Validation of upsilon (Y) zone as pathognomonic ultrasound landmark for chorionicity and amnionicity in triplet pregnancy at any gestational age

Triplet pregnancy is the most common type of high-order multiple gestation, most often occurring after infertility therapy1. Although the number of transferred embryos is generally limited, splitting of one embryo into two may result in multiple pregnancy which is further complicated by monochorionic placentation2, 3. Chorioamnionicity has a significant impact on fetal risk and neonatal outcome in twins and even more so in triplets3, 4. Ultrasound is the key diagnostic tool to determine chorioamnionicity in multiple pregnancy. Although no specific guidelines are available for high-order multiple pregnancies4, it is common practice to determine chorioamnionicity by counting the number of placentae and amniotic sacs and assessing the thickness of the dividing membranes as early as possible in pregnancy, using high-resolution transvaginal ultrasonography5. In 1996, Sepulveda et al. identified for the first time, in 28 triplet pregnancies, a specific ultrasound landmark called the ‘upsilon zone’, which is the junction of the three interfetal membranes, and demonstrated that it is a useful tool for predicting chorioamnionicity in triplet pregnancies, particularly in the first trimester6. We aimed to investigate if the upsilon zone is easily identifiable at any gestational age and to determine its accuracy in establishing chorionicity and amnionicity in triplets using placental histology as reference. We included retrospectively all triplet pregnancies that underwent ultrasound assessment (using a GE Voluson E8 or E10 (GE Healthcare, Zipf, Austria) or Philips IU22 (Philips Healthcare, Andover, MA, USA) ultrasound system) in our tertiary center between 2013 and 2018. All scans and videoclips from 8 + 0 to 36 + 6 weeks' gestation were reviewed independently by two observers who were blinded to the final ultrasound report and histopathology results. For each scan, each observer was requested to identify the upsilon zone, defined as the intersection of the three amniotic sacs, and to determine the chorioamnionicity based on the appearance and thickness of the three interfetal membranes at their intersection site (Figure 1). Chorioamnionicity was confirmed by the placental pathology report which was obtained in all pregnancies delivered in our institution. A total of 55 triplet pregnancies were analyzed (Figure S1). Table 1 shows their baseline characteristics. The upsilon zone was identified in 608/638 (95.3%) scans, with an interobserver agreement of 100%. Based on the upsilon zone, 67.3% (37/55) of pregnancies were classified as trichorionic triamniotic, 25.5% (14/55) as dichorionic triamniotic and 1.8% (1/55) as monochorionic triamniotic. In 5.5% (3/55) of pregnancies, the upsilon zone could not be identified, hence, they were classified as dichorionic diamniotic. In 82.7% (43/52) of cases, placental histology was available and was compared with the observers' results. The sensitivity of the upsilon zone in determining chorioamnionicity was 100% (95% CI, 99.0–100%) from 8 + 0 to 27 + 6 weeks and 96.5% (95% CI, 93.3–98.5%) from 28 + 0 to 36 + 6 weeks. The overall sensitivity and specificity of the upsilon zone were 98.3% (95% CI, 96.9–99.2%) and 100% (95% CI, 80.5–100%), respectively, with an accuracy of 98.3% (95% CI, 97.0–99.2%) (Table S1). This study confirms that the upsilon zone can be identified at any gestational age and that it is an accurate landmark to determine the chorionicity and amnionicity of triplet pregnancies. The strong correlation of the upsilon zone with placental histology was shown for the first time in a large series of triplet pregnancies. The upsilon zone can be identified by retrospective review of ultrasound images, thus, it could be a valuable tool in the not uncommon event of a late referral of a triplet pregnancy to a tertiary center due to delayed diagnosis or complications. The intersection of the intertwin membranes should also be evaluated during routine assessment of the amniotic fluid volume in the sacs as well as during invasive fetal procedures, such as amniocentesis, selective fetal reduction or laser coagulation for twin–twin transfusion syndrome. Additionally, with the advanced ultrasound technology currently available, the upsilon zone can be easily explored using a three-dimensional (3D) approach. The application of 3D techniques and postprocessing of ultrasound volumes acquired at the level of the intersection of the membranes has not been investigated yet in triplet pregnancy and should be considered in future studies (Figure S2). Professionals performing obstetric ultrasound should be familiar with the upsilon zone and presence of this sign should be assessed in all triplet pregnancies as standard practice. The data that support the findings of this study are available from the corresponding author upon reasonable request Figure S1 Description of study population. Figure S2 3D reconstruction of upsilon zone in trichorionic triamniotic pregnancy at 9 weeks' gestation. Table S1 Diagnostic accuracy of upsilon zone using placental histology as reference, overall and according to gestational age Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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