- Email: [email protected]
Age does not influence the effect of embryo fragmentation on successful blastocyst development
Age does not influence the effect of embryo fragmentation on successful blastocyst development We evaluated the rate of blastocyst development in day 3 embryos with appropriate cellular division and investigated whether maternal age modified the effect of embryo fragmentation on blastulation. Our data showed a significant negative correlation between the degree of embryo fragmentation and rate of blastocyst development, but age did not exert an effect on the degree of fragmentation in embryos with appropriate cleaving status, nor did it modify the significant effect embryo fragmentation had on blastocyst formation. (Fertil Steril 2011;95:2778–80. 2011 by American Society for Reproductive Medicine.) Key Words: Embryo fragmentation, blastocyst, in vitro fertilization, age
An age-related decline in female fecundity has been attributed to the impaired quality of aging oocytes, reduced embryo quality, and lower implantation and pregnancy rates (1–3). There is evidence for a direct relationship between the age of women undergoing in vitro fertilization (IVF) and the proportion of anucleate fragmentation in cleavage-stage embryos (4, 5). Although some studies have demonstrated that embryos with more fragmentation have lower rates of reaching blastulation (6, 7) and that patients with increasing maternal age also have lower probability of having blastocysts, it is still unclear whether age plays an additive effect on embryo fragmentation, thus further reducing the chance for blastulation. The objective of the present study was to investigate whether, in embryos with similar degrees of fragmentation, maternal age modifies the effect of embryo fragmentation on successful blastocyst development. A retrospective review of patient and cycle data from all fresh, autologous, and donor-recipient IVF cycles from January 2003 to Diana H. Wu, M.D.a Kasey Reynolds, M.D.b Rose Maxwell, Ph.D.a Steven R. Lindheim, M.D., M.M.M.a Mira Aubuchon, M.D.c Michael A. Thomas, M.D.a a Center for Reproductive Health, University of Cincinnati Academic Health Center, Cincinnati, Ohio b Good Samaritan Hospital, Cincinnati, Ohio c University of Missouri, Columbia, Missouri Received February 14, 2011; revised May 9, 2011; accepted May 10, 2011; published online June 8, 2011. D.H.W. has nothing to disclose. K.R. has nothing to disclose. R.M. has nothing to disclose. S.R.L. has nothing to disclose. M.A. has nothing to disclose. M.A.T. has nothing to disclose. Supported by the Patty Brisben Foundation. Presented at the 57th Annual Meeting of Society for Gynecologic Investigation, Orlando, Florida, March 2010. Reprint requests: Diana H. Wu, M.D., Center for Reproductive Health, University of Cincinnati Academic Health Center, 2123 Auburn Avenue, Suite A-44, Cincinnati, OH 45219 (E-mail: [email protected]).
2778
August 2009 was performed. Institutional Review Board approval was obtained before data collection and analysis. Patients underwent ovarian stimulation with the use of either a luteal-phase GnRH agonist protocol, a GnRH antagonist protocol, or a microdose flare protocol. During these cycles, gonadotropin stimulation was continued until at least two follicles had attained a mean diameter of 18–20 mm. Ultrasound-guided oocyte retrieval was performed 35 hours after administration of either 500 mg of recombinant hCG (Ovidrel; EMD Serono) or 7,500–10,000 IU hCG. After oocyte retrieval, fertilization was performed in IVC-1 media (InVitroCare) by either conventional insemination or intracytoplasmic sperm injection in the presence of male factor infertility. The standard operating procedure during the time frame of data collection was to cryopreserve at least three embryos at the two-pronuclei stage if couples had nine or more fertilized embryos. The remaining six to eight embryos were cultured in IVC-1 medium for the first 72 hours. Embryos were evaluated immediately before transfer on day 3 for cleavage stage and degrees of fragmentation (1 to 4) by the criteria outlined by Veeck, with a fragmentation score of 1 characterized by the absence of cytoplasmic fragments and a score of 4 characterized by significant cytoplasmic fragmentation (8). Depending on female partner age and/or embryo quality, two to three embryos with the best morphology and cleaving status were selected for embryo transfer. Surplus embryos were then transferred to IVC-3 blastocyst medium. These embryos were reevaluated on days 5 and/or 6 for development into suitable blastocysts for cryopreservation. The primary outcome evaluated was the rate of blastocyst formation as a function of fragmentation on day 3 and maternal age. To isolate the effects of fragmentation, only embryos with normal cellular division with six to eight cells on day 3 were included. The rate of blastocyst formation per 6–8-cell cleavage embryo transfered to blastocyst medium was analyzed against the degree of fragmentation on day 3 and maternal age with the use of chi-square test, Pearson correlation, and logistic regression. Age was analyzed with both Pearson correlation as a continuous variable and chi-square test in age groups of <29, 30–34, 35–37, 38–40, and R41 years of age. The division of subjects into age groups for ages R35 years corresponded to groupings used for
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.05.024
both Centers for Disease Control and Society for Assisted Reproductive Technologies data collection, as well as to groupings in American Society for Reproductive Medicine guidelines for the number of embryos to transfer (9). Further division of patients aged <35 years was based on the large proportion of patients in this age group who present for IVF at our center and evidence that an age-related decline in fertility may begin even in the early thirties. An a priori power analysis was performed with an effect size of 11%: the difference reported between the average blastulation rate of embryos with little or no fragmentation (score 1–2) and more fragmented embryos (score 3–4) (7). With power set at 80% and P<.05, a minimum sample size of 177 embryos for each fragmentation score from 1 to 4 was calculated with the use of Epi Info (CDC). A total of 904 cleavage-stage embryos with normal cellular division and fitting other selection criteria were analyzed, which resulted from 285 IVF cycles in 276 patients. The distribution of embryos analyzed per morphology was 158 embryos with a fragmentation score of 1, 328 embryos with a score of 2, 202 embryos with a score of 3, and 216 embryos with a score of 4. Table 1 summarizes the blastocyst formation rate in relation to embryo fragmentation. The average blastulation rate was 36% across all embryos. A significant negative correlation was found between the degree of fragmentation and the rate of blastocyst development (r ¼ 0.28; P<.01). The likelihood of a ‘‘good-quality’’ embryo (scores 1 and 2, with no to minor fragmentation) undergoing normal blastulation was significantly higher than embryos with moderate to significant fragmentation (scores 3 and 4), with an odds ratio of 2.94 (95% confidence interval 2.39–3.85; P<.01). The age of patients included ranged from 20 to 44 years, with a mean SD age of 30.6 4.6 years. Age as a continuous variable did not correlate with embryo fragmentation (r ¼ 0.02; P¼.25), nor did it show an effect when divided into groups (P¼.20). In a multiple regression model with blastocyst formation as the dependent variable and embryo fragmentation as an established predictor, age as an additional variable carried a regression coefficient of 0.03 (P¼.43), showing that age did not further modify the significant relationship between embryo fragmentation and blastocyst formation. When the analysis was expanded to include those abnormal blastocysts which did not meet criteria for cryopreservation (worse than grade 1BB on day 5 or 2BB on day 6; n ¼ 55), the same relationship between age, fragmentation, and blastocyst formation remained. Neither age (r ¼ 0.08; P¼.16) nor degree of fragmentation (r ¼ 0.01; P¼.81) had a significant impact on progression to a normal versus an abnormal blastocyst. Likewise, age (r ¼ 0.08; P¼.11) and degree of fragmentation (r ¼ 0.02; P¼.67) did not influence blastulation occurring on day 5 versus day 6. In this study, we have presented the rate of blastocyst formation and the ultimate fate of supernumerary cleavage-stage embryos from fresh IVF cycles. Our findings suggest that increasing degrees of embryo fragmentation has a direct negative impact on embryo development to the blastocyst stage in appropriate cellular cleavage–stage embryos. This effect of embryo fragmentation is independent of maternal age and may be the most important index of continued embryo viability in extended culture media. These findings are in line with that of previous investigators that the morphologic grade of day 3 embryos is a strong indicator directly correlated with blastocyst formation (6, 7). It has been suggested
Fertility and Sterility
TABLE 1 Blastocyst formation as a function of day 3 embryo morphology. Progression to blastocysta Degree of fragmentation
Total embryos cultured
%
n
1 2 3 4
158 328 202 216
56.3 43.9 27.7 18.5
89 144 56 40
a
P< .01.
Wu. Correspondence. Fertil Steril 2011.
that the degree of fragmentation in cleavage-stage embryos is directly proportional to the incidence of chromosomal abnormality, thereby reducing the capacity of the embryo to reach blastocyst (10–13). Others have suggested that a pervasive pattern of anucleate fragmentation may initiate an apoptotic process in embryos with activation of programmed cell death pathways (5, 14–16). Embryo fragments may also be positioned between blastomeres or along dividing planes and pose mechanical interference with compaction, cavitation, and blastocyst formation (6). Additionally, necrotic fragments can cause a secondary degeneration of the embryo, contributing to the deterioration of the remaining cells. We surmise that these detrimental processes attributed to embryo fragmentation contributing to eventual embryonic demise may all be independent of maternal age. Our findings are strengthened by limiting embryo selection to only those with appropriate cellular division on day 3, thus isolating fragmentation as the sole index of embryo quality. Additional strengths include achieving adequate sample size for assessment based on power analysis and using the same sequential culture media and the same embryologists to perform the embryo quality grading, thus limiting any potential change in laboratory environment over time. Our data are limited by the large proportion (80%) of embryos from patients <35 years of age. This was due in part to the inclusion of embryos from younger oocyte donors, the younger median age of patients who present for IVF at our center, and the few available supernumerary embryos for extended culture in patients R35 years of age. Additionally, by limiting embryo selection to only those with six to eight cells on day 3, one could argue that a selection bias may be present, because many abnormally cleaving embryos from women of increased reproductive age would have been excluded, underscoring the importance of age in embryo viability in vitro. That said, embryos with abnormal cleavage kinetics from younger patients with more available fertilized embryos would equally be excluded, and some limited data do indicate that cleavage rates may not differ significantly based on age alone (17). From a clinical perspective, although improved embryo culture protocols have made blastocyst transfer a well established procedure in assisted reproduction, much debate has occurred regarding cleavage and blastocyst transfers and optimizing clinical outcomes. A Cochrane review comparing day 2–3 embryo transfers with day 5–7 transfers provided updated evidence that blastocyst
2779
transfers can achieve higher live birth rates in ‘‘good-prognosis patients,’’ but couples undergoing blastocyst transfers are more likely to have no embryos available for transfer (18). The present findings provide a better understanding of the relationship between embryo fragmentation on day 3 and blastocyst development, which may
further aid in making the decision to proceed with a cleavagestage or blastocyst transfer. Further studies based on the number of available day 3 embryos and their morphology to determine clinical outcomes can help to establish an optimal criteria and appropriate counseling for blastocyst transfers.
REFERENCES 1. Meldrum DR. Female reproductive aging—ovarian and uterine factors. Fertil Steril 1993;59:1–5. 2. Piette C, de Mouzon J, Bachelot A, Spira A. In-vitro fertilization: influence of women’s age on pregnancy rates. Hum Reprod 1990;5:56–9. 3. Hsu MI, Mayer J, Aronshon M, Lanzendorf S, Muasher S, Kolm P, et al. Embryo implantation in in vitro fertilization and intracytoplasmic sperm injection: impact of cleavage status, morphology grade, and number of embryos transferred. Fertil Steril 1999;72:679–85. 4. 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. 5. Juriscova A, Rogers I, Fasciani A, Casper RF, Varmuza S. Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development. Mol Hum Reprod 1998;4:139–45. 6. 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.
2780
Wu et al.
Correspondence
7. 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. 8. Veeck L. Preembryo grading. In: Atlas of human oocyte and early conceptus, vol. 2. Veeck L, ed. Philadelphia: Lippincott Williams and Wilkins, 1991:121–2. 9. Practice Committee of American Society for Reproductive Medicine. Guidelines on number of embryos transferred. Fertil Steril 2008;90(5 Suppl):S163–4. 10. Bongso A, Ng SC, Lim J, Fong CY, Ratnam S. Preimplantation genetics: chromosomes of fragmented human embryos. Fertil Steril 1991;56: 66–70. 11. 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. 12. Magli MC, Gianaroli L, Ferraretti AP. Chromosomal abnormalities in embryos. Mol Cell Endocrinol 2001;183(Suppl 1):S29–34. 13. Ziebe S, Lundin K, Loft A, Bergh C, Nyboe Andersen A, Selleskog U, et al. FISH analysis for
14.
15.
16.
17.
18.
chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology. Hum Reprod 2003;18: 2575–81. Hardy K, Winston RM, Handyside AH. Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. J Reprod Fertil 1993;98: 549–58. Jurisicova A, Varmuza S, Casper RF. Programmed cell death and human embryo fragmentation. Mol Hum Reprod 1996;2:93–8. 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. Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev 1996;45: 31–7. Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev 2007;(4):CD002118.
Vol. 95, No. 8, June 30, 2011
An age-related decline in female fecundity has been attributed to the impaired quality of aging oocytes, reduced embryo quality, and lower implantation and pregnancy rates (1–3). There is evidence for a direct relationship between the age of women undergoing in vitro fertilization (IVF) and the proportion of anucleate fragmentation in cleavage-stage embryos (4, 5). Although some studies have demonstrated that embryos with more fragmentation have lower rates of reaching blastulation (6, 7) and that patients with increasing maternal age also have lower probability of having blastocysts, it is still unclear whether age plays an additive effect on embryo fragmentation, thus further reducing the chance for blastulation. The objective of the present study was to investigate whether, in embryos with similar degrees of fragmentation, maternal age modifies the effect of embryo fragmentation on successful blastocyst development. A retrospective review of patient and cycle data from all fresh, autologous, and donor-recipient IVF cycles from January 2003 to Diana H. Wu, M.D.a Kasey Reynolds, M.D.b Rose Maxwell, Ph.D.a Steven R. Lindheim, M.D., M.M.M.a Mira Aubuchon, M.D.c Michael A. Thomas, M.D.a a Center for Reproductive Health, University of Cincinnati Academic Health Center, Cincinnati, Ohio b Good Samaritan Hospital, Cincinnati, Ohio c University of Missouri, Columbia, Missouri Received February 14, 2011; revised May 9, 2011; accepted May 10, 2011; published online June 8, 2011. D.H.W. has nothing to disclose. K.R. has nothing to disclose. R.M. has nothing to disclose. S.R.L. has nothing to disclose. M.A. has nothing to disclose. M.A.T. has nothing to disclose. Supported by the Patty Brisben Foundation. Presented at the 57th Annual Meeting of Society for Gynecologic Investigation, Orlando, Florida, March 2010. Reprint requests: Diana H. Wu, M.D., Center for Reproductive Health, University of Cincinnati Academic Health Center, 2123 Auburn Avenue, Suite A-44, Cincinnati, OH 45219 (E-mail: [email protected]).
2778
August 2009 was performed. Institutional Review Board approval was obtained before data collection and analysis. Patients underwent ovarian stimulation with the use of either a luteal-phase GnRH agonist protocol, a GnRH antagonist protocol, or a microdose flare protocol. During these cycles, gonadotropin stimulation was continued until at least two follicles had attained a mean diameter of 18–20 mm. Ultrasound-guided oocyte retrieval was performed 35 hours after administration of either 500 mg of recombinant hCG (Ovidrel; EMD Serono) or 7,500–10,000 IU hCG. After oocyte retrieval, fertilization was performed in IVC-1 media (InVitroCare) by either conventional insemination or intracytoplasmic sperm injection in the presence of male factor infertility. The standard operating procedure during the time frame of data collection was to cryopreserve at least three embryos at the two-pronuclei stage if couples had nine or more fertilized embryos. The remaining six to eight embryos were cultured in IVC-1 medium for the first 72 hours. Embryos were evaluated immediately before transfer on day 3 for cleavage stage and degrees of fragmentation (1 to 4) by the criteria outlined by Veeck, with a fragmentation score of 1 characterized by the absence of cytoplasmic fragments and a score of 4 characterized by significant cytoplasmic fragmentation (8). Depending on female partner age and/or embryo quality, two to three embryos with the best morphology and cleaving status were selected for embryo transfer. Surplus embryos were then transferred to IVC-3 blastocyst medium. These embryos were reevaluated on days 5 and/or 6 for development into suitable blastocysts for cryopreservation. The primary outcome evaluated was the rate of blastocyst formation as a function of fragmentation on day 3 and maternal age. To isolate the effects of fragmentation, only embryos with normal cellular division with six to eight cells on day 3 were included. The rate of blastocyst formation per 6–8-cell cleavage embryo transfered to blastocyst medium was analyzed against the degree of fragmentation on day 3 and maternal age with the use of chi-square test, Pearson correlation, and logistic regression. Age was analyzed with both Pearson correlation as a continuous variable and chi-square test in age groups of <29, 30–34, 35–37, 38–40, and R41 years of age. The division of subjects into age groups for ages R35 years corresponded to groupings used for
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.05.024
both Centers for Disease Control and Society for Assisted Reproductive Technologies data collection, as well as to groupings in American Society for Reproductive Medicine guidelines for the number of embryos to transfer (9). Further division of patients aged <35 years was based on the large proportion of patients in this age group who present for IVF at our center and evidence that an age-related decline in fertility may begin even in the early thirties. An a priori power analysis was performed with an effect size of 11%: the difference reported between the average blastulation rate of embryos with little or no fragmentation (score 1–2) and more fragmented embryos (score 3–4) (7). With power set at 80% and P<.05, a minimum sample size of 177 embryos for each fragmentation score from 1 to 4 was calculated with the use of Epi Info (CDC). A total of 904 cleavage-stage embryos with normal cellular division and fitting other selection criteria were analyzed, which resulted from 285 IVF cycles in 276 patients. The distribution of embryos analyzed per morphology was 158 embryos with a fragmentation score of 1, 328 embryos with a score of 2, 202 embryos with a score of 3, and 216 embryos with a score of 4. Table 1 summarizes the blastocyst formation rate in relation to embryo fragmentation. The average blastulation rate was 36% across all embryos. A significant negative correlation was found between the degree of fragmentation and the rate of blastocyst development (r ¼ 0.28; P<.01). The likelihood of a ‘‘good-quality’’ embryo (scores 1 and 2, with no to minor fragmentation) undergoing normal blastulation was significantly higher than embryos with moderate to significant fragmentation (scores 3 and 4), with an odds ratio of 2.94 (95% confidence interval 2.39–3.85; P<.01). The age of patients included ranged from 20 to 44 years, with a mean SD age of 30.6 4.6 years. Age as a continuous variable did not correlate with embryo fragmentation (r ¼ 0.02; P¼.25), nor did it show an effect when divided into groups (P¼.20). In a multiple regression model with blastocyst formation as the dependent variable and embryo fragmentation as an established predictor, age as an additional variable carried a regression coefficient of 0.03 (P¼.43), showing that age did not further modify the significant relationship between embryo fragmentation and blastocyst formation. When the analysis was expanded to include those abnormal blastocysts which did not meet criteria for cryopreservation (worse than grade 1BB on day 5 or 2BB on day 6; n ¼ 55), the same relationship between age, fragmentation, and blastocyst formation remained. Neither age (r ¼ 0.08; P¼.16) nor degree of fragmentation (r ¼ 0.01; P¼.81) had a significant impact on progression to a normal versus an abnormal blastocyst. Likewise, age (r ¼ 0.08; P¼.11) and degree of fragmentation (r ¼ 0.02; P¼.67) did not influence blastulation occurring on day 5 versus day 6. In this study, we have presented the rate of blastocyst formation and the ultimate fate of supernumerary cleavage-stage embryos from fresh IVF cycles. Our findings suggest that increasing degrees of embryo fragmentation has a direct negative impact on embryo development to the blastocyst stage in appropriate cellular cleavage–stage embryos. This effect of embryo fragmentation is independent of maternal age and may be the most important index of continued embryo viability in extended culture media. These findings are in line with that of previous investigators that the morphologic grade of day 3 embryos is a strong indicator directly correlated with blastocyst formation (6, 7). It has been suggested
Fertility and Sterility
TABLE 1 Blastocyst formation as a function of day 3 embryo morphology. Progression to blastocysta Degree of fragmentation
Total embryos cultured
%
n
1 2 3 4
158 328 202 216
56.3 43.9 27.7 18.5
89 144 56 40
a
P< .01.
Wu. Correspondence. Fertil Steril 2011.
that the degree of fragmentation in cleavage-stage embryos is directly proportional to the incidence of chromosomal abnormality, thereby reducing the capacity of the embryo to reach blastocyst (10–13). Others have suggested that a pervasive pattern of anucleate fragmentation may initiate an apoptotic process in embryos with activation of programmed cell death pathways (5, 14–16). Embryo fragments may also be positioned between blastomeres or along dividing planes and pose mechanical interference with compaction, cavitation, and blastocyst formation (6). Additionally, necrotic fragments can cause a secondary degeneration of the embryo, contributing to the deterioration of the remaining cells. We surmise that these detrimental processes attributed to embryo fragmentation contributing to eventual embryonic demise may all be independent of maternal age. Our findings are strengthened by limiting embryo selection to only those with appropriate cellular division on day 3, thus isolating fragmentation as the sole index of embryo quality. Additional strengths include achieving adequate sample size for assessment based on power analysis and using the same sequential culture media and the same embryologists to perform the embryo quality grading, thus limiting any potential change in laboratory environment over time. Our data are limited by the large proportion (80%) of embryos from patients <35 years of age. This was due in part to the inclusion of embryos from younger oocyte donors, the younger median age of patients who present for IVF at our center, and the few available supernumerary embryos for extended culture in patients R35 years of age. Additionally, by limiting embryo selection to only those with six to eight cells on day 3, one could argue that a selection bias may be present, because many abnormally cleaving embryos from women of increased reproductive age would have been excluded, underscoring the importance of age in embryo viability in vitro. That said, embryos with abnormal cleavage kinetics from younger patients with more available fertilized embryos would equally be excluded, and some limited data do indicate that cleavage rates may not differ significantly based on age alone (17). From a clinical perspective, although improved embryo culture protocols have made blastocyst transfer a well established procedure in assisted reproduction, much debate has occurred regarding cleavage and blastocyst transfers and optimizing clinical outcomes. A Cochrane review comparing day 2–3 embryo transfers with day 5–7 transfers provided updated evidence that blastocyst
2779
transfers can achieve higher live birth rates in ‘‘good-prognosis patients,’’ but couples undergoing blastocyst transfers are more likely to have no embryos available for transfer (18). The present findings provide a better understanding of the relationship between embryo fragmentation on day 3 and blastocyst development, which may
further aid in making the decision to proceed with a cleavagestage or blastocyst transfer. Further studies based on the number of available day 3 embryos and their morphology to determine clinical outcomes can help to establish an optimal criteria and appropriate counseling for blastocyst transfers.
REFERENCES 1. Meldrum DR. Female reproductive aging—ovarian and uterine factors. Fertil Steril 1993;59:1–5. 2. Piette C, de Mouzon J, Bachelot A, Spira A. In-vitro fertilization: influence of women’s age on pregnancy rates. Hum Reprod 1990;5:56–9. 3. Hsu MI, Mayer J, Aronshon M, Lanzendorf S, Muasher S, Kolm P, et al. Embryo implantation in in vitro fertilization and intracytoplasmic sperm injection: impact of cleavage status, morphology grade, and number of embryos transferred. Fertil Steril 1999;72:679–85. 4. 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. 5. Juriscova A, Rogers I, Fasciani A, Casper RF, Varmuza S. Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development. Mol Hum Reprod 1998;4:139–45. 6. 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.
2780
Wu et al.
Correspondence
7. 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. 8. Veeck L. Preembryo grading. In: Atlas of human oocyte and early conceptus, vol. 2. Veeck L, ed. Philadelphia: Lippincott Williams and Wilkins, 1991:121–2. 9. Practice Committee of American Society for Reproductive Medicine. Guidelines on number of embryos transferred. Fertil Steril 2008;90(5 Suppl):S163–4. 10. Bongso A, Ng SC, Lim J, Fong CY, Ratnam S. Preimplantation genetics: chromosomes of fragmented human embryos. Fertil Steril 1991;56: 66–70. 11. 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. 12. Magli MC, Gianaroli L, Ferraretti AP. Chromosomal abnormalities in embryos. Mol Cell Endocrinol 2001;183(Suppl 1):S29–34. 13. Ziebe S, Lundin K, Loft A, Bergh C, Nyboe Andersen A, Selleskog U, et al. FISH analysis for
14.
15.
16.
17.
18.
chromosomes 13, 16, 18, 21, 22, X and Y in all blastomeres of IVF pre-embryos from 144 randomly selected donated human oocytes and impact on pre-embryo morphology. Hum Reprod 2003;18: 2575–81. Hardy K, Winston RM, Handyside AH. Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. J Reprod Fertil 1993;98: 549–58. Jurisicova A, Varmuza S, Casper RF. Programmed cell death and human embryo fragmentation. Mol Hum Reprod 1996;2:93–8. 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. Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev 1996;45: 31–7. Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev 2007;(4):CD002118.
Vol. 95, No. 8, June 30, 2011