Department of Obstetrics and Gynecology and Reproductive Endocrinology, Hôpital Antoine Béclère, Clamart, France
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Abstract |
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Key words: blastocyst/embryo implantation/ultrasound/uterine contractions
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Introduction |
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Yet recent studies of uterine contractility in IVFembryo transfer led us to consider an alternative, and possibly complementary, explanation for the high implantation rates of blastocysts. It has been demonstrated that myometrial contractile activity influences embryo implantation, possibly through mechanical displacement of embryos, in both animals (Pusey et al., 1980; Rogers et al., 1983
) and humans (IJland et al., 1997
,1998
; Fanchin et al. 1998
). Data on the relation between uterine contractility and embryo implantation in humans remained scarce because of the invasiveness of traditional methods that required the introduction of pressure probes into the uterine cavity (Henry et al., 1943
; Hendricks, 1966
; Martinez-Gaudio et al., 1973
).
However, the advent of high resolution ultrasound probes permitted the direct visualization of myometrial activity in ultrasound scans (Oike et al., 1990; Abramowicz and Archer, 1990
; Lyons et al., 1991
; IJland et al., 1997
,1998
). Recently, the reliability of the ultrasound approach for assessing uterine contractions was confirmed by demonstrating the concordance of findings made simultaneously on ultrasound and intrauterine pressure recordings in an experimental model (Bulletti et al., 2000
).
In earlier investigations (Fanchin et al., 1998, 2000a
), we have shown that a considerable fraction of IVFembryo transfer patients have persistently high uterine contraction frequency at the time of non-cavitating embryo transfer, 4 days after human chorionic gonadotrophin (HCG) administration. Moreover, the observation of high-frequency uterine contractions (>5.0 contractions/min) at the time of embryo transfer is associated with markedly lower implantation and pregnancy rates per embryo transfer (4 and 14% respectively) as compared with cases with low-frequency contractions (
3.0 contractions/min; 21 and 53% respectively) (Fanchin et al., 1998
). This may result from the mechanical expulsion of embryos from the uterine cavity.
In addition, more recent work on the hormonal regulation of uterine contractility led us to speculate that utero-relaxation mediated by progesterone during the luteal phase takes a longer time to be fully established in ovarian stimulation compared with the menstrual cycle (Fanchin et al., 2000a). This may constitute a plausible explanation for the high uterine contraction frequency seen at the time of non-cavitating embryo transfer in ovarian stimulation.
Based on this documentation, we decided to investigate the possible advantages of delaying embryo transfer so that embryos reach the uterine cavity when contractility is attenuated and conditions for embryo implantation are improved. For this, we compared uterine contraction characteristics on the day of HCG administration, the day of non-cavitating embryo transfer (HCG + 4), and the day of blastocyst transfers (HCG + 7).
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Materials and methods |
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Ovarian stimulation protocol
A single injection of a time-release gonadotrophin releasing-hormone (GnRH) agonist, triptorelin, (3.0 mg i.m., Decapeptyl®; Ibsen-Biotech Laboratories, Paris, France) was administered on cycle day 2. Eighteen days later, complete pituitary desensitization was confirmed by documenting low plasma oestradiol <40 pg/ml and luteinizing hormone (LH) 2 mIU/ml concentrations. Patients also had a conventional ultrasound examination to exclude ovarian cysts and verify that endometrial thickness was <5 mm. Recombinant FSH therapy (Puregon®; Organon Pharmaceuticals, Saint-Denis, France) was then initiated at a dosage of 225 IU/day for the first 5 days of ovarian stimulation. Further FSH doses and timing of HCG (Gonadotrophine Chorionique Endo®; Organon Pharmaceuticals, 10 000 IU, i.m.) administration were adjusted according to the usual criteria of follicular maturation determined by ultrasound and oestradiol findings. Administration of HCG was performed when at least three follicles exceeded 17 mm in diameter and oestradiol concentrations per mature follicle (
17 mm in diameter) were >300 pg/ml. Oocytes were retrieved 36 h after HCG administration by transvaginal ultrasound-guided aspiration.
All embryo transfers were performed 2 days after oocyte retrieval using Frydman catheters (CCD Laboratories, Paris, France). Luteal phase was supported with progesterone (Crinone® 8%; Ares-Serono SA, Geneva, Switzerland) administered daily by vaginal route starting on the evening of embryo transfer.
Uterine contractility and hormonal assessment
All women underwent three sequential ultrasonographic and hormonal evaluations that took place on the day of HCG administration, just before the actual non-cavitating embryo transfer (4 days after HCG, HCG + 4), and on the day when blastocysts are commonly obtained and transferred (HCG + 7). Two minute ultrasound scans of a sagittal plane of the uterus were performed using a 7.5 MHz transvaginal probe (Siemens Elegra®; Siemens SAS, Saint-Denis, France) at approximately 1100 h by one single operator. Environmental conditions were standardized throughout ultrasound examinations. The present study respected similar methodological characteristics as previously described (Fanchin et al., 1998, 2000a
).
Briefly, images were digitized on-line using a two image/s rate with a computer-assisted image analysis system (IôTEC 3.1.2®; IôDP, Paris, France). As represented in Figure 1, uterine contraction frequency was assessed on time-mode graphs generated electronically using three-dimension reconstruction software (IôTEC 3.1.2®; IôDP). For this, instead of swapping the ultrasound probe as for volume acquisition and three-dimension reconstruction, the probe was kept steady and two-dimension images were acquired over time for 2 min. Hence, in the electronic matrix, the z axis, instead of being the third dimension of volume, was represented by time. In the time-mode graphs, uterine contraction frequency was identified by the number vertical displacements of the myometrialendometrial interface and of the uterine cavity line over time. Precision of uterine contraction frequency measurements, expressed as interassay coefficient of variation, was 8%.
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Serum FSH was measured by an immunometric technique using an Amerlite® kit (Ortho Clinical Diagnostics, Strasbourg, France). Intra-assay and interassay CV were, respectively, 5 and 7% and sensitivity was 0.1 mIU/ml for FSH. Serum progesterone was measured by radioimmunoassay using a I125 Progesterone Coatria® kit (Bio-Mérieux, Paris, France). Sensitivity was 0.05 ng/ml and intra-assay and interassay CV were, respectively, 8 and 11% for progesterone. Serum oestradiol was determined by an immunometric technique using an Estradiol-60 Amerlite® kit (Ortho Clinical Diagnostics). Sensitivity was 14 pg/ml, and intra-assay and interassay CV were 8 and 9% for oestradiol respectively.
Statistics
Measures of central tendency used were means and measures of variability were standard errors. When data distribution was non-parametric, medians and ranges were used. Changes in uterine contraction frequency and ovarian hormone levels were assessed using the paired Student's t-test and repeated-measures analysis of variance (ANOVA) when appropriate. Hormonal influence on uterine contractility was assessed using simple regression. A P value of < 0.05 was considered statistically significant.
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Results |
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Uterine contractility
Changes in uterine contraction (UC) frequency from the day of HCG administration to HCG + 7 are depicted in Figure 2. Mean UC frequency, which reached 4.4 ± 0.2 UC/min (range: 1.58.5) on the day of HCG administration, decreased slightly yet significantly on HCG + 4 at 3.5 ± 0.2 UC/min (range: 1.07.5)(P < 0.003). On HCG + 7, an additional and noticeable reduction of UC frequency was observed (1.5 ± 0.2 UC/min, range: 0.03.5, P < 0.001). The prevalence of each uterine contraction type (cervix-to-fundus or retrograde, fundus-to-cervix or antegrade, antagonistic, and non-propagated) was 57, 32, 9, and 2% respectively on the day of HCG administration. This frequency distribution remained comparable on HCG + 4 (56, 25, 14, and 5% respectively). On HCG + 7, however, prevalence of uterine contraction changed as compared with HCG administration (31, 23, 23, and 25% respectively), with a reduction of retrograde uterine contraction (P < 0.01) and a more pronounced increase in non-propagated UC (P < 0.001).
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On the day of HCG administration, serum progesterone concentrations failed to correlate with uterine contraction frequency. On HCG + 4, as expected, we observed a negative and significant correlation between serum progesterone concentrations and UC frequency (r = -0.43; P < 0.004). Similarly, on HCG + 7, serum progesterone concentrations and UC frequency correlated negatively (r = -0.34; P < 0.03). Serum oestradiol concentrations did not correlate with UC frequency at any time. No statistically significant correlation was identified between direction of uterine contraction and serum progesterone or oestradiol concentrations.
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Discussion |
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Because the number of blastocyst transfers is limited at our institution, it was impractical to enrol enough cases to assess uterine contraction characteristics on the day of actual blastocyst transfers. In an effort to overcome this limitation, we studied a selected population (women aged 38 years, displaying adequate ovarian follicular reserve and with at least two good quality embryos available for embryo transfer on HCG + 4) who had good probability of reaching the blastocyst transfer. Therefore, these methodological characteristics may authorize the extrapolation of present uterine contraction data observed on HCG + 7 (theoretical day of blastocyst transfers) to real blastocyst transfers conditions. However, it is noteworthy that possible direct and local influence of embryos present in the uterine cavity on HCG + 4 and uterine contractility on HCG + 7 could not be ruled out by the present study and need further investigation.
The observed reduction of uterine contraction frequency after HCG administration presumably results from the utero-relaxing properties of progesterone, both secreted by multiple corpora lutea and administered exogenously after embryo transfer. In support of this, the present data indicate that serum progesterone concentrations correlate negatively with uterine contraction frequency not only on HCG + 4, thus confirming our earlier reports (Fanchin et al., 1998, 2000a
), but also on HCG + 7. Conversely, the myo-relaxing action of progesterone may depend on both its absolute circulating levels and the duration of uterine exposure to progesterone. In agreement with this hypothesis, uterine contraction frequency decreases progressively during the luteal phase of the menstrual cycle (Abramowicz and Archer, 1990
; Lyons et al., 1991
). The luteal reduction of uterine contractile activity potentially assists the embryo implantation process. Accordingly, in natural conditions, the entry of the morula into the uterine cavity does not occur before ~7296 h after fertilization (Croxatto et al., 1978
), when uterine activity is already reduced.
Hence, it is conceivable that 2- to 8-cell embryo transfers are often performed too early when utero-relaxation mediated by progesterone has not been fully exerted. Indeed, despite serum progesterone reaching very high mean concentrations on HCG + 4 (>85 ng/ml), the duration of uterine exposure to this hormone at that time remains relatively short (<3 days) (Fanchin et al., 2000b). This brief exposure to progesterone, together with the possibility of persistent utero-stimulating effects of supra-physiological oestradiol concentrations from ovarian stimulation (Fanchin et al., 2000a
), may contribute to the insufficient utero-relaxation observed on the day of non-cavitating embryo transfers (HCG + 4). On HCG + 7, however, duration of uterine exposure to progesterone is much longer and oestradiol-induced utero-excitability may be decreased, which concur to explain utero-relaxation.
Further, the present study showed an increasing prevalence of non-propagated uterine contractions from the day of HCG administration to HCG + 7. Whether or not this phenomenon may be attributed to an action of progesterone as well as its possible role on embryo implantation deserves further elucidation. Finally, it is important to mention that the contractile activity of the uterus is a complex phenomenon that involves either superficial or profound muscle layers with generalized and local consequences. Hence, the complete assessment of all uterine contractility characteristics by ultrasound may sometimes be difficult. This methodological limitation led us to consider only contraction frequency and, to a lesser extent, direction of propagation in our present and past ultrasound studies (Fanchin et al., 1998, 2000a
). Indeed, recent data indicated that, during the luteal phase, the uterine fundus shows only relative quiescence and the residual contractile may be important to blastocyst positioning (Kunz et al., 2000
).
In conclusion, concurring with the putative embryo selection through extended culture, the nearly quiescent status of the uterus reached on the day of blastocyst transfers may avoid embryo displacement in the endometrial cavity and, therefore, assist implantation. Hence, the present data may offer an additional explanation of the high implantation rates reported after blastocyst transfers (Gardner et al., 1998a,b
; Ménézo et al., 1998
; Schoolcraft et al., 1999
; Huisman et al., 2000
). Further, based on these results, extending culture and transferring blastocysts instead on 2- to 8-cell embryos may be opportune in cases of high uterine contraction frequency (
5 contractions/min) (Fanchin et al., 1998
) on the day of non-cavitating embryo transfers.
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Notes |
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References |
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Submitted on October 13, 2000; accepted on February 12, 2001.