This single-centre study included pregnant women who were being followed up by the Obstetrics Service Care Units of the XXI de Santiago de Compostela e Barbanza Foundation, who met the inclusion and exclusion criteria as described in the following paragraphs, and who consented to be included in the study and signed the corresponding informed consent form once they had been provided with the relevant study protocol information sheet and a discussion had been held to explain the information sheet.

The scope of the study included pregnant women over the age of 18 who gave birth between March 1, 2015, and March 1, 2017, and who underwent first-trimester combined screening for chromosomopathies in our hospital.

The inclusion criteria were pregnant women (over 18 years of age) undergoing screening for trisomy 21 and 18 during their first trimester by means of PAPP-A testing of blood at the Santiago de Compostela University Hospital Complex between 11 + 0 and 13 + 6 weeks of gestation, who consented to be included in the study, and who signed the informed consent form for participation in the study once they had been provided with a patient information sheet and this information had been adequately explained to the patient.

The exclusion criteria were multiple pregnancies; incomplete data; abnormal foetal karyotype; previous spontaneous abortion.

During this period, there were 5273 births at our centre. Of these, 4970 births were from singleton pregnancies; 294 were from twin pregnancies; 9 births were from triplet pregnancies. Of the 4970 women with singleton pregnancies, full study data could not be obtained for 734 women. Of the remaining 4236 women, 2794 of them consented to participate in the study by signing the informed consent form.

This population has been selected for the study in order to evaluate the study objectives.

For the data analysis, an epidemiological, observational, analytical, longitudinal, and prospective study was designed. Once permission had been obtained from the Santiago-Lugo Research Ethics Committee (“CEI-SL”), dated February 24, 2015, with registration reference 2015/049, epidemiological and clinical data were obtained by reviewing each patient’s medical records as stored in IANUS (a platform for systems integration and storing of medical information, “Indra”). Clinical data were compiled and coded into the dedicated study Data Collection Logbook (DCL), ensuring patient identities were protected. The collected data could only be accessed by the members of the research team and the health authorities, all of whom were under an obligation to ensure confidentiality.

The project was implemented in accordance with the Declaration of Helsinki (1964) of the World Medical Association as well as ratifications made by subsequent assemblies (in Tokyo, Venice, Hong Kong, and South Africa) on the ethical principles for medical research carried out in humans, as well as Order SCO/256/2007 of February 5, 2007, laying down the principles and detailed guidelines of Good Clinical Practice and the Convention on Human Rights and Biomedicine, agreed in Oviedo on April 4, 1997, as well as subsequent revisions to this order.

In order to analyse the data collected during this study, a descriptive analysis was first carried out of the variables obtained from the patient’s clinical records; this analysis considered the epidemiological and clinical characteristics of the study group.

Continuous variables were expressed as means along with the standard deviation, minimum, and maximum values. Qualitative variables were expressed as absolute frequencies and as percentages.

The chi-square test was applied to verify whether an association existed between qualitative variables, and analysis of variance was applied to compare continuous variables between groups. p values below 0.05 were considered significant.

In order to analyse the relationship between PAPP-A and foetal birth weight, as well as consider the gestational age.

First, different distributions were adjusted for these variables by applying GAMLSS models (generalised additive models for location scale and shape), making it possible to evaluate not only the mean but also other parameters relating to the distribution, such as variance, kurtosis, and asymmetry, as a function of explanatory variables. Goodness of fit for the models was evaluated using the Bayesian Information Criteria as well as graphically using Q-Q plots. Finally, the best-fit distributions were selected, as well as the variables influencing the weight of the newborn and relating to gestational age.

Subsequently, Z-score percentiles were compiled for the screening parameters as a function of the variables of newborn weight and gestational age, based on the GAMLSS regression models. In order to facilitate clinical use, different cut-off points were defined for the 5th, 10th, 25th, 50th, 75th, 90th, and 95th percentiles. In order to carry out the analysis, the “gamlss” package of the R program was used (R statistical software environment, version 4.0.1; R Foundation [11]) (Fig. 1).

Weight percentile according to gestational age and sex

From these percentiles, we established 4 groups:

VERY LOW PAPP-A: PAPP-A value below p2 for GA

LOW PAPP-A: PAPP-A value between p2 and p5 for GA

NORMAL PAPP-A: PAPP-A value between p5 and p95 for GA

HIGH PAPP-A: PAPP-A value above p95 for GA

Serum PAPP-A levels were converted to multiples-of-the-median values (MoM) by correcting for.gestational age, ethnicity, smoking status, maternal weight, and conception method, using an automated chemiluminescent immunometric assay (IMMULITE2000® Siemens).