An increase in the fetal fraction can be associated with birthweight discordance and selective fetal growth restriction

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Identifying those pregnancies likely to be affected by eiher sIUGR or birthweight discordant early can optimize counseling, the timing of interventions and may, in future, allow trials of preventative treatments. Objective The onset of sIUGR often correlates with increased levels of cell-free fetal DNA (cffDNA). Here we tested the hypothesis that the fetal fraction (FF) in twin pregnancies can reflect discrepancies in the birthweights. Study design : This is a retrospective study of 237 twin pregnancies who underwent cffDNA screening for aneuploidy and delivered at the International Peace Maternity and Child Health Hospital in Shanghai between January 2018 and December 2021. The FFs and birthweights of the newborns were collected and analysed. Results The FF was found to be positively correlated with the difference of birthweights (β = 0.004, 95%CI: 0.001 ~ 0.006). For every 1% increase in the FF, the difference in the birthweight between twins increased by 0.4%. A higher FF was significantly associated with an increased risk of birthweight discordance of 20% (adjusted OR: 1.073, 95% CI: 1.009 ~ 1.142), 25% (adjusted OR: 1.092, 95% CI: 1.006 ~ 1.185) and sIUGR (adjusted OR: 1.130, 95% CI: 1.038 ~ 1.231). Using ROC analysis, we obtained optimum cut-off points for FFs ≥ 11.790 and ≥ 14.800 for birthweight discordance of 20% and sFGR, respectively. Compared with women with FFs of < 11.790, there were a 1.091-fold higher risk of birthweight discordance of 20% (adjusted OR: 2.091, 95% CI: 1.218 ~ 3.591) when the FF was ≥ 11.790. There were also significantly increased risks of birthweight discordance of 25% (adjusted OR: 3.045, 95% CI: 1.297 ~ 7.149) and sFGR (adjusted OR: 3.526, 95% CI: 1.443 ~ 8.618) among women with FFs of ≥ 14.800. Conclusions This study showed that the FF was positively correlated with differences in birthweights. In addition, the FF could be used as an indicator to predict birthweight discordance and sIUGR for individualized clinical monitoring of twin pregnancies. twin pregnancy Non-invasive prenatal testing fetal fraction selective fetal growth restriction Figures Figure 1 Introduction The incidence of twin pregnancies has increased exponentially in recent years due to the growing demand of assisted reproductive technologies. Twin pregnancies are more likely to be affected by intrauterine growth restriction (IUGR), preterm birth and perinatal loss. Selective IUGR (sIUGR) is unique to twin pregnancies whereby one twin appears to be compromised while the other grows normally. Twin pregnancies complicated by sIUGR are at increased risk of perinatal mortality and morbidity [ 1 ] . Gestational age at delivery was found to be significantly earlier and the rate of admission to the neonatal unit was higher when compared to those without sIUGR [ 2 ] . The incidence of sIUGR is estimated as 10–20% of twin pregnancies [ 3 ] . Monochronic twins are more likely to be affected by sIUGR than dichorionic twins, with an incidence of 19.7% compared to 10.5% for the latter [ 4 ] . The recent International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) guidance defines sIUGR as a condition in which the estimated fetal weight (EFW) of one twin is less than the 10th centile and an inter-twin EFW discordance greater than 25%. In addition, a discordance cut-off of 20% seems acceptable to distinguish pregnancies at increased risk of adverse outcome [ 5 ] . Identifying those pregnancies likely to be affected by eiher sIUGR or birthweight discordant early can optimize counseling, the timing of interventions and may, in future, allow trials of preventative treatments. Several parameters identifiable in early pregnancy have been shown to be associated with a risk of sIUGR developing later during that pregnancy. Abnormal cord insertion has been found to be associated with sIUGR in twins. However, over 55% of all twin pregnancies have been shown to show discordant cord insertions [ 6 ] . A more specific screening test is urgently needed in clinical practice so as to provide the optimal treatment options for patients as well as health professionals. The onset and prognosis of sIUGR often correlate with differences in placental pathology. sIUGR in MC twins is thought to be caused by an unequal sharing of the placenta and distribution of blood through placental anastomoses [ 7 ] , whereas in DC twins, this can result from placental insufficiency in one of the placentas [ 8 ] . The villous trophoblast of the placenta undergoes continuous turnover throughout gestation resulting in released apoptotic debris and cell-free fetal DNA (cffDNA) into the maternal circulation. It has been suggested that concentrations of cffDNA also may reflect placental pathology. The proportion of cffDNA in the maternal circulation is commonly termed fetal fraction (FF). The FF has been linked to adverse perinatal outcomes, such as IUGR in singleton pregnancies [ 9 ] . Here, we tested the hypothesis that the FF in twin pregnancies would reflect birthweight discrepancies in the newborn. Materials and Methods Study design and data collection We conducted a retrospective cohort study of all twin pregnancies undergoing cffDNA screening for aneuploidy and who delivered at International Peace Maternity and Child Health Hospital in Shanghai, China between January 2018 and December 2021. All women with twin pregnancies were offered a scan to determine chorionicity as well as first-trimester nuchal translucency (NT) at 11 + 0 -13 + 6 weeks. Dichorionic pregnancies were confirmed when ultrasound assessment clearly indicated two placentas. The twin peak sign was used to distinguish chorionicity if only one placenta was visualized. Following this, a thorough genetic consultation was offered to all future parents of the twin cohort. The characteristics of the tests and potential advantages and disadvantages of the different modalities of prenatal genetic testing were discussed. Parents were also be informed that the non-invasive prenatal test (NIPT) included information regarding the detection of only T21, T18 and T13, and that the evidence for using NIPT in twin pregnancies is less than it is in singletons. Therefore, this offered limited information compared with invasive diagnostic testing. We used the platform offered by cffDNA-BGI-Health (Shenzhen, China). The screening was performed specific to our clinical site. We obtained the maternal demographic characteristics, FF, obstetric outcomes and neonatal outcomes from the medical records, and these included maternal age, race, parity, education levels, pre-pregnancy body mass index (BMI), smoking status, gestational age at delivery, neonatal birthweight and the method of conception. Birth discordance was defined as an inter-twin birth weight discordance greater than 20%. Condition in which the birth weight of one twin was less than the 10th centile and an inter-twin birth weight discordance greater than 25% was defined as sIUGR. Cases with chromosome abnormalities were excluded from this study. Other exclusion criteria were the presence of twin-to-twin transfusion syndrome (TTTS), monoamniotic twins, twin anemia–polycythemia sequence (TAPS) and congenital, structural and genetic malformations of the fetus. In order to ensure the placentas would be intact, only MC twin gestations delivered through cesarean section were included in this study. TTTS was diagnosed according to the Quintero criteria and TAPS was diagnosed as a postnatal inter-twin hemoglobin difference of > 8 g/dL. MC twin pregnancies were identified either in the first trimester or early second trimester of pregnancy using the following ultrasonographic criteria: (I) the presence of a single placenta, (II) the presence of a thin dividing membrane, and (III) the absence of a twin peak (lambda) sign. Monochorionicity also was confirmed by obstetricians through postpartum examination of the placenta (ie. the presence of a single placenta with inter-twin anastomoses) [ 10 ] . sIUGR was defined as the presence of (I) a birthweight discordance of > 25% and (II) a IUGR twin with a birthweight less than the 10th percentile. Birthweight discordance was calculated as the difference between the newborns’ weights of the AGA and IUGR twins divided by the weight of the larger twin as follows: [(body weight of the AGA twin − body weight of the sIUGR twin)/ (body weight of the AGA twin)] × 100% [ 11 ] . Statistical Analysis Categorical variables were described by percentage, and continuous variables were described by means and standard deviations. The Kolmogorov-Smirnov test was used to test the normality of the continuous variables. The group differences were examined using the chi-squared, Student t and Mann Whitney U tests where appropriate. The linear regression models were used to test the association of FF with birthweight difference. Logistic regression was used to obtain the odds ratios (ORs) and 95% confidence intervals (CIs) and to examine the effects of FF on birthweight discordance of 20 and 25% and sFGR. The Optimal Cutpoints package was used to perform a receiver operator characteristics (ROC) curve analysis for the FF and birth outcomes that had significance in the above regression analyses, and to determine the optimum cut-off points. Then the FF was converted into a categorical variable according to the optimal cut-off points. The association of the FF (categorical variable) on birth outcomes was then assessed using logistic regression. The multivariate analyzes between the FF and birth outcomes were carried out using the multiple logistic regression and multiple linear regression models. The models were subsequently adjusted for maternal age, weight, primipara, history of abortion, chorionicity, pregnancy via assisted reproductive technology (ART) and physical conditions (gestational hypertension, gestational diabetes). All analyses were performed with the Statistical Package for the Social Sciences (version 24; SPSS Inc, Chicago, IL) and the Optimal Cutpoints and verification packages for R statistical software (version 3.5.1; http://www.R-project.org ). Significance was defined as a 2-tail probability value of < 0.05. Results Study population A total of 237 twin pregnancies who had normal NIPT results and delivered ≥28 weeks were included in our study. 5 cases of intrauterine death of one fetus and 8 structural abnormalities in one or both fetuses were excluded from this study. The proportions of DC and MC twins were 77.6% (174) and 22.3% (50), respectively. Compared to the MC group, the DC group had a higher maternal age ( p = 0.006), longer gestational weeks at delivery ( p < 0.001), larger birthweight of the fetuses ( p = 0.001) and higher percentage of pregnancies conceived by ART ( p < 0.001). The incidence of preterm delivery was higher in the MC group ( p = 0.004). There was no difference in other pregnancy complications such as gestational hypertension, gestational diabetes and thyroid diseases (Table 1 ). Table 1 Demographics of the DC and MC groups of pregnancies. Overall DC MC p Mean ± SD/n (%) Maternal characteristics n = 224 n = 174 n = 50 Age (year), n(%) 0.006 Mean ± SD 31.97 ± 3.75 32.30 ± 3.49 30.82 ± 4.40 < 35 180 (80.4) 137 (78.7) 43 (86.0) ≥ 35 44 (19.6) 37 (21.3) 7 (14.0) Maternal weight, kg 59.09 ± 8.98 59.52 ± 9.38 57.59 ± 7.28 0.228 First gestation, n (%) 0.625 Yes 141 (62.9) 111 (63.8) 30 (60.0) No 83 (37.1) 63 (36.2) 20 (40.0) Primipara, n (%) 0.081 Yes 199 (88.8) 158 (90.8) 41 (82.0) No 25 (11.2) 16 (9.2) 9 (18.0) History of abortion, n (%) 0.404 No 155 (69.2) 118 (67.8) 37 (74.0) Yes 69 (30.8) 56 (32.2) 13 (26.0) Pregnancy via ART, n (%) < 0.001 No 72 (32.1) 42 (24.1) 30 (60.0) Yes 152 (67.9) 132 (75.9) 20 (40.0) Fetal fraction 10.73 ± 4.15 10.57 ± 4.25 11.30 ± 3.78 0.079 Gestational hypertension, n (%) 0.976 No 184 (82.1) 143 (82.2) 41 (82.0) Yes 40 (17.9) 31 (17.8) 9 (18.0) Gestational diabetes, n (%) 0.634 No 180 (80.4) 141 (81.0) 39 (78.0) Yes 44 (19.6) 33 (19.0) 11 (22.0) Thyroid disease, n (%) 0.977 None 191 (85.3) 147 (84.5) 44 (88.0) Subclinical hypothyroidism 5 (2.2) 4 (2.3) 1 (2.0) Hypothyroidism 16 (7.1) 13 (7.5) 3 (6.0) Subclinical hyperthyroidism 7 (3.1) 6 (3.4) 1 (2.0) Hyperthyroidism 5 (2.2) 4 (2.3) 1 (2.0) Fetal characteristics n = 448 n = 348 n = 100 Gestational age at delivery, week 35.87 ± 1.99 36.08 ± 1.85 35.16 ± 2.27 < 0.001 Preterm birth, n (%) 0.004 No 200 (44.6) 168 (48.3) 32 (32.0) Yes 248 (55.4) 180 (51.7) 68 (68.0) Birth weight, g 2413.55 ± 466.54 2456.14 ± 436.53 2265.35 ± 535.05 0.001 Difference of birthweights 0.12 ± 0.11 0.11 ± 0.11 0.12 ± 0.12 0.331 Birthweight discordance of 20%, n (%) 0.795 No 380 (84.8) 296 (85.1) 84 (84.0) Yes 68 (15.2) 52 (14.9) 16 (16.0) Birthweight discordance of 25%, n (%) 0.988 No 412 (92.0) 320 (92.0) 92 (92.0) Yes 36 (8.0) 28 (8.0) 8 (8.0) sFGR, n (%) 0.706 No 416(92.9) 324 (93.1) 92 (92.0) Yes 32 (7.1) 24 (6.9) 8 (8.0) The association of the FF with birthweight discordance and sIUGR As shown in Table 2 , the adjusted model demonstrated that the FF was positively correlated with the difference of birthweight (β = 0.004, 95% CI: 0.001 ~ 0.006). That is, for every 1% increase in the FF, the difference of the birthweight between twins increased by 0.4%. The results of logistic analysis are shown in Table 3 , indicating that a higher FF was significantly associated with an increased risk of birthweight discordance of 20% (adjusted OR: 1.073, 95% CI: 1.009 ~ 1.142), 25% (adjusted OR: 1.092, 95% CI: 1.006 ~ 1.185) and sIUGR (adjusted OR: 1.130, 95% CI: 1.038 ~ 1.231). Table 2 Linear regression analyses of the fetal fraction and difference of birthweights. Outcome β (95%CI) p β (95%CI) a p Difference of birthweights 1.076 (1.046 ~ 1.107) 0.000 1.004 (1.001 ~ 1.006) 0.003 a The model adjusted for maternal age, weight, history of abortion, primipara, pregnancy via ART, chorionicity, gestational hypertension and gestational diabetes Table 3 Logistic regression analyses of the fetal fraction and birthweight discordance and sFGR. Outcome OR (95%CI) p aOR (95%CI) a p Birthweight discordance-20% 1.079 (1.018 ~ 1.144) 0.011 1.073 (1.009 ~ 1.142) 0.025 Birthweight discordance-25% 1.082 (1.005 ~ 1.166) 0.036 1.092 (1.006 ~ 1.185) 0.035 sFGR, n (%) 1.117 (1.035 ~ 1.205) 0.004 1.13 (1.038 ~ 1.231) 0.005 a The model adjusted for maternal age, weight, history of abortion, primipara, pregnancy via ART, chorionicity, gestational hypertension and gestational diabetes The optimum cut-off points for the FF in birthweight discordance and sIUGR were obtained with a ROC analysis (Table 4 and Fig. 1 ). Using ROC analysis, we obtained the optimum cut-off points of the FF ≥ 11.790, ≥ 14.800 and ≥ 14.800 for birthweight discordance of 20% and 25% and sIUGR, respectively. For birthweight discordance of 20%, the AUC was 0.591, the sensitivity was 52.9% and the specificity was 66.3% ( p = 0.017). The AUC of sIUGR was 0.620, with 37.5% sensitivity and 86.5% specificity ( p = 0.024). Table 4 ROC analyses for the fetal fraction and birth outcomes. Outcome Cut-off value AUC (95%CI) p Birthweight discordance of 20% 11.790 0.591 (0.516 ~ 0.666) 0.017 Birthweight discordance of 25% 14.800 0.569 (0.466 ~ 0.673) 0.083 sFGR 14.800 0.620 (0.514 ~ 0.725) 0.024 The FF was further segmented at 11.79 and 14.8. As shown in Table 5 , compared with women with a FF of < 11.790, there was a 1.091-fold higher risk of birthweight discordance of 20% (adjusted OR: 2.091, 95% CI: 1.218 ~ 3.591) among those with FFs ≥ 11.790, and significantly increased risks of birthweight discordance of 25% (adjusted OR: 3.045, 95% CI: 1.297 ~ 7.149) and sIUGR (adjusted OR: 3.526, 95% CI: 1.443 ~ 8.618) among these with FFs ≥ 14.800. Table 5 Logistic regression analyses of the fetal fraction and birthweight discordance and sFGR. Outcome Fetal fraction OR (95%CI) p aOR (95%CI) a p Birthweight discordance of 20% [0, 11.79%) 1 1 [11.79%, 100%] 2.215(1.315 ~ 3.731) 0.003 2.091 (1.218 ~ 3.591) 0.008 Birthweight discordance of 25% [0, 11.79%) 1 1 [11.79%, 14.80%) 0.985 (0.379 ~ 2.559) 0.976 0.843 (0.306 ~ 2.32) 0.741 [14.80%, 100%] 3.167 (1.444 ~ 6.944) 0.004 3.045 (1.297 ~ 7.149) 0.011 sFGR [0, 11.79%) 1 1 [11.79%, 14.80%) 1.286 (0.48 ~ 3.445) 0.617 1.211 (0.43 ~ 3.407) 0.717 [14.80%, 100%] 4.133 (1.815 ~ 9.411) 0.001 3.526 (1.443 ~ 8.618) 0.006 a The model was adjusted for maternal age, weight, history of abortion, primipara, pregnancy via ART, chorionicity, gestational hypertension and gestational diabetes Discussion In this study, we assessed the association between the FF and birthweight discordance and sIUGR. We demonstrated that FF was positively correlated with the difference of birthweight between twins within the pregnancy. For every 1% increase in the FF, the difference of birthweight between the twins increased by 0.4%. A higher FF was significantly associated with the increased risk of birthweight discordance of 20 and 25% as well as sIUGR. Using ROC analysis, we obtained the optimum cut-off points of FF ≥ 11.790, ≥ 14.800 and ≥ 14.800 for birthweight discordance of 20 and 25% and sIUGR, respectively. For a birthweight discordance of 20%, the AUC was 0.591, the sensitivity was 52.9% and the specificity was 66.3% ( p = 0.017). The AUC of sIUGR was 0.620, with 37.5% sensitivity and 86.5% specificity ( p = 0.024). When this was compared with women with a FF of < 11.790, there were a 1.091-fold higher risk of birthweight discordance of 20% among those with FFs of ≥ 11.790, and there significantly increased risks of birthweight discordance of 25% and sFGR among those with FFs of ≥ 14.800. cffDNA is known to originate from placenta [ 12 ] . During normal gestation, cffDNA undergoes a progressive rise, making up approximately 13% of all the cfDNA in the maternal plasma towards the end of pregnancy and then this rapidly drops to undetectable levels post-partum [ 13 ] . The release of cff-DNA is related to the size of the placenta as well as the rate of trophoblast apoptosis [ 14 ] . It is reported that cffDNA induces release of type 1 interferons, IFN-β and IFN-α, and other pro-inflammatory mediators [ 15 ] . Administration of CpG oligonucleotides was shown to cause gestational hypertension and increased vasoconstriction in pregnant rat [ 16 ] . Thus, cffDNA can act as a pro-inflammatory trigger with the unique capability of initiating inflammatory cascades. Studies shown that oxidative stress causes trophoblast apoptosis and increased release of cffDNA [ 17 ] . Other inflammatory factors, such as doxorubicin and high-mobility group box protein-1, can also affect the release of cffDNA. However, lipopolysaccharides did not affect the release of cffDNA in either early or late pregnancy [ 18 ] , which indicated that cffDNA played a role in sterile inflammation rather than during on-going infections progress. Sterile inflammation can contribute to placental dysfunction and this can also result in pregnancy complications. Numerous associations between pregnancy complications and cffDNA plasma concentrations have been made, with the goal of predicting negative outcomes earlier and non-invasively. Higher concentrations of cffDNA during second trimester were found in pregnancies complicated with pre-eclampsia, preterm delivery and IUGR. Peek cffDNA levels have been shown 3 weeks prior to the onset of symptoms [ 19 ] . In twins with birthweight discordant, we were able to see an increase in cffDNA levels which was represented by the FF. Intrauterine surgeries such as laser ablation to treat TTTS can also result in increased cffDNA levels [ 20 ] . Therefore, the increase of cffDNA in twins with birthweight discordance and sFGR may be a consequence of the repair process. The incidence of pre-eclampsia in DC pregnancies affected by discordant growth is as high as 37.5%, compared to 21.9% in MC sFGR pregnancies [ 21 ] . Hence, sIUGR in DC pregnancies was known to be a result of placental insufficiency affecting only one fetus [ 22 ] . After correcting for both pre-eclampsia and gestational diabetes, the increase in cffDNA levels is still associated with the incidence of birthweight discordance and sIUGR, which showed that there may be other pathological mechanisms involved. sIUGR in MC pregnancies maybe related to discordance in the placental share, and the greater the discrepancy in the placental mass in MC pregnancies, the greater the discordance that would be observed. Placentae affected by discordant sharing have more tightly connected fetal circulations with more and larger arterio-arterial anastomoses. These may be beneficial to the growth restricted fetuses, allowing their co-twin to compensate for a degree of their placental insufficiency. However, this close connection can also present special risks to the appropriately grown fetus, which is vulnerable to sudden changes in the smaller co-twin's blood pressure. While unequal placental sharing is the underlying cause of most sFGR in MC pregnancies, their interdependent feto-placental circulations have an important role in the eventual prognosis and perinatal outcome. Glucose transporters, which are essential for glucose transport at maternal-fetal interface, have also been found to be elevated in sFGR, especially in cases with abnormal umbilical arterial Doppler measurements. This may be due to hypo-perfusion. We suspect that the increased levels of cffDNA in MC twins with sIUGR is a consequence of apoptosis in such a hypoxic environment. Further, hypermetabolism may also be a reason for elevated cffDNA since the placenta attempts to increase glucose transport to the fetal circulation as an adaptive response [ 23 ] . Conclusions A high FF is associated with poor placental development and dysfunction, which can be use as potential indicators to predict birthweight discordance and sIUGR. Declarations Ethics approval and consent to participate： This study was approved by the Ethics Review Board of the International Peace Maternity and Child Health Hospital Affiliated to the Shanghai Jiao Tong University School of Medicine (No. GKLW2012-49). As this study is a retrospective study, it will not adversely affect the health of patients, nor will it involve the privacy and personal identity information of patients. The Ethics Committee of the International Peace Maternity and Child Health Hospital Affiliated to the Shanghai Jiao Tong University School of Medicine has waived the requirement of informed consent of patients. Consent for publication ：Not applicable Availability of data and materials： The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding ：This work was supported by a China National Key Research and Development Grant (2018YFC1004600). Acknowledgement The authors would like to thank Dr Dev Sooranna, Imperial College London, for editing the manuscript. References Gremillet L, Netter A, Tosello B, et al. Selective intrauterine growth restriction of monochorionic diamniotic twin pregnancies: What is the neonatal prognosis? J Gynecol Obstet Hum Reprod. 2022;51(3):102304. Parra-Cordero M, Bennasar M, Martinez JM, et al. Cord Occlusion in Monochorionic Twins with Early Selective Intrauterine Growth Restriction and Abnormal Umbilical Artery Doppler: A Consecutive Series of 90 Cases. Fetal Diagn Ther. 2016;39(3):186–91. Sukhwani M, Antolin E, Herrero B, et al. Management and perinatal outcome of selective intrauterine growth restriction in monochorionic pregnancies. J Matern Fetal Neonatal Med. 2021;34(23):3838–43. Coutinho Nunes F, Domingues AP, Vide Tavares M, et al. Monochorionic versus dichorionic twins: Are obstetric outcomes always different? J Obstet gynaecology: J Inst Obstet Gynecol. 2016;36(5):598–601. Khalil A, Rodgers M, Baschat A, et al. ISUOG Practice Guidelines: role of ultrasound in twin pregnancy. Ultrasound Obstet gynecology: official J Int Soc Ultrasound Obstet Gynecol. 2016;47(2):247–63. Cambiaso O, Zhao DP, Abasolo JI, et al. Discordance of cord insertions as a predictor of discordant fetal growth in monochorionic twins. Placenta. 2016;47:81–5. Van Winden KR, Quintero RA, Kontopoulos EV, et al. Decreased Total Placental Mass Found in Twin-Twin Transfusion Syndrome Gestations with Selective Growth Restriction. Fetal Diagn Ther. 2016;40(2):116–22. Queiros A, Blickstein I, Valdoleiros S, et al. Prediction of birth weight discordance from fetal weight estimations at 21–24 weeks' scans in monochorionic and dichorionic twins. J Matern Fetal Neonatal Med. 2017;30(16):1944–7. Rolnik DL, da Silva Costa F, Lee TJ, et al. Association between fetal fraction on cell-free DNA testing and first-trimester markers for pre-eclampsia. Ultrasound Obstet gynecology: official J Int Soc Ultrasound Obstet Gynecol. 2018;52(6):722–7. Morano D, Rossi S, Lapucci C, et al. Cell-Free DNA (cfDNA) Fetal Fraction in Early- and Late-Onset Fetal Growth Restriction. Mol Diagn Ther. 2018;22(5):613–9. Townsend R, Khalil A. Fetal growth restriction in twins. Best Pract Res Clin Obstet Gynecol. 2018;49:79–88. Zhong XY, Wang Y, Chen S, et al. Circulating fetal DNA in maternal plasma is increased in pregnancies at high altitude and is further enhanced by preeclampsia. Clin Chem. 2004;50(12):2403–5. Phillippe M, Adeli S. Cell-free DNA release by mouse placental explants. PLoS ONE. 2017;12(6):e0178845. Wang E, Batey A, Struble C, et al. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn. 2013;33(7):662–6. Barber GN. STING: infection, inflammation and cancer. Nat Rev Immunol. 2015;15(12):760–70. Goulopoulou S, Wenceslau CF, McCarthy CG, et al. Exposure to stimulatory CpG oligonucleotides during gestation induces maternal hypertension and excess vasoconstriction in pregnant rats. Am J Physiol Heart Circ Physiol. 2016;310(8):H1015–25. Tjoa ML, Cindrova-Davies T, Spasic-Boskovic O, et al. Trophoblastic oxidative stress and the release of cell-free feto-placental DNA. Am J Pathol. 2006;169(2):400–4. Kazemi NY, Fedyshyn B, Yelsa I, et al. Increased cell-free fetal DNA release after apoptosis and sterile inflammation in human trophoblast cells. Am J Reprod Immunol. 2021;86(5):e13483. Levine RJ, Qian C, Leshane ES, et al. Two-stage elevation of cell-free fetal DNA in maternal sera before onset of preeclampsia. Am J Obstet Gynecol. 2004;190(3):707–13. Wataganara T, Gratacos E, Jani J, et al. Persistent elevation of cell-free fetal DNA levels in maternal plasma after selective laser coagulation of chorionic plate anastomoses in severe midgestational twin-twin transfusion syndrome. Am J Obstet Gynecol. 2005;192(2):604–9. Sparks TN, Nakagawa S, Gonzalez JM. Hypertension in dichorionic twin gestations: how is birthweight affected? J Matern Fetal Neonatal Med. 2017;30(4):380–5. Wu D, Huang L, He Z, et al. Preeclampsia in twin pregnancies: association with selective intrauterine growth restriction. J Matern Fetal Neonatal Med. 2016;29(12):1967–71. Janzen C, Lei MY, Cho J, et al. Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction. Placenta. 2013;34(11):1072–8. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3942353","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273533589,"identity":"a117dcea-553a-44f9-9a9d-16ae26f95913","order_by":0,"name":"Yiru Shi","email":"","orcid":"","institution":"International Peace Maternity and Child Health Hospital of China Welfare Institution (IPMCH)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiru","middleName":"","lastName":"Shi","suffix":""},{"id":273533590,"identity":"c04723ff-3f9c-4506-a83b-7275d7d4584f","order_by":1,"name":"Yuanqing Xia","email":"","orcid":"","institution":"International Peace Maternity and Child Health Hospital of China Welfare Institution (IPMCH)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanqing","middleName":"","lastName":"Xia","suffix":""},{"id":273533591,"identity":"84f7f7ad-a2e4-41f6-871c-dd7b04523a44","order_by":2,"name":"Shan Wang","email":"","orcid":"","institution":"International Peace Maternity and Child Health Hospital of China Welfare Institution (IPMCH)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Wang","suffix":""},{"id":273533592,"identity":"d625a8d4-9e1d-4d34-b941-aad47b5456c2","order_by":3,"name":"Renyi Hua","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYLCChB//6tlAjA9Ea0nsOZDAB6QZZxBvDduBBDkgxcxDjGLz9t5jEg947uSxMTA/e2ybY8PA396dgFeLzJlzaRIJFs+K2RjYzI1zt6UxSJw5uwGvFgmJHDOJBB5mxjYGHjbp3G2HGQwkcglokX8D1MIG1WK57T8RWiR4QFoOJ4K1MG47QIQWnhxji8SeNGOgX8wke7cl8xD2C/sZw5s/ftjIyTcwP5P4uc1Ojr+9F78WIGCRAFPyD8AUUVHDTHwyGQWjYBSMgpEJAGkTPJGm88/OAAAAAElFTkSuQmCC","orcid":"","institution":"International Peace Maternity and Child Health Hospital of China Welfare Institution (IPMCH)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Renyi","middleName":"","lastName":"Hua","suffix":""},{"id":273533593,"identity":"9938f953-cae4-4693-8d69-6ef61b0295e5","order_by":4,"name":"Jinling Sun","email":"","orcid":"","institution":"International Peace Maternity and Child Health Hospital of China Welfare Institution (IPMCH)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinling","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-02-09 07:51:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3942353/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3942353/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51442866,"identity":"8c1d5c3f-3455-4576-8c4f-e681f475fc14","added_by":"auto","created_at":"2024-02-21 17:54:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":312642,"visible":true,"origin":"","legend":"\u003cp\u003eROC curves for the fetal fraction and birth outcomes.\u003c/p\u003e\n\u003cp\u003eA: ROC curve for the fetal fraction and birthweight discordance of 20%; B: ROC curve for the fetal fraction and birthweight discordance of 25%; C: ROC curve for the fetal fraction and sFGR\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3942353/v1/c7872975b0d08b0be99ce0a7.png"},{"id":87931556,"identity":"2548dcb9-4b40-420a-a52f-40dc24ead672","added_by":"auto","created_at":"2025-07-30 13:39:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1299120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3942353/v1/123e7eac-642f-4d93-82b4-e5859eae05e1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An increase in the fetal fraction can be associated with birthweight discordance and selective fetal growth restriction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe incidence of twin pregnancies has increased exponentially in recent years due to the growing demand of assisted reproductive technologies. Twin pregnancies are more likely to be affected by intrauterine growth restriction (IUGR), preterm birth and perinatal loss. Selective IUGR (sIUGR) is unique to twin pregnancies whereby one twin appears to be compromised while the other grows normally. Twin pregnancies complicated by sIUGR are at increased risk of perinatal mortality and morbidity\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Gestational age at delivery was found to be significantly earlier and the rate of admission to the neonatal unit was higher when compared to those without sIUGR\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe incidence of sIUGR is estimated as 10\u0026ndash;20% of twin pregnancies\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Monochronic twins are more likely to be affected by sIUGR than dichorionic twins, with an incidence of 19.7% compared to 10.5% for the latter\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The recent International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) guidance defines sIUGR as a condition in which the estimated fetal weight (EFW) of one twin is less than the 10th centile and an inter-twin EFW discordance greater than 25%. In addition, a discordance cut-off of 20% seems acceptable to distinguish pregnancies at increased risk of adverse outcome\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Identifying those pregnancies likely to be affected by eiher sIUGR or birthweight discordant early can optimize counseling, the timing of interventions and may, in future, allow trials of preventative treatments. Several parameters identifiable in early pregnancy have been shown to be associated with a risk of sIUGR developing later during that pregnancy. Abnormal cord insertion has been found to be associated with sIUGR in twins. However, over 55% of all twin pregnancies have been shown to show discordant cord insertions\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. A more specific screening test is urgently needed in clinical practice so as to provide the optimal treatment options for patients as well as health professionals.\u003c/p\u003e \u003cp\u003eThe onset and prognosis of sIUGR often correlate with differences in placental pathology. sIUGR in MC twins is thought to be caused by an unequal sharing of the placenta and distribution of blood through placental anastomoses\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, whereas in DC twins, this can result from placental insufficiency in one of the placentas\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The villous trophoblast of the placenta undergoes continuous turnover throughout gestation resulting in released apoptotic debris and cell-free fetal DNA (cffDNA) into the maternal circulation. It has been suggested that concentrations of cffDNA also may reflect placental pathology. The proportion of cffDNA in the maternal circulation is commonly termed fetal fraction (FF). The FF has been linked to adverse perinatal outcomes, such as IUGR in singleton pregnancies\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Here, we tested the hypothesis that the FF in twin pregnancies would reflect birthweight discrepancies in the newborn.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and data collection\u003c/h2\u003e \u003cp\u003eWe conducted a retrospective cohort study of all twin pregnancies undergoing cffDNA screening for aneuploidy and who delivered at International Peace Maternity and Child Health Hospital in Shanghai, China between January 2018 and December 2021. All women with twin pregnancies were offered a scan to determine chorionicity as well as first-trimester nuchal translucency (NT) at 11\u003csup\u003e+\u0026thinsp;0\u003c/sup\u003e-13\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e weeks. Dichorionic pregnancies were confirmed when ultrasound assessment clearly indicated two placentas. The twin peak sign was used to distinguish chorionicity if only one placenta was visualized. Following this, a thorough genetic consultation was offered to all future parents of the twin cohort. The characteristics of the tests and potential advantages and disadvantages of the different modalities of prenatal genetic testing were discussed. Parents were also be informed that the non-invasive prenatal test (NIPT) included information regarding the detection of only T21, T18 and T13, and that the evidence for using NIPT in twin pregnancies is less than it is in singletons. Therefore, this offered limited information compared with invasive diagnostic testing. We used the platform offered by cffDNA-BGI-Health (Shenzhen, China). The screening was performed specific to our clinical site.\u003c/p\u003e \u003cp\u003e We obtained the maternal demographic characteristics, FF, obstetric outcomes and neonatal outcomes from the medical records, and these included maternal age, race, parity, education levels, pre-pregnancy body mass index (BMI), smoking status, gestational age at delivery, neonatal birthweight and the method of conception. Birth discordance was defined as an inter-twin birth weight discordance greater than 20%. Condition in which the birth weight of one twin was less than the 10th centile and an inter-twin birth weight discordance greater than 25% was defined as sIUGR.\u003c/p\u003e \u003cp\u003eCases with chromosome abnormalities were excluded from this study. Other exclusion criteria were the presence of twin-to-twin transfusion syndrome (TTTS), monoamniotic twins, twin anemia\u0026ndash;polycythemia sequence (TAPS) and congenital, structural and genetic malformations of the fetus. In order to ensure the placentas would be intact, only MC twin gestations delivered through cesarean section were included in this study. TTTS was diagnosed according to the Quintero criteria and TAPS was diagnosed as a postnatal inter-twin hemoglobin difference of \u0026gt;\u0026thinsp;8 g/dL. MC twin pregnancies were identified either in the first trimester or early second trimester of pregnancy using the following ultrasonographic criteria: (I) the presence of a single placenta, (II) the presence of a thin dividing membrane, and (III) the absence of a twin peak (lambda) sign. Monochorionicity also was confirmed by obstetricians through postpartum examination of the placenta (ie. the presence of a single placenta with inter-twin anastomoses)\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. sIUGR was defined as the presence of (I) a birthweight discordance of \u0026gt;\u0026thinsp;25% and (II) a IUGR twin with a birthweight less than the 10th percentile. Birthweight discordance was calculated as the difference between the newborns\u0026rsquo; weights of the AGA and IUGR twins divided by the weight of the larger twin as follows: [(body weight of the AGA twin\u0026thinsp;\u0026minus;\u0026thinsp;body weight of the sIUGR twin)/ (body weight of the AGA twin)] \u0026times; 100%\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eCategorical variables were described by percentage, and continuous variables were described by means and standard deviations. The Kolmogorov-Smirnov test was used to test the normality of the continuous variables. The group differences were examined using the chi-squared, Student t and Mann Whitney U tests where appropriate. The linear regression models were used to test the association of FF with birthweight difference. Logistic regression was used to obtain the odds ratios (ORs) and 95% confidence intervals (CIs) and to examine the effects of FF on birthweight discordance of 20 and 25% and sFGR.\u003c/p\u003e \u003cp\u003eThe Optimal Cutpoints package was used to perform a receiver operator characteristics (ROC) curve analysis for the FF and birth outcomes that had significance in the above regression analyses, and to determine the optimum cut-off points. Then the FF was converted into a categorical variable according to the optimal cut-off points. The association of the FF (categorical variable) on birth outcomes was then assessed using logistic regression. The multivariate analyzes between the FF and birth outcomes were carried out using the multiple logistic regression and multiple linear regression models. The models were subsequently adjusted for maternal age, weight, primipara, history of abortion, chorionicity, pregnancy via assisted reproductive technology (ART) and physical conditions (gestational hypertension, gestational diabetes).\u003c/p\u003e \u003cp\u003eAll analyses were performed with the Statistical Package for the Social Sciences (version 24; SPSS Inc, Chicago, IL) and the Optimal Cutpoints and verification packages for R statistical software (version 3.5.1; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.R-project.org\u003c/span\u003e\u003cspan address=\"http://www.R-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Significance was defined as a 2-tail probability value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eA total of 237 twin pregnancies who had normal NIPT results and delivered \u0026ge;28 weeks were included in our study. 5 cases of intrauterine death of one fetus and 8 structural abnormalities in one or both fetuses were excluded from this study. The proportions of DC and MC twins were 77.6% (174) and 22.3% (50), respectively. Compared to the MC group, the DC group had a higher maternal age (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006), longer gestational weeks at delivery (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), larger birthweight of the fetuses (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and higher percentage of pregnancies conceived by ART (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The incidence of preterm delivery was higher in the MC group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). There was no difference in other pregnancy complications such as gestational hypertension, gestational diabetes and thyroid diseases (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographics of the DC and MC groups of pregnancies.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverall\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD/n (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMaternal characteristics\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (year), n(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e180 (80.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e137 (78.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43 (86.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44 (19.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37 (21.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (14.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal weight, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.09\u0026thinsp;\u0026plusmn;\u0026thinsp;8.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.52\u0026thinsp;\u0026plusmn;\u0026thinsp;9.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFirst gestation, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.625\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e141 (62.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e111 (63.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (60.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83 (37.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63 (36.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimipara, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e199 (88.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e158 (90.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41 (82.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 (11.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (9.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (18.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistory of abortion, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.404\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e155 (69.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118 (67.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37 (74.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69 (30.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 (32.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13 (26.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePregnancy via ART, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72 (32.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 (24.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (60.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e152 (67.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e132 (75.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFetal fraction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.73\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.079\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGestational hypertension, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.976\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e184 (82.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e143 (82.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41 (82.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 (17.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31 (17.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (18.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGestational diabetes, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.634\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e180 (80.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e141 (81.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39 (78.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44 (19.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33 (19.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (22.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThyroid disease, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.977\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e191 (85.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e147 (84.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44 (88.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubclinical hypothyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypothyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (7.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (6.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubclinical hyperthyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperthyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFetal characteristics\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGestational age at delivery, week\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreterm birth, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200 (44.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e168 (48.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (32.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e248 (55.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180 (51.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68 (68.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirth weight, g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2413.55\u0026thinsp;\u0026plusmn;\u0026thinsp;466.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2456.14\u0026thinsp;\u0026plusmn;\u0026thinsp;436.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2265.35\u0026thinsp;\u0026plusmn;\u0026thinsp;535.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifference of birthweights\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.331\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eBirthweight discordance of 20%, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.795\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e380 (84.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e296 (85.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84 (84.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68 (15.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52 (14.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (16.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eBirthweight discordance of 25%, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e412 (92.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e320 (92.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92 (92.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36 (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28 (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esFGR, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.706\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e416(92.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e324 (93.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92 (92.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (7.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (6.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eThe association of the FF with birthweight discordance and sIUGR\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the adjusted model demonstrated that the FF was positively correlated with the difference of birthweight (β\u0026thinsp;=\u0026thinsp;0.004, 95% CI: 0.001\u0026thinsp;~\u0026thinsp;0.006). That is, for every 1% increase in the FF, the difference of the birthweight between twins increased by 0.4%. The results of logistic analysis are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, indicating that a higher FF was significantly associated with an increased risk of birthweight discordance of 20% (adjusted OR: 1.073, 95% CI: 1.009\u0026thinsp;~\u0026thinsp;1.142), 25% (adjusted OR: 1.092, 95% CI: 1.006\u0026thinsp;~\u0026thinsp;1.185) and sIUGR (adjusted OR: 1.130, 95% CI: 1.038\u0026thinsp;~\u0026thinsp;1.231).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLinear regression analyses of the fetal fraction and difference of birthweights.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ (95%CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifference of birthweights\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.076 (1.046\u0026thinsp;~\u0026thinsp;1.107)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.004 (1.001\u0026thinsp;~\u0026thinsp;1.006)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003eThe model adjusted for maternal age, weight, history of abortion, primipara, pregnancy via ART, chorionicity, gestational hypertension and gestational diabetes\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLogistic regression analyses of the fetal fraction and birthweight discordance and sFGR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eaOR (95%CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirthweight discordance-20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.079 (1.018\u0026thinsp;~\u0026thinsp;1.144)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.011\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.073 (1.009\u0026thinsp;~\u0026thinsp;1.142)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.025\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirthweight discordance-25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.082 (1.005\u0026thinsp;~\u0026thinsp;1.166)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.036\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.092 (1.006\u0026thinsp;~\u0026thinsp;1.185)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.035\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esFGR, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.117 (1.035\u0026thinsp;~\u0026thinsp;1.205)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.13 (1.038\u0026thinsp;~\u0026thinsp;1.231)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003eThe model adjusted for maternal age, weight, history of abortion, primipara, pregnancy via ART, chorionicity, gestational hypertension and gestational diabetes\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe optimum cut-off points for the FF in birthweight discordance and sIUGR were obtained with a ROC analysis (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Using ROC analysis, we obtained the optimum cut-off points of the FF\u0026thinsp;\u0026ge;\u0026thinsp;11.790, \u0026ge; 14.800 and \u0026ge;\u0026thinsp;14.800 for birthweight discordance of 20% and 25% and sIUGR, respectively. For birthweight discordance of 20%, the AUC was 0.591, the sensitivity was 52.9% and the specificity was 66.3% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). The AUC of sIUGR was 0.620, with 37.5% sensitivity and 86.5% specificity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eROC analyses for the fetal fraction and birth outcomes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCut-off value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAUC (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirthweight discordance of 20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.591 (0.516\u0026thinsp;~\u0026thinsp;0.666)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.017\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirthweight discordance of 25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.569 (0.466\u0026thinsp;~\u0026thinsp;0.673)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esFGR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.620 (0.514\u0026thinsp;~\u0026thinsp;0.725)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.024\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe FF was further segmented at 11.79 and 14.8. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compared with women with a FF of \u0026lt;\u0026thinsp;11.790, there was a 1.091-fold higher risk of birthweight discordance of 20% (adjusted OR: 2.091, 95% CI: 1.218\u0026thinsp;~\u0026thinsp;3.591) among those with FFs\u0026thinsp;\u0026ge;\u0026thinsp;11.790, and significantly increased risks of birthweight discordance of 25% (adjusted OR: 3.045, 95% CI: 1.297\u0026thinsp;~\u0026thinsp;7.149) and sIUGR (adjusted OR: 3.526, 95% CI: 1.443\u0026thinsp;~\u0026thinsp;8.618) among these with FFs\u0026thinsp;\u0026ge;\u0026thinsp;14.800.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLogistic regression analyses of the fetal fraction and birthweight discordance and sFGR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFetal fraction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eaOR (95%CI) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eBirthweight discordance of 20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[0, 11.79%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[11.79%, 100%]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.215(1.315\u0026thinsp;~\u0026thinsp;3.731)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.091 (1.218\u0026thinsp;~\u0026thinsp;3.591)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eBirthweight discordance of 25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[0, 11.79%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[11.79%, 14.80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.985 (0.379\u0026thinsp;~\u0026thinsp;2.559)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.843 (0.306\u0026thinsp;~\u0026thinsp;2.32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.741\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[14.80%, 100%]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.167 (1.444\u0026thinsp;~\u0026thinsp;6.944)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.045 (1.297\u0026thinsp;~\u0026thinsp;7.149)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.011\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esFGR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[0, 11.79%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[11.79%, 14.80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.286 (0.48\u0026thinsp;~\u0026thinsp;3.445)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.211 (0.43\u0026thinsp;~\u0026thinsp;3.407)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.717\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e[14.80%, 100%]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.133 (1.815\u0026thinsp;~\u0026thinsp;9.411)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.526 (1.443\u0026thinsp;~\u0026thinsp;8.618)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003eThe model was adjusted for maternal age, weight, history of abortion, primipara, pregnancy via ART, chorionicity, gestational hypertension and gestational diabetes\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we assessed the association between the FF and birthweight discordance and sIUGR. We demonstrated that FF was positively correlated with the difference of birthweight between twins within the pregnancy. For every 1% increase in the FF, the difference of birthweight between the twins increased by 0.4%. A higher FF was significantly associated with the increased risk of birthweight discordance of 20 and 25% as well as sIUGR. Using ROC analysis, we obtained the optimum cut-off points of FF\u0026thinsp;\u0026ge;\u0026thinsp;11.790, \u0026ge; 14.800 and \u0026ge;\u0026thinsp;14.800 for birthweight discordance of 20 and 25% and sIUGR, respectively. For a birthweight discordance of 20%, the AUC was 0.591, the sensitivity was 52.9% and the specificity was 66.3% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). The AUC of sIUGR was 0.620, with 37.5% sensitivity and 86.5% specificity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024). When this was compared with women with a FF of \u0026lt;\u0026thinsp;11.790, there were a 1.091-fold higher risk of birthweight discordance of 20% among those with FFs of \u0026ge;\u0026thinsp;11.790, and there significantly increased risks of birthweight discordance of 25% and sFGR among those with FFs of \u0026ge;\u0026thinsp;14.800.\u003c/p\u003e \u003cp\u003ecffDNA is known to originate from placenta\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. During normal gestation, cffDNA undergoes a progressive rise, making up approximately 13% of all the cfDNA in the maternal plasma towards the end of pregnancy and then this rapidly drops to undetectable levels post-partum\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The release of cff-DNA is related to the size of the placenta as well as the rate of trophoblast apoptosis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. It is reported that cffDNA induces release of type 1 interferons, IFN-β and IFN-α, and other pro-inflammatory mediators\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Administration of CpG oligonucleotides was shown to cause gestational hypertension and increased vasoconstriction in pregnant rat\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Thus, cffDNA can act as a pro-inflammatory trigger with the unique capability of initiating inflammatory cascades.\u003c/p\u003e \u003cp\u003eStudies shown that oxidative stress causes trophoblast apoptosis and increased release of cffDNA\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Other inflammatory factors, such as doxorubicin and high-mobility group box protein-1, can also affect the release of cffDNA. However, lipopolysaccharides did not affect the release of cffDNA in either early or late pregnancy\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, which indicated that cffDNA played a role in sterile inflammation rather than during on-going infections progress. Sterile inflammation can contribute to placental dysfunction and this can also result in pregnancy complications.\u003c/p\u003e \u003cp\u003eNumerous associations between pregnancy complications and cffDNA plasma concentrations have been made, with the goal of predicting negative outcomes earlier and non-invasively. Higher concentrations of cffDNA during second trimester were found in pregnancies complicated with pre-eclampsia, preterm delivery and IUGR. Peek cffDNA levels have been shown 3 weeks prior to the onset of symptoms\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In twins with birthweight discordant, we were able to see an increase in cffDNA levels which was represented by the FF. Intrauterine surgeries such as laser ablation to treat TTTS can also result in increased cffDNA levels\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Therefore, the increase of cffDNA in twins with birthweight discordance and sFGR may be a consequence of the repair process. The incidence of pre-eclampsia in DC pregnancies affected by discordant growth is as high as 37.5%, compared to 21.9% in MC sFGR pregnancies\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Hence, sIUGR in DC pregnancies was known to be a result of placental insufficiency affecting only one fetus\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter correcting for both pre-eclampsia and gestational diabetes, the increase in cffDNA levels is still associated with the incidence of birthweight discordance and sIUGR, which showed that there may be other pathological mechanisms involved. sIUGR in MC pregnancies maybe related to discordance in the placental share, and the greater the discrepancy in the placental mass in MC pregnancies, the greater the discordance that would be observed. Placentae affected by discordant sharing have more tightly connected fetal circulations with more and larger arterio-arterial anastomoses. These may be beneficial to the growth restricted fetuses, allowing their co-twin to compensate for a degree of their placental insufficiency. However, this close connection can also present special risks to the appropriately grown fetus, which is vulnerable to sudden changes in the smaller co-twin's blood pressure. While unequal placental sharing is the underlying cause of most sFGR in MC pregnancies, their interdependent feto-placental circulations have an important role in the eventual prognosis and perinatal outcome. Glucose transporters, which are essential for glucose transport at maternal-fetal interface, have also been found to be elevated in sFGR, especially in cases with abnormal umbilical arterial Doppler measurements. This may be due to hypo-perfusion. We suspect that the increased levels of cffDNA in MC twins with sIUGR is a consequence of apoptosis in such a hypoxic environment. Further, hypermetabolism may also be a reason for elevated cffDNA since the placenta attempts to increase glucose transport to the fetal circulation as an adaptive response\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA high FF is associated with poor placental development and dysfunction, which can be use as potential indicators to predict birthweight discordance and sIUGR.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate：\u003c/strong\u003eThis study was approved by the Ethics Review Board of the International Peace Maternity and Child Health Hospital Affiliated to the Shanghai Jiao Tong University School of Medicine (No.\u0026nbsp;GKLW2012-49).\u0026nbsp;As this study is a retrospective study, it will not adversely affect the health of patients, nor will it involve the privacy and personal identity information of patients. The Ethics Committee\u0026nbsp;of the International Peace Maternity and Child Health Hospital Affiliated to the Shanghai Jiao Tong University School of Medicine\u0026nbsp;has waived the requirement of informed consent of patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e：Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials：\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e：This work was supported by a China National Key Research and Development Grant (2018YFC1004600).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr Dev Sooranna, Imperial College London, for editing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGremillet L, Netter A, Tosello B, et al. Selective intrauterine growth restriction of monochorionic diamniotic twin pregnancies: What is the neonatal prognosis? J Gynecol Obstet Hum Reprod. 2022;51(3):102304.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParra-Cordero M, Bennasar M, Martinez JM, et al. Cord Occlusion in Monochorionic Twins with Early Selective Intrauterine Growth Restriction and Abnormal Umbilical Artery Doppler: A Consecutive Series of 90 Cases. Fetal Diagn Ther. 2016;39(3):186\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSukhwani M, Antolin E, Herrero B, et al. Management and perinatal outcome of selective intrauterine growth restriction in monochorionic pregnancies. J Matern Fetal Neonatal Med. 2021;34(23):3838\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoutinho Nunes F, Domingues AP, Vide Tavares M, et al. Monochorionic versus dichorionic twins: Are obstetric outcomes always different? J Obstet gynaecology: J Inst Obstet Gynecol. 2016;36(5):598\u0026ndash;601.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil A, Rodgers M, Baschat A, et al. ISUOG Practice Guidelines: role of ultrasound in twin pregnancy. Ultrasound Obstet gynecology: official J Int Soc Ultrasound Obstet Gynecol. 2016;47(2):247\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCambiaso O, Zhao DP, Abasolo JI, et al. Discordance of cord insertions as a predictor of discordant fetal growth in monochorionic twins. Placenta. 2016;47:81\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Winden KR, Quintero RA, Kontopoulos EV, et al. Decreased Total Placental Mass Found in Twin-Twin Transfusion Syndrome Gestations with Selective Growth Restriction. Fetal Diagn Ther. 2016;40(2):116\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQueiros A, Blickstein I, Valdoleiros S, et al. Prediction of birth weight discordance from fetal weight estimations at 21\u0026ndash;24 weeks' scans in monochorionic and dichorionic twins. J Matern Fetal Neonatal Med. 2017;30(16):1944\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRolnik DL, da Silva Costa F, Lee TJ, et al. Association between fetal fraction on cell-free DNA testing and first-trimester markers for pre-eclampsia. Ultrasound Obstet gynecology: official J Int Soc Ultrasound Obstet Gynecol. 2018;52(6):722\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorano D, Rossi S, Lapucci C, et al. Cell-Free DNA (cfDNA) Fetal Fraction in Early- and Late-Onset Fetal Growth Restriction. Mol Diagn Ther. 2018;22(5):613\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTownsend R, Khalil A. Fetal growth restriction in twins. Best Pract Res Clin Obstet Gynecol. 2018;49:79\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong XY, Wang Y, Chen S, et al. Circulating fetal DNA in maternal plasma is increased in pregnancies at high altitude and is further enhanced by preeclampsia. Clin Chem. 2004;50(12):2403\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillippe M, Adeli S. Cell-free DNA release by mouse placental explants. PLoS ONE. 2017;12(6):e0178845.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang E, Batey A, Struble C, et al. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn. 2013;33(7):662\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarber GN. STING: infection, inflammation and cancer. Nat Rev Immunol. 2015;15(12):760\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoulopoulou S, Wenceslau CF, McCarthy CG, et al. Exposure to stimulatory CpG oligonucleotides during gestation induces maternal hypertension and excess vasoconstriction in pregnant rats. Am J Physiol Heart Circ Physiol. 2016;310(8):H1015\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTjoa ML, Cindrova-Davies T, Spasic-Boskovic O, et al. Trophoblastic oxidative stress and the release of cell-free feto-placental DNA. Am J Pathol. 2006;169(2):400\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazemi NY, Fedyshyn B, Yelsa I, et al. Increased cell-free fetal DNA release after apoptosis and sterile inflammation in human trophoblast cells. Am J Reprod Immunol. 2021;86(5):e13483.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevine RJ, Qian C, Leshane ES, et al. Two-stage elevation of cell-free fetal DNA in maternal sera before onset of preeclampsia. Am J Obstet Gynecol. 2004;190(3):707\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWataganara T, Gratacos E, Jani J, et al. Persistent elevation of cell-free fetal DNA levels in maternal plasma after selective laser coagulation of chorionic plate anastomoses in severe midgestational twin-twin transfusion syndrome. Am J Obstet Gynecol. 2005;192(2):604\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparks TN, Nakagawa S, Gonzalez JM. Hypertension in dichorionic twin gestations: how is birthweight affected? J Matern Fetal Neonatal Med. 2017;30(4):380\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu D, Huang L, He Z, et al. Preeclampsia in twin pregnancies: association with selective intrauterine growth restriction. J Matern Fetal Neonatal Med. 2016;29(12):1967\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanzen C, Lei MY, Cho J, et al. Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction. Placenta. 2013;34(11):1072\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"twin pregnancy, Non-invasive prenatal testing, fetal fraction, selective fetal growth restriction","lastPublishedDoi":"10.21203/rs.3.rs-3942353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3942353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTwin pregnancies are more likely to be affected by complications such as selective fetal growth restriction (sIUGR) which increase the risk of perinatal mortality and morbidity. Identifying those pregnancies likely to be affected by eiher sIUGR or birthweight discordant early can optimize counseling, the timing of interventions and may, in future, allow trials of preventative treatments.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe onset of sIUGR often correlates with increased levels of cell-free fetal DNA (cffDNA). Here we tested the hypothesis that the fetal fraction (FF) in twin pregnancies can reflect discrepancies in the birthweights.\u003c/p\u003e\u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003e: This is a retrospective study of 237 twin pregnancies who underwent cffDNA screening for aneuploidy and delivered at the International Peace Maternity and Child Health Hospital in Shanghai between January 2018 and December 2021. The FFs and birthweights of the newborns were collected and analysed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe FF was found to be positively correlated with the difference of birthweights (β\u0026thinsp;=\u0026thinsp;0.004, 95%CI: 0.001\u0026thinsp;~\u0026thinsp;0.006). For every 1% increase in the FF, the difference in the birthweight between twins increased by 0.4%. A higher FF was significantly associated with an increased risk of birthweight discordance of 20% (adjusted OR: 1.073, 95% CI: 1.009\u0026thinsp;~\u0026thinsp;1.142), 25% (adjusted OR: 1.092, 95% CI: 1.006\u0026thinsp;~\u0026thinsp;1.185) and sIUGR (adjusted OR: 1.130, 95% CI: 1.038\u0026thinsp;~\u0026thinsp;1.231). Using ROC analysis, we obtained optimum cut-off points for FFs\u0026thinsp;\u0026ge;\u0026thinsp;11.790 and \u0026ge;\u0026thinsp;14.800 for birthweight discordance of 20% and sFGR, respectively. Compared with women with FFs of \u0026lt;\u0026thinsp;11.790, there were a 1.091-fold higher risk of birthweight discordance of 20% (adjusted OR: 2.091, 95% CI: 1.218\u0026thinsp;~\u0026thinsp;3.591) when the FF was \u0026ge;\u0026thinsp;11.790. There were also significantly increased risks of birthweight discordance of 25% (adjusted OR: 3.045, 95% CI: 1.297\u0026thinsp;~\u0026thinsp;7.149) and sFGR (adjusted OR: 3.526, 95% CI: 1.443\u0026thinsp;~\u0026thinsp;8.618) among women with FFs of \u0026ge;\u0026thinsp;14.800.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study showed that the FF was positively correlated with differences in birthweights. In addition, the FF could be used as an indicator to predict birthweight discordance and sIUGR for individualized clinical monitoring of twin pregnancies.\u003c/p\u003e","manuscriptTitle":"An increase in the fetal fraction can be associated with birthweight discordance and selective fetal growth restriction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 17:54:51","doi":"10.21203/rs.3.rs-3942353/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ce9f390-afe4-43cc-ae1d-c32b05ecaffb","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T13:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-21 17:54:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3942353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3942353","identity":"rs-3942353","version":["v1"]},"buildId":"veTbxFhMMB0_faC6-Wkog","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}