STUDY QUESTION: Is the fetal fraction (FF) of circulating cell-free DNA (cfDNA) affected in pregnancies following ART treatment with either fresh or frozen embryo transfer (ET) compared with natural conception? SUMMARY ANSWER: This study shows a significant reduction in the FF in ART patients compared with naturally conceived pregnancies, which seems to be more pronounced after fresh ET compared with frozen ET. WHAT IS KNOWN ALREADY: Non-invasive prenatal testing (NIPT) is based on cfDNA in maternal blood, of which about 10% is of placental origin and thus represents the fetal karyotype. Validation studies have demonstrated a high sensitivity, specificity and positive predictive value of NIPT for the detection of fetal trisomy 21, 18 and 13. Nevertheless, the FF of cfDNA is an important factor for NIPT test accuracy. Several studies have found a reduction in FF for pregnancies following ART in comparison with natural conception. However, knowledge on how the FF is affected in ART pregnancies after fresh ET compared with frozen ET is very limited. STUDY DESIGN, SIZE, DURATION: The study was designed as a case-control study. A total of 54 women with an ongoing pregnancy following ART treatment were included. After exclusion for different reasons, statistical analyses were based on 23 NIPT samples from pregnant women treated with fresh ET and 26 NIPT samples from pregnant women treated with frozen-thawed ET in a modified natural cycle. Women were included between February 2018 and November 2018. The results were compared with a control group of 238 naturally conceived pregnancies with a high-risk result from the combined first trimester screening (cFTS). PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included women from the Fertility Clinics at Copenhagen University Hospital Hvidovre and Copenhagen University Hospital Rigshospitalet. Blood samples for NIPT analysis were drawn between 11 + 0 and 14 + 2 weeks of gestation and were all analyzed at the NIPT Center at Copenhagen University Hospital Hvidovre. The NIPT-test was performed by massive-parallel whole-genome sequencing. The FF was determined using the SeqFF algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: We found a reduction in FF in ART patients compared with naturally conceived pregnancies, and the reduction was more pronounced for ART pregnancies after fresh ET (mean FF = 0.049) compared with frozen ET (mean FF = 0.063) (multivariate analysis adjusted for maternal BMI, P = 0.02). Another multivariate analysis, adjusted for BMI and multiples of median (MoM) values for pregnancy-associated plasma protein-A (PAPP-A), demonstrated a significantly reduced FF for ART pregnancies (mean FF = 0.056) compared with naturally conceived pregnancies (mean FF = 0.072) (P < 0.0001). We found that FF was significantly reduced with increasing maternal BMI (P < 0.0001) and with decreasing MoM values of PAPP-A (P = 0.003). LIMITATIONS, REASONS FOR CAUTION: A limitation of our study design was the relatively small sample size. Another limitation was that the control group was not matched with the ART-treated women. The majority of the women from the control group had a high risk from cFTS, thereby their biochemical markers were diverging. However, the biochemical markers for the ART-treated women with fresh or frozen ET were not divergent within the subgroups. WIDER IMPLICATIONS OF THE FINDINGS: Concurrent with other studies demonstrating a reduced FF for singleton pregnancies after ART treatment compared with naturally conceived pregnancies, we found a reduction in FF between the two groups. This is one of the first studies to examine FF in ART pregnancies after fresh ET compared with frozen ET, hence the existing knowledge is limited. We find that FF is even more reduced in pregnancies following fresh ET compared with frozen ET, which might possibly reflect the predisposition of being small for gestational age after fresh ET compared with natural cycle frozen ET. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til almene Formaal (the A.P. Møller Foundation for General Purposes). All authors declare no conflicts of interest.NA.