Ovarian Biopsies Research Articles

Morphology of endometrial inflammatory changes in foci of endometriosis and adenomyosis

The high prevalence of endometriosis, its impact on menstrual and reproductive functions, obstetric complications, and the quality of life of women in the reproductive age determines the urgency of the problem.The objective: to assess the prevalence of inflammatory changes in foci of heterotopic endometrium in patients with endometriosis and adenomyosis.Materials and methods. 92 women of reproductive age – from 21 to 46 years old – who applied for surgical treatment for symptomatic adenomyosis (47 women – 1st group) and ovarian endometriosis (45 patients – 2nd group), and 30 practically healthy women of the same age (control group) were examined.Histological examination of adenomyosis and ovarian endometriosis biopsies was performed using a light microscope at magnifications of 40 and 100. For immunohistochemical examination, primary and secondary antibodies were used to determine CD34, CD68, and CD138 markers.Results. In patients with adenomyosis and ovarian endometriosis the following concomitant gynecological pathologies as uterine fibroids, endometrial hyperplasia, chronic endometritis, chronic abnormal uterine bleeding, the frequency of which is significantly higher in adenomyosis, were diagnosed. This may indicate the pathogenetic role of chronic inflammation in the development of these pathological conditions.In patients with adenomyosis the positive expression of CD68 in the epithelial component of heterotopias was determined in 8 (17.0±5.6%) women, and in 4 (8.9±4.2%) women with ovarian endometriosis, which indicated a low phagocytic activity. During the assessment of CD34 expression in vessel walls and the stromal component of endometrioid heterotopias, a positive reaction was determined in 16 (34.0±6.9%) patients with adenomyosis and in 10 (22.2±6.2%) patients with ovarian endometriosis, which reflects the processes of neoangiogenesis and the activity of the pathological process.Positive expression of CD138 in endometrioid heterotopias prevailed in the group of patients with adenomyosis – 12 (25.5±6.4%) cases versus 4 (8.9±4.2%) cases in the group of patients with ovarian endometriosis (p<0.05). This confirmed the histological results and clinical data regarding the presence of chronic endometritis and isthmocele.Conclusions. The identified inflammatory changes, weak phagocytic activity and signs of neoangiogenesis in foci of heterotopic endometrium are more significant in patients with adenomyosis, which is confirmed by clinical and morphological data.

O-076 Novel method to enhance preantral follicle growth by granulocyte colony-stimulating factor administration improves embryos and pregnancy rate in poor ovarian reserve: a randomized controlled trial

Abstract Study question Whether priming with granulocyte colony-stimulating factor (G-CSF) before ART improves embryo and pregnancy rate while increasing anti-Müllerian hormone (AMH) in patients with poor ovarian reserve. Summary answer G-CSF priming improved embryonic development and pregnancy rate in ART with poor ovarian reserve, simultaneously increasing serum AMH. Enhanced preantral follicle growth likely was responsible. What is known already Eleven years ago, we treated repeated implantation failure in 10 women with diminished ovarian reserve by administering G-CSF with embryo transfers. No implantation succeeded directly, but serendipitously 3 of the 10 spontaneously became pregnant 2 months later. One of these pregnancies involved twins in a 45-year-old woman without ovulation induction. We hypothesized that G-CSF administration might have stimulated preantral follicle growth, with improved ovulation after 2 months. In animal models with diminished ovarian reserve, G-CSF administration consistently increased ovarian preantral follicles and AMH. Therefore, we designed this clinical trial. Study design, size, duration Patients were prospectively randomized to priming or control groups. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of IVF/ICSI and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. Participants/materials, setting, methods Hundred ART patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. Main results and the role of chance Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by G-CSF priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts (53% with priming vs. 24% in controls). The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04-7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. The efficacy of G-CSF priming was not associated with genotypes for killer-cell immunoglobulin-like receptors. No adverse effects of priming were observed. Limitations, reasons for caution Enhancement of preantral follicle growth by G-CSF administration needs to be confirmed histologically as this clinical trial could not allow ovarian biopsies for its purpose of infertility treatments. Wider implications of the findings This study proposes a novel, simple, and safe treatment for poor ovarian reserve. In addition, enhancement of preantral follicle growth by G-CSF priming may also improve ovulatory dysfunctions in polycystic ovary syndrome with elevated AMH, reflecting pooling of retarded/arrested growing preantral follicles. Trial registration number UMIN000013956

Ovarian tissue biopsy for cryopreservation by vaginal natural orifice transluminal endoscopic surgery: a new approach for a minimal invasive ovarian biopsy

Vaginal natural orifice transluminal endoscopic surgery (vNOTES) is an emerging surgical procedure that combines the advantages of the vaginal approach with laparoscopic vision and instrumentation. Shorter hospitalization and lesser postoperative pain associated with vNOTES may be explained by the advantages of this innovative surgical approach (e.g., absence of abdominal incisions, shorter operative time, and lower insufflation pressure). Ovarian tissue cryopreservation allows to preserve reproductive and endocrine functions in young women with oncological disease at risk of premature ovarian insufficiency (POI) caused by gonadotoxic treatments. Ovarian tissue biopsy for cryopreservation consists of a large biopsy of 1 or both ovaries that is usually performed by laparoscopy. Then, the removed ovarian tissue is cryopreserved for the future transplant after cancer remission. The volume of ovarian biopsy ranges from 50% of the ovary for women at moderate risk of POI to 70%-100% of it for those at high risk. The inclusion criteria for ovarian tissue cryopreservation are women aged <35 years who cannot delay start of oncological treatments for follicle cryopreservation, with a moderate or high risk of POI and good chance of 5-year survival. Ovarian tissue cryopreservation cannot be performed if tumor treatments include uterine irradiation or for tumors at risk of ovarian metastases (as in the case of ovarian cancer, leukemia, neuroblastoma, or Burkitt lymphoma). Despite widespread adoption of vNOTES in gynecology, ovarian biopsy for cryopreservation has never been performed using this route. Step-by-step explanation of the procedure with descriptive text and narrated video footage. Tertiary-level referral academic center. A 27-year-old patient recently diagnosed with low-grade follicular non-Hodgkin lymphoma was referred to our center for ovarian tissue cryopreservation before chemotherapy. The patient included in this study gave informed consent for publication of the video and posting of the video online including social media, the journal website, scientific literature websites (e.g., PubMed, ScienceDirect, and Scopus), and other applicable sites. Because of the nature of the study, institutional review board approval was not required. Access to the peritoneal cavity was created by a 3-cm posterior colpotomy. The peritoneum was then opened using cold scissors and temporarily fixed to the posterior vaginal wall. The GelPOINT Mini Advanced Access Platform (Applied Medical, Rancho Santa Margarita, CA), with 1 10-mm and 2 5-mm trocars, was used as the vNOTES port. The inner Alexis ring of the GelPOINT was inserted through the colpotomy into the pouch of Douglas. A hysterometer was placed into the uterine cavity to keep the uterus anteverted during the surgery. A pneumoperitoneum was created to a pressure of 8 mm Hg, and the operating table was tilted to a 20° Trendelenburg position. A 10-mm rigid 30° camera was inserted in the inferior and larger trocar, and both ovaries were visualized. Seventy percent of the left ovary was removed with cold scissors to minimize trauma on the surgical specimen. After removal of the GelPOINT cap, ovarian biopsy was immediately picked up by the biologist of our fertility center. The ovary was coagulated with a bipolar instrument. The hysterometer was then replaced by a uterine manipulator to perform tubal patency test, and blue dye passage through both salpinges was observed. Finally, the Alexis retractor and stich on the posterior peritoneum were removed, and the vagina was sutured using interrupted stiches. The total operative time was 25 minutes. Ovarian tissue biopsy for cryopreservation by vNOTES. No intraoperative and postoperative complications were reported, and the patient was discharged after 24 hours from surgery. Vaginal natural orifice transluminal endoscopic surgery may be a feasible alternative approach to laparoscopy for ovarian tissue cryopreservation: it allows an easy access to the ovaries and removal of different tissue volumes. Patients undergoing ovarian cryopreservation may benefit from the vNOTES approach because a rapid postoperative recovery is crucial to start chemotherapy in a short time. As for other vNOTES procedures, accurate selection of patients seems to be crucial for a successful ovarian tissue cryopreservation. We believe that the inclusion and exclusion criteria reported for other gynecologic procedures performed through vNOTES may also be valid for ovarian tissue cryopreservation by vNOTES. Women at high risk of pelvic adhesions (e.g., coexistent endometriosis, previous pelvic surgery, or inflammatory pelvic disease), those with an increased body mass index or enlarged uterus, and those with cervical, vaginal, or uterine cancer cannot be considered for this approach because all these factors are associated with failure of vNOTES. On the other hand, women with no history of surgery, endometriosis, and large myomas may benefit from the vNOTES approach, and these women represent most of patients who undergo ovarian tissue cryopreservation. Further and larger studies are needed to assess the efficacy and safety of this new approach.

Abstract 1066: Colocalization of ovarian cancer-associated biomarkers in tumors, cancer cells and extracellular vesicles

Abstract Introduction: We have previously shown that ovarian cancer can be detected with high specificity and sensitivity by interrogating colocalized membrane-associated cell-surface biomarkers on single tumor-associated extracellular vesicles (EVs). Our blood based Ovarian Cancer Test (OC Test) is composed of 5 cancer-associated biomarkers (BST-2, FOLR1, MUC-1, MUC-16, and sialylated Thomsen-nouveau antigen (sTn)) known to be overexpressed in ovarian cancer relative to healthy tissues. The test employs these 5 biomarkers in 3 combinations to capture and detect EVs from human plasma to distinguish high-grade serous ovarian carcinoma (HGSC) from both benign ovarian tumors and normal samples. Using super-resolution microscopy on ovarian cancer cell lines and EVs and multiplex immunohistochemistry of ovarian benign and tumor tissue, our study aimed to show that the 5 ovarian cancer biomarkers show 1) colocalization on ovarian cancer cells, 2) colocalization on the surface of EVs, and 3) colocalization on cancer cells within ovarian tumor biopsies. Methods: Super-resolution microscopy was employed to visualize stained biomarkers on the cell surface of a collection of ovarian cancer cell lines as well as on the surface of extracellular vesicles released by these cell lines. 58 HGSC (17 Stage I, 30 Stage II, and 10 Stage III) and 17 benign ovarian tumor K2EDTA plasma samples and paired FFPE tissue were sourced from the Ovarian Cancer Research Program (OVCARE, Vancouver BC, Canada). A tumor microarray from identical tissue samples was stained with an Opal multiplex-IHC (mIHC) assay comprised of antibodies to BST2, MUC1, sTn, and pan-cytokeratin and a DAPI counterstain. Machine learning based image analysis was utilized to determine colocalization of the biomarkers at the cellular level within benign and tumor tissue. RNA sequencing was carried out to assess expression levels of the biomarkers in each FFPE tissue sample. OC Tests were run on each plasma sample. Results: Super-resolution microscopy showed colocalization of OC Test combination biomarkers on ovarian cancer cells and EVs derived therefrom. Machine learning-based image analysis of tumor and benign tissue microarrays stained for BST2/MUC1/sTn using mIHC, showed strong evidence for colocalization of these biomarkers on single cells within ovarian tumor tissue, particularly in early-stage tumors, but not on benign tissue. RNA sequencing analysis showed good correlation of biomarker RNA expression levels and with the OC Test results. Conclusions: The results presented here demonstrate that the OC Test biomarkers are colocalized on tumor tissue, ovarian cancer cells, and EVs from ovarian cancer cell lines. RNA and protein expression levels show good correlation with OC Test results. These results provide further evidence that colocalized biomarkers on EVs can be utilized for early detection of cancer. Citation Format: Anthony D. Couvillon, Emily S. Winn-Deen, Shelby Thornton, Shuhong Liu, Michael DeRan, Maciej Pacula, Sanchari Banerjee, Daniel Gusenleitner, Laura T. Bortolin, Jonian Grosha, Timothy Santos-Heiman, Gabrielle Sangiuliano, Kelly M. Biette, Christopher R. Sedlak, Bilal Hamzeh, Delaney M. Byrne, Peter A. Duff, Julie Ho, Dongxia Gao, Amy Jamieson, Lauren T. Cuoco, MacKenzie S. King, Dawn R. Mattoon, Toumy Guettouche, Jessica N. McAlpine, David Huntsman. Colocalization of ovarian cancer-associated biomarkers in tumors, cancer cells and extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1066.

Laparoscopic medulla-sparing ovarian tissue biopsy for cryopreservation: step-by-step surgical technique

To describe a laparoscopic technique for ovarian tissue biopsy (OTB) for fertility preservation. In the last years, the demand for fertility preservation has grown because of the increasing survival rates among patients with cancer and the rising awareness of the importance of quality of life after gonadotoxic therapy. Among fertility-sparing approaches, ovarian tissue cryopreservation is a valid strategy to preserve ovarian endocrine and reproductive function in prepubertal and postpubertal women who will undergo gonadotoxic cancer treatments. Currently, there is no universal consensus regarding ovarian tissue retrieval technique for fertility preservation. Step-by-step description of the surgical technique with narrated video footage. Academic tertiary hospital. Patients with a high risk of premature ovarian insufficiency, usually due to gonadotoxic treatments, who undergo OTB for fertility preservation were included in the study. In this video, we present the clinical case of a 28-year-old patient affected by Hodgkin lymphoma who underwent laparoscopy for OTB before chemotherapy. After exposing the chosen ovary, an incision at the tubal pole of the ovary is made with scissors. Through section and dissection, a large cortical biopsy of the ovary is performed without removing and avoiding any damage to the medulla. At the end of the procedure, hemostasis was achieved with selective coagulation using bipolar coagulation. Step by step educational video. The post-operative course was uneventful and the patient was discharge 24 hours after surgery. Standardization of a step-by-step laparoscopic technique can provide an effective method to optimize ovarian tissue removal while minimizing tissue injury. Medulla-sparing ovarian biopsy allows retrieval of only the cortical part of the ovary, maximizing the number of primordial follicles obtained without damaging the vascular supply of the ovary contained within the medulla. Primordial follicles are resistant to cryoinjury owing to their relatively inactive metabolism, and they are usually found at approximately 0.8 mm below the surface of the cortex. This technique could also reduce the back-table processing time of the ovarian tissue before cryopreservation. One disadvantage could be the difficulty of the technique compared to an oophorectomy because it requires a skilled surgeon that can easily find the cleavage plane between the medulla and the cortex, even in patients submitted to previous chemoradiotherapy or during gonadotropin-releasing hormone analogue therapy.

HDAC6-dependent deacetylation of NGF dictates its ubiquitination and maintains primordial follicle dormancy.

Rationale: Primordial follicles are limited in number and cannot be regenerated, dormant primordial follicles cannot be reversed once they enter a growth state. Therefore, the length of the female reproductive lifespan depends on the orderly progression and selective activation of primordial follicles, the mechanism of which remains unclear. Methods: We used human ovarian cortical biopsy specimens, granulosa cells from diminished ovarian reserve (DOR) patients, Hdac6-overexpressing transgenic mouse model, and RNA sequencing to analyze the crucial roles of histone deacetylase 6 (HDAC6) in fertility preservation and primordial follicle activation. Results: In the present study, we found that HDAC6 was highly expressed in most dormant primordial follicles. The HDAC6 expression was reduced accompanying reproductive senescence in human and mouse ovaries. Overexpression of Hdac6 delayed the rate of primordial follicle activation, thereby prolonging the mouse reproductive lifespan. Short-term inhibition of HDAC6 promoted primordial follicle activation and follicular development in humans and mice. Mechanism studies revealed that HDAC6 directly interacted with NGF, reducing acetylation modification of NGF and thereby accelerating its ubiquitination degradation. Consequently, the reduced NGF protein level maintained the dormancy of primordial follicles. Conclusions: The physiological significance of the high expression of HDAC6 in most primordial follicles is to reduce NGF expression and prevent primordial follicle activation to maintain female fertility. Reduced HDAC6 expression increases NGF expression in primordial follicles, activating their development and contributing to reproduction. Our study provides a clinical reference value for fertility preservation.

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment.

Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). N/A. This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 μM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. N/A.

Abstract B012: Validation of the OncoSignature assay, an ACR-368-tailored response-predictive quantitative multiplexed immunofluorescent assay for prediction of sensitivity to the CHK1/2 inhibitor ACR-368 in individual patients with cancer

Abstract Background: The CHK1/2 kinase inhibitor ACR-368 (Prexasertib) demonstrated durable single-agent efficacy in a subset of patients with high grade serous ovarian, anal and head and neck cancers in past trials. Using a quantitative phospho-proteomics approach, we identified three orthogonal candidate biomarkers tailored to predict tumor response to ACR-368 treatment. The OncoSignature® assay is based on immunofluorescent staining of biomarkers in FFPE tumor sections with image acquisition, analysis, and scoring of biomarkers. The sample is OncoSignature positive if all three biomarker scores are above their thresholds. We validated the OncoSignature® assay to predict response to ACR-368 in cancer cell lines and ovarian cancer PDX models. Here, we further validated OncoSignature® assay in novel cancer indications and in patient biopsy samples from ACR-368 clinical trials. Methods: Multiple cancer types were tested with the OncoSignature® assay for prevalence of potential ACR-368 responders. For validation, endometrial cancer PDX models were tested in anACR-368 efficacy study. Blinded untreated PDX tumors were analyzed in the OncoSignature® assay and results locked prior to unblinding and statistical analysis. Adapted [CS1] Oncosignature assay was applied to frozen pretreatment ovarian cancer biopsies from a single center ACR-368 monotherapy Ph2 trial (NCT02203513). OncoSignature® assay was also applied to FFPE pretreatment ovarian cancer biopsies from a multi-center ACR-368 monotherapy Ph 2 trial (NCT03414047) and from the NCT02203513 trial. After biomarker scores were generated and locked, patient treatment outcomes were unblinded and ACR-368 response was compared with OncoSignature prediction. Results: Evaluation of multiple cancer indications revealed a concordance between OncoSignature-predicted prevalence of ACR-368 responders and results of ACR-368 treatment observed in clinical trials. Metastatic ovarian cancer had 30% of predicted responders and 30% and 14% observed RECIST responses the above-cited trials. Squamous lung cancer had no predicted responders and no RECIST responses in a clinical trial. Endometrial and bladder cancer had a high prevalence of predicted responders with 40% and 36%, respectively. The endometrial cancer PDX efficacy study had multiple complete responses predicted by OncoSignature® assay with an AUC of 0.88. For frozen and FFPE patient biopsy samples from Phase 2 ACR-368 trials, OncoSignature positive groups had highly significant response enrichment to 47% and 57%, respectively, and significant increases in progression free survival time. Conclusions: Bladder and endometrial cancers were identified as novel indications with high prevalence of predicted ACR-368 responders. The OncoSignature® assay significantly predicted response in endometrial PDX models and in samples from ovarian cancer patients treated with ACR-368. OncoSignature® assay driven clinical trial (NCT05548296) in ovarian, endometrial and bladder cancers was initiated to test benefit to patients. Citation Format: Ayesha Murshid, Christina Noe, Kailash Singh, Sibgat Choudhury, Jayakumar Nair, James Dunyak, Michail Shipitsin, Jung-Min Lee, Peter Blume-Jensen. Validation of the OncoSignature assay, an ACR-368-tailored response-predictive quantitative multiplexed immunofluorescent assay for prediction of sensitivity to the CHK1/2 inhibitor ACR-368 in individual patients with cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B012.

Automated imaging and identification of proteoforms directly from ovarian cancer tissue

The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based multiplexed workflow for top-down tandem MS (MS2) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS2 identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer.

Optimizing the Ovarian Tissue Cryopreservation in the 'Oncofertility' Institutional Program at an Italian National Cancer Institute.

The majority of female cancer patients undergoing anticancer treatments are at risk of experiencing 'cancer treatment-related infertility', which can result in permanent damage to their reproductive prospects. Among the fertility preservation methods, ovarian tissue cryopreservation (OTC) has emerged as an alternative for these patients. The Cancer Institute of Bari initiated a research program to assess the feasibility of OTC. This study compares the viability of ovarian cortical fragments cryopreserved using slow freezing (SF) and ultra-rapid freezing (URF) methods. Ovarian cortex biopsies were obtained from 11 fertile women enrolled in our oncofertility service between June 2022 and January 2023. After tissue collection, a histological assessment was performed before cryopreservation. OTC was carried out using both SF and URF methods. Six months later, thawed samples were evaluated for follicle counts and histological integrity. No statistically significant difference was observed in the proportion of intact follicles (means of 31.5% and 73.0% in the SF and URF groups, respectively; p = 0.064). However, there was a significant difference in the number of follicles between the SF group (n = 149) and the URF group (n = 37) (p = 0.046). We assessed the viability of ovarian cortex after freezing and thawing, focusing on the structural integrity of follicles. Our findings suggest that there are no significant differences between the SF and URF methods.

OR19-06 State-of-the-art MALDI Mass Spectrometry Imaging Reveals Unique Spatial Lipid Signatures in Human and Mouse Ovarian Compartments

Abstract Disclosure: N. Pascuali: None. F. Tobias: None. K. Valyi-Nagy: None. S. Salih: None. A. Veiga-Lopez: None. The incidence of infertility has been consistently increasing over the past few decades, with a high proportion of these cases caused by ovarian-related dysfunction. Metabolic disorders such as hyper- or dyslipidemia have been associated with subfertility (e.g., follicular fluid dyslipidemia is associated with lower IVF success). Lipids play a critical role in the structure and function of ovarian tissue. However, while several studies have investigated transcript- and protein-level alterations in ovarian pathologies, less is known about alterations in the lipid landscape. The first step in determining if ovarian disorders are associated with ovarian dyslipidemia - and thus use lipids as potential biomarkers of ovarian pathology - is to understand the physiological distribution of ovarian lipids. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging (MSI) enables label-free in situ analysis of molecules such as lipids and metabolites directly within tissue sections. We aimed to begin mapping the distribution of lipids in human and mouse ovaries using high spatial resolution MALDI MSI. To this end, and after informed consent, two fresh ovarian biopsies from a 26-year-old patient were frozen, cryosectioned into 10-μm sections, and mounted onto ITO-coated slides. A set of 21-day-old mouse ovaries (n=10) were gelatin-embedded and cryosectioned. Slides were sprayed with 1,5-diaminonaphthalene as the matrix. MALDI MSI was performed using a Bruker RapifleX MALDI Tissuetyper (20-μm resolution) in the negative ion mode. Data were processed using the SCiLS Lab software. We identified for the first time in human ovaries over 100 lipid species, with most of them displaying distinct spatial distributions. Specifically, we detected 3 lipids specific to theca cells (m/z: 701.6; 1050.8; 1263.9), 6 lipids specific to granulosa cells (m/z: 279.3; 697.5; 861.7; 883.7; 911.7; 1448.0), 3 lipids specific to the stromal region (m/z 392.1; 753.5; 835.7), and one specific to luteal cells (m/z: 615.3). As for mouse ovaries, we detected 5 lipids specific to follicular cells (theca or granulosa) (m/z: 327.1: 343.2; 552.6; 632.6; 703.5), 5 lipids specific to stromal cells (m/z: 715.6; 729.6; 743.6; 771.7; 799.6), 5 lipids specific to oocytes (m/z: 778.5; 863.7; 1063.8; 1225.7; 1336.0) and 7 lipids specific to the ovarian cortex (m/z: 721.5; 748.5; 776.6; 792.6; 810.6; 869.6; 885.7). To our knowledge, this is the first evaluation of the human ovarian lipidome and characterization of region- or cell-layer-specific lipids. This approach has allowed us to demonstrate that compartments as small as oocytes (∼70 μm in diameter) have a unique lipid signature. Harnessing the unique specificity of the lipid signature per ovarian region and/or cell layer in this study could help pave the way to develop novel biomarkers of ovarian dysfunction. Presentation Date: Saturday, June 17, 2023

Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. N/A.

P-410 First wave of primordial follicle activation during human ovarian tissue manipulation for fertility preservation

Abstract Study question Does ovarian tissue biopsy, transportation and processing for fertility preservation and restoration trigger primordial follicle activation? Summary answer Early manipulation of ovarian tissue is sufficient to trigger follicle activation by stimulating PI3K/Akt and disrupting the Hippo pathway. What is known already Primordial follicle recruitment occurs continuously in physiological conditions by modulation of autocrine and paracrine factors, like the PI3K/Akt and Hippo pathways, to ensure follicle growth over time. During fertility preservation and restoration procedures, follicle activation may be disrupted and follow nonphysiological patterns. The ability to control activation dynamics by up- or downregulation of these pathways may enhance fertility restoration outcomes in a number of ways. Indeed, downregulation of follicle activation shortly after transplantation may protect the ovarian reserve from early depletion. Conversely, ovarian tissue in vitro culture may benefit from upregulation of primordial follicle activation to boost further growth. Study design, size, duration Fresh ovarian tissue was retrieved from nine women undergoing laparoscopic surgery for benign conditions. Three timepoints were investigated. One-third of collected tissue per patient was immediately fixed in the operating room, without any manipulation (time zero, T0). The remaining tissue was transferred to the laboratory and dissected to remove any surplus medulla. It was then cut into small cortical fragments, half of which were fixed after 25 minutes(T25) and the other half after 90 minutes(T90). Participants/materials, setting, methods All cortical fragments were fixed in 4%formaldehyde and embedded in paraffin for histology. In order to explore follicle activation,markers of the PI3K/Akt and Hippo signaling pathways were immunolabeled at each timepoint, targeting: (i) phospho-Akt (p-Akt) in primordial follicles by immunohistochemistry as a marker of early PI3K/Akt pathway activation; and (ii) Yes-associated protein (YAP) cellular localizationin the granulosa cell layer of primordial follicles by immunofluorescence as a marker of Hippo disruption. Main results and the role of chance An upturn in p-Akt expression was observed at T25 (22,34 ± 0.13%; p = 0.0233) and T90 (39,01 ± 0.22%, p = &amp;lt; 0.0001) compared to T0 (2,87 ± 0,03%). In terms of YAP cellular localization, a significant nucleus-to-cytoplasm shift was detected at T25 (1.11 ± 0.09; p = 0.0428) compared to T0 (0.97 ± 0.10), while T90 (1.07± 0.14) values were similar to T25. Our data prove that ovarian tissue manipulation triggers primordial follicle activation very early, involving both the PI3K/Akt and Hippo signaling pathways, which appear to cooperate in primordial-to-primary follicle transition. Our results indicate that the first stages of any fertility preservation or restoration procedure involving ovarian tissue manipulation contribute to dysregulation of the very mechanisms responsible for the ovarian reserve maintenance and follicle growth. Additional strategies are required to gain the control of follicle activation mechanisms in nonphysiological conditions (ex vivo ovarian tissue manipulation), in order to exploit ovarian reserve dynamics to serve the need of patients. Limitations, reasons for caution Analyses in the study were limited to histology and immunolabeling to acquire a descriptive picture of pathway activation kinetics over time. Further investigations using dynamic experimental models are essential to advance our understanding of signaling pathway synergy in vivo. Wider implications of the findings Since dysregulation of follicle activation in nonphysiological conditions appears to be associated with poor oocyte quality, enhancing our ability to control the relevant signaling pathways is crucial to optimizing fertility preservation procedures. Trial registration number not applicable

P-424 Effects of in vitro activation vs fragmentation on human ovarian tissue in culture

Abstract Study question What is the added value of in vitro activation (IVA) protocol compared with fragmentation only in human ovarian tissue culture? Summary answer Although histological assessment shows that the IVA increases follicle survival and growth, IVA and fragmentation stimulate extensive and nearly identical transcriptomic changes in cultured tissues. What is known already Treatments based on activation of the PTEN/PI3K pathway in ovarian tissue in vitro have been administrated to refractory primary ovarian insufficiency (POI) patients and resulted in live births following auto-transplantation of the tissue. However, some studies have shown that mere fragmentation of the tissue produces comparative effects, questioning the added value of chemical stimulation protocol which could potentially activate oncogenic pathways. Study design, size, duration Thirty-three ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section at Karolinska University Hospital Huddinge. The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) +740Y-P (IVA group) during the first 24 h of culture while half were exposed to vehicle only (fragmentation only group). Subsequently, both groups were cultured for additional 6 days. Media change was performed every other day. Participants/materials, setting, methods Effects on follicles were evaluated by counting and scoring follicles before and after the 7-day culture via HE-stained serial sections. Follicle function was assessed by quantification of steroids by UPLC-MS/MS at the end of the culture. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the 24-h initial culture step. Selected differentially expressed genes (DEGs) were validated by qPCR and immunofluorescence in independent culture experiments. Main results and the role of chance Compared to fragmentation only group, significantly higher follicle survival rate, increased secondary follicle number and sizes were found in IVA group. No significant differences were detected in levels of steroids in culture media on day 7 between the two groups. In our RNA-seq data, when comparing the IVA group to fragmentation only group, only 110 DEGs were found with the relaxed cutoff of FDR&amp;lt;0.1. The signaling pathways affected by IVA involved follicle growth but also inflammation and DNA damage. However, we found that the gene expression was profoundly affected in both IVA and fragmentation only groups compared to the fresh tissue: in total 3,676 and 4,223 DEGs were found (FDR&amp;lt;0.001), respectively. The top enriched gene sets in both groups included several pathways that are known to modulate follicle growth, e.g. PI3K/AKT, MTORC1. We also found that classical steroidogenesis genes (e.g. CYP11A1, CYP17A1) were significantly downregulated in both groups after 24-h culture. Moreover, upregulation of genes related to glycolysis, a process that was shown to promote activation of primordial follicles, and its upstream regulator were shown in the RNA-seq data. This effect was validated by qPCR (ENO1, PKM, LDHA, p &amp;lt; 0.0001). Future validation will be performed using western blot and immunofluorescence. Limitations, reasons for caution The study was performed in an in vitro model where tissue was isolated from the regulation of hypothalamic-pituitary-ovarian (HPO) axis. And thus, further experiments with xeno-transplantation may be needed to explore the effect of IVA in vivo in the future. Wider implications of the findings The impact of 24-h culture on gene expression in ovarian tissue far exceeds the effect of IVA. Yet, follicle growth was stimulated by IVA, which may suggest effects on specific cell populations that are diluted in bulk transcriptomics. Cell type-specific impacts need further studies to conclude about effectiveness of IVA. Trial registration number Not applicable

P-425 Biosilk and ovarioids: recombinant silk as a new tool for establishing a 3D-culture system for human ovarian primary cells

Abstract Study question Can Biosilk be used as scaffold for establishing a 3D-culture system to support attachment and growth of primary cells derived from adult ovarian biopsies? Summary answer The use of recombinant spider silk-based scaffold allowed the formation of 3D-structured ovarioids from both cortex and medulla isolated from 5 different patients. What is known already Fertility in women is adversely affected by several factors, such as age, environmental pollutants and diseases requiring gonadotoxic treatments. Understanding ovarian cell composition and organization may pave the way to novel fertility preservation methods with the ultimate goal of being applied to clinics. So far, there are no clinically established methods for in vitro growth and maturation of human ovarian follicles leading to mature competent oocytes. Therefore, exploring new 3D-culture systems for in vitro reconstruction of ovarian somatic cell niche could lead to development of novel tools to support growth of patient-specific follicles. Study design, size, duration Ovarian tissue was collected from gender reassignment patients (GRP) after informed written consent at Karolinska University Hospital Huddinge from 2019 to 2022. After separation of cortex and medulla, the samples were mechanically and enzymatically dissociated into single-cell suspensions and used to evaluate 3D- and 2D-culture methods. Freshly fixed biopsies from cortex and medulla (3x3x1 mm3) were used as control for the transcriptomic analysis and RNA-FISH assay. Participants/materials, setting, methods Tissue was obtained from five patients aged 23-31 years. Dissociated primary somatic cells seeded on Biosilk-foam scaffolds were kept in culture for 2 weeks, followed by detachment, equal division of the foams and suspension culture for additional 4 weeks (BioSilk-Ovarioids, BSO). BSOs were harvested in PFA and RNAlater (n = 6/patient, respectively) for morphological and transcriptomic analysis. Protein ZO1, and cell type-specific marker genes (AMHR2, PDGFRa, CLDN5, GJA4) were evaluated via immunodetection and RNA-FISH assay, respectively. Main results and the role of chance The sizes of BSOs from both cortex and medulla ranged between 0.5-1 mm at the end of the culture, appearing highly compacted under optical microscope. HE-stained BSO sections revealed that cells were distributed throughout the foams, showing good attachment and distribution. Marker genes were selected for specific cell types [AMHR2-granulosa, PDGFRa-stroma, CLDN5-endothelial, GJA4-perivascular cells (Wagner et al. 2020)] and used to study the representation of different somatic cells in the BSOs. The RNA-FISH analysis confirmed the presence of all cell type-specific marker genes, with predominating presence of stromal cells (PDGFRa). Newly formed ZO1-specific gap junctions were detected in both cortex- and medulla-derived BSOs, appearing mainly in the outer part of the structures. Interestingly, cell layers surrounding the original Biosilk scaffold were observed, especially in medulla-BSOs. Transcriptomic profiling of the samples showed clear separation to three main clusters by principal component analysis: freshly fixed tissue, 2D-cultures, and BSO. Moreover, clustering analysis of differentially expressed genes (DEGs) showed the presence of gene clusters in fresh tissues affected by both 3D/2D-cultures. Further analyses will focus on identification of significantly affected gene ontologies and pathways, which will further guide the optimization of the BSO culture system. Limitations, reasons for caution The tissue was derived from GRPs who always receive androgen treatments prior to surgery. As hormonal treatment may affect the ovarian environment, further trials with untreated patient samples will be needed to generalize the model to fertility preservation patients. Wider implications of the findings The establishment and development of the BSO 3D-culture system may enable the construction of patient-specific clinically significant tools for in vitro folliculogenesis. This would open new avenues for fertility restoration in patients who cannot receive auto-transplants and treatment of infertility in premature ovarian insufficiency if residual follicles remain in tissue. Trial registration number Not applicable

Serum Anti-Müllerian Hormone Is an Effective Indicator of Antral Follicle Counts but Not Primordial Follicle Counts.

Serum anti-Müllerian hormone (AMH) is a biomarker for predicting antral follicle counts but there is no clear consensus on whether AMH is indicative of primordial follicle counts in humans. Mice were used as a model species in this study to obtain accurate follicle counts across the reproductive phase of life. Serum AMH was measured in 62 female C57Bl6/J mice aged 25 to 401 days. Primordial and primary follicles were determined by stereological counts and all secondary and antral follicles were counted in serial histological sections. Serum AMH was most strongly correlated with small- and medium-sized antral follicles. Immunohistochemistry and stepwise multiple regression confirmed that these follicle development stages are the key determinants of serum AMH, with little contribution from other stages. Primordial follicles were not found to have strong correlations with serum AMH or antral follicle counts, particularly in younger females, but the strength of the association appeared to increase with age. This result is likely attributed to high interindividual variation in primordial follicle activation and preantral follicle survival rates. Recent large studies in human populations have shown similar results but the primary limitation of these studies was that primordial follicle counts were determined from ovarian cortical biopsies, where regional variation in follicle distribution may affect the quality of the data. In the present study, whole ovaries were surveyed, eliminating this limitation. The findings indicate that primordial follicle counts are not closely related with either serum AMH or antral follicle counts in females in the early phase of the reproductive phase of life.

A rare case of malignancy-associated ascites with no identifiable cause in a 29-year-old female with BRCA1 mutation

Introduction: We present a challenging case of a 29-year-old female with a detected Breast Cancer gene 1 (BRCA1) gene mutation who presented to us with an ascites the cause of which remained undiscovered even after appropriate testing. Case Report: A 29-year-old female presented with a five-month history of worsening intermittent abdominal pain which recently became constant and worsened in intensity without any identifiable trigger. She also had complaints of constipation for the past 2–3 days. Her past medical history was positive for (BRCA1) mutation which was tested after her mother’s diagnosis of breast cancer. Further evaluation revealed our patient had ascites, which was drained to collect 12 liters of fluid. The cytology and culture of ascitic fluid were negative, but total protein concentration or Serum to ascites albumin gradient (SAAG) ratio were concerning for a malignant cause of her ascites. She underwent multiple imaging and biopsies and the cause remain unidentified. Conclusion: By this report we want to shed light on the diagnostic challenges of malignancy-associated ascites in patients without peritoneal carcinomatosis. The high false-negative of ascitic fluid cytology in such cases makes it an unreliable test for evaluation. We performed fresh ovarian biopsies which were benign and highlight the need for further research to assess the false negative rates in frozen versus fresh biopsies while evaluating malignancy-associated ascites in patients with an unknown primary cause.

(179) Incidence of Fertility Preservation Procedures in Prepubertal and Pubertal Individuals with Cancer

Abstract Introduction Fertility Preservation (FP) for children and adolescents with cancer is underutilized. In prepubertal individuals, ovarian and testicular tissue can be frozen; however, this is still considered largely experimental. Objective Identify trends of FP in prepubertal individuals in the United States. Methods We performed a retrospective study of prepubertal children with cancer identified through the Pediatric Health Information System from 2011 to 2020. Children who underwent a testicular or ovarian biopsy were included. Any patients with testicular or ovarian malignancy, or other diagnoses which may have required a gonadal biopsy were excluded. Results A total of 418 boys under 13 and 333 girls under 12 who underwent a gonadal biopsy were identified. There was a total of 66929 new cancer diagnoses in girls and 86001 new cancer diagnoses in boys during this time. The most common cancer diagnosis was hematologic in both boys (50.96%) and girls (36.64%). A concurrent procedure at time of gonadal biopsy was performed in 84% of boys and 62% of girls, with line insertion being the most common. The only predictive variable of receiving a gonadal biopsy was increasing year. Overall, only 0.04% of children had a gonadal biopsy for FP during this time period. Conclusions Gonadal biopsy rates have increased in prepubertal children with cancer, presumably for FP. While recent international guidelines support FP in this group, our findings highlight the need to establish protocols and tracking for FP procedures in the US. Disclosure No

The novel peptide PFAP1 promotes primordial follicle activation by binding to MCM5.

Premature ovarian failure (POF) is a complication of ovarian dysfunction resulting from the depletion or dysfunction of primordial follicles (PFs) in the ovaries. However, residual follicles that have the potential to be activated are present in POF or aged women. Little is known about the mechanisms by which the remaining dormant PFs in POF patients are activated. Using mass spectrometry, we screened differentially generated peptides extracted from the ovarian cortical tissue biopsies of patients with or without POF, during which we identified PFAP1, a peptide that significantly promoted the activation of PFs in the ovaries of 3 dpp mice in vitro. PFAP1 reversed age-related fertility damage in vivo to a certain extent, promoted estrogen (E2) and anti-mullerian hormone (AMH) production (p < .05), and decreased the levels of follicle-stimulating hormone (FSH) (p < .05). In newborn mouse ovaries, PFAP1 could bind to the protein minichromosome maintenance protein 5 (MCM5) and inhibit its ubiquitination and degradation. In addition, PFAP1 promoted the proliferation of GCs, probably by regulating the function and production of MCM5. In conclusion, PFAP1 could promote the activation of PFs in the ovaries of newborn mice, partially restore the ovarian function of aged mice, and increase the proliferation of primary granulosa cells (GCs) by regulating the function of MCM5. PFAP1 is a promising novel peptide that may be developed into a new therapeutic agent for POF and other ovarian diseases.