Oocyte diameter predicts the maturation rate of human immature oocytes collected ex vivo.

PURPOSE: To study the impact of oocyte diameter and cumulus cell mass on the potential for final maturation of immature human oocytes in vitro. METHODS: Immature oocytes (n = 1563) from 75 women undergoing fertility preservation by ovarian tissue cryopreservation (14-41 years) were collected. After preparation of the ovarian cortex for freezing, immature oocytes were collected from the surplus medulla. After collection, IVM was performed according to standard published methods. The mass of cumulus cell surrounding the immature oocyte was grouped according to size. After IVM, each oocyte was photographed, measured, and the diameter was calculated as a mean of two perpendicular measurements. RESULTS: The diameter of the oocytes ranged from 60 to 171 µm with a mean of 115 µm (SD:12.1) and an interquartile range from 107 to 124 µm. The oocyte diameter was positively associated with a higher incidence of MII (p < 0.001). MII oocytes had a significantly larger mean diameter than MI, GV, and degenerated oocytes. The size of the cumulus cell mass was significantly associated with the MII stage (p < 0.001) and larger oocyte diameter (p < 0.001). The results further confirm that the diameter of the fully grown oocyte is reached relatively early in human follicular development and that the factors governing oocyte maturation in vitro are connected to the surrounding cell mass and the oocyte. CONCLUSION: The diameter of the oocyte is a highly determining factor in the nuclear maturation of the human oocyte during in vitro maturation, and the size of the cumulus cell mass is closely positively associated with a larger diameter.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold.

STUDY QUESTION: Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? SUMMARY ANSWER: We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. WHAT IS KNOWN ALREADY: Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. STUDY DESIGN, SIZE, DURATION: Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24 hours followed by 24 hours of 1 mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N = 241) and human (N = 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Incubation in 0.1% SDS for 18-24 hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. STUDY FUNDING/COMPETING INTEREST(S): This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

FERTILITY PRESERVATION: Freezing of ovarian tissue and clinical opportunities.

Ovarian tissue cryopreservation (OTC) is mainly used for fertility preservation in girls and women facing a gonadotoxic treatment. If the woman subsequently becomes menopausal, the ovarian tissue may be transplanted to regain ovarian function, including fertility. The method was developed more than two decades ago and today thousands of women worldwide have undergone OTC. Fewer than 500 patients have had tissue transplanted and close to 100% of those regain ovarian function. Several technical aspects of OTC are now becoming more established, including high quantitative follicle survival, defining the size of the tissue resulting in optimal tissue revascularisation and follicle loss resulting from transport of ovarian tissue prior to freezing. We have used OTC to safeguard fertility in patients with genetic diseases, which for some diagnoses is purely experimental, as no transplantations is yet been performed. Usage of OTC beyond fertility is now also being considered; here, the endocrine function of follicles is the focus. It has been suggested that ovarian tissue stored in the reproductive years may be used to avoid premature ovarian insufficiency (POI) when there is a familial disposition or to postpone menopause in patients with an increased risk of osteoporosis or cardiovascular diseases. The benefit of OTC beyond fertility requires, however, actual clinical studies. The current review includes several recent technical aspects with contributions from Denmark building on some of the early work by Roger Gosden.

A simple method to quantify follicle survival in cryopreserved human ovarian tissue.

STUDY QUESTION: Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically? SUMMARY ANSWER: A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluation and evidence for a consistently high follicle survival in a series of ovarian tissue from 25 Danish girls and women undergoing ovarian tissue cryopreservation (OTC) was obtained. WHAT IS KNOWN ALREADY: Securing follicle survival in cryopreserved ovarian tissue is crucial for proper quality control when centers wish to implement OTC. The only established technique for validation of follicle survival is xenografting of thawed ovarian tissue to immunodeficient mice. However, this functional test is expensive, time consuming, requires animal facilities and only provides a qualitative-not quantitative-measure for follicle survival. STUDY DESIGN SIZE, DURATION: Quantification of follicle survival in human ovarian tissue donated from 30 girls and women having tissue cryopreserved for fertility preservation from 2000 to 2015 at the Laboratory of Reproductive Biology in Copenhagen, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian cortex was donated from 25 girls and young women aged 10-36 years (mean age: 25 years) and the average storage time in liquid nitrogen was 9.1 ± 5.6 years, ranging from 1.6 to 17.9 years. In 12 of the cases, the ovarian tissue was collected from the local hospital and in the other 13 cases the ovarian tissue was transported on ice up to 6 h prior to freezing. Donated fresh ovarian surplus tissue was obtained from five women aged 23-34 years (mean age: 27 years). Ovarian tissues were chopped into small fragments and incubated in culture medium containing 50 mg/ml NR for 3-4 h. Fragments of ovarian tissue containing clearly NR-stained follicles were selected for counting, encapsulated in 4% agar and were processed for histology to calculate a follicular survival rate. MAIN RESULTS AND THE ROLE OF CHANCE: The mean follicle survival rate in the 25 patients after freezing and thawing was 84% ± 11 (mean ±SD), ranging from 50% to 98%. The high follicle survival rate in this clinical series of patients reflects a constant high-quality service performed in our center and confirms the robustness of the slow freezing protocol. No significant association between follicle survival rates and storage time was found using linear regression analysis, suggesting that storage in liquid nitrogen does not affect viability of the tissue. No significant association in follicle survival rates was found between ovarian tissues collected at the local hospital compared to tissues transported on ice prior to freezing, supporting that prolonged cooling does not seem to greatly affect the follicle survival. For the fresh ovarian tissue, the average follicle survival rate was 91% ± 5 (mean ± SD) in five patients, ranging from 81% to 95%. LIMITATIONS, REASONS FOR CAUTION: Even though the NR staining requires active incorporation of the dye, the test is merely a short in situ test that cannot completely replace the functional value of xenografting studies in which the integrity and developmental potential of the ovarian follicles are assessed. WIDER IMPLICATIONS OF THE FINDINGS: OTC is now being employed around the world but to date it has been difficult for centers to evaluate the effectiveness of their program and perform proper quality control. NR staining combined with histological evaluation is the first quantitative method to provide a survival rate for follicles in frozen-thawed human ovarian tissue and offer a valuable and easily applicable tool to validate the cryopreservation procedure when implementing OTC or as routine quality control for the overall freezing performance within tissue banking facilities. STUDY FUNDING/COMPETING INTEREST(S): The Research Pools of Rigshospitalet, the Danish Cancer Foundation, Dagmar Marshalls Foundation, and the Novo Nordic Foundation are thanked for having funded this study. The authors have no conflicts of interest.

Transplantation of frozen-thawed ovarian tissue: an update on worldwide activity published in peer-reviewed papers and on the Danish cohort.

PURPOSE: The purpose of the study is to review all peer-reviewed published reports of women receiving ovarian tissue transplantation (OTT) with frozen/thawed tissue (OTC) with respect to age, diagnosis, transplantation site, fertility outcome, and potential side effects, including data from all women in the Danish program. METHODS: A systematic review of the literature was performed in PubMed combined with results from all patients who had received OTT in Denmark up to December 2017. RESULTS: OTT has been reported from 21 different countries comprising a total of 360 OTT procedures in 318 women. In nine women, malignancy was diagnosed after OTT; none were considered to be directly caused by the OTT. Despite a potential under reporting of cancer recurrence, there is currently no evidence to suggest that OTT causes reseeding of the original cancer. Renewed ovarian endocrine function was reported in 95% of the women. Half of all children born following OTT resulted from natural conception, and newborns were reported to be healthy except for one neonate with a chromosome anomaly with a family disposition. Women who conceived after OTT were significantly younger than those who failed. CONCLUSION: This study found no indications of sufficient numbers of malignant cells present in the ovarian tissue to cause recurrence of cancer after OTT. Further, it is unlikely that OTC affects the well-being of children born. OTC is now an established method of fertility preservation in Denmark with public reimbursement. The current data encourage that women who require gonadotoxic treatment should be offered an individual evaluation considering fertility preservation.

The polymorphic insertion of the luteinizing hormone receptor "insLQ" show a negative association to LHR gene expression and to the follicular fluid hormonal profile in human small antral follicles.

The luteinizing hormone receptor (LHCGR) has a little studied polymorphic 6 bp insertion (rs4539842/insLQ). This study has evaluated the insLQ polymorphism in relation to potential associations with hormonal characteristics of human small antral follicles (hSAFs). In total, 310 hSAFs were collected from 86 women undergoing fertility preservation. Analysis included hormonal profile of 297 follicular fluid (FF) samples and 148 corresponding granulosa cells samples were evaluated by qPCR for selected genes. Significantly reduced and non-detectable mRNA levels of anti-Müllerian hormone receptor II (AMHR2) and LHCGR, respectively, were observed for insLQ/insLQ compared to -/insLQ and the -/- genotypes. Moreover, LHCGR and CYP19a1 together with oestradiol and inhibin-B were significantly increased in -/insLQ compared to the -/- genotype. The homozygous insLQ genotype showed strong significant associations to GC specific genes LHCGR and CYP19a1, which may translate into significant changes in FF hormone profiles and an altered LH signaling.

Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture.

STUDY QUESTION: Can human pre-antral follicles isolated enzymatically from surplus medulla tissue survive and grow in vitro during long-term 3D culture? SUMMARY ANSWER: Secondary human follicles can develop to small antral follicles and remain hormonally active in an alginate-encapsulation culture system for more than 30 days. WHAT IS KNOWN ALREADY: Ovarian tissue cryopreservation followed by transplantation is a promising fertility preservation approach for cancer patients. However, transplantation of cryopreserved tissue to patients may carry the risk of re-implanting malignant cells. Grafting of follicles enzymatically isolated from ovarian tissue or developing a method for follicular culture and maturation in vitro may provide fertility to such patients without the risk of reintroducing the malignancy. However, the growth of pre-antral follicles isolated by enzymatic digestion from medulla tissue during long-term culture has received only little attention. STUDY DESIGN, SIZE, DURATION: Two to ten human pre-antral follicles were encapsulated together within an alginate bead and cultured with or without ovarian interstitial tissue for either 7 days or >30 days. Follicles were cultured in either 20% oxygen or 5% oxygen or encapsulated in a lower concentration of alginate together with a lower concentration of FSH in high oxygen. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 395 pre-antral follicles from 16 cancer patients, aged 9-37 years, were co-cultured for either 7 days or >30 days. A proportion of follicle (64) were removed from culture on Day 7 and assessed for viability using confocal fluorescence microscopy following calcein-AM and ethidium homodimer-1 staining or histology. The remaining follicles (331) were continued in culture for >30 days then assessed for survival and growth. Anti-Müllerian hormone (AMH) and estradiol levels were quantified in the medium. MAIN RESULTS AND THE ROLE OF CHANCE: An optimized protocol for isolation of intact healthy pre-antral follicles from ovarian medulla was developed. After 7 days of culture, secondary follicles had a significantly higher survival rates compared with primary and primordial follicles (70 versus <38%). Primordial and primary follicles did not develop into the antral follicle stage. In contrast, secondary follicles continued to develop in all culture conditions examined. Based on growth rate and morphology, four distinct cohorts of surviving follicles, 'fast growth', 'slow growth', 'no growth' and 'extruded oocyte' were identified. From Day 1 to Day 30, the mean diameter of follicles increased from 184 ± 35 to 661 ± 120 µm (significant from Day 18), 145 ± 19 to 318 ± 68 µm and 136 ± 15 to 162 ± 25 µm (mean ± SD) in the 'fast growth', 'slow growth' and 'no growth' patterns, respectively. The fast growth follicles also contained a larger diameter oocyte than other follicle groups. From the pre-antral follicle to antral stage, follicles became steroidogenically active and secretion of AMH and estradiol increased. No significant difference between the follicles cultured with or without ovarian interstitial tissue was observed. LIMITATIONS, REASONS FOR CAUTION: The number of surviving follicles at the end of study was low in each of the culture conditions therefore whether there is a benefit with any of the conditions is difficult to ascertain. Multiple pre-antral follicles were cultured within the same alginate bead which may affect the in vitro development of the secondary follicles. WIDER IMPLICATIONS OF THE FINDINGS: These findings show that pre-antral follicles, isolated enzymatically from surplus medulla tissue that is normally discarded, possess a developmental potential which may be used to devise safer fertility preservation methods for patients who are at high risk of malignant contamination of their ovarian tissue. STUDY FUNDING/COMPETING INTERESTS: The Child Cancer Foundation in Denmark (2012-26) and the EU interregional project ReproHigh are thanked for having funded this study; and the Key Program of Medical Science and Technology Innovation of Nanjing Military Area Command in China (14ZX06; 11Z010). They had no role in the study design, collection and analysis of data, data interpretation or in writing the report. The authors have no conflicts of interest to disclose.

Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark.

STUDY QUESTION: What are the results of transplanting cryopreserved ovarian tissue? SUMMARY ANSWER: The transplanted ovarian tissue can last up to 10 years, with no relapses following the 53 transplantations, and the chance of a successful pregnancy is currently around one in three for those with a pregnancy-wish. WHAT IS KNOWN ALREADY: Cryopreservation of ovarian tissue is now gaining ground as a valid method for fertility preservation. More than 36 children worldwide have now been born following this procedure. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study of 41 women who had thawed ovarian tissue transplanted 53 times over a period of 10 years, including 1 patient who was lost to follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 41 Danish women, who had in total 53 transplantations, were followed for ovarian function and fertility outcome. Safety was assessed by monitoring relapse in cancer survivors. MAIN RESULTS, AND THE ROLE OF CHANCE: Among 32 women with a pregnancy-wish, 10 (31%) had a child/children (14 children in total); this included 1 woman with a third trimester on-going pregnancy. In addition, two legal abortions and one second trimester miscarriage occurred. A total of 24 clinical pregnancies were established in the 32 women with a pregnancy-wish. The tissue remained functional for close to 10 years in some cases and lasted only a short period in others. Three relapses occurred but were unlikely to be due to the transplanted tissue. LIMITATIONS, REASONS FOR CAUTION: Self-report through questionnaires with only in-one hospital formalised follow-up of transplanted patients could result in unreported miscarriages. The longevity of the tissue may vary by few months compared with those reported because some patients simply could not remember the date when the tissue became non-functional. WIDER IMPLICATIONS OF THE FINDINGS: Cryopreservation of ovarian tissue is likely to become integrated into the treatment of young women, with cancer, who run a risk of losing their fertility. The full functional lifespan of grafts is still being evaluated, because many of the transplanted women have continued to maintain ovarian activity. Some of our first cases have had tissue functioning for ∼ 10 years.

Distribution and function of 3',5'-Cyclic-AMP phosphodiesterases in the human ovary.

The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary. Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found that PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A was not detected. PDE8B was differentially expressed during folliculogenesis. In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone secretion while PDE4 inhibition increased forskolin-stimulated progesterone secretion. In conclusion, we identified PDE3, PDE4, PDE7 and PDE8 as the major PDEs in the human ovary.

Identification of new ovulation-related genes in humans by comparing the transcriptome of granulosa cells before and after ovulation triggering in the same controlled ovarian stimulation cycle.

STUDY QUESTION: Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation? SUMMARY ANSWER: Up-regulated genes in granulosa cells (GC) represented inflammation, angiogenesis, extracellular matrix, growth factors and genes previously associated with ovarian cancer, while down-regulated genes mainly represented cell cycle and proliferation. WHAT IS KNOWN ALREADY: Radical changes occur in the follicle during final follicle maturation after the ovulatory trigger: these range from ensuring an optimal milieu for the oocyte in meiotic arrest to the release of a mature oocyte and remodeling into a corpus luteum. A wide range of mediators of final follicle maturation has been identified in rodents, non-human primates and cows. STUDY DESIGN, SIZE, DURATION: Prospective cohort study including 24 women undergoing ovarian stimulation with the long gonadotrophin-releasing hormone agonist protocol during 2010-2012 at Holbæk Fertility Clinic. Nine paired samples of GC and 24 paired samples of follicular fluid (FF) were obtained before and after recombinant human chorionic gonadotrophin (rhCG) administration. PARTICIPANTS/MATERIALS, SETTING, METHODS: Nine paired (nine arrays before rhCG and nine arrays after rhCG) samples of GC mRNA were amplified and hybridized to Affymetrix Human Gene 1.0 ST GeneChip arrays, compared and bioinformatically analyzed. Eleven selected genes were validated by quantitative reverse transcriptase PCR. FF hormones were analyzed by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven hundred and eighty-six genes were differentially expressed (>2-fold, P<0.0001, false discovery rate <0.0012) when comparing GC isolated before and 36 h after hCG, among those were genes known to be expressed at ovulation, i.e. ADAMTS1 and HAS2. Many new ovulation-related genes were revealed, such as CD24, ANKRD22, CLDN11 and FBXO32. FF estrogen, androstenedione and anti-Müllerian hormone decreased significantly while progesterone increased, accompanied by radical changes in the expression of steroidogenic genes (CYP17A, CYP19A, HSD11B1 and HSD11B2, StAR). Genes related to inflammation, angiogenesis, extracellular matrix formation, growth factors and cancer were up-regulated while cell cycle genes were massively down-regulated. Seventy-two genes previously described in connection with ovarian cancer were among the highly regulated genes. In silico analysis for top upstream regulators of the ovulatory trigger suggested--besides LH--TNF, IGF1, PGR, AR, EGR1 (early growth response 1), ERK1/2 (extracellular signal regulated kinase 1/2) and CDKN1A (cyclin-dependent kinase inhibitor 1A) as potential mediators of the LH/hCG response. LIMITATIONS, REASONS FOR CAUTION: The present dataset was generated from women under hormonal stimulation. However, comparison with a macaque natural cycle whole follicle ovulation dataset revealed major overlap, supporting the idea that the ovulation-related genes found in this study are relevant in the human natural cycle. WIDER IMPLICATIONS OF THE FINDINGS: These data will serve as a research resource for genes involved in human ovulation and final oocyte maturation. Ovulation-related genes might be good candidate biomarkers of follicle and oocyte health. Further, some of the ovulation-related genes may serve as future ovarian cancer biomarkers. STUDY FUNDING/COMPETING INTEREST(S): Grants from the Research Fund of Region Sjælland are gratefully acknowledged. None of the authors declared any conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Which follicles make the most anti-Mullerian hormone in humans? Evidence for an abrupt decline in AMH production at the time of follicle selection.

Anti-Müllerian hormone (AMH) is exclusively produced by granulosa cells (GC) of the developing pre-antral and antral follicles, and AMH is increasingly used to assess ovarian function. It is unclear which size follicles make the most AMH (total content) and are the main contributors to circulating AMH concentrations. To determine AMH gene expression in GC (q-RT-PCR) and follicular AMH production (Elisa and RIA) in relation to follicular development, 87 follicles (3-13 mm diameter) including both GC and the corresponding follicular fluid (FF) were collected in connection with fertility preservation of human ovaries. Further, follicle number and diameter, graded in 1 mm increments, were determined by 3D ultrasound in 113 women in their natural menstrual cycle to determine follicle number and diameter in relation to circulating AMH levels. This study demonstrates for the first time a positive association between AMH gene expression in human and both total follicular fluid AMH (P < 0.02) and follicular fluid AMH concentration (P < 0.01). AMH gene expression and total AMH protein increased until a follicular diameter of 8 mm, after which a sharp decline occurred. In vivo modelling confirmed that 5-8 mm follicles make the greatest contribution to serum AMH, estimated for the first time in human to be 60% of the circulating concentration. Significant positive associations between gene expression of AMH and FSHR, AR and AMHR2 expression (P < 0.00001 for all three) and significant negative association between follicular fluid AMH concentration and CYP19a1 expression were found (P < 0.0001). Both AMH gene expression (P < 0.02) and follicular fluid concentration of AMH (P < 0.00001) correlated negatively with estradiol concentration.

Concentration of activin A and follistatin in follicular fluid from human small antral follicles associated to gene expression of the corresponding granulosa cells.

The present study correlated concentrations of activin A and follistatin in follicular fluid (FF) from human small antral follicles to FF concentrations of AMH, inhibin B, progesterone, and oestradiol and to the mRNA expression of FSH-receptor (FSHR), LH-receptor (LHR), AMH-receptor2 (AMHR2), CYP19a, and androgen-receptor (AR) in the corresponding granulosa cells (GC). FF from 144 follicles (3-12 mm in diameter) was included whereas mRNA expression profiles were established in GC from 66 of the 144 follicles. Levels of follistatin remained constant in relation to follicular diameter, whereas activin A levels increased in follicles exceeding 10 mm in diameter. Levels of activin A and inhibin B showed a highly significant inverse association. Follistatin showed highly significant positive associations with AMH and inhibin B levels and with FSHR and AR gene expression in GC. This study revealed unexpected associations that probably reflect the complicated regulatory mechanisms governing human folliculogenesis.

Preservation of primordial follicles from lions by slow freezing and xenotransplantation of ovarian cortex into an immunodeficient mouse.

Assisted reproductive technology (ART) is considered an important tool in the conservation of endangered species, but often the most limiting factor of ART is the availability of mature oocytes. The aim of the present study was to investigate the feasibility of preserving female germ cells from ovaries of female lions (Panthera leo). Good quality cumulus-oocyte complexes (COCs) were isolated and subjected to in vitro maturation (IVM). In addition, ovarian cortex was obtained and cut into pieces for culture and cryopreservation by slow freezing. The survival of ovarian follicles was assessed by histology. Frozen-thawed samples of ovarian cortex samples were xenotransplanted under the skin of ovariectomized immunodeficient mouse for 28 days. Overall, 178 intact COCs were obtained from 13 lions, but only 28.1% were matured in vitro indicating insufficient IVM conditions. In contrast, almost all follicles within the ovarian cortex survived culture when the original sample was from a young healthy lion collected immediately after euthanasia. Within the xenotransplants, the number of primordial follicles decreased after 28 days by 20%, but the relation between primordial and growing follicles changed in favour of follicular growth. Female gamete rescue from valuable felids may be performed by slow freeze cryopreservation of ovarian cortex. Although the IVM protocol for lions is not yet optimized, mature oocytes may be obtained after long-term xenotransplantation and IVM and could potentially represent one way of salvage of endangered felid species in the future.

No evidence for the presence of oogonia in the human ovary after their final clearance during the first two years of life.

BACKGROUND: Conflicting results of studies on mouse and human have either verified or refuted the presence of oogonia/primordial germ cells in the post-natal ovary. The aim of this study was to trace whether oogonia recognized by immunohistochemical methods in the first trimester human ovary were present also in peri- and post-natal ovaries. METHODS: For this study, 82 human ovaries were collected: 25 from embryos from 5 to 10 weeks post conception (wpc), 2 at 18 wpc, 32 from 32 wpc to 2 years and 23 from 2 to 32 years. Of these, 80 ovaries were fixed and paraffin-embedded and 2 (8 year-old) ovaries were processed for plastic sections. Serial sections were prepared for immunohistochemical detection of markers for oogonia: tyrosine kinase receptor for stem cell factor (SCF)(C-KIT), stage-specific embryonic antigen-4 (SSEA4), homeobox gene transcription factor (NANOG), octamer binding transcription factor 4 (OCT4) and melanoma antigen-4 (Mage-A4), while noting that C-KIT also stains diplotene oocytes. RESULTS: Almost all oogonia exclusively stained for SSEA4, NANOG, OCT4 and C-KIT, whereas MAGE-A4 only stained a small fraction. At birth only a few oogonia were stained. These disappeared before 2 years, leaving only diplotene oocytes stained for C-KIT. From 18 wpc to 2 years, the medulla contained conglomerates of healthy and degenerating oogonia and small follicles, waste baskets (WBs) and oogonia enclosed in growing follicles (FWB). Medulla of older ovaries contained groups of primordial, healthy follicles. CONCLUSIONS: We found no evidence for the presence of oogonia in the human ovary after their final clearing during the first 2 years. We suggest that perinatal medullary WB and FWB give rise to the groups of small, healthy follicles in the medulla.

Anti-Müllerian hormone remains highly expressed in human cumulus cells during the final stages of folliculogenesis.

This study evaluated whether anti-Müllerian hormone (AMH) was differentially expressed in cumulus (CC) and granulosa (GC) cells from large antral and pre-ovulatory follicles collected from individual follicles in women undergoing in-vitro maturation (IVM) or IVF treatment. Expression studies of AMH, AMH receptor 2, FSH receptor, aromatase and androgen receptor were performed in CC in IVM patients where cumulus-oocyte-complex had expanded, CC in IVM patients where cumulus-oocyte-complex remained compacted, GC from immature follicles and CC and GC from IVF patients. Microarray data on corresponding GC and CC from follicles from IVF patients was included. AMH expression was significantly higher in CC than in GC from both mature and immature follicles and in CC from immature follicles than in CC from pre-ovulatory follicles from IVF patients (P < 0.05). AMH expression was significantly higher in CC that remained compacted compared with those that had expanded (P < 0.008). AMH was correlated to the expression of FSH receptor, androgen receptor and AMH receptor 2 but not to aromatase expression. The expression pattern of AMH receptor 2 reflected that of AMH. AMH may exert intrafollicular functions even in human large antral and pre-ovulatory follicles and may be related to follicular health.

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA.

Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24 women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may be augmented by stimulation of androgens via the AR.