosis, eutopic stroma expressed more extensive CD41 staining than ectopic stroma (P < 0.0001). Stroma in the control group exhibited higher TGF-ß1 expression than eutopic and ectopic stroma from adenomyosis patients (P = 0.009 and P < 0.0001). Stroma in the control group also expressed higher VEGF levels than ectopic stroma from patients with adenomyosis (P < 0.001). In patients with adenomyosis, eutopic stroma showed higher VEGF expression than ectopic stroma (P = 0.021). Stroma in ectopic endometrium from adenomyosis patients displayed greater Picrosirius staining compared with both eutopic stroma from adenomyosis patients and stroma in the control group (P < 0.0001). CONCLUSION: The results of this study did not detect a primary role for platelet activation or aggregation in the pathophysiological process of adenomyosis. Higher rates of collagen fibres were found in adenomyotic lesions, likely to be related to a TGF-ß1-independent pathway. Collagen fibre deposition was more extensive in adenomyotic lesions, consistent with fibrosis.

Basal lamina characterization in frozen-thawed and long-term grafted human prepubertal ovarian tissue.

RESEARCH QUESTION: Are there differences in the composition and structure of the basal lamina surrounding follicles in prepubertal versus adult human ovarian tissue? DESIGN: Frozen-thawed human ovarian tissue from six prepubertal and seven adult patients was divided into three fragments in each case: two for non-grafted tissue evaluation and one for long-term xenografting to mice. Collagen IV and laminin expression were investigated by immunohistochemistry before and after grafting. The basal lamina was analysed by transmission electron microscopy on frozen-thawed tissue. RESULTS: In frozen-thawed tissue, collagen IV was significantly less expressed around prepubertal follicles than around adult follicles (primordial, P = 0.02; intermediate/growing follicles, P = 0.03), while laminin was significantly more expressed (primordial, P = 0.03; intermediate, P = 0.01). Collagen IV expression was significantly higher around prepubertal primordial follicles in grafted tissue than in non-grafted tissue, reaching similar levels to those in adult tissue. Ultrastructure analysis showed the basal lamina around follicles in prepubertal frozen-thawed tissue to be rather patchy and thinner than around adult follicles (primordial/intermediate, P = 0.001; primary, P = 0.02). CONCLUSIONS: In frozen-thawed tissue, the basal lamina around prepubertal follicles is less mature than around adult follicles, but it becomes similar in both prepubertal and adult subjects after grafting. Grafting could therefore induce maturation of the basal lamina around prepubertal follicles.

Ovarian tissue transportation: a systematic review.

In recent years, some countries and fertility preservation networks have started adopting 24 h transportation for ovarian tissue, a practice that has the potential to spread very quickly due to the high costs and bureaucracy involved in the establishment of ovarian tissue cryobanks. While pregnancies and live births have been reported after such long periods of transportation, this, however, remains an empirical procedure. This review aims to prompt reflection on ovarian tissue transport, highlighting the lack of knowledge in humans by providing a counterpoint looking into more than 40 studies published in different animal models. By discussing these studies in animals, the findings of various models can be deciphered, and light shed on the patterns identified. Like the development of different assisted reproductive technology procedures, this is an important step in creating guidelines for future studies on human ovarian tissue transportation.

A Draft Map of the Human Ovarian Proteome for Tissue Engineering and Clinical Applications.

Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro, and in vivo,. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, Western blotting, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro, culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general.

Mitochondrial content, activity, and morphology in prepubertal and adult human ovaries.

PURPOSE: To investigate whether mitochondrial content, activity, and morphology differ in prepubertal versus adult ovarian follicles. METHODS: Ovarian tissue was collected from 7 prepubertal girls (age 1-10 years) and 6 adult women (age 20-35 years). Primordial and primary follicles were isolated from frozen-thawed prepubertal and adult ovarian tissue and their viability was assessed. Mitochondrial content was investigated by TOMM20 immunostaining of prepubertal and adult ovarian tissue, while mitochondrial activity in isolated follicles was analyzed by MitoTracker CM-H2XRos and JC-1. Frozen-thawed ovarian tissue from the same patients was also evaluated by transmission electron microscopy to examine mitochondrial morphology. RESULTS: Higher TOMM20 staining was detected in prepubertal follicles compared to their adult counterparts, indicating the presence of more mitochondria in prepubertal follicles. Analysis of mitochondrial activity by MitoTracker showed higher fluorescence intensity in prepubertal follicles, suggesting that follicles in this group are more active than adult follicles. JC-1 analysis did not reveal any statistically significant difference in the inactive/active ratio between the two groups. Moreover, ultrastructural analysis by TEM detected morphological differences in the shape and cristae of prepubertal mitochondria, probably suggesting a mechanism of response to autophagy. CONCLUSION: Differences in the number, activity, and morphology of mitochondria were reported, suggesting that consequential modifications might occur during puberty, which could be the window of opportunity required by mitochondria to undergo changes needed to reach maturity, and hence the capacity for ovulation and fertilization.

New insights into the GDF9-Hedgehog-GLI signaling pathway in human ovaries: from fetus to postmenopause.

RESEARCH QUESTION: Are glioma-associated oncogene homolog 1, 2, and 3 (GLI1, 2, and 3) and protein patched homolog 1 (PTCH1) specific markers for precursor theca cells in human ovaries as in mouse ovaries? DESIGN: To study the GDF9-HH-GLI pathway and assess whether GLI1 and 3 and PTCH1 are specific markers for precursor theca cells in the human ovary, growth differentiation factor 9 (GDF9), Indian Hedgehog (IHH), Desert Hedgehog (DHH), Sonic Hedgehog (SHH), PTCH1 and GLI1, 2 and 3 were investigated in fetal (n=9), prepubertal (n=9), reproductive-age (n=15), and postmenopausal (n=8) human ovarian tissue. Immunohistochemistry against GDF9, IHH, DHH, SHH, PTCH1, GLI1, GLI2, and GLI3 was performed on human ovarian tissue sections fixed in 4% formaldehyde and embedded in paraffin. Western blotting was carried out on extracted proteins from the same samples used in the previous step to prove the antibodies' specificity. The quantitative real-time polymerase chain reaction was performed to identify mRNA levels for Gdf9, Ihh, Gli1, Gli2, and Gli3 in menopausal ovaries. RESULTS: Our results showed that, in contrast to mice, all studied proteins were expressed in primordial follicles of fetal, prepubertal, and reproductive-age human ovaries and stromal cells of reproductive-age and postmenopausal ovaries. Intriguingly, Gdf9, Ihh, and Gli3 mRNA, but not Gli1 and 2, was detected in postmenopausal ovaries. Moreover, GLI1, GLI3, and PTCH1 are not limited to a specific population of cells. They were spread throughout the organ, which means they are not specific markers for precursor theca cells in human ovaries. CONCLUSION: These results could provide a basis for understanding how this pathway modulates follicle development and ovarian cell steroidogenesis in human ovaries.

Adipose tissue-derived stem cells protect the primordial follicle pool from both direct follicle death and abnormal activation after ovarian tissue transplantation.

PURPOSE: To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. CONCLUSION: We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.

GnRH Antagonists with or without Add-Back Therapy: A New Alternative in the Management of Endometriosis?

To evaluate the effectiveness of a new class of medical drugs, namely oral gonadotropin-releasing hormone (GnRH) antagonists, in the management of premenopausal women with endometriosis-associated pelvic pain. We reviewed the most relevant papers (n = 27) on the efficacy of new medical alternatives (oral GnRH antagonists) as therapy for endometriosis. We first briefly summarized the concept of progesterone resistance and established that oral contraceptives and progestogens work well in two-thirds of women suffering from endometriosis. Since clinical evidence shows that estrogens play a critical role in the pathogenesis of the disease, lowering their levels with oral GnRH antagonists may well prove effective, especially in women who fail to respond to progestogens. There is a need for reliable long-term oral treatment capable of managing endometriosis symptoms, taking into consideration both the main symptoms and phenotype of the disease. Published studies reviewed and discussed here confirm the efficacy of GnRH antagonists. There is a place for GnRH antagonists in the management of symptomatic endometriosis. Novel algorithms that take into account the different phenotypes are proposed.