Fresh Ovarian Tissue Research Articles

Vitrified turkey ovarian tissue cultured and assessed through gene expression patterns: A potential screening tool

Biobanking of turkey ovarian tissue has the potential to play a crucial part in preserving female genetics. To date, ovarian tissue has only been vitrified using a standard protocol, with immediate analyses after warming, therefore, long-term cryoinjury is unknown. Long-term cryoinjury was investigated here by in-ovo culturing, fresh (non-vitrified), a purposefully suboptimal poor vitrification (PV), and the standard vitrified (StV) protocol. Assessments were performed via cellular morphological changes and mRNA gene expression differences, immediately (day 0) or after 2, 4, or 6 days of in-ovo culturing. On day 0, the mRNA levels of heat-shock protein A2 (HSPA2) were lowest in the fresh tissue, and increased 5-fold in the StV treatment, and 18-fold in the PV treatment. Whereas, by day 6, growth determining factor 9 (GDF9) mRNA levels within the fresh tissue were over 3-fold and 21-fold higher than StV and PV treatments, respectively. After 6 days of in-ovo culture the follicle density was highest in the fresh ovarian tissue (4701 ± 950 #/mm3), followed by the StV (1601 ± 300 #/mm3), with PV having the lowest density (172 ± 145 #/mm3). This shows that although the density of follicles was higher in StV versus PV, a considerable number (∼65 %) were lost compared to the fresh treatment. Additionally, the HSPA2 expression could be an early screening tool, whereas GDF9 expression could be a late screening tool, used to assess turkey ovarian tissue vitrification protocols. We conclude that the StV protocol should be further optimized to try and improve follicle numbers post-warming.

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

The aim of this study was to investigate the impact of processing human ovarian tissue on follicle activation dynamics. 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. 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. 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.

P-430 Characterization of secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue

Abstract Study question Can we obtain secondary follicles from cultured cryopreserved-thawed human cortical tissue? Summary answer We obtained a comparable percentage of secondary follicles after culture of cryopreserved-thawed human cortical tissue to that reported after culture of fresh human cortical tissue. What is known already The complete in vitro maturation of oocytes starting from primary oocytes (present in unilaminar follicles) until mature MII oocytes has been achieved previously by two different multi-step culture protocols. These protocols were applied to culture fresh ovarian cortical tissue from adult cisgender donors. However, the efficiency of these two protocols for growing unilaminar follicles to secondary follicles starting from cryopreserved cortical tissue from cisgender patients has not been investigated. As the ovarian tissue available for fertility preservation is cryopreserved, it is important to understand the potential of cryopreserved unilaminar follicles to mature in vitro. Study design, size, duration Cryopreserved ovarian cortical tissue from 4 cisgender adult donors was used for in vitro culture. According to the existing culture protocols, cortical ovarian tissue was cultured either for 8 days using the first step medium (Telfer’s medium) reported by M.McLaughlin 2018 (doi: 10.1093/molehr/gay002) or for 7 and 21 days using the first step medium (Xu’s medium) reported by Xu 2021 (doi: 10.1093/humrep/deab003). Participants/materials, setting, methods Ovarian cortical tissue obtained from adult cisgender donors undergoing oophorectomy for fertility preservation purposes was cryopreserved before chemotherapy. The cryopreserved ovarian cortical tissue was thawed and cut into small pieces. Several pieces were fixed immediately (day 0), the others were either cultured in Telfer’s medium or Xu’s medium. After culture, follicle survival, growth and morphology were assessed by histology and immunofluorescence. Main results and the role of chance We performed quantification of the different follicular stages (primordial, primary, secondary and atretic) after culture and observed that the percentage of secondary follicles increased independently of the culture media used. However, the ovarian cortical tissue cultured using Telfer’s protocol resulted in a higher percentage of secondary follicles and lower percentage of atretic follicles compared to that using Xu’s protocol. After culture, the ovarian cortical tissue was further immunostained for TUNEL, PCNA, COLLIV, STAR, AMH and KRT19. We observed that secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium showed more proliferative (PCNA+) FOXL2+ granulosa cells and less apoptotic (TUNEL+) stromal cells. By contrast, ovarian cortical tissue cultured in Xu’s medium showed less proliferative (PCNA+) FOXL2+ granulosa cells and more apoptotic (TUNEL+) stromal cells. Moreover, the expression of AMH was high in granulosa cells of secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium and low in Xu’s medium. Finally, in both Telfer’s and Xu’s medium cultures the expression of KRT19 was low in granulosa cells of secondary follicles present in ovarian cortical tissue. Limitations, reasons for caution The number of donors was limited and the study lacked fresh ovarian cortical tissue as control. Only the first step in Telfers’ and Xu’s protocol was performed, hence further culture is required to determine whether complete maturation of oocytes in vitro is possible starting from cryopreserved human cortical tissue. Wider implications of the findings Our study showed evidence of follicular growth in cryopreserved-thawed human ovarian cortical tissue after a period of in vitro culture. This is an important first step to achieve in vitro maturation of oocytes from cryopreserved-thawed human ovarian cortical tissue that could be used for clinical applications. Trial registration number NOT APPLICABLE

Effect of induced molting on ovarian function remodeling in laying hens

Induced molting (IM) can restore the laying rate of aged laying hens to the peak level of laying and rejuvenate ovarian function for the second laying cycle. To explore the mechanism of ovarian function remodeling during IM in laying hens, in this study, ninety 71-wk-old laying lady hens with 60% laying rate and uniform weight were selected for molting induction by fasting. Samples (serum and fresh ovarian tissue) were collected on the day before fasting (F0), the 3rd and 16th days of fasting (F3, F16), and the 6th, 15th, 32nd days of refeeding (R6, R15, and R32), and the number of follicles in each period was counted. Then, the reproductive hormone levels in serum and antioxidant levels in ovarian tissues were detected at different stages, and the gene expression of the KIT-PI3K-PTEN-AKT pathway and GDF-9 in ovaries was measured by qRT-PCR. The results showed that the laying rate increased rapidly after refeeding and returned to the prefasting level by R32. At F16 and R6, the number of mature follicles significantly decreased; the number of primary and secondary follicles significantly increased; the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (P4) in serum decreased; the relative expression of KIT, PI3K, AKT, and GDF-9 significantly increased; and that of PTEN significantly decreased. At R15 and R32, except for GDF-9, which maintained a high expression state, other indicators showed opposing trends to those observed at F16 and R6. In conclusion, IM activated the KIT-PI3K-PTEN-AKT signaling pathway and promoted the activation of primordial follicles during the fasting period and early resumption of feeding; gonadotropin secretion increased gradually, which promoted the rapid development of primary and secondary follicles to mature follicles and ovulation. This study explained the mechanism of ovarian function remodeling in the process of IM and provided a theoretical basis for improving the ovarian function of laying hens and optimizing the IM program.

Metabolic and secretory recovery of slow frozen-thawed human ovarian tissue in vitro.

Within the options available for fertility preservation, cryopreservation of ovarian cortical tissue has become an important technique. Freezing and thawing procedures have been optimized to preserve tissue integrity and viability. However, the improvement of the tissue retransplantation is currently of great interest. Rapid angiogenesis is needed at the retransplantation site to accomplish sufficient blood supply to provide oxygen and nutrients. Many studies address this issue. However, we need to understand the physiology of the thawed tissue to gain further understanding of the complexities of the procedure. As freezing and thawing generally impairs cellular metabolism, we aimed to characterize the changes in metabolic activity and secretion of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) of frozen-thawed ovarian cortical tissue over time. Biopsy punches of ovarian cortical tissue from patients undergoing fertility preservation were maintained in culture without freezing or after a slow-freezing and thawing procedure. VEGF-A secretion was measured after 48 h by ELISA. To examine temporary changes, metabolic activity was assessed for both fresh and frozen-thawed tissue of the same patient. Metabolic activity and VEGF-A secretion were measured at 0, 24 and 48 h in culture. Thawed ovarian cortical tissue secreted significantly less VEGF-A compared to fresh ovarian cortical tissue within 48 h of culture. After thawing, metabolic activity was significantly reduced compared to fresh ovarian cortex but over the course of 48 h, the metabolic activity recovered. Similarly, VEGF-A secretion of thawed tissue increased significantly over 48 h. Here, we have shown that it takes 48 h for ovarian cortical tissue to recover metabolically after thawing, including VEGF-A secretion.

P-437 Follicle activation in human ovarian tissue: impact of freezing, culture and grafting

Abstract Study question How does freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) affect follicle activation through the PI3K pathway in human ovarian tissue? Summary answer Freezing does not trigger further follicle activation. Although IVC and CAM do induce follicle activation, the process is significantly curbed in the CAM group. What is known already While massive follicle activation has been shown to occur through the PI3K pathway in frozen-thawed human ovarian tissue after both short-term xenotransplantation and IVC, it remains poorly understood in fresh ovarian tissue, raising many questions about the effect of freezing on follicle activation. Furthermore, grafting ovarian tissue to CAM has been found to limit follicle activation in animal ovarian tissue, though its impact on human follicles is yet to be elucidated. Study design, size, duration Thirty fresh cortical fragments from 5 patients and 50 frozen-thawed tissue pieces from another 5 patients were investigated. We first compared fresh fragments (fresh-IVC) with frozen-thawed tissue (FT-IVC) by IVC. These cultured frozen-thawed fragments were then examined against frozen-thawed fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4x2x1 mm) were analyzed on day (D) 0, 1 and 6. Participants/materials, setting, methods Follicle analyses included histology (count and classification), transmission electron microscopy (ultrastructure), immunohistochemistry (Ki67 for follicle proliferation, c-caspase-3 for follicle apoptosis, LC3B for follicle autophagy, p-AKT for PI3K activation and p-rpS6 for PI3K/mTOR activation), and immunofluorescence (FOXO1 for PI3K/FOXO activation). Subcellular localization of FOXO1 was determined in primordial follicles on high-resolution images using structured illumination microscopy. Droplet digital PCR further investigated expression of PI3K pathway-related genes (TSC1 and CDKN1B) and oocyte growth-related genes (GDF9 and LHX8). Main results and the role of chance No differences were detected between the fresh-IVC and the FT-IVC groups in any conducted analyses. After both IVC and CAM grafting, a significant decrease was observed in primordial follicle proportions in all groups on D6 vs D0 (p &amp;lt; 0.01), associated with a substantial increase in intermediate follicle proportions (p &amp;lt; 0.05). In the FT-CAM group, however, the percentage of primordial follicles stabilized between D1 and D6, showing significantly higher rates than the FT-IVC group on D6 (p = 0.04) and reflecting better preservation of the primordial follicle pool in the FT-CAM group. While apoptotic follicle rates increased in all conditions (D0 vs D6, p &amp;lt; 0.03), they remained significantly lower in the FT-CAM group than in the FT-IVC group on D6 (p = 0.01), confirming superior follicle preservation in CAM-grafted tissue. In primordial follicles, significantly lower levels of p-rpS6 were noted over time in all conditions (D0 vs D6, p &amp;lt; 0.009), but there was a significant shift in FOXO1 to a nuclear localization in the FT-CAM group compared to the FT-IVC group on D1 (p = 0.03), indicating inhibition of follicle activation. Limitations, reasons for caution As demonstrated by our results, the CAM model appears to be effective at preserving the follicle pool. However, our study did not investigate the underlying mechanisms behind this improved follicle preservation after thawing. Wider implications of the findings Our data confirm that the freezing procedure itself does not cause follicle burnout. Moreover, the CAM model looks to be capable of preserving the follicle pool, hypothetically thanks to rapid revascularization and circulating embryonic anti-Müllerian hormone. Trial registration number Not applicable

P-445 Recovery of metabolic activity and VEGF secretion after thawing of cryopreserved, slow frozen ovarian tissue

Abstract Study question Is slow-frozen ovarian cortical tissue limited in its metabolical activity after thawing, especially with respect to VEGF secretion? Summary answer Slow-frozen ovarian cortical tissue showed decreased metabolic activity and VEGF directly after thawing, but recovered in the course of 48h in in vitro culture. What is known already Cryopreservation of ovarian cortical tissue has become an important method in fertility preservation. Freezing and thawing processes had been optimized to preserve tissue viability. However, the major obstacle of retransplantation is the rapid angiogenesis to accomplish sufficient blood supply. As freezing and thawing entails constraints in cellular metabolic activity, we aimed to assess the metabolic activity and the secretion of the angiogenic factor VEGF of frozen-thawed ovarian cortical tissue over time. Study design, size, duration Ex vivo gained ovarian tissue was cultured over 48h. Thereby, non-frozen fresh tissue was compared to slow-frozen-thawed ovarian tissue. Participants/materials, setting, methods Provided a signed consent, biopsies of ovarian tissue from patients undergoing fertility preservation were cultured without freezing (n = 13) or after slow-freezing and thawing (n = 12). VEGF secretion during 48h was measured by ELISA. Applying CellTiterBlue assay, metabolic activity was assessed in biopsies without freezing or after thawing from the same patients (n = 4-7) after 0, 24 and 48h. Similarly, VEGF secretion was measured after 0, 24 and 48h as well (n = 7) to capture temporarily changes. Main results and the role of chance Although VEGF secretion varies among patients and biopsies, there was a significant lower VEGF content in supernatant from frozen-thawed tissue compared to fresh ovarian cortical tissue after 48h of culture (p &amp;lt; 0.01). Directly after thawing and 24h thereafter, metabolic activity was significantly reduced (p &amp;lt; 0.05) compared to fresh ovarian cortex. In the course of 48h, the metabolic activity recovered (p &amp;lt; 0.01). Similarly, VEGF secretion increased dramatically during 24h after thawing (p &amp;lt; 0.05). Limitations, reasons for caution This in vitro study might not resemble the metabolic and secretory recovery after retransplantation. Wider implications of the findings We showed that ovarian tissue recovers metabolically over 48h. This recovery included an increase in VEGF secretion. As latter induces angiogenesis a culture step after thawing might support VEGF secretion and angiogenesis. However, the recovery period could also be useful as a temporal bridge until sufficient supplies are available. Trial registration number not applicable

Raman spectroscopic study of benign and malignant ovarian tissues

Ovarian cancer is one of the most common female tumors all over the world, and its mortality rate ranks first among gynecological malignancies. A progressive assessment, diagnosis and remedy are significant for the proper management of the disease process. In this paper, a prompt, non-invasive and highly effective screening test for ovarian cancer was developed based on the Raman spectroscopy (RS) data of fresh ovarian tissues. Raman spectral measurements were performed on fresh ovarian tissue samples from 17 ovarian cancer patients and 14 benign ovarian tumors. We preliminarily identified the Raman peaks in the measured ovarian tissue spectra and summarized their respective characteristic peaks, indicating that specific biomolecules changed among different groups, and their differences were analyzed. The conclusions suggested that the position of the characteristic peaks of Raman spectrum of the ovarian cancer tissues and benign ovarian tissues were different. The relative intensity of ovarian cancer tissue was higher than that of benign ovarian tissue at the 1004, 1155, 1446 cm−1 with phenylalanine, protein and lipids as characteristic peaks, and the difference were significant (P < 0.05). This exploratory work demonstrates that RS may be used as a detection method for screening benign and malignant ovarian tumors.

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Abstract Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies. Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturation in vitro. In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test. Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became mature in vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile. Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse's body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

Fresh and cryopreserved ovarian tissue transplantation for preserving reproductive and endocrine function: a systematic review and individual patient data meta-analysis.

Ovarian tissue cryopreservation involves freezing and storing of surgically retrieved ovarian tissue in liquid or vapour nitrogen below -190°C. The tissue can be thawed and transplanted back with the aim of restoring fertility or ovarian endocrine function. The techniques for human ovarian tissue freezing and transplantation have evolved over the last 20 years, particularly in the context of fertility preservation in pre-pubertal cancer patients. Fresh ovarian tissue transplantation, using an autograft or donor tissue, is a more recent development; it has the potential to preserve fertility and hormonal function in women who have their ovaries removed for benign gynaecological conditions. The techniques of ovarian tissue cryopreservation and transplantation have progressed rapidly since inception; however, the evidence on the success of this intervention is largely based on case reports and case series. The aim of this study was to systematically review the current evidence by incorporating study-level and individual patient-level meta-analyses of women who received ovarian transplants, including frozen-thawed transplant, fresh or donor graft. The review protocol was registered with PROSPERO (CRD42018115233). A comprehensive literature search was performed using MEDLINE, EMBASE, CINAHL and Cochrane Central Register of Controlled Trials from database inception to October 2020. Authors were also contacted for individual patient data if relevant outcomes were not reported in the published manuscripts. Meta-analysis was performed using inverse-variance weighting to calculate summary estimates using a fixed-effects model. The review included 87 studies (735 women). Twenty studies reported on ≥5 cases of ovarian transplants and were included in the meta-analysis (568 women). Fertility outcomes included pregnancy, live birth and miscarriage rates, and endocrine outcomes included oestrogen, FSH and LH levels. The pooled rates were 37% (95% CI: 32-43%) for pregnancy, 28% (95% CI: 24-34%) for live birth and 37% (95% CI: 30-46%) for miscarriage following frozen ovarian tissue transplantation. Pooled mean for pre-transplant oestrogen was 101.6 pmol/l (95% CI: 47.9-155.3), which increased post-transplant to 522.4 pmol/l (95% CI: 315.4-729; mean difference: 228.24; 95% CI: 180.5-276). Pooled mean of pre-transplant FSH was 66.4 IU/l (95% CI: 52.8-84), which decreased post-transplant to 14.1 IU/l (95% CI: 10.9-17.3; mean difference 61.8; 95% CI: 57-66.6). The median time to return of FSH to a value <25 IU/l was 19 weeks (interquartile range: 15-26 weeks; range: 0.4-208 weeks). The median duration of graft function was 2.5 years (interquartile range: 1.4-3.4 years; range: 0.7-5 years). The analysis demonstrated that ovarian tissue cryopreservation and transplantation could restore reproductive and hormonal functions in women. Further studies with larger samples of well-characterized populations are required to define the optimal retrieval, cryopreservation and transplantation processes. Ovarian tissue cryopreservation and transplantation may not only be effective in restoring fertility but also the return of reproductive endocrine function. Although this technology was developed as a fertility preservation option, it may have the scope to be considered for endocrine function preservation.

Comparison between two cryopreservation techniques of human ovarian cortex: morphological aspects and the heat shock response (HSR).

This study was tailored to compare the cryopreservation of the human ovarian cortex using closed metal container vitrification or the slow-freezing technique. Superficial ovarian cortical tissue biopsies were collected from 12 participants who underwent gynaecological videolaparoscopy. The fragmented samples were allocated to three experimental conditions: (a) fresh ovarian tissue, (b) slow-freezing, and (c) vitrification with a metal closed container. After thawing or rewarming, cellular morphological analyses were performed to determine tissue viability. The cellular response to thermal stress was measured by a putative increase in the immune quantification of the heat shock protein 70kDa (heat shock protein 70kDa response - HSR) after a heat challenge (2h exposure at 42°C). Both the total number of intact follicles and the frequency of primordial follicles were higher in fresh ovarian tissue than in the preserved samples, regardless of the technique employed. There was a trend towards an increase in the absolute number of intact follicles in the tissue preserved by vitrification. After cryopreservation, a higher HSR was obtained after slow-freezing. These results indicate that both cryopreservation techniques present advantages and may be used as alternatives to ovarian tissue cryopreservation.

Autologous transplantation of fresh ovarian tissue in the ICR mice model

SUMMARY&#x0D; During the past 20 years, autologous transplantation of either frozen ovarian tissue for cancer patients or ovarian tissue pretreated with curative medications for premature ovarian insufficiency (POI) patients have been achieved worldwide. POI is characterized by amenorrhea before 40 years of age. As a result, juvenile cancer patients and POI patients have increased chances to give healthy birth based on this achievement. It also indicates that mastering the skills of ovarian auto-transplantation is required for researchers and doctors. In this study, we performed orthotopic auto-transplantation of fresh ovarian tissues by transplanting unilateral half ovarian tissue to the contralateral ovary in the ICR (Institute of Cancer Research) strain of outbred, heterogeneous mice to determine if the transplanted tissue could be functional. The transplantation surgery entailed taking partial ovarian tissue from the unilateral ovary of the adult ICR female mouse and then transplanting into the ovarian sac of the contralateral ovary, in which part of the ovarian tissue had been removed in advance. One week after surgery, we found that the freshly transplanted mouse ovarian tissue survived and functional, as histochemical and immunofluorescence assays have shown that not only both follicles at different developing stages and corpus luteum are available, but the morphology of them are properly maintained within the transplanted tissue. This study approved that for those who are interested in ovarian transplantation study, applying mouse model to practice ovarian tissue auto-transplantation is reliable and easy handled.

UNRAVELING THE MECHANISMS THAT DRIVE THECA INTERNA DIFFERENTIATION

Within developing ovarian follicles, oocyte maturation requires coordinated interplay between multiple specialized cells: the oocyte, granulosa cells (GCs), and enveloping stromal cells (mainly the theca externa and interna that provide essential structural and steroidogenic support, respectively). Although numerous studies have focused on explaining the interactions that occur within the oocyte and its surrounding GCs throughout maturation, the mechanisms governing theca interna (TI) specification remain elusive. In this study, we aim to identify the molecular pathways that drive the differentiation of stromal cells into TI, and to characterize and localize intermediate cell types that exist along this trajectory. Stromal (CD55+) and TI (ANPEP+) cells were prospectively sorted from different size antral follicles (n=4, 2-4mm) derived either from fresh ovarian tissue or human ovarian xenografts. Samples were submitted for single-cell RNA sequencing (scRNASeq) and analyzed using the Seurat package in R. Immunolabeling studies were done on cryosections of fresh ovarian tissue. Computational analyses allowed us to delineate a gradual progression from an initial stromal progenitor identity toward a steroidogenic TI cell fate. We identified the transcription factor NR5A1/SF1 as a potential driver of commitment toward steroidogenic cell fate, as its graduated increase correlates with upregulated transcription of steroidogenic genes canonically associated with TI Identity, such as LHCGR, STAR, INSL3, and CYP17A1. This trajectory is also supported by the expression of effectors of Hedgehog (Hh) signaling, which animal studies have identified as an essential mediator of theca differentiation. To attribute temporal information to the inferred differentiation trajectory of TI cells, we used immunolabeling on cryosections of developing follicles and observed TI precursors in the stroma adjacent to transitioning primordial follicles, while definitive TI cells first emerged next to the basement membrane of secondary follicles. At the antral stage, these populations display a heterogeneous distribution within the theca layer, suggesting crosstalk as the follicle develops. Using scRNASeq of stromal populations adjacent to antral follicles, we were able to construct a high-resolution trajectory of differentiation, starting from stromal progenitor cells to steroidogenic TI cells. In part, this trajectory seems to be driven by the transcription factor SF1 and the Hh signaling pathway. We were able to localize discrete populations described along this trajectory to different stages of follicle development, suggesting that TI differentiation is dynamic and intricately related to follicle development.

AUTOLOGOUS CRYOPRESERVED OVARIAN TISSUE TRANSPLANTATION (ACOTT): AN UPDATE FROM OUR PREVIOUS META-ANALYTICAL DATASET AS OF MARCH 2021

Ovarian cryopreservation followed by auto-transplantation has recently been removed from the experimental category for fertility preservation (FP) by the ASRM. However, the current success rates of this FP approach are unknown. Therefore, we sought to perform a meta-analysis with the use of individual patient data, where available, to determine the success rates of autologous cryopreserved ovarian tissue transplantation (ACOTT) with previously cryopreserved tissue. Meta-analysis was performed by reviewing data from 2000, when the first ACOTT procedure was reported, to March 2021. Additional cases were retrieved from meeting abstracts and own database with 7 cases. We selected studies that reported ACOTTs with previously banked tissue in humans. We did not include any cases involving fresh ovarian tissue transplantation or those performed to treat idiopathic premature ovarian failure/insufficiency. Both authors reviewed and selected studies for eligibility, which resulted in 89 full-text studies assessed for eligibility. Cases were extracted from original reports and reviews by the junior author, and the senior author reviewed and verified the extracted data. 42 reports were included for qualitative synthesis. The age at ovarian cryopreservation was reported in 206 women and the mean is 29.26 years (range: 9-40 years, SD: 5.36) with women undergoing the first ACOTT at the mean age of 32.52 (data available in 177 cases, range: 13-45 years, SD: 5.31). In 11 new studies and personal correspondence with authors, detailed data were available to determine live birth plus ongoing (LB + OG) pregnancy and in 12 new studies to calculate endocrine restoration rate. 322 new ACOTTs were performed in 263 new patients, resulting in the birth of 63 children and 11 OG pregnancies. Current cumulative LB+OG pregnancy rate is 26,9%. Since 20002, 518 women had undergone 631 ACOTTs, what resulted in 141 LB plus 11 OG pregnancies (145 singleton and 7 twin pregnancies, respectively) and 148 babies were born. To calculate ovarian activity recovery rate, we identified 315 patients where the information about endocrine function after ACOTT was available. Among those, 241 (76.50%) regained ovarian function with mean graft longevity of 34.49±25.63 months (range: 0-144). ACOTT is a successful procedure for ovarian function restoration, however, there is a need for further improvement in pregnancy rates and longevity.

The Mechanism by Which Chemotherapy with use of Alkylating Agents Cause Follicular Activation: Is there any Further Mode for the Loss of the Primordial Follicles? A Short Communication

A lot of women In their reproductive life go through treatment with a lot of chemotherapeutic drugs of which the alkylating agents which are inclusive of cyclophosphamide, busulphan, along with dacarbazine, more specifically cyclophosphamide, whose utilization is considered for a lot of chemotherapy protocols like In breast cancer, In cases of chronic lymphoblastic leukaemia in children and we encounter survivors of these cancers with usual manifestation of Premature ovarian insufficiency(POI). Thus detailed evaluation was attempted by Shai et al., In case of human ovaries immediately In which fresh ovarian tissue that was obtained from ladies following receipt of alkylating agents, non-alkylating agent in addition to that collected from those who did not get treatment. They corroborated the existence of significant depletion of primordial follicles (PMF’s), in addition to significant escalation of the absolute quantities of growing follicles in contrast to ovaries collected from those who did not get treatment. Staining done for FOXO3A demonstrated reduction in the nuclear existence in the PMF oocytes in cases where ovaries removed were from patients receiving treatment with alkylating agent in the ultra-short period following alkylating agents. No escalation in the expression of the cleaved caspase 3 was observed in PMF’s within the ovaries that got alkylating agents or non-alkylating agents exposure. Marked stromal fibrosis in addition to neovascularization was seen in alkylating agents treatment group only following loss of PMF had occurred earlier (4-6mths). This is a great study despite them unable to demonstrate the phosphorylated FOXO3A. Although it was anticipated that they would further show the part of PI3K/Akt pathway that possesses major part in the follicular activation although they explained in view of immediate preservation of ovaries In formaldehyde in addition to embedding In paraffin. Thus this study goes a long way in explaining the cause of follicular loss following treatment with alkylating agents and aid in the strategies we need to use to avoid the same once feasible.

P–436 Identification of ovarian cell subpopulations by multicolor flow cytometry and its potential impact on ovarian reconstruction programs

Abstract Study question How could multicolor flow cytometry (MFC) help to identify ovarian subpopulations that could be used for ovarian reconstruction with isolated follicles? Summary answer MFC is useful to identify ovarian cell subpopulations in the ovarian cortex. What is known already Ovarian tissue cryopreservation is a fertility preservation option for women before gonadotoxic chemo- and/or radiotherapy. However, graft of cryopreserved ovarian tissue must be performed with caution in women suffering from malignancies that may metastasize to the ovaries. For this purpose, functional ovarian tissue qualification is essential to identify ovarian cell subpopulations that could be used for ovary reconstruction in combination with isolated follicles. Furthermore, ischemic tissue damage occurring after the graft is currently another important issue to be resolved for successful ovarian reuse. Study design, size, duration We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Then, we used MFC for the identification of different cell subpopulations in the cell suspension thus obtained. Participants/materials, setting, methods Human ovarian tissues from patients undergoing surgery for polycystic ovary syndrome were used in this study. Biopsies of ovarian cortex (fresh or frozen-thawed) were dissociated using an automated dissociation method. We used FVS780 and SYTO13 markers to gate viable ovarian cells by MFC. Variable markers were chosen to differentiate and identify cell subpopulations among the viable ovarian cells. Main results and the role of chance The dissociation yield was on average 1.59 ± 1.58 x 106 and 0.78 ± 0.72 x 106 viable ovarian cells per 100 mg of fresh (n = 17) and frozen-thawed (n = 43) ovarian cortical tissue, respectively. On average, 35.4 ± 13.1% of viable ovarian cells were CD34 + (n = 61, stromal phenotype). Concerning endothelial phenotype, 7.8 ± 5.5% of CD31+ cells (n = 51) and 5.3 ± 3.6% of CD144+ cells (n = 29) were identified among viable ovarian cells. Vimentin marker is found in 25.6 ± 10.8% of viable ovarian cells (n = 23) and CD326 (EpCAM expression) in 0.6 ± 0.8% (n = 16). Finally, pericyte phenotype (CD34-/Vimentin-/CD31-/CD146+/ CD140b+) was identified in 4.6 ± 4.3% of viable ovarian cells (n = 7). Limitations, reasons for caution We do not know how these ovarian cell subpopulations could be a factor associated or not with time for ovarian function recovery in vivo after ovarian tissue graft and the impact of these ovarian cells on the ovarian microenvironment of an artificial ovary. Wider implications of the findings: Functional qualification of ovarian tissue can be performed by MFC. MFC is a promising tool for ovarian cortex qualification before reuse of cryopreserved ovarian tissue. Cell sorting could be used to separate and isolate cell subpopulations and add these cells with isolated follicles in an ovarian reconstruction program. Trial registration number Not applicable

O-178 Reproductive and endocrine outcomes after fresh and frozen-thawed ovarian tissue transplantation based on age and anti-cancer therapy: A systematic review and individual patient data meta-analysis

Abstract Study question Do reproductive and endocrine outcomes from fresh and frozen-thawed ovarian transplants differ based on age and anti-cancer therapy before cryopreservation? Summary answer There was a significant difference in reproductive outcomes of women who have their tissue cryopreserved before or at the age of 35 years. What is known already Ovarian tissue cryopreservation (OTC) and transplantation is emerging as a new fertility preservation method. Despite being available for two decades, there is a marked variation in the delivery of this procedure worldwide. Most of the data are based on case reports from specialised centres with expertise in providing this procedure, but there are many unreported cases. Through this review, we aim to collate reproductive and endocrine outcomes from ovarian tissue transplantation. In particular the outcomes in women based on age at cryopreservation and whether they had anti-cancer therapy before cryopreservation were explored. Study design, size, duration This study was a systematic review and individual participant level meta-analysis to synthesize the existing evidence on the use of fresh and cryopreserved ovarian tissue transplantation. The review protocol was registered with PROSPERO (CRD42018115233) in November 2018 and the review was concluded in December 2020, including 87 studies (768 women). Participants/materials, setting, methods Literature search was performed using MEDLINE, EMBASE, CINAHL and Cochrane Central Register of Controlled Trials from inception to October 2020. After screening 20,566 abstracts, 87 studies (768 women) were included in the review. Patient-level data was extracted for 388 women and study-level data for 380 women. Authors were also contacted for data if relevant outcomes were not reported in published manuscripts. Meta-analysis was performed using inverse-variance weighting to calculate summary estimates using a fixed-effects model. Main results and the role of chance Age at cryopreservation was provided for 319 out of 388 (82%) women at participant level data. Of these, 283 (88.7%) had ovarian tissue retrieved at ≤ 35 years of age. A subgroup of four studies that reported data on participants age at cryopreservation and transplantation were included in meta-analysis. Pregnancy rates were higher in participants at ≤ 35 years of age at cryopreservation, with results being statistically significant (OR, 0.35; 95% CI: 0.13 to 0.92; z = 2.13; P 0.03, I2= 0%). Return of hormonal function shown as a decrease in FSH (IU/L) was also lower in this group (MD, 4.38; 95% CI: -4.29 to 13.05; z = 0.99; P 0.32, I2= 0%. Whether a participant had received chemotherapy before cryopreservation was explicitly reported in 122 out of 388 (31%) participants and 56 of them (46%) had received anti-cancer treatment before OTC. Thirty-five pregnancies and twenty-four live births were reported in these women. A further meta-analysis from 5 studies showed that although the results were not statistically significant for return of endocrine function, a decrease in FSH, an increase in oestrogen and increased pregnancy rates were noted in participants who did not receive anti-cancer therapy before cryopreservation. Limitations, reasons for caution Although we gathered 768 cases of ovarian transplants published in the literature, most were case reports and therefore not included in the meta-analysis. Of the studies included in the meta-analysis, information such as age and anti-cancer therapy were not always provided for individual participants but as an aggregate. Wider implications of the findings There was no difference in reproductive and endocrine outcomes for anti-cancer therapy before OTC. Previous chemotherapy alone should therefore not be a deterrent in offering young girls and women OTC. Furthermore, the ideal age to achieve higher pregnancy and live birth rates from OTC is less than 35 years. Trial registration number PROSPERO (CRD42018115233)

An experimental study on autologous transplantation of fresh ovarian tissue in sheep

ObjectiveTo investigate current autologous transplantation methods and sites of ovarian tissues in sheep. MethodsSheep ovaries were resected. Ovarian cortices were sliced and transplanted orthotopically into the ovarian mesangial latum and heterotopically into the greater omentum and under groin skin. The grafts were removed two months after transplantation and examined to evaluate the survival of follicles (hematoxylin-eosin staining) and help determining feasible graft sites and transplantation methods. ResultsGraft nodules were found in the transplanted sites. HE staining of the grafts showed that multiple primordial follicles were able to survive in the grafts on both sides of the ovarian mesangial latum, the right side of the greater omentum, and the left inguinal subcutaneous tissue. Secondary or cystic follicles were found in almost all of the grafts. ConclusionThe ovarian mesangial latum, the greater omentum and the inguinal subcutaneous tissue can be used as autologous transplantation sites, where sheep ovarian tissue can survive and the follicles grow and develop in good condition.

Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. N/A.

Ovaries of patients recently treated with alkylating agent chemotherapy indicate the presence of acute follicle activation, elucidating its role among other proposed mechanisms of follicle loss

To investigate mechanisms of primordial follicle (PMF) loss invivo in human ovaries shortly after alkylating agent (AA) chemotherapy. Cohort study. Tertiary university medical center. Ninety-six women aged 15-39 years who underwent ovarian tissue cryopreservation for fertility preservation. Fresh ovarian tissue samples were harvested from women treated with AA (n = 24) or non-AA (n = 24) chemotherapy <6 months after treatment and age-matched untreated women (n = 48). Differential follicle counts, time from chemotherapy exposure, immunostaining for apoptosis (cleaved caspase-3) and FOXO3A on tissue harvested within ultrashort time intervals (4-12 days), collagen (Sirius red) and neovascularization (CD34). AA-treated ovaries had significant loss of PMFs, and significant increase in absolute numbers of growing follicles compared with untreated control ovaries. The number of growing follicles was inversely correlated with time from chemotherapy. Representative staining for FOXO3A observed decreased nuclear localization in PMF oocytes in AA-treated ovaries removed within the ultrashort time interval compared with untreated ovaries. Neither significant loss of PMFs, increase in growing follicles, nor decrease in nuclear FOXO3A were observed in non-AA-treated ovaries. No increased expression of cleaved caspase-3 was seen in PMFs within the ultrashort time interval after AA or non-AA chemotherapy. Significant stromal fibrosis and neovascularization were observed in AA-treated ovaries only after follicle loss had already occurred (4-6 months). Follicle activation occurs invivo in ovaries of patients treated with AA, indicating a pathologic mechanism which may contribute to chemotherapy-induced follicle loss.