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)? 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. 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. 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. 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. 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. 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. 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. 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. N/A

Objective We aimed to explore fetal cortical brain development by neurosonography in fetuses conceived by assisted reproductive technology (ART), including frozen and fresh embryo transfer (ET), compared with those conceived spontaneously (SC), and to investigate its association with infant neurobehavior at 12 months of age. Methods This was a prospective cohort study of 210 singleton pregnancies, including 70 SC pregnancies, 70 conceived by in-vitro fertilization (IVF) following frozen ET and 70 conceived by IVF after fresh ET. Fetal neurosonography was performed at 32 +/- 2 gestational weeks to assess cortical development. Sulci depths were measured offline and normalized by biparietal diameter (BPD). Ages and Stages Questionnaires (ASQ) were completed postnatally, at 12 +/- 1months of corrected age. Neurosonographic findings were adjusted by regression analysis for maternal age, ethnicity, parity, fetal sex and fetal-weight centile and gestational age at scan, and ASQ scores were adjusted for maternal age, ethnicity, parity, educational level and employment status, gestational age at birth, breastfeeding, infant sex and infant age at the ASQ evaluation. Results Overall, in comparison to the SC fetuses, fetuses conceived by ART showed statistically significant differences in cortical development, with reduced parieto-occipital sulci depth adjusted for BPD (mean +/- SD: fresh ET, 12.5 +/- 2.5 vs frozen ET, 13.4 +/- 2.6 vs SC, 13.4 +/- 2.6, P< 0.001), cingulate sulci depth adjusted for BPD (median (interquartile range (IQR)): fresh ET, 5.8 (4.2- 7.4) vs frozen ET, 5.8 (4.1- 7.5) vs SC, 6.5 (4.8- 7.8), P= 0.001) and calcarine sulci depth adjusted for BPD (median (IQR): fresh ET, 13.5(10.1- 16.1) vs frozen ET, 14.5 (12.1- 15.8) vs SC, 16.4 (14.3- 17.9), P< 0.001), together with lower Sylvian fissure grading score. Changes in cortical development were more pronounced in the fresh ET than in the frozen ET group. ART infants showed lower ASQ scores as compared to SC infants, particularly in the fresh ET group (mean +/- SD global ASQ Z-score: fresh ET, - 0.3 +/- 0.4 vs frozen ET, - 0.2 +/- 0.4 vs SC, 0 +/- 0.4, P< 0.001). Conclusions Fetuses conceived by ART show a distinctive pattern of cortical development and suboptimal infant neurodevelopment, with more pronounced changes in those conceived following fresh ET. These findings support the existence of in-utero brain reorganization associated with ART and warrant follow-up studies to assess its long-term persistence. (c) 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Do spontaneously conceived (SC) fetuses from subfertile couples show the same signs of cardiac remodeling as those observed after IVF treatments? 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. 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). 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%. 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. 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 < 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); <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); <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); <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); <0.001). A sub-analysis including only unexplained infertility cases in subfertile SC and IVF groups showed similar results. 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. 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. 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. N/A.

Female FMR1 (Fragile X mental retardation 1) premutation carriers are at risk for developing fragile X-associated primary ovarian insufficiency (FXPOI), a condition characterized by amenorrhea before age 40 years. Not all women with a FMR1 premutation suffer from primary ovarian insufficiency and nowadays there are no molecular or other biomarkers that can help predict the occurrence of FXPOI. Long non-coding RNAs (lncRNAs) comprise a group of regulatory transcripts which have versatile molecular functions, making them important regulators in all aspects of gene expression. In recent medical studies, lncRNAs have been described as potential diagnostic biomarkers in many diseases. The present study was designed to determine the expression profile of three lncRNAs derived from the FMR1 locus, FMR4, FMR5 and FMR6, in female FMR1 premutation carriers in order: (i) to determine a possible role in the pathogenesis of FXPOI and (ii) to investigate whether they could serve as a biomarker for the diagnosis of FXPOI. FMR4, FMR5 and FMR6 transcripts levels were evaluated in total RNA extracted from peripheral blood by digital droplet PCR and compared between FMR1 premutation carriers with FXPOI and without FXPOI. The diagnostic value of lncRNAs was evaluated by receiver operating characteristic (ROC) analysis. Results revealed a significant association between FXPOI and high expression levels of FMR4. No association was obtained for FMR5 or FMR6. ROC curve analysis revealed that FMR4 can distinguish FMR1 premutation carrier with FXPOI with a diagnostic power of 0.67. These findings suggest a potential role of FMR4 as a possible biomarker for FXPOI.