IN VITRO FERTILIZATION

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1 FERTILITY AND STERILITY VOL. 76, NO. 2, AUGUST 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. IN VITRO FERTILIZATION Embryo fragmentation in vitro and its impact on treatment and pregnancy outcome Thomas Ebner, Ph.D., a Cemil Yaman, M.D., a Marianne Moser, Ph.D., a Michael Sommergruber, M.D., a Werner Pölz, Ph.D., b and Gernot Tews, M.D. a Women s General Hospital, Linz, Austria Received August 30, 2000; revised and accepted April 10, No support of any kind was granted for this study. Presented in part at the 16th Annual Meeting of the European Society of Human Reproduction and Embriology (ESHRE), Bologna, Italy, June 25 28, Reprint requests: Thomas Ebner, Ph.D., Women s General Hospital, IVF-Unit, Lederergasse 47, A-4010 Linz, Austria (FAX: ; thomas. ebner@li.lkh.ooe.gv.at). a IVF-Unit, Women s General Hospital, Linz, Austria. b Department for Statistics, University of Linz, Linz, Austria /01/$20.00 PII S (01) Objective: To determine the impact of embryo fragmentation on pregnancy, obstetric, and perinatal outcome. Design: Retrospective analysis of embryo transfers that were homogeneous in regard to the degree of fragmentation. Setting: Fertility center. Patient(s): A cohort of 460 fresh embryo transfers. Intervention(s): A total of 164 pregnancies were analyzed for the incidence of antepartum complications during gestation, obstetric (multiple pregnancy, preterm delivery, cesarean section), and perinatal outcome (sex, birth weight, admission to neonatal intensive care unit, malformations). Main Outcome Measure(s): Implantation and clinical pregnancy rate, obstetric and perinatal outcome. Result(s): Embryo fragmentation and number of embryos per transfer showed a significant influence on clinical pregnancy and implantation rate. No such relation was found concerning complications, multiple pregnancy rate, incidence of cesarean section, gestation week, birth weight, and average time at the neonatology. On the other hand, pregnancies derived from bad-quality embryos had a significantly higher rate of malformations. Conclusion(s): The higher percentage of malformations found in bad-quality embryos may be due to a higher percentage of apoptotic features and chromosomal disorders. For ethical reasons, the transfer of embryos with 50% fragmentation should be considered only after consultation with the patient. (Fertil Steril 2001;76: by American Society for Reproductive Medicine.) Key Words: Embryo quality, fragmentation, implantation rate, malformation, number of embryos, pregnancy outcome It is well established that the success rate of IVF or intracytoplasmic sperm injection (ICSI) cycles is determined by uterine receptivity and by the number and quality of embryos transferred (1 3). From the beginning of assisted reproduction, embryo quality parameters have remained largely unchanged. The decision of which embryos to transfer is based on the manifestation of the blastomeres and the degree of fragmentation (4). The occurrence of embryo fragmentation in vitro is a common feature and supposed to be associated with a deleterious outcome (2, 3, 5, 6). There have been only a few reports of pregnancies after embryo transfer (ET) of heavily fragmented embryos (4, 6 9). Because most of these studies dealt with heterogeneous ETs, with regard to the degree of fragmentation (4, 7, 8), it can only be assumed which embryo implanted and which did not. Comparison between programs is difficult because evaluation systems that are not standardized are used. Alikani et al. (10) has described a very detailed embryo classification system that implies a close relationship between embryo fragmentation patterns and the incidence of implantation. This, coupled with the fact that a wide range of chromosomal abnormalities are found in fragmented embryos (11 13), led us to retrospectively search our data for homogeneous transfers, consisting exclusively of embryos with the same fragmentation, and to use this information to study the 281

2 impact of embryo fragmentation on the rates of implantation and clinical pregnancy. Because chromosomal abnormalities may harm pregnancies irreversibly or result in loss of pregnancy, we evaluated a possible relationship between embryo fragmentation and the obstetric and perinatal outcome. To date, it has been accepted that there is no difference in obstetric and perinatal outcome between IVF-ICSI and natural cycles (14, 15), for example. Differences in birth weight and preterm delivery are rather the result of multiple pregnancies. MATERIALS AND METHODS Because the present study is dealing with retrospective data of a routine process, approval of the internal institutional review board was obtained. A pool of 1,064 patients was retrospectively searched for homogeneous transfers consisting exclusively of embryos with the same degree of fragmentation (group A: no fragmentation; group B: 25% fragments; group C: 25% 50% fragments; group D: 50% fragments). During this 4-year period, the couples were referred either to the IVF (n 473) or the ICSI program (n 591). A total of 460 ETs were considered homogeneous. The mean age of all these patients was years. Patients with recurrent pregnancy loss were excluded from further analysis. Our criteria for exclusion were at least three consecutive early pregnancy losses. In addition, women suffering from corpus luteum insufficiency or showing an endometrium 8 mm were not included in our study to minimize a possible inhibiting influence on implantation. In all cycles, ovarian stimulation was performed using a long protocol (16) combining a gonadotropin-releasing hormone agonist (Suprecur, Hoechst, Frankfurt, Germany) with a human menopausal gonadotropin treatment (Gonal F, Serono, Italy or Menogon, Ferring, Kiel, Germany). Follicular development was monitored by ultrasonography and serum E 2 measurements. At an appropriate time in stimulation, 5,000 10,000 IU of hcg (Pregnyl, Organon, Oss, The Netherlands) was administered to induce ovulation. The oocyte retrieval was performed transvaginally using ultrasound. The partner s semen sample was collected through masturbation and ejaculation into a sterile specimen container. According to the sperm count, either a swim-up (IVF) or a mini swim-up technique (severe oligoasthenoteratozoospermia) was performed (17). Fertilization and embryo grade were assessed hours and hours after IVF-ICSI. All embryos were graded according to the method of Veeck (4) at a magnification of 200. Embryos that did not reach four-cell stage on the day of transfer were not considered for transfer. Neither assisted hatching nor cryopreservation was performed. All transfers were performed on day 2 using an Edwards- Wallace Catheter (Smiths Industries, Lancing, Kent, United Kingdom). Three thousand IU of hcg (Pregnyl, Organon, Vienna, Austria) were injected on days 2, 5, and 8 for luteal phase. Seventeen days after intrauterine transfer, blood concentration of hcg was measured. Biochemical pregnancy was defined as a significant increase in hcg levels ( 10 mu/ ml). The implantation rate was defined by ultrasound visualization, 4 weeks after embryo transfer, as a gestional sac per embryo transferred. This included subclinical pregnancies, as evidenced by a gestational sac but no fetal heartbeat, and a clinical pregnancy, evidenced by at least one gestational sac with positive heart activity. If patients did not deliver in our clinic, medical records were requested to get information about birth weight, weeks of gestation, complications during pregnancy (vaginal bleeding, contractions), malformations, and transfer to a neonatal care unit. Statistics The 2 test was used to analyze nominal variables in the form of frequency tables. Normally distributed (Kolmogorov-Smirnov Test with Lilliefors correction) metric variables were tested with the t test for independent samples with correction for heteroscedasticity. Ordinal variables or notnormally distributed metric variables were analyzed with the Mann-Whitney U test. If more than two groups had to be analyzed, normally distributed metric variables (Kolmogorov-Smirnov test with Lilliefors significances) with equal variances (Levene test) were examined by means of one-way ANOVA (multiple comparisons are made with Bonferroni test). For not-normally distributed metric variables or for variables with unequal variances, the Kruskal-Wallis one-way ANOVA was employed (multiple comparisons according to Nemenyi) (18). For correlation analysis, Spearman s rank correlation coefficient was used. All tests were two-tailed with a confidence level of 95% (P.05). No adjustments for the P values (such as Bonferroni-corrections) were made, therefore, all P values are only descriptive. RESULTS Treatment Outcome In 196 transfers, no fragmentation was detectable (group A); in 113 transfers, all embryos showed 25% fragments (group B), 76 revealed 25% 50% fragments (group C), and 75 transfers consisted of poor-quality embryos ( 50% fragments; group D). In Figure 1, these groups data are plotted in terms of the impact of number of embryos on the results of homogeneous transfers (P.001). Including 11 biochemical pregnancies, 175 pregnancies 282 Ebner et al. Embryo fragmentation and its impact on outcome Vol. 76, No. 2, August 2001

3 FIGURE 1 Relationship between number of embryos considered for transfer, embryo fragmentation and clinical pregnancy rate. In 196 transfers, no fragmentation was detectable (group 1; u); in 113 transfers, all embryos showed 25% fragments (group 2; ), 76 revealed 25% 50% fragments (group 3; ), and 75 transfers consisted of poor-quality embryos ( 50% fragments, group 4; s). were recorded. The rates of implantation, clinical pregnancy, and multiple pregnancy are shown in Table 1. In 460 ETs, a total of 1,176 embryos was transferred (mean number, embryos per transfer), of which 198 (16.8%) implanted. A significant influence (at least P.05) of fragmentation on implantation rate and clinical pregnancy rate was detected (Table 1), whereas no difference was seen with regard to multiple pregnancy rate and ongoing pregnancy rate. Pregnancy Outcome Between 1994 and 1999, 144 children were born after homogeneous ET (76 female and 68 male). The sex ratio showed no statistical correlation between the four treatment groups. As shown in Table 2, birth weight and weeks of gestation did not correlate with embryo quality in singleton, twin, and triple pregnancies. The rate of complications and cesarean sections could not be analyzed for multiple pregnancies because of the small numbers in these subgroups. For singleton pregnancies, no significant difference was found in this respect. A total of 41 (28.9%) children had to be admitted to the neonatal care unit. The time of admission to this unit was related to multiple pregnancy but not to embryo quality. Significantly more malformations (P.001) were seen in cohorts with transfer of fragmented embryos (groups C and D). In detail, two cases of trisomy 18, one case of hydrocephalus with an anal atresia, and one case of a hydrocele were diagnosed in group D (36.4%), and in cohort C, one trisomy 21 and one case of fibrome were seen (13.3%). Groups A (cleft lip, ventricular septal defect) and B (oval foramen of heart, hydrocele, encephalic cyst) showed only minor malformations (3.2% and 6.3%, respectively). DISCUSSION The accurate evaluation of embryo implantation potential is crucial during IVF-ET treatment to increase clinical- but not multiple-pregnancy rate. The period before implantation is important for reproductive success because the egg undergoes several cleavage divisions to the blastocyst stage. Implantation itself is a symbiotic interaction between such blastocysts and the endometrium. In the present study, the negative influence of an inadequate endometrium on pregnancy (19) could be minimized by including only women with adequately receptive endometria ( 8 mm). Thus, in the present study, differences in rates of implantation or pregnancy rates may be determined by the number and quality of embryos transferred (1). In fact, though all TABLE 1 Impact of embryo fragmentation on implantation and pregnancy rate. Pregnancy Implantation Study group n PR (%) MPR (%) IR (%) Ongoing (%) A (46.9) a 14 (15.2) 109/508 (21.5) c,d 80 (73.4) B (35.4) b 7 (17.5) 48/297 (16.2) 38 (79.2) C (22.4) 3 (17.7) 21/202 (10.4) 15 (71.4) D (20.0) 5 (25.0) 20/169 (11.8) 11 (55.0) Total (35.7) 29 (17.7) 198/1,176 (16.8) 144 (72.7) Note: IR implantation rate; MPR multiple pregnancy rate; n number of homogeneous transfers; PR clinical pregnancy rate. a P.001 (vs. embryo groups C and D). b P.05 (vs. all other groups). c P.05 (vs. group B). d P.001 (vs. groups C and D). FERTILITY & STERILITY 283

4 TABLE 2 Impact of embryo fragmentation on pregnancy outcome. Study group n Birth weight Week CSR (%) Comp (%) Neo A Singletons 51 3, (19.6) 7 (13.7) 8 3 Twins 14 2, (85.7) 4 (57.1) Triplets 15 1, (100) 1 (20) B Singletons 23 3, (34.8) 6 (26.1) Twins 12 2, (50.0) 1 (16.7) Triplets 3 1, (100) 1 (100) 49 0 C Singletons 11 3, (18.2) 5 (45.5) 4 0 Twins 4 2, (50.0) 1 (50.0) 8 0 D Singletons 9 3, (22.2) 4 (44.4) 23 4 Twins 2 1, (100) 1 (100) Comp complications during pregnancy; CSR cesarean section rate; n number of lifeborn children; Neo staying at neonatology. groups in our investigation had a similar mean number of embryos transferred, the outcome was dependent on the number of embryos transferred per recipient (Fig. 1). Some rapid and less-invasive embryo-grading systems have been evaluated for selecting the optimal embryos for transfer. To date, these systems have been mainly based on size and number of blastomeres as well as on the percentage of anucleate fragments (5, 6, 20, 21). More recently, Alikani et al. (10) suggested including the fragmentation pattern in decision making by retrospectively analyzing large series of cases. Though these authors used a more detailed scale for the degree of fragmentation, our data confirm the results concerning implantation behavior. Because we did not consider fragmentation patterns, our results may be preliminary; nevertheless, for the most part our fragmentation class D corresponded to the type IV pattern previously described by Alikani et al. (10) and, consequently, gave the worst results. There are several mechanisms that are known to cause demise of a high number of preimplantation embryos, including uterine factors, immunological rejection, and chromosomal abnormalities. The fact that embryos with moderate to excessive fragmentation may have limited developmental potential both in vivo and in vitro (22) supports the latter finding. Jurisicova et al. (22) detected a number of morphological features associated with apoptosis (e.g., chromatin condensation, cellular shrinkage) in spare human embryos. In contrast to necrosis, which is triggered by chemical or physical injury, apoptosis is caused by external or internal signals (23). Thus, apoptosis is a deliberate, genetically controlled program and can lead to characteristic cellular fragmentation in human embryos (24). Therefore, embryo fragmentation in transferred embryos may reflect different stages of apoptosis (25). The embryo thus maintaining or restoring its viability (10, 26) may eliminate particular blastomeres being affected by anomalies. Our study confirms previous observations that embryo quality has significant impact on pregnancy rate (2, 5, 6, 10, 26) and, consequently, on implantation rate (3, 5, 28). To our knowledge, this study is the first to indicate that multiple implantation can occur in all fragmentation groups. The implantation potential of bad-quality embryos may be underestimated, though their potential to proceed in development may be limited. To date, differences in fetal loss (clinical abortion rate) among groups with different degrees of fragmentation have not been noted (5, 6, 26); this is probably due to the evaluation of a relative small number of pregnancies resulting from transfer of exclusively bad-quality embryos. However, our analysis of more extensive data confirm that clinical abortion rate does not follow any definite trend. Like biochemical pregnancies (26), clinical abortions may reflect the loss of genetically defective embryos because a higher incidence of a wide range of chromosomal abnormalities (including aneuploidy, polyploidy, mosaicism, and premature chromosome condensation) was found in fragmented embryos (11 13) as compared with embryos without fragments. Controlled hyperstimulation of the ovaries and transfer of more than one viable embryo in infertility treatment has a considerable influence on the incidence of higher-order pregnancies. Multiple gestation is associated with prematurity, low birth weight, and increased perinatal morbidity and mortality. In addition, higher rates of maternal complications may be observed, including threatened miscarriage and pre- 284 Ebner et al. Embryo fragmentation and its impact on outcome Vol. 76, No. 2, August 2001

5 term labor. To minimize the influence of multiple pregnancies on the results of obstetric and perinatal outcome, only singleton pregnancies were compared in the present study. Until now, it has been generally accepted that there is no difference in obstetric and perinatal outcome between IVF- ICSI and natural cycles (14, 15). However, ICSI-IVF pregnancies may show significant differences in birth weight and preterm delivery (29 31) as compared with spontaneously conceived pregnancies. For embryo fragmentation, no correlation could be described in this respect. The rate of cesarean sections, which is thought to be higher in IVF-ICSI than in the general population (30), was not affected by the amount of fragments in the present study, and neither was the rate of complications during pregnancy. Admission to the neonatal care unit did not depend on the degree of fragmentation either. In summary, ongoing pregnancies achieved by transfer of bad-quality embryos (our groups C and D) do not necessarily need to be considered high-risk pregnancies because obstetric outcome (complications, cesarean section rate, preterm delivery) for these pregnancies is comparable to that in groups A and B. Nevertheless, a significantly higher percentage of malformations in newborns derived from heavily fragmented embryos (P.001) requires consultation with the patient before the transfer of such embryos. On the basis of our data and considering the legal restrictions of preimplantation diagnosis in some countries, it may be recommendable to exclude bad-quality embryos from transfer. If the gynecologist has informed the patient about these problems and both have decided together to accept a higher risk of major malformations, either amniocentesis should be considered or the intensity and frequency of ultrasound observations should be increased to minimize perinatal morbidity and mortality. Acknowledgment: The authors are grateful to Joann Hofer-Varela for her editorial advice. References 1. 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: Erenus M, Zouves C, Rajamahendran P, Leung S, Fluker M, Gomel V. The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization. Fertil Steril 1991;56: Shulman A, Ben-Nun I, Ghetler Y, Kaneti H, Shilon M, Beyth Y. 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