Variable imprinting of the MEST gene in human preimplantation embryos.

There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.

Quantitative analysis of DNA methylation of imprinted genes in single human blastocysts by pyrosequencing.

We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.

Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro.

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 +/- 0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro.

Comparison of effects of zona drilling by non-contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics.

Use of a non-contact infrared laser (IRL) or acid Tyrode's for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrode's: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrode's biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrode's drilling can also cause damage, and tended to retard blastocyst development.

The predictive value of the "Hull & Rutherford" classification for tubal damage.

OBJECTIVE: This study explores the predictive value for live birth following tubal reconstructive surgery of the "Hull & Rutherford" (H&R) classification system. DESIGN: Retrospective cohort study. SETTING: Tertiary infertility referral service, University of Bristol. POPULATION: Infertile women younger than 40 years with tubal damage undergoing tubal surgery. METHODS: Women (n= 192) were grouped according to three severity grades of disease based on the H&R classification. Essentially, the main features of grade I tubal damage were filmy adhesions, whereas grades II and III referred to unilateral severe damage and bilateral severe damage, respectively. Standard surgical techniques were employed. Pregnancy and live birth rates were calculated and compared using time-specific univariate Kaplan-Maier curves and multivariate Cox's regression analysis. MAIN OUTCOME MEASURES: Pregnancy, ectopic and live birth within three years of surgery. RESULTS: A significant trend towards higher ectopic pregnancy rates (P < 0.001) with increasing severity of tubal damage was noted, but not miscarriage rates. Univariate analysis revealed significant differences in the live birth rates of 69%, 48% and 9% for grades I, II and III, respectively. Multivariate analysis (controlling for age, duration of and primary infertility) confirmed these differences to be significant with risk ratios of 13.7 (95% CI: 4.49-41.9) and 6.54 (95% CI: 2.48-17.24) for grades I and II disease, respectively, compared with grade III disease, used as the reference. CONCLUSIONS: The H&R classification is a simple classification system that is able to distinguish women into three distinct groups giving a favourable, fair and poor prognosis for live birth following tubal surgery.

Karyotyping of human metaphase II oocytes by multifluor fluorescence in situ hybridization.

OBJECTIVE: To quantify aneuploidy in inseminated, injected, and noninjected oocytes from infertility patients using Multifluor fluorescence in situ hybridization (M-FISH). DESIGN: Prospective study. SETTING: Reproductive biology group, academic unit of pediatrics, obstetrics, and gynecology. PATIENT(S): Forty-eight patients undergoing ovarian stimulation and either intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): M-FISH karyotyping of 67 metaphase II oocytes, including noninjected in vitro matured oocytes, and injected inseminated-failed fertilized oocytes. RESULT(S): Thirty-nine percent of oocytes were aneuploid, with nondisjunction of chromosomes in 34% of oocytes and predivision of chromatids in 10%. There was no difference in aneuploidy rates between ICSI noninjected in vitro matured oocytes and injected, failed fertilized oocytes. Chromosomes most frequently involved in aneuploidy were 15, 18, 19, 22, and X. In seven injected ICSI MII oocytes, the prematurely condensed sperm chromatin was karyotyped by M-FISH. CONCLUSION(S): M-FISH was used to diagnose aneuploidy at maternal meiosis I in 39% of oocytes, and M-FISH karyotyping of sperm was demonstrated.

Rates of bacterial vaginosis in women undergoing in vitro fertilisation for different types of infertility.

OBJECTIVE: To assess whether the rate of bacterial vaginosis (BV) is higher in women with tubal factor infertility compared with those with other causes of infertility. DESIGN: Cross-sectional study. SETTING: Assisted conception unit of a teaching hospital in Leeds. POPULATION: Consecutive women undergoing in vitro fertilisation. METHODS: Women undergoing in vitro fertilisation (IVF) had a vaginal smear taken at the time of their egg collection. The smear was Gram-stained and graded as normal, intermediate or BV. MAIN OUTCOME MEASURES: The presence of bacterial vaginosis and the causes of infertility. RESULTS: A total of 749 women were included. The vaginal smears were normal in 63.6%, intermediate in 12.1%, and BV in 24.3%. The rates of BV in women with different types of infertility were 36.4% in tubal factor, 15.6% in male factor, 33.3% in anovulation, 12.5% in endometriosis and 18.9% in unexplained infertility. After controlling for the effects of age and smoking using a multivariate logistic regression model, women with tubal infertility were significantly more likely to have BV than women with endometriosis OR 3.63 (95% CI 1.52-8.67); male factor OR 2.98 (95% CI 1.80-4.90); and unexplained infertility OR 2.20 (95% CI 1.35-3.59). The adjusted figures for the increase of BV in women with anovulation were: endometriosis OR 3.77 (95% CI 1.28-11.08); male factor OR 3.09 (95% CI 1.37-6.96); and unexplained infertility OR 2.29 (95% CI 1.02-5.12). CONCLUSIONS: Women with tubal infertility were three times more likely to have BV than women with endometriosis, male factor or unexplained infertility. These findings support the association between BV, pelvic inflammatory disease (PID) and tubal damage but do not help distinguish between cause and effect. Women with anovulation were also three times more likely to have BV than women with endometriosis or male factor infertility, supporting suggestions of hormonal influence on vaginal flora.

Non-invasive amino acid turnover predicts human embryo developmental capacity.

BACKGROUND: IVF is limited by low success rates and a confounding high multiple birth rate contributing to prematurity, increased neonatal mortality and child handicap. These problems could be overcome if single embryos of known developmental competence could be selected for transfer on day 2/3 of development, but current methods, which rely on morphological appearance, are poor predictors of viability. METHODS: We have measured non-invasively the depletion/appearance (i.e. turnover) of a physiological mixture of 18 amino acids by single human embryos during in-vitro culture using high performance liquid chromatography. RESULTS: From the time of transfer (day 2/3), embryos with future competence to develop to the blastocyst stage (day 5/6) exhibit amino acid flux patterns distinct from those of embryos with similar morphological appearance which arrest. Significantly, the profiles of Ala, Arg, Gln, Met and Asn flux predict blastocyst potentiality at >95%. The amino acid most consistently depleted throughout development by those embryos which form blastocysts was leucine. Of the amino acids which were produced, the most striking was alanine, which appeared in increasing amounts throughout development. CONCLUSIONS: Non-invasive amino acid profiling has the potential to select developmentally competent single embryos for transfer, thereby increasing the success rate and eliminating multiple births in IVF.

A randomized controlled trial of tubal flushing with lipiodol for unexplained infertility.

OBJECTIVE: To test the hypothesis that, in couples with unexplained infertility, tubal flushing with an oil-soluble media (lipiodol) would increase the pregnancy rate within 6 months compared with expectant management. DESIGN: A prospective, randomized, controlled study in which couples were allocated to either a single treatment with lipiodol or no further action. SETTING: Two tertiary referral centers for assisted reproduction. PATIENT(S): Couples with a diagnosis of primary or secondary unexplained infertility based on a normal semen analysis according to World Health Organization criteria, patent fallopian tubes at hysterosalpingography or laparoscopy, and ovulatory menstrual cycles based on midluteal phase progesterone levels or ultrasonic follicle tracking. INTERVENTION(S): In those patients randomized to lipiodol, a single treatment was performed. MAIN OUTCOME MEASURE(S): Biochemical (i.e., positive pregnancy test) and clinical (i.e., fetal heart on ultrasound scan) pregnancy rates. RESULT(S): Seventeen couples were randomized to lipiodol and 17 to expectant treatment. The higher pregnancy rate after lipiodol was statistically significant. There were no complications after lipiodol treatment. CONCLUSION(S): There was a statistically significantly higher pregnancy rate in couples with unexplained infertility randomized to a single tubal flush with lipiodol compared with no treatment.

Value of thyroid function tests in routine screening of women investigated for infertility.

Women presenting with infertility traditionally have been screened for thyroid function, yet recent guidelines have indicated that this is unnecessary in asymptomatic patients. A retrospective analysis of the results of routine thyroid function screening showed that occult thyroid dysfunction is common: 5.1% of the population studied had abnormal thyroid function tests. Therefore, it is recommended that assessment of thyroid function in women attending an infertility clinic should be continued.

Plasma prolactin measurement is not indicated in women in the routine investigation of uncomplicated infertility.

Measurement of plasma prolactin frequently forms part of the routine investigation of infertile couples. In 315 consecutive women attending an infertility clinic in a university teaching hospital, all of those with increased plasma prolactin concentrations would have been detected from their symptoms. All women with prolactin concentrations in the range 500--800 miu l(minus sign1) appeared to be ovulating. These data support previous proposals in guidelines published by both the European Society for Human Reproduction and Embryology (ESHRE, 1996) and the Royal College of Obstetricians and Gynaecologists (RCOG, 1998) that there is no benefit in measuring plasma prolactin in the investigation of infertility in women who are menstruating normally and have no other clinical indications.