STUDY QUESTION Do fetuses from frozen embryo transfer (FET) present signs of cardiac remodeling and suboptimal function similar to those observed in fetuses from fresh embryo transfer (ET)? SUMMARY ANSWER Fetuses from both fresh ET and FET present signs of fetal cardiac remodeling and suboptimal function, with more pronounced changes after fresh ET as compared to FET. WHAT IS KNOWN ALREADY Our group and others have previously demonstrated that fetuses and children conceived by ARTs present cardiac remodeling and suboptimal function. These fetuses show dilated atria, more globular and thicker ventricles, reduced longitudinal motion, and impaired relaxation. Cardiac changes were already present in utero and persisted after birth. Most of the ART fetuses included in previous publications were from fresh ET. However, singletons from FET have different perinatal outcomes compared to those from fresh ET. There are no previous studies comparing cardiac morphology and function between fetuses following fresh and FET. STUDY DESIGN, SIZE, DURATION This is a prospective cohort study of 300 singleton pregnancies recruited from 2017 to 2020, including 100 spontaneously conceived (SC) pregnancies, 100 fetuses conceived by IVF with FET, and 100 fetuses conceived by IVF with fresh ET. Fetal structural and functional echocardiography was performed in all pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS Pregnancies conceived by IVF were recruited from a single assisted reproduction center, ensuring homogeneity in IVF stimulation protocols, endometrial preparation for FET, laboratory procedures, and embryo culture conditions. SC pregnancies from fertile couples were selected from the general population and matched to IVF pregnancies by maternal age. Epidemiological and perinatal outcomes were collected in all cases. Fetal echocardiography was performed at 28–33 weeks of pregnancy to assess cardiac structure and function in all pregnancies. All echocardiographic comparisons were adjusted by maternal age, nulliparity, birthweight centile, preeclampsia, and prematurity. MAIN RESULTS AND THE ROLE OF CHANCE Parental age, ethnicity, body mass index and smoking were similar among the study groups. Median gestational age at echocardiography and estimated fetal weight were similar in all study groups. Both fresh ET and FET groups showed similar fetal echocardiographic changes, with more pronounced features in the fresh ET as compared to FET pregnancies. Fetuses conceived by IVF showed larger atria (right atria-to-heart ratio: fresh ET mean 18.1% (SD 3.2) vs FET 18.0% (3.9) vs SC 17.3% (3.2); linear tendency P-value <0.001), more globular ventricles (right ventricular sphericity index: fresh ET 1.62 (0.29) vs FET 1.61 (0.25) vs SC 1.68 (0.26); <0.001) and thicker myocardial walls (relative wall thickness: fresh ET 0.79 (0.21) vs FET 0.74 (0.22) vs SC 0.65 (0.25); <0.001) as compared to SC pregnancies. Both fresh ET and FET groups also had signs of suboptimal systolic and diastolic function, with reduced tricuspid annular systolic peak velocity (fresh ET 7.17 cm/s (1.22) vs FET 7.41 cm/s (1.19) vs SC 7.58 cm/s (1.32); <0.001) and increased left myocardial performance index (fresh ET 0.53 (0.08) vs FET 0.53 (0.08) vs SC 0.50 (0.09); <0.001) as compared to SC pregnancies. LIMITATIONS, REASONS FOR CAUTION The cardiac changes reported here are subclinical, with most cardiovascular indexes lying within normal ranges. Although echocardiographic changes are recognized as potential cardiovascular risk factors, their association with the long-term cardiovascular disease remains to be proven. The observed milder fetal cardiac features in FET fetuses cannot condition the choice of this technique and must be considered together with the global perinatal results related to these gestations. WIDER IMPLICATIONS OF THE FINDINGS The identification of cardiac remodeling in fetuses conceived by IVF with fresh ET and FET represents an opportunity for early detection. Future studies are warranted to study the potential long-term consequences of these findings. STUDY FUNDING/COMPETING INTEREST(S) This project has been partially funded with support from the Erasmus + Programme of the European Union (Framework Agreement number: 2013-0040). This publication reflects the views only of the author, and the Commission cannot be held responsible for any use, which may be made of the information contained therein. Additionally, the research leading to these results has received funding from ‘la Caixa’ Foundation under grant agreement LCF/PR/GN18/10310003, the Instituto de Salud Carlos III (PI15/00130, PI17/00675, PI18/00073) integrated into the Plan Nacional de I + D+I and cofinanced by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER) ‘Una manera de hacer Europa’, Cerebra Foundation for the Brain Injured Child (Carmarthen, Wales, UK) and AGAUR 2017 SGR grant n° 1531. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A

What are the novel findings of this work?Comparison of brain cortical folding in fetuses conceived by assisted reproductive technology (ART) and fetuses conceived spontaneously showed distinct differences, with reduced sulci depth and lower Sylvian fissure grading score in ART fetuses, and more pronounced changes in the fetuses conceived by ART using fresh embryo transfer (ET) than those conceived using frozen ET. ART infants also showed lower Ages and Stages Questionnaire scores, especially in the fresh ET group. What are the clinical implications of this work?Neurosonography is an appropriate tool to identify subtle brain differences among fetuses conceived by ART. These findings suggest the existence of in-utero brain reorganization associated with ART, and support neurodevelopmental follow-up in offspring conceived by ART.

Stress and anxiety are frequent occurrences among pregnant women. We aimed to evaluate the effects of a Mediterranean diet intervention during pregnancy on maternal stress, well-being, and sleep quality throughout gestation. In a randomized clinical trial, 1221 high-risk pregnant women were randomly allocated into three groups at 19–23 weeks’ gestation: a Mediterranean diet intervention, a Mindfulness-Based Stress Reduction program, or usual care. All women who provided self-reported life-style questionnaires to measure their anxiety (State Trait Anxiety Inventory (STAI), Perceived Stress Scale (PSS)), well-being (WHO Five Well Being Index (WHO-5)), and sleep quality (Pittsburgh sleep quality index (PSQI)) at enrollment and at the end of the intervention (34–36 weeks) were included. In a random subgroup of 106 women, the levels of cortisol and related metabolites were also measured. At the end of the intervention (34–36 weeks), participants in the Mediterranean diet group had significantly lower perceived stress and anxiety scores (PSS mean (SE) 15.9 (0.4) vs. 17.0 (0.4), p = 0.035; STAI-anxiety mean (SE) 13.6 (0.4) vs. 15.8 (0.5), p = 0.004) and better sleep quality (PSQI mean 7.0 ± 0.2 SE vs. 7.9 ± 0.2 SE, p = 0.001) compared to usual care. As compared to usual care, women in the Mediterranean diet group also had a more significant increase in their 24 h urinary cortisone/cortisol ratio during gestation (mean 1.7 ± SE 0.1 vs. 1.3 ± SE 0.1, p < 0.001). A Mediterranean diet intervention during pregnancy is associated with a significant reduction in maternal anxiety and stress, and improvements in sleep quality throughout gestation.

Study question Do spontaneously conceived (SC) fetuses from subfertile couples present signs of cardiac remodeling as those observed after in vitro fertilization (IVF) treatments? Summary answer SC fetuses from subfertile couples do not associate cardiac remodeling, and their cardiac structure and function are similar to those of SC from fertile couples. What is known already Fetuses and children from IVF associate cardiac remodeling and suboptimal function, including dilated atria, more globular and thicker ventricles, reduced longitudinal motion and impaired relaxationin uteroand after birth. Fetal cardiac changes have been demonstrated both after fresh and frozen embryo transfer. The SC fetuses used as ‘controls’ in our previous publications were conceived by fertile couples thus making it difficult to separate the contribution of infertilityper sefrom the IVF procedures on cardiac programming. There are no previous cardiovascular studies investigating the independent effects of infertility in SC fetuses from subfertile couples (time-to-pregnancy (TTP) over 12 months). Study design, size, duration Prospective cohort study of 289 singleton pregnancies recruited from 2017 to 2021, including 96 SC pregnancies from fertile couples (TTP less than 12 months), 97 SC from subfertile couples (TTP over 12 months) and 96 from IVF after fresh ET. Fetal echocardiography was performed in all pregnancies. Epidemiological data and perinatal outcomes were collected in all pregnancies. Participants/materials, setting, methods IVF pregnancies from our centre were identified as eligible at pregnancy diagnosis. Eligible SC pregnancies from fertile and subfertile couples who attended our Maternal-Fetal Unit were invited to participate at third trimester, being matched to the IVF pregnancies by maternal age. Fetal echocardiography was performed at 29-34 weeks of pregnancy to assess cardiac structure and function. Echocardiographic comparisons were adjusted by nulliparity, birthweight centile, gestational age and estimated fetal weight at scan. Main results and the role of chance Parental age, ethnicity, body mass index and smoking exposure, median gestational age and estimated fetal weight were similar in all study groups. There were no significant differences in infertility duration or aetiology between the subfertile and the IVF populations (TTP: subfertile median 30 months [IQR 20-54] versus IVF: 47 [25-61]; p-value=0.052). While both fertile and subfertile SC groups presented similar fetal cardiac results, IVF fetuses showed larger atria (right atria-to-heart ratio: IVF mean 18.9% [SD 3.4] versus subfertile 17.8% [3.5] versus fertile 17.6% [3.3]; adjusted P-value&lt;0.001), more globular ventricles (right ventricular sphericity index: IVF 1.56 [0.25] versus subfertile 1.72 [0.26] versus fertile 1.72 [0.26]; &lt;0.001), and thicker myocardial walls (relative wall thickness: IVF 0.86 [0.22] versus subfertile 0.64 [0.13] versus fertile 0.64 [0.18]; &lt;0.001). Whereas SC fetuses from fertile and subfertile couples had preserved cardiac function, IVF fetuses showed signs of suboptimal systolic and diastolic function with reduced tricuspid ring displacement (IVF 7.26 mm [1.07] versus subfertile 8.04 mm [1.18] versus fertile 7.89 mm [1.51]; &lt;0.001) and increased left myocardial performance index (IVF 0.49 [0.08] versus subfertile 0.45 [0.09] versus fertile 0.45 [0.10]; &lt;0.001). A sub-analysis including only unexplained infertility cases in subfertile SC and IVF groups showed similar results. Limitations, reasons for caution The fetal cardiac changes reported here are subclinical, with most cardiovascular parameters lying within normal ranges. Although echocardiographic changes are recognized as potential cardiovascular risk factors, their association with long-term cardiovascular disease remains to be proven. Wider implications of the findings Subfertility per se does not seem to be associated to fetal cardiac remodeling, which have been previously described in IVF fetuses. Future studies are warranted to further investigate the factors related to fetal cardiac changes associated to ART. Trial registration number Not a trial

HIGHLIGHTS Pregnancies after frozen embryo transfer in programmed cycles have higher rates of hypertensive disorders of pregnancy.  Hemopexin and α 1 -microglobulin act as scavengers that eliminate free heme-groups responsible for the haemoglobin-induced oxidative stress known to contribute to preeclampsia development.  Pregnancies achieved by means of IVF in programmed cycles show more altered maternal hemopexin and α 1microglobulin concentrations suggesting higher levels of oxidative stress in those cases.  The corpus luteum activity during pregnancy could influence the perinatal outcome through lower levels of oxidative stress.Differential concentrations of maternal and fetal hemopexin and α 1 -microglobulin in preeclampsia from IVF pregnancies depending on the presence of corpus luteum at embryo transfer

STUDY QUESTION Do spontaneously conceived (SC) fetuses from subfertile couples show the same signs of cardiac remodeling as those observed after IVF treatments? SUMMARY ANSWER As opposed to fetuses from IVF, SC fetuses from subfertile couples do not show cardiac remodeling and present a similar cardiac structure and function to those of SC fetuses from fertile couples. WHAT IS KNOWN ALREADY Subjects conceived by IVF present signs of cardiac remodeling and suboptimal function in utero and during childhood, including larger atria, more globular and thicker ventricles, reduced longitudinal motion, and impaired relaxation as compared to SC individuals from fertile couples. There are no previous publications investigating the independent cardiac programming effects of infertility in SC fetuses from subfertile couples (with time-to-pregnancy (TTP) over 12 months). STUDY DESIGN, SIZE, DURATION A prospective cohort study of 289 singleton pregnancies exposed and not exposed to subfertility recruited from 2019 to 2021, including 96 SC pregnancies from fertile couples (TTP under 12 months), 97 SC from subfertile couples (TTP over 12 months), and 96 from IVF after fresh embryo transfer. Fetal echocardiography was performed in all pregnancies. Epidemiological data and perinatal outcomes were collected in all pregnancies. The overall attrition rate was 15.7%. PARTICIPANTS/MATERIALS, SETTING, METHODS SC from subfertile couples and IVF pregnancies were identified as eligible at pregnancy diagnosis, and eligible SC pregnancies from fertile couples who attended our maternal-fetal unit were invited to participate at third trimester, being matched to the other groups by maternal age. Fetal echocardiography was performed at 29–34 weeks of pregnancy to assess cardiac structure and function, and results were adjusted by parental age, maternal smoking status, child’s birth order, birthweight centile, gestational age, and estimated fetal weight at scan. MAIN RESULTS AND THE ROLE OF CHANCE Parental age, ethnicity, BMI, and smoking exposure, median gestational age and estimated fetal weight were similar in all study groups. There were no significant differences in infertility duration or etiology between the subfertile and the IVF populations (TTP: subfertile median 35 months (interquartile range 20–48) versus IVF: 47 (25–61); P-value = 0.051). While both fertile and subfertile SC groups presented similar fetal cardiac results, IVF fetuses showed larger atria (right atria-to-heart ratio: IVF mean 18.9% (SD 3.4) versus subfertile 17.8% (3.5) versus fertile 17.6% (3.3); adjusted P-value &lt; 0.001), more globular ventricles (right ventricular sphericity index: IVF 1.56 (0.25) versus subfertile 1.72 (0.26) versus fertile 1.72 (0.26); &lt;0.001), and thicker myocardial walls (relative wall thickness: IVF 0.86 (0.22) versus subfertile 0.64 (0.13) versus fertile 0.64 (0.18); &lt;0.001). Whereas SC fetuses from fertile and subfertile couples had preserved cardiac function, IVF fetuses showed signs of suboptimal systolic and diastolic function, with reduced tricuspid ring displacement (IVF 7.26 mm (1.07) versus subfertile 8.04 mm (1.18) versus fertile 7.89 mm (1.51); &lt;0.001) and increased left myocardial performance index (IVF 0.49 (0.08) versus subfertile 0.45 (0.09) versus fertile 0.45 (0.10); &lt;0.001). A sub-analysis including only unexplained infertility cases in subfertile SC and IVF groups showed similar results. LIMITATIONS, REASONS FOR CAUTION The fetal cardiac changes reported here are subclinical, and most of the cardiovascular parameters were within normal ranges. Although echocardiographic changes are recognized as potential cardiovascular risk factors, their association with long-term cardiovascular disease remains to be demonstrated. WIDER IMPLICATIONS OF THE FINDINGS Subfertility per se does not seem to be associated to fetal cardiac remodeling, which has been previously described in IVF fetuses. Future studies are warranted to further investigate other factors related to the observed fetal cardiac changes associated with ART. STUDY FUNDING/COMPETING INTEREST(S) This project has been partially funded with support from the Erasmus + Programme of the European Union (Framework Agreement number: 2013-0040). This publication reflects the views only of the author, and the Commission cannot be held responsible for any use, which may be made of the information contained therein. Additionally, the research leading to these results has received funding from ‘la Caixa’ Foundation under grant agreement LCF/PR/GN18/10310003, the Instituto de Salud Carlos III (PI15/00130, PI16/00861, PI17/00675, PI18/00073, INT21/00027)—co-funded by the European Union, Cerebra Foundation for the Brain Injured Child (Carmarthen, Wales, UK) and AGAUR 2017 SGR grant no 1531. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Oral communication abstracts Results: Total number of congenital heart diseases (CHD) in the cohort was 182 cases (0.9%). 68 subjects presented SAA of the V sign at the time of first trimester screening. SAA was found in 36.3% of all CHDs in our cohort. Further follow-up showed in this group: 10 cases of aortic arch obstruction, eight cases of d-transposition of great arteries (D-TGA), six cases of D-TGA + VSD, two cases of pulmonary atresia (PA) with intact interventricular septum, three cases of common arterial trunk, seven cases of double outlet right ventricle, four cases of tetralogy of Fallot (TOF), one case of TOF-absent pulmonary valve syndrome, two cases of L-TGA, four cases of PA with ventricular septal defect, two cases of tricuspid atresia, four cases of single ventricle, 16 cases of hypoplastic left heart syndrome and two false positive cases. Conclusions: First trimester SAA of the V sign is an important feature indicating to CHD. It should not be recognised as a specific sign of D-TGA but also of other anomalies. OC01.07 Inclusion of X and V signs in cardiac evaluation at the 11 +0-13 +6 weeks of gestation can improve the detection of outflow tract anomalies

Study question Does the presence of corpus luteum (CL) in in vitro fertilization (IVF) treatments affect maternal and fetal concentrations of hemopexin and α1-microglobulin in preeclampsia? Summary answer Decreased hemopexin and increased α1-microglobulin levels in maternal and fetal blood in IVF pregnancies with absence of CL particularly in pregnancies complicated by preeclampsia. What is known already: Pregnancies after frozen embryo transfer (FET) in programmed cycles have higher rates of hypertensive disorders of pregnancy, suggesting a link between the absence of CL in programmed cycles and adverse maternal outcomes.Cardiovascular function is impaired early in pregnancy in women conceiving by IVF treatments in the absence of CL.Plasma relaxin–2, a potent vasodilator and stimulus of decidualization, has been reported to be undetectable in a non-CL cohort, but markedly elevated in a multiple-CL cohort through pregnancy.Hemopexin and α1-microglobulin act as scavengers that eliminate free heme-groups responsible for hemoglobin-induced oxidative stress known to contribute to preeclampsia development. Study design, size, duration A case-control study of 160 singleton pregnancies recruited from 2016 to 2020, including 54 spontaneous pregnancies from fertile couples, 50 conceived by IVF following fresh embryo transfer (ET) and FET in natural cycle (presence of CL) and 56 IVF after fresh oocyte-donation or FET in programmed cycles (absence of CL). Pregnancies were subclassified according to the presence of preeclampsia in uncomplicated, preeclampsia and severe preeclampsia cases. Participants/materials, setting, methods IVF pregnancies were recruited from a single Assisted Reproduction Center, ensuring homogeneity in IVF stimulation protocols, endometrial preparation, laboratory procedures and embryo culture conditions. Spontaneous pregnancies from fertile couples were randomly selected from our general population and matched to IVF by gestational age at birth. Hemopexin and α1-microglobulin concentrations were measured by ELISA in maternal and cord plasma collected at delivery. All comparisons were adjusted for age, ethnicity, prematurity, birthweight centile, oocyte-donation and FET cycles. Main results and the role of chance Parental ethnicity, body mass index, exposure to aspirin and corticoids during pregnancy, mean gestational age at birth and birthweight were similar in all study groups. While maternal hemopexin levels were lower in treatments without CL, the IVF group with one or several CL showed significantly increased hemopexin concentrations, both in uncomplicated and preeclampsia cases (uncomplicated: spontaneous conceptions median 1520 ug/ml [interquartile range 1054–1746], IVF with CL 1554 [1315–1778], IVF without CL 1401 [1130–1750]; Preeclampsia: spontaneous conceptions 1362 [1121–1667], IVF with CL 1372 [403–2558], IVF without CL 1215 [971–1498]). Maternal α1-microglobulin was significantly higher in the absence of CL in severe preeclamptic cases as compared to spontaneous pregnancies and IVF with CL (spontaneous conceptions median 23 ug/ml [interquantile range 20–24], IVF with CL 24 [24–26], IVF without CL 26 [25–28]). The cord blood profiles were identical to the maternal for both biomarkers. Overall, and in line with previous studies, preeclamptic pregnancies independently of the mode of conception, showed decreased concentrations of hemopexin and increased concentrations of α1-microglobulin both in maternal and fetal plasma, with more pronounced changes in severe preeclampsia cases. Limitations, reasons for caution Infertility factors contribution to the outcome cannot be unraveled from the assisted reproductive technologies procedure itself as we have only included spontaneous pregnancies from fertile couples. Adjustments for oocyte-donation and FET modalities were performed due to the higher proportion of these features in the ET in programmed cycles group. Wider implications of the findings: These findings acknowledge physiological differences between pregnancies following ET in stimulated and natural versus programmed cycles, supporting the hypothesis that the CL activity could influence perinatal results. This approach to perinatal outcomes in IVF patients could lead to changes in ET protocols in order to develop a CL if possible. Trial registration number Not applicable