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Biomedical subjects

C D Matthews

Publications and source records attributed to C D Matthews.

At least 37 records · Page 2Linked to original sources
PubMed1995

Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis (delta F508)

OBJECTIVE: To evaluate direct polymerase chain reaction amplification of mutation on single embryo cells for the routine preimplantation diagnosis of cystic fibrosis. DESIGN: Direct polymerase chain reaction amplification of mutation was performed to identify the cystic fibrosis delta F508 mutation in human blood DNA, single lymphocytes, embryos, and embryo cells obtained by biopsy. Preimplantation diagnosis was performed for a couple who were heterozygous carriers of the delta F508 mutation. SETTING: Laboratory for preimplantation diagnosis in a reproductive medicine unit. MAIN OUTCOME MEASURE: Correct diagnosis of homozygous normal, heterozygous, and homozygous abnormal DNA of the cystic fibrosis delta F508 mutation. RESULTS: 45 blood samples (18 homozygous normal, 17 heterozygous, and 10 homozygous abnormal) and 204 single lymphocytes from known sources showed 100% amplification and were diagnosed correctly. 17 human embryos and 52 normal nucleated embryo cells obtained by single cell embryo biopsy also showed 100% amplification. After a miscarriage of the initial pregnancy (diagnosed at preimplantation to be homozygous normal) in the heterozygous carrier couple, fetal tissue was confirmed to be homozygous normal. CONCLUSION: Direct polymerase chain reaction amplification of mutation is a simple, fast, reliable test for the common cystic fibrosis mutation (delta F508) in blood DNA and single cells and should be applicable to routine programmes of general screening, maternal blood examination, and preimplantation diagnosis.

Base Sequence↗
PubMed1995

Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI).

The assessment of fertilization is an important part of intracytoplasmic sperm injection (ICSI) and oocytes are routinely examined about 17 h after injection using Nomarski differential interference contrast optics. However, it is not possible to conclusively determine the aetiology of fertilization anomalies in this manner, so cytological studies were undertaken to determine the causes of failed and abnormal fertilization after ICSI. Oocytes which exhibited no evidence of fertilization, one pronucleus (PN) or 3 PN were fixed in glutaraldehyde, stained with Hoechst 33342 and examined by fluorescence microscopy to identify PN, metaphase chromosomes, sperm heads and polar bodies. A total of 428 unfertilized oocytes were examined from 170 ICSI cycles. Overall, 82% of these unfertilized oocytes were still at metaphase II (non-activated) while the remaining 18% were activated and had 1 PN and two polar bodies. The majority (71%) of the metaphase II oocytes contained a swollen sperm head, which indicates that the spermatozoon was correctly injected but the oocyte did not activate and complete its second meiotic division. The swollen sperm head was located among the metaphase chromosomes in 4.3% of these oocytes, while in some cases (6.6%), the sperm chromosomes had undergone premature chromosome condensation (PCC). Other aetiologies of failed fertilization in these metaphase oocytes were ejection of the spermatozoon from the oocyte (19%) and complete failure of sperm head decondensation (10%). A similar pattern of anomalies was found in 1 PN oocytes, although the ratios were different (swollen sperm head, 51%; ejection of the spermatozoon, 19%; undecondensed sperm head, 30%). Seventy abnormally fertilized oocytes were also examined, of which 63 had 3 PN and a single polar body, indicating that the unextruded second polar body developed into the third PN. In conclusion, the present study demonstrates that the principal cause of fertilization failure after ICSI is failure of oocyte activation and not ejection of the spermatozoon from the oocyte. It is also apparent that further studies are needed to elucidate the mechanisms that control oocyte activation and sperm head decondensation in injected oocytes.

Adult↗
PubMed1995

Intracytoplasmic sperm injection--clinical results from the reproductive medicine unit, Adelaide.

The clinical results of 391 cycles of intracytoplasmic sperm injection (ICSI) performed between June 1993 and July 1994 are presented in this report. A total of 4797 oocytes were collected, of which 3792 were injected. Of these, 2603 (69%) fertilized, with normal and three pronuclear fertilization rates of 65% and 4% respectively. About 6% of the oocytes were destroyed while denuding and during ICSI. There were 373 (95%) embryo transfers from which 119 pregnancies arose, giving pregnancy rates of 32% per transfer and 30% per cycle, and an implantation rate of 15% per embryo. Of the pregnancies, 98 (82%) were ongoing. Supernumerary embryos were frozen in 44% of the cycles and 61 subsequent transfers of 130 frozen-thawed embryos produced 11 pregnancies (18%). Only 47 (12%) patients had less than 50% of their oocytes fertilized (mean 31%) after ICSI, and of these, 8 had no fertilization of 13 eggs. Nevertheless, 37 of these 47 patients had an embryo transfer and 9 achieved a pregnancy with an implantation rate of 14% per embryo. The percent normal sperm morphology weakly correlated with percent fertilization (r2 = 0.027, P < 0.02) but not with the implantation rate (r2 = 0.003, P > 0.05). Fifty-nine patients with only occasional motile sperm in the ejaculate and 23 patients in whom epididymal sperm were aspirated were treated. The fertilization rates (66% and 70% respectively) and pregnancy rates per transfer (32% and 24% respectively) were comparable in these two subgroups. The overall ICSI results were also compared with 515 cycles of routine in vitro fertilization (IVF) which were performed at the same time.(ABSTRACT TRUNCATED AT 250 WORDS)

Australia↗
PubMed1995

Melatonin response in active epilepsy.

Urinary excretion of 6-sulfatoxymelatonin (aMT.6S), the hepatic metabolite of melatonin, was measured for three consecutive 8-h intervals, beginning at 0600 h, in 30 patients with untreated active epilepsy and in 19 healthy subjects. Excretion of aMT.6S in a 24-h period in patients with active epilepsy was 77.3 +/- 55 nmol (median 68.0, range 8.7-280 nmol), significantly higher (p < 0.05) than that of healthy subjects (49.1 +/- 14 nmol, median 49.0, range 19.7-68.0 nmol). Sequential 8-h urinary aMT.6S excretion rates in patients with active epilepsy were 2.45 +/- 2.8 nmol/h (0600-1400 h), 0.83 +/- 0.5 nmol (1400-2200 h) and 6.38 +/- 5.0 nmol/h (2200-0600 h) as compared with 1.43 +/- 0.8, 1.10 +/- 0.8 and 3.81 +/- 1.3 nmol/h, respectively, in healthy subjects. Analysis of variance (ANOVA) indicated that the difference in total output resulted from greater nocturnal excretion (F = 5.58, p = 0.018). Melatonin production in untreated patients with active epilepsy is increased and has a circadian pattern with a phase difference as compared with that of normal subjects.

Adolescent↗
PubMed1995

Plasma melatonin in the horse: measurements in natural photoperiod and in acutely extended darkness throughout the year.

Plasma melatonin was measured at the winter and summer solstices and the autumn and spring equinoxes in four mares held under natural conditions at 35 degrees S. At all seasons the onset of the nightly elevated melatonin was coincident with or after the time of sunset and the melatonin offset after the time of sunrise. The duration of elevated melatonin was not different from the duration of natural scotophase for each season, with the duration of elevated melatonin longer in winter than the other seasons. Immediately following each 24 hr sampling two mares were resampled in acutely extended darkness to determine the melatonin profile of the endogenous rhythm of the circadian pacemaker, originating from the suprachiasmatic nuclei (SCN). At each season melatonin secretion commenced earlier and decreased later than that measured under the natural photoperiodic condition, suggesting that the expression of the melatonin rhythm is normally gated by natural environmental light both at dusk and dawn. The interval from the onset of melatonin measured under acutely extended darkness to the time of sunset was greater in the spring/summer than the autumn/winter suggesting a possible alternating signal throughout the year. Thus the mare appears to exhibit a similar interaction between endogenous circadian rhythmic activity and the natural photoperiod as the ewe which may underlie the mechanism for timing reproductive activity through the year.

Animals↗
PubMed1995

Is a critical interval of the circadian pacemaker at dusk responsive to light and melatonin responsible for the timing of estrus in the Romney Marsh ewe?

Two experiments, using Romney Marsh ewes, tested for the existence and role of a critical interval of the circadian pacemaker located near dusk that may be integrally involved in the precise timing of the breeding season. Groups of Romney Marsh ewes (n = 6) were provided with exogenous melatonin by injection at dusk (Experiment 1) or by infusion at dawn or subjected to extended darkness at dawn (Experiment 2) from the winter to the summer solstice before being exposed to natural photoperiod at latitude 35 degrees S. Other than the experimental protocols, all animals were held in natural photoperiod. The onset of the breeding season (defined as cyclic ovarian activity as indicated by plasma progesterone monitoring) was normal in those animals treated with morning melatonin but was delayed in those animals treated with melatonin at dusk or extended darkness at dawn compared to controls in natural photoperiod (p < .01). Exogenous melatonin at dusk was associated with a phase advance of the onset of the circadian pacemaker (as measured by endogenous melatonin in acutely extended darkness); additional darkness at dawn was associated with a phase delay of both the onset and the offset of the circadian pacemaker. Exogenous morning melatonin did not change the phase of the circadian pacemaker relative to the controls. The results are consistent with an external coincidence model of seasonal breeding in which a critical interval of the circadian pacemaker requires exposure to light during spring/summer to time estrus correctly. The proposed critical interval appears to be located near dusk in this model and is phase locked to the circadian pacemaker. The effect of the exogenous melatonin on the timing of the breeding season is similar to darkness when administered at dusk but is not equivalent to darkness at dawn. The timing of anestrus was not affected by any of the experimental treatments and may reflect a common response to an environmental influence.

Animals↗
PubMed1994

Sex determination of preimplantation embryos by human testis-determining-gene amplification.

When there is a risk of inherited disease, preimplantation diagnosis gives couples an opportunity to avoid having a child with the disease. Sex determination can be used to exclude the likelihood of a sex-linked disorder. Accuracy of the diagnosis is important. We have tested the reliability of sex determination based on the recognition of a testis-determining gene (SRY) sequence. DNA from the blood of 120 men and women and from 38 single lymphocytes was amplified by polymerase chain reaction (PCR) with the SRY and control (ZP3) gene primers. All results confirmed the correct sex of origin (100%). The test was then used to determine the sex of 21 single embryo cells biopsied from 21 (4-8 cell) human polyspermic embryos. 2 embryo cells recognised at biopsy to have degenerated produced negative results. The other 19 single embryo cells showed 100% PCR amplification. 11 (58%) of the embryos were judged to be "male" and 8 (42%) "female". The SRY and ZP3 gene primers selected are highly specific and give accurate results in sex determination and their use provides a new reliable method for routine preimplantation and general prenatal sex determination in man.

Animals↗
PubMed1994

Assessment of the separation of X- and Y-bearing sperm on albumin gradients using double-label fluorescence in situ hybridization.

OBJECTIVE: To determine the ratio of X- to Y-bearing human spermatozoa in fractions isolated from discontinuous albumin gradients. DESIGN: The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in albumin-separated fractions from the same samples. Two albumin methods were used: a two-layer method (experiment 1) and a three-layer method (experiment 2). X- and Y-bearing sperm were identified simultaneously using chromosome-specific DNA probes and fluorescence in situ hybridization. SETTING: Hospital-based university department. PARTICIPANTS: Healthy donors with normal semen characteristics. MAIN OUTCOME MEASURES: The proportions of haploid cells (X or Y) and cells with two sex chromosomes (XX, YY, or XY) were determined. RESULTS: Labeling efficiencies were > 96% in all samples. Control samples showed a 1:1 ratio of X- to Y-bearing sperm. Fractions isolated on albumin gradients showed a slight, but statistically significant enrichment of X-bearing sperm. This was evident with both albumin methods. CONCLUSIONS: Discontinuous albumin gradients do not enrich Y-bearing sperm as previously reported.

Aneuploidy↗
PubMed1994

Microassisted fertilization in assisted conception: indications and patient impact.

Microassisted fertilization is fast becoming established as a routine clinical technique offering the chance of pregnancy to couples where none existed previously. The exceptional results of SUZI and, more particularly, ICSI now raise the prospect of pregnancy with any sperm in the ejaculate and are bringing to treatment focus some azoospermic men and men without an antegrade ejaculate. The advance in clinical results from SUZI and ICSI over earlier zona drilling and zona breaching techniques appear to be importantly related to the better implantation potential of the embryos. The implications for the way conventional IVF is performed still need to be digested but may be very important and may change our attitude to ovarian stimulation and insemination regimens.

Embryo Transfer↗
PubMed1994

Local experience with zona drilling, zona cutting and sperm microinjection.

Clinical trials were performed between 1987 and 1992 on the use of zona drilling (ZD), zona cutting (ZC) and subzonal sperm microinjection (SZI) for the treatment of severe male infertility. ZD significantly improved the fertilization rate, but embryo morphology was poor and no pregnancies were achieved, so it was abandoned in favour of ZC. The fertilization rate was acceptable in the first trial of ZC but embryo morphology was still poor and no pregnancies were achieved, so a number of protocol changes were instigated. Shrinkage of oocytes in hypertonic sucrose prior to ZC markedly improved embryo quality, whereas transfer of embryos on Day 3 after oocyte retrieval enhanced the pregnancy rate. However, despite these improvements, the overall pregnancy rate in the third ZC trial was still low (16.6% per transfer), so a trial of SZI was initiated in 1992. The overall fertilization rate in 82 SZI cycles was 34.4% and, although the polyspermy rate was high, a clinical pregnancy rate of 30.8% per transfer and an implantation rate of 18.4% per embryo were achieved. These trials demonstrate that SZI is a successful treatment for severe male infertility; under the trial conditions, at least, it was superior to ZD or ZC.

Embryonic and Fetal Development↗
PubMed1994

The contribution of embryo cryopreservation to in-vitro fertilization/gamete intra-fallopian transfer: 8 years experience.

In this paper, the authors summarized their experience with embryo cryopreservation over an 8-year period. The results, therefore, reflect the long-term benefit of embryo cryopreservation to the overall in-vitro fertilization/gamete intra-Fallopian transfer (IVF/GIFT) programme and to the women who had embryos cryopreserved. The stable survival rate of thawed embryos and pregnancy rate, especially over the past 4 years, suggests that the results can reliably be used to evaluate the efficacy of the embryo cryopreservation programme. The ongoing pregnancy rate of frozen/thawed embryo transfer is 10.9%, comparable with the ongoing pregnancy rate of fresh IVF/embryo transfer in our unit over the same period. In addition to those factors known to affect the pregnancy rate in fresh IVF/GIFT cycles, such as age of the recipients and number of embryos transferred, the major factor affecting the efficacy of the cryopreservation programme is the number of oocytes retrieved in the initial stimulation cycle, and the number of embryos available for cryopreservation. The storage time of cryopreserved embryos will also have a significant effect on the realization of the total potential of embryo cryopreservation. Overall the contribution of cryopreservation to our IVF/GIFT programme is substantial, increasing pregnancy rate by 4%, while the greater net benefit, of course, is for the women who had embryos cryopreserved (pregnancy rate increased by 7%), especially for those who returned for frozen/thawed embryo transfer cycles (pregnancy rate increased by 11%).

Adult↗
PubMed1994

The obstetric outcome of singleton pregnancies following in-vitro fertilization/gamete intra-fallopian transfer.

The present study compares 465 singleton live deliveries from in-vitro fertilization/gamete intra-Fallopian transfer (IVF/GIFT) pregnancies with a large control population to evaluate the incidence of pre-term delivery and small for gestational age (SGA) or very small for gestation age (VSGA) babies resulting from IVF/GIFT pregnancies. Overall the incidence of SGA or VSGA from an IVF/GIFT pregnancy is higher than from the normal obstetric population (SGA odds ratio 1.76, 95% confidence interval (CI): 1.38-2.25 and VSGA odds ratio 1.61, 95% CI: 1.05-2.46) particularly among primiparous women (SGA odds ratio 1.99, 95% CI: 1.25-3.16 and VSGA odds ratio 1.97, 95% CI: 1.49-2.62). After stratifying by the cause of infertility, only women with unexplained infertility had a significantly higher proportion of SGA/VSGA babies. There was a significantly higher incidence of pre-term deliveries among the young primiparae (odds ratio 5.02, 95% CI: 3.09-8.13). Thus the excess risk of delivering a SGA/VSGA baby and pre-term delivery from an IVF/GIFT pregnancy seems to be largely confined to women with unexplained infertility and young primiparae.

Adult↗
PubMed1994

Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection.

In this report, we present the results of our first 100 consecutive cycles of intracytoplasmic sperm injection (ICSI). Overall, fertilization occurred in 98% of cycles and embryos were transferred in 94% (2.6 embryos per cycle). About 50% of patients had embryos frozen. The overall fertilization rate was 71%, of which 4% were abnormally fertilized (three pronuclei). A total of 30 clinical pregnancies were established (32% per transfer), resulting in 18 singleton, six twin and one triplet ongoing pregnancies. The implantation rate per embryo was 15%. There were no significant differences in the fertilization or pregnancy rates between patients who had only occasional motile spermatozoa in the ejaculate, semen that was too poor for routine in-vitro fertilization (IVF), or who had failed routine IVF and/or subzonal sperm injection (SUZI). A group of 18 patients were treated with both ICSI and routine IVF on their first cycle because of the high likelihood of failed fertilization due to poor sperm morphology < 20% normal). In this group, ICSI oocytes had a fertilization rate of 76% compared to only 15% for the routine IVF (control) oocytes, and six patients conceived after transfer of ICSI embryos (33%), indicating that ICSI can be used successfully on 50% of the oocytes if fertilization failure is expected. Similarly, patients who had failed to become pregnant with SUZI achieved excellent results after ICSI. There were no significant differences between ICSI and routine IVF in the proportions of grade 1, 2 or 3 embryos on day 3 post-oocyte recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Histopathological analysis of mice born following single cell embryo biopsy.

Single cell embryo biopsy is a useful but invasive technique for preimplantation diagnosis. Biopsy may be performed by physical (direct zona puncture) or chemical methods (zona drilling with acid solution). This study has analysed the safety of a physical method of embryo biopsy in the mouse. Six adult mice (male and female), three from biopsied embryos and three from a control group (non-biopsied) were subjected to histopathological analyses. Macroscopically, the anatomy and morphology of the internal organs in both groups were normal. Microscopic analyses of 15 major organs, which included the brain, heart, lung, liver, kidney, stomach, intestine, voluntary muscle, spleen, pancreas, adrenal, thymus, skin, testis (male) and ovary (female), in both groups were all normal. These results showed that careful single cell embryo biopsy by direct zona puncture performed at the 8-cell embryo stage had no adverse influence on the macroscopic and microscopic structure of the organs. The remaining pluripotential cells of biopsied embryos developed normal microstructure according to the hereditary messages. Ideally, the safety of embryo biopsy requires observation of three stages after embryo biopsy, namely embryonic and fetal development before birth, neonatal assessment and long-term monitoring after birth.

Animals↗