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Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.

We evaluated the efficacy of unilateral versus bilateral laparoscopic ovarian diathermy in infertile women with clomiphene citrate-resistant polycystic ovary syndrome. Recall follow-up 18 to 48 months after laparoscopic ovarian diathermy showed that unilateral ovarian diathermy was as effective and long lasting as bilateral ovarian diathermy in the resumption of menstruation and pregnancy rates.

Summary Pregnancy-associated plasma protein A (PAPP-A) was purified from normal term maternal serum in this study using a three step chromatographic procedure with molecular sieve chromatography, heparin-Sepharose affinity chromatography and DEAE-Sephacel chromatography. Thirty milliliter of the maternal serum samples were applied to the column for sieve chromatography and eluted according to molecular weight. PAPP-A was detected at the high molecular weight fraction area via radioimmunoassay for PAPP-A, and 91% of the total amount of PAPP-A in the maternal samples was recovered. These PAPP-A containing fractions were applied to heparin-Sepharose column and eluted in a stepwise increase of NaCl 0.15, 0.30 and 0.60 M in 0.15 M Tris-HCl buffer, pH 7.8. Ninety-five percent of PAPP-A in the applied samples was recovered after 0.6 M NaCl. The pooled fractions, which were containing PAPP-A after heparin-Sepharose affinity chromatography, were then applied on a DEAE-Sephacel Chromatography and eluted in a stepwise increase of NaCl 0.15, 0.30 and 0.45 M in 0.01 M acetate buffer, pH 5.5. Ninety-two percent of PAPP-A in applied samples was recovered after 0.30 M NaCl. Finally PAPP-A containing fractions were concentrated 10 times and 3.8 IU (1.7 mg) of PAPP-A was isolated. The purification schedule removed approximately 99% of total protein in the maternal serum while 74% of PAPP-A was recovered. The purification factor (fold), which was calculated as the increase in specific activity (mlU:PAPP-A/mg:protein) in comparison with the starting value in maternal serum, was 526. And the purity(mg:PAPP-A/mg:protein) of the final product was 68.4%. Analysis of the final purified material using SDS-PAGE demonstrated a single band of 200 kDa, and western blot analysis showed that purified main protein was PAPP-A. The immunological identity between purified PAPP-A in this study and PAPP-A donated by Teisner et al. was recognized by crossed immunoelectrophoresis and tandem crossed immunoelectrophoresis. The PAPP-A purified in this study by the three step chromatographic procedure will hopefully be utilized for the development of a sensitive and convenient assay for PAPP-A and its standard material, furthermore for the basic study of clarifying the biological function of PAPP-A in human body.

Polymorphisms of the size of heterochromatic centromeric regions of chromosomes have been well documented in the human. They appear to have no phenotypic effects in the carriers. However, they appear to be over-represented in infertile couples and those with repeated miscarriages, and there is now growing evidence that they are involved in meiotic pairing, spindle fibre attachment and chromosome movement. Here an analysis of inheritance is reported for a couple presenting with repeated IVF failure in which several embryos were identified as carriers of a polymorphism of the centromeric region of chromosome 16 (16qh-) following aneuploidy screening by sequential fluorescence in-situ hybridization (FISH), using probes for chromosomes 13, 16, 18, 21, 22, X and Y. Detailed cytogenetic analysis by high-resolution banding and FISH of both parents and grandparents established that the polymorphism was familial and inherited from the maternal grandfather. Furthermore, complete analysis of all embryonic nuclei from carrier embryos and others rejected for transfer because of aneuploidy revealed no abnormalities in the segregation pattern of chromosome 16.

Objective To examine differences in specific protein expression from the surface of the human endometrium with respect to eventual pregnancy in infertile women. Design Laboratory study. Setting University hospital. Patient(s) Thirty-one women presenting for investigation into infertility at an assisted reproductive unit. Intervention(s) Endometrial flushings were collected during the proliferative phase of the menstrual cycle and subjected to electrophoretic separation on the basis of isoelectric point and molecular weight. Computerized analysis of the resulting spots was performed, and the proteins were identified using tandem mass spectrometry. Main Outcome Measure(s) The expression of individual isoforms of leucine-rich α2-glycoprotein (LRG) was compared in nonpregnant patients (n = 25), those who became pregnant as a result of treatment (n = 3), and those who had treatment-independent pregnancies (n = 3). Result(s) A statistically significant difference was found in expression of two LRG isoforms, which were higher in the women who subsequently became pregnant independent of treatment. Conclusion(s) Several indirect lines of evidence suggest a role for LRG in implantation/decidualization. [1] LRG is implicated in transforming growth factor beta signal transduction. [2] Similar sequences have been identified in murine uterine tissues. [3] LRG may be involved in the infiltration of decidua by uterine natural killer cells, given that the murine homolog of LRG supports lymphocyte infiltration into secondary lymphoid tissues. [4] Human uterine natural killer cells differentiate into granular forms during early pregnancy, and LRG is known to support neutrophil granulocytic differentiation in humans.

Objective: To determine the activity of superoxide dismutase (SOD) and the total protein concentration in human preovulatory ovarian follicular fluid (FF) in relation to corresponding serum levels and the fertilization capacity of oocytes. Design: Prospective, observational study. Setting: Academic-based center for reproductive medicine. Patient(s): Twenty-eight female partners of infertile couples, 13 of whom were smokers, undergoing controlled ovarian hyperstimulation for IVF. Intervention(s): Blood and follicular fluid samples were collected 34–36 hours after hCG administration. Main Outcome Measure(s): Levels of SOD activity and total protein concentrations. Result(s): Superoxide dismutase activity was present in all the FF studied and mean levels were statistically significantly higher than in serum. Total protein concentrations in serum were statistically significantly correlated with corresponding concentrations in FF. There was no difference in SOD activity between smokers and nonsmokers. Total protein concentrations in FF were marginally and statistically significantly lower in nonsmokers. Follicular fluid from patients whose oocytes did not become fertilized had a statistically significantly higher level of SOD activity than that from patients whose oocytes did become fertilized. Conclusion(s): Superoxide dismutase activity is present in FF and is higher than in serum. The degree of SOD activity is variable and seems to be inversely related to the fertilization of oocytes.

The technique of doppler ultrasound has revolutionized our understanding of the changes in uteroplacental blood flow impedance that occur in human pregnancy. Several reports in the last few years have examined the use of doppler ultrasound to detect blood velocity changes in maternal (Campbell et al., 1983), umbilical (Trudinger et al., 1986), and fetal vessels (Griffin et al., 1984). Both pulsed (Erskine and Ritchie, 1985) and continuous wave doppler (Trudinger et al., 1985) have been used to study the flow velocity waveforms in normal and abnormal pregnancies. These studies have concentrated on the latter half of pregnancy, in an attempt to assess the potential clinical value of this non invasive test for the screening and subsequent management of high risk pregnancies.