1 University of Colorado Health Sciences Center, Aurora, CO 80010, 2 Walter Reed Army Medical Center, Washington, DC 20003 and 3 Pediatric and Reproductive Endocrinology Branch/National Institute of Child Health and Development/National Institutes of Health, Bethesda, MD 20892, USA
4 To whom correspondence should be addressed at: Advanced Reproductive Medicine, Anschutz Outpatient Pavilion, University of Colorado Health Sciences Center, 1635 N. Ursula Street, Mail Stop F701, Aurora, CO 80010, USA. e-mail: ruben.alvero{at}uchsc.edu
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Abstract |
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Key words: blood/embryo transfer technique/implantation/pregnancy rate/retained embryos
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Introduction |
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Edwards et al. (1984) did not observe a statistically significant effect of catheter contamination with blood on CPR. These results were supported by Visser et al. (1993
). However, Goudas et al. (1998
) and Awonuga et al. (1998
) found that the presence of blood on the transfer catheter after ET was associated with decreased IR and CPR. Visser et al. (1993
) observed that retention of embryos in the transfer catheter significantly reduced the CPR, and that catheters contaminated with blood and cervical mucus indirectly contributed to this effect by increasing the incidence of failed first attempts at ET. In contrast, Nabi et al. (1997
) and Goudas et al. (1998
) observed that retention of embryos in the transfer catheter did not significantly reduce CPR. More recent studies have also found that there was no significant difference in the CPR between transfer procedures in which all embryos were transferred at the first attempt and those which required multiple attempts (Wood et al., 1985
; Egan et al., 1990
; Nabi et al., 1997
).
Although the presence of mucus in the transfer catheter has been associated with an increased incidence of retained embryos at ET, several studies have not demonstrated a significant effect on CPR (Visser et al., 1993; Awonuga et al., 1998
). The presence of both blood and mucus has been implicated in difficult transfers (Awonuga et al., 1998
). Some studies report that difficulty of the ET does not affect pregnancy outcome (Wood et al., 1985
; Egan et al., 1990
; Tur-Kaspa et al., 1998
), while others suggest that difficult transfers adversely affect pregnancy outcome (Leeton et al., 1982
; Mansour et al., 1990
).
In this study, we have evaluated the effect of several factors on pregnancy outcome after ET. Given the strong influence of embryo quality and ultrasound guidance on pregnancy outcome, we have controlled for these variables. We also examined the mean age for each characteristic to make sure that age did not impact outcome.
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Materials and methods |
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Experimental design
Stimulation protocols were individualized to the patient but, in general, patients aged >35 years underwent a microdose flare GnRH agonist protocol (Leondires et al., 1999) and initially received an oral contraceptive containing levonorgestrel and ethinyl estradiol. After cessation of oral contraceptives, patients received either Lupron 20 µg or 40 µg twice daily and gonadotrophins (FSH or a combination of FSH and hMG). Younger women or patients at high risk for hyperstimulation underwent a luteal suppression GnRH agonist protocol with Lupron (TAP Pharmaceuticals, USA) 0.5 mg/day starting in the mid-luteal phase until ovarian quiescence was achieved (serum estradiol <35 pg/ml). The dosage of lupron was then reduced to 0.25 mg s.c. daily and gonadotrophins (FSH or a combination of FSH and hMG) were started. All cycles were monitored with serial follicular ultrasound measurements and serum estradiol levels. hCG was given when the lead follicle had an average diameter of 18 mm and the majority of the cohort measured
14 mm. Oocyte retrieval took place 35 h after the hCG injection.
Semen preparation, oocyte insemination and injection, embryo culture and embryo scoring were performed using our laboratorys standard technique (Scott et al., 2000). Providers adhered to ASRM guidelines for the number of embryos transferred (Society for Assisted Reproductive Technology, 1999
). Cleaving embryos were transferred at day 2 or day 3 and blastocysts were transferred at day 5. The medium used during the ET was the same as that in which the embryos were being cultured. All embryos transfers were performed with the Wallace catheter (SIMS Portex Ltd, UK), and visualized with transabdominal ultrasound (Ultramark 4 with a 5 MHz transducer). The patients started luteal supplementation with progesterone in oil 50 mg i.m. on the night of retrieval and continued until a negative pregnancy test occurred at 2 weeks post-retrieval or until 8 weeks gestation.
Prior to starting an ART cycle, all patients underwent a mock embryo transfer to determine the direction of the endocervical canal and depth of the uterine cavity. If it was anticipated that the transfer would be very difficult on the basis of the pre-cycle mock transfer or other historical reason, the transferring physician might elect to perform a mock transfer immediately prior to the actual procedure. At the discretion of the provider, once cavitary placement was assured, the outer sheath could be left, the inner sheath removed and an inner sheath with embryos loaded could then be placed through the in-situ outer sheath. The embryos were thereby protected from prolonged exposure to the environment and to a possibly traumatic transfer. Patients underwent transfer in the dorsal lithotomy position with a full bladder. A sterile, preheated, bivalve speculum was inserted into the vagina to expose the cervix. The cervix was cleaned with sterile cotton swabs and embryo culture media. Endocervical mucus was removed at the discretion of the transfer physician, but, in general, cervical lavage with media and aspiration with a pipette were not used in this programme. Uterine orientation and the endometrial cavity were identified with abdominal ultrasound prior to the transfer. The embryologist used a sterile 1 ml syringe to flush 1 ml of growth media through the catheter, leaving 0.01 ml intraluminally. The embryos were suctioned from the growth media followed by 5 µl of air. The embryologist passed the loaded catheter to the physician, who carefully deposited the embryos, 1.01.5 cm from the uterine fundus. An air bubble was visualized on ultrasound after deposition of embryos into the endometrial cavity. The plunger of the catheter remained depressed from the point during which the embryos were deposited until the catheter was passed to the embryologist. The embryologist flushed the catheter with growth media and microscopically examined it for the presence of blood, mucus, and retained embryos. Any embryos retained were immediately reloaded by the embryologist and a second transfer performed. The physician who performed the transfer immediately scored the transfer as easy, moderate or difficult, recorded the number of passes required to successfully perform the transfer, whether a mock embryo transfer was performed immediately preceding the ET, and if the transfer was visualized on abdominal ultrasound. After consultation with the embryologist, the physician recorded whether embryos were retained in the transfer catheter, and if blood or mucus were present on or in the catheter.
Statistical analysis
The principal outcome variables were the presence of a clinical pregnancy, defined as a gestational sac visualized on ultrasound at 68 weeks gestation, and IR defined as the number of gestational sacs seen on ultrasound divided by the number of cleaving embryos or blastocysts transferred.
Univariable analysis was used to determine whether the presence of blood or mucus on the transfer catheter, ease of ET, retention of embryos in the transfer catheter, requirement of multiple attempts to achieve successful ET, or requirement of a mock ET immediately preceding ET were associated with IR or CPR. IR data were analysed using Students t-test for all but the difficulty category, which were evaluated using one-way analysis of variance. CPR were analysed using Fishers exact test with the exception of the difficulty category which was evaluated using the 2-test. Multivariable analysis was used to control for confounding in the variables that were studied. Specifically, IR was evaluated using analysis of covariance (ANCOVA) to allow the use of a combination of continuous and categorical variables as the independent variables. Since CPR is a categorical variable, logistic regression was used to quantify the association of this dependent variable with the independent variables.
Statistical Package for the Social Sciences (SPSS), version 10.0 was used for all the statistical calculations in this study. P 0.05 was considered statistically significant.
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Results |
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A total of 10 providers performed the embryo transfers and this analysis is a report of all the practitioners results. An attempt to evaluate individual physicians outcomes did not reveal a statistically significant difference among the providers with regard to CPR, IR, embryo retention rate, and the presence of blood or mucus on the catheter. There were differences in perceived difficulty of transfer (P < 0.0005) and rate of aborted transfers (P < 0.05) among the providers, possibly reflective of the more experienced physicians tending to perform the more challenging procedures.
VF accounted for 437 and ICSI for 147 of the embryo transfers that took place. There were no differences in the outcome variables with regard to type of fertilization procedure performed.
Blood was noted on the transfer catheter after 60 embryo transfers (10.3%). As previously noted, age was not a factor as there was not a statistically significant difference in the mean age of patients in the group with the presence of blood on the transfer catheter after ET (34.9 ± 4.2) compared with the group without the presence of blood (34.1 ± 4.5, P = 0.23, not significant). Overall, CPR were substantially affected by the presence of blood on the catheter, with a 51.7% chance of a gestational sac with fetal pole and cardiac activity if no blood was found, compared to 31.7% if blood was discovered (Table I, P = 0.004). The IR was also affected by the presence of blood on the catheter. The mean IR for those in which no blood was found was 31.3% compared to 19.5% with blood (Table I, P = 0.015).
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There were no significant differences in IR or CPR for presence of mucus on the transfer catheter, ease of ET as judged by the provider, occurrence of aborted attempts to achieve successful ET, or requirement of a mock ET immediately preceding ET (Tables I, II).
Of course it is unlikely that these are completely independent variables. Using IR and CPR as the dependent variables, the aforementioned parameters were analysed using multivariable statistics to assess their relative contribution to outcome. As is seen in Table III, the presence of blood on or in the transfer catheter is the only parameter that is significantly associated with IR when ANCOVA is used to assess the relationship. Retained embryos demonstrate a trend toward being significant but do not actually achieve significance. In Table IV, the results of logistic regression again show that blood significantly compromises CPR whereas the other parameters do not appear to influence outcome.
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Discussion |
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Although Nabi et al. (1997) and Goudas et al. (1998
) observed that retention of embryos in the catheter did not significantly reduce CPR, we found that retention of embryos was associated with a significant reduction in IR. The IR was 31% for ET in which embryos were not retained and 17% for those in which embryos were retained (P = 0.03). As previously noted, CPR is not affected, largely because a greater number of embryos were transferred. There is no easy explanation for why such a significant discrepancy in number of embryos transferred exists.
Although the presence of mucus on the transfer catheter did not affect CPR or IR in our study, it was associated with an increased incidence of retained embryos (data not shown). In agreement with the results of Awonuga et al. (1998), we found that embryos were more likely to be retained when the transfer catheter was contaminated with mucus. Retention of embryos may indicate mechanical blockage of the catheter opening (Leeton et al., 1982
; Egan et al., 1990
; Visser et al., 1993
) and mucus may cause embryos to adhere to the catheter when it is withdrawn (Awonuga et al., 1998
). Our findings lend support to the suggestion of Awonuga et al. (1998
) to remove significant cervical mucus prior to ET in an effort to decrease the incidence of retained embryos. Nevertheless, the presence of mucus does not appear to be a significant factor in ET. The subjective scoring of transfers as difficult did not reduce CPR or IR. The difficulty or ease with which a provider scored an ET is troublesome to assess because of its subjective nature but did not appear to affect outcome. Nevertheless, assessment (and self-assessment) of the providers ET technique and skill acquisition has been an issue of interest to our programme (Hearns-Stokes et al., 2000
; Papageorgiou et al., 2001
). Consciousness of a greater than average number of difficult transfers might induce a provider to retrain in transfer technique.
Our observations were similar to those of Wood et al. (1985
), Egan et al. (1990
) and Nabi et al. (1997
) who also reported that the CPR was not influenced by the number of attempts required to replace embryos, and ET that required multiple attempts were not associated with compromised CPR or IR. In our programme, embryos were returned to the laboratory if the ET could not be completed within 2 min and a mock ET was performed to determine whether cervical obstruction existed, direction of endocervical canal, and depth and direction of endometrial cavity. We have empirically begun this protocol in an effort to minimize the potential for damage or loss of embryos, or trauma to the endometrium.
The results of this retrospective review highlight two possible areas for intervention. If, as was earlier suggested, some women have evidence of blood on the transfer catheter despite no obvious trauma, an endometrial problem may exist. We did not attempt to correlate ultrasound lining characteristics with the incidence of blood on the catheter but this may be worthwhile to assess. This subpopulation of women may fall in the category of an endometrial factor. Although treatment modalities in this area are at present suboptimal, ongoing research will in the future offer practical and effective interventions.
It is impossible to know at this time if there is endometrial bleeding or if cervical bleeding is a marker for significant endometrial trauma due to transfer technique. Cervical trauma may be the more common cause for blood on the catheter tip. There is also the possibility that a subclinical infection, such as bacterial vaginosis, might lead to cervical friability. It is possibly a combination of all of these factors. Regardless of the source, it is clear from this study that blood on the catheter is a marker for reduced success in assisted reproduction. As IVF becomes more successful, it would be prudent to identify the cause for this small but discrete subset of individuals. Possible causes include bleeding disorders, medications or endometrial surface abnormalities.
As with difficult transfers, a large percentage of ET associated with the presence of blood might suggest that a provider reassess ET technique and reassess their training. Much of the previously cited research indicates that a steep learning curve exists for ET. Goudas et al. (1998) suggested that an emphasis on atraumatic ET technique with prevention of bleeding at time of ET should contribute to improved pregnancy outcome in IVF. In our programme, providers are required to perform a significant number of mock transfers and intrauterine inseminations using a soft catheter before they are allowed to transfer embryos. Developing the fine motor skills integral to such a nuanced procedure is critical to success. Moreover, developing the confidence to feel the endocervical pathway rather than forcing the catheter cannot help but decrease traumatic delivery of the embryos to the endometrial cavity.
In summary, avoidance of blood on the transfer catheter and perhaps retained embryos are key to good CPR and IR. Our results support the conclusion that ET technique plays a major role in assisted reproduction outcome. Attention to detail, atraumatic transfers, and frequent assessment of providers technique should positively impact outcome in assisted reproduction.
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FOOTNOTES |
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References |
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Submitted on April 22, 2002; resubmitted on April 22, 2003; accepted on May 16, 2003.