Pathogenesis, developmental consequences, and clinical correlations of human embryo fragmentation

REVIEW Pathogenesis, developmental consequences, and clinical correlations of human embryo fragmentation Victor Y. Fujimoto, M.D.,a Richard W. Browne, Ph.D.,b Michael S. Bloom, Ph.D.,c Denny Sakkas, Ph.D.,d and Mina Alikani, Ph.D.e a

Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, San Francisco, California; b Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, State University of New York, Buffalo, New York; c Departments of Environmental Health Sciences and Epidemiology and Biostatistics, University at Albany, State University of New York, Rensselaer, New York; d Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University, New Haven, Connecticut; and e Tyho-Galileo Research Laboratories, West Orange, New Jersey

This narrative review summarizes the current state of knowledge about human embryo fragmentation during IVF. The clinical relevance of fragmentation is discussed and evidence supporting a central role for the oocyte in the pathogenesis of fragmentation is presented. A mechanism of fragmentation as aberrant cell division involving the cytoskeleton is described along with the novel concept of membrane instability in relation to follicular high-density lipoprotein metabolism and cholesterol transport. (Fertil Steril
2011;95:1197–204. 2011 by American Society for Reproductive Medicine.) Key Words: Embryo fragmentation, oocyte quality, cytoskeleton, apoptosis, HDL metabolism, follicular fluid cholesterol

Fragmentation, or the generation of anucleate cell fragments during early embryo development and after IVF, has been shown to be an important biomarker for implantation potential (1–4). Various embryo grading systems have thus been developed, based on (mostly) subjective assessment of this feature along with cell number, size, nucleation, and symmetry (3, 5, 6), but emphasis has been placed primarily on cell number/cleavage rate and fragmentation. A majority of human IVF embryos display some degree of fragmentation (7, 8). Controlled experiments in enucleated mouse oocytes have definitively shown that oocyte activation is a prerequisite to fragmentation and that fragmentation occurs during the cytokinetic phase of the cell cycle (9). Although these experiments have not been repeated in the human, it is reasonable to assume that the process of fragmentation is similar in the human and involves the cytokinetic apparatus (Fig. 1A and B) (9). Time-lapse photography suggests that fragmentation frequently occurs as early as the first mitotic division, although it is not restricted to that division (10, 11). Fragmentation seems to be a dynamic process, and some fragments can be reincorporated into cells (11, 12), indicating that in some instances, fragmentation may be part of normal embryo development. The visible result of fragmentation is usually reduced cell volume and increased disorganization in the embryo (13–16), but Received July 15, 2010; revised November 12, 2010; accepted November 15, 2010; published online December 13, 2010. D.S. is chief scientific officer and holds stock in Molecular Biometrics Inc., V.Y.F. has nothing to disclose. R.W.B. has nothing to disclose. M.S.B. has nothing to disclose. M.A. has nothing to disclose. Reprint requests: Victor Y. Fujimoto, M.D., Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, 2356 Sutter Street, J707, San Francisco, CA 94115-0916 (E-mail: [email protected]).

0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.11.033

fragmentation clearly has other detrimental consequences. Moderate to extensive fragmentation is often associated with blastomere multinucleation and chromosomal abnormalities, most notably mosaicism (17–28), all of which can lead to failure of implantation. Highly fragmented embryos implant less frequently than embryos with minimal or no fragmentation, whereas those with minimal fragmentation seem to be less compromised (29). Fragmentation during the earliest stages of embryonic development is not unique to humans (30) and occurs both in vitro and in vivo in other mammalian species. However, in murine embryos, which are often used as a model for human embryos, fragmentation is infrequent except under particular experimental conditions (9). Instead, these embryos undergo early cleavage arrest when exposed to suboptimal culture conditions (31). Fragmented human embryos show an erratic distribution of the cell adhesion protein E-cadherin (32), and this abnormal distribution may be associated with abnormal microtubule and mitochondrial distribution (33). Because E-cadherin is vitally important to compaction and blastulation, its altered distribution may partly explain the association between extensive fragmentation and reduced blastocyst formation (34–39). Not only does increasing fragmentation result in reduced blastocyst formation in general, fragmentation apparently can influence allocation of cells during differentiation (37). Cell number was reduced and the reduction was either confined to the trophectoderm (in case of minimal fragmentation) or included the inner cell mass as well (in case of fragmentation exceeding 25%). After blastulation, fragments positioned between the trophoblast and the zona pellucida may interfere with the normal hatching process (40). This may provide another explanation for reduced viability of fragmented embryos. Fragmented embryos may have lower mitochondrial DNA (mtDNA) levels (41) and a different mitochondrial distribution pattern compared with nonfragmented embryos, with mitochondria

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FIGURE 1 Laser scanning confocal (A, B) and differential interference contrast (C) images of cleavage-stage human embryos showing normal cleavage (A), fragmentation and degeneration (B), and type 4 fragments (C). Three normal mitotic spindles are visible in A, and the cytoskeletal structure in the interphase cells is organized. By contrast, the fragmented embryo in B shows disorganized cytoskeletal structure—one abnormal nucleus and one cell in anaphase. (C) Predominantly large fragments (type 4) are interspersed among blastomeres of varying sizes. Green stain is tubulin, and red stain is DNA. Immunofluorescence staining methods are described by Alikani et al. (7, 9).

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remaining concentrated near the center rather than the periphery of the blastomere (42). This pattern may be linked to reduced adenosine triphosphate (ATP) content and reduced developmental potential (43). Reduced ATP content and lower mitochondrial concentrations within the blastomere may also adversely affect membrane integrity, with subsequent cellular lysis occurring via disruption of plasma membrane ion pump function (11). Apart from the degree of fragmentation, the distribution and size of the fragments are considered to be important (13, 44–46). A number of distinct fragmentation patterns have been described (13, 44), including type 1, few small fragments typically associated with only one blastomere; type 2, many small and localized fragments associated with one or more cells; type 3, small and scattered fragments associated with multiple cells; and type 4, large and scattered fragments associated with several unevenly sized cells. It has been shown that fragmentation type is associated with cell number; certain types, particularly those with localized (type 2) or large scattered fragments (type 4; Fig. 1C), have significantly fewer cells than others (8, 13). One study found higher chromosomal abnormalities in embryos with a scattered rather than localized pattern of fragmentation (23).

EMBRYO FRAGMENTATION AND CLINICAL OUTCOMES Studies that involve assessment of relative degrees of fragmentation are difficult to strictly interpret and even more difficult to compare because of their subjective nature. However, although the exact numeric values assigned to fragmentation are subject to inter- and intraobserver variation, the conclusion that the extent of fragmentation and implantation potential are inversely related has been firmly established across all the studies (3, 13, 45, 47–56). Staessen et al. (57) reported a threshold of 20% fragmentation to demonstrate a difference in implantation rates. Alikani and colleagues assessed homogeneous transfers of fragmented embryos and reported that the highest implantation rates were associated with minimally fragmented embryos with 0–15% fragmentation. Implantation was significantly reduced after transfer of embryos with >15% fragmentation, despite removal of the fragments before transfer (8, 13).

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Furthermore, the pattern of fragmentation was found to influence implantation rate, with larger asymmetrical fragments (type 4; Fig. 1C) leading to the most substantial reduction in implantation (8, 13). Other studies have confirmed the relationship between fragmentation and implantation after adjusting for potential confounding variables, such as maternal age (58, 59). Giorgetti et al. (49) evaluated single day-2 ETs and found fragmentation to negatively influence clinical pregnancy rates. The study by Pelinck et al. (58) established a correlation between the degree of fragmentation, cell number on day 2 or day 3, and implantation rate in unselected single ETs from modified natural cycles. The clinical outcome assessed by Pelinck et al. (58) was the presence of a viable intrauterine gestation at 12 weeks. On the basis of these and other clinical studies, it is concluded that cleavage-stage embryos with <10% fragmentation have the highest implantation potential and result in higher clinical pregnancy rates (58, 60–62). Moreover, collectively, these studies demonstrate that significant and sustained fragmentation during early embryo development reduces the viability of IVF embryos and that this reduction can be primarily explained by association of fragmentation with other cytoplasmic and nuclear abnormalities that lead to developmental arrest—most often before blastulation. It is currently unclear whether fragmented embryos increase the risk of clinical miscarriages (49, 54, 56, 57, 59). However, some investigators have suggested that this may indeed be the case (8, 56, 63). An analysis of 43 cases (over the course of more than a decade in one clinic) in which only embryos with >35% fragmentation were transferred after fragment removal showed that biochemical pregnancies and early miscarriages were the most common outcomes (10 of 13 clinical pregnancies, or 77%), although three apparently healthy live births also resulted. By contrast, a large majority of transfers in which the embryos had minimal fragmentation (n ¼ 1,874) led to live birth (73%) (63). In consideration of the risk of physical and psychological trauma associated with unsuccessful pregnancies, the authors suggested that ‘‘patients for whom only embryos with extensive fragmentation are available should be counseled and warned of the high likelihood

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of a negative outcome following transfer.’’ One study suggested that increased fragmentation was also associated with a higher risk of congenital malformations (47); however, it is premature to conclude any association between embryo fragmentation and birth defect risk without further study.

ROLE OF THE OOCYTE IN EMBRYO FRAGMENTATION Several thorough reviews of sperm quality and embryo development have been previously published (64, 65); here we will focus on the role of the oocyte in embryo fragmentation. That oocyte quality is a key determinant of embryo quality is indisputable. Some studies suggest a correlation between oocyte morphology and embryo fragmentation (66–68), but more accurately stated, embryo development is correlated with oocyte cytoplasmic and nuclear competence. One measure of cytoplasmic competence may be mtDNA content (43, 69–72). It has been postulated that normal mitochondrial function and the absence of mitochondrial mutations are associated with better oocyte developmental potential (73, 74). Unfertilized oocytes and fragmented embryos may have reduced mtDNA levels (41, 70); interestingly, increasing maternal age also leads to reduced mtDNA level (42, 75). Given the role of mitochondria in the generation of ATP through electron transport, mitochondrial and ATP content of oocytes seem to be important determinants of development potential (43, 76). Both cytoplasmic and nuclear competence may be influenced by exogenous gonadotropin administration, but it is not clear whether particular protocols lead to higher or lower fragmentation rates. Ziebe et al. (77) reported that 61% of embryos generated after ovarian hyperstimulation with gonadotropins had <10% fragmentation on day 2 of development, compared with 69% of embryos from nonstimulated natural cycles; this difference was not statistically significant. In the MERIT (Menotrophin vs. Recombinant FSH In vitro fertilization Trial) study, an hMG protocol resulted in 83% of embryos with <20% fragmentation, which was significantly greater than the proportion after FSH stimulation (77%) (78). A previous study did not demonstrate substantive differences in embryo quality between hMG and recombinant FSH stimulation (79). Furthermore, Ng et al. (80) did not see differences in embryo quality between high and low responders to gonadotropin stimulation. A number of studies have focused on follicular environment as a potential determinant of oocyte competence and embryo quality. In particular, apoptotic granulosa cells have been suggested to influence embryo fragmentation (81, 82). Follicles with increased apoptotic bodies in both mural and cumulus granulosa cells were associated with reduced embryo quality (82). Furthermore, caspase activity in granulosa cells correlated positively with mean fragmentation rate in cohorts of IVF embryos (81). Apoptotic markers in cumulus and mural granulosa cells seem to be different after GnRH analog and gonadotropin stimulation compared with gonadotropin only or natural cycles (83). Kaneko et al. (83) found increased proportions of apoptotic cells with the GnRH analog approach, suggesting that GnRH analog treatment may adversely influence oocyte quality (83). Although these studies are intriguing, the exact relationship between follicular apoptosis and oocyte quality remains to be better defined. Furthermore, we conclude that there is not enough evidence to clearly link various gonadotropin stimulation regimens with embryo fragmentation outcomes.

PATHOGENESIS OF EMBRYO FRAGMENTATION A number of hypotheses have been proposed to explain the origin of fragmentation in oocytes and embryos with major emphasis having Fertility and Sterility

been placed on programmed cell death or apoptosis (84). Apoptotic gene expression, including Bcl-2, Bclx, Bax, Fas, and various caspases, has been documented in human embryos (85–91), and proapoptotic genes Harakiri and Caspase-3 have been shown to be overexpressed in some human embryos with substantial fragmentation (86). Increased annexin V staining, an early marker of apoptosis, and terminal deoxynucleotidyl transferase mediated X-deoxyuridine triphosphate nick end labeling (TUNEL) staining have been shown in fragmented embryos (21, 84) but not consistently (44). Apoptotic changes have also been observed in the human blastocyst (92). Nonetheless, none of these findings conclusively show that fragmentation represents apoptosis, though they perhaps demonstrate that apoptosis can occur in human embryos. An alternative and more plausible hypothesis is that apoptosis occurs selectively as a consequence of fragmentation (44). Another hypothesis on the origin of fragmentation concerns telomere length. Telomeres are regions of repetitive DNA at the end of chromosomes that function in protecting chromosomes from damage. Keefe and colleagues (93, 94) correlated telomere length in germinal vesicle-stage (GV) human oocytes with mean fragmentation in sibling transferred embryos (93). Maximum telomere lengths in spare GV oocytes predicted 20% of variation in embryo fragmentation after controlling for age and basal FSH level (93). However, the relevance of telomere length differences in GVoocytes to sibling oocyte development is vague, and the telomere length hypothesis remains largely unsubstantiated. Increasing maternal age has been debated as a potential cause of increased fragmentation, with no definitive conclusion having been drawn. In a clinical study using a large embryology database, Alikani et al. (13) suggested an inverse relationship between maternal age and embryo fragmentation, whereas other studies have shown no association (49, 95) or an increase in embryo fragmentation with advancing maternal age (39, 96, 97). Two independent experimental studies in the mouse have pointed to abnormalities in the cytoskeleton and microtubule organization as key features in fragmentation. Alikani (32) and Liu and Keefe (98) used a mouse model to show that meiotic exit and oocyte activation were prerequisites to fragmentation after enucleation (32, 98) or treatment of oocytes with anticancer drugs (98). Furthermore, Alikani et al. (32) were able to induce fragmentation in oocytes during the cytokinetic phase of mitosis I and meiosis II. This led to the conclusion that fragmentation is aberrant division due to cytoskeletal disorder rather than a manifestation of apoptosis, as previously suggested. Liu and Keefe (98), on the other hand, concluded that fragmentation was an apoptotic process resulting from DNA damage and proposed a model of fragmentation according to which the failure of chromosomes and/or centromeres to capture microtubules and form a functional spindle would lead to multiple furrow formation and fragmentation. Although the two studies differ in their conclusions with respect to the cause of fragmentation, they agree that fragmentation primarily involves the cytoskeleton and that the process is inhibited if either actin filaments or microtubules are made to depolymerize. It can be further concluded that fragmentation does not occur if a cell is forced into mitotic arrest. There is controversy surrounding the role of reactive oxygen species in the developmental potential of the human oocyte. Embryos with increased fragmentation exhibit greater oxidative stress (OS) in culture (99, 100). However, recent analyses of follicular fluid (FF) enzymatic activities involved in the glutathione synthesis pathway, an important mechanism counteracting OS, suggest no associations between OS biomarkers and embryo fragmentation (101). Another OS enzyme, paraoxonase (PON) 1,

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identified in FF high-density lipoprotein (HDL) particles, has been shown to correlate with cell number but not fragmentation (102). Although reactive oxygen species detected in culture media positively correlate with fragmentation (99, 100), it is not clear to what extent follicular determinants or in vitro culture influence development. Browne et al. (102) reported a negative correlation between HDL cholesterol, apolipoprotein AI (ApoAI) levels and embryo fragmentation (Fig. 2). Other HDL particle-related enzyme activities, including PON activity, as well as triglyceride and phospholipid levels, were also investigated but not found to be associated with embryo fragmentation. An expanded dataset was subsequently published confirming the inverse relationship between FF HDL cholesterol and embryo fragmentation along with the HDL-associated micronutrients a-tocopherol and b-crytoxanthin (103). These multiple HDL component associations with embryo fragmentation strongly suggest a role for FF HDL particles. After adjusting for HDL cholesterol, no other HDL particle component measurements were predictors of embryo fragmentation, leading to the hypothesis that intrafollicular cholesterol metabolism may be primarily responsible for these observations (104). Follicular fluid sphingosine1-phosphate, another major lipid component of HDL particles, has been shown to induce angiogenesis, offering an indirect explanation

for improved follicular vascularity associated with HDL particles (105). A recent study suggests this membrane lipid may influence in vitro cleavage patterns (106). Although the role of cholesterol metabolism in steroidogenesis has been known (107–111), these data provided the first evidence that intrafollicular cholesterol metabolism may influence oocyte competence. Furthermore, proportional composition of cholesterol and phospholipids within FF HDL particles seems to be important in identifying an oocyte with intact lipid membrane properties (112) (Table 1). It is hypothesized that the maturation of the oocyte membrane may be tightly regulated during follicular maturation (104). The proportional composition of phospholipids and cholesterol in FF HDL particles serves as a potential surrogate marker for cholesterol transport between cumulus granulosa cells and the oocyte. This transport probably occurs concurrent with the LH surge, influencing membrane integrity (112). Although phosphatidylcholine and sphingomyelin are the principal lipids within the cell membrane (113), cholesterol plays a key role in the viability and proliferation of cells (114). Cholesterol trafficking and compartmentalization within the cellular membrane is complex and tightly regulated through de novo synthesis and extracellular cholesterol transport (114). Membrane lipid rafts are essential for cholesterol homeostasis (115) and important for normal oocyte development (116–118). The importance of lipid rafts during early

FIGURE 2 Embryo fragmentation score as a function of unadjusted FF concentrations of HDL particle components, cholesterol, and ApoAI. Reprinted with permission from Fujimoto et al. (104).

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TABLE 1 Proportional HDL particle lipid composition and embryo fragmentation as assessed by Spearman correlations with embryo fragmentation score (rEFS). Parameter

n

Mean

SD

Minimum

Median

Maximum

rEFS

P value

Embryo fragmentation score ApoAI (%) Phospholipids (PL) (%) Total cholesterol (TC) (%) Unesterified cholesterol (UC) (%) Cholesterol esters (%) UC/TC ratio Triglycerides (%) PL/TC ratio

39 39 39 39 31 31 31 38 39

2.05 40.98 30.77 25.24 7.76 18.28 0.29 3.09 1.35

1.00 5.71 3.16 7.01 5.29 6.23 0.18 2.62 0.55

1.00 31.05 24.35 8.42 0.70 6.94 0.03 0.00 0.67

2.00 42.25 30.63 24.23 7.35 17.76 0.29 2.73 1.26

5.00 51.78 37.71 39.33 16.52 33.93 0.60 13.32 3.70

n/a 0.12 0.29 0.35 0.07 0.22 0.03 0.16 0.38

n/a .484 .075 .027a .692 .225 .868 .323 .016a

Note: Modified from Zamah et al. (112), with permission from Elsevier. Study data in this table were obtained with prior approval from the University of California, San Francisco Committee on Human Research. a Significant correlation. Fujimoto. Origins of human embryo fragmentation. Fertil Steril 2011.

embryo cleavage is suggested by the accumulation of cholesterol-rich domains directly adjacent to the cleavage furrow coincident with the appearance of the actin-based contractile ring before cytokinesis (119, 120). It seems that fragmentation occurs primarily near the cleavage furrow in embryos, but it may occur more diffusely away from the main furrow. Several studies have confirmed the incorporation of new membrane during cytokinesis, requiring transport and incorporation of cholesterol and other lipids from organelles (121–123).

Given the role of cholesterol in blastomere membrane biology, the movement of cholesterol and phospholipids within the blastomere membrane is critically important for normal cytokinetic activity (117, 118, 120, 124), with cholesterol depletion resulting in cytokinetic arrest (125). Hence, it is plausible that fragmentation appears with aberrant cholesterol homeostasis in the cholesterol microdomains of the blastomere membrane (126). A physiologic phospholipid/cholesterol ratio in cell membranes is necessary to maintain proper function and membrane fluidity (115). It is an

FIGURE 3 Current understanding of human embryo fragmentation during IVF and the negative clinical consequences associated with this anomaly.

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intriguing observation that this ratio observed in FF HDL particles is correlated with embryo fragmentation (Table 1). Mammalian oocytes do not generate intracellular cholesterol de novo, lacking the necessary biosynthetic enzymes to convert acetate into cholesterol (127). Thus, oocyte membrane maturation occurs through a coordinated process involving the delivery of cholesterol to the membrane from surrounding cumulus cells (127). This lack of intrinsic cholesterol biosynthesis places the burden of oocyte cholesterol incorporation on cholesterol transport between the oocyte and its surrounding cumulus granulosa cells. The upregulation of cholesterol biosynthesis gene expression, including 14 a-demethylase, D14-reductase, and D7-reductase activities, in cumulus cells occurring with the LH surge seems to be important for oocyte meiotic maturation and germinal vesicle breakdown (128, 129). The downstream production of cholesterol may be important to oocyte membrane maturation and stabilization. The efficiency of cholesterol efflux into HDL particles may play an important role in oocyte membrane development, most likely via ABCA1 transporter proteins (104). Cholesterol toxicity in membranes due to poorly regulated intracellular cholesterol metabolism is well documented (130). This could also potentially explain the observed correlations between FF HDL cholesterol levels, FF HDL phospholipid/cholesterol ratios, and embryo fragmentation.

SUMMARY AND CONCLUSIONS This review provides some insights into the problem of embryo fragmentation during therapeutic IVF. It is clear that the presence of significant numbers of fragments, particularly in conjunction with discrepancies in blastomere symmetry, substantially reduces embryo viability and negatively impacts clinical outcome. We believe that the origins of fragmentation may be traced primarily to the oocyte. Figure 3 summarizes our current understanding of embryo fragmentation and its developmental and clinical correlations. Experimental evidence suggests that fragmentation occurs in lieu of normal cytokinesis and coincides with cytoskeletal disorder. Similar to cytokinesis, fragmentation only occurs in meiotically or mitotically active cells and can be inhibited by microtubule and/or microfilament depolymerization. Recent clinical evidence further suggests that fragmentation may result from disruptions in oocyte membrane integrity, which itself may result from aberrant lipid metabolism within the Graafian follicle. It remains to be determined whether these membrane abnormalities lead to specific types of fragmentation or the effect is more general. A better understanding of the interplay between oocyte membrane lipid composition and cytoskeletal dynamics and organization during cytokinesis may lead to novel approaches to the prevention of fragmentation.

REFERENCES 1. Cummins J, Breen T, Harrison K, Shaw J, Wilson L, Hennessey J. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J In Vitro Fert Embryo Transf 1986;3:284–95. 2. Edwards R, Fishel S, Cohen J, Fehilly C, Purdy J, Slater J, et al. Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fert Embryo Transf 1984;1:3–23. 3. Puissant F, Van Rysselberge M, Barlow P, Deweze J, Leroy F. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod 1987;2:705–8. 4. Trounson A, Sathananthan A. The application of electron microscopy in the evaluation of two- to four-cell human embryos cultured in vitro for embryo transfer. J In Vitro Fert Embryo Transf 1984;1:153–65. 5. Steer CV, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum Reprod 1992;7:117–9. 6. Veeck L. Atlas of the human oocyte and early conceptus, Vol. 2. Baltimore, MD: Williams & Wilkins; 1991;41–149. 7. Alikani M. On fragmentation: origin and consequences of abnormal division in human embryos in vitro [dissertation]. Clayton, Australia: Monash University; 2006. 8. Alikani M. The origins and consequences of fragmentation in mammalian eggs and embryos. In: Elder K, Cohen J, editors. Human preimplantation embryo selection. London: Informa Healthcare; 2007. p. 51–78. 9. Alikani M, Schimmel T, Willadsen SM. Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder. Mol Hum Reprod 2005;11:335–44.

1202

Fujimoto et al.

10. Mio Y, Maeda K. Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos. Am J Obstet Gynecol 2008;199:660.e1–5. 11. Van Blerkom J, Davis P, Alexander S. A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos. Hum Reprod 2001;16:719–29. 12. Hardarson T, L€ofman C, Coull G, Sj€ogren A, Hamberger L, Edwards R. Internalization of cellular fragments in a human embryo: timelapse recordings. Reprod Biomed Online 2002;5:36–8. 13. Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott RT. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999;71:836–42. 14. Goyanes VJ, Ron-Corzo A, Costas E, Maneiro E. Morphometric categorization of the human oocyte and early conceptus. Hum Reprod 1990;5:613–8. 15. Hnida C, Ziebe S. Total cytoplasmic volume as biomarker of fragmentation in human embryos. J Assist Reprod Genet 2004;21:335–40. 16. Johansson M, Hardarson T, Lundin K. There is a cutoff limit in diameter between a blastomere and a small anucleate fragment. J Assist Reprod Genet 2003;20:309–13. 17. Balakier H, Cadesky K. The frequency and developmental capability of human embryos containing multinucleated blastomeres. Hum Reprod 1997;12:800–4. 18. Hardy K, Winston RML, Handyside AH. Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. J Reprod Fertil 1993;98:549–58. 19. Kligman I, Benadiva C, Alikani M, Munne S. The presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities. Hum Reprod 1996;11:1492–8. 20. Laverge H, De Sutter P, Verschraegen-Spae MR, De Paepe A, Dhont M. Triple colour fluorescent

Origins of human embryo fragmentation

21.

22.

23.

24.

25.

26.

27.

28.

29.

in-situ hybridization for chromosomes X, Y and 1 on spare human embryos. Hum Reprod 1997; 12:809–14. Levy R, Benchaib M, Cordonier H, Souchier C, Guerin JF. Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos. Mol Hum Reprod 1998;4:775–83. Magli MC, Gianaroli L, Ferraretti AP. Chromosomal abnormalities in embryos. Mol Cell Endocrinol 2001;183:S29–34. Magli MC, Gianaroli L, Ferraretti AP, Lappi M, Ruberti A, Farfalli V. Embryo morphology and development are dependent on the chromosomal complement. Fertil Steril 2007;87:534–41. Marquez C, Sandalinas M, Bahce M, Alikani M, Munne S. Chromosome abnormalities in 1255 cleavage-stage human embryos. Reprod Biomed Online 2000;1:17–26. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64:382–91. Munne S, Chen S, Colls P, Garrisi J, Zheng X, Cekleniak N, et al. Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage-stage embryos. Reprod BioMed Online 2007;14:628–34. Pellestor F, Girardet A, Andreo B, Arnal F, Humeau C. Relationship between morphology and chromosomal constitution in human preimplantation embryo. Mol Reprod Dev 1994;39:141–6. Munne S. Chromosome abnormalities and their relationship to morphology and development of human embryos. Reprod Biomed Online 2006;12:234–53. Staessen C, Nagy ZP, Liu J, Janssenswillen C, Camus M, Devroey P, et al. One year’s experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes. Hum Reprod 1995;10:3305–12.

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30. Formigli L, Roccio C, Belotti G, Stangalini A, Coglitore MT, Formigli G. Non-surgical flushing of the uterus for pre-embryo recovery: possible clinical applications. Hum Reprod 1990;5:329–35. 31. Winston NJ, Johnson MH. Can the mouse embryo provide a good model for the study of abnormal cellular development seen in human embryos? Hum Reprod 1992;7:1291–6. 32. Alikani M. Epithelial cadherin distribution in abnormal human pre-implantation embryos. Hum Reprod 2005;20:3369–75. 33. Neganova IE, Sekirina GG, Eichenlaub-Ritter U. Surface-expressed E-cadherin, and mitochondrial and microtubule distribution in rescue of mouse embryos from 2-cell block by aggregation. Mol Hum Reprod 2000;6:454–64. 34. Alikani M, Calderon G, Tomkin G, Garrisi J, Kokot M, Cohen J. Cleavage anomalies in early human embryos and survival after prolonged culture in-vitro. Hum Reprod 2000;15:2634–43. 35. della Ragione T, Verheyen G, Papanikolaou EG, Van Landuyt L, Devroey P, Van Steirteghem A. Developmental stage on day-5 and fragmentation rate on day-3 can influence the implantation potential of top-quality blastocysts in IVF cycles with single embryo transfer. Reprod Biol Endocrinol 2007;5:2–9. 36. Eftekhari-Yazdi P, Valojerdi M, Ashtiani S, Eslaminejad M, Karimian L. Effect of fragment removal on blastocyst formation and quality of human embryos. Reprod Biomed Online 2006;13:823–32. 37. Hardy K, Stark J, Winston RML. Maintenance of the inner cell mass in human blastocysts from fragmented embryos. Biol Reprod 2003;68:1165–9. 38. Rijnders PM, Jansen CAM. The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 1998;13:2869–73. 39. Stone BA, Greene J, Vargyas JM, Ringler GE, Marrs RP. Embryo fragmentation as a determinant of blastocyst development in vitro and pregnancy outcomes following embryo transfer. Am J Obstet Gynecol 2005;192:2014–9. 40. Sathananthan H, Gunasheela S, Menezes J. Critical evaluation of human blastocysts for assisted reproduction techniques and embryonic stem cell biotechnology. Reprod Biomed Online 2003;7:219–27. 41. Lin DP, Huang CC, Wu HM, Cheng TC, Chen CI, Lee MS. Comparison of mitochondrial DNA contents in human embryos with good or poor morphology at the 8-cell stage. Fertil Steril 2004;81:73–9. 42. Wilding M, Dale B, Marino M, di Matteo L, Alviggi C, Pisaturo ML, et al. Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum Reprod 2001;16: 909–17. 43. Van Blerkom J, Davis PW, Lee J. ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 1995;10:415–24. 44. Antczak M, Van Blerkom J. Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum Reprod 1999;14:429–47. 45. Claman P, Armant D, Seibel M, Wang T, Oskowitz S, Taymor M. The impact of embryo quality and quantity on implantation and the establishment of viable pregnancies. J In Vitro Fert Embryo Transf 1987;4:218–22. 46. Warner CM, Cao W, Exley GE, McElhinny AS, Alikani M, Cohen J, et al. Genetic regulation of egg and embryo survival. Hum Reprod 1998;13:178–90.

Fertility and Sterility

47. Ebner T, Yaman C, Moser M, Sommergruber M, Polz W, Tews G. Embryo fragmentation in vitro and its impact on treatment and pregnancy outcome. Fertil Steril 2001;76:281–5. 48. Erenus M, Zouves C, Rajamahendran P, Leung S, Fluker M, Gomel V. The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization. Fertil Steril 1991;56:707–10. 49. Giorgetti C, Terriou P, Auquier P, Hans E, Spach JL, Salzmann J, et al. Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Hum Reprod 1995;10: 2427–31. 50. Holte J, Berglund L, Milton K, Garello C, Gennarelli G, Revelli A, et al. Construction of an evidence-based integrated morphology cleavage embryo score for implantation potential of embryos scored and transferred on day 2 after oocyte retrieval. Hum Reprod 2007;22:548–57. 51. Roseboom TJ, Vermeiden JPW, Schoute E, Lens JW, Schats R. The probability of pregnancy after embryo transfer is affected by the age of the patient, cause of infertility, number of embryos transferred and the average morphology score, as revealed by multiple logistic regression analysis. Hum Reprod 1995;10:3035–41. 52. Shulman A, Ben-Nun I, Ghetler Y, Kaneti H, Shilon M, Beyth Y. Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertil Steril 1993;60:123–6. 53. Staessen C, Janssenswillen C, van Den Abbeel E, Devroey P, van Steirteghem AC. Avoidance of triplet pregnancies by elective transfer of two good quality embryos. Hum Reprod 1993;8: 1650–3. 54. Visser DS, Fourie FR. The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme. Hum Reprod 1993;8:1719–22. 55. Volpes A, Sammartano F, Coffaro F, Mistretta V, Scaglione P, Allegra A. Number of good quality embryos on day 3 is predictive for both pregnancy and implantation rates in in vitro fertilization/intracytoplasmic sperm injection cycles. Fertil Steril 2004;82:1330–6. 56. Ziebe S, Petersen K, Lindenberg S, Andersen AG, Gabrielsen A, Andersen AN. Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization. Hum Reprod 1997;12:1545–9. 57. Staessen C, Camus M, Bollen N, Devroey P, Van Steirteghem A. The relationship between embryo quality and the occurrence of multiple pregnancies. Fertil Steril 1992;57:626–30. 58. Pelinck MJ, Hoek A, Simons AH, Heineman MJ, van Echten-Arends J, Arts EG. Embryo quality and impact of specific embryo characteristics on ongoing implantation in unselected embryos derived from modified natural cycle in vitro fertilization. Fertil Steril 2010;94:527–34. 59. Shen S, Rosen MP, Dobson AT, McCulloch CE, Rinaudo P, Cedars MI. While day 3 embryo fragmentation significantly impacts implantation, it has no impact on clinical pregnancy loss. Fertil Steril 2005;84:S290–1. 60. Kashyap S, Cedars M, Shen S, Rosen M, Grady D, Wells G. Real data mathematical modeling to predict successful embryo implantation. Fertil Steril 2008;90:S224. 61. Racowsky C, Combelles CMH, Nureddin A, Pan Y, Finn A, Miles L, et al. Day 3 and day 5 morphological predictors of embryo viability. Reprod BioMed Online 2003;6:323–31.

62. Racowsky C, Ohno-Machado L, Kim J, Biggers JD. Is there an advantage in scoring early embryos on more than one day? Hum Reprod 2009;24:2104–13. 63. Alikani M, Tomkin G, Ferry K, Garrisi M. High pregnancy loss rate following implantation of IVF human embryos with extensive fragmentation. Fertil Steril 2008;90(Suppl):S113–4. 64. Barroso G, Valdespin C, Vega E, Kershenovich R, Avila R, Avenda~no C, et al. Developmental sperm contributions: fertilization and beyond. Fertil Steril 2009;92:835–48. 65. Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril 2010;93:1027–36. 66. Ebner T, Moser M, Sommergruber M, Yaman C, Pfleger U, Tews G. First polar body morphology and blastocyst formation rate in ICSI patients. Hum Reprod 2002;17:2415–8. 67. Ebner T, Yaman C, Moser M, Sommergruber M, Feichtinger O, Tews G. Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum Reprod 2000;15:427–30. 68. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod 1997;12:1750–5. 69. Ramalho-Santos J, Varum S, Amaral S, Mota PC, Sousa AP, Amaral A. Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells. Hum Reprod Update 2009;15:553–72. 70. Santos TA, El Shourbagy S, St. John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertil Steril 2006;85:584–91. 71. Van Blerkom J, Davis P, Alexander S. Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence. Hum Reprod 2000;15:2621–33. 72. Van Blerkom J, Davis P, Alexander S. Inner mitochondrial membrane potential (Dym), cytoplasmic ATP content and free Ca2þ levels in metaphase II mouse oocytes. Hum Reprod 2003;18: 2429–40. 73. Cummins JM. Mitochondria: potential roles in embryogenesis and nucleocytoplasmic transfer. Hum Reprod Update 2001;7:217–28. 74. Van Blerkom J. Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction 2004;128:269–80. 75. Seifer DB, DeJesus V, Hubbard K. Mitochondrial deletions in luteinized granulosa cells as a function of age in women undergoing in vitro fertilization. Fertil Steril 2002;78:1046–8. 76. Stojkovic M, Machado SA, Stojkovic P, Zakhartchenko V, Hutzler P, Goncalves PB, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod 2001;64:904–9. 77. Ziebe S, Bangsboll S, Schmidt KLT, Loft A, Lindhard A, Nyboe Andersen A. Embryo quality in natural versus stimulated IVF cycles. Hum Reprod 2004;19:1457–60. 78. Ziebe S, Lundin K, Janssens R, Helmgaard L, Arce JC. MERIT (Menotrophin vs Recombinant FSH in vitro Fertilisation Trial) Group. Influence

1203

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92. 93.

94.

of ovarian stimulation with HP-hMG or recombinant FSH on embryo quality parameters in patients undergoing IVF. Hum Reprod 2007;22:2404–13. Ng E, Lau E, Yeung W, Ho P. HMG is as good as recombinant human FSH in terms of oocyte and embryo quality: a prospective randomized trial. Hum Reprod 2001;16:319–25. Ng EHY, Lau EYL, Yeung WSB, Ho PC. Oocyte and embryo quality in patients with excessive ovarian response during in vitro fertilization treatment. J Assist Reprod Genet 2003;20:186–91. Bencomo E, Perez R, Arteaga MF, Acosta E, Pena O, Lopez L, et al. Apoptosis of cultured granulosa-lutein cells is reduced by insulin-like growth factor I and may correlate with embryo fragmentation and pregnancy rate. Fertil Steril 2006;85:474–80. Nakahara K, Saito H, Saito T, Ito M, Ohta N, Takahashi T, et al. The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 1997;68:312–7. Kaneko T, Saito H, Takahashi T, Ohta N, Saito T, Hiroi M. Effects of controlled ovarian hyperstimulation on oocyte quality in terms of the incidence of apoptotic granulosa cells. J Assist Reprod Genet 2000;17:580–5. Jurisicova A, Varmuza S, Casper RF. Programmed cell death and human embryo fragmentation. Mol Hum Reprod 1996;2:93–8. Jurisicova A, Acton BM. Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development. Reproduction 2004;128:281–91. Jurisicova A, Antenos M, Varmuza S, Tilly JL, Casper RF. Expression of apoptosis-related genes during human preimplantation embryo development: potential roles for the Harakiri gene product and Caspase-3 in blastomere fragmentation. Mol Hum Reprod 2003;9:133–41. Jurisicova A, Latham KE, Casper RF, Varmuza SL. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol Reprod Dev 1998;51:243–53. Liu HC, He ZY, Mele CA, Veeck LL, Davis O, Rosenwaks Z. Expression of apoptosis-related genes in human oocytes and embryos. J Assist Reprod Genet 2000;17:521–33. Metcalfe AD, Hunter HR, Bloor DJ, Lieberman BA, Picton HM, Leese HJ, et al. Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation. Mol Reprod Dev 2004;68:35–50. Spanos S, Rice S, Karagiannis P, Taylor D, Becker DL, Winston RM, et al. Caspase activity and expression of cell death genes during development of human preimplantation embryos. Reproduction 2002;124:353–63. Yang MY, Rajamahendran R. Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro. Anim Reprod Sci 2002;70:159–69. Hardy K. Cell death in the mammalian blastocyst. Mol Hum Reprod 1997;3:919–25. Keefe DL, Franco S, Liu L, Trimarchi J, Cao B, Weitzen S, et al. Telomere length predicts embryo fragmentation after in vitro fertilization in women— toward a telomere theory of reproductive aging in women. Am J Obstet Gynecol 2005;192:1256–60. Liu L, Blasco MA, Trimarchi JR, Keefe DL. An essential role for functional telomeres in mouse germ cells during fertilization and early development. Dev Biol 2002;249:74–84.

1204

Fujimoto et al.

~ 95. Stensen MH, Tanbo T, Storeng R, Abyholm T, Fedorcsak P. Routine morphological scoring systems in assisted reproduction treatment fail to reflect age-related impairment of oocyte and embryo quality. Reprod BioMed Online 2010; 21:118–25. 96. Keltz MD, Skorupski JC, Bradley K, Stein D. Predictors of embryo fragmentation and outcome after fragment removal in in vitro fertilization. Fertil Steril 2006;86:321–4. 97. Ziebe S, Loft A, Petersen JH, Andersen AG, Lindenberg S, Petersen K, et al. Embryo quality and developmental potential is compromised by age. Acta Obstet Gynecol Scand 2001;80:169–74. 98. Liu L, Keefe DL. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum Reprod 2002;17:2678–85. 99. Bedaiwy MA, Falcone T, Mohamed MS, Aleem AA, Sharma RK, Worley SE, et al. Differential growth of human embryos in vitro: role of reactive oxygen species. Fertil Steril 2004;82:593–600. 100. Bedaiwy MA, Mahfouz RZ, Goldberg JM, Sharma R, Falcone T, Abdel Hafez MF, et al. Relationship of reactive oxygen species levels in day 3 culture media to the outcome of in vitro fertilization/intracytoplasmic sperm injection cycles. Fertil Steril 2010;94:2037–42. 101. Shelly WB, Browne RW, Bloom MS, Sandler JR, Huddleston HG, Fujimoto VY. Antioxidant enzymes glutathione peroxidase and glutathione reductase correlate with human embryo cell number. Fertil Steril 2008;90:S101. 102. Browne RW, Shelly WB, Bloom MS, Ocque AJ, Sandler JR, Huddleston HG, et al. Distributions of high-density lipoprotein particle components in human follicular fluid and sera and their associations with embryo morphology parameters during IVF. Hum Reprod 2008;23:1884–94. 103. Browne RW, Bloom MS, Shelly WB, Ocque AJ, Huddleston HG, Fujimoto VY. Follicular fluid high density lipoprotein-associated micronutrient levels are associated with embryo fragmentation during IVF. J Assist Reprod Genet 2009;26:557–60. 104. Fujimoto VY, Kane JP, Ishida BY, Bloom MS, Browne RW. High-density lipoprotein metabolism and the human embryo. Hum Reprod Update 2010;16:20–38. 105. von Otte S, Paletta JRJ, Becker S, Konig S, Fobker M, Greb RR, et al. Follicular fluid high density lipoprotein-associated sphingosine 1-phosphate is a novel mediator of ovarian angiogenesis. J Biol Chem 2006;281:5398–405. 106. Hannoun A, Ghaziri G, Musa AA, Zreik TG, Hajameh F, Awwad J. Addition of sphingosine-1phosphate to human oocyte culture medium decreases embryo fragmentation. Reprod BioMed Online 2010;20:328–34. 107. Azhar S, Reaven E. Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis. Mol Cell Endocrinol 2002;195:1–26. 108. Azhar S, Tsai L, Medicherla S, Chandrasekher Y, Giudice L, Reaven E. Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis. J Clin Endocrinol Metab 1998;83: 983–91. 109. Azhar S, Tsai L, Reaven E. Uptake and utilization of lipoprotein cholesteryl esters by rat granulosa cells. Biochim Biophys Acta 1990;1047:148–60. 110. Glass C, Pittman RC, Weinstein DB, Steinberg D. Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad. Proc Natl Acad Sci U S A 1983;80:5435–9.

Origins of human embryo fragmentation

111. Goldstein JL, Brown MS, Anderson RGW, Russell DW, Schneider WJ. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Ann Rev Cell Biol 1985;1:1–39. 112. Zamah AM, Browne RW, Conti G, Jaggavarapu SR, Sridhar V, Fujimoto VY. High density lipoprotein (HDL) particle size and composition predicts embryo fragmentation. Fertil Steril 2009;92:S96–7. 113. Ohvo-Rekil€a H, Ramstedt B, Leppim€aki P, Slotte JP. Cholesterol interactions with phospholipids in membranes. Prog Lipid Res 2002;41:66–97. 114. Ikonen E. Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 2008;9:125–38. 115. Simons K, Ikonen E. How cells handle cholesterol. Science 2000;290:1721–6. 116. Emoto K, Inadome H, Kanaho Y, Narumiya S, Umeda M. Local change in phospholipid composition at the cleavage rurrow is essential for completion of cytokinesis. J Biol Chem 2005;280:37901–7. 117. Emoto K, Kobayashi T, Yamaji A, Aizawa H, Yahara I, Inoue K, et al. Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis. Proc Natl Acad Sci U S A 1996;93:12867–72. 118. Emoto K, Umeda M. An essential role for a membrane lipid in cytokinesis: regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine. J Cell Biol 2000;149:1215–24. 119. Comiskey M, Warner CM. Spatio-temporal localization of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos. Dev Biol 2007;303:727–39. 120. Ng MM, Chang F, Burgess DR. Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis. Dev Cell 2005;9:781–90. 121. Danilchik MV, Funk WC, Brown EE, Larkin K. Requirement for microtubules in new membrane formation during cytokinesis of xenopus embryos. Dev Biol 1998;194:47–60. 122. Drechsel DN, Hyman AA, Hall A, Glotzer M. A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curr Biol 1997;7:12–23. 123. Shuster CB, Burgess DR. Targeted new membrane addition in the cleavage furrow is a late, separate event in cytokinesis. Proc Natl Acad Sci U S A 2002;99:3633–8. 124. Emoto K, Umeda M. Membrane lipid control of cytokinesis. Cell Struct Funct 2001;26:659–65. 125. Fernandez C, Lobo MdVT, Gomez-Coronado D, Lasuncion MA. Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation. Exp Cell Res 2004;300:109–20. 126. Straight AF, Field CM. Microtubules, membranes and cytokinesis. Curr Biol 2000;10:R760–70. 127. Su YQ, Sugiura K, Wigglesworth K, O’Brien MJ, Affourtit JP, Pangas SA, et al. Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells. Development 2008;135:111–21. 128. Yamashita Y, Nishibori M, Terada T, Isobe N, Shimada M. Gonadotropin-induced D14-reductase and D7-reductase gene expression in cumulus cells during meiotic resumption of porcine oocytes. Endocrinology 2005;146:186–94. 129. Yamashita Y, Shimada M, Okazaki T, Maeda T, Terada T. Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes. Biol Reprod 2003;68:1193–8. 130. Aye IL, Singh AT, Keelan JA. Transport of lipids by ABC proteins: interactions and implications for cellular toxicity, viability and function. Chem Biol Interact 2009;180:327–39.

Vol. 95, No. 4, March 15, 2011