1 Service of Reproductive Medicine, Department of Obstetrics and Gynaecology, Institut Universitari Dexeus, Barcelona and 2 Institut Clinic of Gynaecology, Obstetrics and Neonatology, Faculty of MedicineUniversity of Barcelona, Hospital ClinicInstitut dInvestigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
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Abstract |
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Key words: embryo transfer/frozen-thawed embryos/implantation rates/IVF/ultrasound-guided embryo transfer
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Introduction |
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Improved success rates using ultrasound-guided embryo transfer may be related, at least in part, to the problem of uterine contractions. Uterine junctional zone contractions or endometrial wavelike movements have been reported in both natural and IVF cycles (Ijland et al., 1996; Lesny et al., 1998a
; Eytan et al., 2001
). Increased uterine contractility assessed by ultrasonography at the time of embryo transfer has been shown to be associated with reduced pregnancy and implantation rates in IVF cycles and, importantly, a direct relationship between the embryo transfer technique, uterine contractility and the relocation of embryos towards the cervix has been reported (Fanchin et al., 1998
; Lesny et al., 1998b
, 1999a
, Lesny et al., b
). Thus, atraumatic embryo transfers, with a soft catheter and without touching the uterine fundus, which have no effect on uterine junctional zone contractility and the replaced embryos, would improve pregnancy and implantation rates (Lesny et al., 1999a
,b
). Ultrasonographic guidance at the time of embryo transfer may have several potential advantages in this regard, such as facilitating the placement of soft catheters and avoiding contact with the uterine fundus and endometrial disruption (Schoolcraft et al., 2001
).
The above notwithstanding, mechanical activity of the uterus at the time of embryo transfer depends not only on physical stimulation but also on hormonal influences (Fanchin et al., 1998, 2000
; De Ziegler et al., 2001
; Eytan et al., 2001
). Thus, it has been shown that uterine contractility throughout the IVF cycle is more exaggerated than in spontaneous menstrual cycles (Lesny et al., 1998a
; Epiney et al., 2000
). We have previously reported that ultrasound assistance in fresh embryo transfer significantly improved pregnancy and implantation rates in IVF cycles associated with supraphysiological ovarian steroid levels (Coroleu et al., 2000
). We report here the results of a prospective investigation undertaken to ascertain whether ultrasound guidance could also improve pregnancy rates in transfer of frozenthawed embryos in artificially prepared cycles where estrogen and progesterone profiles mimic the natural cycle.
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Materials and methods |
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The sample size was calculated after considering our previous study comparing ultrasound guidance versus clinical touch for fresh embryo transfer after IVF (Coroleu et al., 2000), where we reported a 16% difference in clinical pregnancy rates in favour of the ultrasound group. Assuming a power of 80% to detect such a difference between groups in the pregnancy rates obtained with a type I risk of 0.05,
90 women per group were needed.
IVF and cryopreservation protocols
Protocols for ovarian stimulation and luteal phase support used in our assisted reproduction programme have been reported in detail elsewhere (Coroleu et al., 2000, 2002
; Martinez et al., 2000
). Briefly, multiple follicular development for IVF and fresh embryo transfer is routinely accomplished using gonadotrophin treatment with FSH under pituitary suppression with leuprolide acetate. In those patients with a previous poor response and/or basal (cycle day 24) FSH serum concentrations >10.5 IU/l which, according to our experience, is associated with poor response cycles (Barri et al., 2000
), we use a flare-up protocol with leuprolide acetate and ovarian stimulation is accomplished with FSH in combination with hMG. The ovulatory injection of hCG was administered when a consistent rise in serum estradiol concentrations was associated with the presence of two or more follicles >18 mm in diameter. Oocyte aspiration was performed by vaginal ultrasonography 3537 h after hCG injection. IVF procedures, including ICSI, used in our programme have also been previously described in detail elsewhere (Calderón et al., 1995
).
Embryos were cryopreserved 48 or 72 h after oocyte retrieval at the 48-cell stage. This was accomplished according to a previously reported protocol (Veiga et al., 1987), where 1,2-propanediol and sucrose solution in phosphate-buffered saline (PBS) are used as cryoprotectants (Testart et al., 1986
). Only embryos having
4 cells on day 2 or
6 cells on day 3 and <30% fragmentation were eligible for freezing. Thawing was performed as reported elsewhere (Testart et al., 1986
; Veiga et al., 1987
). Embryos surviving the freezing procedure (
50% of their initial number of blastomeres intact) were cultured for 24 h and cleaved embryos were preferentially replaced.
Embryo transfer protocol
Patients received programmed cycle endometrial preparation with previous pituitary suppression with a GnRH agonist. A single i.m. injection of 3.75 mg of depot triptorelin (Decapeptyl 3.75 mg; Ipsen Pharma S.A., Barcelona, Spain) was given on days 2022 of the menstrual cycle. At the onset of menses or 1416 days following the injection and after a hypogonadotrophic state (estradiol serum levels <50 pg/ml) was evidenced, endometrial stimulation with estrogen was initiated. The standard estrogen therapy was estradiol valerate (Progynova; Schering España S.A., Madrid, Spain) 8 mg/day in three divided doses for 13 days. On day 14, 200 mg vaginal micronized progesterone, three times daily (Utrogestan; Laboratorio Seid, Barcelona, Spain), was given concomitantly. Estradiol and progesterone serum levels were measured on day 15 for luteal phase evaluation.
Uterine transfer of thawed embryos was carried out on the fourth day of progesterone treatment. One to three embryos per patient were replaced depending upon the age of the patient, the number of previous embryo transfers, and the number and quality of embryos available for replacement. Embryo quality was established according to the number and form of blastomeres and the percentage of cytoplasmic fragmentation, as previously suggested (Plachot and Mandelbaum, 1990). For statistical comparison purposes, and in order to quantify the embryo quality objectively, those three variables were coded according to fixed criteria and then assigned an arbitrary score of 0, 2 or 4 as previously reported (Coroleu et al., 2002
). Embryos having <4 blastomeres on day 2, or <6 blastomeres on day 3, after IVF, were scored 0. In contrast,
4- and
6-cell embryos on days 2 and 3, respectively, were scored 2. Irrespective of the day after IVF, symmetrical cells were scored 4 whereas asymmetrical cells scored 0. Embryos having <15%, 1549.9% and
50% fragmentation, were scored 4, 2 and 0 respectively, both on days 2 and 3. Accordingly, an optimal quality embryo would score 10. For the final analysis of results, the embryo score per patient was considered as the mean value of the scores given to each of the transferred embryos.
The preparation for embryo transfer was the same for both study groups (Coroleu et al., 2000, 2002
). Patients were placed in the lithotomy position and the cervix was exposed using a bivalve speculum. The exocervix was cleaned with a phosphate-buffered saline (PBS) solution (Dulbeccos PBS solution; Irvine Scientific, Santa Anna, CA, USA) and the endocervical mucus was removed by means of a sterile Teflon catheter (Malleable Stylet Wallace, Simcare, Lancing, West Sussex, UK) connected to a syringe.
The EdwardsWallace embryo replacement catheter (SIMS Portex Ltd, Kent, UK) connected to an insulin syringe was used for all embryo transfers. This is a soft silicon catheter possessing a stiffer outer sheath that stabilizes the softer inner cannula, which carries the embryos and actually enters the endometrial cavity for embryo transfers. The catheter was first loaded with transfer medium [50% synthetic serum substitue (Irvine Scientific) and IVF-50 medium (Scandinavian IVF Science, Göthenburg, Sweden)], taking care to avoid air bubbles. The embryos were loaded in the catheter.
The catheter was handed to the clinician who inserted it through the cervical canal. It was at this point that a difference in technique was introduced between the two groups. In the ultrasound-guided group both the insertion and the positioning of the catheter were facilitated by transabdominal (with full bladder) ultrasonography [Tosbee (SSA-240A), convex 3.75 MHz transducer; Toshiba Co., Tokyo, Japan]. The embryo(s) were slowly released when the ultrasound scan showed the catheter to be 1520 mm from the fundus endometrial surface, a depth of embryo replacement which, according to our previous study (Coroleu et al., 2002), is associated with increased implantation rates. In the clinical touch group, the embryo(s) were released according to the clinicians feeling as to the position of the catheter (i.e. advancing the catheter tip into the uterus to a point located at 1520 mm from the fundus according to the ultrasonographic measurement of the uterine cavity performed within 3 months of embryo transfer).
In all transfers, only 30 µl of transfer medium containing the embryos were gently expelled into the uterine cavity. The catheter was gently removed immediately after transfer and then checked under a stereomicroscope to ensure that all embryos had been transferred. At the end of the procedure, patients remained resting in bed for 30 min. Difficult embryo transfer was defined as a procedure involving one or more of the following: cervical resistance leading to a prolonged procedure (>5 min), the need to use increased force or cervical grasping, the need for cervical dilation, the presence of blood on the catheter, or multiple (two or more) sequential attempts because of embryos being retained in the catheter system.
Patients were advised to continue with estrogen and progesterone treatment and were tested for ß-hCG serum concentration 1416 days following embryo transfer. If pregnancy was confirmed, hormonal treatment was maintained until 10 weeks of gestation. Clinical pregnancy was diagnosed by increasing serum concentrations of ß-hCG and the subsequent demonstration of an intrauterine gestational sac by ultrasonography. Only clinical pregnancies were considered for the evaluation of results.
Hormone analysis
Serum estradiol, progesterone and ß-hCG were determined by electrochemiluminescence immunoassay (ECLIA) on the Elecsys 1010 analyser (Roche Diagnostics, Basel, Switzerland). The inter- and intra-assay coefficients of variation were 6.2 and 5.7% for estradiol, 5.4 and 2.4% for progesterone, and 5.1 and 4.5% for ß-hCG respectively.
Statistical analysis
Data were analysed by the Statistical Package for Social Sciences (SPSS, Chicago, IL, USA). We used the 2-test to compare qualitative variables, and Students t-test to compare quantitative variables. The significance level was set at P < 0.05.
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Results |
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Discussion |
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Interestingly, there is now a growing body of evidence indicating that the technique of embryo transfer, as traditionally performed, is inadequate and may contribute to decreased IVF efficiency and success. In fact, although embryo transfer appears deceptively simple, various facets of the transfer procedure may be pivotal in improving implantation rates (Kovacs, 1999; Salha et al., 2001
; Schoolcraft et al., 2001
; Pasqualini and Quintans, 2002
). We and others have recently shown that ultrasonographic guidance is an essential factor associated with successful embryo transfer and implantation after fresh IVF cycles (Coroleu et al., 2000
; Tang et al., 2001
). However, the potential usefulness of ultrasonography in the process of frozenthawed embryo transfer has not been appropriately investigated.
Tang et al. recently reported a randomized controlled trial comparing embryo transfer under ultrasound guidance (n = 400) versus the clinical touch method (n = 400) (Tang et al., 2001). A total of 441 fresh cycles and 359 frozenthawed cycles were included in that study. The overall implantation rate was significantly higher in the ultrasound-guided group. There was no significant improvement in the pregnancy rate, but a consistent trend towards a better pregnancy rate in the ultrasound group in both fresh and frozenthawed cycles was observed (Tang et al., 2001
). In that study, however, frozenthawed embryos were replaced in natural cycles, clomiphene citrate-induced cycles or controlled cycles using pituitary down-regulation followed by hormone replacement therapy. Importantly, a dummy transfer was performed immediately before the real transfer (Tang et al., 2001
). A mock embryo transfer may generate uterine contractility (Lesny et al., 1998b
, 1999b
) and precludes ultrasound-guided embryo transfer from its main advantages, i.e. not touching the endometrium and the uterine fundus with replacement of the embryos in the lumen of the endometrial cavity, which are considered to be the most important factors for successful embryo transfer by most IVF teams (Kovacs, 1999
; Salha et al., 2001
).
The present investigation provides further data favouring the use of ultrasound-guided embryo transfer for improving pregnancy rates and has several original features in this respect. Firstly, the two groups of patients studied were similar with respect to the clinical factors which influence the implantation rate in frozenthawed embryo transfer cycles (Wang et al., 2001), as discussed above. As with patient and fresh IVF cycle characteristics (Tables I and II
), characteristics of the cryopreservedthawed embryo transfer cycles (Table III
) were also similar in groups 1 and 2, thus adding to the validity of our results. Secondly, a mock embryo transfer was not performed in our study. Thirdly, as in our previous study (Coroleu et al., 2002
), all transfers were carried out by the same person, thus avoiding any impact of the physician factor on implantation rates (Karande et al., 1999
; Hearns-Stokes et al., 2000
), and using the most popular embryo transfer catheter (Wood et al., 2000
; Salha et al., 2001
) according to a strictly standardized protocol. Finally, all embryo transfers were performed in artificially prepared cycles with prior pituitary suppression.
This latter point seems important given that controlled ovarian hyperstimulation has been reported to have an adverse effect on the uterine environment and conception outcome (Check et al., 1995, 1999
). Rapid uterine contractions have been observed immediately after oocyte retrieval, which were not noted immediately prior to follicular puncture (Lesny et al., 1998a
). Thus, it has been suggested that there is a release of prostaglandins and/or other inflammatory reaction mediators from the multifollicular/multiluteal ovaries (Lesny et al., 2002
). This effect would persist into the luteal phase and, together with supraphysiological concentrations of serum estradiol and only brief exposure to progesterone, could be responsible for more exaggerated contractions in an IVF cycle compared with natural cycles (Lesny et al., 2002
).
For patients included in the present study there was no deleterious effect of hormonal influences associated with controlled ovarian hyperstimulation, and thus any potential increase in mechanical activity of the uterus at the time of embryo transfer was dependent only on physical stimulation. Futhermore, ultrasound-guided embryo transfer may have two additional advantages over clinical touch embryo transfer when considering that: (i) blind catheter placement has been shown to result in a malposition of the catheter in >25% of cases, thus indicating that tactile assessment of embryo transfer catheter position is unreliable (Woolcott and Stanger, 1997); and (ii) the depth of the embryo replacement into the uterine cavity influences implantation rates, with highest pregnancy rates obtained when embryos are replaced 1520 mm from the fundus endometrial surface (Coroleu et al., 2002
).
All the above supports the notion that ultrasound assistance in embryo transfer is a pivotal tool for improving pregnancy rates in assisted reproduction irrespective of whether embryos are fresh or frozen and replaced in spontaneous, stimulated or artificially prepared cycles. A very recent report showing that ultrasound-guided embryo transfer improves outcome in patients with previous failed IVF cycles (Anderson et al., 2002) provides further evidence in this regard.
In conclusion, ultrasound guidance in frozenthawed embryo transfer significantly increases pregnancy and implantation rates.
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Acknowledgements |
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Notes |
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Submitted on May 14, 2002; accepted on July 17, 2002.