Michael Bonifacio

Prospective evaluation of a first trimester screening program for Down syndrome and other chromosomal abnormalities using maternal age, nuchal translucency and biochemistry in an Australian population

Background:  A combination of maternal age and ultrasound assessment of the nuchal translucency (NT) has been used in the first trimester to screen for chromosomal abnormality. In the United Kingdom, the addition of NT screening was shown to be beneficial.

Production of pregnancy-associated plasma protein-A (PAPP-A) by cultured tumour granulosa cells.

Ten ovarian and 2 cervical tumour cell lines were analysed for the production of pregnancy-associated proteins. Pregnancy-associated plasma protein-A (PAPP-A) was detected by radioimmunoassay in culture media of 2 out of 4 (50%) tumour granulosa cell lines (mean = 104 microIU/10(5) cells/24 h) but not in any ovarian (n = 6) or cervical (n = 2) tumour cell lines. By contrast, human chorionic gonadotrophin (hCG), pregnancy specific beta 1-glycoprotein and alpha-fetoprotein (AFP) were not detected in any of the PAPP-A positive media. Only two cell lines produced hCG (58.5 and 25.5 mIU/10(5) cells/24 h). No AFP was produced by any of these 12 cell lines, whereas placental protein 5 was positive in 7. None of these proteins were detected in the culture media of 4 cell lines. In vitro derived PAPP-A was immunologically indistinguishable from either pregnancy or ovarian follicular PAPP-A. All PAPP-A species interacted reversibly with immobilised heparin and were determined by molecular sieve chromatography to have an apparent molecular weight of 820,000 daltons. Cultured tumour granulosa cells specifically synthesised and secreted a large protein which was immunologically and physicochemically indistinguishable from in vivo (pregnancy and ovarian follicular) derived PAPP-A.

Pregnancy Associated Plasma Protein-A in Human Seminal Plasma

Abstract Human seminal plasma contains a large glycoprotein which is physicochemically and immunologically identical to pregnancy associated plasma protein-A (PAPP-A). Like pregnancy derived PAPP-A, seminal PAPP-A binds reversibly to heparin and to zinc chelate affinity matrices. Seminal plasma PAPP-A concentrations demonstrated no significant correlation with sperm morphology, motility or number, ejaculate volume or seminal plasma zinc levels. Purified PAPP-A has no influence on the mitogenic effect of phytohaemagglutinin on peripheral lymphocytes, but rather specifically inhibits leucocyte elastase. It is suggested that PAPP-A may be involved in protecting the sperm, which are deposited within the female reproductive tract, against the ensuing localised leucocytosis.

Comment on “MeDIP real‐time qPCR of maternal peripheral blood reliably identifies trisomy 21”

We read with interest the recent publication in your journal by Tsaliki et al. titled ‘MeDIP real-time qPCR of maternal peripheral blood reliably identifies trisomy 21’. In a correspondence published in September issue of Nature Medicine, Tong et al. raised technical concerns about this methodology. Experimentally, Tong et al. failed to reproduce the results using 15 normal and 3 trisomy cases. We wish to communicate our preliminary results with colleagues working in this field. We used cell-free circulating DNA extracted from 1.5 to 2mL ofmaternal plasma fromwomen during the first trimester of pregnancy as the starting material. Following the enrichment of methylated DNA using a methodology to be published later by our group, real time Q-PCR was performed using eight molecular markers, EP4–8 and EP10–12, as shown in experiments by Papageorgious and Tong et al. Thirty normal samples and two trisomy 21 cases were examined. We calculated methylation ratios that showed similar ranges for each marker to those presented by Papageorgious et al. We tentatively used the equation published by Papageorgious to calculateD values.Wewere able to correctly identify both trisomy cases and 29 normal cases, although one normal case had a D value of 0.65. We will be able to test the data more accurately once our own equation is developed. We believe that statistically, the equation obtained from work by Papageorgious et al. may only represent the data obtained from their experimental conditions and reagents used. The coefficients in the equation are not necessarily accurate for the data collected in other laboratories unless these coefficients are determined under a standardized system and from a large enough sample size that is statistically able to represent general population of women with first trimester pregnancy. Therefore, we believe that it would be prudent to be cautious when using the equation to explain another set of data. We are not surprised to see that D values varied for the replicates in the reproducibility experiments of Tong et al. Once the coefficients are determined on the basis of a particular set of data, the methylation ratio is an independent variable that ultimately determines theD value. In this context, themethylation ratio is defined as normalized delta Ct for an individual sample against the median normalized delta Ct of a group of known normal samples. In a quantitative polymerase chain reaction (PCR), variation in Ct values amongmultiple replicates of a sample is generally acceptedwithin the half cycle. This allowable, less than half-cycle, difference can produce a range of variation in the D value for any sample given that eight markers contribute to the formation of the D value in this equation. Existence of this variation is logical as long as it does not change the nature of the tested sample that, in this scenario, is trisomy 21 or euploidy. We also tested the reproducibility of methylation enrichment. Within a run, five technical replicates of a single sample were examined. Ct values for five enriched samples were all centered on a value with less than half-cycle difference. With runs on different days, we were able to reach 99–100% consistency for the readings of Ct values when comparing twomeans from each run. In terms of efficiency of enrichment, reproducibility between runs for the eight markers examined is as follows: EP4, 99.30%; EP5, 93.65%; EP6, 98.90%; EP7, 91.14; EP8, 88.89%, EP10, 100%; EP11, 86.96; and EP12, 88.52%. Furthermore, we examined the reproducibility of methylation enrichment using representative markers EP4 and EP5 within a period of 1month, and we are able to reproduce five weekly results with variations of less than half a PCR cycle. Our preliminary results suggest that it is worthwhile to further validate this methodology in a large sample cohort.

The Development of Action Lines in an Australian Maternal Serum Alphafetoprotein Screening Service for Neural Tube Defects

EDITORIAL COMMENT: In this careful study, 6 of 7 mothers carrying fetuses with neural tube defects were found to have raised serum alphafetoprotein levels (more than 2.5 multiples of the median) in the second trimester, out of 3,182 pregnancies tested. In the other 65 patients with elevated AFP levels there were 4 fetuses with major malformations and many with other important clinical complications. All of these fetal malformations were identifiable by ultrasonography. This paper does not consider the association between low AFP levels and Down syndrome, although this is another reason why screening maternal serum AFP levels could be advocated, since it can offer a detection rate of 28% by selecting less than 3% of unaffected pregnancies for amniocentesis, a better yield than using maternal age alone (Cuckle HS, Wald NJ, Thompson SG. Estimating a womans risk of having a pregnancy associated with Downs syndrome using her age and serum alphafetoprotein level. Br J Obstet Gynaecol 1987; 94: 387 ‐ 402 — this review concluded that screening for Down syndrome using both maternal age and serum AFP is more efficient than using age alone and that, where antenatal serum AFP screening programmes are in progress, there is no justification for not using both).

Placental Protein 5 in Human Ovarian Follicular Fluid

By sensitive and specific radioimmunoassays PAPP-A and PP5 were detected in follicular aspirates obtained from women undergoing ovarian hyperstimulation for oocyte harvest prior to in vitro fertilization and embryo transfer. Follicular and pregnancy-derived PAPP-A were immunologically and physicochemically indistinguishable. Similarly, pregnancy- and nonpregnancy-derived PP5 were immunologically indistinguishable. However, in addition to the 18- and 36-K species, a larger species having a molecular size greater than 140K was found in the follicular fluid. Mean follicular PAPP-A and PP5 concentrations were 727 mIU/L and 1376 mAU/L, respectively, with no significant correlation between follicular PAPP-A, PP5, and steroid concentrations. There was, however, a significant but negative relationship with follicular volume. Preliminary in vitro studies indicated that both proteins were synthesized by granulosa cells in preparation for follicular rupture. Follicular PP5, like antithrombin III, interacted reversibly with heparin and thrombin affinity matrices, suggesting a potential biological role as a follicular anticoagulant, whereas PAPP-A, a specific and potent inhibitor of leukocyte elastase, contributes to the maintenance of proteolytic homeostasis and the protection of spermatozoa and embryo against proteolytic attack originating from the maternal leukocytes.

Rare autosomal trisomies: Important and not so rare

Noninvasive prenatal testing (NIPT) can assess chromosomes other than 13, 18, 21, X and Y. These rare autosomal trisomies (RATs) can adversely affect pregnancy outcome.