In the past three decades, in vitro fertilization and embryo transfer (IVF-ET) has become the most effective treatment for infertile couples. Improving the overall therapeutic efficiency of IVF-ET depends on acquiring high quality embryos and implanting them successfully in a receptive endometrium.1 The acquisition of high-quality embryos is not only influenced by controlled ovarian hyperstimulation (COH)2 and the conditions of in vitro culture, but is also influenced by the patients' own gamete quality. Therefore, how to improve the overall embryo utilization rate is very important. It has been reported that endometrial receptivity in response to the high concentrations of estrogen seen in COH cycles is worse than in natural cycles.3 However, natural cycles or hormone replacement cycles similar to natural cycles have been used mostly in frozen-thawed embryo transfer (FET) cycles.4 Therefore, embryo implantation, clinical pregnancy and multiple pregnancy rates might be higher in treatment protocols using FET rather than fresh embryo transfer (ET) cycles. We developed a program of canceling fresh ETs and performing an FET later, starting from 2006. We found that this program could increase the embryo implantation and clinical pregnancy rates, and ultimately improve the embryo utilization rate.
Data on 179 FET cycles in which the fresh ET was canceled and all embryos were frozen after COH followed by an FET cycle, and on 1306 COH and fresh ET cycles were evaluated. The study ran from January 2006 to June 2008 in the Assisted Reproductive Unit of Sir Run Run Shaw Hospital. The women were aged 22–47 years, and the duration of infertility ranged from 1 to 24 years. The causes of infertility included tubal factors, combined female factors, male factors, combinations of female and male factors, and unexplained infertility. The constituent ratios of causes of infertility between two groups were not significantly different. The indications of canceling a fresh ET cycle included the numbers of oocytes retrieved being ≥20, thinness of the endometrium (a double thickness of the endometrium <7 mm), uneven ultrasound images of the endometrium, having a fever, coughing, vaginal bleeding, and social factors. All patients signed informed consent forms before treatment, who were assigned to two groups: group C1, in which the fresh ET was canceled and an FET was performed later (n=179); and group T1 with a fresh ET alone (n=1306). The rates of embryo implantation, clinical pregnancy, multiple pregnancy, miscarriage, and ectopic pregnancy were compared between the two groups. Patient's age and the numbers of oocytes retrieved and embryos transferred were analyzed.
Ovarian stimulation protocol and assessment of embryo quality
Standard programs of COH, embryo culture and transfer were used. A long gonadotropin releasing hormone (GnRH) agonist protocol was used in 64.1% of all cycles and a short protocol was used in 32.0%. The G-1.3 sequential culture system5 was used for embryo culture, and embryo freezing or fresh transfers were performed on the third day after oocyte retrieval. Embryo quality assessment was based on morphology and rate of development in culture. Four grades of embryos were defined: grade 1, embryos had blastomeres of equal size and no cytoplasmic fragmentation; grade 2, embryos had blastomeres of equal or unequal size and cytoplasmic fragmentation to ≤20% of the embryo surface; grade 3, embryos had blastomeres of equal or unequal size and 21%-49% overall cytoplasmic fragmentation and grade 4, embryos had blastomeres of equal or unequal size and cytoplasmic fragmentation to ≥50% of the embryo surface.6 Embryos with a normal cleavage rate (3–4 cells on the second day and 6–8 cells on the third day) and <20% fragmentation were defined as “high quality”.
Embryo freezing and thawing
The classic Testart slow freeze-thaw protocol was used in FET.7,8 A programmable freezer (Planer; Middlesex, UK) and embryo freezing and thawing kits (Irvine Scientific; Santa Ana, CA, USA) were used. ET was performed within 6 hours after thawing. For freezing, embryos needed to have three or more blastomeres on the third day and an embryo assessment grade of 4 or better. Poorer grade embryos were discarded.
Preparation for FET
For women with regular menstrual cycles, the frozen-thawed embryos were replaced in natural cycles. ET was performed three days after ovulation, or four and a half days after the luteinizing hormone (LH) peak. The transfer of frozen-thawed embryos was performed in a hormone replacement cycle or a low-dose human menopausal gonadotrophin (HMG)-stimulated cycle (Menotrophin for Injection, Livzon Pharmaceutical Group, Inc., Guangdong, China) for women with irregular or anovulatory cycles.9
For hormone replacement cycles, we maintained the same dosage of oral estradiol valerate tablets (Progynova; Schering AG, Berlin, Germany) about 6–8 mg/d for 14–28 days. When the double thickness of the endometrium exceeded 8 mm, injections of 80 mg/d of progesterone injection were administered for 3 days before FET and the original dosage was maintained after ET. A serum estradiol level close to 300 ng/L was also necessary for ET to proceed. If the double thickness of the endometrium was still less than 8 mm when the serum estradiol level was close to 300 ng/L, no more estradiol valerate was administered.
For low dose HMG-stimulated cycles, mild stimulation was initiated with a low dose of HMG on days 7–8 of the menstrual cycle. Dose was adjusted according to follicular development as monitored by ultrasound. Injections of 40 mg/d of progesterone were administered for two days after human chorionic gonadotropin (HCG) injection. FET was performed about four and a half days after the HCG injection and 80 mg/d of progesterone was injected for luteal support after ET.
Embryo survival was defined as the retention of at least 50% intact cells.10 Embryo quality was reassessed after thawing. Up to three embryos were transferred transcervically to the middle of the uterine cavity per cycle.
Definition of outcome
Successful pregnancy was confirmed by detecting an increased serum β-hCG concentration 12–14 days after ET. Clinical pregnancy was defined by the observation of a gestational sac with or without a fetal heartbeat on ultrasound evaluation on the 35th day after oocyte retrieval. The number of sacs was taken as the number of implantations.
All statistical calculations were done using SAS 8.1 software (SAS Institute, Cary, NC, USA). Student's t-tests were used for quantitative variables and χ2 tests for qualitative ones. Logistic regression analysis was used to adjust the confounding factors to compare the embryo implantation and clinical pregnancy rates between the two groups. P <0.05 was considered statistically significant.
Comparisons between groups
In group C1, 179 fresh ET attempts were canceled either to prevent serious and potentially fatal complication — ovarian hyperstimulation syndrome (OHSS)11 or in women with endometrial polyps and other clinical problems, and an FET cycle was performed later. Of the 440 embryos thawed, 395 (89.8%) survived. Among these, 5 embryos were discarded because of low quality, and another 7 embryos were re-frozen for seven patients to reduce the multiple pregnancy rate because there were already 2 high quality embryos available for transfer. No FET attempt was canceled because of death of all of the embryos. Among 383 embryos transferred, 252 were of high quality, and the mean number of embryos transferred was 2.1±0.4. In group T1, 1306 fresh ET procedures were performed. Among 2641 embryos transferred, 1836 were of high quality, and the mean number of embryos transferred was 2.0±0.5.
The patients in group C1 were younger than those in group T1 and had more oocytes retrieved (P <0.01). The respective embryo implantation rate (43.6% vs 29.0%), clinical pregnancy rate (63.1% vs 47.0%) and multiple pregnancy rate (46.9% vs 28.5%) were all significantly higher in group C1 than in group T1, but there were no significant differences in miscarriage rate (11.5% vs 8.0%) or ectopic pregnancy rate (1.8% vs 2.6%; Table 1).
Logistic regression analysis confirmed that the embryo implantation and clinical pregnancy rates in group C1 were significantly higher than in group T1, after adjusting for the confounding factors of age, numbers of oocytes retrieved, embryos transferred and high-quality embryos transferred (43.6% vs 29.0%, 63.1% vs 47.0%; P <0.01, respectively; Table 2).
Comparison by age
Comparing the age groups ≥35 or <35 years in groups C1 and T1, the respective embryo implantation rate (41.8% vs 20.6% and 44.0% vs 32.4%) and clinical pregnancy rate (63.0% vs 38.3% and 63.2% vs 50.2%) were consistently higher in group C1 (Table 3).
Comparison by numbers of oocytes retrieved
The clinical pregnancy rates for the number of oocytes retrieved per cycle being ≥15 or <15 were consistently higher in group C1 than in group T1 (66.4% vs 50.3% and 53.3% vs 45.9%, respectively), as was the embryo implantation rate (48.0% vs 33.0% and 31.4% vs 27.7%, respectively). These differences were statistically significant for oocyte numbers ≥15 (Table 3).
Comparison by numbers of embryos transferred
Whether one, two or three high quality embryos were transferred, the clinical pregnancy rate was consistently higher in group C1 than in group T1 (57.1% vs 26.2%, 60.0% vs 50.8%, and 78.1% vs 41.0%, respectively) as was the embryo implantation rate (57.1% vs 26.2%, 45.0% vs 32.3%, and 38.5% vs 16.7%, respectively). These differences were statistically significant for cycles with two or three embryos transferred (Table 3). The embryo implantation rate in single embryo transfer was higher than with two or three embryos transferred in group C1 (57.1% vs 45.0% vs 38.5%, P <0.05, respectively). Because only seven patients underwent single embryo transfer in this series, this was insufficient to evaluate any significant effect of single embryo transfer.
IVF-ET has now been applied for thirty years and the clinical pregnancy rate has increased steadily with the development of advanced embryo culture systems and improved understanding of the endocrinology of COH. It is known that high quality embryos, endometrial receptivity and the need for synchrony between embryo development and endometrial growth are the three most important factors affecting the successful establishment of pregnancy.12 Generating high quality embryos is related to the age of the patient, the COH program and the effectiveness of in vitro culture. When the generation of high quality embryos reaches an optimum in any IVF unit, the synchrony of embryo development with endometrial growth becomes an important factor in the success rate. Thus, excessive ovarian response in COH cycles leads to insufficient secretary transformation of the endometrium, along with discordant glandular and stromal development at a time that coincides with the period of maximum endometrium receptivity.1,13 Differences in the endometrial gene expression profiles for seven days after HCG injection (HCG+7), seven days after LH peak (LH+7) and four days after the LH peak (LH+4) had already been analyzed using gene array technology.14 There was insufficient upregulation or excessive down-regulation of genes at HCG+7 in the endometrium of women undergoing COH and the change in gene expression at LH+4 was less than at LH+7. These results suggested that loss of synchrony of endometrial development in COH cycles might affect embryo implantation directly. FET has been used to prevent serious OHSS and increase the cumulative pregnancy rate since 1983.15–17 FET cycles appear to optimize embryo implantation rate thanks to better endometrial receptivity.18
From this series, we found that performing FET as the first ET attempt after COH was a satisfactory protocol in our infertility treatment program. Because of significant differences in patient's age and the numbers of oocytes retrieved between the two groups, it was impossible to evaluate the clinical pregnancy rate by χ2 tests, so we used Logistic regression analysis. The embryo implantation and clinical pregnancy rates in FET cycles performed as the first ET attempt after COH was higher than in fresh ET attempts, after adjusting for confounding factors. These results confirmed that the poor endometrial receptivity or asynchrony associated with COH appears to reduce the embryo implantation and clinical pregnancy rates and to depress the embryo utilization rate ultimately.
In the present study, the embryo implantation and clinical pregnancy rates were significantly higher in the first FET cycles after canceling a fresh ET cycle than in fresh ET cycles, regardless of the age group (≥35 or <35 years). As most canceled fresh ET cycles had more oocytes retrieved, the data were also analyzed according to this factor. In fact, the embryo implantation and clinical pregnancy rates in the first FET cycles carried out after canceling a fresh ET were higher than in fresh ET cycles no matter what was the number of retrieved oocytes (≥15 or <15) and were statistically significant for the subset with 15 or more oocytes retrieved. These results suggest that high estradiol concentrations influenced endometrial receptivity and synchrony between the embryo and the endometrium in patients with a high ovarian response. Such patients should be advised to cancel their fresh ET and progress to FET to optimize their treatment outcome.
The embryo implantation rate in single embryo transfer cycles was higher than with two or three embryos transferred in group C1. The clinical pregnancy rate reached 57.1% and had no significant difference whether two or three embryos were transferred. However, only seven patients underwent single embryo transfer in this series, so we were unable to evaluate this satisfactorily. And the multiple pregnancy rate in group C1 was very high. This observation suggests that we could carry out FET cycles with single high quality embryos to decrease the multiple pregnancy rate and improve the overall embryo utilization rate. Khalaf et al19 and Shibahara et al20 also suggested that single high quality embryo transfer could reduce the multiple pregnancy rate after IVF while maintaining the overall success rate of their IVF program. However, we need more patients to study this.
The embryo freezing and thawing protocol used in our study was the classic technique of slow freezing and rapid thawing. The embryo survival rate was 89.8% and there was no FET canceled because of the death of all embryos. Such good embryo survival in FET cycles enabled us to optimize the overall embryo utilization rate by allowing the development of good endometrial receptivity.
In this study, a policy of canceling fresh ET and progressing to FET could obtain significantly higher embryo implantation and clinical pregnancy rates, and enhance embryo utilization rate ultimately, especially for those patients with a strong ovarian response.
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