Limited translational genomic research data have been reported on the application of exome sequencing and parallel gene testing for preconception carrier screening (PCS). Here, we present individual-level data from a large PCS program in which exome sequencing was routinely performed on either gamete donors (5,845) or infertile patients (8,280) undergoing in vitro fertilization (IVF) treatment without any known family history of inheritable genetic conditions. Individual-level data on pathogenic variants were used to define conditions for PCS based on criteria for severity, penetrance, inheritance pattern, and age of onset. Fetal risk was defined based on actual carrier frequency data accounting for the specific inheritance pattern (fetal disease risk, FDR). In addition, large-scale application of exome sequencing for PCS allowed a deep investigation of the incidence of medically actionable secondary findings in this population. Exome sequencing achieved remarkable clinical sensitivity for reproductive risk of highly penetrant childhood-onset disorders (1/337 conceptions) through analysis of 114 selected gene-condition pairs. A significant contribution to fetal disease risk was observed for rare (carrier rate < 1:100) and X-linked conditions (16.7% and 41.2% of total FDR, respectively). Subgroup analysis of 776 IVF couples identified 37 at increased reproductive risk (4.8%; 95% CI = 3.4–6.5). Further, two additional couples had increased risk for very rare conditions when both members of a parental pair were treated as a unit and the search was extended to the entire exome. About 2.3% of participants showed at least one pathogenic variant for genes included in the updated American College of Medical Genetics and Genomics v2.0 list of secondary findings. Gamete donors and IVF couples showed similar carrier burden for both carrier screening and secondary findings, indicating no causal relationship to fertility. These translational research data will facilitate development of more effective PCS strategies that maximize clinical sensitivity with minimal counterproductive effects. We provide here crucial information for optimizing the gene-panel design for preconception carrier screening based on the analysis of a large exome sequencing dataset from infertile individuals and gamete donors. Sequencing the entire coding portion of the human genome combined with separate analysis for few relevant genes offers the possibility to detect most of the pathogenetic variants associated with recessive Mendelian diseases and to develop preconception screening strategies that maximise clinical sensitivity with minimal counterproductive effects. Using a large dataset of individual-level exome sequencing data, we have defined gene specific and aggregate fetal risk detectable for conditions selected on discrete criteria of severity, penetrance, inheritance pattern, and age of onset. About 1 out of 300 affected pregnancies can be detected based on a gene-panel of 114 conditions and ~5% of the couples analysed showed an increased risk that warrant consideration from a reproductive viewpoint. These results suggest the use of exome sequencing and parallel gene testing is clinically effective and feasible for preconception carrier screening after proper validation and translational research has been carried out. However, further studies are necessary to define the best framework for clinical implementation and the actual detection rate of at risk couples.