Study Population. The data in the study is based on the registry of CAs located at the University Hospital, City of Pleven, Bulgaria. This is a regional registry, not financially supported by the Ministry of Health, dealing with between 2,000 and 3,000 births per year. The registry is population based; University Hospital is the only hospital in the city and covers all deliveries of resident women within the boundaries of the Pleven region, a region with a decreasing number of inhabitants over the study period (from about 160,000 in 1988 to 140,500 in 2006). Invasive prenatal diagnosis is not available in the Pleven region. Over the period 1988-2006, a total of 47,622 births was surveyed, of which 47,229 (99.2%) were live births and 393 (0.8%) stillbirths.

Case Definition, Classification of Congenital Anomalies, and Ascertainment. The registry records all CAs identified in live births, stillbirths (official statistics of fetal deaths at ³28 weeks gestation until 1992 and at ³24 weeks gestation after 1992) including late miscarriages (fetal deaths of ³20 weeks gestation), and terminations of pregnancy following prenatal diagnosis of CA. Early fetal deaths before the 20th week of gestation were excluded. The case definition anddefinition of CA were taken according to EUROCAT recommendations [6].

The congenital anomalies inclusion list comprised mainly major CAs and some minor anomalies without medical or cosmetic significance, either malformations or deformations (for example, isolated talipes). Some minor anomalies (EUROCAT list of minor anomalies for exclusion) were not recorded [6]. Cases with “isolated” anomalies were classified according to the EUROCAT classification by organ system or body region (e.g., neural system, heart, digestive system). Cases with three or more CAs were classified as “multiple anomalies” and comprised: chromosomal abnormalities, single gene and sporadic malformation syndromes [7], non syndromal dysmorphologic conditions (sequence/association/complex), teratogenic syndromes (environmental abnormalities), multiple anomalies of unknown cause (malformation syndromes with unknown etiology and nosology). The anomalies assigned to the “multifactorial” category included the following isolated anomalies: neural tube defects (NTD); congenital heart disease (CHD); cleft palate and cleft lip; some cases of CAs of the nervous system (Dandy-Walker malformation, hydrocephaly due to congenital stenosis of the aqueduct of Sylvius); esophageal atresia/stenosis/tracheo-esophageal fistula; hypospadias; family cases of pes equinovarus and luxatio coxae congenita; congenital pyloric stenosis.

The criteria for inclusion of CHD cases were as follows: only cases of isolated cardiac defects in which the diagnosis was made by a cardiologist and confirmed by echocardiography or invasive criteria (catheter studies, surgery or pathological examination) were included. Functional or unspecified cardiac murmurs were excluded.

The ascertainment of the cases was mainly (90% of all cases) based on an active searching/screening forCAs conducted by neonatologists, gynecologists and clinical geneticists. Additional sources of information were hospital records (departments of obstetrics and gynecology, neonatology and pediatrics), records of pathological meetings, medical genetics records and stillbirths register. Whatever the source of ascertainment, at the end of every year all registered CA cases (particularly those with multiple anomalies) were carefully reviewed by a clinical geneticist to validate the diagnostic information, to check ID information and to record the final diagnosis. In all cases of CAs and death (in perinatal period or later), the data from pathological examinations were used for diagnostic validity. Maximum age at diagnosis was as follows: for most cases, up to the end of the first week after delivery; for selected malformations (CHD, urogenital), up to the end of the first year of life; in some cases (multiple anomalies) a follow-up was performed.

Along with the physical examination, additional methods were used for identification of the genetic causes of CAs: family history and pedigree; laboratory genetic testing (conventional cytogenetic analysis, biochemical and DNA analysis). In some cases (particularly those with multiple anomalies) dysmorphologic evaluation and search for syndromes, features and references was done by a clinical geneticist, using different sources: dysmorphology literature, “Smith’s recognizable pattern of human malformation” [8], OMIM [7], London Dysmorphology Databases. Environmental causes were identified via interview (a careful history of pregnancy), questionnaire, clinical assessment and laboratory confirmation.

Data Analysis and Statistics. All analyses were based on the number of cases in the anomaly category of interest, not the number of anomalies. The study was approved by the Ethics Committee of the Medical University, Pleven, Buglaria. Informed consent was obtained from the parents in all cases of CAs.

The calculation of the total prevalence and live birth prevalence was conducted using the EUROCAT recommendations [6]. Confidence intervals (95% CIs) were calculated for the total and live birth prevalence. The c2 test for trends was used to compare differences in prevalence over time. A p value of <0.05 was considered to be significant. Statistical analysis was performed using SPSS for Windows, v. 13.1.