Perivitelline threads in cleavage-stage human embryos: observations using time-lapse imaging  Louise Kellam, Laura M. Pastorelli, Angel M. Bastida, Amy Senkbeil, Sue Montgomery, Simon Fishel, Alison Campbell  Reproductive BioMedicine Online  Volume 35, Issue 6, Pages 646-656 (December 2017) DOI: 10.1016/j.rbmo.2017.09.004 Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Classification of perivitelline threads, with timelapse image examples. Threads were classified into three groups, based on their quantity: one or two individual threads (I), three to five threads (II) and more than five threads (III). Class III included observations of large arrays spanning a high proportion of the internal zona. Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 The sequence of events leading to the appearance of perivitelline threads. When the zygote is positioned centrally within the perivitelline space (A) no perivitelline threads are visible; (B) the zygote moves towards the inner edge of the zona pellucida (dashed arrow) making proximal contact; (C) as cytokinesis occurs the threads appear in the newly formed perivitelline space as the embryonic membrane withdraws from the contact area (arrow head); (D) perivitelline threads are seen to extend as cytokinesis progresses. Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Perivitelline threads observed in the perivitelline space as the cytoplasmic membrane withdraws from the zona pellucida. Perivitelline threads appear to follow the line of the cleavage furrow and extend as the membrane moves into the furrow (Supplementary Video 1). Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Before pronuclear fading, and the first cleavage, the membrane withdraws from close proximity to the zona pellucida (black arrows), and an array of perivitelline threads appears, connecting the membrane to the zona pellucida, elongating with time. (Supplementary Video 2b). Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 An array of perivitelline threads appear as a ‘wave’ in the perivitelline space, formed in association with the zygote cytoplasmic membrane (Supplementary Video 2a). Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 (A) Observation of perivitelline threads and their association with fragmentation and cleavage orientation. After pronuclear fading, the zygote moves towards the inner surface of the zona pellucida (dashed arrow) to form a direct contact area (B, arrow). The cleavage furrow is initiated from the most distal region of the zygote (C, arrow head), directly opposite this area of contact (arrow). Perivitelline threads are thought to extend along this axis, directing cleavage in a ‘purse string’ effect drawing the distal cytoplasmic membrane inwards towards this area of membrane – zona pellucida contact (D). As the cleavage furrow deepens the perivitelline threads are observed in the perivitelline space (E). As cytokinesis progresses, the threads are observed attached to extra-embryonic cytoplasm, which is drawn out as fragments (E and F). Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 Magnified images of the cleavage furrow in association with fragments and perivitelline threads. Fragments appear in a purse-string like manner (Supplementary Video 3a). Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 8 Implantation rates for embryos with known implantation status stratified for embryos with (n = 114) and without perivitelline threads (n = 55) and for those with perivitelline threads for each of the classifications of threads (I–III). Class I (n = 41); class II (n = 43); class III (n = 46). Reproductive BioMedicine Online 2017 35, 646-656DOI: (10.1016/j.rbmo.2017.09.004) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions