Morphologic indicators predict the stage of chromatin condensation of human germinal vesicle oocytes recovered from stimulated cycles  Laura Escrich, Ph.D., Noelia Grau, Ph.D., Marcos Meseguer, Ph.D., Antonio Pellicer, M.D., Ph.D., María-José Escribá, Ph.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2557-2564 (May 2010) DOI: 10.1016/j.fertnstert.2009.05.077 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A–D) Contrast-phase microscopy images at ×40 magnification and (E, F) detailed nuclear observation of immature oocytes at the germinal vesicle stage (GV: A–C) or metaphase I stage (MI: D). In images A, B, and C, arrows represent diameters of the (A) oocyte, (B) nucleus, and (C) nucleolus. (A) GV oocyte with a well-defined nuclear envelope (grade 1) and one large eccentric NLB. (B) GV oocyte with an irregular nuclear envelope (grade 2). Nucleus is centrally located in the ooplasm. (C) GV oocyte with a discontinuous nuclear envelope in some areas (grade 3) that defines a nuclear area containing an clearly visible NLB. (D) Immature oocyte at the MI stage. Note the absence of both the nucleus and the first polar body in the perivitelline space. (E) Detailed image of oocyte from A. Note smooth appearance of nucleoplasm. (F) Note rough appearance of nucleoplasm of GV oocyte from C. Fertility and Sterility 2010 93, 2557-2564DOI: (10.1016/j.fertnstert.2009.05.077) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Photographs of human oocytes at the immature stage of either GV (A–D) or MI (E), showing different patterns of chromatin configuration observed with fluorescence microscopy (×100) after Hoestch 33342 staining. (A) Chromatin is dispersed and has a fibrillar appearance in the nucleolar area. (B) Chromatin is more condensed, forming a continuous mass close to the surface of a large NLB with a ring or horseshoe appearance. (C) Similar to B, except that a few chromatin clumps or strands are detected in the nucleoplasm. (D) Chromatin is further condensed into larger clumps and tend to be organized around a single small or absent nucleolus. (E) Chromatin is completely condensed and organized on a meiotic spindle. Fertility and Sterility 2010 93, 2557-2564DOI: (10.1016/j.fertnstert.2009.05.077) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Frequency distribution of GV oocytes according to the continuity of the nuclear envelope grade (grade 1 to grade 3) for the three proposed classification models. (A) Percentage of oocytes of each nuclear envelope grade (grade 1 to grade 3) in GV-DC and GV-CD groups (i.e., chromatin condensation stage: model A). Once again, as the nuclear envelope advanced from grade 1 to grade 3, becoming progressively more disorganized, more eggs of the GV-DC group turned into GV-CD oocytes. At analysis of oocyte frequency within the grade 3 nuclear envelop group, an increased frequency of GV-CD oocytes was evident (P<.05). (B) Percentage of oocytes of each grade of nuclear envelope (grade 1 to grade 3) in GV-SN and GV-NSN classification groups (i.e., chromatin assembly in relation to the nucleolar-like body [NLB]: model B). Within all three nuclear envelope grades, there were high and comparable percentages of GV-SN oocytes; the frequency of GV-NSN oocytes progressively increased as the degree of nuclear envelope increased. However, these differences were not statistically significant. (C) Percentage of oocytes of each grade of nuclear envelope (grade 1 to grade 3). According to GV1, GV2, GV3, or GV4 chromatin classifications (i.e., chromatin condensation and assembly to NLB: model C), grade 1 and grade 2 nuclear envelopes were more frequent among GV1 and GV2 oocytes. As degree of nuclear envelope progressed, the frequency of GV1 and GV2 oocytes diminished and that of GV3 and GV4 oocytes augmented. However, these variations in oocyte frequency distribution did not reach statistical significance. Fertility and Sterility 2010 93, 2557-2564DOI: (10.1016/j.fertnstert.2009.05.077) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions