Five-year study assessing the clinical utility of anti-Müllerian hormone measurements in reproductive-age women with cancer.

RESEARCH QUESTION: An important discussion point before chemotherapy is ovarian toxicity, a side-effect that profoundly affects young women with cancer. Their quality of life after successful treatment, including the ability to conceive, is a major concern. We asked whether serum anti-Müllerian hormone (AMH) measurements before chemotherapy for two most common malignancies are predictive of long-term changes in ovarian reserve? DESIGN: A prospective cohort study measured serum AMH in 66 young women with lymphoma and breast cancer, before and at 1 year and 5 years after chemotherapy, compared with 124 healthy volunteers of the same age range (18-43 years). Contemporaneously, patients reported their menses and live births during 5-year follow-up. RESULTS: After adjustment for age, serum AMH was 1.4 times higher (95% CI 1.1 to 1.9; P < 0.02) in healthy volunteers than in cancer patients before chemotherapy. A strong correlation was observed between baseline and 5-year AMH in the breast cancer group (P < 0.001, regression coefficient = 0.58, 95% CI 0.29 to 0.89). No significant association was found between presence of menses at 5 years and serum AMH at baseline (likelihood ratio test from logistics regression analysis). CONCLUSIONS: Reproductive-age women with malignancy have lower serum AMH than healthy controls even before starting chemotherapy. Pre-chemotherapy AMH was significantly associated with long-term ovarian function in women with breast cancer. At key time points, AMH measurements could be used as a reproductive health advisory tool for young women with cancer. Our results highlight the unsuitability of return of menstruation as a clinical indicator of ovarian reserve after chemotherapy.

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17ß-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved.

Telomere lengths in human pronuclei, oocytes and spermatozoa.

Telomeres are chromosome ends that control functions related to cell division. Short telomeres are proposed to underlie infertility, female reproductive ageing and abnormal embryogenesis, but there is little direct evidence on telomere length in gametes and embryos. The aim of this study was to measure telomere lengths in individual human oocytes, spermatozoa, male and female pronuclei, in order to compare parental contributions to telomere lengths in the human zygote. Quantitative fluorescence in situ hybridization was used to measure average telomere length in pronuclei of oocytes fertilized for research using a known fertile sperm sample. Pronuclei derived from male and female gametes were distinguished by 5-methylcytosine staining. Results were compared with those for unfertilized mature and immature oocytes and individual spermatozoa decondensed in vitro. Fifty unselected men and one sperm donor provided semen samples and 32 women donated oocytes surplus to IVF treatment. Telomeres in mature oocytes and female pronuclei were significantly longer than those in individual spermatozoa and male pronuclei (P < 0.0001). Telomeres were longer in immature oocytes than in mature oocytes (P < 0.04). Sperm telomere length increased with male age (P < 0.05). Neither sperm nor oocyte telomere lengths were significantly associated with clinical parameters or outcome of treatment. In conclusion, telomere length measurements directly comparing human pronuclei under identical conditions show that male-derived telomeres are shorter on average than female-derived telomeres at fertilization. We propose that from this starting point, telomere lengths are probably modified by recombination events in the oocyte until telomerase increases at the blastocyst stage. Our findings do not support the use of gamete telomere lengths as a fertility diagnostic tool.

Who should be offered sperm banking for fertility preservation? A survey of UK oncologists and haematologists.

BACKGROUND: Fertility after cancer therapy is a significant quality-of-life concern for many patients, their partners and families. Authoritative guidance states that men whose fertility may be affected by impending therapies should be offered sperm banking. Yet some patients are not offered this opportunity and are thereby disadvantaged. We sought to understand oncologists' and haematologists' decision making concerning sperm-banking referrals. DESIGN: We surveyed all oncologists and haematologists on the Royal College of Radiotherapists' Faculty of Oncology and British Society for Haematology circulation lists. RESULTS: From 2357 across all specialties, 499 responses were received: 253 haematologists and 246 oncologists (21% response rate). Twenty-one percent of respondents were unaware of local policies on sperm banking and 42% considered that sperm banking should be offered to more patients. Respondents' decisions reveal either assumptions about patients' needs based on characteristics such as age, sexual orientation and severity of illness or the influence of their own moral conclusions upon their patients. The survey identified paucity of training for clinicians, information for patients and systematic recording of discussions about fertility. CONCLUSIONS: A robust care infrastructure supporting male fertility storage is needed urgently to include targeted information for cancer clinicians and patients, identified individuals responsible for coordination and documentation of discussions with patients.

Telomere lengths in human oocytes, cleavage stage embryos and blastocysts.

Telomeres are repeated sequences that protect the ends of chromosomes and harbour DNA repair proteins. Telomeres shorten during each cell division in the absence of telomerase. When telomere length becomes critically short, cell senescence occurs. Telomere length therefore reflects both cellular ageing and capacity for division. We have measured telomere length in human germinal vesicle (GV) oocytes and preimplantation embryos, by quantitative fluorescence in situ hybridization (Q-FISH), providing baseline data towards our hypothesis that telomere length is a marker of embryo quality. The numbers of fluorescent foci suggest that extensive clustering of telomeres occurs in mature GV stage oocytes, and in preimplantation embryos. When calculating average telomere length by assuming that each signal presents one telomere, the calculated telomere length decreased from the oocyte to the cleavage stages, and increased between the cleavage stages and the blastocyst (11.12 versus 8.43 versus 12.22 kb, respectively, P < 0.001). Other methods of calculation, based upon expected maximum and minimum numbers of telomeres, confirm that telomere length in blastocysts is significantly longer than cleavage stages. Individual blastomeres within an embryo showed substantial variation in calculated average telomere length. This study implies that telomere length changes according to the stage of preimplantation embryo development.

Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection?

Prenatal oogenesis produces hundreds of thousands of oocytes, most of which are discarded through apoptosis before birth. Despite this large-scale selection, the survivors do not constitute a perfect population, and the factors at the cellular level that result in apoptosis or survival of any individual oocyte are largely unknown. What then are the selection criteria that determine the size and quality of the ovarian reserve in women? This review focuses on new data at the cellular level, on human prenatal oogenesis, offering clues about the importance of the timing of entry to meiotic prophase I by linking the stages and progress through MPI with the presence or absence of apoptotic markers. The characteristics and responsiveness of cultured human fetal ovarian tissue at different gestational ages to growth factor supplementation and the impact of meiotic abnormalities upon apoptotic markers are discussed. Future work will require the use of a tissue culture model of prenatal oogenesis in order to investigate the fate of individual live oocytes at different stages of development.

Influence of p53 and genetic background on prenatal oogenesis and oocyte attrition in mice.

BACKGROUND: Meiotic progression, and the number of oocytes surviving to birth, determine the ovarian reserve, yet the control of prenatal oogenesis is poorly understood. We investigated the effects of genetic background and p53 upon oogenesis in mice. METHODS: Fetal and neonatal ovaries were analysed in B6CBf1 and B6CBf2 mice from 15.5 to 21 days post-coitum (dpc) and p53 (a tumour suppressor gene) knockout, heterozygous and wild-type mice from 15.5 to 16 dpc. Oocytes in meiotic prophase I (MPI) were identified by labelling synaptonemal complex protein 3, and the specific stage of MPI was classified by the appearance of axial elements. Apoptosis and DNA breaks were assessed by cleaved poly-(ADP-ribose) polymerase (PARP-1) and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL), respectively. RESULTS: The leptotene, zygotene and pachytene stages were earlier in f1 than f2 generations with significant differences at all stages (P< or =0.002). The p53(-/-) oocyte populations and those with the presence of p53 gene differed significantly in terms of: (i) the proportion of oocytes reaching specific stages of MPI on 15.5 and 16 dpc and (ii) the proportion of oocytes having observed abnormalities in synaptonemal complexes (P < 0.001). The absence of p53 resulted in faster progression of oocytes and more with compressed and abnormal axial elements. We observed significant differences between p53(-/-), p53(+/-) and p53(+/+) mice in terms of cleaved PARP-1 staining and TUNEL. CONCLUSION: Genotype has an important impact on prenatal meiosis and oocyte apoptosis. p53 affects the speed of oocyte development and may influence the oocyte selection through apoptosis during MPI.

Human immature oocytes grow during culture for IVM.

BACKGROUND: Oocyte competence for maturation and embryogenesis is associated with diameter in many mammals. We aimed to test whether this relationship exists in humans and to quantify its impact upon in vitro maturation (IVM). METHODS: We used computer-assisted image analysis daily to measure average diameter, zona thickness and other parameters in oocytes. Immature oocytes originated from unstimulated patients with polycystic ovaries, and from stimulated patients undergoing intracytoplasmic sperm injection (ICSI). Some were cultured with meiosis activating sterol (FF-MAS). Matured oocytes were inseminated using ICSI and embryo development was monitored. In vivo matured oocytes were also measured. RESULTS: Immature oocytes were smaller at collection than in vivo matured oocytes. Maturation was related to oocyte diameter and many oocytes grew in culture. FF-MAS stimulated growth in oocytes derived from ICSI patients, but only stimulated growth in PCO derived oocytes if they matured in vitro. Degenerating oocytes showed cytoplasmic shrinkage. Neither zona thickness, perivitelline space, nor the total diameter of the oocyte plus zona were informative regarding maturation capacity. CONCLUSIONS: Immature oocytes grow during maturation culture. FF-MAS promotes oocyte growth in vitro. Oocytes from different sources have different growth profiles in vitro. Measuring oocytes in clinical IVM may provide additional non-invasive information that could potentially avoid the use of growing oocytes.

Growth factors promote meiosis in mouse fetal ovaries in vitro.

Mouse fetal ovaries were cultured to investigate germ cell development in the presence of a combination of the growth factors (GFs) stem cell factor, insulin-like growth factor-1 and leukaemia inhibitory factor. Ovaries were isolated from fetal mice at 13 and 14 days post-coitum (dpc) and cultured to the equivalent of 17 dpc. Culture conditions comprised minimal essential medium-alpha plus 5% fetal calf serum, with or without GFs. Oocytes were assessed using immunofluorescence to illustrate synaptonemal complexes and recombination foci. The proportions of pachytene cells in freshly isolated 13, 14 and 17 dpc ovaries were 0, 8 and 74% respectively. There was a significant (P < 0.0001) increase in the number of pachytene cells after 4 days culture with GFs, with 24% of germ cells from 13 dpc ovaries reaching pachytene. In contrast, no pachytene cells were detected in cultures of 13 dpc ovaries without GFs. After 3 days in culture with GFs, 38% of germ cells from 14 dpc ovaries were at pachytene compared with 19% without GFs. In conclusion, we have demonstrated positive effects of GFs upon oocyte formation by meiosis in vitro. The observed results could be explained by an increased survival of premeiotic oogonia entering meiosis, or by effects on oocytes already in early meiosis.

Different perspectives of patients and health care professionals on the potential benefits and risks of blastocyst culture and multiple embryo transfer.

BACKGROUND: A trade-off exists between the risk of multiple pregnancy and prospects of pregnancy itself in assisted reproduction. Blastocyst culture and embryo transfer after approximately 5 days may be one method of reconciling this dilemma, although a controversial one. METHODS AND RESULTS: We presented a questionnaire to groups of patients, embryologists and clinicians to solicit views on the potential benefits and risks of blastocyst culture and multiple pregnancy. The results indicate that patients are more accepting of multiple pregnancy as a prospective outcome of treatment than those involved in their treatment, despite awareness of the risks. Our data tend to support a genuine difference in values on this point. We also sought views on the patient selection criteria and treatment protocols which should apply in a planned randomized controlled trial comparing blastocyst culture with cleaving embryo transfer (e.g. numbers of embryos to transfer, acceptable levels of risk of twin and triplet pregnancy, the proportion of patients who would be put off from entering the trial by the risk of no embryo transfer). These are presented and discussed with reference to their likely impact on trial recruitment, highlighting differences in perspective between patients and professionals. CONCLUSIONS: We conclude that there are differences among patients, embryologists and clinicians in their perceptions of the desirability of multiple pregnancy, their preferences in certain practical aspects of treatment such as embryo transfer numbers, and their ideas on blastocyst culture and its prospective outcomes and risks.

Factors controlling embryo viability.

Many diverse factors affect embryo viability. The morphological measures routinely used to grade human embryos are of limited use as many of the factors influencing the longterm viability of embryos are genetic or molecular and are undetectable or inconclusive from visualization of living embryos by microscopy. This article presents examples of factors that are known to affect embryo viability, including gamete formation, embryonic genome activation, and im-printing. Aspects of both gamete and embryo development are addressed, and the possibility that various anomalies remain hidden for extended periods before impacting upon a later aspect of development is hypothesized. In future, more detailed and informative assessments of embryo viability before embryo transfer may require invasive approaches to study the composition of embryos at various stages of preimplantation development; however, indirect, non-invasive measures would be preferable.

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries.

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.

Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes?

Three hypotheses were tested: (i) the distance between first and second polar bodies (PB) may relate to embryo morphology, (ii) that the orientation of pronuclei (PN) relative to PB may relate to embryo morphology, (iii) that the placement of a spermatozoon in a fixed plane relative to the first PB [intracytoplasmic sperm injection (ICSI)] may alter PN/PB orientation relative to in-vitro fertilization (IVF). A total of 251 two pronuclear (2PN) embryos (124 ICSI, 127 IVF) from 64 patients was studied. Angles were measured between the PN axis and the nearest PB (alpha), the furthest PB (beta), and between the two PB (gamma). On day 2, the morphological grades of embryos were recorded. gamma ranged from 0 to 150 degrees and was not significantly different for ICSI or IVF embryos of different grades; however, an unusual distribution of gamma suggested different populations of oocytes. The first hypothesis was rejected. alpha and beta ranged from 0 to 90 degrees : alpha did not relate significantly to embryo grade, but beta increased significantly with decreasing quality of ICSI embryos (P < 0.05) and the total group (P < 0.01), supporting hypothesis (ii). The difference in beta between ICSI and IVF embryos was not significant, so hypothesis (iii) was unproven. Significant differences between ICSI and IVF embryos in PN positions, irregular cleavage, and cleavage failure were noted.

Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture.

This study aimed to: (i) determine whether oocytes are present in cultures of human fetal ovary; (ii) identify whether meiotic anomalies are evident; and (iii) assess whether preparation or culture conditions influence oocyte survival and meiotic progression. Ovaries were collected from fetuses after termination at 13-16 weeks. Oocyte assessment utilized antibodies specific for synaptonemal complex proteins (associated with chromosomes only during meiosis), and antibodies to centromeric proteins. Fragments of tissue were cultured in minimal essential medium + 10% serum +/- follicle stimulating hormone (100 mIU/ml). The sera were fetal calf serum (FCS), FCS for embryonic stem cells (ES-FCS) and human female serum. The numbers and stages of oocytes were assessed after 7-40 days, and particular arrangements of chromosome synapsis identified. Results in fresh tissue included oocytes at leptotene, zygotene, pachytene and diplotene in three of five samples. Four specimens remained viable in vitro, and three had detectable oocytes after culture. The numbers of oocytes and the proportions of zygotene and pachytene cells increased with time in culture. The proportion of degenerate cells in culture was initially higher than in fresh samples, but declined subsequently. More oocytes were detected in ES-FCS and human serum than in FCS. We conclude that human oocytes survive in culture and that progression through prophase I continues.

Evidence from human oocytes for a genetic bottleneck in an mtDNA disease.

We have examined oocytes from a patient with Kearn-Sayre syndrome caused by mtDNA rearrangements. In mtDNA diseases, mutant and wild-type mtDNA frequently coexist in affected individuals (the condition of heteroplasmy). The proportion of mutant mtDNA transmitted from mother to offspring is variable because of a genetic bottleneck, and the "dose" of mutant mtDNA received influences the severity of the phenotype. The feasibility of prenatal diagnosis is critically dependent on the nature and timing of this bottleneck. Significant levels of rearranged mtDNA were detectable in the majority of the patient's oocytes, by use of multiplex PCR, with wide variation, in the levels of mutant and wild-type molecules, between individual oocytes. We also used length variation in a homopolymeric C tract, which is often heteroplasmic in normal controls, to identify founder subpopulations of mtDNAs in this patient's oocytes. We present direct evidence that the number of segregating units (n) is three to five orders of magnitude less than the number of mitochondria in the human female oocyte. In some cases, the best estimate of n may correspond to a single mitochondrion, if it is assumed that intergenerational transmission of mtDNA can be treated as a single sampling event. The bottleneck appears to contribute a major component of the variable transmission from mother to oocyte, in this patient and in a control. That this bottleneck had occurred by the time that oocytes were mature advances the prospects for prenatal diagnosis of mtDNA diseases.

In vitro culture of ovarian follicles.

This review offers a practically oriented introduction to follicle culture in vitro, focusing on mouse follicles, but with reference to other species. The main principles of follicle growth are addressed, including the constraints of tissue culture, methods of follicle isolation, and techniques for individual and collective culture of intact follicles. Culture systems that support a spherical or a non-spherical follicular structure in vitro are discussed in terms of follicular and oocyte development, and methods for assessing follicular function in vitro are presented. Oocyte development in most in vitro culture systems is currently suboptimal and the parallel development of oocytes and follicles is discussed, with a view to maintaining the competence of the oocyte. Finally, some potential future applications of follicle growth in vitro are suggested.

Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: support for a genetic bottleneck.

While mtDNA polymorphisms at single base positions are common, the overwhelming majority of the mitochondrial genomes within a single individual are usually identical. When there is a point-mutation difference between a mother and her offspring, there may be a complete switching of mtDNA type within a single generation. It is generally assumed that there is a genetic bottleneck whereby a single or small number of founder mtDNA(s) populate the organism, but it is not known at which stages the restriction/amplification of mtDNA subtype(s) occur, and this uncertainty impedes antenatal diagnosis for mtDNA disorders. Length polymorphisms in homopolymeric tracts have been demonstrated in the large noncoding region of mtDNA. We have developed a new method, T-PCR (trimmed PCR), to quantitate heteroplasmy for two of these tracts (D310 and D16189). D310 variation is sufficient to indicate clonal origins of tissues and single oocytes. Tissues from normal individuals often possessed more than one length variant (heteroplasmy). However, there was no difference in the pattern of the length variants between somatic tissues in any control individual when bulk samples were taken. Oocytes from normal women undergoing in vitro fertilization were frequently heteroplasmic for length variants, and in two cases the modal length of the D310 tract differed in individual oocytes from the same woman. These data suggest that a restriction/amplification event, which we attribute to clonal expansion of founder mtDNA(s), has occurred by the time oocytes are mature, although further segregation may occur at a later stage. In contrast to controls, the length distribution of the D310 tract varied between tissues in a patient with heteroplasmic mtDNA rearrangements, suggesting that these mutants influence segregation. These findings have important implications for the genetic counselling of patients with pathogenic mtDNA mutations.

Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine.

The culture of individual intact follicles in vitro, from small pre-antral to pre-ovulatory stages, will improve our ability to perform controlled experiments studying follicle growth and female gamete development. This study was undertaken to characterize further the conditions required for physiological follicle growth in vitro. We cultured a total of 398 pre-antral follicles of immature mice (aged 26-28 days) with an initial diameter of 140-340 microns for 4 days in vitro, using individual micro-cultures under paraffin oil. Summarizing the results of all groups, 50 follicles were damaged (12.6%) and, of those remaining intact (n = 348), 60 (17.2%) became atretic, 195 (56.0%) became antral and 26 (7.5%) ovulated. The most advanced follicles grew to 400-500 microns diameter. The presence of follicle-stimulating hormone (FSH) in the medium significantly stimulated follicle growth in vitro (P < 0.03), in a manner proportional to the initial diameter over the range of 140-250 microns initial diameter, with larger follicles being refractory. FSH also significantly increased the proportion of follicles forming antra (P < 0.001) and their likelihood of ovulating in vitro (P < 0.01), and reduced the frequency of atresia (P < 0.01). Dibutyryl-cyclic AMP mimicked FSH, significantly stimulating growth of large follicles (P < 0.05) and antrum formation (P < 0.01). Hypoxanthine also stimulated antrum formation (P < 0.01) but did not significantly affect follicle growth. Porcine relaxin had no significant effect on mouse follicle growth or antrum formation. The optimal conditions for mouse follicle growth in vitro have not yet been defined, but selection of follicles of < 250 microns diameter and inclusion of FSH or dibutyryl-cyclic AMP in the culture medium are recommended.

Meiotic progression of mouse oocytes throughout follicle growth and ovulation in vitro.

Intact ovarian follicles can be grown in culture but the normality of their oocyte development has not yet been established. This work examined meiotic progression in oocytes from mouse ovarian follicles grown in vitro and in vivo. Follicles of initial diameter 112-279 microns were isolated from ovaries of B6 CBA F1 mice and cultured individually in 25 microliters minimal essential medium-alpha containing 5% mouse serum, 100 mIU/ml follicle stimulating hormone and various supplements. Oocytes were isolated from follicles of various sizes after culture for < or = 5 days and oocyte chromatin was examined by fluorescence microscopy with Hoechst 33258. The stage of meiosis was classed as prophase I [germinal vesicle (GV) stages I-IV], metaphase I, anaphase I or metaphase II and the results were compared with oocytes from fresh follicles of unprimed or gonadotrophin-primed immature mice, and mature mice in oestrus. Small pre-antral follicles grew in vitro to intact pre-ovulatory follicles and some ovulated spontaneously despite negligible luteinizing hormone (LH). Progression through GV stages I-IV in vitro was related to initial and final follicle diameter, follicle growth rate and antrum formation. All the normal stages of GV development were observed in vitro; however, the cultured follicles were significantly larger than the freshly dissected follicles for stages II and III of GV development. GV breakdown had occurred in 7/11 analysable ovulated oocytes and 3/69 intra-follicular oocytes. We conclude that, under the conditions employed, the majority of oocytes cultured in intact immature follicles retain the GV.(ABSTRACT TRUNCATED AT 250 WORDS)

Pronuclear, cleavage and blastocyst histories in the attempted preimplantation diagnosis of the human hydatidiform mole.

We report a study of fertilization, syngamy and embryonic development in 14 oocytes from a woman with four previous pregnancies involving complete hydatidiform moles. Serial observations of pronuclear movements and syngamy were compared to those in a group of 10 multipronucleate embryos from other patients. One embryo and possibly two others developed normally or near-normally. The others displayed immediate cleavage or had one or three pronuclei. The tripronucleate eggs displayed various anomalous forms of growth. The unipronucleate eggs passed through a double form of syngamy, which might have involved chromosome doubling, and could have developed as androgenetic diploids. We suggest a hypothesis to explain these unusual observations.