Ovarian Cortex Cryopreservation Research Articles

P-359 Ten years’ experience using cryopreserved oocytes from In Vitro Maturation (IVM) or Controlled Ovarian Hyperstimulation (COH) cycles for fertility preservation in oncologic indications

Abstract Study question Is IVM a relevant option for fertility preservation in cancer patients, considering biological/clinical outcomes post-thawing, compared to COH? Summary answer This study shows that despite a significantly altered embryo morphology within the IVM group, the clinical outcomes remain comparable to those of COH. What is known already Fertility Preservation (FP) techniques are proposed to patients with cancer to provide the possibility of childbearing using their own gametes. Currently, oocyte vitrification after COH is the standard option. IVM serves as an alternative when COH is contraindicated, urgent cancer therapy is required, or in conjunction with ovarian cortex cryopreservation. Limited live births have been reported from frozen-thawed oocytes in IVM cycles of cancer survivors. Scarce data are available for comparing biological/clinical outcomes of frozen-thawed oocytes from IVM versus COH cycles in cancer survivors. Study design, size, duration This bicentric retrospective cohort study aimed to analyze the outcomes of all oocyte warming cycles in 73 cancer survivors having undergone oocyte vitrification for FP after COH and/or IVM. All of them had oocyte retrieval before the administration of gonadotoxic treatment and returned after being cured, with their oncologist agreement, for assisted reproduction treatments, between November 2012 and January 2024. Participants/materials, setting, methods Seventy-seven warming attempts followed by ICSI respectively from 37 COH and 40 IVM cycles were analyzed. Four patients benefited from an IVM and COH cycle. Survival, fertilization, top and good-quality embryos, defined at day-2 respectively as 4 and 3-5 adequate-sized blastomeres, without multinucleation with < 20% of cytoplasmic fragments, implantation, biochemical (hCG>100 UI/mL), clinical (intrauterine sac with fetal heartbeat) and live birth rates were compared using appropriate statistical tests. Main results and the role of chance The mean age and antral follicle count at the time of FP was similar in both groups. The indications for FP were breast cancer (n = 61), hematologic malignancies (n = 7), ovarian cancer (n = 3), other (n = 2). The number of cryopreserved oocytes tended to be higher in the COH group, when compared to the IVM group (8.5±7.3 vs 5.9±4.04; p = 0.16). The mean oocyte maturation rate after IVM was 68.6±23.1%. Oocyte survival, and fertilization rates were similar in COH and IVM groups, respectively (76.4±27.0% vs 76.1±22.0%; p = 0.76) and (67.1±34.3% vs 65.3±25.4%; p = 0.36). We reported a significantly better embryo quality at day-2 in the COH group when compared to the IVM group, with respectively top-quality embryos (44.0±34.2% vs 26.1±33.4%, p = 0,04) and good-quality embryos (62.2±38.0% vs 42.8±40.8%, p = 0.049), and also a significantly higher number of useful embryo in the COH group (2.7±2.6 vs 1.5±1.2, p = 0.03). Regarding clinical outcomes, the results were similar in COH and IVM groups, in terms of implantation rates (12.2±30.4% vs 15.5±35.6%; p = 0.86), biochemical (32.3% (10/31) vs 17.9% (6/28); p = 0.39), clinical pregnancies (25.8% (8/31) vs 17.9% (4/28); p = 0.54), live birth rates ( 25.8% (8/31) vs 14.3% (4/28); p = 0.34). Limitations, reasons for caution Statistical power to compare IVF outcomes after COH and IVM is limited by the few women who returned to use their frozen oocytes. The subjective nature of embryo morphology assessment. Wider implications of the findings This study is the largest evaluating frozen-warmed oocytes from IVM cycles in cancer patients, with 5 live births newly reported. A higher oocyte-yield may be necessary in IVM as the number of useful embryos is significantly lower. Novel approaches are being developed to improve maturation rates following IVM. Trial registration number Not applicable

Mesenchymal stem cells promote ovarian reconstruction in mice

BackgroundStudies have shown that chemotherapy and radiotherapy can cause premature ovarian failure and loss of fertility in female cancer patients. Ovarian cortex cryopreservation is a good choice to preserve female fertility before cancer treatment. Following the remission of the disease, the thawed ovarian tissue can be transplanted back and restore fertility of the patient. However, there is a risk to reintroduce cancer cells in the body and leads to the recurrence of cancer. Given the low success rate of current in vitro culture techniques for obtaining mature oocytes from primordial follicles, an artificial ovary with primordial follicles may be a good way to solve this problem.MethodsIn the study, we established an artificial ovary model based on the participation of mesenchymal stem cells (MSCs) to evaluate the effect of MSCs on follicular development and oocyte maturation. P2.5 mouse ovaries were digested into single cell suspensions and mixed with bone marrow derived mesenchymal stem cells (BM-MSCs) at a 1:1 ratio. The reconstituted ovarian model was then generated by using phytohemagglutinin. The phenotype and mechanism studies were explored by follicle counting, immunohistochemistry, immunofluorescence, in vitro maturation (IVM), in vitro fertilization (IVF), real-time quantitative polymerase chain reaction (RT-PCR), and Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) assay.ResultsOur study found that the addition of BM-MSCs to the reconstituted ovary can enhance the survival of oocytes and promote the growth and development of follicles. After transplanting the reconstituted ovaries under kidney capsules of the recipient mice, we observed normal folliculogenesis and oocyte maturation. Interestingly, we found that BM-MSCs did not contribute to the formation of follicles in ovarian aggregation, nor did they undergo proliferation during follicle growth. Instead, the cells were found to be located around growing follicles in the reconstituted ovary. When theca cells were labeled with CYP17a1, we found some overlapped staining with green fluorescent protein(GFP)-labeled BM-MSCs. The results suggest that BM-MSCs may participate in directing the differentiation of theca layer in the reconstituted ovary.ConclusionsThe presence of BM-MSCs in the artificial ovary was found to promote the survival of ovarian cells, as well as facilitate follicle formation and development. Since the cells didn’t proliferate in the reconstituted ovary, this discovery suggests a potential new and safe method for the application of MSCs in clinical fertility preservation by enhancing the success rate of cryo-thawed ovarian tissues after transplantation.

Reproductive health in Turner's syndrome: from puberty to pregnancy.

Turner syndrome (TS) is a genetic pathology that affects about 1/2500 newborn females. Turner's syndrome is characterized by highly variable genetic anomalies that consist in a partial or complete deletion of the X sexual chromosome; it can be present as a monosomy or as a mosaicism with two o three different cellular lines. 50% of the patients with Turner's syndrome has a 45 XO karyotype while the remaining cases have karyotypes with mosaicism or X isochromosome or with partial or whole Y chromosome. This pathology is characterized by multiple anomalies that involve physical and cognitive development and in particular endocrine, cardiovascular, reproductive, auditive and visual systems. Integrity of the X chromosome in essential for fertility. In TS is accelerated germ cells apoptosis. About 30% of TS girls have some pubertal development, 10-20% undergo menarche and 2-8% go through spontaneous pregnancy. Women with TS should be informed about the risk of premature menopause and should be referred, if possible, to a specialist evaluation with a doctor expert in assisted reproductive techniques. In adolescents and in adults, Premature Ovarian Insufficiency (POI) can be evaluated clinically and biochemically with the classic combination of amenorrhea and elevated FSH concentrations (hypergonadotropic hypogonadism). However, in postpubertal adolescents and adult women, reproductive hormones may remain within the normal range before POI is clinically evident, despite significant depletion of the ovarian reserve. Today, reproductive medicine offers the opportunity of fertility preservation in women with premature ovarian insufficiency (POI). Two techniques have been suggested such as ovarian cortex cryopreservation and oocytes cryopreservation.

Current status of fertility preservation in a Spanish tertiary public hospital: multidisciplinary approach and experience in over 1500 patients.

Currently, 15% of gynaecological and 9% of haematological malignancies are diagnosed before the age of 40. The increased survival rates of cancer patients who are candidates for gonadotoxic treatments, the delay in childbearing to older ages, and the optimization of in vitro fertilisation techniques have all contributed to an increased interest in fertility preservation (FP) treatments. This study reviews the experience of the Fertility Preservation Programme (FPP) of a tertiary public hospital with a multidisciplinary approach. This retrospective study included all the available (FP) treatments, performed in patients of childbearing age between 2006 and 2022. 1556 patients were referred to the FPP: 332 oocyte vitrification cycles, 115 ovarian cortex cryopreservation with 11 orthotopic autotransplantations, 175 gonadotropin-releasing hormone (GnRH) agonist treatments, 109 fertility-sparing treatments for gynaecological cancer, and 576 sperm cryopreservation were performed. Malignancy was the main indication for FP (the main indications being breast cancer in women and haematological malignancies in men), although non-oncological pathologies, such as endometriosis and autoimmune diseases, have increased in recent years. Currently, the most widely used FP technique is oocyte vitrification, the increase of which has been associated with a decrease in the use of cortex CP and GnRH agonists. The increase in FP treatment reflects the implementation of reproductive counselling in oncology programmes. A multidisciplinary approach in a tertiary public hospital allows individualised FP treatment for each patient. In recent years, there has been a change in trend with the introduction of new indications for FP and a change in techniques due to their optimisation.

O-082 15-year-experience in oncological fertility preservation: Impact on disease survival and reproductive outcomes

Abstract Study question What is the impact of fertility preservation (FP) procedures and cancer treatment on relapse, survival, ovarian damage and pregnancy outcomes in oncological patients? Summary answer FP technique (including ovarian stimulation for oocyte vitrification) does not affect relapse or survival rates even in hormone dependant tumours. What is known already FP has become a crucial part of oncological evaluation of young women facing cancer given the high survival rates achieved. Oocyte vitrification (OV) and ovarian cortex cryopreservation (OCC) are the main techniques offered to these patients at the moment. However, information on the true incidence of premature ovarian insufficiency (POI), return rates to use the cryopreserved material and the natural pregnancy rates achieved in these patients is still limited. Moreover although there is some data about the safety of these techniques the impact of FP on disease survival is yet to be definitely assessed. Study design, size, duration Prospective cohort study. 695 patients enrolled since 2001 until 2016. Patients referred to FP unit in a public hospital setting (Hospital Peset Valencia 2001-2006 and University Hospital La Fe 2007-2016). After evaluation 556 patients received a FP technique (OV, OCC or embryo vitrification ) and 139 patients did not receive any due to medical reasons or patient’s choice. Minimum follow-up 5 years after enrolment Participants/materials, setting, methods Baseline characteristics including type of cancer and previous chemotherapy at diagnosis and prior to FP technique were recorded followed by risk of chemotherapy treatment received, relapse, survival, POI and poor ovarian reserve (POR) occurrence and pregnancy outcomes. Primary outcome was median survival time after FP in months. Secondary outcomes included relapse rate, POI and POR incidence, usage FP rate, clinical pregnancy and live birth (LB) rates naturally and after FP use. Main results and the role of chance There were no differences in survival comparing patients undergoing FP versus no FP (median 89.67 vs 92.81 months, p = 0.3). However, patients that used their cryopreserved material survived more than those who did not (97.3 vs 89.5, p = 0.012). When assessing survival rates comparing patients that had approval to get pregnant versus those who did not we found a higher survival in the former (98.84 vs 84.79 months, p < 0.001). Breast cancer patients with hormone dependent tumors undergoing ovarian stimulation for OV vs OCC had no differences in survival (95.62 vs 87.38 months, p = 0.37). POI incidence was 20.29% (N = 141). POI patients were significantly older (32.28 vs 29.63, p < 0.001) and had received high-risk chemotherapy more frequently (31.74% vs 2.27%, p < 0.001). Ovarian damage incidence (including also POR) was 48.06% (N = 334). Eighty-six patients (15.47%) used their cryopreserved material. Among the patients with pregnancy wish (N = 266) there were 84 spontaneous live births (31.58%). Patients that conceived naturally were significantly younger (30.71 vs 33.46, p < 0.001) and the chemotherapy received was more frequently low-risk (43.20% vs 23.52%, p = 0.018). There were 37 LB after use of FP (37/86, 43.02%). Patients with a higher ovarian reserve percentile had a higher chance of achieving a natural LBR (OR 1.016, 1.005-1.027, p = 0.004) Limitations, reasons for caution The higher survival found in patients using their cryopreserved material is mediated by the prognosis of the disease itself that limits the chance of pregnancy to those with a stable disease. Wider implications of the findings FP does not have a negative impact on survival even if ovarian stimulation is used. Almost half of the patients had ovarian damage (POI or POR) as a result of treatment; this is higher than previously reported. Among the patients with fertility wish around one third achieved a LB naturally. Trial registration number not applicable

O-115 Fertility preservation or egg donation for hereditary cancer syndromes?

Abstract Individuals with hereditary cancer syndromes are at risk of developing multiple tumours and, therefore, undergo life-long surveillance procedures and risk-reducing and/or therapeutic surgeries. Knowledge and testing for hereditary cancer susceptibility genes is rapidly spreading, often performed at the first cancer diagnosis. Different physical, psychosocial, ethical and economic considerations need to be taken into account when deciding on reproductive options in individuals with a hereditary cancer syndrome. The psychosocial impact of genetic predisposition to cancer may be particularly problematic and still represents an ongoing challenge for the professional taking care of these patients. In fact, worries about passing on a genetic risk for cancer to offspring are concerns expressed by the 65% of female young adult cancer survivors when specifically interviewed. At cancer diagnosis, fertility preservation procedures represent an effort to retain the opportunity to have genetically related children. Unfortunately, oocytes, embryo or ovarian cortex cryopreservation do not guarantee live birth. The success rates of these procedures are highly dependent on age, number of oocytes cryopreserved and may be influenced by other medical, lifestyle and (future) male factors. In addition, carriers of pathogenetic gene variants for cancer predisposition should also be thoroughly counselled about preimplantation genetic testing (PGT), a technique allowing the interruption of passing on the gene associated to cancer risk. However, this option inevitably lowers the efficacy of fertility preservation interventions in terms of future pregnancies. Egg donation is a spreading procedure with higher success rates than fertility preservation techniques. This approach allows individuals to avoid undergoing oocytes retrieval or ovarian tissue cryopreservation surgery, thus avoiding procedural risks (infection, haemorrhage, ovarian hyperstimulation syndrome) and postponement of oncological treatments. On the other side, offspring will not have a genetic link with recipient and the beneficial psychological role of fertility preservation would be missing. In fact, several recent studies have underlined the message of hope that is indirectly perceived by oncological patients when counselled for fertility preservation. Moreover, counselling on the possibility of egg donation is challenging at the time of cancer diagnosis. Making the right reproductive choice in carriers of hereditary cancer syndromes is a dilemma which can’t be solved generalizing. Even patients’ attitude could be different according to the time of counselling, whether is at cancer diagnosis or at the genetic predisposition diagnosis in healthy carriers. Egg donation should be considered in the available armamentarium. The complexity of the topic needs a personalized and multidisciplinary counselling.

P-481 Characterization of the gene expression profile of breast cancer tumours in patients undergoing ovarian stimulation for fertility preservation purposes

Abstract Study question Could controlled ovarian stimulation (COS) protocols with letrozole supplementation induce changes in the molecular footprints of estrogen receptor-positive (ER+) breast cancer (BC) women? Summary answer Ovarian stimulation with letrozole and gonadotropins does not change the gene expression profile of estrogen receptor-positive (ER+) breast malignant tumours as compared to non-stimulated patients. What is known already Fertility preservation (FP) strategies in ER+ BC patients involve modifications of the classical COS protocols to minimise the impact of estrogen levels during ovarian stimulation. The most common approaches are using the aromatase inhibitor letrozole to block the conversion of testosterone into estradiol in the granulosa cells or the use of tamoxifen to block the ER in the breast tissue. Although retrospective studies suggest that these strategies will have no impact on patient’s survival and disease progression, little is known regarding the effect of the ovarian stimulation on the genomic fingerprints of the exposed cancerous tissue. Study design, size, duration Retrospective, non-randomized, comparative study. Gene expression profiles were compared in biopsies at diagnosis and during excision surgery in two groups of ER+ BC patients: Patients undergoing COS for FP before surgery (COS group, n = 10) and patients not undergoing any type of COS (Control group, n = 11). The former received letrozole (5mg/day) supplements during COS. The latter included 7 patients not doing FP and 4 undergoing ovarian cortex cryopreservation. Patients were recruited between 2009 and 2021. Participants/materials, setting, methods Single 5-µm sections of formalin-fixed, paraffin-embedded (FFPE) needle core biopsy (NCB) and breast surgery (BS) tumour samples were obtained prior to starting gonadotoxic therapies. Differential gene expression and gene set analysis (GSA) was performed in tumour samples before (NCB-sample) and after COS (BS-sample). Simultaneous, quantitative detection of 2549 genes associated with tumour biology was performed with the HTG EdgeSeq Oncology Biomarker Panel. Tumour cell proliferation was also assessed by Ki67 staining. Main results and the role of chance Patients were younger in the COS group (COS: 30.2±3.4 vs Control: 34.5±2.3; p = 0.004). None of the patients experienced any relapse during the observation period. The length of COS was 11.5±3.5 days and 10.6±6.8 MII oocytes were vitrified. The exploratory analysis using principal component analysis revealed no relationships between the two BC biopsy samples and gene expression levels in the experimental groups. The differential expression analysis just revealed 6 genes significantly over-expressed after ovarian stimulation (DUSP1, FOS, EGFR1, NR4A1, JUN and CYR61). From these, DUSP1, FOS, and NR4A1 were also significantly upregulated in the unstimulated group. The GSA showed the cytochrome P450 pathway was significantly enriched after COS. No pathways related to cell proliferation were differentially expressed between groups. However, in unstimulated patients, 6 KEGG pathways were upregulated, with the cytokine-cytokine receptor interaction and the Jak-STAT signalling pathway being the most enriched. Ki67 immunostaining showed no differences in cell proliferation after COS (22.8±7.2 vs 24.0±6.9, p = 0.723). The rate of cell proliferation also remained constant in unstimulated patients (22.9±8.4 vs 25.1±8.1, p = 0.560). Limitations, reasons for caution The main limitations of the present study are the retrospective design and the associated risk of bias, although the population is very homogeneous when it comes to clinical characteristics; and the fact that molecular footprints are only interim markers of survival/response to the treatment. Wider implications of the findings The present study provides molecular evidence supporting the safety of COS using letrozole for oocyte vitrification in young BC patients undergoing ovarian stimulation. This information is crucial to support patient guidance during discussions about fertility preservation. Trial registration number Not applicable

Age-related fertility decline: is there a role for elective ovarian tissue cryopreservation?

Age-related fertility decline (ARFD) is a prevalent concern amongst western cultures due to the increasing age of first-time motherhood. Elective oocyte and embryo cryopreservation remain the most established methods of fertility preservation, providing women the opportunity of reproductive autonomy to preserve their fertility and extend their childbearing years to prevent involuntary childlessness. Whilst ovarian cortex cryopreservation has been used to preserve reproductive potential in women for medical reasons, such as in pre- or peripubertal girls undergoing gonadotoxic chemotherapy, it has not yet been considered in the context of ARFD. As artificial reproductive technology (ART) and surgical methods of fertility preservation continue to evolve, it is a judicious time to review current evidence and consider alternative options for women wishing to delay their fertility. This article critically appraises elective oocyte cryopreservation as an option for women who use it to mitigate the risk of ARFD and introduces the prospect of elective ovarian cortex cryopreservation as an alternative.

Fertility preservation techniques in cervical carcinoma.

AstractThe usefulness of this review is to highlight how a fertility preservation (FP) approach is currently feasible for patients diagnosed with uterine cervical cancer. To this regard, a fertility sparing surgery has just overcome its traditional limits, gained acceptance within the major gynecologic oncology societies thanks to the ability to identify the “ideal” candidates to this conservative treatment. On the other hand, the use of other FPs for oocyte and ovarian cortex cryopreservation is still extremely debated. In fact, the existing risk of tumor spreading during oocyte retrieval necessary for oocyte cryostorage for patients’ candidates for neo-adjuvant therapy, as well as the potential hazard of cancer cell dissemination after ovarian tissue replacement in cases of non-squamous type cervical carcinomas should not be underestimated. Therefore, in consideration of the encountered limitations and the need to ensure adequate reproductive health for young uterine cervical cancer survivors, translational research regarding the FP has progressively collected innovative insights into the employment of stemness technology. In this context, the property of ovarian stem cells obtained from the ovarian cortex to generate functional oocytes in women could represent a promising therapeutic alternative to the current procedures for a novel and safer FP approach in cancer survivors.

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

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

In Vitro Maturation of Oocytes Retrieved from Ovarian Tissue: Outcomes from Current Approaches and Future Perspectives.

In vitro maturation (IVM) of transvaginally aspirated immature oocytes is an effective and safe assisted reproductive treatment for predicted or high responder patients. Currently, immature oocytes are also being collected from the contralateral ovary during laparoscopy/laparotomy and even ex vivo from the excised ovary or the spent media during ovarian tissue preparation prior to ovarian cortex cryopreservation. The first live births from in vitro-matured ovarian tissue oocytes (OTO-IVM) were reported after monophasic OTO-IVM, showing the ability to achieve mature OTO-IVM oocytes. However, fertilisations rates and further embryological developmental capacity appeared impaired. The introduction of a biphasic IVM, also called capacitation (CAPA)-IVM, has been a significant improvement of the oocytes maturation protocol. However, evidence on OTO-IVM is still scarce and validation of the first results is of utmost importance to confirm reproducibility, including the follow-up of OTO-IVM children. Differences between IVM and OTO-IVM should be well understood to provide realistic expectations to patients.

Impact of chemotherapy on risk of diminished ovarian reserve in premenopausal women subjected to ovarian cortex cryopreservation

Background: Chemotherapy damage ovarian reserve and may lead to premature ovarian failure (POF). Ovarian tissue cryopreservation has been proposed as one of the alternatives to fertility preservation. Anti-mullerian hormone (AMH) testing has become a useful tool for assessing the iatrogenic damage to the ovarian reserve.

P–699 Multi-scale study of the architecture, topography and mechanics of the human ovary from prepuberty to menopause: a blueprint for next-generation bioengineering and diagnosis

Abstract Study question Does the ovarian ECM have a precise and unique biophysical phenotype, specific to each age, from prepuberty to menopause? Summary answer Differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. What is known already Ovarian engineering has recently emerged to respond to patient needs and offer reliable models for basic research. It has relied on synthetic and natural biomaterials and microfluidics. However, these techniques were designed based on knowledge acquired from 2D cell culture and animal models. Our lack of information on the human ovary hampers our ability to mimic the main features of this organ, for clinical applications. The complex composition and hierarchical structure of its ECM complicates the design of truly biomimetic constructs, notably: fiber morphology, interstitial and perifollicular fiber orientation, porosity, topography, and viscoelasticity, which all play a role in mechanotransduction. Study design, size, duration Ovarian biopsies were taken from prepubertal (mean age [±SD]=7±3 years, n = 21), reproductive age (mean age [±SD]=27±5, n = 26 ) and menopausal (mean age [±SD]=61±6 years, n = 29) patients after obtaining their informed consent. All participating adult subjects were undergoing laparoscopic surgery for benign gynecological diseases not affecting the ovaries. Prepubertal tissue was derived from young cancer patients scheduled for ovarian cortex cryopreservation as a fertility preservation strategy, before being subjected to acute gonadotoxic cancer treatments. Participants/materials, setting, methods All samples were cryopreserved by slow freezing and kept frozen until the day of their analysis. Tissues provided from the same patients (n = 5 per age group) were investigated by scanning electron microscopy (SEM) (fiber, pore and topography analyses) and atomic force microscopy (AFM). A larger number of paraffin-fixed biopsies (prepubertal, n = 16, reproductive-age, n = 21, and menopausal, n = 24) obtained from the biobank of St-Luc’s Hospital were used to conduct computed fiber orientation analysis. Main results and the role of chance Our results revealed a unique ECM architecture at reproductive age, where fibers of intermediate diameter are assembled into thickest bundles compared to prepubertal and menopausal tissues(p < 0.0001). Indeed, during prepuberty the bundles assemble into a tight network with high number of small pores while reproductive-age ovary gain more porosity(p < 0.0001). However, at menopause tissue pore number and area change significantly(p < 0.001). These pore geometry and distribution changes contribute to diffusion and access of key molecules to/from cells, which can be translated into changes in permeability and molecule selectivity with age. Fiber directionality around follicle borders at preantral stages revealed that before and after puberty, secondary follicles appear to modify their microenvironment arrangement locally compared to follicles at earlier stages of development (p < 0.01), by reorienting the majority of collagen fibers below 50°.This could indicate that follicles at this stage require higher fiber contact and adhesion signaling to complete their development and maturation towards ovulation. AFM evidenced a relatively rigid ovarian tissue at prepuberty, softening significantly at reproductive age, then stiffening considerably upon menopause. These differences(p < 0.01) are not only structure-dependent, but also related to biochemical differences in ECM composition, as previously demonstrated in our follow-up of variations in elastic matrisome components from prepuberty to menopause. Limitations, reasons for caution The samples represent single time points from each age group which could present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. Wider implications of the findings: Our study provides the first conclusive proof of a link between ECM biophysics and fertility by comparing different stages of ovarian transformation related to a woman’s reproductive life, which will oriente new strategies for infertility prognoses based on ECM biophysics and may become a blueprint for designing functional engineered ovaries. Trial registration number Not applicable

Human Ovarian Cortex biobanking: A Fascinating Resource for Fertility Preservation in Cancer.

Novel anti-cancer treatments have improved the survival rates of female young patients, reopening pregnancy issues for female cancer survivors affected by the tumor treatment-related infertility. This condition occurs in approximately one third of women of fertile age and is mainly dependent on gonadotoxic protocols, including radiation treatments. Besides routine procedures such as the hormonal induction of follicular growth and subsequent cryopreservation of oocytes or embryos, the ovarian protection by gonadotropin-releasing hormone (GnRH) agonists during chemotherapy as well as even gonadal shielding during radiotherapy, other innovative techniques are available today and need to be optimized to support their introduction into the clinical practice. These novel methods are hormone stimulation-free and include the ovarian cortex cryopreservation before anti-cancer treatments and its subsequent autologous reimplantation and a regenerative medicine approach using oocytes derived in vitro from ovarian stem cells (OSCs). For both procedures, the major benefit is related to the prompt recruitment and processing of the ovarian cortex fragments before gonadotoxic treatments. However, while the functional competence of oocytes within the cryopreserved cortex is not assessable, the in vitro maturation of OSCs to oocytes, allows to select the most competent eggs to be cryopreserved for fertility restoration.

Optimizing ovarian tissue quality before cryopreservation: comparing outcomes of three decortication methods on stromal and follicular viability

To evaluate whether specific ovarian decortication techniques vary in promoting ovarian cortex cryopreservation and transplant outcomes. Experimental design. University hospital. Nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) female mice. Human ovarian biopsy samples allocated to one of the following decortication procedures: scratching with scalpel blade (B), cutting with microsurgical scissors (M), separation with slicer (S), or no-separation (control, C). Parallel, invivo experiment: decortication techniques combined with slow freezing (SF) and vitrification (VT) before xenograft into immunodeficient mice. Follicular counts, apoptosis, shear stress, Hippo pathway and inflammation. Invivo: recovered grafts analyzed for follicular counts, angiogenesis, proliferation, and fibrosis. There were no differences in follicular density or number of damaged follicles between the decortication techniques in the invitro study. Nevertheless, the M samples showed statistically significantly increased stromal damage compared with the controls and S samples, and up-regulation of Hsp60 shear stress gene expression. Decortication by both M and S inhibited the Hippo pathway, promoting gene expression changes. In the 21-day xenograft, total follicular density statistically significantly decreased compared with the nongrafted controls in all groups. Nevertheless, no differences were observed between the decortication techniques. Ovarian stroma vascularization was increased in the vitrified samples, but among the slow-freezing samples, the B samples had the lowest microvessel density. The M decorticated xenografts had increased fibrosis. Decortication with a slicer causes less damage to ovarian tissue than other commonly used methods although microsurgical scissors seem to preserve slightly increased follicular numbers. Nevertheless, blade decortication seems to be a reliable technique for maintaining acceptable follicular conditions without inducing serious stromal impairment.

Breast cancer: an update on treatment-related infertility.

Breast cancer (BC) is the most common malignancy in women with a significant increasing incidence during the reproductive life. However, based on the newest anti-cancer molecular targeting drugs, successful treatments lead to the disease healing particularly in young patients, thus refreshing their motherhood programs. However, as effect of the BC treatment, a premature depletion of the ovarian follicle reserve occurs in more than one-third of patients resulting in permanent infertility. To prevent the cancer treatment-related infertility (CTRI), several options are today utilized. Besides the ovary suppression by gonadotropin-releasing hormone agonist (GnRHa), other procedures include either oocytes or embryos cryopreservation as well as ovarian cortex cryopreservation that are currently adopted before anti-cancer therapies. These modern techniques appear variably successful in terms of pregnancy rate though their safety concerning the hormonal stimulation to promote the folliculogenesis is still debated in relation to the potential oncogenic risk in patients bearing hormone-sensitive tumors as BC, while the ovarian cortex re-implantation often results in a low number of regenerated follicles including oocytes of unknown quality. Recent studies on ovarian stem cells (OSCs) suggest their use for future application in CTRI. In fact, OSCs from ovarian cortex have been shown to differentiate in vitro into oocyte-like cells (OLCs) and express molecular markers of mature oocytes. Once the OSC technology will be optimized and translated to clinical use, oocytes derived from these cells will be molecularly assessed before fertilization to assure their best embryo quality resulting in a safe procedure to treat CTRI in patients as young women with BC.

Knowledge, attitude, intention, and religion aspect toward fertility preservation among obstetrics and gynecology residents in Indonesia: A cross-sectional study.

BackgroundThe lack of knowledge from healthcare providers regarding fertility preservation will certainly affect the patient's knowledge, attitude, behavior, and also perspective. Obstetrics and Gynecology (OB/GYN) residents may most likely be the first line professionals to integrate fertility preservation technologies into their practice which plays an important task in giving an understanding of the relationship between age and fertility for patients.ObjectiveThis study aims to assess OB/GYN resident knowledge and beliefs regarding age-related fertility decline, intentions, and religion aspect toward fertility preservation.Materials and MethodsA cross-sectional study was conducted on 92 Indonesian OB/GYN residents at the Cipto Mangunkusomo Hospital between November and December 2017. Data were collected using a questionnaire which had four sections, knowledge, attitude, intention, and religion aspect toward fertility preservation.ResultsThe majority of participants believed that an OB/GYN should encourage discussions about potential childbearing desires (96.74%) and age-related fertility decline (94.57%) with patients, of which 79.34% believed that these discussions should be part of a woman's annual health examination. Cancer patients are likely to undergo oocyte cryopreservation than people who choose career as priority. From the religion aspect, fertility preservation options such as sperm, oocyte, embryo, and ovarian cortex cryopreservation were accepted by most residents with varied religions, while oocyte and sperm donor methods were unacceptable (48% and 57%, respectively) because of the belief that oocyte/sperm should only be given to legitimate partners, but many still do not know that oocyte and sperm donor were prohibited by all religions.ConclusionAge-related fertility decline and frozen egg storage should be discussed during annual woman wellness examinations by OB/GYN specialists.

Oncofertilité et cancer du sein

Over the past decades, progresses in oncology have improved the recovery rates after numerous malignant diseases, including breast cancer, that strike young adults in childbearing age. Quality of life of young cancer survivors has become a major issue. However, anticancer therapies can have a detrimental impact on fertility. It is now well-established that all patients should receive information about the fertility risks associated with their cancer treatment and the fertility preservation options available. These techniques aim to limit the negative impact of chemotherapy on the ovaries or to preserve gametes before treatment. Currently, oocyte or embryo freezing after controlled ovarian hyperstimulation represents the most effective method for preserving female fertility. Over the past years innovative protocols of ovarian stimulation have been developed to enable breast cancer patients to undergo oocyte or embryo cryopreservation irrespective of the phase of the cycle or without exogenous follicle-stimulating hormone related increase in serum estradiol levels. When controlled ovarian hyperstimualtion cannot be implemented, other techniques such as cryopreservation of ovarian cortex, in vitro maturation or the use of GnRH agonists may be proposed. However, it is important to inform patients that all these fertility preservation techniques do not represent a guarantee of pregnancy.

Ovarian damage from chemotherapy and current approaches to its protection.

BackgroundAnti-cancer therapy is often a cause of premature ovarian insufficiency and infertility since the ovarian follicle reserve is extremely sensitive to the effects of chemotherapy and radiotherapy. While oocyte, embryo and ovarian cortex cryopreservation can help some women with cancer-induced infertility achieve pregnancy, the development of effective methods to protect ovarian function during chemotherapy would be a significant advantage.Objective and rationaleThis paper critically discusses the different damaging effects of the most common chemotherapeutic compounds on the ovary, in particular, the ovarian follicles and the molecular pathways that lead to that damage. The mechanisms through which fertility-protective agents might prevent chemotherapy drug-induced follicle loss are then reviewed.Search methodsArticles published in English were searched on PubMed up to March 2019 using the following terms: ovary, fertility preservation, chemotherapy, follicle death, adjuvant therapy, cyclophosphamide, cisplatin, doxorubicin. Inclusion and exclusion criteria were applied to the analysis of the protective agents.OutcomesRecent studies reveal how chemotherapeutic drugs can affect the different cellular components of the ovary, causing rapid depletion of the ovarian follicular reserve. The three most commonly used drugs, cyclophosphamide, cisplatin and doxorubicin, cause premature ovarian insufficiency by inducing death and/or accelerated activation of primordial follicles and increased atresia of growing follicles. They also cause an increase in damage to blood vessels and the stromal compartment and increment inflammation. In the past 20 years, many compounds have been investigated as potential protective agents to counteract these adverse effects. The interactions of recently described fertility-protective agents with these damage pathways are discussed.Wider implicationsUnderstanding the mechanisms underlying the action of chemotherapy compounds on the various components of the ovary is essential for the development of efficient and targeted pharmacological therapies that could protect and prolong female fertility. While there are increasing preclinical investigations of potential fertility preserving adjuvants, there remains a lack of approaches that are being developed and tested clinically.

In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification.

Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.