Immune system regulation of physiological and pathological aspects of the ovarian follicle pool throughout the female reproductive lifespan.

The immune system plays a major role in ovarian physiology by regulating the ovarian follicle pool through complex signaling of different growth factors, cytokines, and chemokines. These may promote follicle activation and further growth but could also trigger follicle atresia and clearance of aging or damaged cells within the ovarian cortex. Moreover, extraglandular steroidogenesis potentially occurring in different immune cells like macrophages and natural killer cells might be another way of modulating follicle growth. Ovarian macrophages have recently been found to contain two different populations, namely resident macrophages and monocyte-derived cells, with potentially different roles. The immune system also plays a role in the development of pathological conditions, including premature ovarian insufficiency (POI). Indeed, autoimmune activation against various ovarian antigen targets results in lymphocytic oophoritis mainly targeting early growing follicles, but later leading to complete follicle pool depletion. Immune-mediated ovarian damage may also be caused by viral infection or be the consequence of iatrogenic damage. Certain novel cancer immunotherapies like checkpoint inhibitors have recently been shown to induce ovarian reserve damage in a murine model. Studies are needed to corroborate these findings and further investigate the potential of newly developed immunotherapies to treat POI. Technological advances such as single-cell analyses of less represented cell populations like immune cells inside the ovary are now contributing to valuable new information, which will hopefully lead to the development of new therapeutic strategies for women with fertility issues

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

To what extent does regenerative medicine with stem cell therapy help to address infertility issues for future clinical application? 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. 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. 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. 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. Preclinical studies exploring MSCs from different animal origins and tissue sources in specific conditions were selected (n = 112), including: 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 and 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. While preclinical results look promising in terms of protecting the ovarian reserve in different experimental models (especially those using various mammal experimental models and 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. 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. 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. N/A

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

P-483 Improving <i>in vitro</i> culture of isolated human ovarian primordial-primary follicles by adding adipose derived stem cells

Abstract Study question Could adipose derived stem cells (ADSc) secrete survival- and growth-promoting factors able to improve in vitro culture (IVC) of human isolated follicles? Summary answer Co-encapsulation of isolated human primordial-primary follicles with ADSc enhances follicular survival, growth and maturation after 7 days of IVC. What is known already The ability to obtain in vitro mature oocyte from earliest follicular stage may help to preserve fertility in female cancer patients. Unfortunately, human primordial follicles show low rates of growth in vitro. The only study that obtained mature human oocytes from primordial follicle used an IVC multi-step method with a success rate lower than 1% and abnormal polar body. Recently, several studies have shown that, in mice, ADSc promote survival, antrum formation and oocyte maturation in vitro by secreting factors important for follicular development. To our knowledge, no IVC studies on human ovarian follicles have been attempted using ADSc. Study design, size, duration Fragments of frozen-thawed human ovarian tissue (n = 5) have been mechanically and enzymatically digested to isolate primordial-primary follicles. Eight-to-ten isolated unilaminar follicles have been embedded in alginate-matrigel, half with 20.000 ADSc and half alone. Alginate beads were then cultured in vitro for 7 days. At day (D) 7, we compared isolated follicles co-cultured with ADSc to isolated follicles cultured alone. Participants/materials, setting, methods Every two days, IVC medium has been collected to perform hormonal (estradiol, progesteron, anti-Müllerian hormone, androgen, dehydroepiandrosterone) and growth factors measurement (VEGF, bFGF, IGF, HGF) by enzyme-linked immunosorbent assay (ELISA) to study follicle and ADSc interactions. Follicle diameter have been measured at D0 and at D7 of IVC. At D0 and at the end of the IVC (D7), follicle viability (live dead assay) and ultrastructure (transmission electron microscopy [TEM]) have been investigated. Main results and the role of chance A total of 373 unilaminar follicles were obtained after enzymatic isolation. At the end of IVC the percentage of viable follicles (with &amp;lt;10% of dead granulosa cells) was found to be significantly higher in follicles co-cultured for 7 days with ADSc (63.3%) compared to follicles in vitro cultured alone (45.5%) (p &amp;lt; 0.01). A significant increase in follicle size was observed in both groups after culture (p &amp;lt; 0.0001). This increase was statistically higher in follicles co-cultured with ADSc than follicles cultured alone (p &amp;lt; 0.005), reflecting better follicle growth in the follicle-ADSc group. Preliminary results by TEM show ADSc did not affect the ultrastructure of isolated follicles after 7 days of IVC. Limitations, reasons for caution As demonstrated by our results, ASCs appears to improve IVC conditions and boost follicle activation and survival preserving the follicle ultrastructure. Our study, however, did not investigate the underlying mechanisms behind this improved follicle growth and survival after IVC. Wider implications of the findings Co-encapsulation of human unilaminar follicles with ADSCs promotes early-stage ovarian follicle development and survival, possibly because of bidirectional interactions between follicles and ADSCs. This represents a promising first step for fertility preservation in female cancer patients who cannot benefit from cryopreserved ovarian tissue transplantation. Trial registration number not applicable </jats:sec

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

To what extent does regenerative medicine with stem cell therapy help to address infertility issues for future clinical application? 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. 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. 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. 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. Preclinical studies exploring MSCs from different animal origins and tissue sources in specific conditions were selected (n = 112), including: 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 and 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. While preclinical results look promising in terms of protecting the ovarian reserve in different experimental models (especially those using various mammal experimental models and 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. 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. 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. N/A

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

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? 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. 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. 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. 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. 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]. The impact of specific CHT protocols could not be investigated since the group of patients with previous CHT was highly heterogeneous. 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. 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. N/A

O-038 Role of apoptosis and autophagy in ovarian follicle pool decline from birth to late reproductive age and after gonadotoxic chemotherapeutic treatments

Abstract Study question Which biological mechanisms are responsible for physiological age-dependent and non-physiological ovarian reserve decline throughout a woman’s life? Summary answer Ovarian follicle pool size is modulated by both apoptosis and autophagy, the latter playing a major role in its decline before puberty. What is known already Among different pathways of controlled cell death, apoptosis and autophagy are both involved in follicle death. Apoptosis participtes in eliminating damaged follicles, like those affected by chemotherapy (CHT)-induced DNA damage, but its role in physiological age-related follicle decline is less 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 Eighty-four patients aged between 1 and 35 years, with ovarian tissue stored for histological analyses at the time of cryopreservation (between 2012 and 2021) at the Université Catholique de Louvain (Belgium) were included in retrospective evaluations. Participants/materials, setting, methods Ovarian fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; caspase-3 immunostaining to identify follicle apoptosis; and microtubule-associated proteins 1A/1B light chain 3B (LC3B) immunolabeling to detect follicle autophagy. Ovarian fragments from 9 patients were assigned for transmission electron microscopy to investigate ultrastructural features of oocytes and granulosa cells. All analyses were conducted stratifying patients by age, menarchal status (premenarchal=32; postmenarchal=52), and potentially gonadotoxic CHT before cryopreservation (n = 14). Main results and the role of chance Premenarchal patients had a larger follicle pool in terms of total follicle density (4979.98±4952.16 vs 918.8±903.86, p &amp;lt; 0.0001), but higher rates of morphologically abnormal (8.52±6.48% vs 3.54±4.54%, p &amp;lt; 0.0001) and atretic follicles (15.8±9.14% vs 10.6±9.43%, p = 0.01) than postmenarchal subjects. Apoptosis rates did not change with increasing age (27.94±28.78% in prepubertal subjects and 29.5±33.58% in postpubertal subjects). Autophagic follicles were around 10 times more common in premenarchal compared to postmenarchal subjects (10.21±16.% vs 1.34±4.02%, p &amp;lt; 0.0001), playing a crucial role in age-related follicle decline and elimination of ‘abnormal follicles’, that are rarely seen after menarche. The impact of previous CHT varied according to age. In premenarchal patients with previous CHT, significantly more apoptotic (40.22±40.44% vs 26.79±25.35%, p = 0.04) and fewer abnormal (3.84±3.91% vs 9.83±6.5%, p = 0.02) follicles were detected than in those 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, lower quiescent follicle rates were observed compared to patients with no CHT before tissue freezing (70.1±36.36% vs 85.89±12.61%, p = 0.04), suggesting accelerated follicle activation and growth. Limitations, reasons for caution The present study could not investigate the impact of specific CHT protocols, since the group of patients with previous CHT was highly heterogeneous. However, these patients could not be excluded a priori, since the effect of follicle pool depletion has not been fully elucidated. Wider implications of the findings This study yields a deeper understanding of follicle pool decline regulation, evidencing for the first time that (i) different cell death pathways are involved in physiological and non-physiological follicle depletion, and (ii) the follicle pool responds differently to gonadotoxic damage according to age and ovarian reserve size. Trial registration number N/A </jats:sec

Management of adnexal masses during the third trimester of pregnancy: a case report in twin-pregnancy and review of the literature

The occurrence of ovarian masses during pregnancyis uncommon, nevertheless the correct diagnosis and management, either surgical or obstetric, may represent an issue. The clinical management has to take into consideration aspects both related to the mass (symptoms of torsion, rupture or occlusion and malignant potential) and to the foetal risks. A 36-year-old woman with a twin pregnancy at 29 weeks of gestation was diagnosed with an ovarian cyst with suspicious ultrasonographic features (diameter of 15 cm and enhanced blood flow). An expectant management until a safer gestational age for the twins was established. At 32 weeks of gestation symptoms of bowel obstruction and abdominal pain required a caesarean section and the removal of the affected adnexum. The histological analysis revealed a mucinous borderline tumour with intraepithelial carcinoma. When an adnexal mass is diagnosed during third trimester of pregnancy the ultrasonographic evaluation has to be done to assess the potential of malignancy. The clinical management needs a multidisciplinary approach has to be balanced between the risk of malignancy or other issues related to the mass and the foetal health