Quantification of follicles in human ovarian tissue using image processing software and trained artificial intelligence†.

Cancer survival rates in prepubertal girls and young women have risen in recent decades due to increasingly efficient treatments. However, many such treatments are gonadotoxic, causing premature ovarian insufficiency, loss of fertility, and ovarian endocrine function. Implantation of donor ovarian tissue encapsulated in immune-isolating capsules is a promising method to restore physiological endocrine function without immunosuppression or risk of reintroducing cancer cells harbored by the tissue. The success of this approach is largely determined by follicle density in the implanted ovarian tissue, which is analyzed manually from histologic sections and necessitates specialized, time-consuming labor. To address this limitation, we developed a fully automated method to quantify follicle density that does not require additional coding. We first analyzed ovarian tissue from 12 human donors between 16 and 37 years old using semi-automated image processing with manual follicle annotation and then trained artificial intelligence program based on follicle identification and object classification. One operator manually analyzed 102 whole slide images from serial histologic sections. Of those, 77 images were assessed by a second manual operator, followed with an automated method utilizing artificial intelligence. Of the 1181 follicles the control operator counted, the comparison operator counted 1178, and the artificial intelligence counted 927 follicles with 80% of those being correctly identified as follicles. The three-stage artificial intelligence pipeline finished 33% faster than manual annotation. Collectively, this report supports the use of artificial intelligence and automation to select tissue donors and grafts with the greatest follicle density to ensure graft longevity for premature ovarian insufficiency treatment.

The role of the indoleamine 2,3-dioxygenase gene in preventing ovarian transplant rejection in rats†.

Indoleamine 2,3-dioxygenase (IDO) plays important roles in maternal immune tolerance. Female Sprague Dawley rats (9-11 weeks old) were randomly divided into an autoplastic transplantation group (n = 75) and an allograft transplantation group (n = 300) was further divided into subgroups of ovarian transplantation, allograft ovarian transplantation, allograft ovarian transplantation with cyclosporine A treatment, allograft ovarian transplantation and transfection with IDO-expressing lentiviruses, and allograft ovarian transplantation and transfection with control lentiviruses. IDO was successfully transfected into the transplanted ovarian tissue. The survival rate, success rate of ovarian transplantation, period until estrous cycle restoration, and estrogen levels of rats that received IDO-expressing lentiviruses were significantly different from those of rats that underwent allograft transplantation and with control transfection (all P < 0.05), but not significantly different from those rats that received autoplastic transplantation (all P > 0.05). The number of ovarian follicles in the transplanted ovarian tissue of rats that received IDO-expressing lentiviruses was also significantly higher. The expression level of IDO protein detected by immunohistochemistry and western blotting was especially high in ovaries that had received IDO-containing lentiviruses. Naturally pregnant rats were found in each group postoperatively. These results indicated that IDO-expressing lentiviruses were successfully transfected into transplanted ovarian tissues of rats and that IDO was stably expressed within a certain time. These findings suggest that the expression level of IDO protein is associated with an enhanced success rate of ovarian tissue transplantation and a short restoration period of endocrine function.

Exploring the effect of platelet-rich plasma on vascularization and survival of follicles in xenotransplanted human ovarian tissue.

RESEARCH QUESTION: Do platelet-rich plasma (PRP) products, specifically human platelet lysate (hPL) and umbilical cord plasma, enhance vascularization and follicular survival in human ovarian tissue transplanted to immunodeficient mice? DESIGN: Human ovarian tissue was transplanted to subcutaneous pockets in nude mice, followed by daily injections for 6 days of PRP or saline at the transplantation sites. After a grafting period of 3 and 6 days, vascularization was assessed using CD-31 quantification, and gene expression of angiogenic markers (VEGF/Vegf) together with apoptosis-related genes (BAX/BCL-2), oxidative stress markers (HMOX-1/Hmox-1) and pro-inflammatory markers (Il-1ß/Il-6/Tnf-α) was quantitively analysed. Follicle density was analysed in the grafts after 4 weeks. Additionally, a pilot study was conducted exploring the suitability of ultrasound scanning for assessing survival and vascularization in ovarian tissue xenografted to mice. RESULTS: Although there was a significant increase in the CD-31 area from day 3 to day 6 post-grafting, there were no significant differences between the hPL and control groups. Gene expression analysis revealed significant down-regulation of VEGF from day 3 to day 6 for both the hPL and control groups, and significant up-regulation of BAX/BCL-2 in the hPL group compared with the controls. The follicle density showed no significant differences in the hPL group and UCP groups compared with the controls. Furthermore, ultrasound biomicroscopy provided valuable insights into graft morphology, necrotic areas and blood flow, suggesting its potential as a monitoring tool. CONCLUSIONS: Despite the angiogenic properties of PRP, this study was unable to demonstrate a significant impact of hPL on vascularization or of hPL and UCP on follicular survival in xenotransplanted human ovarian tissue.

First report on successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe.

RESEARCH QUESTION: Cryopreservation of ovarian tissue is one feasible option to preserve female fertility prior to cancer treatment. The slow freezing protocol represents the current standard approach, while vitrification has been suggested as a promising alternative. This paper reports the follow-up and first successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe. DESIGN: After the patient received a diagnosis of breast cancer, ovarian tissue was removed laparoscopically and sent via overnight transportation to University Hospital Bonn for vitrification on site. The patient was treated with chemotherapy, leading to ovarian failure. After 2 years, retransplantation of the vitrified, rapid warmed tissue was conducted on site. RESULTS: Two months after grafting, the patient reported regular menstrual cycles. After 1 further month a clinical pregnancy occurred, which ended in a spontaneous abortion at the 8th week of pregnancy. Six months after grafting, another naturally conceived pregnancy was determined, resulting in the birth of a healthy boy 14 months after retransplantation of the ovarian tissue. CONCLUSIONS: Complementing the successful deliveries reported by the groups of Suzuki (Japan) and Silber (USA) regarding vitrified tissue, the current results confirm the high potential of this cryopreservation method in a clinical routine setting as an alternative approach to the widespread slow freezing method.

Modeling delay of age at natural menopause with planned tissue cryopreservation and autologous transplantation.

BACKGROUND: Ovarian tissue cryopreservation has been proven to preserve fertility against gonadotoxic treatments. It has not been clear how this procedure would perform if planned for slowing ovarian aging. OBJECTIVE: This study aimed to determine the feasibility of cryopreserving ovarian tissue to extend reproductive life span and delay menopause by autotransplantation near menopause. STUDY DESIGN: Based on the existing biological data on follicle loss rates, a stochastic model of primordial follicle wastage was developed to determine the years of delay in menopause (denoted by D) by ovarian tissue cryopreservation and transplantation near menopause. Our model accounted for (1) age at ovarian tissue harvest (21-40 years), (2) the amount of ovarian cortex harvested, (3) transplantation of harvested tissues in single vs multiple procedures (fractionation), and (4) posttransplant follicle survival (40% [conservative] vs 80% [improved] vs 100% [ideal or hypothetical]). RESULTS: Our model predicted that, for most women aged <40 years, ovarian tissue cryopreservation and transplantation would result in a significant delay in menopause. The advantage is greater if the follicle loss after transplant can be minimized. As an example, the delay in menopause (D) for a woman with a median ovarian reserve who cryopreserves 25% of her ovarian cortex at the age of 25 years and for whom 40% of follicles survive after transplantation would be approximately 11.8 years, but this extends to 15.5 years if the survival is 80%. As another novel finding, spreading the same amount of tissue to repetitive transplants significantly extends the benefit. For example, for the same 25-year-old woman with a median ovarian reserve, 25% cortex removal, and 40% follicle survival, fractionating the transplants to 3 or 6 procedures would result in the corresponding delay in menopause (D) of 23 or 31 years. The same conditions (3 or 6 procedures) would delay menopause as much as 47 years if posttransplant follicle survival is improved to 80% with modern approaches. An interactive Web tool was created to test all variables and the feasibility of ovarian tissue freezing and transplantation to delay ovarian aging (here). CONCLUSION: Our model predicts that with harvesting at earlier adult ages and better transplant techniques, a significant menopause postponement and, potentially, fertile life span extension can be achieved by ovarian tissue cryopreservation and transplantation in healthy women.

Safety of ovarian cryopreservation and transplantation in patients with acute leukemia: a case series.

BACKGROUND: With increased success, ovarian tissue cryopreservation has recently become a standard technique for fertility preservation. However, malignant cell introduction through ovarian tissue transplantation remains a major concern for patients with acute leukemias. OBJECTIVE: This study aimed to investigate the safety of performing autologous ovarian tissue transplantation in survivors of acute leukemia. STUDY DESIGN: Clinical, histopathological, and molecular data of 4 women with acute myeloid leukemia and 2 women with acute lymphoblastic leukemia who underwent ovarian tissue cryopreservation and transplantation were analyzed in this case series. Following cryopreservation of 66% to 100% of an ovarian cortex with a slow freezing method, all women received high-dose multiagent alkylating preconditioning chemotherapy for allogeneic hematopoietic stem cell transplantation. Before the ovarian tissue transplantation, (1) antral follicle counts, serum antimüllerian hormone and follicle-stimulating hormone levels were assessed to confirm primary ovarian insufficiency; (2) all recipients were cleared by their hematologist-oncologists; (3) representative cortical strips were screened for leukemia infiltration by histologic (hematoxylin and eosin staining), immunohistochemical (CD3, CD20, CD34, CD68, CD117, CD163, PAX-5, Tdt, lysozyme, and MPO), and molecular marker evaluation (BCR/ABL p190 and AML1/ETO) where appropriate. RESULTS: The median age was 20 years (interquartile range, 15-32) at ovarian tissue cryopreservation. Before undergoing hematopoietic stem cell transplantation, all patients received induction or consolidation chemotherapy that included cytarabine + daunorubicin or Berlin-Frankfurt-Munich-95 protocol and were in remission. The mean serum antimüllerian hormone was 1.9±1.7 ng/mL before ovarian tissue cryopreservation. In all cases, ovarian tissue screening for leukemic cells was negative. Ovarian transplantation was performed laparoscopically with or without robotic assistance, after a median of 74.5 months (interquartile range, 41-120) after ovarian tissue cryopreservation. Ovarian function resumed in all patients after a median of 3.0 months (range, 2.5-4.0), and 2 women had 1 live birth each. The median graft longevity was 35.5 months (interquartile range, 18-57) after ovarian tissue transplantation. After a median follow-up of 51 months (interquartile range, 20-74), all patients remained relapse-free. In 1 patient, the graft was removed during cesarean delivery and was negative for immunochemical leukemia markers. CONCLUSION: Our long-term follow-up demonstrated no evidence of disease relapse after ovarian tissue transplantation in patients with acute leukemia who received allogeneic hematopoietic stem cell transplantation. This safety profile may be explained by the fact that these patients are induced into remission by nongonadotoxic induction chemotherapy before undergoing ovarian tissue cryopreservation. We propose that ovarian tissue cryopreservation should not be excluded as a fertility preservation option for young women with leukemia who are due to receive preconditioning chemotherapy before allogeneic hematopoietic stem cell transplantation.

Erythropoietin effects on cryopreserved/transplanted cat ovarian tissue: A comparison of two incubation methods.

Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.

Heterotopic ovarian transplantation instead hormonal replacement therapy after radical hysterectomy for cervical cancer: case report and review of literature.

PURPOSE: Only a few case reports have described heterotopic ovarian tissue transplantation (OTT) with the only objective of restoring ovarian function. METHODS: Case report and review of the literature for reporting cases of heterotopic OTT with the only aim of restoring ovarian endocrine function. In a cancer survivor woman with a history of hysterectomy and bilateral oophorectomy for cervical cancer and because she poorly tolerated hormone replacement therapy (HRT), we performed a heterotopic OTT in a pelvic subcutaneous "pocket" after an OT cryostorage of 17 years. RESULTS: A cyclic ovarian endocrine function started 3 months after OTT with an immediate patient self-described improvement of her quality of life. A second OTT was performed 19 months after, due to hot flushes recurrence and FSH increase. Despite a cyclic endocrine function, progesterone levels have always been low, resulting in a relative hyperoestrogenism state. CONCLUSION: In the future, the indications of heterotopic OTT could be spread in alternative to HRT. However, our data suggest that the heterotopic graft environment is less favorable to corpus luteum development, and further studies are needed to assay the best site of heterotopic graft, the optimal number of ovarian cortex fragments to graft, and the potential risk of relapse in case of malignant residual disease.

Ovarian tissue cryopreservation for fertility preservation before hematopoietic stem cell transplantation in patients with sickle cell disease: safety, ovarian function follow-up, and results of ovarian tissue transplantation.

PURPOSE: To describe the experience of performing ovarian tissue cryopreservation (OTC) before hematopoietic stem cell transplantation (HSCT), among girls/women with severe sickle cell disease (SCD)(SS or S/ß0-thalassemia) who are, besides the usual surgical risk, at risk of SCD-related complications during the fertility preservation procedure for improving their counseling and management. METHODS: This retrospective study included 75 patients (girls/women) with SCD who have had OTC before myeloablative conditioning regimen (MAC) for HSCT. Characteristics of patients and data on OTC, ovarian status follow-up, and results of ovarian tissue transplantation (OTT) were collected in medical records. RESULTS: At OTC, the median (IQR 25-75; range) age of the patients was 9.6 (6.9-14.1; 3.6-28.3) years, 56/75 were prepubertal, and no SCD or surgery-related complications occurred. The median follow-up post-HSCT was > 9 years. At the last follow-up, among prepubertal patients at HSCT, 26/56 were ≥ 15 years old and presented with a premature ovarian insufficiency (POI), except 2, including the patient who had received an OTT to induce puberty. Eight were 13-15 years old and presented for POI. The remaining 22 patients were under 13. Among the 19 patients who were menarche at HSCT, 2 died 6 months post-HSCT and we do not have ovarian function follow-up for the other 2 patients. All the remaining patients (n = 15) had POI. Five patients had OTT. All had a return of ovarian function. One patient gave birth to a healthy baby. CONCLUSION: OTC is a safe fertility preservation technique and could be offered before MAC independent of the patient's age.

Current Fertility Preservation Steps in Young Women Suffering from Cancer and Future Perspectives.

Childhood cancer incidence, especially in high-income countries, has led to a focus on preserving fertility in this vulnerable population. The common treatments, such as radiation and certain chemotherapeutic agents, though effective, pose a risk to fertility. For adult women, established techniques like embryo and egg freezing are standard, requiring ovarian stimulation. However, for prepubescent girls, ovarian tissue freezing has become the primary option, eliminating the need for hormonal preparation. This review describes the beginning, evolution, and current situation of the fertility preservation options for this young population. A total of 75 studies were included, covering the steps in the current fertility preservation protocols: (i) ovarian tissue extraction, (ii) the freezing method, and (iii) thawing and transplantation. Cryopreservation and the subsequent transplantation of ovarian tissue have resulted in successful fertility restoration, with over 200 recorded live births, including cases involving ovarian tissue cryopreserved from prepubescent girls. Despite promising results, challenges persist, such as follicular loss during transplantation, which is attributed to ischemic and oxidative damage. Optimizing ovarian tissue-freezing processes and exploring alternatives to transplantation, like in vitro systems for follicles to establish maturation, are essential to mitigating associated risks. Further research is required in fertility preservation techniques to enhance clinical outcomes in the future. Ovarian tissue cryopreservation appears to be a method with specific benefits, indications, and risks, which can be an important tool in terms of preserving fertility in younger women.

The impact of heterotopic ovarian tissue transplantation with varied volumes on reproductive endocrine function in oophorectomized rats.

BACKGROUND: This study aimed to investigate the effects of different volumes of ovarian tissue transplantation on the reproductive endocrine function of rats after oophorectomy. METHODS: Female rats were selected to establish a castration model and then underwent different volumes of ovarian tissue transplantation. Group I served as the sham operation group. The transplantation group was divided into five subgroups based on the calculated ratio of ovarian weight to body weight in normal female rats, δ = (2.52 ± 0.17) ×10-4: Group II: transplanted ovarian volume was δ; Group III: 0.75δ; Group IV: 0.5δ; Group V: 0.25δ; Group VI: without ovarian transplantation. The post-transplant oestrous cycle recovery was observed, and blood samples were collected every 2 weeks to measure serum hormone levels. Histological evaluation was performed at the end of the observation period. RESULTS: Rats in Group V exhibited disrupted oestrous cycles after transplantation, which were significantly longer than those in Group I. Rats in Groups II, III, and IV showed no cyclic changes. At 6 weeks post-transplantation, rats in Group V had lower E2 and AMH levels and higher FSH levels compared to Group I. The uterine wet weight and the number of normal follicles in Group V were significantly lower than those in Group I, but the number of atretic follicles was higher than in Group I. CONCLUSION: The larger ovarian tissue transplantation resulted in a faster recovery with a higher survival rate of the uterus and normal follicles, compared to smaller ovarian tissue transplantation.

With advancements in science and technology, ovarian transplantation techniques have become increasingly mature. However, there are still many questions that need to be addressed. For instance, the large size of the transplanted ovarian tissues may cause over-recruitment of the primordial follicles. When the transplanted ovarian tissue is too small, it can only exert limited functionality and may not meet the patient's needs. This study aimed to investigate the effects of different volumes of ovarian tissue transplantation on the reproductive endocrine function in rats after oophorectomy, and to provide a theoretical basis for determining the minimum effective volume of heterotopic ovarian tissue transplantation.

Research progress on mechanism of follicle injury after ovarian tissue transplantation and protective strategies. / 移植卵巢组织卵泡损伤机制及保护策略.

Ovarian tissue cryopreservation and transplantation is the only way to preserve fertility for female cancer patients in prepubertal ages and those who cannot delay radiotherapy or chemotherapy. However, the success rate of cryopreservation and transplantation of ovarian tissue is still low at present due to the risk of ischemia and hypoxia of the grafted tissues. Abnormal activation of primordial follicles and ischemia-reperfusion injury after blood supply recovery also cause massive loss of follicles in grafted ovarian tissues. Various studies have explored the use of different drugs to reduce the damage of follicles during freezing and transplantation as well as to extend the duration of endocrine and reproductive function in patients with ovarian transplantation. For example, melatonin, N-acetylcysteine, erythropoietin or other antioxidants have been used to reduce oxidative stress; mesenchymal stem cells derived from different tissues, basic fibroblast growth factor, vascular endothelial growth factor, angiopoietin 2 and gonadotropin have been used to promote revascularization; anti-Müllerian hormone and rapamycin have been used to reduce abnormal activation of primordial follicles. This article reviews the research progress on the main mechanisms of follicle loss after ovarian tissue transplantation, including hypoxia, ischemia-reperfusion injury and associated cell death, and abnormal activation of follicles. The methods for reducing follicle loss in grafted ovarian tissues are further explored to provide a reference for improving the efficiency of ovarian tissue cryopreservation and transplantation.

Comparison of melatonin, oxytetracycline, and N-acetylcysteine pre-treatments in autologous intraperitoneal ovarian transplantation in rats.

This study was aimed at investigating the effects of melatonin, oxytetracycline and N-acetylcysteine on the ovarian follicle reserves and surface epithelium in autologous intraperitoneal ovarian transplantation in rats. Thirty adult female Wistar Albino were selected and randomly divided into six groups (n = 5). Group 1, which was the control group, only had their abdomens opened and closed while Group 2 underwent ovarian transplantation. Group 3, 4, 5 and 6 received 20 µg/kg/IM melatonin, 10 mg/kg/IM oxytetracycline, 150 mg/kg/IP N-Asetil sistein (NAC) and 1% ethanol respectively 15 min before the ovarian transplantation. Vaginal cytology was performed to monitor the estrus phase and the follicle reserve and changes in the surface epithelium were histopathologically evaluated during the preparations. Moreover, cellular apoptosis in tissues was evaluated with immunofluorescence staining of Bcl-2 and Bax. The Bax/Bcl-2 ratio was then calculated as the mean fluorescence intensity (MFI) of Bax and Bcl-2 MFI. Dysplastic change was found only significantly higher in the transplantation group (G2) (p < 0.01). Histopathologically, it was found that the follicle reserve was preserved significantly in the oxytetracycline and melatonin treated group (G3, G4) (p < 0.01). It was also observed that the oxytetracycline treated group (G4) were able to show better preventive effects against dysplastic changes of the surface epithelium. Moreover, the melatonin treated group depicted a low Bax/Bcl-2 ratio compared to the group that only underwent transplantation (G2) (p < 0.01). This study indicated that oxytetracycline and melatonin might be more effective than N-acetylcysteine in protecting against oxidative stress during ovarian transplantation.

Determinants of transplantation success with cryopreserved ovarian tissue: data from 196 women of the FertiPROTEKT network.

STUDY QUESTION: What are the pregnancy and live birth rates for ovarian tissue transplantation and which factors are associated with the success rate? SUMMARY ANSWER: Pregnancy and live birth rates per transplanted woman are 32.7% and 26.5% and success rate is associated with female age and first versus repeated transplantation. WHAT IS KNOWN ALREADY: Live birth rates after ovarian tissue transplantations have been reported to be between around 24% and 41% per patient. Success rates seem to be negatively associated with increasing female age at the time of tissue cryopreservation and with pelvic radiation. Success rates are apparently not reduced after overnight transportation of ovarian tissue before freezing. STUDY DESIGN, SIZE, DURATION: Registry analysis of 244 transplantations in 196 women, performed by 26 FertiPROTEKT network centres from 2007 to 2019 with follow-up till December 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Orthotopic ovarian tissue transplantations were performed in 196 women, 191 with previous malignant and 5 with previous non-malignant diseases. Size of transplanting centres varied between 1 and 100 transplantations per centre (median: 2). Factors possibly associated with success rate such as female age, first and repeated transplantation, experience of the transplanting centre and overnight transportation of the ovarian tissue before freezing were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Average age of all 196 transplanted women was 31.3 years (SD 5.2; range 17-44) at the time of cryopreservation of tissue and 35.9 years (SD 4.8; range 23-47) at the time of transplantation. Pregnancy rate was 30.6% (95% CI, 24.2-37.6%) per first transplantation and 32.7% (95% CI, 26.1-39.7%) per patient. Pregnancy rate was higher after first transplantation (30.6% (95% CI, 24.2-37.6%)) compared to second and subsequent transplantations (11.8% (95% CI, 3.3-27.5%)). Live birth rate per first transplantation was 25.0% (95% CI, 19.1-31.7%) and per patient 26.5% (95% CI, 20.5-33.3%). Success rate decreased with increasing age at the time of ovarian tissue freezing. Live birth rate was 28.2% (95% CI, 20.9-36.3%) in women <35 years and 16.7% (95% CI, 7.9-29.3%) in women >35 years. Pregnancy rates after first transplantation were higher in centres who had performed ≥10 transplantations (35.1%) compared to centres with <10 transplantation (25.4%) (P = 0.12). Corresponding live birth rates were 27.0% and 18.6%. Success rates were not different in women with and without overnight transportation of tissue before cryopreservation. LIMITATIONS, REASONS FOR CAUTION: The data were drawn from a registry analysis. Data such as ovarian reserve and premature ovarian insufficiency were not available for all women. Data might be influenced by different follow-up policies of the centres. WIDER IMPLICATIONS OF THE FINDINGS: The study reveals the high potential of ovarian tissue freezing and transplantation, but only if freezing is performed in younger women. The study suggests focus should be placed on the first and not on repeated transplantations. It also opens the discussion of whether transplantation should rather be performed by experienced centres. STUDY FUNDING/COMPETING INTEREST(S): No funding. No competing interests. TRIAL REGISTRATION NUMBER: N/A.

Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. METHODS: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. RESULTS: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. CONCLUSION: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.

Strategies to safely use cryopreserved ovarian tissue to restore fertility after cancer: a systematic review.

Ovarian tissue cryopreservation and subsequent autotransplantation is a successful technique for fertility preservation in oncological patients. However, there are concerns regarding safety, as the graft may contain malignant cells that could lead to the reintroduction of cancer. To circumvent this problem several experimental strategies are being pursued. This systematic review was conducted to provide an overview of the strategies aiming to safely use cryopreserved human ovarian tissue to restore fertility after cancer. Thirty-one studies were included, covering five different experimental strategies: (i) in-vitro maturation of oocytes, (ii) constructing an artificial ovary as a scaffold for reseeding pre-antral follicles, (iii) purging strategies aimed at the eradication of contaminating malignant cells, (iv) maturation of oocytes by xenotransplantation, and (v) stem cell-based oogenesis. These strategies to circumvent the reintroduction of cancer cells through ovarian tissue autotransplantation are being developed, but so far have not reached the stage of clinical trials. Further research is required to establish their risks and effectiveness while the ethical aspects associated with these strategies also need to be discussed. Despite the fact that these experimental procedures are still under development, they might provide safe fertility restoration options for oncological patients in the future.

In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity.

RESEARCH QUESTION: Is it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? DESIGN: A total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. RESULTS: Every woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. CONCLUSIONS: The authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight 'core genes' control primordial follicle recruitment and development.

Revascularization of human ovarian grafts is equally efficient from both sides of the cortex tissue.

RESEARCH QUESTION: Does revascularization of human ovarian grafts in a mouse model occur with equal efficiency from both sides of the cortex tissue? DESIGN: Twenty-four frozen-thawed ovarian cortex pieces from 12 women were transplanted to immunodeficient mice, for 8 days to analyse graft revascularization using immunohistochemical detection of murine CD31, or for 8 weeks to evaluate follicle density (follicles/mm3). The CD31-positive vessel area and density were quantified using a custom-designed application. Three regions of interest (ROI) were defined in each tissue section: the cortical side, the centre and the medullary side. Vessels were subdivided into three categories according to size: microvessels (<300 µm2), small vessels (300-1000 µm2) and large vessels (>1000-3000 µm2). RESULTS: No significant difference in the mean percentage of the CD31-positive vessel area was found between the three ROI (cortical side: 3.9% ± 0.2%; centre: 3.5% ± 0.2%; medullary side: 4.0% ± 0.3%; P = 0.17), but a significantly lower density of vessels was found in the centre of the human ovarian grafts compared with the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; centre: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; P < 0.001). Microvessels comprised 89-91% of all vessels in the three ROI. Follicle density in ungrafted cortex pieces was 51.8 ± 17.3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks of xenografting, resulting in a follicle survival rate of 28%. CONCLUSIONS: Host revascularization was established equally efficiently from both sides of transplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft could potentially facilitate faster graft revascularization.

Transplantation of small ovarian tissue fragments using pipelle device is effective: method evaluation and reproductive outcomes.

PURPOSE: To assess the feasibility, effectiveness, and reproductive outcomes of transplantation of tiny cryopreserved ovarian pieces through a pipelle cannula during laparoscopic surgery. METHODS: A retrospective study of patients who underwent ovarian tissue transplantation for fertility restoration between 2004 and 2022. The "pipelle group" had their ovarian cortex cut into tiny pieces of ~ 1-2 mm3 before cryopreservation. The pieces were too small to be handled and transplanted via standard laparoscopic tools. Transplantation was performed using a pipelle cannula during laparoscopic surgery. The "control group" underwent transplants of ovarian cortex pieces 1-2 mm thick, measuring approximately 25-50 mm2 pieces, using standard procedures. RESULTS: The pipelle group consisted of 4 patients aged 19, 21, 27, and 28 years old at ovarian tissue cryopreservation (OTC). The control group consisted of 14 patients aged 21-30 years old. All pipelle patients restored their endocrine activity, and all of them conceived. FSH levels dropped during the first 3 months following the pipelle transplant. IVF cycle outcomes were similar for both groups. All patients from the pipelle group conceived, resulting in 5 pregnancies and 4 live births (one patient had 2 deliveries, and one additional pregnancy is ongoing), compared to the control group, where 8 patients achieved a total of 20 pregnancies and 18 live births. CONCLUSION: Pipelle transplantation for tiny cryopreserved ovarian pieces is feasible and effective. This study opens a door for patients who had their ovaries cut into small pieces and may even simplify the procedure in some instances, making ovarian transplant more accessible. TRIAL REGISTRATION: (#6531-19-SMC) [18/09/2019].

Functional survey of decellularized tissues transplantation for infertile females.

Numbers of women worldwide face infertility, which will have a significant impact on a couple's life. As a result, assisting with the treatment of these individuals is seen as a critical step. Successful births following uterus and ovary donation have been reported in recent. When immunosuppressive drugs are used in patients who receive donated tissues, there are always problems with the drugs' side effects. In recent years, tissue engineering has mainly been successful in treating infertility using decellularization techniques. Engineered uterus and ovary prevent immunological reactions and do not require immunosuppressive drugs. The most important aspect of using decellularized tissue is its proper function after transplantation. These tissues must be able to produce follicles, secrete hormones and cause pregnancy. This study aimed to investigate research on decellularized tissues and transplanted into the female reproductive system. In this study, just tissues that, after transplantation, have the proper function for fertility were investigated.