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Jacques Donnez,1,2 Luciana Cacciottola,3 Jean-Luc Squifflet,4 Marie-Madeleine Dolmans3,4 1Department of Gynaecology, Université Catholique de Louvain, Brussels, Belgium; 2Société de Recherche pour l’Infertilité (SRI), Brussels, Belgium; 3Gynecology Research Laboratory, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium; 4Gynecology Department, Cliniques Universitaires St-Luc, Brussels, BelgiumCorrespondence: Marie-Madeleine Dolmans, Gynecology Research Unit, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, Brussels, 1200, Belgium, Tel +32 02 764 5237, Fax +32 02 764 9507, Email marie-madeleine.dolmans@uclouvain.beAbstract: Estrogens play a critical role in the pathogenesis of endometriosis and it is logical to assume that lowering estradiol levels with oral gonadotropin-releasing hormone (GnRH) antagonists may prove effective, especially in women who fail to respond to progestogens. Indeed, due to progesterone resistance, oral contraceptives and progestogens work well in two-thirds of women suffering from endometriosis, but are ineffective in 33% of women. Oral GnRH antagonists have therefore been evaluated for management of premenopausal women with endometriosis-associated pelvic pain. The first publication on these drugs reported the efficacy of elagolix. The present paper is a narrative review of linzagolix, which is an orally administered GnRH receptor antagonist with low pharmacokinetic/pharmacodynamic variability. It binds to and blocks the GnRH receptor in the pituitary gland, resulting in a dose-dependent drop in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) production. This reduction in LH and FSH levels in turn leads to a dose-dependent decline in estrogen. Phase 2 and 3 trials are reviewed and discussed here. There is a place for GnRH antagonists in the management of symptomatic endometriosis, and linzagolix with or without add-back therapy (ABT) is one option that can be used at low doses, avoiding the need for ABT, which is contraindicated in some patients.Keywords: endometriosis, pelvic pain, dysmenorrhea, progesterone resistance, GnRH antagonist

Objective: To create a novel, more advanced in vitro model of human endometrium, using so-called assembloids, looking to explore endometrial receptivity in adenomyosis., Design: Evaluation of assembloid responsiveness to hormonal stimulation by immunohistochemistry, ELISA and scanning electron microscopy (SEM)., Setting: University-based research unit in gynecology., Subjects: Twelve women, 6 of whom were affected by adenomyosis., Intervention: Organoids (in the form of glandular fragments) and stromal fibroblasts were collected from endometrial biopsies. The two populations were combined inside an extracellular matrix to create 3D assembloids, which were then exposed to hormonal stimulation (β-estradiol for 48 hours, followed by β-estradiol/progesterone/cyclic adenosine monophosphate for 72 hours) to mimic the window of implantation., Main Outcome Measures: Glycodelin, leukemia inhibitory factor (LIF) and homeobox A10 (HOXA10) expression, prolactin secretion and pinopode development., Results: Endometrial organoids and stromal cells were successfully isolated from women with and without adenomyosis and combined to generate the assembloid model. Upon stimulation, assembloids from both groups acquired a more secretory phase-like phenotype, as demonstrated by histology, and were shown to be positive for glycodelin, LIF and HOXA10 by immunohistochemistry. Adenomyotic assembloids expressed significantly lower levels of LIF and HOXA10 within the stromal compartment after stimulation than did healthy assembloids in the same condition. ELISA revealed prolactin secretion in vitro, showing an upward trend in hormonally treated assembloids from both healthy and affected women. By SEM, fully formed pinopodes were discerned on the epithelial surface of healthy assembloids after stimulation, but they were absent in case of adenomyosis., Conclusion: Primary assembloids can be generated from endometrial biopsies from both healthy subjects and women affected by adenomyosis. These assembloids are amenable to hormonal stimulation and mimic secretory phase-specific characteristics of endometrial tissue in vivo, including glycodelin, LIF and HOXA10 expression, and pinopode formation. Assembloids from adenomyosis appear to be less sensitive to hormonal treatment, showing reduced expression of LIF and HOXA10 in the stromal compartment and failing to form pinopodes. All in all, endometrial assembloids may serve as an advanced preclinical model of adenomyosis-related impaired endometrial receptivity, opening up new horizons in understanding and treating the condition., (Copyright © 2024. Published by Elsevier Inc.)

Background/Objectives : While it is known that adenomyosis is associated with poor reproductive outcomes, the underlying mechanisms are unclear, and to date, there is no standard treatment protocol for these patients. Endometrium from adenomyosis patients is characterized by several abnormalities, potentially resulting in impaired receptivity and subsequent implantation failure. Methods : Endometrial biopsies were collected from 26 women with adenomyosis and 26 control subjects. Immunohistochemistry was performed to evaluate the expression of markers of endometrial receptivity, namely the progesterone receptor (PR), glycodelin, leukemia inhibitory factor (LIF), homeobox A10 (HOXA10), integrin beta chain beta 3 (integrin β3) and osteopontin. Scanning electron microscopy was used to observe pinopodes on the surface of mid-secretory endometrial epithelium. Results : PR, LIF and osteopontin expression were all found to be weaker in secretory-phase stroma from adenomyosis patients than in healthy controls. HOXA10 expression was decreased in adenomyosis during the secretory phase, and also the proliferative phase, where it reached statistical significance in both epithelial and stromal compartments. Glycodelin and integrin β3 levels did not differ between diseased and healthy tissues in any of the cycle phases. Pinopodes were fewer and at later developmental stages in adenomyosis compared to those on the surface of healthy endometrium from the same time period of the menstrual cycle. Conclusions : Endometrium from adenomyosis patients is characterized by abnormal expression of various receptivity markers. The stromal compartment appears to be affected most, showing reduced expression of PR, LIF and osteopontin in the secretory phase and lower levels of HOXA10 during both proliferative and secretory phases. Decreased receptivity due to impaired stromal decidualization may contribute to poor reproductive outcomes in adenomyosis patients.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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-β1), 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-β1 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-β1 could be paracrine mediators underlying the beneficial effects., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Fibroids are a common pathology and increasingly observed in women seeking medical treatment for infertility. The longer reproductive horizon because of improvements in medical care and current trend for women to postpone childbearing are making fibroid-related infertility increasingly common. This review aimed to critically analyze the association between uterine fibroids and infertility, mechanisms by which uterine fibroids may impair fertility, and management of myoma-related infertility. The association of fibroids with infertility is a source of controversy. As the focus of this review is infertility, it is crucial to analyze the mechanisms by which fertility may be impaired by the presence of fibroids. Current management strategies involve mainly surgical interventions, including myomectomy by hysteroscopy, laparotomy, or laparoscopy, and nonsurgical approaches, such as uterine artery embolization and focused ultrasound performed under radiologic or echographic guidance. The risks and benefits of each option should be discussed with patients, and several factors need to be considered, including the skills of surgeons and availability of different resources in various centers. Concerning the efficacy of oral gonadotropin-releasing hormone antagonists (i.e., elagolix, relugolix, and linzagolix), they were shown to have a rapid impact on heavy menstrual bleeding (HMB) in >70% of women. When used without add-back therapy, these drugs cause a significant reduction in fibroid volume, namely, approximately 50% from baseline to week 24. Further studies are required to determine the best protocol and optimal dosage if a reduction in myoma volume is the main goal, as in case of myoma-related infertility., Competing Interests: Declaration of Interests J.D. is a member of the Scientific Advisory Board of ObsEva, Theramex, and Gedeon Richter, outside the submitted work. H.S.T. reports funding from AbbVie and honoraria from Netscape and was president of the American Society for Reproductive Medicine, outside the submitted work. L.M. has nothing to disclose. M.-M.D. reports honoraria from Theramex and Gedeon Richter, outside the submitted work., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

A better understanding of uterine fibroid-related pathogenesis and symptoms like uterine bleeding and infertility is mandatory., Competing Interests: Declaration of Interests M.M.D. has nothing to disclose. J.D. has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of Interests M.Z. reports funding from the Fonds National de la Recherche Scientifique de Belgique (FC 52391) outside the submitted work. D.-Y.L. has nothing to disclose. C.A.S. has nothing to disclose. L.C. has nothing to disclose. M.-M.D. reports funding from the Fonds National de la Recherche Scientifique de Belgique–FSR (5/4/150/5) outside the submitted work.

Fibroids are benign uterine tumors characterized by the proliferation of uterine smooth muscle cells, embedded in an abundant extracellular matrix. Their prevalence is estimated to be >50% in women aged >45 years. Fibroids represent a considerable health burden. It is time to acquire a deeper mechanistic understanding of uterine fibroid-related etiology and pathogenesis, which may help pinpoint new strategies and an individualized approach. There is a need to gather prospective data and conduct studies to compare alternative approaches and assess long-term outcomes with respect to quality of life, recurrence of symptoms (bleeding and bulk symptoms), fertility, and even complications The goal of this review was to evaluate the widely accepted pathogenesis and identify risks factors and future directions for clinical and basic research into fibroids., Competing Interests: Declaration of Interests M.-M.D. has nothing to disclose. F.P. has nothing to disclose. W.H.C. has nothing to disclose. J.D. has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Abnormal uterine bleeding (AUB) is a bleeding from the uterine corpus that is abnormal in regularity, volume, frequency or duration. It encompasses heavy menstrual bleeding, irregular menstrual bleeding and intermenstrual bleeding, which are common symptoms among women of reproductive age, impacting their overall well-being. Menstruation involves interactions between endometrial epithelial and stromal cells, immune cell influx, and changes in endometrial vasculature. These events resemble an inflammatory response with increased vessel permeability, tissue breakdown, and the arrival of innate immune cells. However, the mechanisms of menstrual cessation are poorly understood. AUB can be related to structural causes (polyp, adenomyosis, leiomyoma, malignancy/hyperplasia) and nonstructural conditions (coagulopathy, ovulatory dysfunction, endometrial, iatrogenic). While transvaginal ultrasound is the primary method for the screening of intracavitary lesions, saline infusion sonohysterography is more accurate to detect endometrial polyps and submucous leiomyomas, while hysteroscopy with biopsy remains the reference method for a definitive diagnosis. The main goals in managing AUB are addressing and correcting the underlying primary cause, if possible, and establishing a regular bleeding pattern or amenorrhea, which can be done with antifibrinolytic agents, progestins, gonadotropin-releasing hormone agonists and antagonists, or surgical interventions, each one with specific indications and limitations. Further research is necessary to assess the effectiveness and the long-term effects of various medical and surgical treatments. Meanwhile, the availability of diagnostic methods such as transvaginal ultrasound and hysteroscopy and the universal distribution of medical treatments for AUB should be prioritized by policymakers to minimize the diagnostic and treatment delay and thus reduce the risk of AUB-related anemia and the need of hysterectomy.

Competing Interests: J.D. was a member of the scientific advisory board of ObsEva until August 2022 and received fees from Gedeon Richter, ObsEva, and Theramex. M.-M.D. has received fees from Gedeon Richter and Theramex.

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., Competing Interests: Declaration of competing interest The authors have nothing to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ovarian tissue cryopreservation (OTC) and subsequent transplantation (OTT) is a fertility preservation technique widely offered to prepubertal girls and young fertile women who need to undergo oncological treatment but are at a high risk of infertility. However, OTT is not considered safe in patients with certain diseases like leukemia, Burkitt's lymphoma, and ovarian cancer because of the associated risk of malignant cell reintroduction. In vitro follicle development has therefore emerged as a promising means of obtaining mature metaphase II (MII) oocytes from the primordial follicle (PMF) pool contained within cryopreserved ovarian tissue, without the need for transplantation. Despite its significant potential, this novel approach remains highly challenging, as it requires replication of the intricate process of intraovarian folliculogenesis. Recent advances in multi-step in vitro culture (IVC) systems, tailored to the specific needs of each follicle stage, have demonstrated the feasibility of generating mature oocytes (MII) from early-stage human follicles. While significant progress has been made, there is still room for improvement in terms of efficiency and productivity, and a long way to go before this IVC approach can be implemented in a clinical setting. This comprehensive review outlines the most significant improvements in recent years, current limitations, and future optimization strategies.

Deep endometriosis and uterine adenomyosis are two frequently encountered conditions affecting approximately 200 million women worldwide. They are closely related, showing similar histological patterns and multiple common pathogenic features, and share the same symptoms. It is therefore not surprising that they are often thought to have a common developmental origin. Indeed, both deep endometriosis and adenomyosis appear to derive from estrogen-dependent overproliferation of endometrial tissue and its subsequent implantation in ectopic sites. Although the scientific community has shown increasing interest in these diseases over recent years, neither pathogenesis has yet been elucidated, so there are currently no efficient treatment options. Understanding the mechanisms underlying disease development, as well as discerning their relationship, are key to improving clinical management for millions of patients. The aims of this review are to summarize current knowledge on deep endometriosis and adenomyosis pathogeneses and discuss the possibility that these two entities are actually differential phenotypes of the same disease., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: None of the authors have any competing interest to disclose., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

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., (Copyright © 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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 β-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., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To study the impact of aneuploid granulosa and stromal cells on folliculogenesis of small ovarian follicles from patients with mosaic Turner syndrome (TS) using a murine xenograft model., Design: Laboratory study., Setting: University hospital., Patient(s): Ovarian cortical tissue was obtained by laparoscopic surgery from 18 patients with mosaic TS (aged 5-19 years) and 13 controls (aged 5-18 years)., Intervention(s): Part of each tissue fragment was used to karyotype ovarian cells in nongrafted tissue by fluorescence in situ hybridization. The remaining tissue was xenografted to severe combined immunodeficient mice for 5 months. Grafted tissue was analyzed for aneuploidy, and follicle density and morphology were determined. Expressions of proliferating cell nuclear antigen and anti-Müllerian hormone were investigated by immunohistochemistry., Main Outcome Measure(s): The impact of aneuploid granulosa and stromal cells on folliculogenesis. Fluorescence in situ hybridization of ovarian tissue before grafting was performed to determine the level of aneuploidy in stromal cells and oocytes and granulosa of small follicles. After xenografting, the level of aneuploidy of the newly formed layers of granulosa cells was again determined in secondary and antral follicles., Result(s): Follicle density in ovarian tissue from patients with TS was significantly lower than in controls before grafting. Fluorescence in situ hybridization analysis confirmed that 101 of 104 oocytes from nongrafted tissue of patients with TS showed normal X chromosome content, whereas granulosa and stromal cells were mainly 45,X. Fragments from 12 patients with TS contained follicles at all stages after xenografting, including secondary and antral follicles. Follicle density in patients with TS and controls decreased significantly after grafting. Moreover, a shift from high to low proportions of 45,X granulosa cells was observed during folliculogenesis. Expression of proliferating cell nuclear antigen in follicles from patients with TS increased significantly during grafting. Secretion of anti-Müllerian hormone was impaired before grafting in peripubertal/postpubertal girls with TS, but recovered after grafting., Conclusion(s): Our study showed that small follicles from patients with mosaic TS undergoes folliculogenesis, despite the presence of aneuploid granulosa and stromal cells. Ovarian tissue cryopreservation could therefore be a valid option to preserve fertility in young patients with mosaic TS if sufficient numbers of follicles are present, thus preferably before the age of 12., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining natural immunodeficiency up to day 17 postfertilization, making it ideal to study short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

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., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Hydatidiform Mole (HM) is the most common form of gestational trophoblastic disease. Dilatation and curettage is the classical treatment of this affection. Hysteroscopic resection (HsR) is an alternative for the treatment of intra-uterine pathology. Objective: To describe the feasibility of HsR for the management of HM. Result: Case series of patients who had a complete or partial HM confirmed by histological examination of the trophoblastic tissue resected by operative hysteroscopy between 2007 and 2019. After approval of our ethics committee, we evaluated 36 patients who underwent hysteroscopic resection for molar pregnancy. Histological analysis showed partial HM in 28 patients (77.8%) and complete HM in 8 (22.2%). Main surgical complications were uterine perforation in one patient and glycine resorption in 10 patients with two cases of hyponatremia corrected by standard treatment. We performed an ultrasound control 1 month after the intervention in 19 patients (52.8%) as they had slow decrease of HCG or bleeding complaints and found retained product of conception (RPOC) in six patients (16.7%). Conclusion: This first report on a small number of patients demonstrate that hysteroscopic resection is a feasible procedure for the management of molar pregnancy. Direct visualization of the procedure helps the surgeon to control the resection. Further studies are mandatory to compare this technique with D&C in term of RPOC and fertility outcomes as it remains the standard treatment., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2023.)

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., (Copyright © 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To study the effect of freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) on follicle outcomes in human ovarian tissue., Design: An experimental study., Setting: University-based research laboratory., Patients: Fresh and cryopreserved ovarian tissue from 10 patients was donated to research with their consent and institutional review board approval., Interventions: Fresh and frozen-thawed ovarian cortical pieces were in vitro-cultured and compared (fresh-IVC vs FT-IVC). The FT-IVC fragments were then examined against fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4×2×1 mm 3 ) were analyzed on days 0, 1, and 6., Main Outcome Measures: Follicle analyses included histology (count and classification) and immunohistochemistry (Ki67 [proliferation], caspase-3 [apoptosis], 1A and 1B light chain 3B [autophagy], p-Akt, FOXO1, and p-rpS6 [PI3K activation]). Droplet digital polymerase chain reaction further explored expression of PI3K pathway- and oocyte-related genes in tissue sections., Results: No major differences were detected between fresh-IVC and FT-IVC tissues in any conducted analyses. Although a significant drop was observed in primordial follicle (PF) proportions in the fresh-IVC and FT-IVC groups (d0 vs. d6, P<.002), they held steady in the FT-CAM group (d0 vs. d6, P>.05). The PF rates were also significantly higher in the FT-CAM group than the FT-IVC group on d6 (P=.02). Importantly, avian erythrocytes were already present in 30% of implants from d1. Apoptotic and autophagic follicle rates increased during IVC (P<.008), but remained significantly lower in the FT-CAM group (P<.01), confirming superior follicle preservation in CAM-grafted tissue. Upregulation of the PI3K/FOXO pathway was established in the IVC groups, demonstrating PF activation, whereas significant pathway downregulation was detected in the FT-CAM group (P<.03). The droplet digital polymerase chain reaction tests confirmed oocyte growth during IVC and follicle autophagy in all groups; however, the PI3K pathway appeared to be differentially modulated in tissues and follicles., Conclusions: In vitro culture induces PF depletion with no additional impact of freezing. Grafting to CAM preserves the PF pool by curbing follicle activation, apoptosis, and autophagy, probably thanks to rapid graft revascularization and/or the circulating embryonic antimüllerian hormone. These findings highlight the importance of enhancing neoangiogenesis in ovarian grafts and investigating the potential benefits of administering antimüllerian hormone to prevent PF burnout., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Endometriosis is a chronic inflammatory disease causing distressing symptoms and requiring a life-long management strategy. The objective of this review is to evaluate endometriosis-related pathways and identify novel therapies to treat it. We focused on the crucial role of inflammation and inflammatory molecules in order to define new perspectives for non-hormonal treatment of the disease by targeting inflammation, nuclear factor kappa B and cytokines, or reactive oxygen species, apoptotic and autophagic pathways, regulators of epithelial-mesenchymal transition, and angiogenesis and neuroangiogenesis. Novel non-steroidal therapies targeting these pathways for endometriosis were explored, but multiple challenges remain. While numerous agents have been investigated in preclinical trials, few have reached the clinical testing stage because of use of inappropriate animal models, with no proper study design or reporting of preclinical strategies. Targeting estrogens is still the best way to control endometriosis progression and inflammation.

Abnormal uterine bleeding (AUB) is a clinical entity which can lead to iron deficiency anemia. Classification according to the acronym PALM-COEIN (polyp, adenomyosis, leiomyoma, malignancy, and hyperplasia; coagulopathy, ovulatory dysfunction, endometrial, iatrogenic, and not otherwise classified) provides a structured approach to establish the cause of AUB. The goal of this review is to discuss the different mechanisms and the relationship between uterine disorders and AUB. Heavy menstrual bleeding, a subgroup of AUB, is more closely related to the presence of uterine fibroids. The relationship between heavy menstrual bleeding and uterine fibroids remains poorly characterized, particularly the understanding of endometrial function in women with structural myometrial features such as leiomyomas. A number of theories have been proposed in the literature and are discussed in this review. Uterine adenomyosis is also a frequent cause of AUB, and its pathogenesis is still far from being fully elucidated. The mechanisms contributing to its development are multifactorial. Many theories lean toward invasion of the myometrium by endometrial cells. Both clinical and basic studies favor the theory of direct invasion, although de novo development of adenomyosis from Müllerian rests or stem cells has not been ruled out. Development of adenomyotic lesions involves repeated tissue injury and repair. In addition, this review describes the other causes of AUB such as endometrial polyps, cesarean scar defects, and uterine vascular abnormalities. Endometrial polyps are often asymptomatic, but approximately 68% of women have concomitant AUB. Histologic alterations in the lower uterine segment in patients who had undergone cesarean sections were identified and may explain the cause of AUB., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Iron deficiency (ID) and iron deficiency anemia (IDA) are highly prevalent among women across their life span, especially during the reproductive age. An iron-deficient state has been associated with a number of adverse health consequences, affecting all aspects of the physical and emotional well-being of women. Heavy menstrual bleeding, pregnancy state, and the postpartum period are the major causes of ID/IDA. However, despite the high prevalence and the impact on quality of life, ID/IDA among fertile age women remains underdiagnosed and undertreated. The present Views and Reviews provides an overview on IDA in women's health, describing the background on iron metabolism, heavy menstrual bleeding pathogenetic mechanisms, including a focus on uterine disorders, such as uterine fibroids and adenomyosis. Iron replacement therapies, patient blood management, and treatment options for uterine disorders also are explored., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We report successful clinical outcomes after transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary in a patient diagnosed with stage IIIb rectal adenocarcinoma. Whole ovary cryopreservation was proposed as a fertility preservation strategy in 2006 prior to radiotherapy, chemotherapy and oncological surgery. To allow for minimal residual disease screening before ovarian reimplantation, the whole ovary was thawed and dissected into cortical strips. While awaiting the results, the majority of the cortical strips were refrozen. These refrozen-rethawed cortical strips were laparoscopically grafted to 2 sites: the previously irradiated pelvic cavity and the non-irradiated extrapelvic cavity. Ovarian function resumption was assessed by recovery of menses, hormone levels, ultrasound and oocyte pick-up following controlled ovarian stimulation (COS). Restoration of ovarian function occurred 6 months after reimplantation, with recovery of menses and estradiol secretion. A total of 12 cycles were followed by the IVF department. A second reimplantation was performed 1.5 years later, since the grafts were found to have stopped functioning for >3 consecutive months. Overall, 3 fertilizable oocytes were retrieved transabdominally from the extrapelvic graft following COS, yielding 2 embryos and culminating in one fresh embryo transfer, but no pregnancy. Concerning the reimplantation site, no ovarian activity was detected in the graft placed in the previously irradiated pelvic cavity. Indeed, only fibrotic-looking tissue was observed in the pelvic site at second laparoscopy 1.5 years later, while ovarian activity was noted in the extrapelvic graft, showing a large antral follicle. All in all, transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary can lead to ovarian function resumption and embryo development if grafted to a non-irradiated field.

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)