Binucleated embryos at the two-cell stage show higher blastocyst formation rates and higher pregnancy and live birth rates compared to non-multinucleated embryos.

STUDY QUESTION: How does nucleus status at the two-cell stage predict blastocysts formation and clinical outcome after single blastocyst transfer? SUMMARY ANSWER: Binucleated embryos at the two-cell stage (2BI) show higher rates of good quality blastocyst formation, pregnancy and live birth compared to those with one nucleus in each blastomere (2MONO), whereas true multinucleated embryos at the two-cell stage (2MULTI) show lower rates of good quality blastocyst formation and pregnancy compared to 2MONO embryos. WHAT IS KNOWN ALREADY: The introduction of time-lapse culture has made it possible to study nucleus status at the two-cell stage more consistently and it shows that multinucleation at the two-cell stage (2MN) is a common event. The effect of 2MN is still unclear. High numbers of 2MN with the potential to develop to blastocysts that become clinical pregnancies and result in birth of healthy babies with no impaired perinatal outcome have been reported. However, some studies have found 2MN to be associated with impaired implantation and live birth. Furthermore, knowledge on how the different subgroups of multinucleation affects the IVF outcome is limited. STUDY DESIGN SIZE DURATION: A non-interventional retrospective study was performed in a public fertility clinic. Blastocyst formation data from 223 women attending their first IVF cycle between May 2016 and December 2018, and clinical outcome data from 1314 single blastocyst transfers between May 2014 and December 2018 were used for the study. Fresh and frozen-thawed embryo transfers were included. PARTICIPANTS/MATERIALS SETTING METHODS: Embryos were cultured until the blastocyst stage in a time-lapse incubator and nucleus status at the two-cell stage, the Gardner score and other morphokinetic parameters were annotated. We compared blastocyst development and clinical outcome, including positive hCG, ongoing pregnancy and live birth, of embryos with 2BI and/or 2MULTI blastomeres to 2MONO embryos. MAIN RESULTS AND THE ROLE OF CHANCE: Embryos with 2BI in one blastomere (2BI1) were twice as likely to develop to good quality blastocysts (odds ratio (OR) 2.54, 95% CI 1.30-4.95, P = 0.006) compared to 2MONO embryos. Embryos with 2MULTI in both blastomeres (2MULTI2) were significantly less able to develop to good quality blastocysts (OR 0.38, 95% CI 0.23-0.63, P < 0.001) compared to 2MONO embryos. Embryos with 2BI in both blastomeres (2BI2) had a significantly better chance of resulting in a positive hCG (OR 2.40, 95% CI 1.11-5.20, P = 0.027), ongoing pregnancy (OR 2.79, 95% CI 1.29-6.04, P = 0.009) and live birth (OR 3.16, 95% CI 1.43-6.95, P = 0.004) compared to 2MONO blastocysts after single blastocyst transfer. In contrast, 2MULTI2 embryos were significantly less likely to result in a positive hCG (OR 0.58, 95% CI 0.35-0.97, P = 0.036) and ongoing pregnancy (OR 0.51, 95% CI 0.28-0.94, P = 0.030) compared to 2MONO blastocysts. LIMITATIONS REASONS FOR CAUTION: Discrepancies among the existing studies regarding the definition of multinucleation may lead to different conclusions. Even though the distinction between binucleation and true multinucleation was a strength in our study design, a further distinction between true multinucleated and micronucleated embryos could be interesting to investigate in future studies. Also, we included any anucleated embryos in the 2MONO group. For the study of clinical outcomes, the patients were allowed to be included with more than one transfer cycle. Both fresh and thawed transfers were included. WIDER IMPLICATIONS OF THE FINDINGS: We find it important to discriminate between binucleation and true multinucleation when evaluating embryo nucleus status at the two-cell stage. Embryos displaying 2BI1 and 2BI2 have significantly better good quality blastocyst formation rates and clinical outcome after single blastocyst transfers, respectively. 2MULTI2 embryos have impaired blastocyst development potential and poorer clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: H.S.N. received an unrestricted grant from Merck for 3 months' normal salary for a medical Doctor (A.L.T.) to write the manuscript. Merck was not involved in the study design, analysis, interpretation of data, writing the paper or the decision to submit the manuscript for publication. H.S.N. has received speaker's fees from Ferring Pharmaceuticals, Merck Denmark A/S, Astra Zeneca, Cook Medical and Ibsa Nordic (outside the submitted work). N.l.C.F. has received a grant from Gedeon Richter (outside the submitted work). The other authors did not report any potential conflicts of interest. All authors declared no conflicts of interest regarding this work. TRIAL REGISTRATION NUMBER: N/A.

Fetal fraction of cell-free DNA in pregnancies after fresh or frozen embryo transfer following assisted reproductive technologies.

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. TRIAL REGISTRATION NUMBER: NA.