Abstract
We analyzed the incidence of ectopic pregnancy in frozen-thawed embryo transfer (FET) as a function of natural and hormone replacement cycles and ectopic pregnancy-related factors. In this study, there were 4,034 FET cycles performed in our center between January 2005 and December 2010, and the rates of ectopic pregnancy were compared between natural and hormone replacement cycles. The analysis of ectopic pregnancy-related factors in FET was performed with 1:4 age-matched chi-square tests. The rate of ectopic pregnancy was lower in natural FET cycles (1.46%) than in hormone replacement FET cycles (3.31%) with a statistical significance (P < 0.05). Many factors were associated with ectopic pregnancy in FET, but only treatment protocols were considered as a controllable factor. We conclude that the incidence of ectopic pregnancy is significantly lower in natural FET cycles than in hormone replacement FET cycles. The application of exogenous sex hormones in assisted reproductive cycles may be an important factor to cause ectopic pregnancy in FET. This suggests that care should be taken when selecting the treatment protocol in order to avoid ectopic pregnancy.
Introduction
Many kinds of ovarian stimulation protocols have been used in assisted reproductive technology, but ovarian stimulation has many disadvantages. These include, complex drug regimens, high pharmaceutical fees, complex luteal support protocols, and the observed high incidence of ovarian hyperstimulation syndrome. Therefore, an increasing number of reproductive centers tend to adopt embryo freezing, and a great deal of attention has been given to frozen-thawed embryo transfer (FET) in recent years. It is known that ectopic pregnancy is a risk of assisted reproductive technology with an incidence of 2-5% [ Lesny et al. Citation1999; Strandell et al. Citation1999; Sowter and Farquhar Citation2004]. There have been conflicting reports regarding whether there is a difference in the incidence of ectopic pregnancy between fresh and frozen-thawed cycles in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) [Clayton et al. Citation2006; Check et al. Citation2005; Jun and Milki Citation2007; Keegan et al. Citation2007]. Little research has been carried out regarding the comparison of the incidence of ectopic pregnancy between natural and hormone replacement cycles in FET. In this study, we compared the incidence of ectopic pregnancy between 2,198 natural and 1,836 hormone replacement cycles in FET, evaluated the risk of ectopic pregnancy in FET, and analyzed the possible causes of ectopic pregnancy.
Results
Comparison of related factors between the patients with ectopic pregnancy in FET and the women with normal pregnancy
Of the 4,034 FET cycles, clinical pregnancy occurred in 1,090 and ectopic pregnancy in 26 with an ectopic pregnancy rate of 2.39%. Related factors were analyzed between the patients with ectopic pregnancy in FET and the women with normal pregnancy using a 1:4 age-matched chi-square test ().
Table 1. Comparison of related factors between the patients with ectopic pregnancy and the women with normal pregnancy.
The 1:4 age-matched chi-square analyses indicated that there were statistical differences in history of salpingoplasty or hydrotubation, history of salpingitis, history of pelvic inflammation, history of abdominal surgery, history of curettage/ induced labor, history of ectopic pregnancy, secondary infertility, and treatment protocols (all p < 0.05). These were the risk factors of ectopic pregnancy in FET. Salpingoplasty or hydrotubation was related to salpingitis, and abdominal surgery was related to pelvic inflammation; but direct causation was not observed. A history of curettage/ induced labor or a history of ectopic pregnancy had causation with secondary infertility. Except for treatment protocols, other factors could not be controlled.
General conditions and the distributions of related risk factors between natural and hormone replacement cycles
There were no statistical differences in general conditions and embryo quality between the two groups (P > 0.05, ). In addition there were no statistical differences in the distribution of these ectopic pregnancy-related risk factors including history of salpingoplasty or hydrotubation, history of salpingitis, history of pelvic inflammation, history of abdominal surgery, history of curettage/ induced labor, history of ectopic pregnancy, and secondary infertility between natural and hormone replacement cycles (P > 0.05, ).
Table 2. General conditions in natural cycles and hormone replacement cycles.
Table 3. The distributions of ectopic pregnancy-related risk factors between natural cycles and hormone replacement cycles.
Ectopic pregnancy in natural and hormone replacement FET cycles
Of the 4,034 cycles, 2,198 underwent natural FET, 547 were pregnant including 8 ectopic pregnancies with a rate of ectopic pregnancy of 1.46%. In comparison 1,836 underwent hormone replacement FET, 543 were pregnant including 18 ectopic pregnancies with a rate of ectopic pregnancy of 3.31%. The rate of ectopic pregnancy was significantly lower in natural cycles than in hormone replacement cycles (P < 0.05, ).
Table 4. Ectopic pregnancy in natural cycles and hormone replacement cycles.
Discussion
With the development of assisted reproductive technology, the success rate of pregnancy is increasing every year. Attention is now being given to ectopic pregnancy. It is reported that the incidence of ectopic pregnancy was 2.1-9.4% in IVF-ICSI-ET, 3.5-8.6% in FET, and 1.9% in natural pregnancy [Lozeau and Potter Citation2005; Rosman et al. Citation2009]. In this study, the rate of ectopic pregnancy in FET was 2.39% which is higher than that of natural pregnancy.
Factor(s) leading to the increased rate of ectopic pregnancy in FET
In this study, we analyzed the risk factors of ectopic pregnancy in FET using 1:4 age-matched chi-square tests. Our results indicated that a history of salpingoplasty or hydrotubation, a history of salpingitis, a history of pelvic inflammation, a history of abdominal surgery, a history of curettage/ induced labor, a history of ectopic pregnancy, secondary infertility, and treatment protocols were the risk factors of ectopic pregnancy in FET. In the risk factors above, tubal pathological changes were basal, although a history of abnormal pregnancy may lead to tubal pathological changes. Comparing treatment natural cycles and hormone replacement cycles, revealed one of the risk factors of ectopic pregnancy in FET. This study indicated that the rate of ectopic pregnancy in FET was significantly higher in hormone replacement cycles (3.31%) than in natural cycles (1.46%) with a statistical significance of P < 0.05.
Clayton et al. [2006] have reported that tubal factor is the main risk factor of ectopic pregnancy in IVF-ET. Weigert et al. [2009] has also described that the risk of ectopic pregnancy is increased in IVF-ET for the patients with a history of ectopic pregnancy, which is consistent with the results presented in this study. At present, most studies assessing risk factors of ectopic pregnancy are performed in fresh embryo transfer. However, little research has confirmed whether the risk factors of ectopic pregnancy in FET are the same as that in fresh embryo transfer. We assume that the transferred embryos may move between the uterine cavity and the fallopian tubes, and corpus luteum can allow most embryos to go back to the uterine cavity. However, in patients with pathological tubal changes, embryos may remain in the fallopian tubes, leading to ectopic pregnancy. This study indicated that in FET, the rate of ectopic pregnancy was significantly higher in hormone replacement cycles than in natural cycles, which is reported for the first time. Estrogen and progestogen play an important role in embryo implantation. They can synchronize endometrium and embryo development to ensure successful embryo implantation. In hormone replacement cycles, the application of estrogen and progestogen may cause changes in motility intensity, frequency, and direction of tubal peristalsis to result in the retrogression of transferred embryos into fallopian tubes. On one hand, high levels of estrogen can decrease the function of the estrogen receptor [Zou et al. Citation2006], reducing endometrial glandular proliferation and promoting stromal hyperplasia which permits interstitial development to be earlier than glandular development. On the other hand, an abnormal ratio of progesterone (P) to estradiol (E2) can cause endometrial glandular development to be later than interstitial development, decreasing endometrial receptivity for embryos. The two factors above may cause a high incidence of ectopic pregnancy in hormone replacement cycles.
This study discovered that in FET, the incidence rate of ectopic pregnancy was significantly higher in hormone replacement cycles than in natural cycles. Additional data from other centers is required to confirm this new finding. However, the application time and dose of exogenous estrogen remains to be explored in future studies. FET cycles mainly include natural cycles and hormone replacement cycles. In the patients with regular menstrual cycles and normal ovulation, natural cycles are commonly adopted because it is closer to the endocrine status of natural pregnancy. However, in partial patients with abnormal ovulation such as polycystic ovary syndrome or endometrial dysplasia, hormone replacement cycles are usually adopted. Based on the results reported in the above the rate of ectopic pregnancy was significantly higher in hormone replacement cycles than in natural cycles. The application of large-dose exogenous sex hormones should be carefully considered in order to avoid ectopic pregnancy. In summary, in frozen-thawed embryo transfer, the difference in the incidence of ectopic pregnancy between natural cycles and hormone replacement cycles should be considered. When possible natural cycles should be adopted to decrease the risk of ectopic pregnancy.
Materials and Methods
All study methods were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All the subjects enrolled into the study gave written formal consent to participate.
Subjects
We retrospectively analyzed 4,034 FET cycles including 2,198 natural cycles and 1,836 hormone replacement cycles performed in our Reproductive Medical Center between January, 2005 and December, 2010. The mean age of women was 30.2 ± 4.5 y (range 20-47). The duration of infertility was 2-19 y. Infertility causes included oviducal factors, endometriosis, pelvic adhesions, and unexplained infertility. All of the 4,034 cycles underwent day 3-embryo FET.
Ovarian stimulation
Short-acting gonadotropin releasing hormone agonist (GnRH-α, 0.05-0.1 mg, 0.1 mg/ampule) was subcutaneously injected once a day during the midluteal phase. After attaining the standards of down regulation, gonadotrophin (Gn) was injected until the time of human chorionic gonadotropin (HCG) administration. Gn was highly purified recombinant follicle stimulating hormone (r-FSH, 75IU/ampule) or HMG (75U/ampule). The dose of Gn was adjusted according to the specific status of patients. Oocyte collection, insemination, embryo transfer, and luteal support were performed according to the routine methods of our center [Sun et al. Citation2002].
Embryo transfer
Natural cycle was used for the patients with regular menstrual cycle of 25-35 d. The follicle was monitored with B ultrasound on the seventh or eighth day of menstruation. When follicular diameter was 14 mm or over, the level of luteinizing hormone (LH) in urina sanguinis was determined. When LH peak (LH value was more than two times as much as basal LH value) occurred, 10,000 IU of HCG was injected and the levels of LH, E2, and P in blood were determined. On the next day, follicular rupture was monitored. The blood P level was again determined on the day of follicular rupture, and embryo transfer was performed 3 d after follicular rupture. Hormone replacement cycle was used for the patients with irregular menstrual cycle or poor folliculogenesis. If B ultrasound indicated that pelvic cavity, uterus, and ovaries were normal, 2-4 mg/d of estrogen (progynova, estradiol valerate tablets, one milligram/tablet) was orally given on the third day of post-hormone withdrawal menstruation for 4 d, estrogen dose might be increased to 6-8 mg if necessary. When the endometrium was 8 mm or over, 60 mg/d of progesterone was given for 4 d on the thirteenth to sixteenth day of menstruation followed by abdominal B ultrasound-guided ET. The number of transferred embryos was 3 or under.
Assessment of pregnancy
Blood β-HCG was detected 14 d after ET to determine biochemical pregnancy. It was diagnosed as clinical pregnancy when B-mode ultrasound showed gestational sac and fetal heart beat 35 d after ET. It was diagnosed as ectopic pregnancy when B-mode ultrasound was able to resolve a gestation sac with or without an embryo bud and heart tube beat, or ectopic pregnancy lesion was found during operation 35-50 d after ET.
Grouping
Frozen-thawed embryo transfer cycles were divided into natural cycles and hormone replacement cycles according to whether estrogen was used. The incidence of ectopic pregnancy was compared between natural cycles and hormone replacement cycles.
Statistical treatment
Analysis of ectopic pregnancy-related factors in FET
A 1:4 matching chi-square analysis was performed using Excel according to the age distribution of patients with ectopic pregnancy in FET. The 26 patients with ectopic pregnancy served as a case group. Based on the age of each patient with ectopic pregnancy, 4 women with normal pregnancy of similar age were selected as the control, so the control group included 104 women with normal pregnancy. A 1:4 age-matched chi-square test is more suitable to this study because of the lower incidence of ectopic pregnancy. The 1:4 age-matched chi-square tests were used for analysis of ectopic pregnancy-related factors with EPI Info software. Statistical significance was established at P < 0.05.
Comparison between natural and hormone replacement cycles
Statistical analysis was performed with SPSS13.0 software. A t test was used for comparison of age, duration of infertility, the number of transferred embryos, the rate of high-quality embryos, and the survival rate of thawed embryos between the two groups. The χ2 test was used for comparison of ectopic pregnancy between the two groups. Statistical significance was established at P < 0.05.
Declaration of interest: The authors have no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
Author contributions: Conceived and designed the study: Y-lZ, Y-pS; Performed the study: Y-cS; Analyzed the data: Y-hG; Wrote the manuscript: Y-lZ, JS.
Abbreviations
| FET: | = | frozen-thawed embryo transfer |
| IVF: | = | in vitro fertilization |
| ICSI: | = | intracytoplasmic sperm injection |
| ET: | = | embryo transfer |
| HCG: | = | human chorionic gonadotropin |
| LH: | = | luteinizing hormone |
| E2: | = | estradiol |
| P: | = | progesterone. |
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