Anticancer treatments, particularly chemotherapy, induce ovarian damage and loss of ovarian follicles. There are limited options for fertility restoration, one of which is pre-chemotherapy cryopreservation of ovarian tissue. Transplantation of frozen-thawed human ovarian tissue from cancer survivors has resulted in live-births. There is extensive follicular loss immediately after grafting, probably due to too slow graft revascularization. To avoid this problem, it is important to develop methods to improve ovarian tissue neovascularization. The study’s purpose was to investigate if treatment of murine hosts with simvastatin or/and embedding human ovarian tissue within fibrin clots can improve human ovarian tissue grafting (simvastatin and fibrin clots promote vascularization). There was a significantly higher number of follicles in group A (ungrafted control) than in group B (untreated tissue). Group C (simvastatin-treated hosts) had the highest levels of follicle atresia. Group C had significantly more proliferating follicles (Ki67-stained) than groups B and E (simvastatin-treated hosts and tissue embedded within fibrin clots), group D (tissue embedded within fibrin clots) had significantly more proliferating follicles (Ki67-stained) than group B. On immunofluorescence study, only groups D and E showed vascular structures that expressed both human and murine markers (mouse-specific platelet endothelial cell adhesion molecule, PECAM, and human-specific von Willebrand factor, vWF). Peripheral human vWF expression was significantly higher in group E than group B. Diffuse human vWF expression was significantly higher in groups A and E than groups B and C. When grafts were not embedded in fibrin, there was a significant loss of human vWF expression compared to groups A and E. This protocol may be tested to improve ovarian implantation in cancer survivors.

Turner syndrome (TS) is characterized by a complete or partial absence of one X chromosome (1). The most frequent chromosome constitution is 45X0, although approximately half of patients have a mosaic chromosome complement, the most common being 45X0/46XX (15%) and 46XXq or 46XXp deletions (6%). The chromosome constitution is important because patients with deletions on Xp have short stature and congenital malformations, whereas those with a deletion on Xq only have gonadal dysfunction. There are also rare cases of female TS patients with 46XY 45X0/46XY karyotypes.

The improved survival of young cancer patients results often in follicle destruction. The infertility level varies by age, with alkylating agents considered the most gonadotoxic. One option for fertility preservation is ovarian cryostorage, often conducted post-chemotherapy. Large number of follicles remain in ovaries of pediatric patients post-chemotherapy. Anti-Mullerian hormone (AMH), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are ovarian markers. Their ovarian expression in post-chemotherapy girls might provide an indication of follicle normality, and if it is worthwhile to cryopreserve such tissue. AMH, GDF9 and BMP15 protein expression in human ovarian samples from 49 girls pre-and-post-chemotherapy was evaluated by regular immunohistochemistry (IHC) and immunofluorescence (IF) for confocal laser microscopy. Protein expression for the three growth factors in follicles of pediatric patients pre-and-post-chemotherapy (including alkylating agent exposure) was identified by both methods from primordial stages onwards in oocytes and granulosa cells, and in irregularly shaped follicles. In the pre-chemotherapy group, IHC AMH staining seemed strongest at age ≤ 8 years, and was more abundant in pubertal-than-pre-pubertal patients by IF. Post-chemotherapy patients had significantly more immunostaining for AMH and BMP15 in atretic follicles after exposure to alkylating agents. IHC immunostaining for all three proteins seemed fuller before than after chemotherapy; there were more follicles containing growth factor-expressing granulosa cells post-chemotherapy; BMP15 staining seemed stronger pre-than-post-chemotherapy. The growth factor distribution post-chemotherapy as well as the high numbers of remaining follicles, suggests that it is worthwhile to cryostore ovarian tissue from pediatric patients even after chemotherapy initiation, including alkylating agent exposure.

INTRODUCTIONOptions for preserving fertility in children and adolescents with cancer depend on patient age, the available time frame, and the treatment regimen. Ovarian stimulation with mature oocyte preservation is often the optimal method in post-menarcheal adolescents. We describe a case of a 17-year-old girl with vaginal soft-tissue Ewing sarcoma in whom transvaginal oocyte collection for fertility preservation was ruled out by the large tumor. To overcome the limitations of the transabdominal approach, we applied a novel method of laparoscopically-assisted ultrasound-guided percutaneous transabdominal oocyte collection. In this manner, we were able to both perform oophorectomy and obtain superficial and deep ovarian follicles for cryopreservation.

AimTo determine the effect of nonylphenol-ethoxylate-10 (NP-10) on the ovarian reserve in a mouse model.DesignFemale mice were maintained on purified water or exposed to NP-10 from 3-7-weeks of age. At 7-weeks they were stimulated, mated and the zygotes were cultured in-vitro. Three and 7-weeks old mice were untreated controls. Identical groups were sacrificed without stimulation. Ovaries were analysed for follicular composition. Respiratory-chain (RC) activity and reactive-oxygen-species (ROS) production were measured in brains and livers.ResultsSeven-weeks-old mice produced fewer oocytes/embryos than 3-week-old mice. At 7-weeks, mice exposed to NP-10 produced more oocytes/embryos the controls. Their ovaries contained more primordial/primary follicles, with a lower rate of proliferation and fewer antral follicles. There were no differences in follicular apoptosis, RC-activity or ROS production.ConclusionsIn this model, exposure to NP-10 inhibited the spontaneous follicular recruitment, the first report of successful inhibition of physiologic ovarian aging, to the best of our knowledge.

Anti-cancer therapy, particularly chemotherapy, damages ovarian follicles and promotes ovarian failure. The only pharmacological means for protecting the ovaries from chemotherapy-induced injury is gonadotrophin-releasing hormone agonist, but its efficiency remains controversial; ovarian transposition is used to shield the ovary from radiation when indicated. Until the late 1990s, the only option for fertility preservation and restoration in women with cancer was embryo cryopreservation. The development of other assisted reproductive technologies such as mature oocyte cryopreservation andin vitromaturation of oocytes has contributed to fertility preservation. Treatment regimens to obtain mature oocytes/embryos have been modified to overcome various limitations of conventional ovarian stimulation protocols. In the last decades, several centres have begun cryopreserving ovarian samples containing primordial follicles from young patients before anti-cancer therapy. The first live birth following implantation of cryopreserved-thawed ovarian tissue was reported in 2004; since then, the number has risen to more than 130. Nowadays, ovarian tissue cryopreservation can be combined within vitromaturation and vitrification of oocytes. The use of cryopreserved oocytes eliminates the risk posed by ovarian implantation of reseeding the cancer. Novel methods for enhancing follicular survival after implantation are presently being studied. In addition, researchers are currently investigating agents for ovarian protection. It is expected that the risk of reimplantation of malignant cells with ovarian grafts will be overcome with the putative development of an artificial ovary and an efficient follicle class- and species-dependentin vitrosystem for culturing primordial follicles.

INTRODUCTION:Advances in cancer therapy have improved the long-term survival of cancer patients. Concerns about fertility represent a major issue for young cancer patients. The emergent discipline of oncofertility, an intersection between oncology and fertility, is a new concept that describes an integrated network of clinical resources that focus on fertility preservation from both clinical and research perspectives. Patients and methods: In this article we describe our designated multidisciplinary program for fertility preservation in pediatric and young adult populations. The program is also designed to serve as a prospective platform for the evaluation of reproductive outcomes in this patient cohort.RESULTS:We have observed considerably higher referral rates following launching the program and earlier referral of chemonaïve patients that concedes maximal fertility preservation. Two hundred and thirty five patients were referred to the program over a period of 3 years.CONCLUSIONS:Our program demonstrates that multidisciplinary programs that encompass relevant specialists, skilled laboratory resources and a facilitated path that drives the process in the shortest time, maximizes the yield.

To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the “improvement protocol” of host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. Human ovarian tissue was preserved by needle-immersed vitrification or slow-freezing and transplanted into immunodeficient mice, either untreated (groups A and C, respectively) or treated with the improvement protocol (groups B and D, respectively). Grafted and ungrafted slices were evaluated by follicle counts, apoptosis assay and immunohistochemistry for Ki67 and platelet endothelial cell adhesion molecule (PECAM). Follicle number in the recovered grafts was limited. The number of atretic follicles was significantly higher after vitrification with/without the improvement protocol and slow-freezing than that after slow-freezing + the improvement protocol. Stroma cell apoptosis was the lowest in the group D. PECAM staining showed a peripheral and diffuse pattern in the group D (mostly normal follicular morphology) and a diffuse pattern in all other groups (few follicles, mostly atretic), with significantly higher diffuse levels in the vitrification groups. Ki67 staining was identified in all normal follicles. Follicles did not survive transplantation in the vitrification groups. Ovarian sample preparation with slow-freezing + the improvement protocol appears to yield better implantation outcomes than needle-immersed vitrification with/without the improvement protocol. The real quality of frozen tissue can be assessed only after grafting and not after thawing/warming.

How chemotherapy affects dormant ovarian primordial follicles is unclear. The 'burnout' theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using 4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 mu mol/l/10 mu mol/l) (treated samples) for 24-48 h. Follicular counts and classification, Ki67 and anti-Mullerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17 beta-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured (P = 0.04 to P < 0.0005) and uncultured controls (P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twice the levels of AMH and 17 beta-oestradiol in treated compared with untreated samples (AMH with 4hc 3 mu mol/l; P = 0.04). All follicles stained positively for AMH included treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro. Study findings support the 'burnout' theory as the mechanism of chemotherapy-induced ovarian toxicity. (C) 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The possibility of maturing human primordial follicles in vitro would assist fertility restoration without the danger of reseeding malignancies. Leukaemia inhibitory factor (LIF) and certain culture matrices may promote human follicular growth. The present study compared human primordial follicular growth on novel culture matrices, namely human recombinant vitronectin (hrVit), small intestine submucosa (SIS), alginate scaffolds and human recombinant virgin collagen bioengineered in tobacco plant lines (CollPlant). The frozen-thawed ovarian samples that were used had been obtained from girls or young women undergoing fertility preservation. In the first part of the study, 20 samples were cultured for 6 days on hrVit or SIS with basic culture medium alone or supplemented with one of two concentrations of LIF (10ngmL-1 and 100ngmL-1), with and without LIF-neutralising antibody. In the second part of the study, 15 samples were cultured for 6 days on alginate scaffolds or CollPlant matrices with basic culture medium. Follicular development was assessed by follicular counts and classification, Ki67 immunohistochemistry and 17β-oestradiol and anti-Müllerian hormone measurements in spent media samples. Primordial follicular growth was not enhanced by LIF. Despite some significant differences among the four matrices, none appeared to have a clear advantage, apart from significantly more Ki67-stained follicles on alginate and CollPlant matrices. Further studies of other culture matrices and medium supplements are needed to obtain an optimal system.

Current evidence indicates sub-optimal incidence of fertility preservation (FP) in eligible patients. We present herein our designated multidisciplinary program for FP in pediatric and adolescent population and present our data on FP in female patients.

STUDY QUESTION Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? SUMMARY ANSWER Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. WHAT IS KNOWN ALREADY Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. STUDY DESIGN, SIZE, DURATION A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. PARTICIPANTS/MATERIALS, SETTING, METHODS The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. MAIN RESULTS AND ROLE OF CHANCE Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). LIMITATIONS, REASONS FOR CAUTION There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. WIDER IMPLICATIONS OF THE FINDINGS Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. STUDY FUNDING/COMPETING INTERESTS The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. TRIAL REGISTRATION NUMBER N/A.

The neurotrophin family consists of nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), in addition to brain-derived neurotrophic factor (BDNF) and the neuronal growth factors, glial cell line-derived neurotrophic factor (GDNF) and vasointestinal peptide (VIP). Although there are a few literature reviews, mainly of animal studies, on the importance of neurotrophins in the ovary, we aimed to provide a complete review of neurotrophins as well as neuronal growth factors and their important roles in normal and pathological processes in the ovary. Follicular assembly is probably stimulated by complementary effects of NGF, NT4/5 and BDNF and their receptors. The neurotrophins, GDNF and VIP and their receptors have all been identified in preantral and antral follicles of mammalian species, including humans. Transgenic mice with mutations in the genes encoding for Ngf, Nt4/5 and Bdnf and their tropomyosin-related kinase β receptor showed a reduction in preantral follicles and an abnormal ovarian morphology, whereas NGF, NT3, GDNF and VIP increased the in vitro activation of primordial follicles in rats and goats. Additionally, NGF, NT3 and GDNF promoted follicular cell proliferation; NGF, BDNF and VIP were shown to be involved in ovulation; VIP inhibited follicular apoptosis; NT4/5, BDNF and GDNF promoted oocyte maturation and NGF, NT3 and VIP stimulated steroidogenesis. NGF may also exert a stimulatory effect in ovarian cancer and polycystic ovarian syndrome (PCOS). Low levels of NGF and BDNF in follicular fluid may be associated with diminished ovarian reserve and high levels with endometriosis. More knowledge of the roles of neuronal growth factors in the ovary has important implications for the development of new therapeutic drugs (such as anti-NGF agents) for ovarian cancer and PCOS as well as various infertility problems, warranting further research.

BACKGROUND:In vitro maturation (IVM) of immature oocytes is an important technology for selected clinical indications. We previously described a pregnant woman with a history of renal transplantation who underwent oocyte aspiration during cesarean section (CS) for fertility preservation and future surrogacy.CASE:A 27-year-old pregnant woman was diagnosed with neck rhabdomyosarcoma at 37 weeks' gestation. CS was performed with direct aspiration of small follicles from one ovary and oophorectomy of the other. Twenty-one identified oocyte-cumulus complexes were cultured, and 12 mature oocytes and 14 ovarian cortex strips were cryopreserved.CONCLUSION:Aspirating competent oocytes during CS may serve as an additional means of fertility preservation in pregnant women. The procedure may also be offered to patients with an IVF pregnancy who are scheduled for elective CS.

Premature ovarian failure (POF), also known as premature menopause, is defined as at least six months of amenorrhea before the age of 40 with elevated gonadotrophins and depressed 17-β-oestrogen (E2) levels (Dixit et al., 2010, Fonesca et al., 2014). POF affects some approximately 1% of women (Chatterjee et al., 2007). Previous studies have demonstrated a functional association between POF and polymorphisms in the promotor of bone morphogenetic protein 15 (BMP15). The BMP family is the largest group in the transforming growth factor β superfamily (Abir and Fisch, 2011; Margulis et al., 2009; Shimasaki et al., 2004).

PURPOSE:To improve human primordial follicle culture.METHODS:Thin or thick ovarian slices were cultured on alginate scaffolds or in PEG-fibrinogen hydrogels with or without bpV (pic), which prevents the conversion of phosphatidylinositol-trisphosphate (PIP3) to phosphatidylinositol-bisphosphate (PIP2) or 740Y-P which converts PIP2 to PIP3. Follicular growth was evaluated by follicular counts, Ki67 immunohistochemistry, and 17β-estradiol (E2) levels.RESULTS:BpV (pic) had a destructive effect on cultured follicles. Thawed-uncultured samples had more primordial follicles than samples cultured in basic medium and fewer developing follicles than samples cultured in PEG-fibrinogen hydrogels with 740Y-P. There were more atretic follicles in samples cultured on alginate scaffolds than in PEG-fibrinogen hydrogels, and in samples cultured in PEG-fibrinogen hydrogels with 740Y-P than in PEG-fibrinogen hydrogels with basic medium. Ki67 staining was higher in PEG-fibrinogen hydrogels than on alginate scaffolds. E2 levels were higher in thick than in thin slices.CONCLUSIONS:PEG-fibrinogen hydrogels appear to have an advantage over alginate scaffolds for culturing human primordial follicles. Folliculogenesis is not increased in the presence of substances that enhance PIP3 production or with thin rather than thick sectioning.