Incidence and development of zygotes exhibiting abnormal pronuclear disposition after identification of two pronuclei at the fertilization check

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1 Incidence and development of zygotes exhibiting abnormal pronuclear disposition after identification of two pronuclei at the fertilization check David E. Reichman, M.D., Katharine V. Jackson, B.A., and Catherine Racowsky, Ph.D. Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women s Hospital, Boston, Massachusetts Objective: To determine the incidence, developmental potential, and clinical implications of embryos having one pronucleus (1PN) or three pronuclei (3PN) at early cleavage, despite exhibiting 2PN at the fertilization check. Design: Retrospective cohort study. Setting: Hospital-based academic medical center. Patient(s): All IVF cycles from January 2006 through May 2008 having 2PN zygotes that subsequently transitioned to 1PN or 3PN before cleavage, matched to cycles having 2PN zygotes progressing to cleavage without intervening abnormal pronuclear disposition. Intervention(s): Standard IVF protocol. Main Outcome Measure(s): Incidence, day 3 development, and implantation rates of 2PN zygotes transitioning to 1PN and 3PN states before cleavage, compared with normal embryos. Result(s): The incidences of 1PN and 3PN zygotes were 2.9% and 0.4%, respectively. Both types of abnormal zygote showed slower day 3 cleavage, although only the 1PNs exhibited higher fragmentation and asymmetry compared with controls. The 1PN zygotes had a 6.4% implantation rate and viable pregnancy rate of 1.3%. Of the nine 3PN zygotes transferred, none implanted. Conclusion(s): Two-pronuclear zygotes transitioning through 1PN or 3PN states tend to develop into poorer-quality embryos than 2PN control zygotes. Patients should be counseled regarding the very low likelihood of viable pregnancy after transfer of these abnormally developing zygotes. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Unipronuclear, 1PN, tripronuclear, 3PN, early cleavage, IVF, ICSI, zygote Several studies have investigated the incidence, origin, development, and chromosomal composition of abnormal pronucleate zygotes (1 3). Single-pronucleate (1PN) zygotes are thought to be secondary to either parthenogenetic activation of the oocyte or abnormal syngamy of pronuclei, whereas tripronucleate (3PN) zygotes result from either dispermy or failure of extrusion of the second polar body. Although such embryos are usually discarded in IVF programs, healthy children have been born after transfer of 1PN embryos (4). It has become the practice of several centers to transfer such embryos in the absence of suitable alternatives. Studies, however, have focused on zygotes which, at hours after insemination, exhibited abnormal pronuclear number without ever being identified as having a normal two-pronucleate (2PN) state. In our experience, we have occasionally noted zygotes which exhibited evidence of normal fertilization with two pronuclei and later went on to display one pronucleus or three pronuclei when checking for early cleavage (24 27 hours after insemination). We hypothesized that such zygotes should be classified as abnormal Received February 16, 2009; revised and accepted April 8, 2009; published online May 23, D.E.R. has nothing to disclose. K.V.J. has nothing to disclose. C.R. has nothing to disclose. Reprint requests: Catherine Racowsky, Ph.D., Brigham & Women s Hospital, 75 Francis Street, ASB 1þ3, Rm 08, Boston, MA (FAX: ; cracowsky@partners.org). and would have poorer developmental potential and implantation rates than zygotes with two pronuclei. Upon our review of the existing literature, we could find only one study mentioning this phenomenon in a single fertilized oocyte (5) and no studies that examined the incidence, developmental fate, or suitability for transfer of these zygotes. Therefore, the purpose of the present study was to discover the incidence of these abnormally developing zygotes in our standard IVF population, to track their development based on cell number, fragmentation, and asymmetry on day 3, and to determine their suitability for transfer. MATERIALS AND METHODS This study was approved by the Partners Healthcare Institutional Review Board for chart review. Cycle Inclusion Criteria The index cycles identified for this study were culled after review of all IVF cycles (either standard insemination or intracytoplasmic sperm injection [ICSI]) performed in our program from January 2006 through May 2008 in which the embryos were evaluated for early cleavage (23.7 to 26.9 hours after insemination, mean time 25.1 hours) and for which development was followed in culture to day 3 (n ¼ 2,859 cycles) /$36.00 Fertility and Sterility â Vol. 94, No. 3, August doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 Clinical Protocols The patients typically had normal clomiphene citrate challenge testing (generally FSH levels <10 miu/ml) and underwent controlled ovarian stimulation with luteal down-regulation using leuprolide acetate (Lupron; TAP Pharmaceuticals, Lake Forest, IL). Leuprolide acetate was begun either 1 week after urinary LH surge or the day after midluteal P determination, and was continued until at least day 2 of menses. Baseline ultrasonography and blood testing were performed to document no ovarian cysts >3cm,E 2 <50 pg/ml, and P <1.5 ng/ml. Alternatively, in patients with histories of poor gonadotropin response or high levels of FSH (R10 miu/ml), poor responder protocols were implemented. Although these protocols varied, such protocols were typically either microdose lupron with 0.05 mg SC leuprolide acetate twice daily starting cycle day 1 after an oral contraceptive (OC) lead-in with baseline ultrasound testing performed on day 2, or a GnRH antagonist (GnRH-a) protocol using OC pills for 3 weeks with baseline ultrasound testing cycle day 2 and GnRH-a starting at a dose of 0.25 mg/day beginning stimulation day 6. When baseline criteria were met, gonadotropin therapy (either Gonal-F [Serono Laboratories, Rockland, MA] or Follistim [Organon, Roseland, NJ]) with or without hmg (Humegon [Organon], Pergonal, or Repronex [Ferring Pharmaceuticals, Suffern, NY]) was begun. Stimulation was accomplished using either a single dose or divided daily doses of between 2 and 8 ampules/day, depending on patient age and anticipated response. Monitoring of follicles was achieved using ultrasound. Serum E 2 levels were measured, in general, on stimulation day 6 and then every 1 3 days as indicated. A 10,000 IU dose of hcg (Profasi; Serono) was administered IM when the two lead follicles had a mean diameter of >16.5 mm and the E 2 concentration was >500 pg/ml. Retrieval was performed transvaginally 36 hours after hcg administration in the standard fashion using either IV or regional anesthesia. One day after oocyte retrieval, luteal P supplementation was begun using one of three regimens: 1) daily IM P (50 mg); 2) daily 8% P vaginal gel (Crinone; Wyeth-Ayerst, Madison, NJ); or 3) twice daily vaginal P suppositories ( mg). Embryo transfer was performed with a Wallace catheter (Marlow/Cooper Surgical, Shelton, CT). For difficult transfers, a Marrs no. 4 or Marrs no. 5 embryo transfer catheter (Cook Ob/Gyn, Spencer, IN) was occasionally used. Laboratory Protocols All gametes and embryos were maintained at 37 C in a humidified atmosphere of 5% CO 2 in air. Within 4 6 hours of retrieval, oocytes were inseminated in groups of 3 5 in 1 ml Ham F10 medium supplemented with 5% human serum albumin (Invitro Care, Frederick, MD) or were fertilized (within 3 5 hours after retrieval) via ICSI. Zygotes having 2PN at the fertilization check hours after insemination or ICSI were cultured individually in 25 ml of growth medium (G1.2, G1.3 [Scandinavian IVF Science/Vitrolife, Gothenburg, Sweden], or Life Global [IVF Online, Toronto, Canada]) overlaid with 8 ml oil in Falcon 1007 culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ). Early cleavage check was carried out approximately hours after insemination to assist in identifying those embryos with maximal implantation potential (6 8). On day 3, the morphology of each embryo was assessed using standard criteria, with fragmentation and asymmetry being graded with numerical scores (9). Fragmentation scores of 0 through 4 were awarded to each embryo, where each score correlated to 0, 1% 9%, 10% 25%, 26% 50%, or >50% fragmentation, respectively. Blastomere asymmetry was graded using a numerical score of 1 through 3 according to uniformity in size and shape, where a score of 1 represented perfect symmetry, 2 moderate asymmetry, and 3 severe asymmetry. A team of six to eight embryologists, who participate in proficiency testing to ensure consistency in grading, performed the embryo assessments in our lab. Embryos with the lowest percentage of fragmentation, lowest asymmetry, and optimal number of cells were selected for transfer. Preference was given to eight-cell embryos when available, other characteristics being equal. Number of embryos transfered was determined according to number and quality of available embryos, patient age, and prior clinical history. Index Zygotes All 2PN zygotes at the fertilization check (n ¼ 23,293) were screened to identify those with one pronucleus or three pronuclei at early cleavage. Control Zygotes Two types of control groups of zygotes were assessed for each of the 1PN and 3PN datasets to compare embryo quality, implantation rates, and pregnancy rates with each type of index zygote: 1) internal control groups, composed of those 2PN zygotes from the same cycles as either the 1PN or 3PN zygotes but not exhibiting abnormal pronuclear number at early cleavage check; and 2) external matched-pair control groups, composed of zygotes from cycles performed during the same time period but with no 1PN or 3PN zygotes. These external control groups were obtained by matching each cycle having a 1PN or 3PN zygote with a control cycle for patient age, date of embryo retrieval, number of eggs retrieved, and number of embryos transferred. For accurate comparison, only control zygotes exhibiting 2PN or 0PN (rather than two or more cells) at early cleavage were selected for analysis. By creating this matched external control group we hoped to eliminate a source of bias, given the possibility that patients who produce 1PN or 3PN zygotes might not be representative of a normal IVF population. Outcome Variables Assessed Index zygotes were compared with zygotes in the internal and external control groups for distributions according to cell 966 Reichman et al. Abnormal pronuclei number late on day 1 Vol. 94, No. 3, August 2010

3 TABLE 1 Demographic comparison: index population (1PN) versus matched controls. 1PN Control No. cycles Age Attempt no Day 3 FSH (miu/ml) No. ampules Peak E 2 (pg/ml) 2, , No. eggs No. MII % 2PN/MII No. good-quality a * embryos No. embryos frozen y No. embryos transfered Note: Values represent mean SD. 1PN ¼ single pronucleus; MII ¼ metaphase II. a R8 cells with <10% fragmentation on day 3. * P<.004. y P<.02. number, fragmentation and asymmetry scores on day 3. In addition, implantation rates and pregnancy rates were compared for those zygotes with known developmental fate. Implantation rate was defined as the number of sacs detected by ultrasound at 5 6 weeks out of the total number of embryos transfered. Viable implantation rate was defined as the number of viable fetuses detected by ultrasound at 8 weeks out of the total number of embryos transferred. Pregnancy rate was defined as the number of transfers resulting in at least one viable fetus (by ultrasound to at least 8 weeks) out of all transfers performed. Statistical Analysis Differences between the index populations and matched-pair populations were determined using the Mann-Whitney U test. The data were analyzed using chi-squared with Fisher exact test, where P<.05 was considered to be statistically significant. RESULTS 1PN Zygotes Incidence and cycle demographics Of the 23,293 2PN zygotes evaluated at early cleavage, 671 (2.9%) exhibited 1PN. These 671 1PN zygotes arose from 445 patients and 512 cycles. The demographics of the cycles, along with those of the matched control group, are shown in Table 1. Comparison of the 1PN zygote population to the zygotes in the matched control cycles revealed significant differences only for the number of good-quality embryos (R8-cell with <10% fragmentation; P<.004]), and in number of embryos frozen (P<.02). No statistical significance among the index population was found between conventional IVF and ICSI in terms of number of 1PN zygotes obtained. Day 3 development of 1PN zygotes Figure 1 depicts the distribution of embryos according to cell number on day 3 for the index population compared with that for the internal and external control groups. Other than a higher number of 14-cell embryos in the internal control group, no statistical significance was uncovered regarding cell number between the two control groups. Therefore, for simplicity of presentation, statistical comparisons for cell number were made between the 1PN embryos and only those in the internal controls. The 1PN embryos showed a significant delay in cleavage, as reflected by significant increases in the percentage of embryos having 1 4 cells, and significant decreases in those with 6 8 or 10 cells (Fig. 1). The quality of these embryos was further assessed by comparing fragmentation and asymmetry scores with those of the two control groups. Significantly fewer 1PN embryos exhibited <10% fragmentation compared with either control group (42.1% for index vs. 54.9% for internal control [P<.0007] and vs. 54.2% for external control [P<.002]), with a corresponding significant increase in the proportion having 10% 25% fragmentation (42.1% for index vs. 34.0% for internal control [P<.0245] and 32.5% for external control [P<.007]). A trend toward increased asymmetry was observed for 1PN embryos, although this did not reach statistical significance. Developmental competency of 1PN zygotes In total, 98 of the 671 index embryos were transferred. Determining a precise implantation rate was obscured, given that 20 of the affected embryos were transfered along with unaffected embryos in cycles resulting in pregnancy but with less than 100% implantation. However, for 78 of the affected transferred embryos, developmental fate was certain. The implantation rate was 6.4% (5 sacs out of 78 embryos transfered). The viable implantation rate was 1.3% (1 viable fetus at 8 weeks out of 78 embryos transfered). Of the 76 transfers involving these 78 embryos of known developmental fate, one resulted in a viable pregnancy (1.3% pregnancy rate). In comparison, for the matched control group, the viable implantation rate was 20.7% (271 fetuses out of 1,309 embryos transfered) and the pregnancy rate was 40.4% (207 out of 512 transfers with at least one viable fetus). Of note, the one pregnancy that resulted from the transfer of a single affected embryo culminated in the delivery at 39 weeks of a healthy female weighing 3,316 g. 3PN Zygotes Incidence and cycle demographics Of the 23,293 2PN zygotes evaluated at early cleavage, 99 (0.42%) exhibited 3PN. These 3PN zygotes arose from 89 patients and 94 cycles. The demographics of the cycles, along with those of the matched control group, are shown in Table 2. Comparison of 3PN embryos to those in the matched control cycles revealed significant differences only for number of embryos Fertility and Sterility â 967

4 FIGURE 1 Number of cells on day 3 of zygotes with aberrant single-pronucleate (1PN) state at early cleavage compared with control groups. frozen (P<.01). No statistical significance among the index population was found between conventional IVF and ICSI regarding number of 3PN zygotes obtained. Day 3 development of 3PN zygotes Figure 2 depicts the distribution of embryos according to cell number on day 3 for the index population compared with those for the internal and external control groups. Again, our two control groups revealed consistent patterns for embryo cell number; other than a higher number of 11-cell embryos in the matched pair group, no statistical significance was found regarding cell number between the two groups. The 3PN embryos revealed a marked trend toward an increased percentage of embryos having %4 cells, with a corresponding significant decrease in those exhibiting 8 cells (P¼.0001). The quality of these embryos was further assessed by comparing fragmentation and asymmetry scores to those of the two control groups. However, no statistically significant differences were detected between index embryos and control groups regarding fragmentation or asymmetry, and no trend was discernible (data not shown). Developmental competency of 3PN zygotes In total, 9 of the 99 3PN embryos were transferred. Unlike their 1PN counterparts, none of the 3PN embryos implanted. In comparison, the viable implantation rate and pregnancy rate for the corresponding matched control group were 18.4% (45 fetuses out of 244 embryos transfered) and 40.4% (38 pregnancies with at least 1 viable fetus out of 94 transfers), respectively. DISCUSSION In this study, we set out to assess the development potential and clinical implications of embryos with one pronucleus or three pronuclei at early cleavage check despite their having exhibited two pronuclei at the time of fertilization. We discovered that these relatively rare phenomena resulted in embryos that exhibited diminished embryo quality for both 1PN and 3PN zygotes, as well as markedly decreased implantation and pregnancy rates compared with our control assisted reproductive technologies population. The process of pronuclear fusion has been well described in the literature. Syngamy is characterized by close association of maternal and paternal pronuclei with occasional occurrence of interdigitation of the opposing nuclear membranes (10). However, fusion of the two pronuclei occurs only after breakdown of the nuclear membranes (11). The 2PN zygote thereby progresses to a one-cell embryo lacking any overt evidence of pronuclei before proceeding through interphase of the first mitotic division, ultimately to undergo cytokinesis and form the two-cell embryo. Although embryos with abnormal pronuclear number have been described, such embryos typically represent events of 968 Reichman et al. Abnormal pronuclei number late on day 1 Vol. 94, No. 3, August 2010

5 TABLE 2 Demographic comparison: index population (3PN) versus matched controls. 3PN Control No. cycles Age Attempt no Day 3 FSH (miu/ml) No. ampules Peak E 2 (pg/ml) 2,260 1,057 2, No. eggs No. MII %2PN/MII No. good-quality a embryos No. embryos frozen * No. embryos transfered Note: Values represent mean SD. 3PN ¼ three pronuclei; MII ¼ metaphase II. a R8 cells with <10% fragmentation on day 3. * P<.01. abnormal fertilization, whether via parthenogenetic activation of the oocyte, dispermy, or failure of extrusion of the second polar body (1, 12). These rare embryos are typically discarded at the time of the fertilization check, given their abnormal chromosomal makeup and dismal prospects for reproductive success. Rarely, however, embryos are noted to appear normally fertilized, exhibiting 2PN status, but subsequently deviate from normal development. To our knowledge, only one article has previously addressed the type of embryos studied in the present paper, without a subsequent examination of their developmental integrity or role in assisted reproduction (5). Checking zygotes late on day 1 for early cleavage has been reported to improve embryo selection by identifying those embryos with the greatest implantation potential (6 8). Doing so, however, has revealed zygotes such as those described in this paper, whose development deviates from the norm. Although such embryos were noted to occur relatively rarely in our lab, identifying their developmental potential is useful, especially when patients have few high-quality embryos for transfer and the embryos of the seemingly highest quality have transitioned through abnormal pronuclear milestones. The question arises as to whether, in the absence of high-quality normal embryos, an embryo which at early cleavage check exhibited an abnormal number of pronuclei but subsequently developed into a symmetric, unfragmented eight-cell embryo should be transferred preferentially over a traditional embryo with less optimal parameters. The findings we present here show that the implantation potential of such deviant embryos is <2%, indicating that transfer of these embryos should be avoided when possible. The etiology of these embryos remains unclear. It would be informative to karyotype such embryos to identify whether their unusual development is associated with aneuploidy, as has been done with abnormally pronucleated zygotes of the more traditional variety (i.e., having not first achieved a complement of two pronuclei [4, 13]). Although dispermy, parthenogenetic oocyte activation, or asynchronous dissolution of the pronuclear membrane may, respectively, explain the appearance of 3PN or 1PN zygotes identified at the fertilization check, an alternative explanation must be found for the appearance of these pronuclear stages after normal 2PN development. It is of interest that both types of abnormal zygotes occurred in cycles with unusually high yields of oocytes (mean SD: and , respectively; Tables 1 and 2). In our program, we retrieve ten oocytes on average. Although it was not the focus of the present study, this finding raises the question whether cycles with high oocyte yield, or patients predisposed to higher oocyte yield, may be especially susceptible to forming such abnormal zygotes. In reviewing the infertility diagnoses for our 1PN and 3PN groups, we discovered that 11.7 % and 9.6% of patients, respectively, carried the diagnosis of PCOS or anovulatory dysfunction, a diagnosis correlated with robust ovarian response during superovulation. Perhaps further studies could elucidate whether this association bears any causal relationship. Tripronucleate embryos occurred more rarely and displayed poorer developmental fate regarding cell number on day 3 of culture. Single-pronucleate embryos, on the other hand, more closely followed the development of normal embryos, while still resulting in a statistically significant decrease in the number of highest-quality embryos. This study is not without limitations. Although the scoring of embryos was performed by well trained embryologists in a - major academic center, this grading is subject to human error. However, given the abnormal cleavage rates exhibited by both the 1PN and 3PN populations, it is likely that PN scoring was accurate in at least the majority of cases. This study takes a retrospective approach; however, given the rare nature of these embryos and an inability to predict their occurrence, performing a prospective study would be both resource and time intensive. It is our hope that the present findings may aid centers of reproductive medicine in offering the highest chances of success to IVF patients. By elucidating the developmental fate and reproductive potential of the abnormal zygotes in this study, we hope to assist embryologists in selecting those embryos with the highest implantation potential while excluding, as in this study, those embryos whose deviation from the norm results in suboptimal rates of successful pregnancy. CONCLUSION In this study, we examined the incidence, developmental fate, and implantation rate of zygotes noted to have two pronuclei at the fertilization check but to have transitioned to an abnormal PN number at early cleavage check late on day 1. The Fertility and Sterility â 969

6 FIGURE 2 Number of cells on day 3 of zygotes with aberrant tripronucleate (3PN) state at early cleavage compared with control groups. present results indicate that such 1PN and 3PN zygotes at early cleavage are both relatively rare, with 1PN zygotes being the more common of the two. Such embryos were noted to have poorer overall development characteristics compared with control groups as well as a marked decrease in both implantation potential and pregnancy rate. Patients should therefore be counseled regarding the very low likelihood of viable pregnancy after transfer of these abnormally developing zygotes in the event that no other suitable embryos are available for transfer. Further studies are needed to elucidate the origin of abnormal pronuclear stages after normal fertilization. Acknowledgment: We thank all of the embryologists at Brigham and Women s Hospital for their expertise in embryo grading, and their tireless commitment to maintaining the highest standards of care to our patients. REFERENCES 1. Balakier H, Squire J, Casper RF. Characterization of abnormal one pronuclear human oocytes by morphology, cytogenetics and in-situ hybridization. Hum Reprod 1993;8: Balakier H. Tripronuclear human zygotes: the first cell cycle and subsequent development. Hum Reprod 1993;8: Munne S, Tang Y, Grifo J, Cohen J. Origin of single pronucleated human zygotes. J Assist Reprod Genet 1993;10: Staessen C, Janssenswillen C, Devroey P, Van Steirteghem AC. Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization. Hum Reprod 1993;8: Nagy ZP, Liu J, Joris H, Devroey P, Van Steirteghem A. Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod 1994;9: Shoukir Y, Campana A, Farley T, Sakkas D. Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod 1997;12: Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum Reprod 1998;13: Neuber E, Rinaudo P, Trimarchi JR, Sakkas D. Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development. Hum Reprod 2003;18: Racowsky C, Combelles CM, 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: Xu KP, Greve T. A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes. J Reprod Fertil 1988;82: Levron J, Munne S, Willadsen S, Rosenwaks Z, Cohen J. Male and female genomes associated in a single pronucleus in human zygotes. Biol Reprod 1995;52: Grossman M, Calafell JM, Brandy N, Vanrell JA, Rubio C, Pellicer A, et al. Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum Reprod 1997;12: Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod 1987;37: Reichman et al. Abnormal pronuclei number late on day 1 Vol. 94, No. 3, August 2010