Abstract
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 INTEREST(S)
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.
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.
STUDY FUNDING/COMPETING INTEREST(S)
The Child Cancer Foundation in Denmark (2012–26) and the EU interregional project ReproHigh are thanked for having funded this study. They had no role in the study design, collection and analysis of data, data interpretation or writing of the report. The authors have no conflict of interest to disclose.
Abstract
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 INTEREST(S)
The work was funded by ESHRE. None of the authors has a conflict of interest.
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.
Abstract
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.
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.
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.
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.
BACKGROUND
Cryopreservation of ovarian tissue for fertility preservation is based on the ovarian cortex that contains the vast majority of the follicular reserve, while the remaining tissue, the ...medulla is discarded. The present study describes the development of a gentle method for isolating pre-antral follicles from human ovarian medulla and evaluating its follicular content.
METHODS
Medulla was collected from 40 girls/women aged 3–35 years undergoing cryopreservation of the ovarian cortex. Follicle density was assessed for all patients and pre-antral follicles were isolated from 22 patients. On the basis of the neutral red (NR) staining of follicles and enzymatic digestion with a mixture of Collagenase IV and Liberase Thermolysin Medium, viable pre-antral follicles were isolated.
RESULTS
NR accumulated in follicles resulting in a distinct red staining within the medulla. Follicle density of the medulla varied from 0 to 9824 follicles/gram of medulla and was significantly higher (P< 0.001) in the 3–9-year age group when compared with older groups (10–35 years). Enzymatic digestion combined with follicle identification by NR yielded a high output of isolated and viable pre-antral follicles from medulla, of which, 3607 follicles were collected and classified. The percentage of primordial and growing follicles decreased and increased, respectively, with age (P< 0.0001 and <0.0007).
CONCLUSIONS
Discarded medulla contained a considerable pool of pre-antral follicles, especially in young girls. Our new method allowed the isolation of viable pre-antral follicles from human ovarian medulla and provides a unique opportunity for basic scientific studies and for culture and grafting purposes.
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.
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