In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility.

BACKGROUND: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential. METHODS: In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 µg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section. RESULTS: In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3.1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age. CONCLUSIONS: NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries.

Whole ovary cryopreservation and transplantation has been proposed as a method for preserving long-term ovarian function. This work reports ovarian function 6years post transplantation of frozen-thawed whole sheep ovaries. Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide organs for the experiments. After perfusing with cold University of Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were cryopreserved using unidirectional solidification freezing technology. After thawing, ovaries were re-perfused and re-transplanted orthotopically by microvascular re-anastomosis, to the contralateral ovarian pedicle after removing the remaining ovary. Six years following transplantation and after inducing superovulation, the sheep were killed and the ovaries analysed. Two ovaries had normal size and shape showing some recent corpora lutea, while the third showed atrophic changes. A total of 36 antral follicles were counted by transillumination and four germinal vesicle oocytes were aspirated and matured in vitro to metaphase II. Serum progesterone concentrations were indicative of ovulatory activity in one of the three sheep. Histological evaluations revealed normal tissue architecture, intact blood vessels and follicles at various stages. Currently, this is the longest recorded ovarian function after cryopreservation and re-transplantation. Cryopreservation of whole ovaries, using directional freezing combined with microvascular anastomosis, is a promising method for preserving long-term reproductive capacity and endocrine function.

The oocyte population is not renewed in transplanted or irradiated adult ovaries.

BACKGROUND: According to conventional theory, the oocyte population is not renewed in mammalian ovaries after birth. A new hypothesis proposes that oocytes are generated continuously from haematopoietic progenitor cells. There is, however, no evidence that they can ovulate, although they may partially restore fertility by organizing 'helper follicles'. The hypothesis that follicles can form de novo in adult ovaries has been tested in a transplant model. METHODS: Ovaries from adult mice were transplanted under the kidney capsule or into the ovarian bursa of histocompatible, transgenic CAG::H2B-EGFP host animals. Some donors were sterilized before transplantation by X-irradiation to ensure 'empty niches' were available for repopulation. The phenotype of follicular oocytes at 2, 4 and 8 weeks post-transplantation was scored by epifluorescence. RESULTS: A total of 819 oocytes were examined in 30 ovarian grafts. None expressed green fluorescence, as would be predicted if they had formed de novo from germ cell progenitors in the systemic circulation of the host. Furthermore, small follicles eliminated by irradiation were not replaced in transplanted ovaries, and the few growing follicles present were apparently survivors of the original population. CONCLUSIONS: No evidence was found to support the hypothesis that progenitor cells from extra-ovarian sources can repopulate the adult ovary. The findings are consistent with the conventional view that a limited number of oocytes are formed before birth and declines with age. The study did not, however, rule out the possibility that germline stem cells may reside in the adult ovary.

A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.

BACKGROUND: A series of monozygotic (MZ) twin pairs discordant for premature ovarian failure presented an unusual opportunity to study ovarian transplantation. METHODS: Ten MZ twin pairs requested ovarian transplantation and eight have undergone transplantation with cryopreservation of spare tissue. Seven had a fresh cortical tissue transplant, one of whom received a second frozen-thawed transplant after the first ceased functioning at three years. One had a fresh microvascular transplant. RESULTS: All recipients reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 77-142 days. Six have already conceived naturally (one twice). Currently, two healthy babies have been delivered, and another three pregnancies are ongoing. The oldest transplant functioned for 36 months, resulting in one child and one miscarriage. She conceived again after a frozen-thawed secondary transplant. There was no apparent difference in return of ovarian function between the eight fresh ovarian grafts and the one frozen graft. CONCLUSIONS: Ovarian transplantation appears to restore ovulatory function robustly. Successful pregnancies, including one after cryopreservation, bode well for application to fertility preservation.

Prevalence of premature ovarian failure in monozygotic and dizygotic twins.

BACKGROUND: Premature ovarian failure (POF) before 40 years of age from natural causes affects approximately 1% of adult women, with minor variations between ethnic groups. A recent case of ovarian transplantation between young monozygotic (MZ) twins in which one had undergone unexplained POF at 14 years has prompted a study of the prevalence of POF. METHODS: Menopausal ages of 832 Australian and UK female twin-pairs were extracted from volunteer national twin registry databases containing medical, reproductive and lifestyle data surveyed by mail questionnaire. Surgical menopause was an exclusion criterion. RESULTS: The prevalence of POF in both MZ and dizygotic (DZ) twins was similar in both registries and 3- to 5-fold greater than the general population at age thresholds 40 and 45 years. No specific factors were found to account for the higher risk of early menopause. Some twins of both zygosities were highly discordant for menopausal age (>or=10 years). Nevertheless, there was significant intra-twin dependence, especially for MZ twins, and the average age difference at last menses was greater in DZ twin-pairs. CONCLUSION: Both MZ and DZ twins are at higher risk of POF. Despite some striking differences within MZ twin-pairs, menopausal ages were more concordant than for DZ twin-pairs, confirming that the timing of menopause has a heritable component.

Cryopreservation of murine embryos, human spermatozoa and embryonic stem cells using a liquid nitrogen-free, controlled rate freezer.

A Stirling Cycle Cryocooler has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. Three types of samples from two species (murine embryos, human spermatozoa and embryonic stem cells), each requiring different cooling protocols, were cryopreserved in the Stirling Cycle freezer. For comparison, cells were also frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, the rates of survival of viable cells were generally greater than 50% for mouse embryos and human embryonic stem cells, based on morphology (mouse embryos) and staining and colony formation (human embryonic stem cells). Survival rates of human spermatozoa frozen in the Stirling Cycle freezer, based on motility and dead cell staining, were similar to those of samples frozen in a conventional controlled rate freezer using liquid nitrogen.

Assessment of ovarian reserve. Ovarian biopsy is not a valid method for the prediction of ovarian reserve.

The evaluation of ovarian reserve, often critical for the elderly infertile woman, is notoriously difficult and inaccurate. The place of ovarian biopsy in this evaluation has been hotly disputed for three decades, but not resolved. To examine the feasibility of ovarian biopsy for this purpose, a project was designed to estimate the total number of oocytes in a human ovary and investigate whether any biopsy regimen is representative of the follicular reserve in an individual. Ovaries removed from patients of reproductive age during operations not involving ovarian pathology were utilized to count the number and type of follicles found in multiple biopsies of 2 and 5 mm and in the whole ovary. Representative results taking into account the total number of follicles found in the whole ovary showed that predicted values based on the biopsies were extremely varied. We concluded that due to the huge variation in the distribution of follicles across the surface of the ovary, there is no place for this procedure in clinical evaluation of reproductive ageing in the individual patient.

Spindles, mitochondria and redox potential in ageing oocytes.

Studies of human oocytes obtained from women of advanced reproductive age revealed that spindles are frequently aberrant, with chromosomes sometimes failing to align properly at the equator during meiosis I and II. Chromosomal analyses of donated and spare human oocytes and cytogenetic and molecular studies on the origin of trisomies collectively suggest that errors in chromosome segregation during oogenesis increase with advancing maternal age and as the menopause approaches. Disturbances in the fidelity of chromosome segregation, especially at anaphase I, leading to aneuploidy are prime causes of reduced developmental competence of embryos in assisted reproduction, as well as being responsible for the genesis of genetic disease. This review provides an overview of spindle formation and chromosome behaviour in mammalian oocytes. Evidence of a link between abnormal mitochondrial function in oocytes and somatic follicular cells, and finally disturbances in chromosome cohesion and segregation, and cell cycle control in aged mammalian oocytes, are also discussed.

Expression of mRNAs for DNA methyltransferases and methyl-CpG-binding proteins in the human female germ line, preimplantation embryos, and embryonic stem cells.

Recent evidence indicates that mammalian gametogenesis and preimplantation development may be adversely affected by both assisted reproductive and stem cell technologies. Thus, a better understanding of the developmental regulation of the underlying epigenetic processes that include DNA methylation is required. We have, therefore, monitored the expression, by PCR, of the mRNAs of DNA methyltransferases (DNMTs), methyl-CpG-binding domain proteins (MBDs), and CpG binding protein (CGBP) in a developmental series of amplified cDNA samples derived from staged human ovarian follicles, oocytes, preimplantation embryos, human embryonic stem (hES) cells and in similar murine cDNA samples. Transcripts of these genes were detected in human ovarian follicles (DNMT3A, DNMT3b1, DNMT3b4, DNMT1, MDBs1-4, MeCP2, CGBP), germinal vesicle (GV) oocytes (DNMT3A, DNMT3b1, DNMT1, MDBs1-4, MeCP2, CGBP), mature oocytes (DNMT3A, DNMT3b1, DNMT1, CGBP), and preimplantation embryos (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD2, MDB4, CGBP). Differential expression of DNMT3B gene transcripts in undifferentiated (DNMT3b1) and in vitro differentiated human ES cells (DNMT3b3) further demonstrated an association of the DNMT3b1 transcript variant with totipotent and pluripotent human cells. Significantly, whilst the murine Dnmt3L gene is both expressed and essential for imprint establishment during murine oogenesis, transcripts of the human DNMT3L gene were only detected after fertilisation. Therefore, the mechanisms and/or the timing of imprint establishment may differ in humans.

Oocyte biology and genetics revelations from polar bodies.

The enormous volume of the fertilized egg is attributable to the suppression of cleavage during oocyte growth and the unequal cleavages during the first and second meiotic divisions. The two products of these divisions are the diminutive polar bodies (PB), which contain a redundant set of chromosomes/chromatids plus cytoplasmic organelles. The PB have strictly limited but differential life spans; while viable they possess the genetic potential to support normal embryonic development after transfer to a cytoplast. In addition to the theoretical possibility of using this non-cloning technique to generate more embryos, polar bodies can be used for genetic testing. By cytogenetic analysis of both PB using fluorescent in-situ hybridization (FISH) or chromosome painting, partial or full chromosomal status in the oocyte can be predicted; this approach finds particular application for women of advanced reproductive age as well as with maternally inherited translocations and single gene defects. By studying both of the PB, potential problems of interpretation arising from allele dropout can be reduced; a heterozygous first polar body provides the least ambiguous result. Mitochondria segregate randomly during meiotic cleavages providing an opportunity also to use the PB to screen for mitochondrial mutations and deletions. Thus, the PB can serve useful diagnostic purposes, especially where pre-fertilization screening or avoidance of embryo biopsy is desirable.

Gonadal tissue cryopreservation and transplantation.

Transplantation of ovarian and testicular tissue has been practised for over a century, mainly for experimental purposes. It is now being considered as a potential strategy for preserving fertility in young patients, including children, undergoing sterilizing treatment for cancer and other diseases. Ovarian tissue biopsies can be stored at liquid nitrogen temperatures indefinitely so that, after thawing, they can be returned as either ortho- or heterotopic grafts to the original patient. A different approach is needed for preserving male germ cells to restore fertile potential. Experimental studies have shown that spermatogonial stem cells injected into the rete testis/seminiferous tubules can re-initiate spermatogenesis after sterilizing treatment with alkylating agents; alternatively, in prepubertal cases, testicular biopsies that have been cryopreserved can be grafted subcutaneously to generate enough spermatozoa for intracytoplasmic sperm injection (ICSI). These strategies have been demonstrated in animal models and are now undergoing clinical testing.

Isolation, characterization and expression of the human Factor In the Germline alpha (FIGLA) gene in ovarian follicles and oocytes.

The Factor In the Germline alpha (FIGalpha) transcription factor regulates expression of the zona pellucida proteins ZP1, ZP2 and ZP3 and is essential for folliculogenesis in the mouse. Using the published mouse Figla sequence, BLAST searches identified a human chromosome 2 BAC clone with high sequence identity. Using PCR primers derived from this clone, amplicons derived from ovarian follicles and mature oocytes revealed 100% identity with the appropriate human BAC clone, the expected homology with the mouse Figla gene sequence, and homology on translation with the FIGalpha protein identified in the Japanese rice fish, medaka (Oryzias latipes). PCR expression profiling of this transcript revealed FIGLA mRNA expression in cDNA derived from ovarian follicles (5/5 samples from the primordial through to the secondary stage) mature oocytes (6/9 samples), and less frequently in preimplantation embryos (2/7 samples). Subsequent BLAST searches revealed the predicted full length coding sequence of the human FIGalpha protein which demonstrates 68 and 25% similarity overall to mouse and medaka proteins respectively, with 96 and 57% identity respectively within the basic helix-loop-helix region. This confirms our identification of the human homologue for this gene which maps to chromosome 2p12. Further work is required to understand its role in normal human oocyte development and the potential involvement in human infertility.

Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts.

Retrieval, extracorporal storage and autotransplantation of testicular tissue could become an important strategy for preserving male gonadal function. The present study used syngeneic and immunodeficient nude mice as hosts, and immature and adult mice, neonatal and adult photoregressed Djungarian hamsters and neonatal marmosets to identify the potential of testicular tissue grafting to maintain the morphological and functional integrity of the testis. Testicular tissue was grafted s.c. either as fresh tissue or after cryopreservation into adult, orchidectomized hosts. The mice that received rodent testis tissue were autopsied 50 days later, and blood samples were collected. Sixty-five per cent of mouse isografts contained morphologically normal testicular tissue and seminiferous tubules with some degree of spermatogenic recovery. Mature spermatozoa were recovered after enzymatic disaggregation. Although the recovery of spermatogenesis was limited in adult mouse and hamster tissue, complete spermatogenesis was observed in grafts from immature rodents. Testicular tissue from neonatal marmosets developed up to the stage of spermatocytes at day 135 after xenografting. Androgen concentrations were comparable in intact control mice and in mice receiving fresh mouse and hamster grafts, slightly lower in mice receiving cryopreserved grafts and adult photoregressed hamster tissue, and low in castrated control mice and in mice receiving marmoset tissue. These results show that isografts and xenografts of immature and adult testicular tissue become functionally active as a s.c. graft in the mouse and that this approach might be useful in combination with cryopreservation as a tool for storage and activation of the male germ line and androgen replacement therapy in patients.

Oogenesis as a foundation for embryogenesis.

The majority of oocytes in postnatal ovaries are small, non-growing and reside in primordial follicles. They have to undergo a prolonged phase of growth and differentiation before nuclear and cytoplasmic maturation enables them to resume meiosis and undergo fertilization. A better knowledge of this phase of oogenesis is essential for understanding causes of oocyte pathology and optimizing methods for growing oocytes in vitro and for cryopreservation. There could also be spin-off discoveries for contraceptive strategies and pharmacologically controlling oocyte maturation. During oocyte growth, a molecular programme for development is assembled for the timely expression of mRNAs, some of which are expressed throughout oogenesis while others are 'masked' until or after meiotic maturation. Masking and stability in storage are largely due to a truncated poly(A) tail, controlled by regulatory sequences on the 3'-untranslated region (UTR) of the mRNA. Most maternal RNAs are degraded early in cleavage, there being a narrow overlap between persisting maternal mRNAs and activation of the embryonic genome. Accumulation of RNAs and proteins are not, however, the only major changes taking place during oogenesis. Cytoplasmic organelles multiply and redistribute, and there are epigenetic modifications of DNA for monoallelic expression of imprinted genes. The granulosa cells are obligatory for they provide physical support, nutrients and mediate the regulatory influences of gonadotrophic hormones. On the other hand, the oocyte actively influences the growth and differentiation of its granulosa cells. Thus, healthy embryos reflect the quality of both the oocyte and the granulosa cells.

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization.

OBJECTIVE: To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body. DESIGN: Prospective case study. SETTING: Research laboratories, university hospital. PATIENT(S): A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI. MAIN OUTCOME MEASURE(S): Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured to metaphase II in vitro. RESULT(S): Oocyte hyperploidy was diagnosed by M-FISH to be 23, X +15 cht +19 cht +22 cht. The correspond- ing polar body was hypoploid, with a karyotype of 23, X -15 cht -19 cht -22 cht. This was due to unbalanced predivision at meiosis I. Reprobing confirmed karyotype assignments for chromosomes X, 13, 18, and 21. CONCLUSION(S): The mechanism involved in maternally derived aneuploidy can be defined by using M-FISH to simultaneously karyotype both oocyte and first polar body chromosomes at metaphase II. Multifluor FISH may be useful for investigative studies of maternally derived aneuploidy, which is a major cause of preimplantation waste in natural and assisted reproduction.

Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for autotransplantation.

BACKGROUND: Ovarian failure is a common sequel to chemo/radiotherapy in patients successfully treated for cancer. Harvesting, cryopreserving and subsequently re-implanting ovarian cortical grafts can be used to re-establish reproductive potential in women with cancer. The safety issue, however, is of great concern because residual disease in autografted ovarian tissues might cause recrudescence of disease. METHODS: A total of 30 non-obese diabetic severe combined immunodeficient (NOD/LtSz-SCID) mice were individually xenografted s.c. with frozen-thawed ovarian tissue from 18 patients with lymphoma [13 Hodgkin's lymphoma (HL) and 5 non-Hodgkin's lymphoma (NHL)]. The animals were autopsied at 16 weeks, or earlier if cachectic. The xenograft, liver, spleen, sternum, para-aortic lymph nodes and thymus were prepared for histology, immunohistochemistry and human DNA microsatellite analysis. RESULTS: None of the animals grafted with ovarian tissue from lymphoma patients developed disease. However, all 3 animals grafted with lymph node tissue from an NHL patient developed B-cell lymphomas that were confirmed as human in origin by DNA microsatellite analysis. CONCLUSION: Ovarian tissue harvested before high-dose chemotherapy for HL or NHL may not carry a risk of disease transmission by autotransplantation, although the possibility is difficult to exclude completely.

Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma.

BACKGROUND: Infertility is a common late effect of chemotherapy and radiotherapy, and has a substantial effect on the quality of life for young survivors of cancer. For men, semen cryopreservation is a simple way of preserving reproductive potential but for women, storage of mature eggs rarely proves successful, and the alternative-immediate in vitro fertilisation with cryopreservation of embryos-is not always appropriate. Reimplantation of cryopreserved ovarian tissue has been shown to restore natural fertility in animals. We applied this technique in a woman who had received sterilising chemotherapy for lymphoma. METHODS: A 36-year-old woman underwent a right oophorectomy with cryopreservation of ovarian cortical strips before receiving high-dose CBV chemotherapy for a third recurrence of Hodgkin's lymphoma. 19 months later, when serum sex steroid analysis confimed a postmenopausal state, two ovarian cortical strips were thawed and reimplanted-one onto the left ovary and another at the site of the right ovary. FINDINGS: 7 months after reimplantation of ovarian cortical strips, the patient reported resolution of hot flashes and, for the first time, oestradiol was detected in the serum. This finding was associated with a decrease in the concentrations of follicle-stimulating hormone and luteinising hormone, and ultrasonography revealed a 10 mm thick endometrium, a poorly visualised left ovary, and a 2 cm diameter follicular structure to the right of the midline. The patient had one menstrual period, but by 9 months after the implantation, her sex steroid concentrations had returned to those seen with ovarian failure. INTERPRETATION: Orthotopic reimplantation of frozen/thawed ovarian cortical strips is a well tolerated technique for restoring ovarian function in women treated with sterilising chemotherapy for cancer.