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Does it really matter how far from the fundus embryos are transferred?
European Journal of Obstetrics & Gynecology and Reproductive Biology 162 (2012) 62–66
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Does it really matter how far from the fundus embryos are transferred? Peter Kovacs a,*, Attila Sajgo a, Ferenc Rarosi b, Steven G Kaali a a b
Kaali Institute IVF Center, Budapest, Hungary Department of Medical Physics and Informatics, Bolyai Institute, University of Szeged, Hungary
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 October 2011 Received in revised form 18 January 2012 Accepted 20 February 2012
Objective: Embryo transfer (ET) is an important last step during the process of IVF. Over the years much has been learned about the importance of the details of this procedure including the potential impact of transfer depth on outcome. The objective of our study was to evaluate whether transfer depth assessed by air bubble location after ET is associated with clinical outcome. Study design: Retrospective analysis of the association between transfer distance from the fundus (assessed by air bubble location after ET) and IVF outcome based on cycles (N = 409) of patients with good prognosis undergoing infertility treatment for various indications. Treatments followed standard stimulation, IVF-ICSI and ET procedures. The distance of the air bubble as a surrogate marker of embryo location after transfer was measured and was correlated with implantation (IR) and pregnancy rates (PR) after day 3 and day 5 ET. Univariate comparisons were performed by nonparametric methods and multiple logistic regression was used to further evaluate the association between pregnancy rate and those factors that might influence outcome. Results: The distance of the bubble was similar in those cycles that led to a pregnancy and those that did not (6.7 vs 6.5 mm; p = 0.48) and PR were comparable when the transfer was in the upper or middle third of the cavity. The IR did not differ when embryos were transferred into the upper, middle or lower third of the uterine cavity. Outcome was analyzed separately based on the day of transfer (cleavage vs blastocyst stage) and the IR did not differ based on the location of the transfer. Conclusions: Transfer depth does not affect implantation and pregnancy rates when the ET is in the middle or upper third of the uterus. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Embryo transfer depth Air bubble Implantation rate Pregnancy rate Blastocyst transfer
1. Introduction There are several critical steps during the process of in vitro fertilization (IVF). One of them is the embryo transfer (ET), as all the preparations during IVF can be ruined by a poorly executed procedure. A recent review found that the use of trial transfer, soft catheters, removal of cervical mucus prior to ET, atraumatic transfer, ultrasound (US) guidance and transfer to the mid- to lower-midportion of the uterus were predictors of successful ET [1]. Studies evaluating the ideal location for ET have reported controversial results, however. Some suggested that ET into the upper cavity resulted in superior outcome, others favored transfer into the mid to lower half of the cavity and yet others found no association [2–8]. As the embryo itself cannot be visualized by US, most studies used the location of the air bubble deposited with the embryo at
* Corresponding author at: Kaali Institute IVF Center, Istenhegyi ut 54 a, 1125 Budapest, Hungary. Tel.: +36 1 202 2802; fax: +36 1 214 6050. E-mail address: [email protected] (P. Kovacs). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2012.02.018
the time of ET as a surrogate marker for embryo location. It has been reported that the bubble does not move after the ET [9] and that the majority of the embryos implant where they are transferred [10]. Many centers routinely use US instead of ‘clinical touch’ guidance for ideal embryo placement. A recent Cochrane review showed a significantly higher ongoing pregnancy rate (PR) with US guidance but found no significant benefit when live birth rates were evaluated [11]. We do not use US guidance for ET and before changing the practice we decided to look at the impact of ET location on clinical outcome. The aim of our study was to assess whether embryo placement affects IVF results. Since uterine contractility changes from day 3 ET to day 5 ET, a separate analysis taking the day of ET into account was also performed. 2. Materials and methods All fresh non-donor egg IVF cycles that ended with ET undertaken at the Kaali Institute IVF Center in Budapest, Hungary in 2009 were considered for the analysis. In the final dataset only good prognosis cycles (age 38 years, day 3FSH 12 IU/l, 1st or 2nd IVF cycle, transfer of at least one good quality embryo) were
P. Kovacs et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 162 (2012) 62–66
included. Due to the retrospective nature of the study, IRB approval was not required according to the institutional protocol. Standard protocols for controlled ovarian hyperstimulation (COH) were employed. Ovarian suppression strategies either utilized gonadotropin-releasing hormone agonist (GnRHa, Buserelin, Suprefact, Aventis; luteal long or follicular short protocol) or flexible GnRH antagonist (cetrorelix 0.25 mg, Cetrotide, Merck Serono) [12]. Recombinant FSH (Gonal F, Merck Serono), human menopausal gonadotropin (hMG, Merional, IBSA), hpHMG (Menopur, Ferring) or their combination were used for COH. Human chorionic gonadotropin (hCG, 250 mg rHCG, Ovitrelle, Merck Serono) was used 35–36 h prior to the transvaginal retrieval. Fertilization was achieved using IVF or intracytoplasmic sperm injection (ICSI) [13]. Embryos were cultured in G1.5 culture medium (Vitrolife, Scandinavia) until the transfer. For blastocyst stage transfer, embryos were transferred into Vitrolife G2.5 (Vitrolife, Scandinavia) media on day 4. Embryo quality was assessed on the day of transfer. Cleavage-stage embryos were scored based on the blastomere number and fragmentation (>6cells and <20% fragmentation indicated good quality) [14]. Blastocysts were assessed based on the quality of the inner cell mass, outer cell layer and the coelomic cavity [14,15]. The number of embryos transferred was decided based on the age, order of treatment cycle and embryo availability and was discussed with the patient. Our transfer policies follow international guidelines [16]. ET was performed on either day 3 or day 5 using Wallace catheters. The patient was placed in the dorsal lithotomy position. Full bladder was not required. Prior to ET, endometrial thickness (EMT) and length of the uterine cavity and cervix were measured on US. The length of the uterine cavity was measured by setting the calipers at the myometrial–endometrial interface at the fundus and at the internal cervical os. Cervical length was measured by setting the calipers at the internal os and external os. The sum of these distances guided ET depth and the aim was to transfer in the upper- to mid-third of the cavity. The transfer catheter was loaded by bracketing a small volume of culture medium (15–30 ml) containing the embryo(s) between two air bubbles. A bivalve speculum was used to visualize the cervix, which was first cleaned using sterile saline. An empty catheter was advanced to the internal cervical os, then the inner catheter was removed and a second inner part loaded with the embryo(s) was threaded through. An insulin syringe was used to gently push the embryo(s) out before the catheter was slowly withdrawn. At the completion of the transfer the embryologist flushed the catheter to check for retained embryo(s). Finally an US was performed to document the position of the air by bubble (distance from fundus and whether upper-, mid- or lower-third of the cavity) by photographing it for
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the medical records. The distance of the upper edge of the bubble from the fundus was measured. The luteal phase was supported with vaginal micronized progesterone (3200 mg Utrogestan, Lab Besius Int., France). Two weeks after ET, cycle outcome was determined by assessing serum bHCG; level >10 IU/l was considered as consistent with a biochemical pregnancy. Clinical pregnancy (CP) was defined as evidence of an intrauterine gestation sac on US. Implantation rate (IR) was calculated (#gestational sacs/#ET 100). A pregnancy was considered ongoing when fetal heartbeat was noted around week 8 and the patient was discharged into the care of the primary obstetrician. Data were collected for patient characteristics (age, ovarian reserve as reflected by baseline FSH, first vs repeat ART cycle), COH parameters (stimulation protocol, gonadotropin dose, number of eggs retrieved, IVF vs ICSI fertilization, number of embryos transferred, number of good quality embryos transferred, day of ET [D3 vs D5], EMT on day of ET, bubble distance from fundus) and outcome of IVF cycles (IR, PR). Univariate comparisons were performed by nonparametric methods Mann–Whitney U test, Kruskal–Wallis test or Chi-square test for continuous or categorical variables, respectively. A multiple logistic regression was used to further evaluate the association between PR and those factors that might influence outcome. The independent factors studied were dose of gonadotropin, number of embryos transferred, proportion of good quality embryos transferred, EMT and distance of the air bubble from the fundus. A p-value <0.05 was considered to be statistically significant. 3. Results Data based on 409 cycles were included in the analysis. The PR in the whole study population was 44.0% (180/409), while the ongoing PR was 36.9% (151/409). The baseline characteristics of the patients who achieved an ongoing pregnancy, those who did not and for the entire study population are shown in Table 1. Age, baseline FSH and the number of embryos transferred were comparable but those with an ongoing pregnancy used on average 200 IU less gonadotropins. The order of the treatment cycle, the use of different stimulation protocols and the proportion of IVF vs ICSI fertilization did not differ between cycles with or without ongoing pregnancy (data not shown). The endometrium in cycles with ongoing pregnancy was 0.6 mm thicker on the day of ET (11.5 vs 10.9 mm; p = 0.0002). However, the mean distance of the air bubble from the fundus after ET was similar for both groups (6.7 vs 6.5 mm; p = 0.48). In 285 cases ET was into the upper third, in 119 cases into the middle third and despite efforts to transfer into the mid to upper
Table 1 Baseline and stimulation parameters in cycles that resulted in ongoing pregnancy, those that did not and for the entire study population. Variable*
Age (yrs) CD3 FSH (IU/l) Gonadotropin dose (IU) # of oocytes # of embryos # of embryos transferred # of good quality embryos transferred Endometrial thickness (mm) Bubble distance from fundus (mm) * **
All cycles
31.7 2.8 6.8 2.0 1649 677 10.1 5.9 6.4 3.6 2.1 0.5 1.9 0.5 11.1 1.4 6.5 2.5
Variables are reported as value standard deviation (SD). Difference between cycles resulting in ongoing pregnancy and those that did not.
p value**
Ongoing pregnancy Yes
No
(N = 151)
(N = 258)
32.1 2.5 6.8 1.8 1523 528 10.5 4.8 6.8 3.5 2.0 0.4 1.9 0.5 11.4 1.5 6.7 2.5
31.5 2.9 6.7 2.1 1723 741 9.9 6.5 6.2 3.7 2.1 0.5 1.9 0.5 10.9 1.4 6.5 2.5
0.085 0.762 0.043 0.064 0.056 0.327 0.539 0.0002 0.485
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P. Kovacs et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 162 (2012) 62–66
Table 2 The effect of transfer location based on day of ET. Values are expressed mean SD and as percentages. None of these differences reached statistical significance using non parametric analysis.
Distance from fundus (mm) Number and percent of cycles when ET into upper 1/3 of cavity Number and percent of cycles when ET into middle 1/3 of cavity
Day 3 ET into mid or upper third of cavity (N = 253)
Day 5 ET into mid or upper third of cavity (N = 151)
Ongoing pregnancy (N = 81)
No pregnancy (N = 172)
Ongoing pregnancy (N = 69)
No pregnancy (N = 82)
6.6 2.6 55 (67.9%)
6.3 2.4 127 (73.8%)
6.7 2.3 47 (68.1%)
6.8 2.7 56 (68.3%)
26 (32.1%)
45 (26.2%)
22 (31.9%)
26 (31.7%)
third in 5 cases into the lower third. The number of cases when the ET was into the lower third is very low and therefore meaningful comparisons cannot be made with it. An IR was calculated for these cycles as well but the data are not reported in the tables as the comparisons could be misleading due to the low number of cases. The absolute number of pregnancies was higher when the embryos were transferred into the upper third due to more cases with ET to this region. However the proportion of successful and unsuccessful cycles when the embryo was transferred into the upper third (68% of ongoing pregnancies vs 72% of cycles without pregnancy) or middle third of the cavity (32% of ongoing pregnancies vs 28% of cycles without pregnancy) was similar (p = 0.59). To account for any differences in uterine contractility and/or receptivity between cleavage-stage and blastocyst-stage ET the outcome was analyzed separately based on the developmental stage of the embryos at ET (Table 2). The mean distance of the bubble from the fundus was similar in cycles with different clinical outcomes on day 3 (ongoing pregnancy: 6.6 mm 2.6 SD vs no ongoing pregnancy: 6.3 mm 2.4 mm, p = NS) and on day 5 (ongoing pregnancy: 6.7 mm 2.3 SD vs no ongoing pregnancy: 6.8 mm 2.7 mm, p = NS) as well (Fig. 1). The overall IR in the study population was 25.4%. The IR did not differ when embryos were transferred into the upper, middle or lower third of the cavity (25.7% vs 24.7% vs 25.0%, p = NS). The IR however was significantly higher with the transfer of blastocysts (day 5: 33.3% vs day 3: 20.8%; p = 0.0006). However, when analyzed separately based on the day of transfer (cleavage- vs
Fig. 1. Mean distance of the air bubble from the fundus in successful and nonsuccessful cycles based on the day of ET.
blastocyst-stage) the IR did not differ based on the location of the transfer (upper, middle or lower third). We also compared the patient and cycle characteristics based on the transfer location (upper vs middle third) to see if any of those could affect the outcome. None of the patient (age, baseline FSH, estradiol, cycle number) or cycle characteristics (gonadotropin dose, number of eggs/embryos/transferred embryos, endometrial thickness, day 3 vs day 5 ET) differed significantly (Table 3). Logistic regression analysis controlling for the dose of gonadotropin, number of embryos transferred, the proportion of good quality embryos and EMT still failed to find a significant impact of the distance of the air bubble from the fundus on PR and ongoing PR (OR: 1.028; 95% CI: (0.951–1.111, p value: 0.483). 4. Comments Our analysis based on over 400 cycles did not find an association between ET depth (measured by the distance of the air bubble from the fundus) and treatment outcome. We included cycles with good prognosis and in which good quality embryos were transferred to minimize the impact of patient and embryonic factors on outcome. The majority of the transfers were in the upper or middle third of the cavity. Transfer into the lower portion occurred in only a small percentage of the cycles. We looked at day 3 and day 5 ET outcomes separately but found no impact of transfer location on IR or ongoing PR in either case. ET is the last step of IVF treatment. If poorly executed, all the efforts put into the treatment up until the ET can be ruined within minutes. A recent reviewed suggested that the use of soft catheters, the removal of cervical mucus, gentle technique and the use of USguidance all increase the success rate [1]. The same review also suggested that transfer to the mid- to lower-mid-portion of the uterus is a predictor of successful ET [1]. As the embryo cannot be visualized by US most studies use the location of the air bubble deposited with the embryo at the time of ET as a surrogate marker for embryo location. This assumes that the embryo does not move from its place of deposition. The uterus is known to have contractions that change in intensity and direction during the spontaneous menstrual cycle as well as in stimulated cycles. During the stimulation phase of an IVF cycle contractions are mainly cervico-fundal in direction with peak intensity on the day of hCG injection. Contraction intensity decreases thereafter [17]. Fanchin et al. noted that PR and IR decreased with increasing uterine contraction frequency [18]. In a separate study, they observed progressively decreasing uterine contractility after the retrieval, with minimal contractility by day 7 at the time of blastocyst ET [19]. Therefore, if we want to use the air bubble as a marker for embryo placement it is important to study what happens to the air bubble following ET. Woolcott and Stanger found that in 94% of cases the air bubble did not move from its immediate after-ET position [9]. Baba et al. using three-dimensional ultrasound found
P. Kovacs et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 162 (2012) 62–66
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Table 3 Comparison of patient and cycle characteristics based on the transfer location (upper vs middle 1/3). None of these differences reached statistical significance using non parametric analysis. Parameter (mean SD)
ET into upper 1/3 of the cavity (N = 285)
ET into middle 1/3 of the cavity (N = 119)
p-Value
Age (yrs) Cycle day 3 FSH (IU/l) Cycle day 3 estradiol (pmol/l) Order of the treatment cycle Gonadotropin dose (IU) # of oocytes # of embryos # of transferred embryos # and proportion of day 3 transfers (%) # and proportion of day 5 transfers (%) Endometrial thickness (mm)
31.7 2.6 6.8 2.1 146 78 1.7 0.7 1661 703 9.9 6.4 6.4 3.8 2.1 0.5 182 (63.8%) 103 (36.1%) 11.0 1.4
31.7 3.1 6.6 1.7 136 75 1.8 0.7 1622 616 10.5 4.7 6.6 3.3 2.1 0.4 71 (59.6%) 48 (40.3%) 11.3 1.4
0.98 0.90 0.24 0.46 0.38 0.27 0.39 0.63 0.42 0.42 0.27
that 81% of the pregnancies implanted where the embryos were transferred [10]. The literature is not uniform regarding this issue as Confino et al. reported random bubble movement following ET, with no movement in only 11% of the cases [20]. Lacking a better marker, most studies use the position of the air bubble as an indicator of embryo location following ET. The literature is rather divided on the issue of the best location for ET. Lambers et al. found significantly higher PR when the bubble was closer to the fundus. PR almost doubled when the ET was in the upper half of the cavity compared to the lower half (43% vs 24.4%) [2]. Friedman et al. reported improved PR when the ET was close to the fundus (<10 mm) with blastocyst stage ET [3]. Tiras et al. reported the highest PR when the embryos were transferred between 10 and 20 mm from the fundus [21]. Pacchiarotti et al. found a significantly higher PR with ET distance 10–15 mm from the fundus when compared to less than 10 mm (27.7% vs 14%, respectively) [6]. Pope et al. reported an increase of 11% in the PR for each mm of embryo deposition distance away from the fundus at the time of ET [4]. Coroleu et al. randomized patients to ET at 10, 15 and 20 mm away from the fundus. They reported a significant increase in IR and PR when ET was at 15 and 20 mm compared to the group where it was closer to the fundus [7]. Frankfurter et al., in a prospective cohort study, found improved IR and PR with transfer into the middle to lower uterine segment [5]. Since uterine contractility progressively decreases after retrieval, cycle outcome needs to be analyzed separately for cleavage-stage vs blastocyst-stage transfers. The only study that looked at this association was the study by Friedman et al. and they found better results with ET closer to the fundus [3]. We could not confirm this finding because even when the day 5 ETs were analyzed separately, we still did not find an association between air bubble location and clinical PR. A retrospective analysis has its weaknesses. Not all factors affecting IR/PR can be controlled for. We therefore tried to choose a homogeneous population to minimize the effect of patient-related factors. In addition we included only cycles where at least one good quality embryo was transferred. One of the weaknesses of this study is that we could not adequately study the impact of transfer into the lower-third as it only occurred in a few percent of the cycles. Therefore we could only analyze the association between absolute distance from the fundus and outcome, and the impact of transfer into the upper- or middle-third of the cavity. According to our analysis when transfer depth is treated as a continuous variable in a logistic regression model it is not a significant predictor for ongoing pregnancy (p = 0.483). Based on our data the coefficient of transfer depth on the odds ratio of clinical pregnancy was extremely small, 1.028, and we believe that this difference is clinically irrelevant. We have performed a power analysis for the logistic regression model considering an odds ratio of 1.4 (for clinical pregnancy) as a clinically relevant effect size.
Average probability of ongoing pregnancy was 0.37 in our study and this odds ratio means that we would be able to detect an increase in ongoing pregnancy to 0.45 using our sample size with 90% power [22]. Using the effect size based on our data we would need to collect information on over 6000 cycles to show a significant impact of embryo transfer depth on clinical pregnancy rate with a 90% power. By including this many cycles, however, even small, clinically meaningless differences may come up as statistically significant. Such a small difference however is unlikely to affect the clinical practice. The use of US during ET complicates the whole process. Its use requires the presence of an additional person performing the scan and sometimes it is time-consuming to localize the endometrial stripe. While the literature is divided on the issue of ideal location of ET there seems to be sufficient support for US guidance during ET [1]. In order to allow adequate visualization of the endometrial stripe, the patient is required to have a full bladder. This may be associated with additional benefits as the full bladder straightens the cervico-uterine angle and makes the transfer easier. It is possible that this, and not the sonographic guidance of the embryo deposition itself, leads to the improved PR. US guidance offers benefits in certain cases (e.g. tortuous cervix). US guidance could also help to avoid touching the fundus and inducing contractions that may expel the embryo. With adequate visualization one can avoid transfer into the fallopian tube. US guidance also helps with training physicians to perform transfers. Since the literature is rather split on the issue of ideal location we question whether US guidance should be used routinely. It certainly should be reserved for the more challenging cases. Acknowledgements To authors wish to thank Dr. Krisztina Boda for her comments regarding the statistical analysis. We also thank Mrs. Erika Oravetz and Ms. Bernadett Buzas for their help during the data collection. References [1] Mains L, Van Voorhis BJ. Optimizing the technique of embryo transfer. Fertil Steril 2010;94:785–90. [2] Lambers MJ, Dogan E, Lens JW, Schats R, Hompes PG. The position of transferred air bubbles after embryo transfer is related to pregnancy rate. Fertil Steril 2007;88:68–73. [3] Friedman BE, Lathi RB, Henne MB, Fisher SL, Milki AA. The effect of air bubble position after blastocyst transfer on pregnancy rates in IVF cycles. Fertil Steril 2011;95:944–7. [4] Pope CS, Cook EK, Arny M, Novak A, Grow DR. Influence of embryo transfer depth on in vitro fertilization and embryo transfer outcomes. Fertil Steril 2004;81:51–8. [5] Frankfurter D, Trimarchi JB, Silva CP, Keefe DL. Middle to lower uterine segment embryo transfer improves implantation and pregnancy rates compared with fundal embryo transfer. Fertil Steril 2004;81:1273–7. [6] Pacchiarotti A, Mohamed MA, Micara G, et al. The impact of the depth of embryo replacement on IVF outcome. J Assist Reprod Genet 2007;24:189–93.
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[7] Coroleu B, Barri PN, Carreras O, et al. The influence of the depth of embryo replacement into the uterine cavity on implantation rates after IVF: a controlled, ultrasound-guided study. Hum Reprod 2002;17:341–6. [8] Franco Jr JG, Martins AM, Baruffi RL, et al. Best site for embryo transfer: the upper or lower half of endometrial cavity. Hum Reprod 2004;19:1785–90. [9] Woolcott R, Stanger J. Ultrasound tracking of the movement of embryoassociated air bubbles on standing after transfer. Hum Reprod 1998;13:2107–9. [10] Baba K, Ishihara O, Hayashi N, Saitoh M, Taya J, Kinoshita K. Where does the embryo implant after embryo transfer in humans. Fertil Steril 2000;73:123–5. [11] Brown J, Buckingham K, Abou-Setta AM, Buckett W. Ultrasound versus ‘clinical touch’ for catheter guidance during embryo transfer in women. Cochrane Database Syst Rev 2010;(January (1)):CD006107. [12] Mochtar MH. Dutch Ganirelix Study Group. The effect of an individualized GnRH antagonist protocol on folliculogenesis in IVF/ICSI. Hum Reprod 2004;19:1713–8. [13] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8. [14] Baczkowski T, Kurzawa R, Głabowski W. Methods of embryo scoring in in vitro fertilization. Reprod Biol 2004;4:5–22.
[15] Gardner DK, Phil D, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards single blastocyst transfer. Fertil Steril 2000;73:1155–8. [16] Practice Committee of the American Society for Reproductive Medicine. Practice Committee of the Society for Assisted Reproductive Technology. Guidelines on number of embryos transferred. Fertil Steril 2009;92:1518–9. [17] Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Uterine junctional zone contractions during assisted reproduction cycles. Hum Reprod Update 1998;4:440–5. [18] Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in vitro fertilization. Hum Reprod 1998;13:1968–74. [19] Fanchin R, Ayoubi JM, Righini C, Olivennes F, Scho¨nauer LM, Frydman R. Uterine contractility decreases at the time of blastocyst transfers. Hum Reprod 2001;16:1115–9. [20] Confino E, Zhang J, Risquez F. Air bubble migration is a random event post embryo transfer. J Assist Reprod Genet 2007;24:223–6. [21] Tiras B, Polat M, Korucuoglu U, Zeyneloglu HB, Yarali H. Impact of embryo replacement depth on in vitro fertilization and embryo transfer outcomes. Fertil Steril 2010;94:1341–5. [22] Hsieh FY. Sample size tables for logistic regression. Stat Med 1989;8:795–802.
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Does it really matter how far from the fundus embryos are transferred? Peter Kovacs a,*, Attila Sajgo a, Ferenc Rarosi b, Steven G Kaali a a b
Kaali Institute IVF Center, Budapest, Hungary Department of Medical Physics and Informatics, Bolyai Institute, University of Szeged, Hungary
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 October 2011 Received in revised form 18 January 2012 Accepted 20 February 2012
Objective: Embryo transfer (ET) is an important last step during the process of IVF. Over the years much has been learned about the importance of the details of this procedure including the potential impact of transfer depth on outcome. The objective of our study was to evaluate whether transfer depth assessed by air bubble location after ET is associated with clinical outcome. Study design: Retrospective analysis of the association between transfer distance from the fundus (assessed by air bubble location after ET) and IVF outcome based on cycles (N = 409) of patients with good prognosis undergoing infertility treatment for various indications. Treatments followed standard stimulation, IVF-ICSI and ET procedures. The distance of the air bubble as a surrogate marker of embryo location after transfer was measured and was correlated with implantation (IR) and pregnancy rates (PR) after day 3 and day 5 ET. Univariate comparisons were performed by nonparametric methods and multiple logistic regression was used to further evaluate the association between pregnancy rate and those factors that might influence outcome. Results: The distance of the bubble was similar in those cycles that led to a pregnancy and those that did not (6.7 vs 6.5 mm; p = 0.48) and PR were comparable when the transfer was in the upper or middle third of the cavity. The IR did not differ when embryos were transferred into the upper, middle or lower third of the uterine cavity. Outcome was analyzed separately based on the day of transfer (cleavage vs blastocyst stage) and the IR did not differ based on the location of the transfer. Conclusions: Transfer depth does not affect implantation and pregnancy rates when the ET is in the middle or upper third of the uterus. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Embryo transfer depth Air bubble Implantation rate Pregnancy rate Blastocyst transfer
1. Introduction There are several critical steps during the process of in vitro fertilization (IVF). One of them is the embryo transfer (ET), as all the preparations during IVF can be ruined by a poorly executed procedure. A recent review found that the use of trial transfer, soft catheters, removal of cervical mucus prior to ET, atraumatic transfer, ultrasound (US) guidance and transfer to the mid- to lower-midportion of the uterus were predictors of successful ET [1]. Studies evaluating the ideal location for ET have reported controversial results, however. Some suggested that ET into the upper cavity resulted in superior outcome, others favored transfer into the mid to lower half of the cavity and yet others found no association [2–8]. As the embryo itself cannot be visualized by US, most studies used the location of the air bubble deposited with the embryo at
* Corresponding author at: Kaali Institute IVF Center, Istenhegyi ut 54 a, 1125 Budapest, Hungary. Tel.: +36 1 202 2802; fax: +36 1 214 6050. E-mail address: [email protected] (P. Kovacs). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2012.02.018
the time of ET as a surrogate marker for embryo location. It has been reported that the bubble does not move after the ET [9] and that the majority of the embryos implant where they are transferred [10]. Many centers routinely use US instead of ‘clinical touch’ guidance for ideal embryo placement. A recent Cochrane review showed a significantly higher ongoing pregnancy rate (PR) with US guidance but found no significant benefit when live birth rates were evaluated [11]. We do not use US guidance for ET and before changing the practice we decided to look at the impact of ET location on clinical outcome. The aim of our study was to assess whether embryo placement affects IVF results. Since uterine contractility changes from day 3 ET to day 5 ET, a separate analysis taking the day of ET into account was also performed. 2. Materials and methods All fresh non-donor egg IVF cycles that ended with ET undertaken at the Kaali Institute IVF Center in Budapest, Hungary in 2009 were considered for the analysis. In the final dataset only good prognosis cycles (age 38 years, day 3FSH 12 IU/l, 1st or 2nd IVF cycle, transfer of at least one good quality embryo) were
P. Kovacs et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 162 (2012) 62–66
included. Due to the retrospective nature of the study, IRB approval was not required according to the institutional protocol. Standard protocols for controlled ovarian hyperstimulation (COH) were employed. Ovarian suppression strategies either utilized gonadotropin-releasing hormone agonist (GnRHa, Buserelin, Suprefact, Aventis; luteal long or follicular short protocol) or flexible GnRH antagonist (cetrorelix 0.25 mg, Cetrotide, Merck Serono) [12]. Recombinant FSH (Gonal F, Merck Serono), human menopausal gonadotropin (hMG, Merional, IBSA), hpHMG (Menopur, Ferring) or their combination were used for COH. Human chorionic gonadotropin (hCG, 250 mg rHCG, Ovitrelle, Merck Serono) was used 35–36 h prior to the transvaginal retrieval. Fertilization was achieved using IVF or intracytoplasmic sperm injection (ICSI) [13]. Embryos were cultured in G1.5 culture medium (Vitrolife, Scandinavia) until the transfer. For blastocyst stage transfer, embryos were transferred into Vitrolife G2.5 (Vitrolife, Scandinavia) media on day 4. Embryo quality was assessed on the day of transfer. Cleavage-stage embryos were scored based on the blastomere number and fragmentation (>6cells and <20% fragmentation indicated good quality) [14]. Blastocysts were assessed based on the quality of the inner cell mass, outer cell layer and the coelomic cavity [14,15]. The number of embryos transferred was decided based on the age, order of treatment cycle and embryo availability and was discussed with the patient. Our transfer policies follow international guidelines [16]. ET was performed on either day 3 or day 5 using Wallace catheters. The patient was placed in the dorsal lithotomy position. Full bladder was not required. Prior to ET, endometrial thickness (EMT) and length of the uterine cavity and cervix were measured on US. The length of the uterine cavity was measured by setting the calipers at the myometrial–endometrial interface at the fundus and at the internal cervical os. Cervical length was measured by setting the calipers at the internal os and external os. The sum of these distances guided ET depth and the aim was to transfer in the upper- to mid-third of the cavity. The transfer catheter was loaded by bracketing a small volume of culture medium (15–30 ml) containing the embryo(s) between two air bubbles. A bivalve speculum was used to visualize the cervix, which was first cleaned using sterile saline. An empty catheter was advanced to the internal cervical os, then the inner catheter was removed and a second inner part loaded with the embryo(s) was threaded through. An insulin syringe was used to gently push the embryo(s) out before the catheter was slowly withdrawn. At the completion of the transfer the embryologist flushed the catheter to check for retained embryo(s). Finally an US was performed to document the position of the air by bubble (distance from fundus and whether upper-, mid- or lower-third of the cavity) by photographing it for
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the medical records. The distance of the upper edge of the bubble from the fundus was measured. The luteal phase was supported with vaginal micronized progesterone (3200 mg Utrogestan, Lab Besius Int., France). Two weeks after ET, cycle outcome was determined by assessing serum bHCG; level >10 IU/l was considered as consistent with a biochemical pregnancy. Clinical pregnancy (CP) was defined as evidence of an intrauterine gestation sac on US. Implantation rate (IR) was calculated (#gestational sacs/#ET 100). A pregnancy was considered ongoing when fetal heartbeat was noted around week 8 and the patient was discharged into the care of the primary obstetrician. Data were collected for patient characteristics (age, ovarian reserve as reflected by baseline FSH, first vs repeat ART cycle), COH parameters (stimulation protocol, gonadotropin dose, number of eggs retrieved, IVF vs ICSI fertilization, number of embryos transferred, number of good quality embryos transferred, day of ET [D3 vs D5], EMT on day of ET, bubble distance from fundus) and outcome of IVF cycles (IR, PR). Univariate comparisons were performed by nonparametric methods Mann–Whitney U test, Kruskal–Wallis test or Chi-square test for continuous or categorical variables, respectively. A multiple logistic regression was used to further evaluate the association between PR and those factors that might influence outcome. The independent factors studied were dose of gonadotropin, number of embryos transferred, proportion of good quality embryos transferred, EMT and distance of the air bubble from the fundus. A p-value <0.05 was considered to be statistically significant. 3. Results Data based on 409 cycles were included in the analysis. The PR in the whole study population was 44.0% (180/409), while the ongoing PR was 36.9% (151/409). The baseline characteristics of the patients who achieved an ongoing pregnancy, those who did not and for the entire study population are shown in Table 1. Age, baseline FSH and the number of embryos transferred were comparable but those with an ongoing pregnancy used on average 200 IU less gonadotropins. The order of the treatment cycle, the use of different stimulation protocols and the proportion of IVF vs ICSI fertilization did not differ between cycles with or without ongoing pregnancy (data not shown). The endometrium in cycles with ongoing pregnancy was 0.6 mm thicker on the day of ET (11.5 vs 10.9 mm; p = 0.0002). However, the mean distance of the air bubble from the fundus after ET was similar for both groups (6.7 vs 6.5 mm; p = 0.48). In 285 cases ET was into the upper third, in 119 cases into the middle third and despite efforts to transfer into the mid to upper
Table 1 Baseline and stimulation parameters in cycles that resulted in ongoing pregnancy, those that did not and for the entire study population. Variable*
Age (yrs) CD3 FSH (IU/l) Gonadotropin dose (IU) # of oocytes # of embryos # of embryos transferred # of good quality embryos transferred Endometrial thickness (mm) Bubble distance from fundus (mm) * **
All cycles
31.7 2.8 6.8 2.0 1649 677 10.1 5.9 6.4 3.6 2.1 0.5 1.9 0.5 11.1 1.4 6.5 2.5
Variables are reported as value standard deviation (SD). Difference between cycles resulting in ongoing pregnancy and those that did not.
p value**
Ongoing pregnancy Yes
No
(N = 151)
(N = 258)
32.1 2.5 6.8 1.8 1523 528 10.5 4.8 6.8 3.5 2.0 0.4 1.9 0.5 11.4 1.5 6.7 2.5
31.5 2.9 6.7 2.1 1723 741 9.9 6.5 6.2 3.7 2.1 0.5 1.9 0.5 10.9 1.4 6.5 2.5
0.085 0.762 0.043 0.064 0.056 0.327 0.539 0.0002 0.485
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Table 2 The effect of transfer location based on day of ET. Values are expressed mean SD and as percentages. None of these differences reached statistical significance using non parametric analysis.
Distance from fundus (mm) Number and percent of cycles when ET into upper 1/3 of cavity Number and percent of cycles when ET into middle 1/3 of cavity
Day 3 ET into mid or upper third of cavity (N = 253)
Day 5 ET into mid or upper third of cavity (N = 151)
Ongoing pregnancy (N = 81)
No pregnancy (N = 172)
Ongoing pregnancy (N = 69)
No pregnancy (N = 82)
6.6 2.6 55 (67.9%)
6.3 2.4 127 (73.8%)
6.7 2.3 47 (68.1%)
6.8 2.7 56 (68.3%)
26 (32.1%)
45 (26.2%)
22 (31.9%)
26 (31.7%)
third in 5 cases into the lower third. The number of cases when the ET was into the lower third is very low and therefore meaningful comparisons cannot be made with it. An IR was calculated for these cycles as well but the data are not reported in the tables as the comparisons could be misleading due to the low number of cases. The absolute number of pregnancies was higher when the embryos were transferred into the upper third due to more cases with ET to this region. However the proportion of successful and unsuccessful cycles when the embryo was transferred into the upper third (68% of ongoing pregnancies vs 72% of cycles without pregnancy) or middle third of the cavity (32% of ongoing pregnancies vs 28% of cycles without pregnancy) was similar (p = 0.59). To account for any differences in uterine contractility and/or receptivity between cleavage-stage and blastocyst-stage ET the outcome was analyzed separately based on the developmental stage of the embryos at ET (Table 2). The mean distance of the bubble from the fundus was similar in cycles with different clinical outcomes on day 3 (ongoing pregnancy: 6.6 mm 2.6 SD vs no ongoing pregnancy: 6.3 mm 2.4 mm, p = NS) and on day 5 (ongoing pregnancy: 6.7 mm 2.3 SD vs no ongoing pregnancy: 6.8 mm 2.7 mm, p = NS) as well (Fig. 1). The overall IR in the study population was 25.4%. The IR did not differ when embryos were transferred into the upper, middle or lower third of the cavity (25.7% vs 24.7% vs 25.0%, p = NS). The IR however was significantly higher with the transfer of blastocysts (day 5: 33.3% vs day 3: 20.8%; p = 0.0006). However, when analyzed separately based on the day of transfer (cleavage- vs
Fig. 1. Mean distance of the air bubble from the fundus in successful and nonsuccessful cycles based on the day of ET.
blastocyst-stage) the IR did not differ based on the location of the transfer (upper, middle or lower third). We also compared the patient and cycle characteristics based on the transfer location (upper vs middle third) to see if any of those could affect the outcome. None of the patient (age, baseline FSH, estradiol, cycle number) or cycle characteristics (gonadotropin dose, number of eggs/embryos/transferred embryos, endometrial thickness, day 3 vs day 5 ET) differed significantly (Table 3). Logistic regression analysis controlling for the dose of gonadotropin, number of embryos transferred, the proportion of good quality embryos and EMT still failed to find a significant impact of the distance of the air bubble from the fundus on PR and ongoing PR (OR: 1.028; 95% CI: (0.951–1.111, p value: 0.483). 4. Comments Our analysis based on over 400 cycles did not find an association between ET depth (measured by the distance of the air bubble from the fundus) and treatment outcome. We included cycles with good prognosis and in which good quality embryos were transferred to minimize the impact of patient and embryonic factors on outcome. The majority of the transfers were in the upper or middle third of the cavity. Transfer into the lower portion occurred in only a small percentage of the cycles. We looked at day 3 and day 5 ET outcomes separately but found no impact of transfer location on IR or ongoing PR in either case. ET is the last step of IVF treatment. If poorly executed, all the efforts put into the treatment up until the ET can be ruined within minutes. A recent reviewed suggested that the use of soft catheters, the removal of cervical mucus, gentle technique and the use of USguidance all increase the success rate [1]. The same review also suggested that transfer to the mid- to lower-mid-portion of the uterus is a predictor of successful ET [1]. As the embryo cannot be visualized by US most studies use the location of the air bubble deposited with the embryo at the time of ET as a surrogate marker for embryo location. This assumes that the embryo does not move from its place of deposition. The uterus is known to have contractions that change in intensity and direction during the spontaneous menstrual cycle as well as in stimulated cycles. During the stimulation phase of an IVF cycle contractions are mainly cervico-fundal in direction with peak intensity on the day of hCG injection. Contraction intensity decreases thereafter [17]. Fanchin et al. noted that PR and IR decreased with increasing uterine contraction frequency [18]. In a separate study, they observed progressively decreasing uterine contractility after the retrieval, with minimal contractility by day 7 at the time of blastocyst ET [19]. Therefore, if we want to use the air bubble as a marker for embryo placement it is important to study what happens to the air bubble following ET. Woolcott and Stanger found that in 94% of cases the air bubble did not move from its immediate after-ET position [9]. Baba et al. using three-dimensional ultrasound found
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Table 3 Comparison of patient and cycle characteristics based on the transfer location (upper vs middle 1/3). None of these differences reached statistical significance using non parametric analysis. Parameter (mean SD)
ET into upper 1/3 of the cavity (N = 285)
ET into middle 1/3 of the cavity (N = 119)
p-Value
Age (yrs) Cycle day 3 FSH (IU/l) Cycle day 3 estradiol (pmol/l) Order of the treatment cycle Gonadotropin dose (IU) # of oocytes # of embryos # of transferred embryos # and proportion of day 3 transfers (%) # and proportion of day 5 transfers (%) Endometrial thickness (mm)
31.7 2.6 6.8 2.1 146 78 1.7 0.7 1661 703 9.9 6.4 6.4 3.8 2.1 0.5 182 (63.8%) 103 (36.1%) 11.0 1.4
31.7 3.1 6.6 1.7 136 75 1.8 0.7 1622 616 10.5 4.7 6.6 3.3 2.1 0.4 71 (59.6%) 48 (40.3%) 11.3 1.4
0.98 0.90 0.24 0.46 0.38 0.27 0.39 0.63 0.42 0.42 0.27
that 81% of the pregnancies implanted where the embryos were transferred [10]. The literature is not uniform regarding this issue as Confino et al. reported random bubble movement following ET, with no movement in only 11% of the cases [20]. Lacking a better marker, most studies use the position of the air bubble as an indicator of embryo location following ET. The literature is rather divided on the issue of the best location for ET. Lambers et al. found significantly higher PR when the bubble was closer to the fundus. PR almost doubled when the ET was in the upper half of the cavity compared to the lower half (43% vs 24.4%) [2]. Friedman et al. reported improved PR when the ET was close to the fundus (<10 mm) with blastocyst stage ET [3]. Tiras et al. reported the highest PR when the embryos were transferred between 10 and 20 mm from the fundus [21]. Pacchiarotti et al. found a significantly higher PR with ET distance 10–15 mm from the fundus when compared to less than 10 mm (27.7% vs 14%, respectively) [6]. Pope et al. reported an increase of 11% in the PR for each mm of embryo deposition distance away from the fundus at the time of ET [4]. Coroleu et al. randomized patients to ET at 10, 15 and 20 mm away from the fundus. They reported a significant increase in IR and PR when ET was at 15 and 20 mm compared to the group where it was closer to the fundus [7]. Frankfurter et al., in a prospective cohort study, found improved IR and PR with transfer into the middle to lower uterine segment [5]. Since uterine contractility progressively decreases after retrieval, cycle outcome needs to be analyzed separately for cleavage-stage vs blastocyst-stage transfers. The only study that looked at this association was the study by Friedman et al. and they found better results with ET closer to the fundus [3]. We could not confirm this finding because even when the day 5 ETs were analyzed separately, we still did not find an association between air bubble location and clinical PR. A retrospective analysis has its weaknesses. Not all factors affecting IR/PR can be controlled for. We therefore tried to choose a homogeneous population to minimize the effect of patient-related factors. In addition we included only cycles where at least one good quality embryo was transferred. One of the weaknesses of this study is that we could not adequately study the impact of transfer into the lower-third as it only occurred in a few percent of the cycles. Therefore we could only analyze the association between absolute distance from the fundus and outcome, and the impact of transfer into the upper- or middle-third of the cavity. According to our analysis when transfer depth is treated as a continuous variable in a logistic regression model it is not a significant predictor for ongoing pregnancy (p = 0.483). Based on our data the coefficient of transfer depth on the odds ratio of clinical pregnancy was extremely small, 1.028, and we believe that this difference is clinically irrelevant. We have performed a power analysis for the logistic regression model considering an odds ratio of 1.4 (for clinical pregnancy) as a clinically relevant effect size.
Average probability of ongoing pregnancy was 0.37 in our study and this odds ratio means that we would be able to detect an increase in ongoing pregnancy to 0.45 using our sample size with 90% power [22]. Using the effect size based on our data we would need to collect information on over 6000 cycles to show a significant impact of embryo transfer depth on clinical pregnancy rate with a 90% power. By including this many cycles, however, even small, clinically meaningless differences may come up as statistically significant. Such a small difference however is unlikely to affect the clinical practice. The use of US during ET complicates the whole process. Its use requires the presence of an additional person performing the scan and sometimes it is time-consuming to localize the endometrial stripe. While the literature is divided on the issue of ideal location of ET there seems to be sufficient support for US guidance during ET [1]. In order to allow adequate visualization of the endometrial stripe, the patient is required to have a full bladder. This may be associated with additional benefits as the full bladder straightens the cervico-uterine angle and makes the transfer easier. It is possible that this, and not the sonographic guidance of the embryo deposition itself, leads to the improved PR. US guidance offers benefits in certain cases (e.g. tortuous cervix). US guidance could also help to avoid touching the fundus and inducing contractions that may expel the embryo. With adequate visualization one can avoid transfer into the fallopian tube. US guidance also helps with training physicians to perform transfers. Since the literature is rather split on the issue of ideal location we question whether US guidance should be used routinely. It certainly should be reserved for the more challenging cases. Acknowledgements To authors wish to thank Dr. Krisztina Boda for her comments regarding the statistical analysis. We also thank Mrs. Erika Oravetz and Ms. Bernadett Buzas for their help during the data collection. References [1] Mains L, Van Voorhis BJ. Optimizing the technique of embryo transfer. Fertil Steril 2010;94:785–90. [2] Lambers MJ, Dogan E, Lens JW, Schats R, Hompes PG. The position of transferred air bubbles after embryo transfer is related to pregnancy rate. Fertil Steril 2007;88:68–73. [3] Friedman BE, Lathi RB, Henne MB, Fisher SL, Milki AA. The effect of air bubble position after blastocyst transfer on pregnancy rates in IVF cycles. Fertil Steril 2011;95:944–7. [4] Pope CS, Cook EK, Arny M, Novak A, Grow DR. Influence of embryo transfer depth on in vitro fertilization and embryo transfer outcomes. Fertil Steril 2004;81:51–8. [5] Frankfurter D, Trimarchi JB, Silva CP, Keefe DL. Middle to lower uterine segment embryo transfer improves implantation and pregnancy rates compared with fundal embryo transfer. Fertil Steril 2004;81:1273–7. [6] Pacchiarotti A, Mohamed MA, Micara G, et al. The impact of the depth of embryo replacement on IVF outcome. J Assist Reprod Genet 2007;24:189–93.
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