Telomere length heterogeneity in placenta revealed with high-resolution telomere length analysis.

INTRODUCTION: Telomeres, are composed of tandem repeat sequences located at the ends of chromosomes and are required to maintain genomic stability. Telomeres can become shorter due to cell division and specific lifestyle factors. Critically shortened telomeres are linked to cellular dysfunction, senescence and aging. A number of studies have used low resolution techniques to assess telomere length in the placenta. In this study, we applied Single Telomere Length Analysis (STELA) which provides high-resolution chromosome specific telomere length profiles to ask whether we could obtain more detailed information on the length of individual telomeres in the placenta. METHODS: Term placentas (37-42 weeks) were collected from women delivering at University Hospital of Wales or Royal Gwent Hospital within 2 h of delivery. Multiple telomere-length distributions were determined using STELA. Intraplacental variation of telomere length was analysed (N = 5). Telomere length distributions were compared between labouring (N = 10) and non-labouring (N = 11) participants. Finally, telomere length was compared between female (N = 17) and male (N = 20) placenta. RESULTS: There were no significant influences of sampling site, mode of delivery or foetal sex on the telomere-length distributions obtained. The mean telomere length was 7.7 kb ranging from 5.0 kb to 11.7 kb across all samples (N = 42) and longer compared with other human tissues at birth. STELA also revealed considerable telomere length heterogeneity within samples. CONCLUSIONS: We have shown that STELA can be used to study telomere length homeostasis in the placenta regardless of sampling site, mode of delivery and foetal sex. Moreover, as each amplicon is derived from a single telomeric molecule, from a single cell, STELA can reveal the full detail of telomere-length distributions, including telomeres within the length ranges observed in senescent cells. STELA thus provides a new tool to interrogate the relationship between telomere length and pregnancy complications linked to placental dysfunction.

Placental expression of imprinted genes varies with sampling site and mode of delivery.

UNLABELLED: Imprinted genes, which are monoallelically expressed by virtue of an epigenetic process initiated in the germline, are known to play key roles in regulating fetal growth and placental development. Numerous studies are investigating the expression of these imprinted genes in the human placenta in relation to common complications of pregnancy such as fetal growth restriction and preeclampsia. This study aimed to determine whether placental sampling protocols or other factors such as fetal sex, gestational age and mode of delivery may influence the expression of imprinted genes predicted to regulate placental signalling. METHODS: Term placentas were collected from Caucasian women delivering at University Hospital of Wales or Royal Gwent Hospital within two hours of delivery. Expression of the imprinted genes PHLDA2, CDKN1C, PEG3 and PEG10 was assayed by quantitative real time PCR. Intraplacental gene expression was analysed (N = 5). Placental gene expression was compared between male (N = 11) and female (N = 11) infants, early term (N = 8) and late term (N = 10) deliveries and between labouring (N = 13) and non-labouring (N = 21) participants. RESULTS: The paternally expressed imprinted genes PEG3 and PEG10 were resilient to differences in sampling site, fetal sex, term gestational age and mode of delivery. The maternally expressed imprinted gene CDKN1C was elevated over 2-fold (p < 0.001) in placenta from labouring deliveries compared with elective caesarean sections. In addition, the maternally expressed imprinted gene PHLDA2 was elevated by 1.8 fold (p = 0.01) in samples taken at the distal edge of the placenta compared to the cord insertion site. CONCLUSION: These findings support the reinterpretation of existing data sets on these genes in relation to complications of pregnancy and further reinforce the importance of optimising and unifying placental collection protocols for future studies.

Rectovaginal endometriosis -- a frequently missed diagnosis.

To assess the proportion of women found to have rectovaginal endometriosis who underwent a previous laparoscopy with negative findings, a 5-year retrospective observational study was carried out at the University Hospital of Wales, Cardiff UK, from 2001 to 2005. A total of 61 cases with potential symptoms of rectovaginal endometriosis who underwent laparoscopy were identified. Rectovaginal endometriosis was identified in 16 of these cases. Previous laparoscopy was carried out in 33 of these 61 cases. In the group of women found to have rectovaginal endometriosis, 14 cases of rectovaginal endometriosis were not identified by pre-referral laparoscopy. This study supports the anecdotal idea that rectovaginal endometriosis is an often missed diagnosis at the time of laparoscopy. Diagnostic laparoscopy by generalist gynaecologists frequently fails to diagnose rectovaginal endometriosis. The routine use of rectal probes at laparoscopy is recommended to increase diagnostic accuracy.

Predicting the presence of rectovaginal endometriosis from the clinical history: a retrospective observational study.

Rectovaginal endometriosis is a severe variant of endometriosis. Common presenting symptoms for endometriosis include dysmenorrhoea, pelvic pain and dyspareunia. It is now recognised that there are other less traditional symptoms of endometriosis that are also relatively common. The aim of this study is to assess the relative strength of each of the potential symptoms of rectovaginal endometriosis and compare these with the laparoscopic and histological findings. In this retrospective, observational study the overall prevalence of rectovaginal endometriosis in the group was 31.4%. The presence of dyschesia gave a likelihood ratio of 1.27 (95% CI: 0.56 - 2.89) with a predictive prevalence of rectovaginal endometriosis of 37%. Apareunia and nausea or abdominal bloating were particularly strong markers for rectovaginal disease with a predictive prevalence of 87% and 89%, respectively. The classical symptoms often attributed to irritable bowel syndrome are also common in women with rectovaginal disease.

Self-administration of vaginal misoprostol after mifepristone for termination of pregnancy: patient acceptability.

This was a questionnaire survey involving women who self-administered vaginal misoprostol in the hospital setting following oral mifepristone for medical termination of pregnancy. The sample number was 89 with a median gestational age of 9 weeks; median dose of misoprostol used was 1600 mug and median induction abortion interval was 5.3 h. The success rate was 100% with the majority finding it easy to self-administer vaginal misoprostol and two-thirds did not mind doing this. Only one-third experienced adverse effects of the medication and 83% were satisfied with the procedure. Only one-third was willing to try it at home in future if necessary. Self-administration of vaginal misoprostol for termination of pregnancy in the hospital is safe and effective. Although women were comfortable in self administering the pessaries in the hospital, they do not appear to be keen to do it at home without any supervision. However, as this is the first study in the UK involving women expressing their views regarding this issue, added research in this area is required.

Self-administration of misoprostol for termination of pregnancy: safety and efficacy.

We conducted a retrospective study to determine the efficacy and safety of self-administration of vaginal misoprostol (following oral mifeprestone) for medical termination of pregnancy. This study revealed that self-administration was accepted by the majority of the patients (90%) and the success rate (98.4%) and duration of hospital stay was not altered significantly compared to our previous year's data, where women were administered vaginal misoprostol by the staff. Based on this study's results, we are of the opinion that this regimen not only demedicalises the problem but also decreases the workload for the medical staff.

Training in endoscopic surgery in gynaecology.

Over the last decade there has been an explosion of endoscopic surgical procedures that is now having profound effects on training in gynaecology. As trainees adopt these new procedures, they should obtain formal training supplemented by appropriate senior supervision which has been shown to reduce the risk of iatrogenic endoscopic complications.

Persistent ectopic pregnancy following conservative surgery for tubal pregnancy.

OBJECTIVE: To audit the incidence and management of persistent ectopic pregnancy following conservative tubal surgery performed at laparotomy and via the laparoscope. DESIGN: A retrospective analysis of the case records. SETTING: The Birmingham and Midland Hospital for Women. PARTICIPANTS: Two hundred and fourteen women who received surgical treatment for ectopic pregnancy between October 1991 and December 1994. RESULTS: Of the 85 women who underwent conservative tubal surgery, nine were diagnosed as having persistent ectopic pregnancy on the basis of hCG values. The incidence after laparoscopy was no higher than after laparotomy. A second surgical procedure was indicated in only four cases. CONCLUSIONS: Post-operative surveillance of serum hCG remains mandatory. Patients who remain symptom free may be managed conservatively The threshold for a second-look laparoscopy should be relatively high and be based on the presence of symptoms rather than changes in hCG values.

Fluorescent in-situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes.

Fluorescent in-situ hybridization (ISH) to interphase nuclei of human preimplantation embryos has been demonstrated with the X and Y chromosome-specific DNA probes, pBamX7 and pHY2.1, respectively. Assigning the sex on the basis of the number of hybridization signals in the majority of nuclei, the efficiencies with both probes to nuclei from male embryos were considerably higher than those previously reported for pHY2.1 detected by isotopic or conventional non-isotopic methods. Only approximately 15% of nuclei from male embryos failed to hybridize with these probes. With pBamX7, a high incidence (18%) of nuclei with two (or more) signals in embryos classified as males and four signals in a female embryo was observed. In some cases, the double spot nuclei were larger than those with single spots, providing evidence of tetraploidy. The feasibility of using fluorescent ISH for sexing biopsied embryos in couples at risk of X-linked disease and for the preimplantation diagnosis of chromosome abnormalities is discussed.

Cytogenetic analysis of unfertilized oocytes retrieved after treatment with the LHRH analogue, buserelin.

Chromosome analysis is reported on 155 oocytes from an in-vitro fertilization (IVF) programme in which the LHRH analogue, buserelin, was used in the superovulation regime. Seventy-one oocytes had the normal number of metaphase II chromosomes. Hyperhaploidy was apparent in four cases only; doubling this figure to include the reciprocally formed hypohaploid oocytes gave an aneuploidy rate due to non-disjunction of 10%. This is close to the figure for naturally occurring aneuploidy which may be calculated from data on recognized conceptions at a comparable maternal age. Taken together with the suggestion of a maternal age effect in our series these data suggest that follicular stimulation regimes which precede IVF do not necessarily add to the naturally high aneuploidy rate of the human species. Thirteen oocytes had failed to form the first polar body and the presence of diploid mitotic or sperm chromosomes provided evidence of fertilization and arrested development in 15. These figures for chromosomal anomalies following buserelin treatment are not significantly different from those obtained from comparable surveys following clomiphene citrate stimulation, providing no evidence that the improved pregnancy rate with buserelin is due to chromosomal factors.

Developmental profile of chromogranin, hormonal peptides, and 5-hydroxytryptamine in gastrointestinal endocrine cells.

In this immunocytochemical study, we have analyzed the developmental profile and phenotypic expression of the endocrine cell antigens chromogranin, 5-hydroxytryptamine, gastrin/cholecystokinin, cholecystokinin (9-20), somatostatin, somatostatin 28 (1-14), somatostatin cryptic peptide, glucagon, glucagonlike peptides 1 and 2, glicentin, peptide YY, glucose-dependent insulinotropic peptide, secretin, neurotensin, and substance P in human fetal stomach and intestine. All currently identifiable endocrine cell types were detected by 10 wk of gestation. Immunostaining for the endocrine cell marker chromogranin revealed abundant endocrine cells in the earliest specimens (8 wk of gestation) with a relatively higher frequency in both proximal duodenum and distal colon/rectum compared with other areas. Quantification of endocrine cells showed an increase with age that was roughly parallel to the growth of the gut as a whole. These studies show that the diversity of the endocrine component of the gut appears to be established by 10 wk of gestation and that gut activity is preceded by the development of a fully differentiated endocrine component, which may subserve or even initiate the onset of functional maturity.

Rapid sexing of human embryos by non-radioactive in situ hybridization: potential for preimplantation diagnosis of X-linked disorders.

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidin-linked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.

Fetal liver alanine: glyoxylate aminotransferase and the prenatal diagnosis of primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the hepatic peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT, EC 2.6.1.44) (Danpure and Jennings, FEBS Lett., 201, 20-24, 1986). The activity of AGT has been measured in fetal livers of gestational age 14-21 weeks. Activity increases up to 17 weeks and then levels off between 17 and 21 weeks. At this time, the mean AGT activity is about 30 per cent of the mean normal postnatal level. As in adult liver, the AGT enzyme activity and the AGT immunoreactive protein are peroxisomal. Prenatal diagnosis has been performed by measuring AGT enzyme activity and immunoreactive AGT protein on liver biopsies from two fetuses at risk for primary hyperoxaluria type 1. One was unaffected and one was affected.

Biopsy of human preimplantation embryos and sexing by DNA amplification.

A single cell was removed, through a hole made in the zona pellucida, from each of 30 human embryos at the 6-10 cell cleavage stage three days after in-vitro fertilisation. A normal proportion of the embryos (37%) developed to the blastocyst stage by day six in culture and 6 hatched from the zona. Each male embryo was sexed from the DNA by amplification of a repeated sequence specific for the Y chromosome. In 15 embryos with the normal two pronuclei the sex was determined also by in-situ hybridisation with a Y specific probe or fluorescent chromosome staining to detect metaphase Y chromosomes; the results of Y specific amplification were confirmed. This approach may be valuable for couples at risk of transmitting X-linked disease.