- Email: [email protected]
Factors Influencing Pregnancy Outcome Following Slow Cooling Cryoembryo Transfer and Risk of Multiple Conception
Journal of Reproduction & Contraception
http://www.RandC.cn
2008 Jun; 19(2):93-100
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Factors Influencing Pregnancy Outcome Following Slow Cooling Cryoembryo Transfer and Risk of Multiple Conception Su-ying LIU, Jin-lan HAN, Bin TENG, Zhu-di LU, Yu ZHENG, Ying CAO, Xiang CAO, Ning-yi WANG, Bin HUANG, Jing-ming YAN Shanghai Jiai Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University, Shanghai 200011, China
Objective To investigate the factors that influence the potential for cryoembryo implantation and multiple pregnancy. Methods In this retrospective study, a total of 937 thawing cycles (859 couples) in which 3286 d 3-embryos were thawed. Rates of implantation, clinical pregnancy and multiple conception following FET were observed. Results There were significant differences in female age (P<0.05) and number of good quality embryos (P<0.05) between cycles that resulted in pregnancy and those did not. There was a trend toward decreasing rates of implantation, clinical pregnancy and multiple pregnancy with increasing female age. Compared with transferring 1 good quality embryo, clinical pregnancy rate of transferring 2 and 3 good quality embryos was increased significantly (P<0.001), there was no significant difference in clinical pregnancy rate between transferring 2 and 3 good quality embryos. Multiple pregnancy rate was increased significantly in the group of transferring 3 good quality embryos (P<0.05),but there was no significant differences in multiple pregnancy rate between transferring 1 and 2 good quality embryos.Younger women ( ≤ 30 years) also had a significantly higher multiple pregnancy rates (28.13%) than the older ones(>35 years) (13.64%).With an increase in age from ≤ 30 years to>40 years, clinical pregnancy rate declined from 45.61% to 25.00%. Conclusion Female age and the number of good quality embryos transferred are important factors influencing the clinical and multiple pregnancy rate, reducing the number of good quality embryos transferred may decrease the rate of multiple pregnancy but do not affect the clinical pregnancy rate. Key words: in vitro fertilization; cryopreservation; embryo transfer; clinical pregnancy; multiple pregnancy Corresponding author: Jin-lan HAN; Tel: +86-21-63459977*215; E-mail: [email protected] 93
Pregnancy, without consideration of obstetric and neonatal outcomes, is no longer the objective. Practitioners readily acknowledge the significant contribution of multiple pregnancy to the risk and complications of assisted reproductive technology. It is universally recognized that multiple gestation, and its attendant prematurity, is associated with increased mortality and morbidity, both for mothers and fetuses[1]. A twin gestation involes a perinatal outcome with a 15 fold increase risk of complications, compared with a singleton[2]. Since the development of assisted reproduction techniques, most countries have witnessed increased rates of multiple pregnancy. Despite the guidelines proposed by various scientific societies, these rates continue to be abnormally high. Embryo cryopreservation provided additional safety in the presence of ovarian hyperstimulation and contributed to lowering the risk of multiple conception by reducing the need to transfer multiple fresh embryos. However, frozen embryo transfer is not free from the risk of multiple conception. Effective strategies must be established to prevent multiple pregnancy without reducing overall pregnancy rates. The pregnancy rate following FET has been shown to be related to the number of blastomeres and morphological appearance of embryos prior to freezing[3,4], the extent of embryo damage after thawing[5]. The effects of embryological parameters on pregnancy rate after FET have also been addressed in several studies. To date, few attempts have been made to assess the number of good quality embryos transferred on the FET outcome, therefore, the current retrospective study was to evaluate the impact of clinical and embryological parameters on the FET outcome.
Materials & Methods Materials From January 2005 to December 2006, a total of 937 thawing cycles on 859 couples were studied retrospectively. Rates of implantaion, clinical pregnancy and multiple conception following FET according to patient age at the time of embryo freezing , the number of embryos transferred, endometrium thickness, and the number of good quality embryos transferrd were observed. The indications for IVF (ICSI) and embryo transfer included tuble factor, male factor, endometriosis and unexplained factor of infertility. Cryopreservation Cryopreservation procedure was performed using a slow-freeze protocol[6]. Embryos were equilibrated in 1.5 mol/L propanediol (PROH) at room temperature for 10 min, then transferred to 1.0 mol/L propanediol/0.1 mol/L sucrose and loaded into plastic straws. Straws were put in a controlled-rate freezer at a rate of -2℃/min to -7℃/min, at which point seeding was performed manually with liquid nitrogen-cooled forceps. Cooling was then continued at rates of -0.3℃/min to -30℃/min and rapidly cooled at a rate of -50℃/min to -140℃/min before plunging and storage in liquid nitrogen. 94
Thawing Embryos were thawed rapidly by removing straws from nitrogen, exposure to air for 30 s, then embryos were sequentially incubated in each of the following solutions for 5 min: 1.0 mol/L propanediol/0.2 mol/L sucrose, 0.2 mol/L sucrose and sucrose-free Dulbecco’s phosphate-buffered saline (DPBS). Embryos were assessed for numbers of remaining blastomeres, then were transferred to culture medium at 37℃ in 5% CO2 until the time of transfer. Post-thaw survival of cryopreserved embryos was defined as ≤ 50% blastomere loss. Freezing and thawing solutions consisted of cryoprotectants in DPBS supplemented with 20% serum protein substitute(SPSS). Routine examination of embryo quality included the number of blastomeres, the degree of fregmentation, the uniformity of blastomeres. Embryo morphology was scored as follows: grade 1, no fragments and equal blastomeres; grade 2, <20% fragmentation, equal blastomeres; grade 3, equal or unequal blastomeres, 20%-50% fragments; grade 4, equal or unequal blastomeres, >50% fragments. ≥7 blastomeres, grade 1 and grade 2 were defined as good quality embryo. Embryo transfer was performed 1 h later after thawing. To precisely control the uterine environment for embryo transfer, most of transfers were performed in hormone replacement treatment(HRT) cycles, natural cycles were performed only in those who had spontaneous ovulation. The ovarian steroid replacement protocol consisted of oral estradiol valerate and progesterone.On d 3 of the replacement cycle, estradiol was commenced daily. Luteal phase support was initiated 3 d before the embryo transfer by daily administration of progesterone and estradiol 4-6 mg. Biochemical pregnancy was confirmed by a positive blood hCG test 2 weeks after the embryo transfer. Clinical pregnancy was diagnosed by the presence of intrauterine gestational sac and heart beat on vaginal ultrasound 4 weeks after the embryo transfer. A maximum of three selected thawed embryos were transferred. All pregnant women continued to have estradiol and progesterone, both administered daily until week 10 of gestation. Statistical analysis Statistical analysis was performed using SPSS12.0 software, Student’s t-test and χ2 test. Data were presented as －x ± s. P<0.05 was considered statistically significant.
Results A total of 937 thawing cycles in which 3 286 embryos were thawed, 2 853 embryos survived, the survival rate was 86.8%. Totally 2 575 embryos were transferred, a maximum of 3 embryos were transferred. The mean maternal age in pregnant and non pregnant group were 31.0 ± 3.9 years and 31.7 ± 3.9 years, respectively (P<0.05). The mean numbers of embryos transferred were 2.7 ± 0.5 and 2.7 ± 0.5 (P>0.05), the good quality embryos transferred were 1.9 ± 0.8 and 1.6 ± 1.0 (P<0.05), the thickness of the endometrium were 9.8 ± 0.8 mm and 9.7 ± 2.1 mm (P>0.05) (Table 1). 95
Table 1 Characteristics between pregnancy and nonpregnancy cycles Item
Pregnant group
Number of cycles
Nonpregnant group
P
395
542
31.0 ± 3.9
31.6 ± 3.9
0.035
Tubal factor n(%)
181(45.93)
241(44.46)
>0.05
Male factor n(%)
169(42.89)
230(42.44)
>0.05
14(3.55)
23(4.24)
>0.05
Age(year) Indications of ART
Unexplained n(%)
31(7.88)
48(8.86)
>0.05
No. of embryo transferred
Other n(%)
2.7 ± 0.5
2.7 ± 0.5
0.11
No. of good quality embryo transferred
1.9 ± 0.8
1.6 ± 0.9
<0.01
Endometrial thickness(mm)
9.7 ± 2.1
9.7 ± 2.1
0.63
There was a trend toward decreasing rates of implantation, clinical pregnancy and multiple pregnancy with increasing female age. The implantation, clinical pregnancy and multiple pregnancy rates in the two younger age groups (≤ 30 and 31-35 years) were not significantly different from each other. The implantation rate in the group of ≤30 years was significantly higher than that in women aged 36-40 years and >40 years. Younger women also had a significantly higher multiple pregnancy rate than the older women. With an increase in age from ≤30 years to >40 years, clinical pregnancy rate declined from 45.61% to 25.00%. There were no multiple pregnancy in women aged >40 years old(Table 2). Table 2 Rate of clinical pregnancy, multiple pregnancy according to patient age Age (years)
Number of cycles
Embryo transferred
Implantation rate Clinical (%) pregnancy rate (%)
Multiple pregnancy rate (%)
≤ 30
421
2.5 ± 0.5
21.14(252/1 192)
45.61(192/421)
31-35
380
2.5 ± 0.5
19.57(202/1 032)
40.26(153/380)
27.45(42/153)
37.93(44/116)
13.64(6/44)*
36-40 >40
**
116
2.5 ± 0.5
16.56(50/302)
20
2.5 ± 0.5
9.80(5/51)
25.00(5/20)
28.13(54/192)
0
∗: P<0.05, **: P<0.001, compared with age of ≤ 30 years old
Clinical pregnancy rates was increased with the number of good quality embryos increased. Compared with transfering 1 good quality embryos, the clinical pregnancy rates were significantly increased in both the group of transferring 2 and 3 good quality embryos, but no difference was noticed between the groups. Multiple pregnancy rate of transfering 3 good quality embryos was increased significantly compared with tranferring 1 good quality embryo. Transferring 2 good quality embryos resulted in similar pregnancy rate but significantly 96
reduced the overall incidence of multiple gestations when compared with transferring 3 good quality embryos (24.3% vs 37.6%). Twin gestations was decreased from 29.36% to 23.2%, and triplets significantly decreased from 8.25% to 1.11%. The elective transfer of 2 good quality embryos resulted in similar pregnancy rates and significantly reduced multiple gestation rates when compared with the elective transfer of 3 good quality embryos (Table 3). Table 3 Rate of clinical pregnancy, multiple pregnancy according to numbers of good embryo quality transferred No. of good quality embryo transferred Item
0
1
2
3
No. of cycles
88
260
384
205
No. of embryo transfered
2.67 ± 0.43
2.60 ± 0.57
2.63 ± 0.45
2.61 ± 0.55 **
52.20(109/205)**
Clinical pregnancy rate(%)
26.14(23/88)
31.20(81/260)
4 7 .1 0 ( 1 8 1 / 3 8 4 )
Multiple pregnancy rate(%)
4.30(1/88)
19.80(16/260)
24.30(44/384)
37.60(41/205)*∆
Twin(%)
4.30(1/88)
17.28(14/260)
23.2(42/384)
29.36(32/205)
Triplet(%)
0
2.47(2/260)
1.11(2/384)
8.25(9/205)∆
*: P<0.05, **: P<0.001, compared with transferring 1 good quality embryo ∆: P<0.05, compared with 2 good quality embryos transferred
Discussion Maternal age and FET The maternal age-related decline in female fertility has been attributed to a variety of causes including progressive oocyte depletion, meiotic irregularities cell cycle regulation, and mitochondrial dysfunction[7]. The implantation and pregnancy rate following frozen embryo transfer was decreased with increasing female age, the effect of age on implantation rate seemed to be independent[8,9]. More and more young women are delaying childbearing. Older women comprise an increasing portion of patients entering assisted reproduction programmes. A retrospective analysis of 1 217 IVF cycles in women aged 40 years and older showed that success rates declined with each year after age 40; pregnancy and delivery rates were 13.9% and 9.1% at age 40 and 2.8% and 0.7% at age 45[10]. There were no deliveries at age 45[10]. Unrealistic expectations may be avoided if accurate data are provided regarding delivery rates per year after age 40. The pregnancy rate per cycle showed a significant decrease with increasing maternal age[11]. According to the data presented here, the implantation, clinical pregnancy and multiple pregnancy rates in the two younger age groups (≤30 and 31-35) were not significantly different from each other. The implantation rates in the group of ≤ 30 years was 21.14%, 97
which was significantly higher than that of women aged 36-40 years (16.56%). Younger women also had a significantly higher multiple pregnancy rates (28.13%) than the older women (13.64%). With an increase age from ≤ 30 years to >40 years, clinical pregnancy rate declined from 45.61% to 25.00%. There were no multiple pregnancy in women aged > 40 years old. This finding is consisitent with earlier reports[8,9]. Number of good quality embryos and FET Cryopreservation can affect the implantation potential of early cleavage-stage embryos if cryodamage occurs. It has been shown that partially intact thawed cleavage-stage embryos can result in pregnancies, the implantation potential drops in parallel with increasing degrees of blastomere lysis, but the intact thawed embryos have the same implantation potential as equivalent to fresh embryos[5,12]. It has been shown that not all human embryos survive the cryopreservation procedure. The survival rate based on reviews from the literature, appears to be highly variable, ranging from 56%-92%[13,15]. In this study, there was a high embryonic survival rate after thawing (86.8%). The pregnancy rate following FET has been shown to be related to the number of blastomeres and morphological appearance of embryos prior to freezing, the extent of embryo damage after thawing. A retrospective study of single ET(embryo transfer) in infertile women with a decreased egg reserve showed significantly higher clinical pregnancy rate with the transfer of a single embryo with 6-8-blastomere than 4-5-cell embryo (40.4% vs 6.6% clinical), degree of fregmentation did not predict outcome nearly as well as blastomere number [16]. Another retrospective analysis of 6 916 cryopreserved d 2-embryo transfer procedures, transfer of two cryopreserved embryos resulted in higher clinical pregnancy rates when compared with transfer of a single thawed embryo but was also associated with elevated multiple pregnancy rates (26.7% in women under 36). In cryopreserved 4-cell stage embryos, loss of a single blastomere did not reduce implantation potential[17]. Dowling, et al. reported that the elective transfer of 2 embryos resulted in similar pregnancy rate but significantly reduced the overall incidence of multiple gestations (20% vs 39%) when compared with the elective transfer of 3 embryos[18]. Twin gestations decreased from 28% to 19%, and triplets significantly decreased from 9% to 1%. The elective transfer of 2 embryos resulted in similar pregnancy rates and significantly reduced multiple gestation rates when compared to the elective transfer of 3 embryos. It is recommended that a maximum of 2 embryos are transferred in young women with good quality embryos at the time of transfer, the transfer of 3 embryos is only recommended in women ≥ 38 years who have one or no good quality embryos available at the time of transfer. Given the promising potential of eSBT(elective single blastocyst transfer) to markedly reduce the risk of twin gestation without a significant compromise to pregnancy outcomes, an active attempt should 98
be made to consider and use eSBT in the young, favorable-prognosis patient who has goodquality embryos available for transfer and cryopreservation[19]. Similar to the results mentioned above, comparing with transfering 1 good quality embryo, clinical pregnancy rate of transfering 2 and 3 good quality embryos increased significantly (P<0.001), there was no significant difference in clinical pregnancy rate between transfering 2 and 3 good quality embryos. Multiple pregnancy rate increased significantly in the group of transfering 3 good quality embryos (P<0.05), but there was no significant difference in multiple pregnancy rate between transferring 1 and 2 good quality embryos. With 3 good quality embryos transferred, the clinical pregnancy rate increased slightly than 2 good quality embryos transfer, but the risk of multiple conception increased significantly. Several factors should be considered before frozen embryo transfer regarding the chance of pregnancy and risk of multiple conception. In conclusion, female age, the embryo quality and the number of good quality embryos are the most important factors that influence the rate of implantation, clinical pregnancy and multiple pregnancy following frozen embryo transfer. The expectation of pregnancy and the risk of multiple conception are important issues for both the clinician and the couple. The approach should be individualized. For younger patients (<40 years old), if there are enough good qualty embryos, transfering 2 good quality embryos and freezing surplus embryos would not compromise the expectation of pregnancy while reducing the risk of multiple conception.
References 1.
Wennerholm UB.Obstetric and neonatal in complications multiple gestation. In Healthy DL, Kovacs, GT, McLachlan R, et al. (eds) Reproductive Medicine in the Twenty-First Century. Parthenon, London: Proceedings of the 17th World Congress on Fertility and Sterility, 2002:182-93.
2.
Hurst T, Shafir E, Lancaster P. Assisted conception Australia and NewZealand 1997. AIHW cat. No PER 10. Sydney: Australia Institute of Health and Welfare National Perinatal Statistics Unit (Assisted Conception Series 1999; no 4).
3. Hartshorne GM, Wick K, Elder K, et al. Effect of cell number at freezing upon survival and viability of cleaving embryos generated from stimulated IVF cycles. Hum Reprod, 1990, 5(7):857-61. 4. Schalkoff M, Oskowitz S, Powers R. A multifactorial analysis of the pregnancy outcome in a succssful embryo cryopreservation program. Fertil Steril, 1993, 59(5):1 070-4. 5. Edgar DH, Bourne H, Speirs AL, et al. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod, 2000, 15(1):175-9. 6. Testart J, Lassalle B, Belaisch-Allart J, et al. High pregnancy rate after early human embryo freezing. Fertil Steril, 1986, 46(2):268-72. 7. Steuerwald NM, Bermudez MG, Wells D, et al. Maternal age-related differential global expression profiles observed in human oocytes. Reprod Biomed Online, 2007, 14(6):700-8 8. Wang JX, Yap YY, Mattews CD. Frozen-thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception. Hum Reprod, 2001, 16(11):2 316-9.
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9. Richter KS, Bugge KR, Bromer JG, et al. Relationship between endometrial thickness and embryo implantation, based on 1 294 cycles of in vitro fertilization with transfer of two blastocyst-stage embryos. Fertil Steril, 2007, 87(1):53-9. 10. Tsafrir A, Simon A, Revel A, et al. Retrospective analysis of 1 217 IVF cycles in women aged 40 years and older. Reprod Biomed Online, 2007, 14(3):348-55. 11. Chuang CC, Chen CD, Chao KH, et al. Age is a better predictor of pregnancy potential than basal folliclestimulating hormone levels in women undergoing in vitro fertilization. Fertil Steril, 2003, 79(1):63-8. 12. Guerif F, Bidault R, Cadoret V, et al. Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal. Hum Reprod, 2002, 17(5):1 321-6. 13. Fugger EF, Bustillo M, Katz LP, et al. Embryonic development and pregnancy from fresh and cryopreserved sibling pronuclear human zygotes. Fertil Steril, 1988, 50(2):273-7. 14. Cohen J, DeVane GW, Elsner CW, et al. Cryopreservation of zygotes and early cleaved human embryos. Fertil Steril, 1988, 49(2):283-9. 15. Pantos K, Athanasiou V, Stefanidis K, et al. Influence of advanced age on the blastocyst development rate pregnancy rate and implantation rate. Fertil Steril, 1999, 71(6):1 144-6. 16. Check JH, Summers-Chase D, Yuan W, et al. Effect of embryo quality on pregnancy outcome following single embryo transfer in women with a diminished egg reserve. Fertil Steril, 2007, 8(4)7:749-56. 17. Edgar DH, Archer J, McBain J, et al. Embryonic factors affecting outcome from single cryopreserved embryo transfer. Reprod Biomed Online, 2007, 14(6):718-23. 18. Dowling-Lacey D, Jones E, Mayer J, et al. Elective transfer of two embryos: reduction of multiple gestations while maintaining high pregnancy rates. J Assist Reprod Genet, 2007, 24(1):11-5. 18. Karlstrom PO, Bergh C. Reducing the number of embryos transferred in Sweden-impact on delivery and multiple birth rates. Hum Reprod, 2007, 22(8): 2 202-7. (Received on January 14, 2008)
100
http://www.RandC.cn
2008 Jun; 19(2):93-100
[email protected]
Factors Influencing Pregnancy Outcome Following Slow Cooling Cryoembryo Transfer and Risk of Multiple Conception Su-ying LIU, Jin-lan HAN, Bin TENG, Zhu-di LU, Yu ZHENG, Ying CAO, Xiang CAO, Ning-yi WANG, Bin HUANG, Jing-ming YAN Shanghai Jiai Genetics & IVF Institute, Obstetrics & Gynecology Hospital, Fudan University, Shanghai 200011, China
Objective To investigate the factors that influence the potential for cryoembryo implantation and multiple pregnancy. Methods In this retrospective study, a total of 937 thawing cycles (859 couples) in which 3286 d 3-embryos were thawed. Rates of implantation, clinical pregnancy and multiple conception following FET were observed. Results There were significant differences in female age (P<0.05) and number of good quality embryos (P<0.05) between cycles that resulted in pregnancy and those did not. There was a trend toward decreasing rates of implantation, clinical pregnancy and multiple pregnancy with increasing female age. Compared with transferring 1 good quality embryo, clinical pregnancy rate of transferring 2 and 3 good quality embryos was increased significantly (P<0.001), there was no significant difference in clinical pregnancy rate between transferring 2 and 3 good quality embryos. Multiple pregnancy rate was increased significantly in the group of transferring 3 good quality embryos (P<0.05),but there was no significant differences in multiple pregnancy rate between transferring 1 and 2 good quality embryos.Younger women ( ≤ 30 years) also had a significantly higher multiple pregnancy rates (28.13%) than the older ones(>35 years) (13.64%).With an increase in age from ≤ 30 years to>40 years, clinical pregnancy rate declined from 45.61% to 25.00%. Conclusion Female age and the number of good quality embryos transferred are important factors influencing the clinical and multiple pregnancy rate, reducing the number of good quality embryos transferred may decrease the rate of multiple pregnancy but do not affect the clinical pregnancy rate. Key words: in vitro fertilization; cryopreservation; embryo transfer; clinical pregnancy; multiple pregnancy Corresponding author: Jin-lan HAN; Tel: +86-21-63459977*215; E-mail: [email protected] 93
Pregnancy, without consideration of obstetric and neonatal outcomes, is no longer the objective. Practitioners readily acknowledge the significant contribution of multiple pregnancy to the risk and complications of assisted reproductive technology. It is universally recognized that multiple gestation, and its attendant prematurity, is associated with increased mortality and morbidity, both for mothers and fetuses[1]. A twin gestation involes a perinatal outcome with a 15 fold increase risk of complications, compared with a singleton[2]. Since the development of assisted reproduction techniques, most countries have witnessed increased rates of multiple pregnancy. Despite the guidelines proposed by various scientific societies, these rates continue to be abnormally high. Embryo cryopreservation provided additional safety in the presence of ovarian hyperstimulation and contributed to lowering the risk of multiple conception by reducing the need to transfer multiple fresh embryos. However, frozen embryo transfer is not free from the risk of multiple conception. Effective strategies must be established to prevent multiple pregnancy without reducing overall pregnancy rates. The pregnancy rate following FET has been shown to be related to the number of blastomeres and morphological appearance of embryos prior to freezing[3,4], the extent of embryo damage after thawing[5]. The effects of embryological parameters on pregnancy rate after FET have also been addressed in several studies. To date, few attempts have been made to assess the number of good quality embryos transferred on the FET outcome, therefore, the current retrospective study was to evaluate the impact of clinical and embryological parameters on the FET outcome.
Materials & Methods Materials From January 2005 to December 2006, a total of 937 thawing cycles on 859 couples were studied retrospectively. Rates of implantaion, clinical pregnancy and multiple conception following FET according to patient age at the time of embryo freezing , the number of embryos transferred, endometrium thickness, and the number of good quality embryos transferrd were observed. The indications for IVF (ICSI) and embryo transfer included tuble factor, male factor, endometriosis and unexplained factor of infertility. Cryopreservation Cryopreservation procedure was performed using a slow-freeze protocol[6]. Embryos were equilibrated in 1.5 mol/L propanediol (PROH) at room temperature for 10 min, then transferred to 1.0 mol/L propanediol/0.1 mol/L sucrose and loaded into plastic straws. Straws were put in a controlled-rate freezer at a rate of -2℃/min to -7℃/min, at which point seeding was performed manually with liquid nitrogen-cooled forceps. Cooling was then continued at rates of -0.3℃/min to -30℃/min and rapidly cooled at a rate of -50℃/min to -140℃/min before plunging and storage in liquid nitrogen. 94
Thawing Embryos were thawed rapidly by removing straws from nitrogen, exposure to air for 30 s, then embryos were sequentially incubated in each of the following solutions for 5 min: 1.0 mol/L propanediol/0.2 mol/L sucrose, 0.2 mol/L sucrose and sucrose-free Dulbecco’s phosphate-buffered saline (DPBS). Embryos were assessed for numbers of remaining blastomeres, then were transferred to culture medium at 37℃ in 5% CO2 until the time of transfer. Post-thaw survival of cryopreserved embryos was defined as ≤ 50% blastomere loss. Freezing and thawing solutions consisted of cryoprotectants in DPBS supplemented with 20% serum protein substitute(SPSS). Routine examination of embryo quality included the number of blastomeres, the degree of fregmentation, the uniformity of blastomeres. Embryo morphology was scored as follows: grade 1, no fragments and equal blastomeres; grade 2, <20% fragmentation, equal blastomeres; grade 3, equal or unequal blastomeres, 20%-50% fragments; grade 4, equal or unequal blastomeres, >50% fragments. ≥7 blastomeres, grade 1 and grade 2 were defined as good quality embryo. Embryo transfer was performed 1 h later after thawing. To precisely control the uterine environment for embryo transfer, most of transfers were performed in hormone replacement treatment(HRT) cycles, natural cycles were performed only in those who had spontaneous ovulation. The ovarian steroid replacement protocol consisted of oral estradiol valerate and progesterone.On d 3 of the replacement cycle, estradiol was commenced daily. Luteal phase support was initiated 3 d before the embryo transfer by daily administration of progesterone and estradiol 4-6 mg. Biochemical pregnancy was confirmed by a positive blood hCG test 2 weeks after the embryo transfer. Clinical pregnancy was diagnosed by the presence of intrauterine gestational sac and heart beat on vaginal ultrasound 4 weeks after the embryo transfer. A maximum of three selected thawed embryos were transferred. All pregnant women continued to have estradiol and progesterone, both administered daily until week 10 of gestation. Statistical analysis Statistical analysis was performed using SPSS12.0 software, Student’s t-test and χ2 test. Data were presented as －x ± s. P<0.05 was considered statistically significant.
Results A total of 937 thawing cycles in which 3 286 embryos were thawed, 2 853 embryos survived, the survival rate was 86.8%. Totally 2 575 embryos were transferred, a maximum of 3 embryos were transferred. The mean maternal age in pregnant and non pregnant group were 31.0 ± 3.9 years and 31.7 ± 3.9 years, respectively (P<0.05). The mean numbers of embryos transferred were 2.7 ± 0.5 and 2.7 ± 0.5 (P>0.05), the good quality embryos transferred were 1.9 ± 0.8 and 1.6 ± 1.0 (P<0.05), the thickness of the endometrium were 9.8 ± 0.8 mm and 9.7 ± 2.1 mm (P>0.05) (Table 1). 95
Table 1 Characteristics between pregnancy and nonpregnancy cycles Item
Pregnant group
Number of cycles
Nonpregnant group
P
395
542
31.0 ± 3.9
31.6 ± 3.9
0.035
Tubal factor n(%)
181(45.93)
241(44.46)
>0.05
Male factor n(%)
169(42.89)
230(42.44)
>0.05
14(3.55)
23(4.24)
>0.05
Age(year) Indications of ART
Unexplained n(%)
31(7.88)
48(8.86)
>0.05
No. of embryo transferred
Other n(%)
2.7 ± 0.5
2.7 ± 0.5
0.11
No. of good quality embryo transferred
1.9 ± 0.8
1.6 ± 0.9
<0.01
Endometrial thickness(mm)
9.7 ± 2.1
9.7 ± 2.1
0.63
There was a trend toward decreasing rates of implantation, clinical pregnancy and multiple pregnancy with increasing female age. The implantation, clinical pregnancy and multiple pregnancy rates in the two younger age groups (≤ 30 and 31-35 years) were not significantly different from each other. The implantation rate in the group of ≤30 years was significantly higher than that in women aged 36-40 years and >40 years. Younger women also had a significantly higher multiple pregnancy rate than the older women. With an increase in age from ≤30 years to >40 years, clinical pregnancy rate declined from 45.61% to 25.00%. There were no multiple pregnancy in women aged >40 years old(Table 2). Table 2 Rate of clinical pregnancy, multiple pregnancy according to patient age Age (years)
Number of cycles
Embryo transferred
Implantation rate Clinical (%) pregnancy rate (%)
Multiple pregnancy rate (%)
≤ 30
421
2.5 ± 0.5
21.14(252/1 192)
45.61(192/421)
31-35
380
2.5 ± 0.5
19.57(202/1 032)
40.26(153/380)
27.45(42/153)
37.93(44/116)
13.64(6/44)*
36-40 >40
**
116
2.5 ± 0.5
16.56(50/302)
20
2.5 ± 0.5
9.80(5/51)
25.00(5/20)
28.13(54/192)
0
∗: P<0.05, **: P<0.001, compared with age of ≤ 30 years old
Clinical pregnancy rates was increased with the number of good quality embryos increased. Compared with transfering 1 good quality embryos, the clinical pregnancy rates were significantly increased in both the group of transferring 2 and 3 good quality embryos, but no difference was noticed between the groups. Multiple pregnancy rate of transfering 3 good quality embryos was increased significantly compared with tranferring 1 good quality embryo. Transferring 2 good quality embryos resulted in similar pregnancy rate but significantly 96
reduced the overall incidence of multiple gestations when compared with transferring 3 good quality embryos (24.3% vs 37.6%). Twin gestations was decreased from 29.36% to 23.2%, and triplets significantly decreased from 8.25% to 1.11%. The elective transfer of 2 good quality embryos resulted in similar pregnancy rates and significantly reduced multiple gestation rates when compared with the elective transfer of 3 good quality embryos (Table 3). Table 3 Rate of clinical pregnancy, multiple pregnancy according to numbers of good embryo quality transferred No. of good quality embryo transferred Item
0
1
2
3
No. of cycles
88
260
384
205
No. of embryo transfered
2.67 ± 0.43
2.60 ± 0.57
2.63 ± 0.45
2.61 ± 0.55 **
52.20(109/205)**
Clinical pregnancy rate(%)
26.14(23/88)
31.20(81/260)
4 7 .1 0 ( 1 8 1 / 3 8 4 )
Multiple pregnancy rate(%)
4.30(1/88)
19.80(16/260)
24.30(44/384)
37.60(41/205)*∆
Twin(%)
4.30(1/88)
17.28(14/260)
23.2(42/384)
29.36(32/205)
Triplet(%)
0
2.47(2/260)
1.11(2/384)
8.25(9/205)∆
*: P<0.05, **: P<0.001, compared with transferring 1 good quality embryo ∆: P<0.05, compared with 2 good quality embryos transferred
Discussion Maternal age and FET The maternal age-related decline in female fertility has been attributed to a variety of causes including progressive oocyte depletion, meiotic irregularities cell cycle regulation, and mitochondrial dysfunction[7]. The implantation and pregnancy rate following frozen embryo transfer was decreased with increasing female age, the effect of age on implantation rate seemed to be independent[8,9]. More and more young women are delaying childbearing. Older women comprise an increasing portion of patients entering assisted reproduction programmes. A retrospective analysis of 1 217 IVF cycles in women aged 40 years and older showed that success rates declined with each year after age 40; pregnancy and delivery rates were 13.9% and 9.1% at age 40 and 2.8% and 0.7% at age 45[10]. There were no deliveries at age 45[10]. Unrealistic expectations may be avoided if accurate data are provided regarding delivery rates per year after age 40. The pregnancy rate per cycle showed a significant decrease with increasing maternal age[11]. According to the data presented here, the implantation, clinical pregnancy and multiple pregnancy rates in the two younger age groups (≤30 and 31-35) were not significantly different from each other. The implantation rates in the group of ≤ 30 years was 21.14%, 97
which was significantly higher than that of women aged 36-40 years (16.56%). Younger women also had a significantly higher multiple pregnancy rates (28.13%) than the older women (13.64%). With an increase age from ≤ 30 years to >40 years, clinical pregnancy rate declined from 45.61% to 25.00%. There were no multiple pregnancy in women aged > 40 years old. This finding is consisitent with earlier reports[8,9]. Number of good quality embryos and FET Cryopreservation can affect the implantation potential of early cleavage-stage embryos if cryodamage occurs. It has been shown that partially intact thawed cleavage-stage embryos can result in pregnancies, the implantation potential drops in parallel with increasing degrees of blastomere lysis, but the intact thawed embryos have the same implantation potential as equivalent to fresh embryos[5,12]. It has been shown that not all human embryos survive the cryopreservation procedure. The survival rate based on reviews from the literature, appears to be highly variable, ranging from 56%-92%[13,15]. In this study, there was a high embryonic survival rate after thawing (86.8%). The pregnancy rate following FET has been shown to be related to the number of blastomeres and morphological appearance of embryos prior to freezing, the extent of embryo damage after thawing. A retrospective study of single ET(embryo transfer) in infertile women with a decreased egg reserve showed significantly higher clinical pregnancy rate with the transfer of a single embryo with 6-8-blastomere than 4-5-cell embryo (40.4% vs 6.6% clinical), degree of fregmentation did not predict outcome nearly as well as blastomere number [16]. Another retrospective analysis of 6 916 cryopreserved d 2-embryo transfer procedures, transfer of two cryopreserved embryos resulted in higher clinical pregnancy rates when compared with transfer of a single thawed embryo but was also associated with elevated multiple pregnancy rates (26.7% in women under 36). In cryopreserved 4-cell stage embryos, loss of a single blastomere did not reduce implantation potential[17]. Dowling, et al. reported that the elective transfer of 2 embryos resulted in similar pregnancy rate but significantly reduced the overall incidence of multiple gestations (20% vs 39%) when compared with the elective transfer of 3 embryos[18]. Twin gestations decreased from 28% to 19%, and triplets significantly decreased from 9% to 1%. The elective transfer of 2 embryos resulted in similar pregnancy rates and significantly reduced multiple gestation rates when compared to the elective transfer of 3 embryos. It is recommended that a maximum of 2 embryos are transferred in young women with good quality embryos at the time of transfer, the transfer of 3 embryos is only recommended in women ≥ 38 years who have one or no good quality embryos available at the time of transfer. Given the promising potential of eSBT(elective single blastocyst transfer) to markedly reduce the risk of twin gestation without a significant compromise to pregnancy outcomes, an active attempt should 98
be made to consider and use eSBT in the young, favorable-prognosis patient who has goodquality embryos available for transfer and cryopreservation[19]. Similar to the results mentioned above, comparing with transfering 1 good quality embryo, clinical pregnancy rate of transfering 2 and 3 good quality embryos increased significantly (P<0.001), there was no significant difference in clinical pregnancy rate between transfering 2 and 3 good quality embryos. Multiple pregnancy rate increased significantly in the group of transfering 3 good quality embryos (P<0.05), but there was no significant difference in multiple pregnancy rate between transferring 1 and 2 good quality embryos. With 3 good quality embryos transferred, the clinical pregnancy rate increased slightly than 2 good quality embryos transfer, but the risk of multiple conception increased significantly. Several factors should be considered before frozen embryo transfer regarding the chance of pregnancy and risk of multiple conception. In conclusion, female age, the embryo quality and the number of good quality embryos are the most important factors that influence the rate of implantation, clinical pregnancy and multiple pregnancy following frozen embryo transfer. The expectation of pregnancy and the risk of multiple conception are important issues for both the clinician and the couple. The approach should be individualized. For younger patients (<40 years old), if there are enough good qualty embryos, transfering 2 good quality embryos and freezing surplus embryos would not compromise the expectation of pregnancy while reducing the risk of multiple conception.
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9. Richter KS, Bugge KR, Bromer JG, et al. Relationship between endometrial thickness and embryo implantation, based on 1 294 cycles of in vitro fertilization with transfer of two blastocyst-stage embryos. Fertil Steril, 2007, 87(1):53-9. 10. Tsafrir A, Simon A, Revel A, et al. Retrospective analysis of 1 217 IVF cycles in women aged 40 years and older. Reprod Biomed Online, 2007, 14(3):348-55. 11. Chuang CC, Chen CD, Chao KH, et al. Age is a better predictor of pregnancy potential than basal folliclestimulating hormone levels in women undergoing in vitro fertilization. Fertil Steril, 2003, 79(1):63-8. 12. Guerif F, Bidault R, Cadoret V, et al. Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal. Hum Reprod, 2002, 17(5):1 321-6. 13. Fugger EF, Bustillo M, Katz LP, et al. Embryonic development and pregnancy from fresh and cryopreserved sibling pronuclear human zygotes. Fertil Steril, 1988, 50(2):273-7. 14. Cohen J, DeVane GW, Elsner CW, et al. Cryopreservation of zygotes and early cleaved human embryos. Fertil Steril, 1988, 49(2):283-9. 15. Pantos K, Athanasiou V, Stefanidis K, et al. Influence of advanced age on the blastocyst development rate pregnancy rate and implantation rate. Fertil Steril, 1999, 71(6):1 144-6. 16. Check JH, Summers-Chase D, Yuan W, et al. Effect of embryo quality on pregnancy outcome following single embryo transfer in women with a diminished egg reserve. Fertil Steril, 2007, 8(4)7:749-56. 17. Edgar DH, Archer J, McBain J, et al. Embryonic factors affecting outcome from single cryopreserved embryo transfer. Reprod Biomed Online, 2007, 14(6):718-23. 18. Dowling-Lacey D, Jones E, Mayer J, et al. Elective transfer of two embryos: reduction of multiple gestations while maintaining high pregnancy rates. J Assist Reprod Genet, 2007, 24(1):11-5. 18. Karlstrom PO, Bergh C. Reducing the number of embryos transferred in Sweden-impact on delivery and multiple birth rates. Hum Reprod, 2007, 22(8): 2 202-7. (Received on January 14, 2008)
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