Frozen–thawed embryo transfer: influence of clinical factors on implantation rate and risk of multiple conception

J.X. Wang,1, Y.Y. Yap and C.D. Matthews

Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville, South Australia 5011, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Frozen embryo transfer is an important supplementary procedure in the treatment of infertility. While general information concerning the outcome of frozen embryo transfer has been documented, few studies have addressed the potential of embryo implantation in particular clinical situations. Importantly, the risk of multiple conception following frozen embryo transfer has been poorly documented compared with the information available for fresh embryo transfer. METHODS: This is a retrospective study analysing 3570 frozen embryo transfer cycles (1438 couples) with a view to increasing our understanding of the clinical circumstances that influence the potential for embryo implantation. RESULTS: The overall implantation rate was 9.1%. The characteristics associated with a more favourable implantation rate were the success of the previous fresh embryo transfer cycle, age < 40 years and non-tubal factor aetiology of infertility. Such women had an increased risk of multiple conception. CONCLUSION: Female age, the aetiology of infertility and the outcome of fresh embryo transfer are the most important factors influencing the implantation rate following frozen embryo transfer. A prognostic table has been constructed that may assist with the determination of the optimal number of embryos to be replaced in frozen embryo transfer to provide better individualized counselling and to secure an optimal chance of pregnancy while reducing the risk of multiple conception.

Key words: frozen embryo transfer/infertility/multiple conception


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the first successful report (Trounson et al., 1983Go) of frozen embryo transfer, the cryopreservation of embryos has been an important supplementary procedure in the treatment of infertility. Couples undertaking IVF, gamete intra-Fallopian transfer (GIFT) or intracytoplasmic sperm injection (ICSI), are commonly encouraged to have supplementary embryos cryopreserved for future use (Mandelbaum et al., 1998Go). Frozen embryo transfer procedures contribute an additional 25–50% chance of pregnancy for those couples who have embryos cryopreserved (Toner et al., 1991aGo; Wang .et al, 1994Go). Embryo cryopreservation has also provided additional clinical 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 (Matson et al., 1995Go; Tanbo et al., 1995Go). However, frozen embryo transfer is not free from the risk of multiple conception even though the implantation rate of frozen–thawed embryos is usually lower than that of fresh transferred embryos. Indeed, the multiple conception rate following frozen embryo transfer as reported by the Australian and New Zealand registry is 13% (Hurst et al., 1999Go), which is 15 times higher than that in spontaneous conceptions. It is well recognized that certain subgroups of women have both a higher implantation potential and risk of multiple conception following fresh embryo transfer (Nijs et al., 1993Go; Grochowski et al., 1997Go). This study of the outcomes of frozen embryo transfer has assessed the impact of a number of factors known to influence the results of fresh embryo transfer, including the age of the female partner, the aetiology of infertility and the outcome of the fresh cycle and the objective being to recognize better the potential for implantation following frozen embryo transfer and to provide more precise outcome advice for couples.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This was a retrospective cohort study, with data obtained from a tertiary referral centre, the Reproductive Medicine Unit (RMU), University of Adelaide, which provides frozen embryo transfer treatment at the Queen Elizabeth Hospital and the Wakefield Clinic in South Australia together with Darwin Private Hospital in Darwin, Northern Territory. The data traced couples who received frozen embryo transfer treatment from January 1990 to December 1998, during which time the programme protocols have been relatively constant. During this period of time, 1724 couples undertook a total of 4289 frozen embryo transfer cycles. However, cycles were excluded if the embryos were derived from donated gametes or if the embryos transferred were derived from more than one fresh cycle 6 months apart from each other. The final number of frozen embryo transfer cycles satisfying the criteria and included in the study was 3570 (83% of all cycles).

The general procedures associated with the programmes of assisted reproduction have been previously described (Kerin et al., 1984Go; Sathanandan et al., 1989Go; Matthews et al., 1991Go; Norman et al., 1991Go) and embryo cryopreservation by Wang et al. (Wang et al., 1994Go). Following fertilization and the transfer of fresh embryos/oocytes on either day 2 or day 3 after oocyte retrieval, the remaining embryos were assessed and embryos classified grade I or II according to a standard classification system (Mandelbaum et al., 1987Go) were cryopreserved. Embryos were generally cryopreserved at the early cleavage stages. Since 1988 a standard freezing–thawing protocol based on propanediol as cryoprotectant has been used (Testart et al., 1986Go). At the time of frozen embryo transfer, the frozen embryos were thawed and surviving embryos were transferred within 24 h depending on the stage of development at cryopreservation. Survival was defined as at least 50% of the blastomeres being intact. Confirmation of a successful implantation was performed by detecting an increased serum human chorionic gonadotrophin (HCG) concentration (>25 IU/l) 14 days post-frozen embryo transfer. The number of embryonic sac(s) was determined by ultrasound scanning 4–6 weeks after the thawed embryo transfer. Both ectopic pregnancy and miscarriage prior to ultrasound were considered as having a single embryo implantation. The implantation rate was calculated as the number of successful implanted embryos divided by the total number of embryos transferred.

The major factors analysed included: maternal age at the time of transfer; aetiology of infertility; classified as tubal factor and non-tubal factor (endometriosis, male factor and unexplained infertility); type of assisted reproduction employed in the fresh cycle; outcome of the fresh embryo transfer (pregnant or not); number of previous fresh cycle attempts; embryo storage time defined as from the day of storage (fresh stimulation cycle) to the day of frozen embryo transfer.

Statistical analyses
Instat and SAS programmes were used to perform statistical analyses. The results were analysed using {chi}2 test or Fisher's exact test whenever there was a cell with small number (n <5). A two-sided P value of < 0.05 was considered as statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 1438 couples (mean female age 33.5, range 19–46) and their 3570 frozen embryo transfer cycles were included in the study (mean number of cycles: 2.5). In these cycles, 10 075 frozen embryos were thawed, 6975 embryos survived the thawing process (69% survival rate) and 6965 were transferred. The median time of cryopreservation for the frozen–thawed embryos was 128 days (range 28–3505 days). The mean number of embryos transferred was two per cycle (range 1–3). Following the thawing and transfer of 6965 embryos in 3570 cycles, 631 embryos implanted (9.1%). The overall pregnancy rate per transfer cycle was 15.7%. Table IGo shows the clinical data of the frozen embryo transfer cycles stratified by five female age groups.


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Table I. Clinical outcome data of frozen embryo transfer cycles stratified by female age
 
The implantation and pregnancy rates in the three younger age groups (<30, 30–34 and 35–39 years) were not significantly different from each other. The overall pregnancy rate of the younger women (<40) was 16.4% [95% confidence interval (CI): 15.1–17.9%], which was significantly higher than those women aged 40–44 years (7.5%; 95% CI: 4.9–10.4%). Younger women also had a significantly higher implantation rate (9.5%; 95% CI: 8.7–10.2%) than the older women (4.0%; 95% CI: 2.6–6.1%). Only five frozen embryo transfer cycles (11 embryos) were performed in women aged >=45 years and no pregnancy was recorded.

Multiple conception was also markedly higher in the groups with the higher implantation rate. In the three younger groups, 17.7% of subjects had a multiple conception compared with only 4.8% of women aged >40 years old. This result was not significant possibly due to the small sample size.

In addition to age, both the aetiology of the infertility and the outcome of the fresh embryo transfer at the time the embryos were generated also had significant influences on the implantation rate of the frozen–thawed embryos (Table IIGo). Younger women (<40 years) with non-tubal infertility had a significantly higher implantation rate than similar-aged women with a tubal cause for their infertility. However, for older women (>=40 years) the implantation rate was low, and did not differ between tubal and non-tubal factor infertility. An increased rate of embryo implantation after frozen embryo transfer was evident in younger women who became pregnant in their fresh assisted reproductive technology cycles compared with those who did not. This increase was of the order of 65% (P < 0.001) and was even more marked in older women (>40 years) who established a pregnancy in the fresh cycle for whom a 144% increase was observed. However, this latter observation failed to reach statistical significance (P = 0.055). A slight, albeit non-significant (P = 0.06), increase in the implantation rate was also associated with a longer storage time (8.9% for <12 months, 10.3% for 1–2 years and 11.4% for >2 years), but this result was probably due to the confounding effect of those women who achieved a pregnancy following the fresh cycle. Patients previously treated by GIFT also had a better implantation rate (19%) but <10% of women were in this category. The number of oocytes recovered in the fresh treatment cycle and the number of fresh cycle attempts was also analysed but failed to show significant impact on the implantation rate.


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Table II. Influence of female age, aetiology of infertility and the outcome of the previous fresh embryo transfer cycle on frozen embryo transfer implantation rate
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The overall implantation rate of embryos following frozen embryo transfer, recorded in this study was 9.1%, which is comparable with results obtained from other large study groups (8–11%) (al-Shawaf et al., 1993; Frederick et al., 1995Go; Selick et al., 1995Go; Salat Baroux et al., 1996) but was lower than another report (18%) for frozen–thawed embryos following ICSI (Hu et al., 1999Go). Even with this rate of implantation, the frequency of multiple conception was unacceptably high in the younger women (15–22%). It is a concern, since multiple conception is associated with a higher incidence of an adverse outcome (Bergh et al., 1999Go).

Similar to previous findings following the transfer of fresh embryos, a reduced pregnancy rate following frozen embryo transfer was recorded with increasing female age, with the major reduction occurring after 40 years of age (P < 0.01). The effect of age on implantation rate seemed to be independent of the other two major factors, i.e. infertility aetiology and the outcome of the fresh cycle. A relatively consistently high implantation rate in women aged <40 years implies that this group of patients will benefit most from frozen embryo transfer. However, women >40 years had a poor prognosis of a successful pregnancy from frozen embryo transfer and even when the number of embryos transferred was increased from two to three, the chance of pregnancy was not improved. As previous noted (Toner et al., 1991bGo), the chance of implantation and pregnancy following frozen embryo transfer was higher if conception occurred in the fresh cycle. Increasing the number of embryos transferred in these subjects was more likely to increase the chance of multiple conception rather than their chance of pregnancy. The third major factor found to affect the implantation rate in our frozen embryo transfer cycles was the aetiology of the infertility. Patients with tubal blockage had a significantly lower implantation rate than average. This finding is consistent with earlier reports of lower general fecundity in this group of patients (Templeton and Morris, 1998Go). The lack of an effect of the time of embryo storage was reassuring.

The expectation of pregnancy and the risk of multiple conception are important issues for both the clinician and the couple. Indeed, efforts to reduce multiple conception have demanded a great deal of recent attention (Bronson et al., 1997Go; Coetsier and Dhont, 1998Go; Roest, 1998Go). Ideally, the approach should be individualized; however, there has been a lack of useful up-to-date data associated with frozen embryo transfer. The use of a prognostic table to show the risk of an adverse event has been documented in a number of medical fields (Anderson et al., 1991Go), including cardiology (Jackson, 1999Go). However, to date, this approach has not been applied in reproductive medicine. Using the current data, a similar table was constructed to document the expectation of pregnancy and importantly the risk of multiple conception.

Using the factors that were shown to influence the implantation rate in frozen embryo transfer, women could be stratified into five groups depending on their estimated chance of pregnancy per frozen embryo transfer cycle.

In Table IIIGo, the chance of pregnancy following one-, two- or three-embryo transfer is shown. The risk of multiple implantation was documented as well as the chance of pregnancy. If a single frozen–thawed embryo was transferred, the pregnancy rate would be between 10 and 14% in younger patients (<40 years old) who had conceived on their fresh cycle. No multiple implantation was recorded. For other subjects with a single embryo transferred the expectation of pregnancy would be much lower, <10%. If two embryos were transferred the expectation of pregnancy could be >20% in younger women who had achieved pregnancy in the fresh cycle and their risk of multiple conception was 15–29%. With three embryos transferred, the pregnancy chance would not be higher than two-embryo transfer in the majority of subjects but the risk of multiple conception would be increased. Using a prognostic table similar to that described here several factors could be considered before frozen embryo transfer regarding the chance of pregnancy and risk of multiple conception. Such easier-to-understand summarized information would allow improved patient decision making.


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Table III. The prognosis of pregnancy and multiple conception following single, two and three embryo transfer (ET) based on maternal age, aetiology of infertility and the outcome of the previous fresh embryo transfer cycle. Data are percentages
 
One limitation of the current study is that no precise account was able to be taken of embryo quality, which has been shown to be important in both fresh or frozen embryo transfer (Salat Baroux et al., 1996; Van den Abbeel et al., 1997Go). However all embryos transferred in the present study had at least 50% surviving blastomeres which indicated a level of quality assessment.

In conclusion, female age, the aetiology of infertility and the outcome of the fresh cycle are the most important factors so far analysed that influence the implantation rate following frozen embryo transfer. Based on our data, it has been possible to construct a prognostic table that may assist in determining the optimal number of embryos to replace in frozen embryo transfer to provide better individualized counselling and secure an optimal expectation of pregnancy while reducing the risk of multiple conception.


    Notes
 
1 To whom correspondence should be addressed. E-mail: jim.wang{at}adelaide.edu.au Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on March 28, 2001; accepted on June 28, 2001.