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Biomedical subjects

H Newton

Publications and source records attributed to H Newton.

At least 19 recordsLinked to original sources
PubMed2000

Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice.

OBJECTIVE: To determine the long-term survival of frozen-thawed human ovarian tissue as xenografts in severe-combined-immunodeficiency (SCID) mice. DESIGN: Animal study. SETTING: Animal and laboratory facilities at an academic center. PATIENT(S): Ovarian tissue obtained from a 27-year-old woman. INTERVENTION(S): Grafting of frozen-thawed ovarian tissue in SCID mice for 22 weeks. MAIN OUTCOME MEASURE(S): Follicle counts and growth by morphology and PCNA staining in frozen-thawed grafts and fresh controls. RESULT(S): All three grafts were recovered intact after 22 weeks. Their stroma was devoid of necrotic cells and contained healthy follicles. The ratio of primordial-total follicles decreased significantly after grafting (0.94 +/- 0.02 to 0.87 +/- 0. 01, control vs. grafting). Compared with controls, after 22 weeks of grafting, a higher percentage of follicles had initiated growth (5.6 +/- 2.4 vs. 12.5 +/- 1.9), but there was still a significant number of primordial follicles/graft (75 +/- 6.8). Follicle stages were similar between two groups; only primordial and one-layer follicles were seen in the xenografts. In the controls, except for one two-layer follicle, the most advanced follicle was at the one-layer stage. CONCLUSION(S): Human primordial follicles survive freeze-thaw and long-term xenografting procedures and retain their capacity to initiate growth. These findings encourage future attempts for human autologous ovarian transplantation.

Adult↗
PubMed1999

In vitro growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue.

A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.

Animals↗
PubMed1999

Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocytes.

Controlled ovarian stimulation during an in vitro fertilization cycle usually produces large numbers of oocytes and, consequently, it is likely that more embryos will be generated than can be transferred in a given cycle. It is desirable to freeze-bank surplus oocytes before insemination to avoid the ethical and legal complications of disposing of or storing embryos. Although many attempts have been made to cryopreserve human oocytes, to date, post-thaw survival has been poor, and viable pregnancies after in vitro fertilization have been rare. A possible explanation for the lack of success is that the freezing methods have been adapted from animal studies but have not been optimized for the human oocyte. In this study, video microscopy was used to determine the volumetric responses of mature human oocytes to changes in osmolarity during preparation for freezing. A Boyle van't Hoff plot of data collected in static experiments with fresh human oocytes gave a value of 0.19 +/- 0.01 (mean +/- SEM) for the osmotically inactive volume. Dynamic measurements during exposure to dimethyl sulphoxide at room temperature (22 degrees C) were analysed by a two-parameter transport model and produced values of 1.30 x 10(-6) cm atm-1 s-1 for the hydraulic conductivity of the plasma membrane and 3.15 x 10(-5) cm s-1 for dimethyl sulphoxide permeability (chi-squared = 0.43, df = 20) of fresh human oocytes. Oocytes that had failed to fertilize had a slightly lower hydraulic conductivity and dimethyl sulphoxide permeability and, after exposure to 1.5 mol dimethyl sulphoxide l-1, these cells appeared to become permeable to normally impermeable solutes. These permeability properties have been used to design a protocol for the addition and removal of dimethyl sulphoxide to control the magnitude of volumetric changes.

Cell Membrane↗
PubMed1998

Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?

OBJECTIVE: To review the potential for cryopreserving immature follicles either in situ or after isolation from ovarian stroma and to consider the options for fertility by transplantation or in vitro follicle growth. DESIGN: The problems of storing embryos and mature (metaphase II) oocytes were considered in light of the needs of patients to protect fertility before undergoing potentially sterilizing therapy for cancer. The evidence from the experimental biology literature showing that immature oocytes (prophase I) in primordial follicles can be cryopreserved successfully and transplanted to produce fertile eggs was reviewed. The review, which was compiled from MEDLINE and other bibliographic databases, is intended to emphasize the practical opportunities for this technology and the need for future research rather than to be a comprehensive treatment of the subject. CONCLUSION(S): The disappointing results obtained with the cryopreservation of oocytes at metaphase II and ethical concerns about embryo storage are giving impetus to the banking of ovarian tissue for patients who require conservation of fertility. The numbers of needy patients are growing as long-term survivorship after high-dose chemotherapy and bone marrow transplantation rises. More speculatively, if ovarian tissue banking becomes a proven effective method, young, healthy women may request storage of ovarian biopsy samples to keep their reproductive options open in midlife when oocyte fertility is declining. Although the cryotechnology is not yet perfected, the major question now is how to use the tissue most effectively after thawing. For the present, ovarian tissue cryopreservation is still at the experimental stage, but it holds the promise of valuable applications.

Cellular Senescence↗
PubMed1998

Conservation of the follicular population in irradiated rats by the cryopreservation and orthotopic autografting of ovarian tissue.

AIM: To preserve the follicular population in rats prior to abdominal irradiation by the orthotopic autografting of ovarian cortical slices (frozen/thawed) in one of two cryoprotective agents (CPAs). The proportion of follicular survival will be quantified at autopsy and a comparison will be made of the two CPAs. METHOD: Thirty Wistar rats, aged 38-39 days, were unilaterally ovariectomised. The ovaries of twenty animals were slow-cooled in either 1.5 M dimethylsulphoxide (DMSO; group B) or 1.5 M ethylene glycol (EG; group C). The ovaries of the remaining ten animals (group A) were fixed and histologically examined to determine a fresh follicle count. The contralateral ovary was irradiated with a single dose of 2.5 Gy. Frozen tissue (from groups B and C) was thawed rapidly and autografted into the irradiated ovarian bursa. All animals were mated eighteen days after grafting and were sacrificed eighteen days later. At autopsy, the number of embryos was counted and the ovarian weights recorded, before the tissue was prepared histologically for quantification of follicular survival. RESULTS: There was no significant difference in the pregnancy rate in the three groups and, although there was a higher number of embryos in the irradiated control group A compared with groups B and C, the difference was not significant. The follicle count in groups B and C was significantly higher than in group A. In group A, the percentage of growing follicles was higher than in groups B and C and the fresh ovary. CONCLUSIONS: Autografting frozen/thawed ovarian tissue preserved a proportion of the follicular population in irradiated rats. There was no significant difference in the numbers of follicles surviving cryopreservation in the two CPAs. Since the irradiated rats were not totally sterilised, we cannot conclude that cryopreserved grafts can restore fertility.

Animals↗
PubMed1998

Interpretive risks: the use of the Hopkins Symptom Checklist 90-Revised (SCL 90-R) with brain tumour patients.

Patients with brain tumours often report distress. Interpretive problems ensue when measures normed on healthy persons are utilized to quantify distress. This study investigated potentially spurious elevations on the Hopkins Symptom Checklist 90 Revised (SCL 90-R). Responses of 17 patients were obtained prior to aggressive chemotherapy. Traditional interpretation indicated that 47% of the patients endorsed clinical levels of somatization, 53% obsessive-compulsive and 59% psychotic disorders. Elevations were attributable to common consequences of brain tumours, medication and the emotional reaction to prognosis. Conventional interpretation would lead to inappropriate classifications. The majority of SCL 90-R item endorsements were significantly different than those of the norm group. Appropriate interpretation of scores is discussed.

Adult↗
PubMed1998

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.

Adult↗
PubMed1998

Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone.

In contrast to the many detailed studies of Graafian follicles, the biology of small follicles in the human ovary is poorly understood and the trigger for follicular growth initiation remains unknown. No practical model exists to study preantral follicle growth in the human because of their slow growth rate and lack of an effective culture system. We therefore tested ovarian xenografts as a new strategy to study the early stages of ovarian follicular growth in vivo. Mice homozygous for severe combined immunodeficiency (SCID) and hypogonadism (hpg) received human ovarian xenografts under their kidney capsules. Follicle growth was assessed by morphology and proliferating cell nuclear antigen (PCNA) immunostaining. The grafts were recovered after 11 (short-term) and 17 weeks (long-term), and serially sectioned. During the last 6 weeks of long-term grafting, mice were randomized to receive either placebo or 1 IU of purified follicle stimulating hormone (FSH) s.c. on alternating days. After 11 weeks of grafting, the most advanced follicles had a maximum of two granulosa cell layers. In the absence of FSH administration, follicles did not progress beyond the two-layer stage even after 17 weeks of grafting, and the oestradiol levels remained undetectable. In the FSH-treated long-term grafts, follicles had grown to antral stages and resulted in oestradiol levels as high as 2070 pmol/l. Growth initiation indices did not differ between control and FSH-treated grafts. This study demonstrates that follicles can survive and grow in human ovarian tissue grafted under the renal capsules of immunodeficient mice for at least 17 weeks, and indicate that xenograft models are potentially useful for studying human follicle development. Using this physiological model, we showed that FSH is required for follicle growth beyond the two-layer stage, although growth initiation is independent of gonadotrophin stimulation.

Animals↗
PubMed1998

Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts.

Ovarian cortical tissue cryopreservation with subsequent autografting is a potential strategy for the preservation of fertility in patients undergoing systemic chemotherapy and pelvic radiotherapy. Non-vascular implants are first subjected to a period of ischaemia before revascularization and are, therefore, vulnerable to ischaemia-reperfusion injury from reactive oxygen species. Ischaemia-reperfusion injury was investigated during the first week after surgery in murine ovarian grafts and human ovarian xenografts in mice with severe combined immune deficiency (SCID) by measuring total lipid peroxides and malondialdehyde concentrations with a colorometric assay. The effects of administering an antioxidant, vitamin E, on these concentrations were also tested. Products of lipid peroxidation were higher in non-supplemented murine autografts compared with control ovaries (P < 0.05), and were significantly reduced on day 3 by vitamin E administration (P < 0.05). Similarly, in human xenografts, there was a significant reduction in lipid peroxidation with vitamin E administration. These results correspond to a significantly greater total follicle survival in the murine grafts of the supplemented group (45 versus 72%; P < 0.05). They suggest that antioxidant treatment improves the survival of follicles in ovarian grafts by reducing ischaemia-reperfusion injury.

Adult↗
PubMed1997

Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue.

OBJECTIVE: To develop an efficient isolation technique for human primordial follicles. DESIGN: Prospective, experimental study of ovarian biopsies collected from healthy women undergoing elective cesarean section. Ovarian blocks either were fixed for histology and follicle counting or partially disaggregated with type 1A collagenase before or after cryopreservation. After partial disaggregation, follicles were isolated by microdissection. SETTING: Leeds General Infirmary. MAIN OUTCOME MEASURE(S): Follicle viability was assessed with live-dead stains using 5-(and 6-) carboxyfluorescein diacetate, succinimidyl ester and propidium iodide, respectively, and using electron microscopy. The numbers recovered were expressed as a percentage of the numbers of primordial follicles in comparable blocks of tissue and the viability of the whole follicle and oocyte were scored separately. RESULT(S): On average, 18.0 +/- 3.8 and 15.9 +/- 2.2 (mean +/- SEM) follicles per block were recovered from fresh and cryopreserved ovarian tissue, respectively, corresponding to recovery rates of 57.9% +/- 8.8% and 56.2% +/- 16.7%. In the fresh group, the percent viability of whole follicles and oocytes were 71.6% +/- 2.4% and 91.3% +/- 2% compared with 71.5% +/- 4.7% and 95% +/- 4.3% in the frozen-thawed group. Electron microscopy confirmed that the majority of the cells lacked ultrastructural signs of damage after isolation and cryopreservation. CONCLUSION(S): Primordial follicles can be isolated from fresh and cryopreserved human ovarian tissue with similar high efficiency and viability rates.

Adult↗
PubMed1996

[Follicle freezing and autografting. A new method of medically assisted procreation?].

Although frozen human follicles were shown to be viable after thawing, more than forty years, the low yield of primordial follicles after cryopreservation has greatly limited research in this area. The aim of freezing follicles is to stock a patient's ovocytes before potentially destructive therapy for cancer then to reimplant the follicular tissue after successful treatment. The patient's ovulatory capacity would then theoretically be restored, allowing natural or possibly in vitro fertilization. As have other teams, we recently renewed work in cryopreservation of follicles in experimental models in search for a better cryopreservation agent and have had encouraging results suggesting that primordial human follicles or ovarian fragments can be grafted successfully after cryopreservation. Questions still under study include the optimal site for implantation, the survival time for grafts and the quantity of follicles needed to achieve pregnancy. Work on large animal models or xenografts of human tissues on immunotolerant animals may provide further insight. The possibilities of grafting primordial follicles, either alone or within ovarian tissue, may raise hope for many women, although new questions such as an age limit for reimplantation and use of allografts requiring preparation with major immunosuppressive therapy require further debate.

Animals↗
PubMed1996

Low temperature storage and grafting of human ovarian tissue.

Ovarian tissue storage at low temperatures is a promising new method for protecting young cancer patients from the sterilizing effects of chemotherapy and/or radiotherapy. Tissue can be stored and returned to the body in due course as a thin cortical graft since the primordial follicles are distributed peripherally and are relatively resistant to ischaemia. Slices of tissue donated by healthy patients were trimmed to a uniform size and preserved by slow freezing to liquid nitrogen temperatures for up to 2 months in one of the following cryoprotectants: dimethylsulphoxide, ethylene glycol, glycerol, propylene glycol. Their viability was assessed by counting follicles in histological sections 18 days after grafting under the kidney capsules of severe combined immunodeficiency (SCID) mice, and the results were expressed as percentages of the numbers in comparable pieces of ungrafted tissue. While only 10% of the total number of follicles was found in the glycerol group compared to controls, significantly higher percentages (44-84%) survived cryopreservation in the other media. The tissues were sterile when frozen and thawed without a cryoprotectant. These results suggest that if comparable results could be achieved by autografting, a patient's fertility should be safeguarded from cytotoxic treatment.

Adolescent↗
PubMed1991

Stereotypes of pilots and apprehension about flying with them: a study of commercial aviation scenarios.

This study explored the effects of gender, experience, and performance of a commercial aviation pilot on concern of passengers about flying with him or her. It used scenarios that represented two aspects of pilot behavior: skill and judgment. We cast 112 adult subjects in the role of passengers. They read vignettes that described their captain and the flight in a 2 x 2 x 2 factorial design (male or female, newly promoted or veteran captain, strong or weak performance). Results showed that of the three manipulated variables, only pilot performance had a statistically significant main effect upon apprehension about making a continuation flight with the pilot (F = 41.52; p less than or equal to 0.001 for the skill scenario and F = 41.73; p less than or equal to 0.001 for the judgment scenario). Statistical interactions showed that the level of passenger concern was not based entirely on pilot performance. For instance, on the skill scenario, when a female pilot performed well, her rating was higher than that of an equally performing male, but when she performed poorly, her rating was lower than his (F = 5.38; p less than or equal to 0.02).

Adult↗
PubMed1990

Effect of hydrocephalus on prostaglandins and thromboxane B2 in ventricular cerebrospinal fluid.

The concentrations of prostaglandin F2 alpha, prostaglandin E2, 6-ketoprostaglandin F1 alpha (prostacyclin metabolite), and thromboxane B2 were assayed in ventricular cerebrospinal fluid obtained from 28 patients with hydrocephalus (17 obstructive, 11 communicating). Seven patients received dexamethasone or hydrocortisone on the day of sampling. No patient received nonsteroidal anti-inflammatory compounds for 48 hours before sampling. The median values did not differ significantly between the two types of hydrocephalus or from the concentrations in lumbar cerebrospinal fluid obtained from patients without intracranial pathology during lumbar myelography for possible lumbar disc disease. Hence, there is no evidence that eicosanoids accumulate in the ventricles in hydrocephalus, and it is unlikely that they have a significant role in its symptomatology.

6-Ketoprostaglandin F1 alpha↗