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Prognostic value of pronuclear morphology
test of any difference between the two groups, regardless of the direction of the difference. One-tailed tests require proof in only one direction, and the full 5% probability falls in one tail of the curve rather than being split between the upper and lower limits of the confidence interval. A preplanned, single-sided significance test (one tail) is defensible for certain types of study designs or when it is impossible for the results to go in the opposite direction. There are few studies comparing rFSH to hMG that are unidirectional. The most notable exception was the European and Israeli study comparing highly purified hMG (HP-hMG) (Menopur; Ferring Pharmaceuticals, Suffern, NY) with recombinant FSH (rFSH) (Gonal-F; Serono Pharmaceuticals, Rockland, MA). The latter was a randomized trial with ongoing pregnancy rates as the primary outcome (3). It was designed as a noninferiority trial, with HP-hMG as the new drug to be compared with rFSH as the standard or reference compound. The study design and statistical hypothesis for the study were set up to prove that treatment with HP-hMG was not inferior to rFSH in terms of pregnancy rates achieved. The power analysis for the study was scripted to be able to conclude a “clinically negligible difference,” predefined as 10% between rFSH and HP-hMG with a one-sided 95% confidence limit. The use of unidirectional study designs and statistical measures that permit some inferiority in the new product compared with the reference standard are important for the development and approval of new drugs. In this context, the new drug can be approved if it retains a substantial fraction of the effect of the active control and might have better toxicity. If it is felt that a one-sided test is appropriate, then this decision and a clear statement attesting to this decision should be made at the beginning of the study and before the data are analyzed. The decision to use a unidirectional test of significance must not depend on the results obtained after the study is complete. With a one-sided test it is usually easier to get a P value smaller than .05. The area of 5% situated in one tail is a larger target than 2.5% in one tail and 2.5% in the other tail. However some studies analyzed as being one way are often judged so after the analyst has looked at the numbers. This is obviously bending the rules and akin to betting on a horse after the race is over. For this reason, it is vital that the protocol of a trial or meta-analysis designed to use a unidirectional test of significance contains a clear statement that this is its explicit intention. In this case, the studies cited by the Dr. Daya are all bidirectional in design and analysis. His comments, although focused on the upper limits of the confidence interval, serve to illustrate that the test statistic is a bidirectional continuum with 2.5% of the probability statistic in each end. Letters such as the one from Dr. Steiner that search for methodologic flaws in a study are one of the most important aspects of peer review. It is a segment of peer review that has no “statute of limitations.” The give and take of the “Letters
to the Editor” section of the journal is designed to challenge cozy assumptions and provide a stimulus for fresh perspectives. Unfortunately, the time to hard copy publication is slow. Perhaps cyberspace and rapid on-line electronic responses as currently practiced by the British Medical Journal can speed up the response time for the reader. Paul G. McDonough, M.D. Associate Editor, Letters Medical College of Georgia Augusta, Georgia March 30, 2004
References 1. Daya S. Methodologic pitfalls in assessing the efficacy of recombinant follicle-stimulating hormone versus human menopausal gonadotropin in assisted reproduction. Fertil Steril 2003;80:1100 –4. 2. Goldfarb JM, Desai N. Follitropin-␣ versus human menopausal gonadotropin in an in vitro fertilization program. Fertil Steril 2003;80:1094 – 99. 3. The European and Israeli Study Group on Highly Purified Menotropin versus Recombinant Follicle-Stimulating Hormone. Efficacy and safety of highly purified menotropin versus recombinant follicle-stimulating hormone in in vitro fertilization/intracytoplasmic sperm injection cycles: a randomized comparative trial. Fertil Steril 2002;78:520 –8.
doi:10.1016/j.fertnstert.2004.04.012
Prognostic value of pronuclear morphology To the Editor: The article by Dr. Gianaroli et al. (1) presents interesting observations and data supporting the already established value of pronuclear (PN) scoring as an aid used to identify pre-embryos with optimal development and implantation potential for IVF-ET (2, 3). It also highlights an important concept, which is the combined observation of zygote characteristics, such as size, alignment, and orientation of PN/ nuclear precursor bodies (NPB) relative to the polar bodies (PB). It is my opinion that if the embryos are observed in a consistent and detailed manner, they will “tell” the observer which are the ones with the best potential for continued development and implantation. Thus, these observation events could be considered as a screening process. As indicated by the authors, the A1␣ configuration is the optimal one for obtaining quality embryos on the day of ET. However, it might be that not all the zygotes are undergoing the various intracellular events at the same time relative to each other. For example, A2 and A3 patterns could still be rotating to achieve proper alignment with the PBs and simultaneously undergoing NPB arrangement. This observation, combined with the presence of a cytoplasmic halo, might be indicative of these transition events, which might be confirmed by observing the zygotes up to the expected time when syngamy occurs to determine whether complete NPB rearrangement took place. Furthermore, the data indicate that if a zygote has already reached the PN morphology type A but has not yet completed the proper NPB migration Vol. 82, No. 1, July 2004
258
(␣ configuration), then the zygote is “locked” in that alignment, with a higher probability of subsequent developmental defects (i.e., asymmetric and irregular blastomeres, fragmentation, and chromosomal aberrations). If the scattered NPB arrangement represents a transition event toward the NPB-␣ patterns, the ones exhibiting the extreme ␥ configuration might have a long way to go before reaching the “lock” configuration (A1␣). Another comment that the authors should consider discussing regards the timing of the assumed fertilization events and whether there is a difference between the conventional IVF and intracytoplasmic sperm injection (ICSI) cases. Because the timing of sperm entry is known in ICSI, could there be a difference with conventional IVF, where the interaction of sperm/cumulus-oocyte requires additional temporal events? In addition, in conventional IVF the oocyte maturational status is not necessarily assessed as in oocytes directly observed for maturational status before ICSI. Evidence regarding these factors seems to be conflicting (4 – 6), but it could be worthwhile investigating whether such timing effects exist. Juan R. Correa-Pe´ rez, Ph.D. Centro de Fertilidad del Caribe Rı´o Piedras, Puerto Rico March 1, 2004
References 1. Gianaroli L, Magli MC, Ferraretti AP, Fortini D, Grieco N. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril 2003;80:341–9. 2. Zollner U, Zollner KP, Hartl G, Dietl J, Steck T. The use of a detailed zygote score after IVF/ICSI to obtain good quality blastocysts: the German experience. Hum Reprod 2002;17:1327–33. 3. Scott L. Pronuclear scoring as a predictor of embryo development. Reprod Biomed Online 2003;6:201–14. 4. Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:1014 –9. 5. Van de Velde H, De Vos A, Joris H, Nagy ZP, Van Steirteghem AC. Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:3160 –4. 6. Goud P, Goud A, Van Oostveldt P, Van der Elst J, Dhont M. Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity. Fertil Steril 1999;72: 245–52.
doi:10.1016/j.fertnstert.2004.04.013
Reply of the Authors: Although part of a not yet clearly understood mechanism in human oocytes, early signs of polarity are already evident at the pronuclear stage and have been related to the embryo chromosomal status (1). The results obtained in our study designated some configurations for which the proportion of chromosomally normal embryos was significantly higher compared with others, whereas other configurations were strongly predictive of a chromosomally abnormal complement. FERTILITY & STERILITY威
As pointed out by Dr. Correa, the morphology of pronuclear zygotes varies progressively with time. Pronuclei are barely visible during initial chromatin condensation but become more and more evident as nuclear envelopes inflate. Concomitantly, they progress toward the central region of the oocyte, where syngamy occurs. At the same time, nuclear precursor bodies (NPB) merge into nucleoli that fuse in a cycle-dependent manner: several nucleoli are evident at the beginning of the G1 phase, whereas only one or two nucleoli remain at the S phase (2). These observations suggest that differences in the timing of these sequential events could exist among zygotes, thus implying that some configurations could be transitory and indicative of a polarization occurring later in development. As far as this delay remains within an acceptable time lapse, the corresponding configurations could still have good chances of developing viable embryos, although the risk of developmental errors occurring in the remaining steps cannot be excluded. Possibly, synchrony in pronuclear development, besides the absolute speed needed to complete the process of polarization, could have important effects on embryo viability, as part of a process with a precise timing and coordinated sequences of events. In agreement with other reports (3,4), we detected no significant differences between intracytoplasmic sperm injection (ICSI) and IVF zygotes. However, because most of the analyzed zygotes derived from ICSI (70%), this finding could be related to the size of the studied samples. Theoretically, a different timing in the events after fertilization could be expected when the two insemination techniques are compared. We fully agree with Dr. Correa that investigation of this aspect should be expanded. Additionally, the possible effect of sperm quality and the type of male infertility on pronuclear zygote morphology should be investigated. Previously reported results indicate a correlation between the severity of the male factor condition and the proportion of chromosomal abnormalities in the resulting embryos (5). All these aspects must be considered when attempts are made to evaluate the effects related to ICSI on pronuclear zygote morphology compared with conventional IVF. Luca Gianaroli, M.D. M. Cristina Magli, M.Sc. Anna P. Ferraretti, M.D, Ph.D. S.I.S.Me.R., Reproductive Medicine Unit Bologna, Italy March 29, 2004
References 1. Gianaroli L, Magli MC, Ferraretti AP, Fortini D, Grieco N. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril 2003;80:341–9. 2. Tesarik J, Kopecny V. Developmental control of the human male pronucleus by ooplasmic factors. Hum Reprod 1989;4:962–8. 3. Garello C, Baker H, Rai J, Montgomery S, Wilson P, Kennedy CR, et al. Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in vitro fertilization: further evidence for polarity in human oocytes? Hum Reprod 1999;14:2588 –95. 4. Scott L, Alvero R, Leondires M, Bradley M. The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod 2000;15:2394 –403.
259
to the Editor” section of the journal is designed to challenge cozy assumptions and provide a stimulus for fresh perspectives. Unfortunately, the time to hard copy publication is slow. Perhaps cyberspace and rapid on-line electronic responses as currently practiced by the British Medical Journal can speed up the response time for the reader. Paul G. McDonough, M.D. Associate Editor, Letters Medical College of Georgia Augusta, Georgia March 30, 2004
References 1. Daya S. Methodologic pitfalls in assessing the efficacy of recombinant follicle-stimulating hormone versus human menopausal gonadotropin in assisted reproduction. Fertil Steril 2003;80:1100 –4. 2. Goldfarb JM, Desai N. Follitropin-␣ versus human menopausal gonadotropin in an in vitro fertilization program. Fertil Steril 2003;80:1094 – 99. 3. The European and Israeli Study Group on Highly Purified Menotropin versus Recombinant Follicle-Stimulating Hormone. Efficacy and safety of highly purified menotropin versus recombinant follicle-stimulating hormone in in vitro fertilization/intracytoplasmic sperm injection cycles: a randomized comparative trial. Fertil Steril 2002;78:520 –8.
doi:10.1016/j.fertnstert.2004.04.012
Prognostic value of pronuclear morphology To the Editor: The article by Dr. Gianaroli et al. (1) presents interesting observations and data supporting the already established value of pronuclear (PN) scoring as an aid used to identify pre-embryos with optimal development and implantation potential for IVF-ET (2, 3). It also highlights an important concept, which is the combined observation of zygote characteristics, such as size, alignment, and orientation of PN/ nuclear precursor bodies (NPB) relative to the polar bodies (PB). It is my opinion that if the embryos are observed in a consistent and detailed manner, they will “tell” the observer which are the ones with the best potential for continued development and implantation. Thus, these observation events could be considered as a screening process. As indicated by the authors, the A1␣ configuration is the optimal one for obtaining quality embryos on the day of ET. However, it might be that not all the zygotes are undergoing the various intracellular events at the same time relative to each other. For example, A2 and A3 patterns could still be rotating to achieve proper alignment with the PBs and simultaneously undergoing NPB arrangement. This observation, combined with the presence of a cytoplasmic halo, might be indicative of these transition events, which might be confirmed by observing the zygotes up to the expected time when syngamy occurs to determine whether complete NPB rearrangement took place. Furthermore, the data indicate that if a zygote has already reached the PN morphology type A but has not yet completed the proper NPB migration Vol. 82, No. 1, July 2004
258
(␣ configuration), then the zygote is “locked” in that alignment, with a higher probability of subsequent developmental defects (i.e., asymmetric and irregular blastomeres, fragmentation, and chromosomal aberrations). If the scattered NPB arrangement represents a transition event toward the NPB-␣ patterns, the ones exhibiting the extreme ␥ configuration might have a long way to go before reaching the “lock” configuration (A1␣). Another comment that the authors should consider discussing regards the timing of the assumed fertilization events and whether there is a difference between the conventional IVF and intracytoplasmic sperm injection (ICSI) cases. Because the timing of sperm entry is known in ICSI, could there be a difference with conventional IVF, where the interaction of sperm/cumulus-oocyte requires additional temporal events? In addition, in conventional IVF the oocyte maturational status is not necessarily assessed as in oocytes directly observed for maturational status before ICSI. Evidence regarding these factors seems to be conflicting (4 – 6), but it could be worthwhile investigating whether such timing effects exist. Juan R. Correa-Pe´ rez, Ph.D. Centro de Fertilidad del Caribe Rı´o Piedras, Puerto Rico March 1, 2004
References 1. Gianaroli L, Magli MC, Ferraretti AP, Fortini D, Grieco N. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril 2003;80:341–9. 2. Zollner U, Zollner KP, Hartl G, Dietl J, Steck T. The use of a detailed zygote score after IVF/ICSI to obtain good quality blastocysts: the German experience. Hum Reprod 2002;17:1327–33. 3. Scott L. Pronuclear scoring as a predictor of embryo development. Reprod Biomed Online 2003;6:201–14. 4. Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:1014 –9. 5. Van de Velde H, De Vos A, Joris H, Nagy ZP, Van Steirteghem AC. Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13:3160 –4. 6. Goud P, Goud A, Van Oostveldt P, Van der Elst J, Dhont M. Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity. Fertil Steril 1999;72: 245–52.
doi:10.1016/j.fertnstert.2004.04.013
Reply of the Authors: Although part of a not yet clearly understood mechanism in human oocytes, early signs of polarity are already evident at the pronuclear stage and have been related to the embryo chromosomal status (1). The results obtained in our study designated some configurations for which the proportion of chromosomally normal embryos was significantly higher compared with others, whereas other configurations were strongly predictive of a chromosomally abnormal complement. FERTILITY & STERILITY威
As pointed out by Dr. Correa, the morphology of pronuclear zygotes varies progressively with time. Pronuclei are barely visible during initial chromatin condensation but become more and more evident as nuclear envelopes inflate. Concomitantly, they progress toward the central region of the oocyte, where syngamy occurs. At the same time, nuclear precursor bodies (NPB) merge into nucleoli that fuse in a cycle-dependent manner: several nucleoli are evident at the beginning of the G1 phase, whereas only one or two nucleoli remain at the S phase (2). These observations suggest that differences in the timing of these sequential events could exist among zygotes, thus implying that some configurations could be transitory and indicative of a polarization occurring later in development. As far as this delay remains within an acceptable time lapse, the corresponding configurations could still have good chances of developing viable embryos, although the risk of developmental errors occurring in the remaining steps cannot be excluded. Possibly, synchrony in pronuclear development, besides the absolute speed needed to complete the process of polarization, could have important effects on embryo viability, as part of a process with a precise timing and coordinated sequences of events. In agreement with other reports (3,4), we detected no significant differences between intracytoplasmic sperm injection (ICSI) and IVF zygotes. However, because most of the analyzed zygotes derived from ICSI (70%), this finding could be related to the size of the studied samples. Theoretically, a different timing in the events after fertilization could be expected when the two insemination techniques are compared. We fully agree with Dr. Correa that investigation of this aspect should be expanded. Additionally, the possible effect of sperm quality and the type of male infertility on pronuclear zygote morphology should be investigated. Previously reported results indicate a correlation between the severity of the male factor condition and the proportion of chromosomal abnormalities in the resulting embryos (5). All these aspects must be considered when attempts are made to evaluate the effects related to ICSI on pronuclear zygote morphology compared with conventional IVF. Luca Gianaroli, M.D. M. Cristina Magli, M.Sc. Anna P. Ferraretti, M.D, Ph.D. S.I.S.Me.R., Reproductive Medicine Unit Bologna, Italy March 29, 2004
References 1. Gianaroli L, Magli MC, Ferraretti AP, Fortini D, Grieco N. Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril 2003;80:341–9. 2. Tesarik J, Kopecny V. Developmental control of the human male pronucleus by ooplasmic factors. Hum Reprod 1989;4:962–8. 3. Garello C, Baker H, Rai J, Montgomery S, Wilson P, Kennedy CR, et al. Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in vitro fertilization: further evidence for polarity in human oocytes? Hum Reprod 1999;14:2588 –95. 4. Scott L, Alvero R, Leondires M, Bradley M. The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod 2000;15:2394 –403.
259