to find a more reliable scoring system to associate embryo morphology and pregnancy outcome.

Transcription

1 Embryo quality is more important for younger women whereas age is more important for older women with regard to in vitro fertilization outcome and multiple pregnancy Tsung-Hsien Lee, M.D., M.Sc., a,b,c Chin-Der Chen, M.D., a Yi-Yi Tsai, M.Sc., a Li-Jung Chang, M.Sc., a Hong-Nerng Ho, M.D., a and Yu-Shih Yang, M.D., Ph.D. a a Department of Obstetrics and Gynecology and b Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; and c Department of Obstetrics and Gynecology, Taipei County Hospital, Sanchong, Taiwan Objective: To determine the efficiency of embryo scoring systems for multiple pregnancy in women undergoing IVF procedures. Design: Retrospective record analysis. Setting: University hospital, tertiary medical center. Patient(s): Three hundred one patients undergoing controlled ovarian stimulation, IVF/intracytoplasmic sperm injection (ICSI), and day 3 embryo transfer. Intervention(s): IVF/ICSI and embryo transfer. Main Outcome Measure(s): Rate of pregnancy and rate of multiple pregnancy. Result(s): The score of the best three embryos (Top3) was more correlated with IVF outcome than were the number of good embryos (P.009) or the cumulative embryo score (P.038). In the logistic regression model, Top3 was more relevant to IVF outcome and multiple pregnancy for younger patients than was age (P.05). For older patients, age was more correlated with IVF outcome and multiple pregnancy than was embryo morphology (P.05). Conclusion(s): The embryo morphology criteria can help reduce the number of embryos transferred into younger patients. We could use the age of patients as an indicator to determine the number of embryos transferred into older patients. (Fertil Steril 2006;86: by American Society for Reproductive Medicine.) Key Words: IVF, multiple pregnancy, embryo morphology, age Multiple pregnancy is a common consequence of the IVF procedure, because multiple embryos are often transferred to increase the pregnancy rate per cycle (1). Twin and highorder multiple pregnancies are associated with higher rates of preterm birth and perinatal morbidity, and higher costs for medical care (2). Many efforts have been tried to reduce the number of transferred embryos so as to decrease the rate of multiple pregnancy (3 5). However, IVF is expensive and the cost is not covered by health insurance in some areas. People in these areas are still struggling to find a way to maximize the rate of pregnancy while limiting the rate of multiple pregnancy (6). The major clinical factors related to successful pregnancy in IVF cycles include the following: age of the patient (7), embryo morphology (8), number of unsuccessful prior attempts, and number of embryos transferred (9). Several methods or scoring systems for embryo grade have been used to determine embryo morphology (10 12). In this study, we tried Received June 30, 2005; revised and accepted November 30, Reprint requests: Yu-Shih Yang, M.D., Ph.D., Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7, Chung- Shan South Road, 100, Taipei, Taiwan (FAX: ; ysyang@ha.mc.ntu.edu.tw). to find a more reliable scoring system to associate embryo morphology and pregnancy outcome. The guideline for suggested numbers of transferred embryos proposed by the American Society of Reproductive Medicine (ASRM) in 2004 divided patients into groups by age (13). We adopted this guideline and further analyzed the efficiency of these factors with groups of patients of different age. We tried to develop an individualized algorithm to determine the number of embryos transferred in IVF cycles. MATERIALS AND METHODS Study Subjects This was a retrospective study of the results of IVF outcomes based on the medical records of patients undergoing IVF treatment at National Taiwan University Hospital. A total of 584 embryo transfer cycles performed at National Taiwan University Hospital from August 2001 to January 2004 were included in this study. The patients with the following characteristics were excluded from analysis: ultralong or ultrashort protocol, GnRH antagonist protocol, and cycles for oocyte donation. Only the first or second stimulated cycles for the patients were in- 64 Fertility and Sterility Vol. 86, No. 1, July /06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 cluded for analysis during the study period. If both the first and second cycles were performed during the study period, only the first cycle was included for this analysis. In addition, only the cycles with more than three embryos available for transfer after in vitro culture for 72 hours (D3ET) were included. As a result, a total of 301 D3ET procedures were analyzed in this study. Institutional review board approval was not required, because our IVF unit is licensed and regulated by the Human and Fertilization Authority: Bureau of Health Promotion, in the Department of Health, Taiwan. Besides, there was no intervention other than those for standard IVF treatments. Ovarian Stimulation Protocols Two stimulation cycles were used in this work: the long and the short protocol for GnRH agonist administration. The formal long protocol for controlled ovarian stimulation conducted herein has been described previously (14). Briefly, the patient s stimulation procedure commenced with a daily administration of buserelin (Supromon; Hoechst, Frankfurt, Germany) by means of a nasal spray in midluteal phase. When an appropriate level of pituitary suppression (serum E 2 level 50 pg/ml) had been achieved, hmg (150 IU/ day) and recombinant FSH (rfsh) (150 IU/ day) were administered by IM injection in each of the first 4 days of stimulation. As for the short protocol, the buserelin was used from day 2 of the menstruation cycle. The hmg (150 IU/ day) and rfsh (150 IU/ day) were administered by IM injection on days 5 and 6 for the treatment cycle. Thereafter, for both the long and short protocols, rfsh was discontinued and the dose of HMG was adjusted according to the observed ovarian response by means of serial ultrasonography and serum levels of E 2. When two leading follicles (sized at least 18 mm in diameter) appeared to be present, hmg was withdrawn and a dose of 10,000 IU of hcg was administered IM. Transvaginal oocyte retrieval was performed under ultrasound guidance 34 hours after hcg administration. Subsequent to oocyte collection and IVF/ICSI, the embryos were graded morphologically by the same technician. Embryo transfer was performed 72 hours after oocyte retrieval. The patient s luteal phase was supported with 1,500 IU of hcg given IM on days 4, 7, and 10 following oocyte retrieval, together with the daily IM administration of 25 mg of progesterone (in oil) for the first 14 days subsequent to ET. When the serum level of E 2 was elevated above 3,600 pg/ml or the number of retrieved oocytes exceeded 20, hcg was withdrawn and 50 mg of progesterone (in oil) was used for 14 days. Serum hcg was checked 14 days after ET, and a level above 50 IU/L was considered to constitute a state of pregnancy. Ultrasound examination was performed 1 week and then 3 weeks later to determine, respectively, the number of intrauterine gestational sacs present and fetal viability. Embryo Scoring Systems The embryos were classified according to the criteria proposed by Steer et al. in 1992 (11), namely: score 4: equally sized blastomeres, no fragmentation; score 3: equally or unequally sized blastomeres, 20% overall fragmentation; score 2: equally or unequally sized blastomeres, 20% 50% fragmentation; and score 1: equally or unequally sized blastomeres, 50% fragmentation. The cumulative embryo score (CES) was calculated by the summation of quality scores of all the embryos transferred, which resulted from the morphologic score times the number of the blastomeres in the embryos. The embryos with score 4 or score 3, together with 6 to 8 blastomeres on day 3 were considered as good-quality embryos. Statistical Analysis The various biologic parameters germane to the IVF/ICSI cycles of the data were analyzed by Student t test, Fisher exact test, 2 test, or one-way analysis of variance (ANOVA), determined by respective conditions. All the analyses were performed by the Statistical Package for the Social Sciences (version 9.0; SPSS, Chicago, IL). When significant change was detected by ANOVA, the Bonferroni test was used for subsequent post hoc multiple comparison. A confidence level of P.05 was considered to constitute the limit of statistical significance for comparison purposes. Receiver operating characteristic (ROC) curve analysis was used to estimate the predictive power of the measured variables. The relative ability of scoring systems for embryo morphology to predict the IVF outcome were compared by calculating the areas under the ROC curve (AUCs) and their 95% confidence intervals (95% CIs). MedCalc software (version 6.14; MedCalc, Broekstraat, Belgium) was used to compare the areas under two ROC curves. Logistic regression was performed to determine the independent effect of individual variables. RESULTS The clinical parameters regarding the outcome of IVF procedures for different age groups are summarized in Table 1. The patients of the younger groups revealed significantly higher numbers of retrieved oocytes and higher rates of pregnancy, implantation, and multiple pregnancy. The youngest group contributed most to the multiple pregnancies (49 of 67, 73.1%). The patients with fewer than two retrieved oocytes or fertilized embryos wanted to receive the embryos transferred totally. The situations were compatible with the so-called compulsory double embryos transfer (DET), and therefore these patients were not included in this analysis. We analyzed only those patients with three or more embryos available for transfer. The logistic regression was used to evaluate the clinical factors of multiple pregnancy. Fertility and Sterility 65

3 TABLE 1 Outcome of different aged groups participating in controlled ovarian stimulation and IVF/ICSI. Age 35 (n 184) 35 < Age 40 (n 88) Age >40 (n 29) Number of retrieved oocytes a ICSI (%) 104/184 (56.5) 51/88 (58.0) 19/29 (65.5) Number of transfered embryos Number of good embryos Cumulative embryo scores b Score of the best three embryos Mean of embryo scores Pregnancy rate (%) c 102/184 (55.4) 38/88 (43.2) 4/29 (13.8) Implantation rate (%) d 172/902 (19.1) 59/422 (14.0) 5/127 (3.9) Multiple pregnancy rate (%) e 49/184 (26.6) 17/88 (19.3) 1/29 (3.4) Note: The data are presented a mean SD or with percentage. Significance determined by one-way ANOVA test. a P.001. b P.019. c P.001. d P.001. e P.015. Variable embryo scoring systems were used to represent the quality of embryo morphology. In this analysis, we chose four embryo morphology systems number of good embryos (NGE), cumulative embryo score (CES), mean embryo score (MES), and CES for the best three embryos (Top3) to represent the factor of embryo morphology. Comparisons of the AUC of the ROC curve were performed to evaluate the predictability of the four types of parameters. Table 2 shows that the Top3 score is a better predictor of pregnancy than is NGE or CES (P.009 and P.038, respectively). For the patients of the younger group, MES or Top3 combined with age is better correlated with pregnancy outcome than is age alone (P.034 and P.015, respectively). For the older group, none of the four types of embryo morphology score is a better factor than age alone. However, the correlation with multiple pregnancy reveals no difference among these scores of embryo morphology. We divided the patients into two age groups ( 35 and 35 years) and then analyzed the data according to the age of the patients, the number of embryos transferred, and the embryo morphology (Top3). The results are summarized in Table 3. The embryo morphology (represented by Top3 TABLE 2 Comparison of areas under the ROC curves of pregnancy. Predictive factors with (age plus one of the following) Age 35 (n 184) Age >35 (n 117) Total (n 301) Number of good embryos (NGE) c Cumulative embryo scores (CES) d Mean of embryo scores (MES) a Score of the best three embryos (Top3) b c,d Age alone a,b Note: The data are presented as the 95% confidence interval. a P.034. b P.015. c P.009. d P Lee et al. Embryo quality for multiple pregnancy Vol. 86, No. 1, July 2006

4 TABLE 3 Coeffeicent for conditional logistic regression model based on the age of patients and the score of the best three embryos (Top3). Age 35 (n 184) Age >35 (n 117) Total (n 301) For pregnancy Age Top For multiple pregnancy Age Top Note: The significant coefficients are shown as the 95% confidence interval. Final prediction models: Total patients: fertility index (pregnancy) age Top3; fertility index (multiple pregnancy) Top3; younger patients: fertility index (pregnancy) Top3; fertility index (multiple pregnancy) Top3; older patients: fertility index (pregnancy) age; fertility index (multiple pregnancy) age. The probability (p) of pregnancy and multiple pregnancy can be calculated through p e fertility index /(1 e fertility index ). score) is a more important factor than age for pregnancy outcome and multiple pregnancy for the younger patients (P.05), whereas age is a more important factor than embryo morphology for patients 35 years (P.05). The model by logistic regression analysis is shown in Table 3. The model of younger patients is given in Figure 1. When the Top3 score is 48, the probabilities for pregnancy and multiple pregnancy are 49.5% and 23.1%, respectively. The model of patients 35 years is pictured in Figure 2. If the patient is 40 years old, the estimated rates of pregnancy and multiple pregnancy are 25.8% and 7.7%, respectively. FIGURE 1 The logistic regression model of pregnancy and multiple pregnancy for patients 35 years old, by the score of the best three embryos (Top3). DISCUSSION In this retrospective analysis, the probability of pregnancy could be calculated successfully by the age of patients and the score of embryo morphology. Among the commonly used scoring systems for embryo morphology, the score of the best three embryos (Top3) was more applicable to figure the probability of pregnancy than NGE and CES. Several factors have been associated with the pregnancy outcome of IVF cycles. In 1995, Roseboom et al. (9) indicated that 14 variables were associated with the probability of pregnancy. However, it is impossible and impractical to FIGURE 2 The logistic regression model of pregnancy and multiple pregnancy for patients 35 years old, by age. Fertility and Sterility 67

5 combine all these parameters to develop an algorithm to figure the outcome and decrease the number of transferred embryos. The age of the patient, the number of embryos transferred, and embryo morphology score were included for logistic regression analysis in the present study. However, the number of embryos transferred failed to correlate with pregnancy in this model (data not shown). This was probably because the number of embryos transferred in our center was already affected by the previous analysis, which suggested four embryos transferred could achieve the maximal pregnancy rate (14). If the patients got only one or two embryos available for transfer, the situation was viewed as compulsory single embryo transfer (SET) or DET. Compulsory SET cycles in IVF have been evaluated, and the results showed that the embryos were characterized by low implantation potential and poor pregnancy outcome (15). It is highly possible that the rates of pregnancy and multiple pregnancy for compulsory DET would be just a little higher than those for compulsory SET. We analyzed only those cycles with more than three embryos available for transfer, because these cycles needed urgently a rule to decrease the number of transferred embryos and subsequently the rate of multiple pregnancy. We analyzed the data and tried to find a more reliable scoring system for embryo morphology. The CES combined the information of embryo quality and the number of embryos transferred into one figure. Wu et al. in 1996 (14) suggested the use of CES for patients undergoing day 2 tubal embryo transfer. The MES emphasizes embryo quality over the number of embryos transferred. Hu et al. in 1998 (10) recommended the use of MES for day 3 embryo transfer. Both of them were used successfully to determine the optimal number of embryos transferred to maximize the pregnancy rate and to limit high-order multiple pregnancies (10, 14). Cleavage speed and fragmentation are the two major parameters involved in the embryo morphology score. If those cycles with all the embryos transferred resulted in ongoing pregnancy, then the embryo characteristics can be used for analysis of implantation potential. For example, heterozygotic twin pregnancy in cycles with two embryos transferred (16) and singleton pregnancy in single embryo transfer cycles (17) could be used to evaluate implantation potential of embryos. Another approach able to describe the implantation potential is to analyze the cycles with only transfers of embryos with identical cleavage stage and identical fragmentation (12). The observation of implanted embryos cultured to day 3 also concluded that the top-quality embryos were characterized with seven or more cells on day 3, 20% anucleated fragments, and absence of multinucleated blastomeres (16). This proved indirectly that the embryo quality or embryo morphology score has a positive relationship to implantation potential of embryos and the pregnancy rate for IVF cycles. A later study suggested that the number of good-quality embryos was also an important factor to predict the probability of pregnancy for IVF cycles (18). Our data revealed that the Top3 embryo score contributed most accurately to the pregnancy outcome of a mixture of embryos of different types. Elective SET may be the final and the best way to decrease the rate of multiple pregnancy. However, a successful elective SET depends on optimal selection criteria for the patients and an efficient cryopreservation program for the embryos (17, 19). Individual centers have to establish their criteria for embryo and patient selection to decrease the rate of multiple pregnancy while maintaining the pregnancy rate. Almost 60% of the patients in our analysis are suitable ( 35 years old) for reducing the number of transferred embryos. This indicates that by applying the selection criteria, the rate of multiple pregnancy will be lowered to a significant degree. If the patients have more than three good embryos available for transfer, the Top3 score is 48 in this system. All three embryos transferred would result in a chance of pregnancy 49.5% and a possible rate of multiple pregnancy 23.1%. We suggest that only two embryos should be transferred into these patients to decrease the rate of multiple conception. If the cumulative score of the best two embryos exceeds 48 for young patients for the first-time IVF procedure, SET may be performed after careful discussion with the patients. The relationship between age and natural fertility is neither linear nor exponential (20). The age of patients and the outcome of IVF were suggested to have a biphasic association (21). We divided the patients into different age groups according to the guideline of ASRM and then analyzed the data by a logistic regression model to see if the age of patients and the score of embryo morphology have different effects on pregnancy outcome. For younger patients (age 35 years), the effect of age was not significant; only the embryo morphology was strongly associated with the rates of pregnancy and multiple conception, whereas age contributed more than embryo morphology did to multiple pregnancy for patients 35 years. It has been determined that the embryo morphology does not deteriorate with increased maternal age (22), although aneuploidy increases with maternal age (23). The number of good embryos, the CES, and the Top3, as shown on Table 1, revealed no significant difference among different age groups. Furthermore, aneuploidy occurred more frequently in embryos with good morphology and development rate than in embryos developing poorly (23). This may explain in part why the embryo morphology in cleavage stage was not a statistically significant factor for pregnancy and multiple conception for older patients in this study. 68 Lee et al. Embryo quality for multiple pregnancy Vol. 86, No. 1, July 2006

6 Instead of embryo morphology, further parameters that could actually define the implantation potential for older patients may be required to decrease the number of embryos transferred. Preimplantation genetic diagnosis may be one of the choices (24). In conclusion, the age of the patient and the embryo morphology score are important factors to predict IVF outcome and multiple pregnancy. The CES of the best three embryos can be used to decrease the number of embryos transferred into younger patients. However, we need other parameters to determine the implantation potential and to lower the number of embryos transferred in older patients. Before the establishment of these parameters, we suggest following the ASRM guideline on the number of embryos transferred. REFERENCES 1. Schieve LA, Peterson HB, Meikle SF, Jeng G, Danel I, Burnett NM, et al. Live-birth rates and multiple-birth risk using in vitro fertilization. JAMA 1999;282: Jain T, Hornstein MD. To pay or not to pay. 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