Ovarian Cortex Transplantation Research Articles

Fertility Preservation in the Era of Immuno-Oncology: Lights and Shadows.

In recent years, immuno-oncology has revolutionized the cancer treatment field by harnessing the immune system's power to counteract cancer cells. While this innovative approach holds great promise for improving cancer outcomes, it also raises important considerations related to fertility and reproductive toxicity. In fact, most young females receiving gonadotoxic anti-cancer treatments undergo iatrogenic ovarian exhaustion, resulting in a permanent illness that precludes the vocation of motherhood as a natural female sexual identity. Although commonly used, oocyte cryopreservation for future in vitro fertilization and even ovarian cortex transplantation are considered unsafe procedures in cancer patients due to their oncogenic risks; whereas, ovarian stem cells might support neo-oogenesis, providing a novel stemness model of regenerative medicine for future fertility preservation programs in oncology. Recent scientific evidence has postulated that immune checkpoint inhibitors (ICIs) might in some way reduce fertility by inducing either primary or secondary hypogonadism, whose incidence and mechanisms are not yet known. Therefore, considering the lack of data, it is currently not possible to define the most suitable FP procedure for young patients who are candidates for ICIs. In this report, we will investigate the few available data concerning the molecular regulation of ICI therapy and their resulting gonadal toxicity, to hypothesize the most suitable fertility preservation strategy for patients receiving these drugs.

P–451 Improving neovascularization and follicle viability in cryopreserved bovine ovarian tissue transplants

Abstract Study question Does cryopreservation and transplantation of bovine ovarian medulla-containing cortex tissue improve the viability and vascularization of the graft? Summary answer: Transplantation of bovine ovarian cortex containing medulla has a positive effect on follicular viability and neovascularization of the graft compared to cortex transplantation alone. What is known already For female fertility protection, cryopreservation and retransplantation of ovarian tissue is a widely used method. During cryopreservation, ovarian tissue is exposed to mechanical and hypoxic stress resulting in follicular loss. Moreover, after retransplantation tissue vitality and follicle survival is limited due to ischemia. As follicular viability is of major importance for fertility and hormonal activity, the main focus is on improving vitality and viability of the grafts. In current protocols, ovarian medulla is discarded and merely cortex tissue is preserved. However, medulla tissue predominantly contains blood vessels, thereby obtaining high potential for revascularization processes and thereby supporting tissue vitality. Study design, size, duration This experimental laboratory work was performed during a period of ten months. The rapidly vascularized chorioallantoic-membrane (CAM) of fertilized chicken eggs was used as model system to investigate neovascularization, follicle survival and tissue vitality of different bovine ovarian grafts. In four independent experimental rows four different tissue types (isolated cortex, thick medulla-containing cortex (8 x 10 x 3 mm), thin medulla-containing cortex (5 x 10 x 3 mm) and sole medulla tissue were compared. Participants/materials, setting, methods Out of four bovine ovaries preserved from the slaughterhouse, in total 117 samples of the four different tissue types were primed and cryopreserved by the common slow-freezing protocol. After thawing, grafts were transplanted on separate CAMs at day four of fertilized eggs. After four days of incubation, blood vessels growing towards the grafts were counted. Subsequently, grafts were harvested, digested with collagenase and stained with Neutral Red® to determine the total amount of vital follicles. Main results and the role of chance To investigate the neovascularization, all graft-supplying blood vessels were determined and distinguished between small and thick vessels. Compared to sole cortex, there were more small vessels in the medulla-containing grafts (9,72 vs. 8,65). Especially thin medulla- containing cortex pieces exhibited the highest number of small vessels (9,90). Also in isolated medulla tissue an increased amount of small vessels was observed (9,79). However, the average number of big vessels was not significantly different in all four test groups (Cortex: 2,12; thin medulla-containing cortex: 1,69; thick medulla-containing cortex: 1,5; medulla: 2). The total number of all vessels differed from 10,76 (sole cortex) to 11,75 (medulla-containing grafts), indicating a support of neoangiogenesis by medulla tissue. To further examine whether medulla tissue also alters the amount of vital follicles, Neutral Red® stained vital follicles were determined in all different sample groups. Indeed, in medulla-containing cortex samples was an augmented average number of vital follicles (342,4) compared to sole cortex tissue (256,11). Most vital follicles were detectable in the thick medulla-containing cortex tissue (346,61), closely followed by the thin medulla-containing cortex grafts (338,19). As expected, there was just a rare amount of vital follicles in sole medulla grafts (8,13). Limitations, reasons for caution As the ovarian reserve in cattle is very individual, the prepared ovaries are different in their follicle amount. These individual differences may influence the number of counted follicles. Furthermore, the CAM model is only a short term experimental approach to investigate neovascularization and follicle survival. Wider implications of the findings: According to our results, transplantation of human medulla-containing cortex appears promising. Keeping medulla tissue on the graft seems to improve both follicle viability and revascularization. Our findings need to be proven with human tissue, but might change the preparation of human ovarian tissue for fertility preservation in future. Trial registration number Not applicable

Anti-Müllerian Hormone (AMH) regulates BRCA1 and BRCA2 gene expression after ovarian cortex transplantation

Objective To test whether recombinant anti-Müllerian hormone (rAMH) could exert an inhibitory function on BRCA1/2 expression in human ovarian cortex. Methods Pilot study on ovariectomized nude mice xenotransplanted with human vitrified/warmed ovarian cortex and treated with rAMH via infusion pump. Twelve nude mice were ovariectomized and Alzet pumps delivering 1.23 mcg rAMH/day to reach a serum concentration of 17.5 ng/mL, or placebo (controls), were inserted intraabdominally. Previously vitrified/warmed 2x2 mm ovarian cortex fragments were transplanted on day 7 and then harvested on day 14 after pump placement. PCR analyses determined mRNA levels for BRCA1 and BRCA2 in the human ovarian cortex. Results In mice treated with rAMH, BRCA1 expression was significantly lower (0.196 fg/µg RNA, IQR 0.158, 0.236) than in controls (0.544 fg/µg RNA, IQR 0.458, 0.554; p = .030), while BRCA2 expression remained similar in rAMH mice (5.355 fg/µg RNA, IQR 4.479, 6.230) and in controls (4.011 fg/µg RNA, IQR 3.650, 4.182; p = .327). Conclusion Administration of rAMH in the peri-transplant period caused downregulation of BRCA1, but not of BRCA2 expression, in human ovarian cortex. These results help our understanding of DNA repair mechanism in the ovarian cortex and identify AMH’s possible protective effect on ovarian reserve in BRCA1 mutation carriers.

In-vitro fragmentation of ovarian tissue activates primordial follicles through the Hippo pathway.

STUDY QUESTIONWhat is the role of the Hippo and PI3K/Akt pathway in follicles during ovarian tissue culture in tissue derived from oncological patients and transgender men?SUMMARY ANSWERResults highlight a Hippo pathway driven primordial follicle activation in vitro, predominantly from Day 0 to Day 4.WHAT IS KNOWN ALREADY In-vitro ovarian tissue culture aims at activating and maturing primordial follicles for fertility restoration in patients with a threatened ovarian reserve. Not all patients are eligible for ovarian cortex transplantation and therefore several groups are attempting to culture ovarian tissue in-vitro. Cortex fragmentation disrupts the Hippo pathway, leading to increased expression of downstream growth factors and follicle growth. The PI3K/Akt pathway is considered the intracellular pathway to where different extracellular factors involved in primordial follicle activation in-vivo converge. In order to optimise current ovarian tissue culture models, information on progression of these pathways during tissue culture is mandatory.STUDY DESIGN, SIZE, DURATIONThe first step of a multistep cortex culture system was performed using 144 ovarian cortex pieces from a total of six patients. Per patient, 24 cortical strips were cultured for 6 days and six pieces per patient were collected for downstream analysis of follicle development and Hippo and PI3K/Akt pathway targets every second day.PARTICIPANTS/MATERIALS, SETTING, METHODSOvarian tissue was obtained from oncological (N = 3; 28.67 ± 4.51 years) and transgender (N = 3; 23.33 ± 1.53 years) patients. Follicles were analysed using haematoxylin-eosin staining and pathways were studied using immunohistochemistry and precise follicle excision by laser capture micro-dissection for RT-qPCR analysis. MIQE guidelines for RT-qPCR were pursued. Reference gene selection (GAPDH, RPL3A, 18s rRNA) was performed using GeNorm Reference Gene Selection Kit. Statistical analysis was conducted with IBM SPSS Statistics 23 (Poisson regression, negative binomial regression, ANOVA and paired t-test).MAIN RESULTS AND THE ROLE OF CHANCEImmunohistochemical analysis confirmed a Hippo pathway driven primordial follicle activation due to mechanical manipulation of the cortical strips. Ovarian tissue preparation and culture induced the inhibitory phosphorylated Yes-associated protein (pYAP) to disappear in granulosa cells of primordial follicles on Day 2. The stimulatory YAP on the contrary appeared in primordial granulosa cells over increasing culture days. Looking at the YAP target connective tissue growth factor (CTGF), a significantly up-regulated CTGF was noted in primordial follicles when comparing Day 2 and Day 4 (ratio Day 2/4 = 0.082; P < 0.05), clearly showing an effect on the Hippo pathway in primordial follicles during tissue culture. Follicle classification showed a significant drop in estimated primordial follicle counts in the oncological cohort (−78%; P = 0.021) on Day 2 and in the transgender cohort on Day 4 (−634%; P = 0.008). Intermediate follicle counts showed a non-significant increasing trend to during culture and this follicle recruitment and growth resulted in a significant rise in estimated primary follicle counts on Day 6 in oncological patients (170%; P = 0.025) and, although limited in absolute numbers, a significant increase in secondary follicles on Day 4 (367%; P = 0.021) in the transgender cohort. Subsequent antral follicle development could not be observed.LIMITATIONS, REASONS FOR CAUTIONA limitation is the small sample size, inherent to this study subject, especially as a large amount of tissue was needed per patient to reduce inter-patient variation in different downstream analysis techniques. A particular and specific weakness of this study is the inability to include an age-matched control group.WIDER IMPLICATIONS OF THE FINDINGSThese findings support an adapted tissue preparation for Hippo pathway disruption and a shorter first phase of tissue culture. This work may also have implications for transplantation of cryopreserved tissue as larger strips (and thus slower burnout due to less Hippo pathway disruption) could be a benefit.STUDY FUNDING/COMPETING INTEREST(S)This research was financially supported by the Foundation Against Cancer (Stichting tegen Kanker, TBMT001816N), the Flemish Foundation of Scientific Research (FWO Vlaanderen, FWO G0.065.11N10) and the Gender Identity Research and Education Society (GIRES) foundation. The authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A.

Ovarian Cortex Transplantation

To describe a laparoscopic technique for the transplantation of a cryopreserved ovarian cortex. Educational video. University Hospital of Strasbourg, France. A 28-year-old nulliparous woman presented with anaplastic T lymphoma and was then treated with chemotherapy. Before the treatment, the ovarian cortex was collected by laparoscopy to preserve fertility. Remission was achieved, but the patient suffered from premature ovarian failure. At the age of 32 years, she wished to become pregnant. The patient was thus included in the research protocol Development of Ovarian Tissue Autograft in Order to Restore Ovarian Function, and the transplantation site was chosen accordingly. The cortex was stored in liquid nitrogen at -196°C after slow congelation. To restore ovarian function and because of pregnancy desire, we transplanted the cryopreserved ovarian cortex in the right ovary and inside a pocket of the peritoneum of the left ovarian fossa. The first step included adhesiolysis to treat small adhesions developed after the first surgery. On the right, the ovarian cortex was opened by an antimesial incision with cold scissors. The cryopreserved ovarian cortex was placed through the cortex of the right ovary and fixed with stitches. On the left side, the peritoneum of the ovarian fossa was opened, and a subperitoneal pocket was dissected. The cortex was inserted. It was then closed with absorbable sutures or with a hemostatic pad. Six months after her surgery, the patient had natural cycles. We monitored an ovulation of both the sides. She underwent 3 in vitro fertilizations but with failures of embryo transfer. She conceived spontaneously a year after the surgery. She gave birth to a healthy child weighing 3300 g. For patients who have suffered from premature ovarian failure owing to chemotherapy, ovarian cortex transplantation can restore the ovulatory function, allow in vitro fertilization, and permit, as in our case, a spontaneous pregnancy.

Enhancing the safety of ovarian cortex autotransplantation: cancer cells are purged completely from human ovarian tissue fragments by pharmacological inhibition of YAP/TAZ oncoproteins.

Is it possible to eliminate metastasized cancer cells from ovarian cortex fragments prior to autotransplantation without compromising the ovarian tissue or follicles? Ex vivo pharmacological inhibition of YAP/TAZ by Verteporfin enabled us to efficiently eradicate experimentally induced small tumours, derived from leukaemia and rhabdomyosarcoma, from human ovarian tissue fragments. Autotransplantation of ovarian tissue fragments that contain metastasized tumour cells may reintroduce the malignancy to the recipient. In order to enhance safety for the patient there is a strong need for protocols that effectively purges the ovarian tissue from malignant cells ex vivo prior to transplantation, without compromising ovarian tissue integrity. Tumour foci were experimentally induced in human ovarian cortex tissue fragments derived from at least three patients by micro-injection of cancer cell lines. Next, the tissue fragments were cultured to allow formation of metastasis-like structures followed by a 24 h ex vivo treatment with the YAP/TAZ inhibitor Verteporfin to eradicate the cancer cells. A control treatment was included in all experiments. The purged ovarian cortex fragments were cultured for an additional 6 days to allow any possibly surviving cancer cells to establish new metastatic foci. Human ovarian tissue was obtained after female-to-male sex reassignment surgery. Human rhabdomyosarcoma, leukeamia, breast cancer and Ewing's sarcoma cell lines were utilized for the induction of tumour foci. Tumour specific (immuno)histochemistry and RT-PCR were used for the detection of residual cancer cells after ex vivo treatment. Ovarian tissue and follicle integrity after exposure to Verteporfin was evaluated by histology, a follicular viability assay and a glucose uptake assay. Metastasized rhabdomyosarcoma and leukaemia cells could be effectively purged from ovarian cortex tissue by a 24 h ex vivo treatment with Verteporfin, while breast cancer and Ewing's sarcoma did not respond to this treatment. Ovarian tissue integrity was not affected by purging, as no statistically significant difference (P > 0.05) was observed in the percentage of morphologically normal follicles, percentage of follicles with apoptotic cells, follicular viability or glucose uptake between the control treated ovarian cortex and Verteporfin treated ovarian cortex. Our tumour model is based on growth of human cancer cell lines. It is unclear whether these cells reflect the behaviour of malignant cells that have metastasized to the ovary during natural disease progression. Furthermore, the functionality of the ovarian tissue after ex vivo treatment requires further investigation in vivo. The results indicate that ex-vivo tumour cell purging of human ovarian cortex fragments intended for fertility preservation purposes is feasible by short-term pharmacological treatment. Effective purging of the ovarian cortex tissue enhances safety of ovarian cortex autotransplantation for the patient. This increases the likelihood that this form of fertility restoration may become an option for patients with malignancies for which ovarian cortex transplantation is currently considered unsafe. Unconditional funding was received from Merck B.V. The Netherlands (Number 2016-FERT-1) and the foundation 'Radboud Oncologie Fonds' (Number KUN 00007682). The authors have no conflicts of interest. NA.

Oocyte vitrification versus ovarian cortex transplantation in fertility preservation for adult women undergoing gonadotoxic treatments: a prospective cohort study

To compare the efficacy of oocyte vitrification (OV) with that of ovarian cortex cryopreservation and transplantation (OCT) in women undergoing gonadotoxic treatments. Prospective observational cohort study. Not applicable. Candidates for chemo-/radiotherapy who joined our fertility preservation (FP) program were included in this study between 2005 and 2015. One cohort included 1,024 patients undergoing OV; the other cohort included 800 patients undergoing OCT. OV using the cryotop device and OCT using a slow freezing protocol. Live-birth rate (LBR) and clinical pregnancy rate (CPR). Basal antimüllerian hormone levels of the patients revealed no differences in ovarian reserve before FP (OV, 11.6 pM [5.4-24.7]; OCT, 11.8 pM [6.4-21.9]). In the OV cohort, 49 patients used the vitrified oocytes after a mean storage time of 3.9years. In the OCT cohort, 44 sought pregnancy after a mean storage time of 5.5years. A trend toward higher CPR and LBR (per patient) was observed in the OV group (risk ratio [RRCPR], 1.31 [95% confidence interval, 0.90-1.92]; RRLBR 1.39 [95% confidence interval, 0.95-2.03]), although differences were not statistically significant. In the OCT group, 46.7% of pregnancies occurred spontaneously and no pregnancy was achieved when the tissue was harvested beyond the age of 36years. All patients except three undergoing OCT resumed or improved endocrine ovarian function. Although we observed a trend toward higher LBR after OV, OCT is a very effective method to preserve fertility, allows for natural pregnancy, and restores ovarian function. In clinical scenarios where OV is not feasible, OCT remains the FP technique of choice and should no longer be considered experimental.

Chapter 13 Human Ovarian Tissue Vitrification.

Ovarian freezing and transplantation has garnered increasing interest as a potential way of preserving fertility in cancer patients as well as for women who just wish to delay childbearing. This chapter spells out our techniques of ovarian cortex vitrification and results for frozen compared to fresh ovarian cortex transplantation (in one single series from one center for the sake of consistency), as well as potentially provides insight into the mechanism behind ovarian follicle recruitment. This represents an effort to simplify and popularize an approach that has yielded favorable results (all cases recovered ovulation and 75% had successful spontaneous pregnancy) in one single, disciplined study. It should be clear that this is a review for the more general reader of our original scientific papers published in Reproductive BioMedicine Online, New England Journal of Medicine, Fertility and Sterility, Human Reproduction, Molecular Human Reproduction, and Journal of Assisted Reproduction and Genetics (JARG).

Ovarian tissue cryopreservation and transplantation: scientific implications.

After fresh or frozen ovary transplantation, FSH levels return to normal, and menstrual cycles resume by 150 days, coincident with anti-Müllerian hormone rising to higher than normal levels. AMH then returns to well below normal levels by 240 days, remaining as such for many years with nonetheless normal ovulation and fertility. To date, 20 babies have been born in our program from 11 fresh and 13 cryopreserved ovary transplant recipients with a live baby rate of over 70 % (11 babies from fresh and 9 from frozen). Globally, over 70 live births have been reported for both fresh and frozen ovary transplants with an approximate 30 % live birth rate. Given the rapid rise of AMH after the fall of FSH, with a subsequent AMH decrease with retention of ovarian function, it is tempting to speculate the existence of a shared mechanism controlling primordial follicle recruitment, fetal oocyte meiotic arrest, and recruitment in the adult ovary. With the massive recruitment of primordial follicles observed after human ovarian cortical tissue transplantation, which subsides to an extremely low recruitment rate, we will discuss how this phenomenon suggests a unifying theory implicating ovarian cortical tissue rigidity in the regulation of both fetal oocyte arrest and recruitment of follicles in the adult ovary. As the paper by Winkler-Crepaz et al. in this issue demonstrates, our in vivo results are consistent with the in vitro demonstration that primordial follicles in the fetal cortex are “locked” in development, resulting in meiotic arrest, which spares the oocytes from being rapidly lost all at once (Winkler-Crepaz et al., J Assist Reprod Genet, 1). Winkler-Crepaz et al. demonstrate that follicle loss after ovarian cortex transplantation is unlikely due to ischemic apoptosis, but rather from a “burst” of primordial follicle recruitment. In vivo, primordial follicles are normally resistant to further development or activation to prevent oocyte depletion. The dense fibrous ovarian cortex, through as yet unresolved mechanisms, arrests the further continuation of meiosis and also prevents a sudden depletion of all resting follicles in the adult ovary. Intrinsic tissue pressure is released after cortical tissue transplantation, temporarily resulting in a rapid follicle depletion. These results are consistent with the observation that once the ovarian reserve is reduced in the graft, the rate of recruitment diminishes and the ovarian tissue exhibits a relatively long duration of function.

Anti-Mullerian hormone (AMH) regulates BRCA1 and BRCA2 gene expression in an ovarian cortex transplantation model

Anti-Mullerian hormone (AMH) regulates BRCA1 and BRCA2 gene expression in an ovarian cortex transplantation model

How ovarian transplantation works and how resting follicle recruitment occurs: a review of results reported from one center.

Ovarian freezing and transplantation has garnered increasing interest as a potential way of preserving fertility in cancer patients. This special report aims to identify the success rate of frozen compared with fresh ovarian cortex transplantation (in one single series from one center for the sake of consistency), as well as potentially provides insight into the mechanism behind ovarian follicle recruitment. A comparison of fresh versus frozen transplantation techniques is presented, highlighting the similarity and differences between the fresh and frozen transplantation procedures. Much of the literature is scattered case reports with different patient populations and different techniques. This represents an effort to simplify and popularize an approach that has yielded favorable results (all cases recovered ovulation and 75% had successful spontaneous pregnancy) in one single, disciplined study.

Anti-Mullerian hormone (AMH) may inhibit oocyte maturation and follicular vascularization in human ovarian cortex

AMH inhibits follicle recruitment [1, 2]. In fact, in the absence of AMH, primordial follicles are recruited at a faster pace, a common complication after transplantation of ovarian cortex blamed on a defective re-vascularization. VEGF stimulates neovascularization and GDF9 is essential for oocyte-dependent ovarian follicle development [3]. We tested the hypothesis that administration of recombinant AMH to ovarian cortex fragments would inhibit follicular development by modulating GDF9 and VEGF expression.

Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice.

This paper describes the success and expansion of ovarian tissue cryopreservation and transplantation as a fertility restoration procedure, with the largest series of 60 live births worldwide reported. By repeating the procedure, ovarian activity can be restored for more than 11 years.

Influence of mouse strain on ovarian tissue recovery after engraftment with angiogenic factor.

BackgroundFor women facing gonadotoxic treatment, cryopreservation of ovarian tissue with subsequent retransplantation during remission is a promising technique for fertility preservation. However, follicle loss within grafted ovarian tissue can be caused by ischemia and progressive revascularization. Several xenograft models using different immunodeficient rodent lines are suitable for studying ovarian tissue survival and follicular viability after frozen-thawed ovarian cortex transplantation. SCID mice, which are deficient for functional B and T cells, are the most commonly used mice for ovarian xenograft studies. However, due to incomplete immunosuppression, NOD-SCID mice displaying low NK cell function and an absence of circulating complement might be more appropriate. The present study aims to define the most appropriate immunodeficient mouse strain for ovarian tissue xenotransplantation by comparing ovarian graft recovery in SCID and NOD-SCID mice following engraftment in the presence of isoform 111 of vascular endothelial growth factor.MethodsSheep ovarian cortex fragments were embedded in a collagen matrix, with or without VEGF111, before being stitched onto the ovaries of SCID and NOD-SCID mice. Transplants were recovered after 3 days to study early revascularization or after 3 weeks to evaluate follicle preservation and tissue fibrosis through histological analyses.ResultsAt day 3, vessels were largely reorganized in the ovarian grafts of both mouse strains. After 3 weeks, the cortical tissue was clearly identifiable in SCID mice but not in NOD-SCID mice. Upon VEGF111 treatment, vascularization was significantly improved 3 days after transplantation in SCID mice. This increase in vessel density was correlated with better follicular preservation in SCID mice 3 weeks after transplantation. Fibrosis was not decreased by VEGF treatment in either mouse strain.ConclusionsTissue architecture and follicular morphology were better preserved in ovarian tissues grafted in SCID mice in comparison with NOD-SCID mice. Moreover, tissue revascularization was improved in SCID mice by VEGF111 graft treatment. Thus, we consider SCID mice to be the best murine model for studying ovarian tissue xenografts.

Mesenchymal stem cells enhance angiogenesis and follicle survival in human cryopreserved ovarian cortex transplantation

Since the main obstacle of the cryopreserved ovarian tissue transplantation is massive follicle loss as a result of ischemia in the process of transplantation, this study is aimed to investigate whether mesenchymal stem cells (MSCs) could be applied to enhance the angiogenesis and follicle survival in human cryopreserved ovarian cortex transplantation.

Live birth after allografting of ovarian cortex between monozygotic twins with Turner syndrome (45,XO/46,XX mosaicism) and discordant ovarian function

To report the restoration of ovarian function in a woman with Turner syndrome by allografting ovarian cortex from her monozygotic twin. Case report. Gynecology research unit in a university hospital. A 37-year-old woman with Turner syndrome who had suffered premature ovarian failure (POF) at the age of 15. In this woman, we performed ovarian cortex transplantation. The donor was her monozygotic twin who had not experienced POF and who had had two normal deliveries. Restoration of ovarian activity and pregnancy. Restoration of ovarian function was achieved 3 months after transplantation. After 7 months of regular cycles, spontaneous pregnancy occurred. The patient delivered a healthy baby girl weighing 2,850 g at 38 weeks of gestation. Girls with Turner syndrome suffer accelerated loss of ovarian follicles and only 2%-5% experience spontaneous menses with the potential to achieve pregnancy without medical intervention. This manuscript reports the first live birth after allografting between monozygotic twins with 45,XO/46,XX mosaicism and discordant growth pattern and ovarian function.

Ovarian cortex transplantation in the baboon: comparison of four different intra-abdominal transplantation sites

Several sites have been used for ovarian cortex transplantation (OCT) in humans. The present study was designed to evaluate different intra-abdominal transplantation sites in the baboon to gain further knowledge about alternative transplantation sites in a human setting. Autologous fresh OCTs were performed in 12 baboons (Papio anubis). Four different sites were tested: the free portion of the omentum (OMF), the portion of the omentum adjacent to the spleen (OMS), the pouch of Douglas (D) and the pelvic wall on the psoas muscle (PW). Cortex survival, follicle density, cyclicity and hormonal levels were compared between the different sites, 3 and 6 months after transplantation. Macroscopically, antral follicles were only found in the OMS and OMF locations, which also showed a higher proportion of follicle-containing cortex at light microscopy (OMF 71.4%, OMS 83.3% versus PW 58.8% and D 40%, P< 0.05). Higher densities of primordial [OMF: 3.54 (0-13.18) follicles/grid, OMS: 3.85 (0-8.53), PW: 0 (0-13.25), D 0 (0-1.33), P< 0.05] and primary follicles [OMF: 3.54 (0-18.52), OMS: 3.85 (0-1), PW: 0 (0-4.58), D 0 (0-0.25), P< 0.05] was also found in the omental locations. Omental locations provide a better site, in terms of follicle survival, for intra-abdominal OCT in the baboon compared with the pelvic wall and the D.

Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapy – A technique in its infancy but already successful in fertility preservation

Increasing survival rates in young cancer patients, new reproductive techniques and the growing interest in quality of life after gonadotoxic cancer therapies have placed fertility preservation as an important issue to oncologists, fertility specialists and patients. Several techniques are now available for fertility preservation in these patients. A new promising method is cryopreservation and transplantation of ovarian cortex. Ovarian tissue can be extracted by laparoscopy without any significant delay of gonadotoxic therapy. The tissue can be cryopreserved by specialised centres of reproductive medicine and transplanted in case the women experience premature ovarian failure (POF). This review summarises the European expertise on cryopreservation and transplantation of ovarian tissue, following around 30 reported transplantations globally, resulting in six live births and several ongoing pregnancies. It emphasises that fertility preservation by the cryopreservation of ovarian tissue is a new but already a successful clinical option, which can be considered for selected cancer patients.

ORIGINAL ARTICLE: Development of Human Graafian Follicles Following Transplantation of Human Ovarian Tissue into NOD/SCID/γcnull Mice

Problem Transplantation of human ovarian cortex into host mice may permit various kinds of challenges in reproductive medicine. A novel immunodeficient mouse strain (NOD/SCID/γcnull: NOG) has been developed as a host of transplantation of human tissue.Method of study Human ovarian cortex was transplanted into various sites of NOG mice and human follicular development was examined by immunohistochemistry.Results Transplantation of human ovarian tissue into NOG mice resulted in approximately similar tissue survival and follicle growth as did transplantation into non‐obese diabetic‐severe combined immunodeficient mice. The human Graafian follicule from NOG mouse expressed the same steroidogenic enzymes as observed in human Graafian follicles, which developed in the human body. The NOG mice’s ovarian bursa was better placed for transplantation than the back skin or kidney capsule.Conclusion These results represent the successful generation and biological confirmation of the human Graafian follicles from the human ovarian cortex in the NOG mice.

Fresh Human Orthotopic Ovarian Cortex Transplantation: Long-term Results

An increasing number of young women are being diagnosed with cancer, and most cancer treatments have irreversibly negative effects on reproduction. Ovarian stimulation and preservation of oocytes or embryos is far from an ideal solution, in part because hormonal treatment may adversely affect some cancers. Another possibility is cryopreservation of ovarian tissue for later autotransplantation, but the ischemic damage caused by the procedure can lead to follicle loss. Using the ovarian medulla for orthotopic ovarian cortex transplantation might limit the risk of ischemia because the ovarian artery would assure a good blood supply. In addition, the medulla may have a role in follicular development. This prospective case-control study examined the effects of ischemia on long-term ovarian function in 12 premenopausal women who had abdominal hysterectomy for uterine disease and also bilateral fresh orthotopic transplantation of the entire ovarian cortex. Five control women had only hysterectomy. Serum levels of follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH) were monitored over 2 years, and ovulatory cycles were identified by vaginal ultrasonography and estimating serum progesterone. Bilateral cortex resection and transplantation took about 30 minutes and caused no complications. Ovulation returned in 11 of the 12 grafted patients (91.7%). The mean number of ovulations was 9.3, compared to 12.0 in control women. Nine study patients remained ovulatory 2 years after surgery. Five of the 12 women in the study group had the same pattern of FSH secretion as the control women. In six cases the serum FSH increased postoperatively and then declined to less than 20 IU/liter after 5 months. Two patients ultimately had menopausal levels of FSH. In only one grafted patient did ovarian function cease after surgery. AMH levels correlated less closely with normal ovarian function than did FSH levels. Serum AMH tended to decline after surgery, but was higher in women whose FSH patterns suggested normal ovarian function. Although this is a small-scale study, the results indicate that fresh orthotopic ovarian cortex transplantation maintains normal ovarian function for at least 2 years in a majority of women, and also may protect against ovarian ischemia.