Article Outcome of 1114 ICSI and embryo transfer cycles of women 40 years of age and over

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1 RBMOnline - Vol 13. No Reproductive BioMedicine Online; on web 15 August 2006 Article Outcome of 1114 ICSI and embryo transfer cycles of women 40 years of age and over H Nadir Çiray obtained his MD (1987) and Histologist & Embryologist (1990) degrees from Ankara University. He received his PhD (1995) from Uppsala University, Sweden under the supervision of the late Prof. Ulf Ulmsten. During PhD studies, he employed electrophysiology and dye-coupling techniques for studying human myometrial gap junction communication. His interest led him join the IVF team in Uppsala afterwards. He joined the German Hospital and Bahceci Clinic in Istanbul in 1999 as Director of the Embryology Laboratory. The laboratory currently performs approximately 4000 embryo transfer cycles per year. He became an Associate Professor in His research focuses on identification of the embryo to implant. Dr H Nadir Çiray H Nadir Çiray, Ulun Ulug, Suleyman Tosun, Halit F Erden, Mustafa Bahceci 1 Bahçeci Women s Health Care Centre, and German Hospital in Istanbul, Istanbul 1 Correspondence: Azer Is Merkezi, Abdi Ipekci Cad 44/17 Kat 5, Nisantasi, Istanbul, Turkey. Fax: ; mbahceci@superonline.com Abstract The purpose of this study was to contribute to the development of strategies to obtain acceptable outcomes in assisted reproduction treatments in women over 40 years of age. A retrospective study was carried out on the database of the German Hospital in Istanbul using data from the years 1997 to A total of 1114 embryo transfer cycles were assessed. The pregnancy, implantation and delivery rates of the assessed population were 18.2, 8.3 and 10.9% respectively. The results showed that the demographics and outcome of cycles of women at 40 years differed significantly from those over 40. Cycles in which six or more oocytes were retrieved displayed better characteristics and outcome than those with five or fewer. The clinical pregnancy and delivery rates after transfer of three embryos were similar to four or more. Therefore, women over 40 years with a good ovarian response and at least three embryos available for transfer have an acceptable pregnancy and delivery rate with a low multiple pregnancy risk. Keywords: advanced maternal age, ICSI outcome, implantation rate, pregnancy rate 516 Introduction The introduction of assisted reproduction technologies has enabled many couples to overcome infertility problems in older age, when the fecundity of women decreases (Padilla and Garcia, 1989). However, the success rate of assisted conception treatments is affected by the women s age (Cittadini and Palermo, 1989; Dew et al., 1998; Moon et al., 2000). Low pregnancy, implantation and high miscarriage rates have been shown for women >40 years after intracytoplasmic sperm injection (ICSI) (Devroey et al., 1996; Lass et al., 1998; Szamatowicz and Grochowski, 1998; Nikolettos et al., 2000; Ron-El et al., 2000; Jansen 2003). The cumulative pregnancy rate is also lower in women between 40 and 43 years compared with those aged 37 to 39 (Osmanagaoglu et al., 2002). Development of strategies to obtain acceptable outcomes in women over 40 years requires data on a large series of patients. The low implantation rate observed in older women can partly be attributed to the high incidence of aneuploidy, which can be detected by preimplantation genetic diagnosis (PGD) (Kuliev and Verlinsky, 2003). Hence, PGD for aneuploidy screening could be one approach to improve outcome in older women. Transfer of a single genetically normal embryo has been shown to yield an acceptable pregnancy rate in women of advanced age, compared with those without PGD (Obasaju et al., 2001). In contrast, in a randomized controlled clinical trial, no benefit of PGD in advanced maternal age was found when the number of transferred blastocysts was not restricted (Staessen et al., 2004). However, poorer blastocyst development has been shown for women at 40 years of age as compared with younger women, yielding lower pregnancy and implantation rates (Pantos et al., 1999). Alternative strategies such as cytoplasm and germinal vesicle transfer have been suggested to overcome the effects of aged cytoplasm on the meiotic division; however, the effectiveness of these strategies remains to be assessed (Krey et al., 2001). The most frequently used approach to improve the outcome in women over 40 years is to increase the number of embryos

2 transferred. It has been shown that ovarian reserve has a higher impact than age in predicting the outcome of assisted reproduction treatments (Lawson et al., 2003; Kailasam et al., 2004). In accordance, women at advanced maternal age having at least four embryos available for transfer yielded a better prognosis than those with three or fewer (Adonakis et al., 1997; Alrayyes et al., 1997). Therefore, many countries do not limit the number of embryos transferred to women over 39 years (e.g. Belgian legislation; Gordts et al., 2005). The present study evaluated the outcome of 1114 ICSI embryo transfer cycles performed in women 40 years of age and over. Data were obtained from cycles in which embryos were not screened for aneuploidy and not subjected to blastocyst culture. The results were related to the number of retrieved oocytes, and the number of embryos transferred. The findings are expected to contribute to developing strategies to improve the outcome of ICSI cycles in older women. These strategies may also be useful in countries where donation programmes are illegal. Materials and methods The database of the IVF Unit of The German Hospital in Istanbul from January 1997 to October 2004 was retrospectively analysed. A total of 15,340 oocyte retrieval cycles were recruited. Of these, 1281 cycles involved women who were at least 40 years of age at the time of oocyte retrieval. In all cases, ICSI with the husbands spermatozoa was the method of insemination. Only fresh embryo transfer cycles were included in the study. PGD cycles were not recruited, since they were out of the scope of the survey. The aetiology of infertility in the assessed cycles was diminished ovarian reserve (40%), male (28%), tubal (5%) or endometrial factors (4%), unexplained infertility (8%), and a combination of these (15%). Two types of ovarian stimulation protocols were employed. The long protocol began with pituitary desensitization using a gonadotrophin-releasing hormone (GnRH) agonist (Lucrin; Abbott, France) in the mid-luteal phase of the preceding menstrual period. Administration of gonadotrophins (Gonal F ; Serono, Aubonne, Switzerland) was initiated on day 3 of the commencing cycle. The microdose flare-up protocol began with low-dose oral contraceptive (Desolett ; Organon, France), starting on day 1 of the previous menstrual cycle, for 21 days. Leuprolide acetate (Lucrin ; Abbott; 40 µg s.c. per day) was initiated on day 2 of menstruation, followed by gonadotrophins, which were administered on cycle day 3. Gonadotrophin dosage was tailored according to individual ovarian responses, with initial doses ranging from 300 to 600 IU per day. When the leading follicle reached a diameter of 18 mm, human chorionic gonadotrophin (HCG; Pregnyl ; Organon) 10,000 IU was administered to trigger ovulation. The embryology procedures were performed as described previously (Çıray et al., 2005). Briefly, embryos were cultured in 30 µl drops of human tubal fluid-based media (Vitrolife, Sweden or SAGE-Biopharma, USA) under sterile mineral oil. Embryo transfers were performed on day 3. Embryo quality was scored according to the criteria introduced by Steer et al. (1992). According to the quality and number of available embryos, up to six embryos were transferred. Cycles in which only poor quality embryos were present (uneven blastomeres with >50% fragmentation), transfers were cancelled after the patient s consent was obtained. Clinical pregnancy was defined as presence of an intrauterine gestational sac by ultrasonography 30 days after transfer. Implantation rate was defined as the ratio of gestational sacs to the total of transferred embryos. Pregnancies beyond 20 weeks of gestation were regarded as on-going pregnancies. Statistical differences between the groups were analysed by one-way analysis of variance (ANOVA) followed by an appropriate post-hoc test (Tukey Kramer) and a chi-squared analysis or Fisher s exact test, where applicable. A probability of <0.05 was considered statistically significant. Results From a total of 1281 cycles in which at least one oocyte was retrieved, 1114 resulted in transfer (cancellation rate 13%; in 31 cycles there were no mature oocytes for insemination, in 42 cycles there were no normally fertilized oocytes, in 53 cycles transfer was cancelled due to poor embryo development, and in 41 cycles due to poor quality endometrium, in which case obtained embryos were frozen for a subsequent thaw cycle). The characteristics of the embryo transfer cycles are given in Table 1. Of the 1114 embryo transfer cycles, 203 clinical pregnancies were obtained of which 121 delivered (10.9% of transfers). There were 20 twin deliveries (16.5% of deliveries). There were no triplet deliveries; however, three cycles displaying triplet sacs on ultrasonography were subjected to fetal reduction and resulted in twins, and one cycle with triplet sacs resulted in a miscarriage. The cycle demographics of women who delivered at 40 years of age or older are presented according to age group in Table 2. The oldest woman who had a clinical pregnancy and gave birth to a healthy baby was 45 years old. As there was only one pregnancy and delivery in women over 44 years, the outcome parameters (pregnancy, implantation and delivery rates) of these cycles were not taken into statistical evaluation. Women at 40 years of age displayed a higher peak oestradiol concentration than women over 40 years, although day 3 FSH concentrations and the number of ampoules administered were similar. A significantly lower number of oocytes was retrieved in women over 44 years compared with those aged (P < 0.01). Similarly, the mean number of transferred embryos was similar between groups, with the exception of women over 44 years in which significantly fewer embryos were transferred as compared with women aged years (P < 0.01). The following outcomes were significantly higher in women at 40 years compared with the other age groups: clinical pregnancy (P < 0.01 versus 42 44), implantation and delivery rates (both P < 0.05 versus 41; P < 0.01 versus 42 44) (Figure 1). Cycles of women 40 years and over were assessed according to the number of retrieved oocytes and then assigned into one of two groups; those in which five or fewer oocytes were retrieved and those in which six or more oocytes were retrieved. The results are given in Table 3. The groups differed from each other regarding mean age of women, day 3 FSH concentration, peak oestradiol concentration, number of ampoules administered, number of retrieved, MII, and fertilized oocytes, number of 517

3 Table 1. Outcome of the assessed parameters of 1114 intracytoplasmic sperm injection and embryo transfer cycles in women 40 years of age and over. Parameter Value Age (years) 41.6 ± 0.1 Day 3 FSH concentration (miu/ml) 8.7 ± 0.1 Peak oestradiol concentration (pg/ml) 1540 ± 34 Ampoules of gonadotrophin administered (75 IU/each) 59 ± 1 No. of oocytes (n) 7.7 ± 0.2 (8586) % MII oocytes (n) 82.2 ± 0.6 (6846) % 2PN (n) 73.8 ± 1.0 (4745) Total no. of cleaved embryos 4380 No. of embryos transferred (n) 2.9 ± 0.01 (3195) % G1 + G2 embryos a 69.0 % clinical pregnancy per embryo transfer (n) 18.2 (203) % implantation rate (n) 8.3 (265) % delivery/cycle (n cycles; n deliveries) 10.9 (121; 141) Values are means ± SEM unless otherwise stated. a Steer et al. (1992). MII = metaphase II; PN = pronucleate. Table 2. Demographics of 1114 intracytoplasmic sperm injection and embryo transfer cycles of women 40 years and over according to age group. Parameter Age (years) >44 Number of cycles Day 3 FSH (miu/ml) 8.6 ± ± ± ± ± ± 0.6 Peak oestradiol 1771 ± ± ± ± ± ± 102 concentration (pg/ml) 1 Ampoules of 59 ± 1 60 ± 1 61 ± 1 59 ± 2 59 ± 2 58 ± 2 gonadotrophin administered (75 IU/each) No. of oocytes (n) ± ± ± ± ± ± 0.5 (3238) (1911) (1631) (912) (524) (362) 4 % MII oocytes (n) 82.0 ± ± ± ± ± ± 2.4 (2577) (1543) (1264) (727) (432) (296) % 2PN (n) 74.1 ± ± ± ± ± ± 2.6 (1812) (1052) (877) (516) (273) (209) Total no. of cleaved embryos Embryos transferred (n) ± ± ± ± ± ± 0.2 (1171) (731) (606) (337) (188) (158) 4 % G1 + G2 embryos a Values are means ± SEM, unless otherwise stated. Superscripts 1 and 3 denote comparison between all age groups by one-way analysis of variance; superscripts 2, 4, 5, 6 denote appropriate post-hoc tests. P-values are given below. 1 P < 0.001, 2 P < 0.05 versus all groups; 3 P < 0.001, 4 P < 0.01 versus 40 42; 5 P < versus all groups; 6 P < 0.05 versus 41 and 42. a Steer et al. (1992). MII = metaphase II; PN = pronuclei. 518

4 Figure 1. Clinical pregnancy (PR), implantation (IR), and delivery (DEL) rates of women 40 years and over according to age groups. a P = 0.07 versus 41, P < 0.01 versus b P < 0.05 versus 41, P < 0.01 versus *Not taken into statistical evaluation. Table 3. Outcome of the assessed parameters in women 40 years and over according to the number of retrieved oocytes. Parameter No. of retrieved oocytes (no. of cycles) P-value 5 6 No. of cycles Age of women (years) 41.9 ± ± 0.1 < Day 3 FSH concentration (miu/ml) 10.0 ± ± 0.1 < Peak oestradiol concentration (pg/ml) 802 ± ± 1120 < Ampoules of gonadotrophin administered 62 ± 1 57 ± 1 < (75 IU each) No. of oocytes (n) 3.1 ± 0.1 (1550) 11.6 ± 0.2 (7036) < % MII oocytes (n) 86.5 ± 0.8 (1309) 78.5 ± 0.7 (5537) < % 2PN (n) 79.3 ± 1.1 (977) 69.2 ± 1.6 (3768) < Total no. of cleaved embryos < No. of embryos transferred (n) 1.8 ± 0.0 (917) 3.8 ± 0.1 (2278) < % G1+ G2 embryos a Clinical pregnancy (%ET) 56 (11.0) 147 (24.3) < Implantation rate (%) 71 (7.7) 194 (8.5) NS % delivery/cycle (n cycles; n deliveries) 5.9 (30; 34) 15.0 (91; 107) < Values are means ± SEM, unless otherwise stated. a Steer et al. (1992). MII = metaphase II; PN = pronucleate; %ET = % of embryos transferred; NS = not significant. embryos transferred, clinical pregnancy and delivery rates. Cycles in which five or fewer oocytes were retrieved comprised women who had a higher mean age than those in which six or more were retrieved, and consequently displayed poorer cycle characteristics, which yielded a poorer outcome. Cycles were grouped according to the number of transferred embryos (Table 4). The poorest cycle characteristics and outcome were observed in groups in which one and two embryos were transferred. Women in these groups displayed a similar mean age; however, a lower day 3 FSH and a higher peak oestradiol concentration were observed in the two-embryo transfer group. The clinical pregnancy, implantation and delivery rates were higher in the two-embryo transfer cycles compared with one embryo transfer, as shown in Figure 2. The peak oestradiol concentration and the number of retrieved oocytes increased gradually in the groups in parallel with the number of embryos transferred (Table 4). Women in the three to five or more embryos transferred groups displayed a similar mean age, which was lower than the one- and two-embryo transfer groups. Groups in which three to five or more embryos were transferred displayed similar cycle demographics, with the exception of peak oestradiol concentrations and number of retrieved oocytes. The clinical pregnancy and delivery rates were also similar between these groups (Figure 2). 519

5 Table 4. The cycle demographics of women 40 years and over according to the number of transferred embryos. Parameter No. of embryos transferred Total number of cycles Age of women (years) ± ± ± ± ± 0.1 Day 3 FSH concentration 10.1 ± ± ± ± ± 0.3 (miu/ml) 4 Peak oestradiol 837 ± ± ± ± ± 84 concentration (pg/ml) 7 Ampoules of 61 ± ± ± 1 55 ± 1 57 ± 1 gonadotrophin administered (75 IU each) 12 No. of oocytes retrieved ± 0.2 (945) ± 0.2 (1119) ± 0.4 (1827) ± 0.4 (2198) ± 0.4 (2497) % MII oocytes (n) 80.5 ± 1.4 (687) 81.8 ± 1.3 (854) 83.0 ± 1.1 (1479) 83.7± 1.0 (1801) 82.6 ± 1.1 (2025) % 2PN (n) ± 1.8 (395) ± 1.7 (526) 74.2 ± 1.5 (1030) 76.3 ± 1.3 (1321) 79.9 ± 4.8 (1473) Total no. of cleaved embryos % G1 + G2 embryos a Values are means ± SEM, unless otherwise stated. Superscripts 1, 4, 7, 12, 15 and 20 denote comparison between all groups by one-way analysis of variance; all other superscripts denote appropriate post-hoc tests. P-values are given below. 1 P < 0.001; 2 P < versus 3, 4 and 5; 3 P < 0.05 versus 4 and 5; 4 P < 0.001; 5 P < 0.05 versus 2, P < versus 3, 4 and 5; 6 P < versus 4 and P < 0.05 versus 5; 7 P < 0.001; 8 P < 0.01 versus 2, P < versus 3, 4 and 5; 9 P < versus 3, 4 and 5; 10 P < 0.05 versus 4, P < versus 5; 11 P < versus 5; 12 P < 0.001; 13 P < 0.01 versus 4; 14 P < 0.05 versus 3 and 5, P < versus 4; 15 P < 0.001; 16 P < versus 2, 3, 4 and 5; 17 P < versus 3, 4 and 5; 18 P < versus 4 and 5; 19 P < versus 5; 20 P < 0.05; 21 P < 0.05 versus 5, 22 P < 0.05 versus 1 and 2, P < 0.01 versus 4, P < versus 5; 23 P < 0.05 versus 2, P < 0.05 versus 4. a Steer et al. (1992). MII = metaphase II; PN = pronucleate. Figure 2. Clinical pregnancy (PR), implantation (IR), and delivery (DEL) rates of women 40 years and over undergoing intracytoplasmic sperm injection and embryo transfer according to the number of embryos transferred. a P < versus all groups; b P < 0.05 versus 3 and 4; P < 0.01 versus 5; c P < 0.05 versus 1; d P < 0.05 versus 3, P < 0.01 versus 5; e P < 0.01 versus 1 and

6 Discussion The findings of the present study showed that: (i) the characteristics and outcome of cycles of women at 40 years of age were different from those of women over 40 years, (ii) in women 40 years and over, the characteristics and outcome of cycles in which six or more oocytes retrieved were better than those in which five or fewer oocytes were retrieved, and (iii) transfer of three embryos resulted in similar clinical pregnancy and delivery rates compared with transfer of four or more embryos. So far as is known, the current study reports the largest set of data on the demographics and outcome of cycles in women 40 years of age and over from a single embryology laboratory. The clinical pregnancy, implantation, delivery and miscarriage rates obtained from the present study (18.2, 8.3, 10.9 and 40.4% respectively) are comparable with those reported earlier (Devroey et al., 1996; Lass et al., 1998; Szamatowicz and Grochowski, 1998; Nikolettos et al., 2000; Ron-El et al., 2000; Osmanagaoglu et al., 2002; Jansen 2003; Combelles et al., 2005). A delivery rate per transfer of 7 8.5% (Devroey et al., 1996; Szamatowicz and Grochowski, 1998) and cumulative delivery rate of 12% have been reported in women over 40 years (Osmanagaoglu et al., 2002). In those studies, the pregnancy rate per transfer varied between 11.6 and 12.8%, the implantation rate between 4.9 and 7.4, and the miscarriage rate between 27 and 50% (Lass et al., 1998; Szamatowicz and Grochowski, 1998; Nikolettos et al., 2000; Ron-El et al., 2000; Jansen 2003). Multiple pregnancies occurred mostly as twins, and no triplet pregnancies (Lass et al., 1998) and no deliveries after 44 years (Grimbizis et al., 1998; Ron-El et al., 2000; Jansen 2003) have been reported, except in one study (Dal Prato et al., 2005). In the current study, four cycles resulted in triplet gestational sacs, of which three cycles were reduced to twin pregnancies and resulted in healthy babies, and one resulted in miscarriage at 8 weeks. Three of these women were 40 years old. One had three embryos transferred (miscarriage), and the other two had five embryos transferred. The fourth triplet pregnancy was in a 43-year-old woman, who had two embryos transferred and delivered two healthy babies after reduction. The oldest woman who delivered a healthy baby in the present study was 45 years, and had a basal FSH value of 11 miu/ml; 19 oocytes were retrieved and six embryos were transferred. Determining the best strategy to achieve acceptable implantation and delivery rates in women over 40 years requires accumulation of comparative data on a large series of patients with similar demographics. Embryo transfer policies are different for cycles in women at advanced maternal age compared with younger patients. The number of embryos to be transferred to women over 39 years of age has not been restricted in the legislation of many countries (Gordts et al., 2005). Adonakis et al. (1997) reported that women of advanced maternal age having at least four embryos transferred yield a higher clinical pregnancy rate than those having three or fewer transferred. Alrayyes et al. (1997) found that women aged 37 years and over with good ovarian response, producing more than three embryos suitable for transfer, had a pregnancy rate similar to younger patients. The outcome of cycles was similar when five or more embryos were available in women older than 39 years, irrespective of embryo transfer policy (selective, i.e. not all embryos transferred, or non-selective, i.e. all embryos transferred) (Grimbizis et al., 1998). The impact of ovarian response on the outcome was also analysed in the present study as the number of retrieved oocytes and the number of embryos transferred related to the results. For example, women with a lower day 3 FSH concentration who showed a higher peak oestradiol concentration in response to similar dose of gonadotrophins, and subsequently had two embryos transferred, displayed a significantly higher clinical pregnancy and delivery rate than those having one embryo transferred (P < 0.001; Table 4 and Figure 2). A recent study (Combelles et al., 2005) showed an optimal number of five embryo transfers to be beneficial to the clinical outcome for women >40 years of age. The difference between the results may be caused by embryo transfer strategies as more embryos (up to 11) were transferred in that study compared with the present. Of particular interest was the data obtained from the demographics and outcome of cycles of women at 40 years, which differed significantly from those over 40. This may emphasize the need to reconsider the transfer policy on these patients. Moreover, the finding that transfer of more than three embryos did not increase the clinical pregnancy and delivery rates requires attention for future assessments, which may propose application of restrictions on the number of embryos transferred to women at advanced maternal age. Improvements in the laboratory and in-vitro culture conditions should also be taken into consideration in updating embryo transfer policies in these patients. Egg donation programmes can be proposed to women at advanced age (Antinori et al., 2003). Other strategies in improving the outcome of cycles in women at advanced age rely on the selection of embryos with minimal risk of chromosomal disorders for transfer. The relatively low success rates compared with younger women can be attributed to age related aneuploidies, which contribute significantly to implantation failure and spontaneous abortions (Kuliev and Verlinsky, 2003; Rubio et al., 2005). Ooplasmic anomalies may account for the nuclear problem, and hence application of novel techniques, such as ooplasmic injection and germinal vesicle transfer have been under debate to improve outcome of assisted reproduction treatments in women of advanced age (Krey et al., 2001). Extended culture has also been suggested, but poorer blastocyst development has been shown at 40 years compared with younger women, yielding low pregnancy and implantation rates (Pantos et al., 1999). PGD has been offered as a tool to identify abnormal embryos in women of advanced age, which in turn will enable fewer embryos to be transferred and reduce the risk of multiple pregnancies (Kuliev and Verlinsky, 2003). According to the findings of the present and previous studies (Lass et al., 1998), the risk of multiple pregnancies for women over 40 years is questionable. In one study, transfer of a single genetically normal embryo resulted in an acceptable pregnancy rate compared with the transfers without PGD in women at advanced age (Obasaju et al., 2001). The delivery rates have not been reported in these cycles and, according to calculations, the mean age of these groups were significantly different. Of the 27 cycles from 23 patients (mean age 42.2 ± 1.3 years), 17 had normal embryos transferred, which resulted in six pregnancies, compared with 69 cycles (and transfers) from 54 women (mean age 43.3 ± 1.9 years), which resulted in 19 pregnancies. Others (Staessen et al., 2004) reported no benefit 521

7 522 of PGD in an unrestricted number of blastocyst transfers in advanced maternal age. In conclusion, women over 40 years with a good ovarian response and at least three embryos available for transfer have an acceptable pregnancy and delivery rate with a low multiple pregnancy risk. Further studies are required to support the findings of the present data that may require certain restrictions on the number of embryos transferred to be applied for patients over 40 years, or at least for those aged 40 years. References Adonakis G, Camus M, Joris H et al The role of the number of replaced embryos on intracytoplasmic sperm injection in women over the age of 40. Human Reproduction 12, Alrayyes S, Fakih H, Khan I 1997 Effect of age and cycle responsiveness in patients undergoing intracytoplasmic sperm injection. Fertility and Sterility 68, Antinori S, Versaci C, Cerusico F et al Obstetric and prenatal outcome in menopausal women: a 12-year-old study. 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