Anti-Müllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma.

STUDY QUESTION: What is the involvement of ovarian stroma in the anti-Müllerian hormone (AMH) signaling pathway and which stromal cells are involved? SUMMARY ANSWER: Mouse and human ovaries show high expression of AMH receptor II (AMHR2) in the stromal fibroblasts surrounding the follicles and activation of the post-AMHR2 pathway by recombinant AMH was evidenced by increased phosphorylation of SMAD1,5 and 9, increased expression AMHR2 and upregulation of αSMA, suggesting fibroblast activation to initiate myofibroblast differentiation. WHAT IS KNOWN ALREADY: AMH secreted by small growing follicles, regulates ovarian activity. It suppresses initial primordial follicle (PMF) recruitment and FSH-dependent growth. AMH signal transduction is mediated by AMHR2, activating intracellular SMAD proteins and other signaling cascades to induce target-gene expression. Although AMHR2 expression has been reported within the follicle unit, there is evidence suggesting it may be identified in the stroma as well. STUDY DESIGN, SIZE, DURATION: Fresh murine ovaries were extracted from BALB/c mice (6 weeks old; n = 12 and 21 days old; n = 56). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18-35, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine (6 weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1α/αSMA/VCAM1, with additional vimentin staining in mice. Murine (21 days old) and human donor ovaries were used for fibroblast isolation and subsequent 7-day cultures. Prior to assessing AMH effects on isolated fibroblast culture, purity validation tests were implemented to ensure the absence of epithelial, immune, endothel, granulosa, and theca ovarian cell populations. The fibroblast culture's homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for αSMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200 ng/ml) for 0-72 h, with an additional time point of 96 h for human samples, followed by RT-qPCR, western blot, and immunocytochemistry (ICC) for AMHR2 expression. AMHR2 post-receptor signaling was examined by pSMAD1,5,9 levels via western blot. Activated fibroblast marker, αSMA, was assessed via western blot and ICC. MAIN RESULTS AND THE ROLE OF CHANCE: Immunostaining of mouse and human ovarian tissue showed that stromal cells around follicles at all developmental stages exhibit high AMHR2 expression, while granulosa cells of growing follicles show considerably lower levels. The majority of these AMHR2-positive stromal cells were identified as fibroblasts (Collagen1α in mice and human; vimentin in mice). RT-qPCR, western blot, and immunostaining were performed on cultured mouse and human fibroblasts, confirming that they consisted of a pure fibroblast population (αSMA/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94 ± 1.34 cells/field in mice) and 100% (average 19.20 ± 1.39 cells/field in human samples) of these fibroblasts expressed AMHR2 (ICC). rAMH treated cultured fibroblasts showed increased pSMAD1,5 and 9 levels, demonstrating the effects of AMH on its downstream signaling pathway. pSMAD1,5 and 9 expression increased, as detected by western blot: 1.92-fold in mice (48 h, P = 0.026) and 2.37-fold in human samples (48 h, P = 0.0002). In addition, rAMH treatment increased AMHR2 protein expression, as observed in ICC (human): a 2.57-fold upregulation of AMHR2 Mean Fluorescence Intensity (MFI) (96 h, P = 0.00036), and western blot, showing a 4.2-fold time-dependent increase (48 h, P = 0.026) in mice and 2.4-fold change (48 h, P = 0.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72 h, P = 0.0137) in mice and a 7.87-fold change (72 h, P < 0.0001) in humans. rAMH treatment induced fibroblast activation (αSMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. αSMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96 h, P = 0.000067), and in western blot with a 5.12-fold increase in mice (48 h, P = 0.0345) and a 2.69-fold increase in humans (48 h, P ≤ 0.0001). Activated AMHR2-positive stained fibroblast fractions were solely located around growing follicles, in both human and mice. In addition, a small population of AMHR2-positive stained theca cells (VCAM1 positive) was observed. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ex vivo, fibroblast gene expression might be changed by adhesion to the tissue-culture plate. Nevertheless, cultured fibroblasts (with and without rAMH) are subjected to the same conditions. Observations or significant differences can therefore be considered reliable. In addition, the presented effect of rAMH on fibroblasts is not directly linked to the known inhibitory effect of AMH on follicle activation. WIDER IMPLICATIONS OF THE FINDINGS: Clarifying the populations of AMH-responsive cells in the ovary provides a foundation for further investigation of the complex AMH signaling across the ovary. The composition of AMH-releasing and -responsive cells can shed light on the communication network between follicles and their environment, which may elucidate the mechanisms behind the AMH inhibitory effect on PMF activation. STUDY FUNDING/COMPETING INTEREST(S): This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study. TRIAL REGISTRATION NUMBER: N/A.

Fertility preservation parameters in patients with haematologic malignancy: a systematic review and meta-analysis.

Patients with haematologic malignancies represent one of the most common groups referred for fertility preservation before gonadotoxic oncological treatment. The aim of this systematic review and meta-analysis was to evaluate the effect of haematologic cancer on ovarian reserve and response to ovarian stimulation compared with healthy controls. A total of eight observative studies were included in the final quantitative analysis. Despite a younger age (mean difference -4.17, 95% CI -6.20 to -2.14; P < 0.0001), patients with haematologic malignancy had lower serum anti-Müllerian hormone levels compared with the control group (MD -1.04, 95% CI -1.80 to -0.29; P = 0.007). The marginally higher total recombinant FSH dose (MD 632.32, 95% CI -187.60 to 1452.24; P = 0.13) and significantly lower peak oestradiol serum level (MD -994.05, 95% CI -1962.09 to -26.02; P = 0.04) were demonstrated in the study group compared with the healthy controls. A similar number of retrieved oocytes were achieved in both groups (MD 0.20, 95% CI -0.80 to 1.20; P = 0.69). In conclusion, haematologic malignancies may detrimentally affect ovarian function manifesting in decreased AMH serum levels despite a younger age compared with healthy controls. This effect can be overcome by the application of relevant IVF protocols and stimulation doses to achieve an adequate oocyte yield.

Dual suppression of follicle activation pathways completely prevents the cyclophosphamide-induced loss of ovarian reserve.

STUDY QUESTION: To what extent and how does combined administration of the follicle activation pathway suppressive agents temsirolimus (Tem) and c-terminus recombinant anti-Müllerian hormone (rAMH) protect against chemotherapy-induced ovarian reserve loss? SUMMARY ANSWER: Combined administration of Tem and rAMH completely prevents cyclophosphamide (Cy)-induced follicle depletion and protects the ovarian reserve in mice, primarily via primordial follicle (PMF) suppression of activation and to a lesser degree by reducing apoptosis. WHAT IS KNOWN ALREADY: There is conflicting evidence regarding the contributory roles of apoptosis and follicle activation in chemotherapy-induced PMF loss. Tem, a mammalian target of rapamycin (mTOR) inhibitor, reduces activity of the phosphoinositide 3-kinases-phosphatase and tensin homolog (PI3K-PTEN) pathway which provides intrinsic regulation of PMF activation. Anti-Müllerian hormone (AMH), secreted by early growing follicles, is an extrinsic regulator of PMF activation. STUDY DESIGN, SIZE, DURATION: Whole ovaries of 12-day-old mice were cultured ex vivo for 7 days in the presence of Cy ± rAMH or Tem. Eight-week-old mice were randomized into eight treatment groups: vehicle control/rAMH/Tem/Cy/Tem + rAMH/Cy + Tem/Cy + rAMH/Cy + Tem + rAMH. Twelve hours after treatment, ovaries were removed for DNA damage analysis, and 24 h after treatment either for analysis of PI3K pathway proteins or to be fixed and immunostained for analyses of proliferation and apoptosis. Three or 21 days following treatment, ovaries were fixed and sectioned for follicle counting. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hematoxylin and eosin staining was used for differential follicle counts of primordial, primary, and secondary follicles in ex vivo (n = 16-18 ovaries per group) and in vivo ovaries (n = 8 mice per group). Histological analyses were carried out to measure proliferation by quantifying Ki-67-positive granulosa cells in primary follicles (n = 4 mice per group). DNA damage and apoptosis were measured by quantification of phosphorylated form of histone 2AX (γH2AX) and cleaved poly (ADP-ribose) polymerase (cPARP)-positive PMF oocytes, respectively (n = 8 mice per group). Protein extracts from whole ovaries were analyzed by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: In vivo experiments show that treatment with Cy alone caused significant loss of PMF reserve (32 ± 2.12 versus 144 ± 2.8 in control, P < 0.001), and this was significantly attenuated by treatment with either Tem (P < 0.001) or rAMH (P < 0.001). Combined cotreatment with Cy + Tem + rAMH provided complete protection of the PMF reserve, with no significant difference in numbers of PMF versus untreated animals. Similar results were demonstrated in the ex vivo experiments. Proliferation marker Ki-67 staining was significantly reduced in granulosa cells of primary follicles in the Cy + Tem + rAMH group compared with Cy alone group (after 24 h in vivo administration of Cy, 16% versus 65%, respectively; P < 0.001). Protein analysis demonstrated not significant increased phosphorylation of follicle activation proteins rpS6 and mTOR with in vivo administration of Cy alone (1.9 and 1.4 times the control ovaries, respectively), and this was reduced to below control levels in the Cy + Tem + rAMH group (P < 0.01). The Cy + Tem + rAMH combined cotreatment protected the follicle reservoir via inhibition of Cy-induced upregulation of the PI3K signaling pathway, together with replacement of AMH suppression of PMF activation with rAMH, implying a complementary effect of the two inhibitors. The DNA damage marker γH2AX was highly positive in PMF oocytes from Cy-treated ovaries 12 h after treatment, compared with controls (94% versus 59%, respectively, P < 0.001) and was significantly reduced to (69%) in Cy + Tem + rAMH cotreated ovaries (P < 0.001). However, only 22% of PMF oocytes of the Cy group showed apoptosis at 24 h, and this was significantly reduced (12%) in ovaries after treatment with Cy + Tem + rAMH (P < 0.01). This suggests that it is not possible to equate DNA damage with oocyte death, and also indicates that less than one-third of the total PMF loss can be attributed to apoptosis, implying that most of the PMF depletion results from PMF activation but that both mechanisms play a significant role. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The experimental design was limited by the selection of one time point for analysis of PMF activation and apoptosis (i.e. 24 h after Cy administration), although DNA damage was measured at 12 h after Cy administration and any impact on short-term follicle dynamics at 3 days after treatment. Protein analysis was conducted on whole ovary lysates therefore the protein changes identified cannot be localized to specific cells within the ovary. However, this complementary assay showed that there was activation in the ovary through massive reduction in the phosphorylation of key proteins in the PI3K cascade (rpS6 and mTOR), which is consistent with the sequence of events after Cy administration. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the complementary nature of different follicle activation pathways and the impact of their suppression in prevention of chemotherapy-induced ovotoxic damage, as well as their involvement in DNA damage inhibition, provides an interesting direction for future research, and the potential for noninvasive pharmacological fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Morris Kahn Foundation. The authors declare no conflicts of interest.

Evaluation of ovarian reserve in young females with non-iatrogenic ovarian insufficiency to establish criteria for ovarian tissue cryopreservation.

RESEARCH QUESTION: Can ovarian reserve parameters predict the outcome of ovarian tissue cryopreservation (OTCP) in patients ≤18 years with non-iatrogenic premature ovarian insufficiency (POI)? DESIGN: Retrospective cohort analysis carried out in a single tertiary hospital between August 2010 and January 2020. Thirty-seven patients ≤18 years with non-iatrogenic POI (27 with Turner syndrome, six with POI of unknown aetiology, three with galactosemia and one with blepharophimosis, ptosis, epicanthus inversus syndrome) were included. Three parameters were used to evaluate ovarian reserve: anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and transabdominal antral follicle count. Fertility preservation (most commonly OTCP) was offered if ovarian reserve was diminished and one or more parameters was positive. Follicles were counted in ovarian samples obtained at the time of OTCP. RESULTS: Ovarian reserve was diminished in 34 patients and 19 of them had one or more positive parameter. Fourteen (11 aged ≥12 years and 3 aged <12) underwent OTCP, one (14 years old) underwent ovarian stimulation and oocyte cryopreservation and four declined fertility preservation. Follicles were detected in 11 of 14 patients who underwent OTCP with one or more positive parameters (79%), and in all those (100%) who had two or three positive parameters. The median number of follicles was 27 (range 5-64) and 48 (range 21-75) in patients ≥12 years and those <12 years, respectively. CONCLUSION: This study shows that if OTCP is performed in patients with one or more positive parameters of ovarian activity, a 79% positive predictive value is achieved for the detection of follicles. The incorporation of this criterion for OTCP will minimize the risk of harvesting ovarian tissue with a low number of follicles.

Overactivation or Apoptosis: Which Mechanisms Affect Chemotherapy-Induced Ovarian Reserve Depletion?

Dormant primordial follicles (PMF), which constitute the ovarian reserve, are recruited continuously into the cohort of growing follicles in the ovary throughout female reproductive life. Gonadotoxic chemotherapy was shown to diminish the ovarian reserve pool, to destroy growing follicle population, and to cause premature ovarian insufficiency (POI). Three primary mechanisms have been proposed to account for this chemotherapy-induced PMF depletion: either indirectly via over-recruitment of PMF, by stromal damage, or through direct toxicity effects on PMF. Preventative pharmacological agents intervening in these ovotoxic mechanisms may be ideal candidates for fertility preservation (FP). This manuscript reviews the mechanisms that disrupt follicle dormancy causing depletion of the ovarian reserve. It describes the most widely studied experimental inhibitors that have been deployed in attempts to counteract these affects and prevent follicle depletion.

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].

FERTILITY PRESERVATION: Follicle reserve loss in ovarian tissue transplantation.

Ovarian tissue cryopreservation and transplantation (OTCP-TP) has progressed over the past decade from a revolutionary experimental procedure to a well-accepted treatment in many centers for young patients with a high risk of ovarian failure after cancer treatment. The procedure is remarkably successful, with studies reporting return of ovarian function in up to 95% of graft recipients and pregnancy rates of between 30 and 50%. The most significant limitation of OTCP-TP is the massive loss of follicles that occurs following transplantation, which is primarily attributed to ischemic damage and follicle activation. We review the current approaches to reducing follicle loss and maximizing graft lifespan via pharmacological agents which reduce ischemic damage and follicle activation. We further discuss the value and disadvantage of inducing follicle activation in the graft as a means of generating mature follicles in the immediate short term.

Pharmacological administration of recombinant human AMH rescues ovarian reserve and preserves fertility in a mouse model of chemotherapy, without interfering with anti-tumoural effects.

PURPOSE: To determine whether pharmacological administration of recombinant human anti-Mullerian hormone (rAMH) protects the ovarian reserve and preserves fertility without interfering with anti-tumoural cytotoxic action of chemotherapy. METHODS: Intraperitoneal delivery of rAMH and ovarian post-receptor activity were assessed with immunohistochemistry and western blot. Differential follicle counts and reproductive outcomes were assessed after cyclophosphamide (Cy) administration, with/without concurrent administration of rAMH. Interference of rAMH with Cy chemotoxicity was assessed on a human breast cancer cell line and an in vivo mouse model of human leukaemia. RESULTS: rAMH reached the ovary after intraperitoneal injection and demonstrated post-receptor bioactivity. Cy administration in mice caused primordial follicle activation, as shown by a decrease in primordial follicle population accompanied by an increase in early growing follicles and granulosa cell proliferation. Co-administration of rAMH reduced follicle activation, thereby protecting the primordial follicle reserve, and improving long-term fertility and reproductive outcomes. rAMH co-administration did not interfere with the cytotoxic actions of Cy in vitro on breast cancer cell line or in vivo in a model of human leukaemia. CONCLUSION: This study demonstrates that rAMH is bioactive in the ovary for a limited time, and that pharmacological administration of rAMH during chemotherapy treatment reduces follicle activation and primordial follicle loss and significantly improves reproductive outcomes in a mouse model, and does not interfere with the therapeutic actions of the treatment. Further investigation is necessary to determine whether it has similar protective effects in the human ovary.

Patient-centered elective egg freezing: a binational qualitative study of best practices for women's quality of care.

PURPOSE: How can elective egg freezing (EEF) be made patient centered? This study asked women to reflect on their experiences of EEF, which included their insights and recommendations on the optimal delivery of patient-centered care. METHODS: In this binational, qualitative study, 150 women (114 in the USA, 36 in Israel) who had completed at least one cycle of EEF were recruited from four American IVF clinics (two academic, two private) and three in Israel (one academic, two private) over a two-year period (June 2014-August 2016). Women who volunteered for the study were interviewed by two medical anthropologists. Interviews were audio recorded, transcribed, and entered into a qualitative data management program (Dedoose) for analysis. RESULTS: The majority (85%) of women were without partners at the time of EEF, and thus were undertaking EEF alone in mostly couples-oriented IVF clinics. Following the conceptual framework known as "patient-centered infertility care," we identified two broad categories and eleven specific dimensions of patient-centered EEF care, including (1) system factors: information, competence of clinic and staff, coordination and integration, accessibility, physical comfort, continuity and transition, and cost and (2) human factors: attitude and relationship with staff, communication, patient involvement and privacy, and emotional support. Cost was a unique factor of importance in both countries, despite their different healthcare delivery systems. CONCLUSIONS: Single women who are pursuing EEF alone in the mostly couples-oriented world of IVF have distinct and multifaceted needs. IVF clinics should strive to make best practices for patient-centered EEF care a high priority.

Follicle activation is a significant and immediate cause of follicle loss after ovarian tissue transplantation.

PURPOSE: Extensive follicle loss has been demonstrated in ovarian grafts post transplantation, reducing their productivity and lifespan. Several mechanisms for this loss have been proposed, and this study aims to clarify when and how the massive follicle loss associated with transplantation of ovarian tissue graft occurs. An understanding of the mechanisms of follicle loss will pinpoint potential new targets for optimization and improvement of this important fertility preservation technique. METHODS: Frozen-thawed marmoset (n = 15), bovine (n = 37), and human (n = 46) ovarian cortical tissue strips were transplanted subcutaneously into immunodeficient castrated male mice for 3 or 7 days. Histological (H&E, Masson's trichrome) analysis and immunostaining (Ki-67, GDF9, cleaved caspase-3) were conducted to assess transplantation-associated follicle dynamics, with untransplanted frozen-thawed tissue serving as a negative control. RESULTS: Evidence of extensive primordial follicle (PMF) activation and loss was observed already 3 days post transplantation in marmoset, bovine, and human tissue grafts, compared to frozen-thawed untransplanted controls (p < 0.001). No significant additional PMF loss was observed 7 days post transplantation. Recovered grafts of all species showed markedly higher rates of proliferative activity and progression from dormant to growing follicles (Ki-67 and GDF9 staining) as well as higher growing/primordial (GF/PMF) ratio (p < 0.02) and higher collagen levels compared with untransplanted controls. CONCLUSIONS: This multi-species study demonstrates that follicle activation plays an important role in transplantation-induced follicle loss, and that it occurs within a very short time frame after grafting. These results underline the need to prevent this activation at the time of transplantation in order to retain the maximal possible follicle reserve and extend graft lifespan.

Outcome of immature oocytes collection of 119 cancer patients during ovarian tissue harvesting for fertility preservation.

PURPOSE: Few clinical options for fertility preservation are available to females with cancer, and data about clinical outcomes is limited. Potential supplementary approaches to fertility preservation include retrieval of immature oocytes followed by in vitro maturation (IVM) and storage. The aim of this study was to evaluate post-thawing outcomes of immature oocytes collected both by transvaginal aspiration and from excised ovarian tissue. METHODS: We conducted a retrospective cohort study of patients treated in a single tertiary center. We reviewed the records of 119 cancer patients who underwent ovarian tissue cryopreservation and immature oocyte harvesting for fertility preservation. All embryos and oocytes that were frozen and thawed were included in the study. Post-thawing outcomes were evaluated. RESULTS: Thirty-five stored embryos from eight patients were thawed. Twenty-nine embryos survived (82% survival rate) and were transferred. Six oocytes were thawed, two oocytes survived, and no oocytes were fertilized. Only one PCOS patient became pregnant, resulting in the normal delivery of a healthy baby. CONCLUSIONS: Although a relatively high number of mature oocytes and embryos can be stored with the combined procedure, the limited rate of pregnancies represents a poor reproductive outcome. Therefore, this approach should be reserved for special groups with limited options.

Ten pathways to elective egg freezing: a binational analysis.

PURPOSE: What are the specific pathways that lead women to freeze their eggs? In this binational study, women were asked directly about the life circumstances that led them on the path to elective egg freezing (EEF). METHODS: From June 2014 to August 2016, 150 women (114 in the USA, 36 in Israel) who had completed at least 1 cycle of EEF were interviewed by two medical anthropologists. Study participants were recruited through four American IVF clinics (two academic, two private) and three in Israel (one academic, two private). Interviews were audio-recorded, transcribed verbatim, and entered into a qualitative data management program (Dedoose) for analysis. RESULTS: The majority (85%) of women in the study were without partners, while 15% had partners at the time of EEF. Six pathways to EEF were found among women without partners (being single, divorced, broken up, deployed overseas, single mother, career planner), with career planning being the least common pathway to EEF. Among women with partners, four pathways to EEF were found (relationship too new or uncertain, partner not ready to have children, partner refusing to have children, or partner having multiple partners). With only one exception, the pathways and their frequencies were similar in both countries. CONCLUSIONS: Partnership problems, not career planning, lead most women on pathways to EEF. These pathways should be studied in a variety of national settings, and fertility clinics should offer patient-centered care for single women pursuing EEF in the couples-oriented world of IVF.

Medical egg freezing: the importance of a patient-centered approach to fertility preservation.

PURPOSE: This binational qualitative study of medical egg freezing (MEF) examined women's motivations and experiences, including their perceived needs for patient-centered care in the midst of fertility- and life-threatening diagnoses. METHODS: Forty-five women who had undertaken MEF were interviewed in the USA (33 women) and in Israel (12 women) between June 2014 and August 2016. Interviews lasted approximately 1 h and were conducted by two senior medical anthropologists, one in each country. Women were recruited from four American IVF clinics (two academic, two private) and two Israeli clinics (both academic) where MEF is being offered to cancer patients and women with other fertility-threatening medical conditions. RESULTS: Women who undertake MEF view their fertility and future motherhood as important components of their identities and recovery and, thus, are grateful for the opportunity to pursue fertility preservation. However, women who undergo MEF have special needs, given that they tend to be a "vulnerable" population of young (age < 30), unmarried, resource-constrained women, who are facing not only fertility loss but also the "double jeopardy" of cancer. Through in-depth, qualitative interviews, these women's MEF stories reveal 10 dimensions of care important to fertility preservation, including five "system factors" (information, coordination and integration, accessibility, physical comfort, cost) and five "human factors" (adolescent issues, male partner involvement, family involvement, egg disposition decisions, emotional support). Together, these dimensions of care constitute an important framework that can be best described as "patient-centered MEF." CONCLUSIONS: Women pursuing MEF have special medical needs and concerns, which require particular forms of patient-centered care. This study outlines 10 dimensions of patient-centered fertility preservation that are appropriate for MEF patients. This approach may help IVF clinics to be better prepared for delivering top-quality care to mostly young, single women facing the daunting prospect of fertility loss and life-threatening medical diagnoses.

Optimizing outcomes from ovarian tissue cryopreservation and transplantation; activation versus preservation.

Ovarian tissue cryopreservation and transplantation (OTCP) is gaining increasing traction in the field of fertility preservation as a result of accumulated successes. We now have a decade of experience with the technique, with tens of live births and greater than 90% return of ovarian function in graft recipients. Recently, a novel method of OTCP has been described, termed in vitro activated OTCP which proposes significant changes to the standard protocol. This method aims to stimulate activation of dormant follicles within the grafts prior to transplantation and ensure that mature oocytes can be generated in the immediate short term after transplantation. By contrast, conventional OTCP seeks to maintain dormancy and thus preserve the follicle reserve in the graft with the aim of maximizing graft lifespan. This opinion paper will compare the two methods of OTCP, highlighting their respective advantages and disadvantages, and provide suggestions as to when to apply either one of these methods in a clinical setting.

Follicle activation and 'burn-out' contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness.

STUDY QUESTION: What are the effects of thin ovarian grafts compared with grafts of the standard thickness on follicle loss post-transplantation? SUMMARY ANSWER: Transplantation of reduced-thickness ovarian grafts led to intense activation and 'burn-out' a short time after transplantation resulting in significant folllicle loss. WHAT IS KNOWN ALREADY: Transplantation of fresh and frozen-thawed ovarian tissue has been proved successful, but techniques vary and are not optimised, often resulting in significant follicular loss. Follicle loss is mostly related to the freezing-thawing process and to post-transplantation hypoxia. STUDY DESIGN, SIZE, DURATION: Bovine ovarian tissue strips (n = 55) were prepared in two groups of conventional-thickness strips (1-2 mm) or thin strips (0.5-0.9 mm). Fresh or frozen-thawed samples were xenotransplanted into sterilized immune-deficient mice (n = 49). Non-transplanted conventional size fresh samples were used as controls (n = 6). Grafts from all study groups were recovered after 7 days for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Morphometric differential counting of follicle classes was performed by two observers. Immunohistochemistry was conducted for proliferation (Ki67), cortical fibrosis (Masson tri-chrome) and blood-vessel density (CD31). Results were expressed as the mean number of dormant or growing follicle (GF) type per section or total follicle counts per graft. Blood-vessel density was calculated per mm(2). P-values <0.05 were considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: The loss of all follicle types, and most noteably of primordial follicles (PMFs), was observed 7 days post-transplantation (P < 0.05). The relatively high number of GFs and the positive Ki67 staining in all recovered grafts indicated that follicle activation was depleting the resting follicle pool. The reduced graft thickness had an adverse effect on the number of recovered follicles, especially on the resting non-GFs in the fresh, and more so in the frozen-thawed, samples (P < 0.05). Extensive stromal fibrosis and high blood-vessel density were observed in all grafts with no advantage in the thin prepared grafts. LIMITATIONS, REASONS FOR CAUTION: This study used only one species of ovaries (bovine) for xenotransplantation. The immediate post-transplantation events were not visualized directly nor were the molecules involved in follicle activation studied. WIDER IMPLICATIONS OF THE FINDINGS: Follicle activation and 'burn-out' appear to be important in follicle loss after transplantation. Reducing graft thickness in an attempt to improve freezing conditions and reduce post-transplantation ischemia has adverse effects on the graft follicle pool due to increased activation and loss. Agents which prevent 'burn-out' will potentially improve follicle pool survival. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by research grants from the Israeli Science Foundation (No. 1675/10), the Israeli Jack Craps foundation and the Israel Cancer Research Fund (ICRF No. 12-3081). The authors have no competing interest to declare.

Ovarian minimal residual disease in chronic myeloid leukaemia.

The options for fertility preservation include cryopreservation of ovarian tissue. Although transplantation of cryopreserved-thawed ovarian tissue in cancer survivors has resulted in live births, there is evidence of malignancy involvement in ovarian tissue, especially in leukaemia. The objectives of this study were to investigate the involvement of chronic myeloid leukaemia (CML) in ovaries by both pathological/immunohistochemical methods and PCR for the identification of the Philadelphia chromosome (BCR-ABL transcripts). The patient was a survivor of paediatric CML whose ovaries were cryopreserved. The patient became infertile and requested ovarian reimplantation in adulthood. Pathological examinations of ovarian tissue with immunohistochemical stainings, quantitative PCR and two-step nested PCR were applied to identify BCR-ABL transcripts. Despite the lack of positive pathological/immunohistochemical evidence, PCR and two-step nested PCR revealed that the ovary was contaminated by malignant minimal residual CML. Survivors of childhood CML may harbour minimal residual disease in the ovaries. This finding stresses the danger of reseeding cancer by ovarian grafting, especially in patients with leukaemia. If ovarian grafting is considered, reimplantation should be preceded by examination of ovarian samples both pathologically and by molecular techniques. On the basis of molecular findings, ovarian autografting was not recommended in this case report.

Fertility preservation in young females with hematological malignancies.

Impaired reproductive function and possible infertility are major concerns in long-term survivors of hematological malignancies. The ongoing increase in the survival rates of these patients is therefore accompanied with a growing demand for effective, safe and specifically tailored fertility preservation options. When approaching patients facing hematological malignancy, an individual evaluation of potential infertility risks and possible preventive or preserving measures should be performed. This review aims to provide up-to-date knowledge on female reproductive risks, and ovarian, uterine and genital injuries associated with therapy regimens currently used in hemato-oncological disorders. Recent progress in fertility preservation methods including ovarian tissue cryopreservation and transplantation, egg and embryo freezing, ovarian transposition and their specific role in hematological disorders are presented. The efficacy of these methods, possible risks and future challenges are critically discussed.

Use of titanium spiral tacks for long-term oophoropexy before pelvic irradiation.

BACKGROUND: Ovarian transposition before planned pelvic irradiation can preserve ovarian function in young patients with pelvic malignancies. The transposed ovaries are fixed to the posterolateral abdominal wall. We described the use of a titanium spiral tack as a fixation device and compared it with other methods of oophoropexy. METHODS: Medical and surgical records of all consecutive patients who underwent oophoropexy in our institution between 2007 and 2013 were reviewed. Demographic and clinical data were summarized; follicle-stimulating hormone values, recorded; and imaging scans, reviewed. RESULTS: Oophoropexy was performed in 30 patients: 28 with cervical carcinomas and 2 with pelvic sarcomas. The procedure was done through laparoscopy in 13 patients and through laparotomy in 17. Titanium spiral tack was used for ovarian fixation in 14 patients, Vicryl suturing in 14, and in 2 cases the ovaries were pulled up through a retroperitoneal tunnel and fixed to the peritoneum with sutures. Titanium spiral tack fixation took a few seconds to perform. There were no immediate intraoperative or postoperative complications. Ovarian function was preserved in 15 patients (7/14 with spiral tack, 6/14 with sutures, and in both patients with retroperitoneal tunneling). Postoperative imaging results showed that all ovaries retained their extrapelvic location for a median period of 11.6 months (range, 2.3-63 months). CONCLUSIONS: Spiral tack is a simple, reliable method for oophoropexy before pelvic irradiation. Its efficacy is comparable with that of suture fixation, with the added advantage of ultrashort operative time. It is therefore worth considering as an alternative to suturing.

Recommendations for fertility preservation in patients with lymphoma, leukemia, and breast cancer.

Fertility issues should be addressed to all patients in reproductive age before cancer treatment. In men, cryopreservation of sperm should be offered to all cancer patients in reproductive age regardless of the risk of gonadal failure. In women, the recommendation of fertility preservation should be individualized based on multiple factors such as the urgency of treatment, the age of the patient, the marital status, the regimen and dosage of cancer treatment.

The neglected members of the family: non-BRCA mutations in the Fanconi anemia/BRCA pathway and reproduction.

BACKGROUND: BReast CAncer (BRCA) genes are extensively studied in the context of fertility and reproductive aging. BRCA proteins are part of the DNA repair Fanconi anemia (FA)/BRCA pathway, in which more than 20 proteins are implicated. According to which gene is mutated and which interactions are lost owing to the mutation, carriers and patients with monoallelic or biallelic FA/BRCA mutations exhibit very different phenotypes, from overt FA to cancer predisposition or no pathological implications. The effect of the so far neglected non-BRCA FA mutations on fertility also deserves consideration. OBJECTIVE AND RATIONALE: As improved treatments allow a longer life expectancy in patients with biallelic FA mutations and overt FA, infertility is emerging as a predominant feature. We thus reviewed the mechanisms for such a manifestation, as well as whether they also occur in monoallelic carriers of FA non-BRCA mutations. SEARCH METHODS: Electronic databases PUBMED, EMBASE and CENTRAL were searched using the following term: 'fanconi' OR 'FANC' OR 'AND' 'fertility' OR 'pregnancy' OR 'ovarian reserve' OR 'spermatogenesis' OR 'hypogonadism'. All pertinent reports in the English-language literature were retrieved until May 2021 and the reference lists were systematically searched in order to identify any potential additional studies. OUTCOMES: Biallelic FA mutations causing overt FA disease are associated with premature ovarian insufficiency (POI) occurring in the fourth decade in women and with primary non-obstructive azoospermia (NOA) in men. Hypogonadism in FA patients seems mainly associated with a defect in primordial germ cell proliferation in fetal life. In recent small, exploratory whole-exome sequencing studies, biallelic clinically occult mutations in the FA complementation group A (Fanca) and M (Fancm) genes were found in otherwise healthy patients with isolated NOA or POI, and also monoallelic carrier status for a loss-of-function mutation in Fanca has been implicated as a possible cause for POI. In those patients with known monoallelic FA mutations undergoing pre-implantation genetic testing, poor assisted reproduction outcomes are reported. However, the mechanisms underlying the repeated failures and the high miscarriage rates observed are not fully known. WIDER IMPLICATIONS: The so far 'neglected' members of the FA/BRCA family will likely emerge as a relevant focus of investigation in the genetics of reproduction. Several (rather than a single) non-BRCA genes might be implicated. State-of-the-art methods, such as whole-genome/exome sequencing, and further exploratory studies are required to understand the prevalence and mechanisms for occult FA mutations in infertility and recurrent miscarriage.