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Biomedical subjects

G M Hartshorne

Publications and source records attributed to G M Hartshorne.

At least 19 recordsLinked to original sources
PubMed2002

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.

Attitude of Health Personnel↗
PubMed2002

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.

Adaptor Proteins, Signal Transducing↗
PubMed2001

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.

Aging↗
PubMed2001

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.

Adult↗
PubMed1999

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.

Cell Count↗
PubMed1999

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.

Cell Nucleus↗
PubMed1998

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.

DNA Primers↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1994

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)

Animals↗
PubMed1994

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.

Animals↗
PubMed1992

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.

Adult↗
PubMed1991

Role of embryonic factors in implantation: recent developments.

The embryonic factors influencing implantation have been studied extensively in laboratory and domestic animals, but not in primates, including humans. Species differences make extrapolation inadvisable. Embryonic factors affecting implantation include intrinsic features of the embryo, such as its genetic constitution, morphology and hatching. Abnormal genetic constitutions or unsuccessful transitions from maternal to embryonic transcription could account for many failures of early embryonic growth and implantation. Morphology per se does not greatly influence implantation, except when it reflects an abnormal genetic constitution, e.g. in severe fragmentation, although subtle effects may be detected as experimental techniques are refined. The initiation of differentiation and intraembryonic communication between cells and cell types has been studied in animal embryos. Signals must be exchanged between the embryo and the mother to ensure satisfactory implantation. These could include platelet activating factor, prostaglandins, histamine related factors, steroids, proteins, metabolic products and immune-active factors. No one factor seems to be totally responsible for alerting the mother to the presence of an embryo, and a concerted action of these and other agents is probably responsible. The process of implantation itself is poorly understood because of a lack of adequate experimental models. The expression of complementary proteins and the role of specific enzymes and markers of endometrial and embryonic competence are factors well worthy of further study. Knowledge about human implantation is increasing because of recent developments in assisted reproductive technology, and concepts arising from many years of research in animals should find clinical applications in understanding and controlling human reproduction.

Animals↗
PubMed1991

The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts.

Blastocysts from 198 patients were frozen using glycerol as cryoprotectant. No difference in the post-thaw survival of blastocysts or implantation rates was found between 177 patients (122 transfers) with all surplus embryos cultured to blastocysts before freezing and 20 patients (12 transfers) whose embryos were considered unsuitable for freezing during cleavage and were then frozen as blastocysts. Nineteen pregnancies were achieved, of which six aborted. Pre-freezing morphology was similar in blastocysts of patients in groups 1 and 2 and did not relate to their survival after cryopreservation. A significantly lower proportion with suspected damage after thawing was present among patients becoming pregnant after transfers of single blastocysts (P less than 0.01) and implanting embryos were in general more expanded at the time of transfer. No differences were detected between blastocysts resulting in normal development and those leading to abortion. The developmental consequences of damage to human blastocysts are discussed.

Adult↗
PubMed1991

Dynamic and phagocytotic activity of human granulosa cells in vitro.

This study has examined the interaction of human granulosa cells in culture with inert latex particles to determine whether an active role in phagocytosis may be contemplated. The report shows that granulosa cells exhibit various types of movement in culture. They also interact with inert latex particles which are actively phagocytosed. Uptake of an individual bead took approximately 30 min and was affected by agents modulating levels of divalent cations. The physiological relevance of this property of granulosa cells is not known.

Cells, Cultured↗
PubMed1990

In vitro fertilization and development of one of two human oocytes with fused zonae pellucidae: case report.

This report describes a pair of human oocytes of differing maturities but fused at the zona pellucida, believed to have originated from a binovular follicle. After insemination in vitro, one oocyte fertilized and developed into an expanded blastocyst, whereas the other, which contained a germinal vesicle, degenerated. A biopsy was aspirated from the blastocyst, which subsequently re-expanded but did not hatch.

Adult↗
PubMed1990

Binding proteins for insulin-like growth factors in the human ovary: identification, follicular fluid levels and immunohistological localization of the 29-32 kd type 1 binding protein, IGF-bp1.

The occurrence of pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29-32 kd insulin-like growth factor binding protein, now termed type 1 or IGF-bp1, has been examined in the human ovary by monoclonal and polyclonal antibody based radioimmunoassay and immunohistological techniques. Follicular fluids aspirated from 51 follicles of 32 women undergoing hyperstimulation involving buserelin or clomiphene-based protocols contained 35.5-276.0 ng/ml (mean 101.0 mg/ml) of immunoreactive IGF-bp1. Mean fluid concentrations were three times the level of IGF-bp1 detected in paired serum samples, available for 21 women. Immunoreactive IGF-bp1 in follicular fluid exhibited similar dose-response curves to purified protein and amniotic fluid and immunoreactive IGF-bp1 coeluted in gel filtration with a peak of [125I]-IGF-1 binding corresponding to the elution profile of purified IGF-bp1. Gel filtration also revealed the presence in follicular fluid of a greater than 100 kd binding protein with a binding capacity equal to IGF-bp1 under the conditions employed. A highly significant correlation (P less than 0.001) was found between follicular fluid progesterone and IGF-bp1 and a correlation of lower significance was found between oestradiol and IGF-bp1 (P less than 0.05). However, only low levels of immunoreactive IGF-bp1 were detected in supernatant media of granulosa cells in culture (range undetectable to 2.3 ng/ml). Employing monoclonal antibody-based immunohistology, immunoreactive IGF-bp1 was consistently associated with luteinized granulosa cells of corpora lutea rather than paraluteal cells and its intensity of reactivity appeared to reflect luteal phase steroid hormone profiles. No consistent reactivity was detected in preovulatory follicles and granulosa cells in culture, although reactivity was associated with primordial oocytes. Immunoreactive IGF-bp1 was detected in six of nine supernatant media of explants of luteal tissue obtained from five corpora lutea, with levels ranging from undetectable to greater than 200 ng/ml. These observations suggest that IGF-bp1 is primarily related to luteinization of the granulosa and the resultant luteal cells, and if produced by the luteal cells, additional exogenous factors are required to induce production by granulosa cells in vitro.

Adult↗