Early compaction on day 3 may be associated with increased implantation potential

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1 Early compaction on day 3 may be associated with increased implantation potential Christine C. Skiadas, M.D., Katharine V. Jackson, B.S., and Catherine Racowsky, Ph.D. Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts Objective: To determine whether day 3 embryos exhibiting early compaction have an improved implantation potential compared to embryos without compaction. Design: A retrospective cohort study. Setting: Hospital-based academic medical center. Patient(s): Women 38 years of age undergoing IVF cycles between November 2001 and December 2004 having a day 3 transfer of one or two embryos with 8 cells. Intervention(s): Standard IVF protocol. Main Outcome Measure(s): Compaction grading and implantation rates of 1,047 embryos as related to fragmentation of 8-cell embryos in patients with either 0% or 100% implantation. Result(s): Compaction grading was strongly associated with implantation potential; however, the direction of this effect depended on the degree of fragmentation. In embryos with 10% fragmentation, implantation rates increased with the degree of compaction (grade 1, 25%; grade 2, 33%; and grade 3, 47%); in embryos with 10% fragmentation, the effect was reversed (grade 1, 38%; grade 2, 20%; and grade 3, 9%). Conclusion(s): Assessing the degree of compaction can be a valuable addition to traditional morphologic assessment in identifying optimal embryos for transfer on day 3. (Fertil Steril 2006;86: by American Society for Reproductive Medicine.) Key Words: Embryo compaction, embryo fragmentation, IVF, implantation rates Attempts to maximize IVF pregnancy rates (PR) were initially achieved by transferring greater numbers of embryos. This approach had the negative side effect of increasing the incidence of high-order multiple gestations (1 5). To maintain PRs, and still reduce high-order multiple gestations, there has been a progressive move toward decreasing the number of day 3 embryos transferred (6), as well as performing day 5 transfers involving only one or two blastocysts (7, 8). However, blastocyst transfer may not be ideal in all cases, and may compromise a successful outcome that would otherwise be achieved after a day 3 transfer (8, 9). Therefore, continuing to optimize selection of the most developmentally competent embryos for day 3 transfer remains an important goal in improving IVF implantation rates. Routine morphologic assessment on day 3 includes cell number (10), extent of fragmentation (11), and degree of asymmetry (12), all of which have been shown to be correlated with PRs after day 3 transfer. Although these correlations are well established (12), many embryos that appear viable on day 3 by traditional assessment fail to implant (13). To supplement traditional means of assessment, several studies have investigated embryonic developmental milestones as an additional means to determine developmental competence. Received November 9, 2005; revised and accepted March 21, Reprint requests: Catherine Racowsky, Ph.D., Department of Obstetrics and Gynecology, Brigham and Women s Hospital, 75 Francis Street, ASB 1 3, Rm 082, Boston, MA (FAX: ; cracowsky@partners.org). Embryos that undergo early cleavage have been associated with higher implantation rate and PR (3, 14, 15). Nucleolar alignment (16) and pronuclear morphology (16 19) have also been postulated as important developmental markers. In addition, combined methods of scoring with differential weighting of these morphologic features have been proposed (20, 21) and correlated with improved implantation rate and PR (20) when compared with traditional assessment (21, 22). Another potential marker of embryonic developmental competence is the early formation of tight junctions as the process of compaction gets underway. Compaction typically occurs on day 4 as the embryo proceeds to the morula stage (23). Because the degree of compaction on day 4 has been correlated with implantation potential (24), the possibility exists that early compaction on day 3 may also represent a predictor of enhanced developmental competency. Therefore, the purpose of the present study was to determine whether or not day 3 embryos that exhibit early compaction have an improved implantation potential compared to those embryos with little or no compaction. MATERIALS AND METHODS Study Entry Criteria Entry criteria for the cycles included in this study were as follows: IVF cycles between November 2001 and December 2004, using G1.2 or G1.3 media for embryo culture from days 1 3, maternal age 38 years, and day 3 transfer of one 1386 Fertility and Sterility Vol. 86, No. 5, November /06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 or two embryos having 8 cells on day 3. To assess definitively the fate of each transferred embryo, only cycles resulting in either 0% or 100% implantation were included. Implantation was defined as the presence of a viable fetal heart at 12 weeks of gestation. Therefore, all embryos could be included in the analysis, even in cases where the transferred embryos exhibited different compaction grades. After appropriate institutional review board approval for chart review was obtained, a total of 611 cycles were identified as meeting these inclusion criteria. Stimulation Protocols Patients with normal clomiphene citrate (CC) challenge testing (generally FSH levels 10 miu/ml) underwent controlled ovarian stimulation with luteal down-regulation using leuprolide acetate (LA; Lupron; TAP Pharmaceuticals, Lake Forest, IL). Leuprolide acetate was begun either a week after documentation of urinary LH surge or the day after a midluteal P determination, and was continued until at least day 2 of menses. Baseline ultrasonography and blood testing were then performed to document that no cysts 3 cm were present, E 2 was 50 pg/ml, and P was 1.5 ng/ml. Alternatively, in patients with histories of poorer gonadotropin responses or FSH levels 10 miu/ml, poor responder protocols were used. The most usual protocol used was either a microdose lupron protocol with lupron 0.05 mg SC twice a day started cycle day 1 of a period after oral contraceptive (OC) pill lead in and baseline ultrasound testing performed on day 2 or, alternatively, a GnRH antagonist (GnRH-a) protocol using an OC pill for 3 weeks, then baseline ultrasound testing cycle day 2, with GnRH-a initiation at a dose of 0.25 mg/day SC starting stimulation day 6. When baseline criteria were met, gonadotropin therapy (either Gonal-F, Serono Laboratories, Inc., Rockland, MA or Follistim, Organon, Roseland, NJ) with or without hmg (Humegon: Organon, Pergonal, or Repronex: Ferring Pharmaceuticals Inc., Suffern, NY) was begun. Stimulation was generally achieved using single or divided daily dosing of between 2 and 8 ampules/day, as appropriate, depending on patient age and anticipated response. Monitoring of follicle growth was achieved using ultrasound, and serum E 2 levels were measured starting on stimulation day 6 and then every 1 3 days as indicated. A dose of 10,000 IU of hcg (Profasi: Serono) was administered IM when two follicles reached a maximum diameter of 20 mm (mean 16.5 mm) and the E 2 concentration was 500 pg/ml. Transvaginal oocyte retrieval was performed 36 hours after hcg administration in the standard fashion with IV general anesthesia or, in some cases, spinal anesthesia as indicated. Oocyte Fertilization, Embryo Culture, Transfer, and Outcome Assessment Within 4 6 hours of retrieval, oocytes were inseminated in groups of 3 5 in 1 ml Ham s F10 medium supplemented with 5% human serum albumin (InvitroCare Inc., Frederick, MD) or were injected with a single sperm. After identification of two pronuclei (PN) at the fertilization check hours after insemination or intracytoplasmic sperm injection (ICSI) on day 1, zygotes with 2 PNs were cultured individually in 25 L of growth medium (G1.2 or G1.3; Scandinavian IVF Science/Vitrolife, Gothenburg, Sweden) overlaid with 8 ml of oil in Falcon 1007 culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ). All cultures were maintained at 37 C in a humidified atmosphere of 5% CO 2 in air. On day 3, the morphology of each embryo was assessed using standard criteria (12) hours after insemination ( hours after insemination, mean SD). Fragmentation was graded as 10%, 10% 25%, and 25% of the blastomere volume; blastomere asymmetry was graded according to uniformity in size and shape of the blastomeres as exhibiting no asymmetry, moderate asymmetry, and severe asymmetry. Embryos having the optimal cell number, the lowest percentage of fragmentation, and the lowest asymmetry in a given cohort were selected for transfer. Other characteristics being equal, preference was given to embryos having eight cells than to those having more than eight cells. The number of embryos transferred to a given patient was determined by the number and quality of embryos she had available, the patient s age, and her prior clinical history. Transferred embryos were photographed within the 2 hours before transfer and photographs were stored in the medical record. Luteal P supplementation was initiated the day after oocyte retrieval and was achieved using one of three regimens: [1] daily IM P (50 mg); [2] daily vaginal gel (8% P [Crinone; Wyeth-Ayerst, Madison, NJ]); or [3] twice daily 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. Clinical pregnancies were identified by the presence of a gestational sac on ultrasonography 5 weeks after oocyte retrieval. The implantation rate was calculated as the number of fetal hearts present at 12 weeks of gestation, divided by the number of embryos transferred, multiplied by 100. Compaction Grading Embryo images (n 1,047) in archived photographs were evaluated retrospectively for degree of compaction by an experienced embryologist who was blinded to pregnancy outcomes. Compaction was graded into one of three categories as follows: grade 1 (no compaction: blastomeres spherical with no evidence of membrane fusion); grade 2 (some compaction: some membrane fusion evident but the number of blastomeres easily countable); and grade 3 (full compaction: extreme membrane fusion making it very difficult to count the number of blastomeres present). Embryos were Fertility and Sterility 1387

3 FIGURE 1 Representative images of embryos exhibiting the three compaction grades. Original magnification, 300. from each group to be regraded by the same embryologist, who was blind to previous compaction scores. Of the 42 embryos, only one was graded differently on the second assessment, representing a variance in compaction grading of 2.4%. Statistical Analysis Data were analyzed using 2 with Fisher s exact test, with P.05 considered statistically significant. RESULTS Of the 1,047 embryos, the majority (69%) exhibited 10% fragmentation, with the remaining 31% exhibiting 10%. The distribution of compaction grades within each fragmentation group is depicted in Table 1. As shown, the distribution of embryos across compaction grades was statistically significant regardless of fragmentation group. Furthermore, when comparing the proportion of embryos exhibiting each compaction grade between the two fragmentation groups, we found significant differences for compaction grades 1 and 2 (0.01 and 0.05, respectively), but not for compaction grade 3. further stratified according to their degree of fragmentation into those having 10% fragmentation, and those with 10% fragmentation. This stratification was chosen based on previous studies showing that embryos with 10% fragmentation have significantly decreased implantation rates, compared with those with 10% fragmentation (5, 12). Figure 1 shows examples of embryos exhibiting each compaction grade within each of these two fragmentation groups. To determine the extent of intraobserver variance in compaction grading, 42 representative embryos were selected Compaction grading was strongly associated with implantation potential; however, the direction of this effect (either positive or negative) varied with the degree of fragmentation. In embryos with 10% fragmentation, the overall implantation rate was 31%, with embryos having significantly higher implantation rates as the extent of compaction increased (grade 1: 25% implantation; grade 2: 33% implantation, and grade 3: 47% implantation; P.004; Fig. 2). However, an inverse relationship between compaction and implantation rate was observed in embryos with 10% fragmentation, (grade 1: 38% implantation, grade 2: 20% implantation, and grade 3: 9% implantation; P.0005; Fig. 2). In addition, we analyzed the relationship between compaction grade and implantation rate in the subset of embryos that were derived from cycles with 2 embryos available for transfer (n 928), as it is this group in whom compaction grading might be relevant as an additional tool in embryo selection. The same relationship between compaction grade TABLE 1 Distribution of embryos according to compaction grade Distribution of embryos Compaction grade Fragmentation <10% (n 721) P.001 Fragmentation 10% (n 326) P (39.9%) a 98 (30.1%) b (53.5%) c 196 (60.1%) d 3 47 (6.5%) 32 (9.8%) Note: Groups with different superscript letters are significantly different: a vs. b, P.01; c vs. d, P Skiadas et al. Embryo compaction and implantation Vol. 86, No. 5, November 2006

4 FIGURE 2 Implantation rate by compaction grade. The stippled columns represent embryos exhibiting 10% fragmentation, the red columns represent embryos with 10% fragmentation for the entire study dataset. Columns denoted by different letters are statistically significant: a vs. b, P.05; a vs. c, P.005; A vs. B, P.002; A vs. C, P.003. and implantation rate was observed for each fragmentation group within this subcohort (Fig. 3). DISCUSSION Preimplantation development follows a programmed timeline during which an organized series of critical events take place. After fertilization, the embryo undergoes first cleavage between 20 and 27 hours after insemination (25 27), and usually reaches the four-cell stage at approximately 48 hours, and then the eight-cell stage by approximately 72 hours. Compaction is expected to occur on day 4 (23), with embryos undergoing onset of blastocyst formation early on day 5, with completion of blastulation by late day 5 (28). All of these developmental stages have been evaluated both with regard to selecting embryos with optimal morphology, and in terms of identifying the best time for embryo transfer. Blastocyst transfer has held out hope as a way to optimize implantation rates with the fewest embryos transferred; however, this approach may not be ideal for all patients (8). Therefore, a need still exists for further optimization of embryo selection for day 3 transfers. Numerous studies have combined key morphologic criteria for predicting developmental competency of cleavage stage embryos. The vast majority of these studies have used the conventional characteristics of cell number, fragmentation, and symmetry immediately before transfer on day 3 (1, 2, 21, 29), with some more recent studies also combining characteristics on day 1 and day 2, into a cumulative scoring system (14, 20, 22, 30, 31). However, to our knowledge, only one article has addressed compaction as part of a combined score system. In this system, Desai et al. (32) evaluated day 3 embryos for cell number, presence of equal sized blastomeres, blastomere expansion (blastomeres touching the zona with minimal perivitelline space), cellular cytoplasm clear of vacuoles, presence of cytoplasmic pitting, signs of compaction, and the pattern of fragmentation. Using this methodology, the PR was found to increase with the transfer of a compacting embryo. However, this relationship did not reach statistical significance and the degree of compaction was not independently assessed. FIGURE 3 Group analysis of implantation rate by compaction grade. Embryos are derived from the cohort of cycles where 2 embryos were available for selection before embryo transfer. Columns denoted by different letters are statistically significant: a vs. b, P.025; A vs. B, P.05. Fertility and Sterility 1389

5 The data reported in the present work suggest that compaction grading should be combined as part of a cumulative scoring system to help predict which embryos are optimal for transfer. However, in this paradigm, the present results indicate that compaction would be added favorably to scores of minimally fragmented embryos ( 10%), whereas it would negatively impact the cumulative score in embryos exhibiting greater fragmentation ( 10%). It was unexpected that the effect of compaction on implantation varied depending on the degree of fragmentation. The explanation for these observations remains to be determined. However, the possibility exists that in minimally fragmented embryos, compaction represents the normal transition to the next stage of development, whereas in embryos with 10% fragmentation, either the fragments may impair the normal process of compaction, or fragmentation could represent a mechanism of apoptosis. Further studies are needed to investigate these possibilities. There are several limitations to this study. First and foremost, all compaction grading was done retrospectively using only two-dimensional photographs, as opposed to grading embryos prospectively by evaluating them under the microscope. It is possible that compaction grading would have been altered by prospective assessment. Second, the study was confined only to patients 38 years of age. It is possible, albeit unlikely, that the results obtained may not be applicable to patients across all age groups. Finally, all compaction grading was performed by a single embryologist. Although it was considered important to provide such standardization for the present study, the reproducibility of this morphological assessment needs to be evaluated among multiple embryologists and across several IVF laboratories to ensure concordance with the grading system. Nevertheless, in view of the present data, we have incorporated compaction assessment in our current embryo grading system. In conclusion, in combination with traditional morphologic assessment of cell number and percent fragmentation, the present results indicate that early compaction can be a valuable tool in selecting which 8-cell embryos to transfer on day 3. In embryos with 8 cells and with minimal fragmentation, early compaction portends a favorable prognosis, whereas in embryos with 8 cells and with a higher degree of fragmentation, compaction is negatively associated with implantation potential. Acknowledgments: The authors thank the entire embryology team at Brigham and Women s Hospital for their expertise in embryo grading and Ms. Lauren Racowsky for assistance with extensive data entry. REFERENCES 1. Puissant F, Van Rysselberge M, Barlow P. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod 1987;2: Cummins J, Breen T, Harrison K. 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