Human Reproduction, Vol.29, No.6 pp. 1238 –1243, 2014 Advanced Access publication on March 23, 2014 doi:10.1093/humrep/deu058
ORIGINAL ARTICLE Infertility
Uterine peristalsis before embryo transfer affects the chance of clinical pregnancy in fresh and frozen-thawed embryo transfer cycles Department of Reproductive Medicine, Xiangya Hospital, Central-south University, Changsha City, Hunan Province, P.R. China *Correspondence address. Department of Reproductive Medicine, Xiangya Hospital, 87 Xiangya Road, Changsha City, Hunan Province, P.R. China. E-mail: [email protected]
Submitted on November 30, 2013; resubmitted on February 22, 2014; accepted on February 26, 2014
study question: Does uterine peristalsis influence the chance of clinical pregnancy in an embryo transfer cycle? summaryanswer: The uterine peristaltic wave frequency before embryo transfer is inversely related to the clinical pregnancy rates in fresh and frozen-thawed embryo transfer cycles.
what is known already: Uterine peristalsis participates in regulating fluid migration after mock embryo transfer, but whether it could potentially influence pregnancy outcomes had remained unclear. study design, size, duration: This prospective cohort study included a total of 292 infertile women and was conducted between March 2013 and August 2013.
participants/materials, setting, methods: Patients underwent fresh embryo transfer in a fresh stimulation cycle with a long down-regulation protocol, a natural frozen-thawed embryo transfer cycle or an artificial frozen-thawed embryo transfer cycle. Uterine peristaltic activity was assessed before embryo transfer by transvaginal ultrasonography.
main results and the role of chance: The uterine peristaltic wave frequencies of most patients were between 1.1 and 3.0 waves/min before embryo transfer (ET). The clinical pregnancy rate was the highest when ,2.0 waves/min was observed and it decreased with an increasing wave frequency thereafter, with an especially dramatic decrease with .3.0 waves/min. Uterine peristaltic wave frequencies of the non-pregnant patient group were higher than that of the clinically-pregnant patient group in all types of transfer, fresh embryo transfer, natural FET or artificial FET cycle. Binary logistic regression analysis demonstrated that the association between uterine peristaltic wave frequency before embryo transfer and clinical pregnancy was independently significant (odds ratio: 0.49; 95% confidence interval: 0.34 –0.70, P , 0.001). limitations, reasons for caution: Uterine peristalsis after embryo transfer was not observed in case any possible negative effect of the observation disturbed embryo implantation or caused psychological stress. Uterine peristalsis after embryo transfer may differ from that before embryo transfer. Another limitation of the present study was the lack of uterine peristaltic wave type analysis which is also an important parameter to assess uterine activity. wider implications of the findings: Patients with uterine peristalsis of ,3.0 waves/min before embryo transfer had a higher chance of pregnancy compared with those with higher frequencies. This could be a promising quantitative marker of uterine receptivity and pregnancy outcome in an embryo transfer cycle. The predictive validity of the cut-off value needs to be tested in further study. study funding/competing interest(s): The study is supported by Hunan Provincial Innovation Foundation for Postgraduates. The authors declare that they have no competing interests in this study. Key words: uterine peristalsis / uterine receptivity / clinical pregnancy / assisted reproductive technology
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
L. Zhu, H.S. Che, L. Xiao, and Y.P. Li*
1239
Uterine peristalsis and pregnancy rates
Introduction
Materials and Methods Patients From March 2013 to August 2013, all patients undergoing IVF/ICSI-ET and FET treatment at the Department of Reproductive Medicine at Xiangya Hospital of Central-south University were offered participation in the study. To limit the interference of confounding variables affecting embryo quality and/or uterine receptivity, patients were only selected if they (i) were 20 – 35 years of age; (ii) had a normal uterus; (iii) had two good-quality day-3 cleavage stage embryos available for transfer (good-quality day-3 embryos were defined by at least seven cells, ,20% fragmentation, and no apparent morphological abnormalities); and (iv) underwent easy embryo transfer (i.e. the catheter was smoothly inserted without touching the fundus, no cervix tenaculum was used and the catheter was clean of blood). After excluding 13 patients due to cancellation of embryo transfer (because of ovarian hyperstimulation syndrome or hydrohystera), a change to blastocyst transfer, or the use of another drug intervention, a total of 166 patients underwent fresh embryo transfer and 126 patients received either natural FET (n ¼ 69) or artificial FET (n ¼ 57) were analysed. Written informed consent was obtained from each patient before inclusion. The study was approved by the institutional review board (Clinical Trial Registration Number: ChiCTR-OCH-13003105).
Cryopreservation cycles In the natural FET cycles, the development of the dominant follicle and endometrium was monitored from Day 10 by regular vaginal ultrasound, urine LH tests and serum LH, E2 and progesterone measurements until ovulation. If the diameter of the dominant follicle was ,14 mm, the patient was asked to return every second day. If the dominant follicle was ≥14 mm, a urine LH test was performed and the patient was asked to return the next day. When the diameter of the dominant follicle was ≥18 mm or the urine LH test was .45 mIU/ml, serum LH, E2 and progesterone concentrations were measured to help estimate the maturation of the dominant follicle, predict ovulation and find out whether the follicle was luteinized. If the serum progesterone concentration was .1.0 ng/ml and the follicle was still unruptured, which was usually accompanied by heterogeneous echo of the follicular fluid, luteinized unruptured follicle was identified and that day was taken as the ovulation day. To preclude luteal phase defect, luteal phase support was provided from the day of ovulation day progesterone injection 40 mg/day and added progesterone soft capsules at a dose of 200 mg/night from the day of embryo transfer. In the artificial FET cycles, patients received increasing doses (6 mg for days 3 – 7, 8 mg for 2 days, and 10 mg for 2 days thereafter) of estradiol valerate (Progynova, Bayer) in order to prepare the endometrium for embryo transfer. Endometrial thickness was monitored by transvaginal ultrasound every time before adjusting the doses of estradiol valerate, and when the endometrium reached or exceeded 7 mm, increasing doses (20 mg for 1 day, 40 mg for 1 day and 60 mg for 1 day thereafter) of progesterone were given intramuscularly once per day. Progesterone soft capsules at a dose of 200 mg/ night were administered orally from the day of embryo transfer.
Embryo transfer Two good-quality embryos were transferred under abdominal ultrasound guidance 3 days after oocyte retrieval in a fresh cycle, 2 days after ovulation in a natural FET cycle and 3 days after progesterone administration in an artificial FET cycle, respectively. All of the embryo transfers were performed by an experienced doctor (Y.P.L.), who was blind to the frequency of uterine peristalsis, using the same type of catheter (CCD131230301, Ultrasoft Frydman Set with Guide, Laboratoire C.C.D., France). Blood samples were collected on the day of embryo transfer to determine the serum E2 and P using electrochemiluminescence immunoassays. Approximately 80% of all fresh cycles were conducted as IVF and 20% were conducted as ICSI/half-ICSI. Patients were confirmed to be clinically pregnant by ultrasonographic visualization of a gestational sac 2 – 3 weeks after a positive pregnancy test. Luteal support was continued either until 1 month after confirmation of intrauterine pregnancy or until pregnancy was ruled out by a negative serum HCG measurement, in all types of treatment cycles.
Fresh cycles
Observation and assessment of uterine peristalsis
Patients underwent ovarian stimulation in a long down-regulation protocol using urinary FSH (Urofollitropin, Livzon, China), recombinant follitropin alfa (Gonal-F, Merck Serono) or recombinant follitropin beta (Puregon,
Transvaginal ultrasonography scans (Mindray DC-6 Expert, 5 – 8 MHz transducer) of uterine peristalsis were performed by a single examiner (L.Z.) about 1 h before embryo transfer while the patient was lying relaxed in a
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
Controlled ovarian stimulation improvements and technological advancements in the laboratory have provided adequate oocytes and high-quality embryos to infertile couples, leading to steadily increased success rates for IVF over the years. However comparatively speaking, considerably fewer efforts have been devoted to improving uterine receptivity, especially in clinical practice. In contrast with an invasive endometrium biopsy, uterine peristalsis can be directly visualized by ultrasonography and has therefore broadened the horizon for exploring uterine receptivity in human reproduction. During the luteal phase in a natural cycle, uterine activity reaches an absolute minimum and the reduced uterine peristalsis is believed to facilitate embryo implantation (Ijland et al., 1997a; Bulletti et al., 2000; Bulletti and de Ziegler, 2006). Studies of mock embryo transfer (ET) have also testified to the important role of uterine peristalsis in controlling the migration of embryo analogues. Lesny et al. (1998) observed that the transferred fluid could be expelled out of the uterine cavity by strong uterine contractions after touching the uterine fundus with the catheter. Our previously published study also found that uterine peristaltic wave frequency was positively correlated with the distance that fluid moved after it was deposited in the uterine cavity (Zhu et al., 2014). Since uterine peristalsis can exert an influence on the drift of an embryo analogue during the implantation window, we hypothesized that it may also have an effect on the subsequent clinical pregnancy outcome in a real embryo transfer cycle. This inspired us to comprehensively analyse the relationship between uterine peristalsis just before embryo transfer and the clinical pregnancy rates under different conditions, including fresh embryo transfer, natural frozen-thawed embryo transfer (FET) and artificial FET cycles.
Merck Sharp & Dohme). FSH doses were adjusted individually according to the patient’s ovarian response as assessed by ultrasound and hormonal measurements performed every 1 – 3 days. A dose of 5000 – 10 000 IU of human chorionic gonadotrophin (HCG, Livzon, China) was administered when at least three follicles were ≥18 mm in diameter. Oocyte retrieval was conducted transvaginally 34 – 36 h after the HCG trigger. The luteal phase was supported with progesterone injections at a dose of 60 mg/day and progesterone soft capsules at a dose of 200 mg/night from the day of oocyte retrieval.
1240
Zhu et al.
lithotomy position. The probe was introduced into the vagina as gently as possible to avoid stimulating the cervix. After scanning the mid-sagittal plane of the uterus, the probe was fixed as steady as possible while a 5 min video of uterine peristalsis was recorded simultaneously. The records were analysed at 4× regular speed using a VLC media player 1.1.11 (VideoLAN team) by two independent experienced observers (L.Z. and L.X.). The uterine peristaltic wave frequency was the mean of the results from each observer. Both observers were blind to the treatment condition (fresh cycle, natural FET cycle or artificial FET cycle) of all patients.
Statistical analysis
Results The mean age of this population was 29.1 + 3.5 years and the duration of infertility ranged from 1–15 years (mean + SD: 4.5 + 2.9). A total of 144 patients had primary infertility and 148 had secondary infertility. The intraclass correlation coefficient was 0.988 for inter-observer
Table I Demographic comparison of pregnancy group versus non-pregnancy group. Characteristic
Clinical pregnancy (n 5 156)
Non-pregnancy (n 5 136)
P-value
............................................................................................................................................................................................. Age (year)
29.1 + 3.4
29.0 + 3.7
0.720a
Duration of infertility (year)
4.2 + 2.9
4.8 + 3.0
0.102a
Fresh cycle
95 (60.9)
71 (52.2)
0.084b
Natural FET cycle
38 (24.4)
31 (22.8)
Treatment cycle (%)
Artificial FET cycle
23 (14.7)
34 (25.0)
Basal serum FSH (IU/l)
6.4 + 2.2
6.3 + 2.0
0.790a
Basal serum LH (IU/l)
5.2 + 3.2
5.5 + 4.0
0.473a
Basal serum E2 (pg/ml)
41.2 + 29.7
40.5 + 38.1
0.854a
Serum E2 on embryo transfer day (pg/ml)
1068.3 + 973.0
947.1 + 903.6
0.273a
Serum P on embryo transfer day (ng/ml)
56.3 + 11.5
52.8 + 15.2
0.030a
Fresh cycle
58.3 + 9.3
59.0 + 7.2
0.582a
Natural FET cycle
53.2 + 13.6
48.4 + 16.4
0.188a
Artificial FET cycle
53.0 + 14.5
43.8 + 20.0
0.049a
Endometrium thickness on embryo transfer day (mm)
11.3 + 2.7
11.2 + 3.0
0.761a
Good-quality embryo rate (%)
68.6 + 22.9
72.9 + 21.9
0.104a
a
Two-sample t-test. Pearson x 2.
b
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
Statistical analyses were carried out using SPSS version 19.0. Continuous data were presented as mean and standard deviation (SD). A two-sample t-test was used to determine whether the uterine peristaltic wave frequency on embryo transfer day differs in the pregnancy and non-pregnancy patients. Student – Newman – Keuls (SNK) test was used in comparison of every combination of group pairs (fresh versus natural FET cycle, fresh versus artificial FET cycle and natural FET versus artificial FET cycle) for uterine peristaltic wave frequency as well as serum progesterone. Inter-observer agreement of uterine peristaltic wave frequency was evaluated with intraclass correlation coefficient. Categorical variables were compared by the x 2 test. Binary logistic regression analysis (enter) was conducted to test for association between demographic variables and clinical pregnancy outcome. The cut-off value of uterine peristalsis in predicting clinical pregnancy outcome was identified by ROC curve analysis.
agreement of uterine peristaltic wave frequency, meaning that the agreement between the two readers was very good. The demographic characteristics of the study subjects according to clinical pregnancy outcomes are detailed in Table I. No difference was found between the two groups, except for the serum progesterone concentration on the day of embryo transfer. All of the patients were subjectively divided into five groups according to uterine peristaltic wave frequency on embryo transfer day: ≤1.0, 1.1 – 2.0, 2.1–3.0, 3.1– 4.0 and .4.0 waves/min. Most patients were distributed into group 1.1 –2.0 and 2.1– 3.0. The clinical pregnancy rate was the highest when no more than 2.0 waves/min were observed before embryo transfer and it decreased with increasing wave frequency thereafter, with a dramatic decrease when .3.0 waves/min were observed (Table II). As shown in Table III, uterine peristaltic wave frequencies of nonpregnancy patients were significantly higher than that of clinical pregnancy patients no matter whether they underwent fresh embryo transfer, natural FET or artificial FET cycles, respectively. Additionally, the uterine peristaltic wave frequency before embryo transfer seemed to be a little lower in the fresh cycles (1.80 + 0.77 waves/min) than in FET cycles (natural FET 2.05 + 0.80 waves/min and artificial FET 2.15 + 0.81 waves/min). But the differences in the comparison of every combination of group pairs were not significantly different. Eight patients were diagnosed with ectopic pregnancy in our study, and the uterine peristaltic wave frequency of those patients showed a higher trend compared with those with intrauterine pregnancy (2.21 + 1.20 versus 1.73 + 0.65 waves/min), although the difference did not reach statistical significance (P ¼ 0.29). Binary logistic regression analysis revealed that the uterine peristaltic wave frequency on the day of embryo transfer was inversely related to the clinical pregnancy rate. Women with high uterine peristaltic wave frequency were less likely to get pregnant than those with a relatively low
1241
Uterine peristalsis and pregnancy rates
Table II Clinical pregnancy outcomes of different uterine peristaltic wave frequency groups. Uterine peristaltic wave frequency (waves/min)
n
Clinical pregnancy
Non-pregnancy
Clinical pregnancy rate (%)
x2
P
............................................................................................................................................................................................. ≤1.0
39
23
16
58.97
1.1– 2.0
118
75
43
63.55
2.1– 3.0
117
57
60
48.71
3.1– 4.0
16
1
15
6.25
2
0
2
0.00
.4.0
Cycles
Uterine peristaltic wave frequency (waves/min)
and
b
1.69 + 0.69
Natural FET
1.82 + 0.66
1.95 + 0.85 2.34 + 0.86
Odds ratio
95% CI
P
........................................................................................ Age
Non-pregnancy (n 5 136)
........................................................................................ Fresh embryo transfer
Table IV Binary logistic regression analysis of factors potentially associated with the occurrence of clinical pregnancy.
P
.....................................................
Clinical pregnancy (n 5 156)
,0.001
0.033
Duration of infertility
0.033
1.03
0.96– 1.12
0.405
20.072
0.93
0.85– 1.02
0.125
Basal serum FSH
20.005
1.00
0.88– 1.13
0.942
Basal serum LH
20.046
0.96
0.89– 1.03
0.230
0.006
Basal serum E2
0.002
1.00
0.99– 1.01
0.649
Serum E2 on embryo transfer day
0.000
1.00
1.00– 1.00
0.261
Serum P on embryo transfer day
0.016
1.02
1.00– 1.04
0.139
Endometrium thickness on embryo transfer day
0.033
1.03
0.94– 1.14
0.492
Artificial FET
1.82 + 0.59
2.38 + 0.87
0.010
Total
1.74 + 0.67
2.14 + 0.88
,0.001
wave frequency. The other demographic characteristics included in the analysis were not significantly associated with the chance of clinical pregnancy (Table IV). The cut-off value of uterine peristalsis was 2.45 waves/ min in predicting clinical pregnancy outcome. The follow-up showed that up to now, of the 156 clinically pregnant cases, 70 cases ended up with live birth, 52 pregnancies were still in ongoing, 9 cases were lost of follow-up, 8 cases had surgery to terminate ectopic pregnancies, 13 cases underwent spontaneous abortion and 4 cases had induced abortion because of fetal malformation or complication.
Discussion In this prospective cohort study, we found a significant, independent association between uterine peristaltic wave frequency on the day of embryo transfer and clinical pregnancy outcomes. To the best of our knowledge, this is the first comprehensive study which included both fresh cycles, natural FET cycles and artificial FET cycles to further explore the possible role of uterine peristalsis in embryo implantation. The demographic characteristics of the study subjects were comparable (P . 0.05) between the pregnancy and non-pregnancy groups, except for serum progesterone levels on the day of embryo transfer (P ¼ 0.030) (Table I). Although the serum progesterone level was significantly different between the groups, they were both .40 ng/ml which is much higher than the physiological level of mid-luteal phase (5.97– 14.15 ng/ml) in a natural cycle (John et al., 2008). Furthermore, logistic regression analysis showed that the serum progesterone level on the day of embryo transfer did not influence the clinical pregnancy rate in our
Good-quality embryo rate
20.006
0.99
0.98– 1.01
0.277
Types of ART cycles
20.251
0.78
0.49– 1.23
0.279
Uterine peristaltic wave frequency on embryo transfer day
20.714
0.49
0.34– 0.70
,0.001
Clinical pregnancy outcome was the dependent binary variable with clinical pregnancy coded as 1 (positive) and non-pregnancy coded as 0 (negative). b , 0 and OR , 1 means clinical pregnancy outcome is inclined to be negative.
study, implying that the luteal support was adequate for pregnancy occurrence (Table IV). Before embryo transfer, uterine peristalsis of most patients was maintained at a low level of ,3.0 waves/min, similar to the phenomenon observed in a natural cycle (IJland et al., 1997a,b; Bulletti et al., 2000). In line with this, the clinical pregnancy rate was much higher in these patients compared with those with a frequency of .3.0 waves/min, indicating a beneficial effect of low frequency uterine peristalsis on pregnancy (Table II). Our result is in accordance with the study of Fanchin et al. (1998), who also found a stepwise decrease in clinical pregnancy rates with increasing uterine contractions before embryo transfer (53, 46, 23 and 14% for group ≤3.0, 3.1–4.0, 4.1–5.0 and .5.0, respectively). The discrepancy in grouping may be attributed to inter-subject, observer and cycle differences. It is noteworthy that in our previous study (Zhu et al., 2014), the uterine peristalsis of those who expelled the transferred fluid was also .3.0 waves/min. Thus, the result obtained in this study, together with previously published studies, suggested that patients with the uterine
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
Table III Comparison of uterine peristaltic wave frequencies between clinical pregnancy non-pregnancy women in different cycles.
23.0
1242
system (Kunz et al., 2006). Therefore, we have reason to believe that multiple corpora luteum in fresh cycles secreted more progesterone which was transferred into the uterus via the utero-ovarian countercurrent system, providing more effective function than the FET cycles which relied on systemic progesterone. The uterine peristaltic wave frequency before embryo transfer in the non-pregnant group was 2.14 + 0.88 waves/min, which seemed to be moderate for embryo implantation. Nevertheless, if the embryo transfer procedure is taken into consideration, uterine peristalsis might be enhanced thereafter (Lesny et al., 1998; Zhu et al., 2014). In the present study, we did not observe uterine peristalsis after embryo transfer in case any possible negative effect disturbed embryo implantation or caused psychological stress for the patients. In our previous study, an approximate 36% increase in uterine peristalsis was observed after mock embryo transfer, no matter whether the fluid was kept inside the uterine cavity or extruded outside (Zhu et al., 2014). When we take a rough estimate of the uterine peristalsis after embryo transfer in the present study accordingly, it would be 2.91 waves/min. ROC curve analysis showed that the cut-off value of uterine peristalsis was 2.45 waves/ min in predicting clinical pregnancy outcome, which was a medium between 2.14 and 2.91 waves/min. This deduction sheds light on the predictive validity of the cut-off value and places it as a promising quantitative marker of uterine receptivity and pregnancy outcome in an embryo transfer cycle. Moreover, binary logistic regression analysis demonstrated that the association between uterine peristaltic wave frequency before embryo transfer and clinical pregnancy was independently significant (odds ratio: 0.49; 95% confidence interval: 0.34 –0.70, P , 0.001). We believe that further investigations are necessary to testify to the effectiveness of uterine peristalsis in predicting clinical pregnancy before definitive conclusions can be drawn. Subjective and objective methods were both applied in uterine peristaltic wave counting. Although several studies (Fanchin et al., 1998; Oczeretko et al., 2006; Meirzon et al., 2011) have explored objective methods to reduce the influence of the subjective factor, the complicated approach and professional technology requirement prohibits their widespread use and application. It is also noteworthy that computer analysed results do not deserve complete trust since a subtle movement of the ultrasound probe may induce a false wave for analysis which could be avoided by dynamic analysis of uterine peristalsis video. Furthermore, comparisons of counting uterine peristaltic wave frequencies of accelerated images and other objective methods have shown that they are equivalent (Ayoubi et al., 2001; Nakai et al., 2004). Thus, with the rich experience accumulated in our preliminary experiments and previous studies, we chose the classic and convenient method to assess uterine peristalsis. One limitation of the present study was the lack of uterine peristaltic wave type analysis which is also an important parameter to assess uterine activity. Previous studies (Kunz et al., 1996; Leyendecker et al., 1996; IJland et al., 1997a; Fanchin et al., 1998; Bulletti et al., 2000; Nakai et al., 2008; Zhu et al., 2012) have reached a consensus on the percentage distribution of uterine peristalsis wave types in menstrual cycle, i.e. cervicofundal and fundocervical waves took a predominant and second position, respectively, with other types showing lower proportions. Nevertheless, it was too complicated to tell the definite or even possible function of each type. We speculate that the cervicofundal waves may counteract the effect of waves of other types and function predominantly. Thus, we focused on the frequency of uterine peristalsis in the present
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
peristalsis of ,3.0 waves/min before embryo transfer have a better chance of becoming pregnant. Our current findings also confirmed that embryo transfer cycles which ended with successful pregnancy showed less uterine peristalsis before embryo transfer than non-pregnant cycles (Table III), consistent previous research (IJland et al., 1997a). It may be because the intense uterine peristalsis adversely affects embryo migration after embryo transfer and extrudes it out of the uterine cavity, resulting in implantation failure or ectopic pregnancy. Recently, the application of oxytocin antagonists to selectively decrease the uterine activity was applied with success (Pierzynski et al., 2007; Moraloglu et al., 2010; Pierzynski, 2011). Other research has also tried to improve pregnancy rates by suppressing uterine peristalsis with various agents (Moon et al., 2004; Nakai et al., 2008; Kido et al., 2009; Xu et al., 2013). However molecular mechanisms modulating uterine peristalsis around the implantation window are awaiting further study which may contribute to an improvement in endometrial receptivity and pregnancy rates. The incidence of ectopic pregnancy in this population was 2.7%, similar to the reported ectopic rate following assisted reproductive technologies (Schippert et al., 2012). Although the uterine peristaltic wave frequency was a little higher in the ectopic pregnancy group than those with intrauterine pregnancy, the difference was not significant. This may be due to the uneven distribution of sample size in the ectopic pregnancy group (8 cases) and intrauterine pregnancy group (148 cases). However, it was contrary to our expectation that the uterine peristaltic wave frequency on the day of embryo transfer showed an uptrend in fresh, natural FET and artificial FET cycles, although the differences for comparison of each combination of group pairs did not reach statistical significance. Previous investigators have found that progesterone had an inhibitory action on mediating uterine peristalsis (Fanchin et al., 1998; Mueller et al., 2006; Zhu et al., 2012). In the present study, serum progesterone levels on the day of embryo transfer were 58.6 + 8.5, 51.0 + 15.0 and 47.5 + 18.4 ng/ml for fresh, natural FET and artificial FET cycles. Statistical analysis showed that the serum progesterone levels in the fresh cycles were significantly different from that in the natural FET and artificial FET cycles, while the difference between the natural FET and artificial FET cycles was not significant. The decreasing supraphysiological serum progesterone may only partly account for the uptrend of uterine peristalsis, since we found that uterine peristalsis was not correlated with supraphysiological concentrations of progesterone in our previous study (Zhu et al., 2012). Another reasonable explanation might be the endogenous corpus luteal function in different cycles. In a fresh cycle, controlled ovarian stimulation was applied to obtain a cohort of co-dominant follicles, and multiple corpora lutea were formed after the collection of oocytes. But in a natural FET cycle, only one corpus luteum produces progesterone after ovulation; an artificial cycle is totally dependent upon on exogenous luteal support. In our study, even though high doses of progesterone were given to the patients of all cycles reaching supraphysiological concentrations of serum progesterone, the function of local progesterone derived from corpus luteum could not be underestimated. The utero-ovarian counter-current system (Einer-Jensen, 1988; Kunz et al., 1998) plays an important role in local endocrine regulation, especially in guiding sperm transport preferentially into the oviduct ipsilateral to the dominant follicle (Kunz et al., 1996, 1998; Wildt et al., 1998; Kissler et al., 2004). In addition, gestational sacs inclined to locate within the uterine horn ipsilateral to the corpus luteum are also closely related to the utero-ovarian counter-current
Zhu et al.
Uterine peristalsis and pregnancy rates
study and did not analyse the uterine peristalsis wave types. In order to clarify the function of uterine peristalsis of different wave types, we think it would be better to design a more rigorous study in the future. In conclusion, the uterine peristaltic wave frequency before embryo transfer was inversely related to the clinical pregnancy in fresh and frozen-thawed embryo transfer cycles. Uterine peristalsis of ,3.0 waves/min before embryo transfer indicates a favourable pregnancy outcome. This may represent a convenient, although invasive, indicator of uterine receptivity and pregnancy outcome in an embryo transfer cycle.
Authors’ roles
Funding The study is supported by Hunan Provincial Innovation Foundation for Postgraduates.
Conflict of interest The authors declare that they have no competing interests in this study.
References Ayoubi JM, Fanchin R, Kaddouz D, Frydman R, de Ziegler D. Uterorelaxing effects of vaginal progesterone: comparison of two methodologies for assessing uterine contraction frequency on ultrasound scans. Fertil Steril 2001;76:736 – 740. Bulletti C, de Ziegler D. Uterine contractility and embryo implantation. Curr Opin Obstet Gynecol 2006;18:473 – 484. Bulletti C, de Ziegler D, Polli V, Diotallevi L, Del Ferro E, Flamigni C. Uterine contractility during the menstrual cycle. Hum Reprod 2000;15:81 – 89. Einer-Jensen N. Countercurrent transfer in the ovarian pedicle and its physiological implications. Oxf Rev Reprod Biol 1988;10:348 – 381. 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 – 1974. IJland MM, Evers JL, Dunselman GA, Volovics L, Hoogland HJ. Relation between endometrial wavelike activity and fecundability in spontaneous cycles. Fertil Steril 1997a;67:492 – 496. IJland MM, Evers JL, Hoogland HJ. Velocity of endometrial wavelike activity in spontaneous cycles. Fertil Steril 1997b;68:72– 75. John OS, Joseph IS, Lisa MH, Barbara LH, Karen DB, Gary C. Reproductive Endocrinology. Williams Gynecology. 2008. McGraw-Hill companies. Kido A, Togashi K, Hatayama H, Nakayama T, Yamamoto A, Kataoka M, Tulandi T. Uterine peristalsis in women with repeated IVF failures: possible therapeutic effect of hyoscine Bromide. J Obstet Gynaecol Can 2009;31:732 – 735. Kissler S, Siebzehnruebl E, Kohl J, Mueller A, Hamscho N, Gaetje R, Ahr A, Rody A, Kaufmann M. Uterine contractility and directed sperm transport assessed by hysterosalpingoscintigraphy (HSSG) and intrauterine pressure (IUP) measurement. Acta Obstet Gynecol Scand 2004;83:369 – 374.
Kunz G, Beil D, Deininger H, Wildt L, Leyendecker G. The dynamics of rapid sperm transport through the female genital tract: evidence from vaginal sonography of uterine peristalsis and hysterosalpingoscintigraphy. Hum Reprod 1996;11:627 – 632. Kunz G, Herbertz M, Noe M, Leyendecker G. Sonographic evidence for the involvement of the utero-ovarian counter-current system in the ovarian control of directed uterine sperm transport. Hum Reprod Update 1998; 4:667 – 672. Kunz G, Beil D, Huppert P, Leyendecker G. Control and function of uterine peristalsis during the human luteal phase. Reprod Biomed Online 2006; 13:528– 540. Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer— can we learn anything new from the observation of junctional zone contractions? Hum Reprod 1998;13:1540– 1546. Leyendecker G, Kunz G, Wildt L, Beil D, Deininger H. Uterine hyperperistalsis and dysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patients with endometriosis and infertility. Hum Reprod 1996;11:1542 – 1551. Meirzon D, Jaffa AJ, Gordon Z, Elad D. A new method for analysis of non-pregnant uterine peristalsis using transvaginal ultrasound. Ultrasound Obstet Gynecol 2011;38:217– 224. Moon HS, Park SH, Lee JO, Kim KS, Joo BS. Treatment with piroxicam before embryo transfer increases the pregnancy rate after in vitro fertilization and embryo transfer. Fertil Steril 2004;82:816– 820. Moraloglu O, Tonguc E, Var T, Zeyrek T, Batioglu S. Treatment with oxytocin antagonists before embryo transfer may increase implantation rates after IVF. Reprod Biomed Online 2010;21:338– 343. Mueller A, Siemer J, Schreiner S, Koesztner H, Hoffmann I, Binder H, Beckmann MW, Dittrich R. Role of estrogen and progesterone in the regulation of uterine peristalsis: results from perfused non-pregnant swine uteri. Hum Reprod 2006;21:1863 – 1868. Nakai A, Togashi K, Kosaka K, Kido A, Hiraga A, Fujiwara T, Koyama T, Fujii S. Uterine peristalsis: comparison of transvaginal ultrasound and two different sequences of cine MR imaging. J Magn Reson Imaging 2004; 20:463– 469. Nakai A, Togashi K, Kosaka K, Kido A, Kataoka M, Koyama T, Fujii S. Do anticholinergic agents suppress uterine peristalsis and sporadic myometrial contractions at cine MR imaging? Radiology 2008;246:489–496. Oczeretko E, Swiatecka J, Kitlas A, Laudanski T, Pierzynski P. Visualization of synchronization of the uterine contraction signals: running crosscorrelation and wavelet running cross-correlation methods. Med Eng Phys 2006;28:75–81. Pierzynski P. Oxytocin and vasopressin V (1A) receptors as new therapeutic targets in assisted reproduction. Reprod Biomed Online 2011;22:9– 16. Pierzynski P, Reinheimer TM, Kuczynski W. Oxytocin antagonists may improve infertility treatment. Fertil Steril 2007;88:213.e19 – 22. Schippert C, Soergel P, Staboulidou I, Bassler C, Gagalick S, Hillemanns P, Buehler K, Garcia-Rocha GJ. The risk of ectopic pregnancy following tubal reconstructive microsurgery and assisted reproductive technology procedures. Arch Gynecol Obstet 2012;285:863 – 871. Wildt L, Kissler S, Licht P, Becker W. Sperm transport in the human female genital tract and its modulation by oxytocin as assessed by hysterosalpingoscintigraphy, hysterotonography, electrohysterography and Doppler sonography. Hum Reprod Update 1998;4:655 – 666. Xu A, Li Y, Zhu L, Tian T, Hao J, Zhao J, Zhang Q. Inhibition of endometrial fundocervical wave by phloroglucinol and the outcome of in vitro fertilization. Reprod Biol 2013;13:88 – 91. Zhu L, Li Y, Xu A. Influence of controlled ovarian hyperstimulation on uterine peristalsis in infertile women. Hum Reprod 2012;27:2684 – 2689. Zhu L, Xiao L, Che HS, Li YP, Liao JT. Uterine peristalsis exerts control over fluid migration after mock embryo transfer. Hum Reprod 2014; 29:279– 285.
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
L.Z. contributed to the study design, transvaginal ultrasonographic scans, data analysis and drafting of the manuscript. H.S.C. performed the follow-up studies and statistical analysis of the data. L.X. participated in the acquisition and analysis of data. Y.P.L. participated in the study conception and design as well as the embryo transfer procedure. All authors contributed to the production of the manuscript.
1243
ORIGINAL ARTICLE Infertility
Uterine peristalsis before embryo transfer affects the chance of clinical pregnancy in fresh and frozen-thawed embryo transfer cycles Department of Reproductive Medicine, Xiangya Hospital, Central-south University, Changsha City, Hunan Province, P.R. China *Correspondence address. Department of Reproductive Medicine, Xiangya Hospital, 87 Xiangya Road, Changsha City, Hunan Province, P.R. China. E-mail: [email protected]
Submitted on November 30, 2013; resubmitted on February 22, 2014; accepted on February 26, 2014
study question: Does uterine peristalsis influence the chance of clinical pregnancy in an embryo transfer cycle? summaryanswer: The uterine peristaltic wave frequency before embryo transfer is inversely related to the clinical pregnancy rates in fresh and frozen-thawed embryo transfer cycles.
what is known already: Uterine peristalsis participates in regulating fluid migration after mock embryo transfer, but whether it could potentially influence pregnancy outcomes had remained unclear. study design, size, duration: This prospective cohort study included a total of 292 infertile women and was conducted between March 2013 and August 2013.
participants/materials, setting, methods: Patients underwent fresh embryo transfer in a fresh stimulation cycle with a long down-regulation protocol, a natural frozen-thawed embryo transfer cycle or an artificial frozen-thawed embryo transfer cycle. Uterine peristaltic activity was assessed before embryo transfer by transvaginal ultrasonography.
main results and the role of chance: The uterine peristaltic wave frequencies of most patients were between 1.1 and 3.0 waves/min before embryo transfer (ET). The clinical pregnancy rate was the highest when ,2.0 waves/min was observed and it decreased with an increasing wave frequency thereafter, with an especially dramatic decrease with .3.0 waves/min. Uterine peristaltic wave frequencies of the non-pregnant patient group were higher than that of the clinically-pregnant patient group in all types of transfer, fresh embryo transfer, natural FET or artificial FET cycle. Binary logistic regression analysis demonstrated that the association between uterine peristaltic wave frequency before embryo transfer and clinical pregnancy was independently significant (odds ratio: 0.49; 95% confidence interval: 0.34 –0.70, P , 0.001). limitations, reasons for caution: Uterine peristalsis after embryo transfer was not observed in case any possible negative effect of the observation disturbed embryo implantation or caused psychological stress. Uterine peristalsis after embryo transfer may differ from that before embryo transfer. Another limitation of the present study was the lack of uterine peristaltic wave type analysis which is also an important parameter to assess uterine activity. wider implications of the findings: Patients with uterine peristalsis of ,3.0 waves/min before embryo transfer had a higher chance of pregnancy compared with those with higher frequencies. This could be a promising quantitative marker of uterine receptivity and pregnancy outcome in an embryo transfer cycle. The predictive validity of the cut-off value needs to be tested in further study. study funding/competing interest(s): The study is supported by Hunan Provincial Innovation Foundation for Postgraduates. The authors declare that they have no competing interests in this study. Key words: uterine peristalsis / uterine receptivity / clinical pregnancy / assisted reproductive technology
& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
L. Zhu, H.S. Che, L. Xiao, and Y.P. Li*
1239
Uterine peristalsis and pregnancy rates
Introduction
Materials and Methods Patients From March 2013 to August 2013, all patients undergoing IVF/ICSI-ET and FET treatment at the Department of Reproductive Medicine at Xiangya Hospital of Central-south University were offered participation in the study. To limit the interference of confounding variables affecting embryo quality and/or uterine receptivity, patients were only selected if they (i) were 20 – 35 years of age; (ii) had a normal uterus; (iii) had two good-quality day-3 cleavage stage embryos available for transfer (good-quality day-3 embryos were defined by at least seven cells, ,20% fragmentation, and no apparent morphological abnormalities); and (iv) underwent easy embryo transfer (i.e. the catheter was smoothly inserted without touching the fundus, no cervix tenaculum was used and the catheter was clean of blood). After excluding 13 patients due to cancellation of embryo transfer (because of ovarian hyperstimulation syndrome or hydrohystera), a change to blastocyst transfer, or the use of another drug intervention, a total of 166 patients underwent fresh embryo transfer and 126 patients received either natural FET (n ¼ 69) or artificial FET (n ¼ 57) were analysed. Written informed consent was obtained from each patient before inclusion. The study was approved by the institutional review board (Clinical Trial Registration Number: ChiCTR-OCH-13003105).
Cryopreservation cycles In the natural FET cycles, the development of the dominant follicle and endometrium was monitored from Day 10 by regular vaginal ultrasound, urine LH tests and serum LH, E2 and progesterone measurements until ovulation. If the diameter of the dominant follicle was ,14 mm, the patient was asked to return every second day. If the dominant follicle was ≥14 mm, a urine LH test was performed and the patient was asked to return the next day. When the diameter of the dominant follicle was ≥18 mm or the urine LH test was .45 mIU/ml, serum LH, E2 and progesterone concentrations were measured to help estimate the maturation of the dominant follicle, predict ovulation and find out whether the follicle was luteinized. If the serum progesterone concentration was .1.0 ng/ml and the follicle was still unruptured, which was usually accompanied by heterogeneous echo of the follicular fluid, luteinized unruptured follicle was identified and that day was taken as the ovulation day. To preclude luteal phase defect, luteal phase support was provided from the day of ovulation day progesterone injection 40 mg/day and added progesterone soft capsules at a dose of 200 mg/night from the day of embryo transfer. In the artificial FET cycles, patients received increasing doses (6 mg for days 3 – 7, 8 mg for 2 days, and 10 mg for 2 days thereafter) of estradiol valerate (Progynova, Bayer) in order to prepare the endometrium for embryo transfer. Endometrial thickness was monitored by transvaginal ultrasound every time before adjusting the doses of estradiol valerate, and when the endometrium reached or exceeded 7 mm, increasing doses (20 mg for 1 day, 40 mg for 1 day and 60 mg for 1 day thereafter) of progesterone were given intramuscularly once per day. Progesterone soft capsules at a dose of 200 mg/ night were administered orally from the day of embryo transfer.
Embryo transfer Two good-quality embryos were transferred under abdominal ultrasound guidance 3 days after oocyte retrieval in a fresh cycle, 2 days after ovulation in a natural FET cycle and 3 days after progesterone administration in an artificial FET cycle, respectively. All of the embryo transfers were performed by an experienced doctor (Y.P.L.), who was blind to the frequency of uterine peristalsis, using the same type of catheter (CCD131230301, Ultrasoft Frydman Set with Guide, Laboratoire C.C.D., France). Blood samples were collected on the day of embryo transfer to determine the serum E2 and P using electrochemiluminescence immunoassays. Approximately 80% of all fresh cycles were conducted as IVF and 20% were conducted as ICSI/half-ICSI. Patients were confirmed to be clinically pregnant by ultrasonographic visualization of a gestational sac 2 – 3 weeks after a positive pregnancy test. Luteal support was continued either until 1 month after confirmation of intrauterine pregnancy or until pregnancy was ruled out by a negative serum HCG measurement, in all types of treatment cycles.
Fresh cycles
Observation and assessment of uterine peristalsis
Patients underwent ovarian stimulation in a long down-regulation protocol using urinary FSH (Urofollitropin, Livzon, China), recombinant follitropin alfa (Gonal-F, Merck Serono) or recombinant follitropin beta (Puregon,
Transvaginal ultrasonography scans (Mindray DC-6 Expert, 5 – 8 MHz transducer) of uterine peristalsis were performed by a single examiner (L.Z.) about 1 h before embryo transfer while the patient was lying relaxed in a
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
Controlled ovarian stimulation improvements and technological advancements in the laboratory have provided adequate oocytes and high-quality embryos to infertile couples, leading to steadily increased success rates for IVF over the years. However comparatively speaking, considerably fewer efforts have been devoted to improving uterine receptivity, especially in clinical practice. In contrast with an invasive endometrium biopsy, uterine peristalsis can be directly visualized by ultrasonography and has therefore broadened the horizon for exploring uterine receptivity in human reproduction. During the luteal phase in a natural cycle, uterine activity reaches an absolute minimum and the reduced uterine peristalsis is believed to facilitate embryo implantation (Ijland et al., 1997a; Bulletti et al., 2000; Bulletti and de Ziegler, 2006). Studies of mock embryo transfer (ET) have also testified to the important role of uterine peristalsis in controlling the migration of embryo analogues. Lesny et al. (1998) observed that the transferred fluid could be expelled out of the uterine cavity by strong uterine contractions after touching the uterine fundus with the catheter. Our previously published study also found that uterine peristaltic wave frequency was positively correlated with the distance that fluid moved after it was deposited in the uterine cavity (Zhu et al., 2014). Since uterine peristalsis can exert an influence on the drift of an embryo analogue during the implantation window, we hypothesized that it may also have an effect on the subsequent clinical pregnancy outcome in a real embryo transfer cycle. This inspired us to comprehensively analyse the relationship between uterine peristalsis just before embryo transfer and the clinical pregnancy rates under different conditions, including fresh embryo transfer, natural frozen-thawed embryo transfer (FET) and artificial FET cycles.
Merck Sharp & Dohme). FSH doses were adjusted individually according to the patient’s ovarian response as assessed by ultrasound and hormonal measurements performed every 1 – 3 days. A dose of 5000 – 10 000 IU of human chorionic gonadotrophin (HCG, Livzon, China) was administered when at least three follicles were ≥18 mm in diameter. Oocyte retrieval was conducted transvaginally 34 – 36 h after the HCG trigger. The luteal phase was supported with progesterone injections at a dose of 60 mg/day and progesterone soft capsules at a dose of 200 mg/night from the day of oocyte retrieval.
1240
Zhu et al.
lithotomy position. The probe was introduced into the vagina as gently as possible to avoid stimulating the cervix. After scanning the mid-sagittal plane of the uterus, the probe was fixed as steady as possible while a 5 min video of uterine peristalsis was recorded simultaneously. The records were analysed at 4× regular speed using a VLC media player 1.1.11 (VideoLAN team) by two independent experienced observers (L.Z. and L.X.). The uterine peristaltic wave frequency was the mean of the results from each observer. Both observers were blind to the treatment condition (fresh cycle, natural FET cycle or artificial FET cycle) of all patients.
Statistical analysis
Results The mean age of this population was 29.1 + 3.5 years and the duration of infertility ranged from 1–15 years (mean + SD: 4.5 + 2.9). A total of 144 patients had primary infertility and 148 had secondary infertility. The intraclass correlation coefficient was 0.988 for inter-observer
Table I Demographic comparison of pregnancy group versus non-pregnancy group. Characteristic
Clinical pregnancy (n 5 156)
Non-pregnancy (n 5 136)
P-value
............................................................................................................................................................................................. Age (year)
29.1 + 3.4
29.0 + 3.7
0.720a
Duration of infertility (year)
4.2 + 2.9
4.8 + 3.0
0.102a
Fresh cycle
95 (60.9)
71 (52.2)
0.084b
Natural FET cycle
38 (24.4)
31 (22.8)
Treatment cycle (%)
Artificial FET cycle
23 (14.7)
34 (25.0)
Basal serum FSH (IU/l)
6.4 + 2.2
6.3 + 2.0
0.790a
Basal serum LH (IU/l)
5.2 + 3.2
5.5 + 4.0
0.473a
Basal serum E2 (pg/ml)
41.2 + 29.7
40.5 + 38.1
0.854a
Serum E2 on embryo transfer day (pg/ml)
1068.3 + 973.0
947.1 + 903.6
0.273a
Serum P on embryo transfer day (ng/ml)
56.3 + 11.5
52.8 + 15.2
0.030a
Fresh cycle
58.3 + 9.3
59.0 + 7.2
0.582a
Natural FET cycle
53.2 + 13.6
48.4 + 16.4
0.188a
Artificial FET cycle
53.0 + 14.5
43.8 + 20.0
0.049a
Endometrium thickness on embryo transfer day (mm)
11.3 + 2.7
11.2 + 3.0
0.761a
Good-quality embryo rate (%)
68.6 + 22.9
72.9 + 21.9
0.104a
a
Two-sample t-test. Pearson x 2.
b
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
Statistical analyses were carried out using SPSS version 19.0. Continuous data were presented as mean and standard deviation (SD). A two-sample t-test was used to determine whether the uterine peristaltic wave frequency on embryo transfer day differs in the pregnancy and non-pregnancy patients. Student – Newman – Keuls (SNK) test was used in comparison of every combination of group pairs (fresh versus natural FET cycle, fresh versus artificial FET cycle and natural FET versus artificial FET cycle) for uterine peristaltic wave frequency as well as serum progesterone. Inter-observer agreement of uterine peristaltic wave frequency was evaluated with intraclass correlation coefficient. Categorical variables were compared by the x 2 test. Binary logistic regression analysis (enter) was conducted to test for association between demographic variables and clinical pregnancy outcome. The cut-off value of uterine peristalsis in predicting clinical pregnancy outcome was identified by ROC curve analysis.
agreement of uterine peristaltic wave frequency, meaning that the agreement between the two readers was very good. The demographic characteristics of the study subjects according to clinical pregnancy outcomes are detailed in Table I. No difference was found between the two groups, except for the serum progesterone concentration on the day of embryo transfer. All of the patients were subjectively divided into five groups according to uterine peristaltic wave frequency on embryo transfer day: ≤1.0, 1.1 – 2.0, 2.1–3.0, 3.1– 4.0 and .4.0 waves/min. Most patients were distributed into group 1.1 –2.0 and 2.1– 3.0. The clinical pregnancy rate was the highest when no more than 2.0 waves/min were observed before embryo transfer and it decreased with increasing wave frequency thereafter, with a dramatic decrease when .3.0 waves/min were observed (Table II). As shown in Table III, uterine peristaltic wave frequencies of nonpregnancy patients were significantly higher than that of clinical pregnancy patients no matter whether they underwent fresh embryo transfer, natural FET or artificial FET cycles, respectively. Additionally, the uterine peristaltic wave frequency before embryo transfer seemed to be a little lower in the fresh cycles (1.80 + 0.77 waves/min) than in FET cycles (natural FET 2.05 + 0.80 waves/min and artificial FET 2.15 + 0.81 waves/min). But the differences in the comparison of every combination of group pairs were not significantly different. Eight patients were diagnosed with ectopic pregnancy in our study, and the uterine peristaltic wave frequency of those patients showed a higher trend compared with those with intrauterine pregnancy (2.21 + 1.20 versus 1.73 + 0.65 waves/min), although the difference did not reach statistical significance (P ¼ 0.29). Binary logistic regression analysis revealed that the uterine peristaltic wave frequency on the day of embryo transfer was inversely related to the clinical pregnancy rate. Women with high uterine peristaltic wave frequency were less likely to get pregnant than those with a relatively low
1241
Uterine peristalsis and pregnancy rates
Table II Clinical pregnancy outcomes of different uterine peristaltic wave frequency groups. Uterine peristaltic wave frequency (waves/min)
n
Clinical pregnancy
Non-pregnancy
Clinical pregnancy rate (%)
x2
P
............................................................................................................................................................................................. ≤1.0
39
23
16
58.97
1.1– 2.0
118
75
43
63.55
2.1– 3.0
117
57
60
48.71
3.1– 4.0
16
1
15
6.25
2
0
2
0.00
.4.0
Cycles
Uterine peristaltic wave frequency (waves/min)
and
b
1.69 + 0.69
Natural FET
1.82 + 0.66
1.95 + 0.85 2.34 + 0.86
Odds ratio
95% CI
P
........................................................................................ Age
Non-pregnancy (n 5 136)
........................................................................................ Fresh embryo transfer
Table IV Binary logistic regression analysis of factors potentially associated with the occurrence of clinical pregnancy.
P
.....................................................
Clinical pregnancy (n 5 156)
,0.001
0.033
Duration of infertility
0.033
1.03
0.96– 1.12
0.405
20.072
0.93
0.85– 1.02
0.125
Basal serum FSH
20.005
1.00
0.88– 1.13
0.942
Basal serum LH
20.046
0.96
0.89– 1.03
0.230
0.006
Basal serum E2
0.002
1.00
0.99– 1.01
0.649
Serum E2 on embryo transfer day
0.000
1.00
1.00– 1.00
0.261
Serum P on embryo transfer day
0.016
1.02
1.00– 1.04
0.139
Endometrium thickness on embryo transfer day
0.033
1.03
0.94– 1.14
0.492
Artificial FET
1.82 + 0.59
2.38 + 0.87
0.010
Total
1.74 + 0.67
2.14 + 0.88
,0.001
wave frequency. The other demographic characteristics included in the analysis were not significantly associated with the chance of clinical pregnancy (Table IV). The cut-off value of uterine peristalsis was 2.45 waves/ min in predicting clinical pregnancy outcome. The follow-up showed that up to now, of the 156 clinically pregnant cases, 70 cases ended up with live birth, 52 pregnancies were still in ongoing, 9 cases were lost of follow-up, 8 cases had surgery to terminate ectopic pregnancies, 13 cases underwent spontaneous abortion and 4 cases had induced abortion because of fetal malformation or complication.
Discussion In this prospective cohort study, we found a significant, independent association between uterine peristaltic wave frequency on the day of embryo transfer and clinical pregnancy outcomes. To the best of our knowledge, this is the first comprehensive study which included both fresh cycles, natural FET cycles and artificial FET cycles to further explore the possible role of uterine peristalsis in embryo implantation. The demographic characteristics of the study subjects were comparable (P . 0.05) between the pregnancy and non-pregnancy groups, except for serum progesterone levels on the day of embryo transfer (P ¼ 0.030) (Table I). Although the serum progesterone level was significantly different between the groups, they were both .40 ng/ml which is much higher than the physiological level of mid-luteal phase (5.97– 14.15 ng/ml) in a natural cycle (John et al., 2008). Furthermore, logistic regression analysis showed that the serum progesterone level on the day of embryo transfer did not influence the clinical pregnancy rate in our
Good-quality embryo rate
20.006
0.99
0.98– 1.01
0.277
Types of ART cycles
20.251
0.78
0.49– 1.23
0.279
Uterine peristaltic wave frequency on embryo transfer day
20.714
0.49
0.34– 0.70
,0.001
Clinical pregnancy outcome was the dependent binary variable with clinical pregnancy coded as 1 (positive) and non-pregnancy coded as 0 (negative). b , 0 and OR , 1 means clinical pregnancy outcome is inclined to be negative.
study, implying that the luteal support was adequate for pregnancy occurrence (Table IV). Before embryo transfer, uterine peristalsis of most patients was maintained at a low level of ,3.0 waves/min, similar to the phenomenon observed in a natural cycle (IJland et al., 1997a,b; Bulletti et al., 2000). In line with this, the clinical pregnancy rate was much higher in these patients compared with those with a frequency of .3.0 waves/min, indicating a beneficial effect of low frequency uterine peristalsis on pregnancy (Table II). Our result is in accordance with the study of Fanchin et al. (1998), who also found a stepwise decrease in clinical pregnancy rates with increasing uterine contractions before embryo transfer (53, 46, 23 and 14% for group ≤3.0, 3.1–4.0, 4.1–5.0 and .5.0, respectively). The discrepancy in grouping may be attributed to inter-subject, observer and cycle differences. It is noteworthy that in our previous study (Zhu et al., 2014), the uterine peristalsis of those who expelled the transferred fluid was also .3.0 waves/min. Thus, the result obtained in this study, together with previously published studies, suggested that patients with the uterine
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
Table III Comparison of uterine peristaltic wave frequencies between clinical pregnancy non-pregnancy women in different cycles.
23.0
1242
system (Kunz et al., 2006). Therefore, we have reason to believe that multiple corpora luteum in fresh cycles secreted more progesterone which was transferred into the uterus via the utero-ovarian countercurrent system, providing more effective function than the FET cycles which relied on systemic progesterone. The uterine peristaltic wave frequency before embryo transfer in the non-pregnant group was 2.14 + 0.88 waves/min, which seemed to be moderate for embryo implantation. Nevertheless, if the embryo transfer procedure is taken into consideration, uterine peristalsis might be enhanced thereafter (Lesny et al., 1998; Zhu et al., 2014). In the present study, we did not observe uterine peristalsis after embryo transfer in case any possible negative effect disturbed embryo implantation or caused psychological stress for the patients. In our previous study, an approximate 36% increase in uterine peristalsis was observed after mock embryo transfer, no matter whether the fluid was kept inside the uterine cavity or extruded outside (Zhu et al., 2014). When we take a rough estimate of the uterine peristalsis after embryo transfer in the present study accordingly, it would be 2.91 waves/min. ROC curve analysis showed that the cut-off value of uterine peristalsis was 2.45 waves/ min in predicting clinical pregnancy outcome, which was a medium between 2.14 and 2.91 waves/min. This deduction sheds light on the predictive validity of the cut-off value and places it as a promising quantitative marker of uterine receptivity and pregnancy outcome in an embryo transfer cycle. Moreover, binary logistic regression analysis demonstrated that the association between uterine peristaltic wave frequency before embryo transfer and clinical pregnancy was independently significant (odds ratio: 0.49; 95% confidence interval: 0.34 –0.70, P , 0.001). We believe that further investigations are necessary to testify to the effectiveness of uterine peristalsis in predicting clinical pregnancy before definitive conclusions can be drawn. Subjective and objective methods were both applied in uterine peristaltic wave counting. Although several studies (Fanchin et al., 1998; Oczeretko et al., 2006; Meirzon et al., 2011) have explored objective methods to reduce the influence of the subjective factor, the complicated approach and professional technology requirement prohibits their widespread use and application. It is also noteworthy that computer analysed results do not deserve complete trust since a subtle movement of the ultrasound probe may induce a false wave for analysis which could be avoided by dynamic analysis of uterine peristalsis video. Furthermore, comparisons of counting uterine peristaltic wave frequencies of accelerated images and other objective methods have shown that they are equivalent (Ayoubi et al., 2001; Nakai et al., 2004). Thus, with the rich experience accumulated in our preliminary experiments and previous studies, we chose the classic and convenient method to assess uterine peristalsis. One limitation of the present study was the lack of uterine peristaltic wave type analysis which is also an important parameter to assess uterine activity. Previous studies (Kunz et al., 1996; Leyendecker et al., 1996; IJland et al., 1997a; Fanchin et al., 1998; Bulletti et al., 2000; Nakai et al., 2008; Zhu et al., 2012) have reached a consensus on the percentage distribution of uterine peristalsis wave types in menstrual cycle, i.e. cervicofundal and fundocervical waves took a predominant and second position, respectively, with other types showing lower proportions. Nevertheless, it was too complicated to tell the definite or even possible function of each type. We speculate that the cervicofundal waves may counteract the effect of waves of other types and function predominantly. Thus, we focused on the frequency of uterine peristalsis in the present
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
peristalsis of ,3.0 waves/min before embryo transfer have a better chance of becoming pregnant. Our current findings also confirmed that embryo transfer cycles which ended with successful pregnancy showed less uterine peristalsis before embryo transfer than non-pregnant cycles (Table III), consistent previous research (IJland et al., 1997a). It may be because the intense uterine peristalsis adversely affects embryo migration after embryo transfer and extrudes it out of the uterine cavity, resulting in implantation failure or ectopic pregnancy. Recently, the application of oxytocin antagonists to selectively decrease the uterine activity was applied with success (Pierzynski et al., 2007; Moraloglu et al., 2010; Pierzynski, 2011). Other research has also tried to improve pregnancy rates by suppressing uterine peristalsis with various agents (Moon et al., 2004; Nakai et al., 2008; Kido et al., 2009; Xu et al., 2013). However molecular mechanisms modulating uterine peristalsis around the implantation window are awaiting further study which may contribute to an improvement in endometrial receptivity and pregnancy rates. The incidence of ectopic pregnancy in this population was 2.7%, similar to the reported ectopic rate following assisted reproductive technologies (Schippert et al., 2012). Although the uterine peristaltic wave frequency was a little higher in the ectopic pregnancy group than those with intrauterine pregnancy, the difference was not significant. This may be due to the uneven distribution of sample size in the ectopic pregnancy group (8 cases) and intrauterine pregnancy group (148 cases). However, it was contrary to our expectation that the uterine peristaltic wave frequency on the day of embryo transfer showed an uptrend in fresh, natural FET and artificial FET cycles, although the differences for comparison of each combination of group pairs did not reach statistical significance. Previous investigators have found that progesterone had an inhibitory action on mediating uterine peristalsis (Fanchin et al., 1998; Mueller et al., 2006; Zhu et al., 2012). In the present study, serum progesterone levels on the day of embryo transfer were 58.6 + 8.5, 51.0 + 15.0 and 47.5 + 18.4 ng/ml for fresh, natural FET and artificial FET cycles. Statistical analysis showed that the serum progesterone levels in the fresh cycles were significantly different from that in the natural FET and artificial FET cycles, while the difference between the natural FET and artificial FET cycles was not significant. The decreasing supraphysiological serum progesterone may only partly account for the uptrend of uterine peristalsis, since we found that uterine peristalsis was not correlated with supraphysiological concentrations of progesterone in our previous study (Zhu et al., 2012). Another reasonable explanation might be the endogenous corpus luteal function in different cycles. In a fresh cycle, controlled ovarian stimulation was applied to obtain a cohort of co-dominant follicles, and multiple corpora lutea were formed after the collection of oocytes. But in a natural FET cycle, only one corpus luteum produces progesterone after ovulation; an artificial cycle is totally dependent upon on exogenous luteal support. In our study, even though high doses of progesterone were given to the patients of all cycles reaching supraphysiological concentrations of serum progesterone, the function of local progesterone derived from corpus luteum could not be underestimated. The utero-ovarian counter-current system (Einer-Jensen, 1988; Kunz et al., 1998) plays an important role in local endocrine regulation, especially in guiding sperm transport preferentially into the oviduct ipsilateral to the dominant follicle (Kunz et al., 1996, 1998; Wildt et al., 1998; Kissler et al., 2004). In addition, gestational sacs inclined to locate within the uterine horn ipsilateral to the corpus luteum are also closely related to the utero-ovarian counter-current
Zhu et al.
Uterine peristalsis and pregnancy rates
study and did not analyse the uterine peristalsis wave types. In order to clarify the function of uterine peristalsis of different wave types, we think it would be better to design a more rigorous study in the future. In conclusion, the uterine peristaltic wave frequency before embryo transfer was inversely related to the clinical pregnancy in fresh and frozen-thawed embryo transfer cycles. Uterine peristalsis of ,3.0 waves/min before embryo transfer indicates a favourable pregnancy outcome. This may represent a convenient, although invasive, indicator of uterine receptivity and pregnancy outcome in an embryo transfer cycle.
Authors’ roles
Funding The study is supported by Hunan Provincial Innovation Foundation for Postgraduates.
Conflict of interest The authors declare that they have no competing interests in this study.
References Ayoubi JM, Fanchin R, Kaddouz D, Frydman R, de Ziegler D. Uterorelaxing effects of vaginal progesterone: comparison of two methodologies for assessing uterine contraction frequency on ultrasound scans. Fertil Steril 2001;76:736 – 740. Bulletti C, de Ziegler D. Uterine contractility and embryo implantation. Curr Opin Obstet Gynecol 2006;18:473 – 484. Bulletti C, de Ziegler D, Polli V, Diotallevi L, Del Ferro E, Flamigni C. Uterine contractility during the menstrual cycle. Hum Reprod 2000;15:81 – 89. Einer-Jensen N. Countercurrent transfer in the ovarian pedicle and its physiological implications. Oxf Rev Reprod Biol 1988;10:348 – 381. 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 – 1974. IJland MM, Evers JL, Dunselman GA, Volovics L, Hoogland HJ. Relation between endometrial wavelike activity and fecundability in spontaneous cycles. Fertil Steril 1997a;67:492 – 496. IJland MM, Evers JL, Hoogland HJ. Velocity of endometrial wavelike activity in spontaneous cycles. Fertil Steril 1997b;68:72– 75. John OS, Joseph IS, Lisa MH, Barbara LH, Karen DB, Gary C. Reproductive Endocrinology. Williams Gynecology. 2008. McGraw-Hill companies. Kido A, Togashi K, Hatayama H, Nakayama T, Yamamoto A, Kataoka M, Tulandi T. Uterine peristalsis in women with repeated IVF failures: possible therapeutic effect of hyoscine Bromide. J Obstet Gynaecol Can 2009;31:732 – 735. Kissler S, Siebzehnruebl E, Kohl J, Mueller A, Hamscho N, Gaetje R, Ahr A, Rody A, Kaufmann M. Uterine contractility and directed sperm transport assessed by hysterosalpingoscintigraphy (HSSG) and intrauterine pressure (IUP) measurement. Acta Obstet Gynecol Scand 2004;83:369 – 374.
Kunz G, Beil D, Deininger H, Wildt L, Leyendecker G. The dynamics of rapid sperm transport through the female genital tract: evidence from vaginal sonography of uterine peristalsis and hysterosalpingoscintigraphy. Hum Reprod 1996;11:627 – 632. Kunz G, Herbertz M, Noe M, Leyendecker G. Sonographic evidence for the involvement of the utero-ovarian counter-current system in the ovarian control of directed uterine sperm transport. Hum Reprod Update 1998; 4:667 – 672. Kunz G, Beil D, Huppert P, Leyendecker G. Control and function of uterine peristalsis during the human luteal phase. Reprod Biomed Online 2006; 13:528– 540. Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer— can we learn anything new from the observation of junctional zone contractions? Hum Reprod 1998;13:1540– 1546. Leyendecker G, Kunz G, Wildt L, Beil D, Deininger H. Uterine hyperperistalsis and dysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patients with endometriosis and infertility. Hum Reprod 1996;11:1542 – 1551. Meirzon D, Jaffa AJ, Gordon Z, Elad D. A new method for analysis of non-pregnant uterine peristalsis using transvaginal ultrasound. Ultrasound Obstet Gynecol 2011;38:217– 224. Moon HS, Park SH, Lee JO, Kim KS, Joo BS. Treatment with piroxicam before embryo transfer increases the pregnancy rate after in vitro fertilization and embryo transfer. Fertil Steril 2004;82:816– 820. Moraloglu O, Tonguc E, Var T, Zeyrek T, Batioglu S. Treatment with oxytocin antagonists before embryo transfer may increase implantation rates after IVF. Reprod Biomed Online 2010;21:338– 343. Mueller A, Siemer J, Schreiner S, Koesztner H, Hoffmann I, Binder H, Beckmann MW, Dittrich R. Role of estrogen and progesterone in the regulation of uterine peristalsis: results from perfused non-pregnant swine uteri. Hum Reprod 2006;21:1863 – 1868. Nakai A, Togashi K, Kosaka K, Kido A, Hiraga A, Fujiwara T, Koyama T, Fujii S. Uterine peristalsis: comparison of transvaginal ultrasound and two different sequences of cine MR imaging. J Magn Reson Imaging 2004; 20:463– 469. Nakai A, Togashi K, Kosaka K, Kido A, Kataoka M, Koyama T, Fujii S. Do anticholinergic agents suppress uterine peristalsis and sporadic myometrial contractions at cine MR imaging? Radiology 2008;246:489–496. Oczeretko E, Swiatecka J, Kitlas A, Laudanski T, Pierzynski P. Visualization of synchronization of the uterine contraction signals: running crosscorrelation and wavelet running cross-correlation methods. Med Eng Phys 2006;28:75–81. Pierzynski P. Oxytocin and vasopressin V (1A) receptors as new therapeutic targets in assisted reproduction. Reprod Biomed Online 2011;22:9– 16. Pierzynski P, Reinheimer TM, Kuczynski W. Oxytocin antagonists may improve infertility treatment. Fertil Steril 2007;88:213.e19 – 22. Schippert C, Soergel P, Staboulidou I, Bassler C, Gagalick S, Hillemanns P, Buehler K, Garcia-Rocha GJ. The risk of ectopic pregnancy following tubal reconstructive microsurgery and assisted reproductive technology procedures. Arch Gynecol Obstet 2012;285:863 – 871. Wildt L, Kissler S, Licht P, Becker W. Sperm transport in the human female genital tract and its modulation by oxytocin as assessed by hysterosalpingoscintigraphy, hysterotonography, electrohysterography and Doppler sonography. Hum Reprod Update 1998;4:655 – 666. Xu A, Li Y, Zhu L, Tian T, Hao J, Zhao J, Zhang Q. Inhibition of endometrial fundocervical wave by phloroglucinol and the outcome of in vitro fertilization. Reprod Biol 2013;13:88 – 91. Zhu L, Li Y, Xu A. Influence of controlled ovarian hyperstimulation on uterine peristalsis in infertile women. Hum Reprod 2012;27:2684 – 2689. Zhu L, Xiao L, Che HS, Li YP, Liao JT. Uterine peristalsis exerts control over fluid migration after mock embryo transfer. Hum Reprod 2014; 29:279– 285.
Downloaded from http://humrep.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on May 19, 2014
L.Z. contributed to the study design, transvaginal ultrasonographic scans, data analysis and drafting of the manuscript. H.S.C. performed the follow-up studies and statistical analysis of the data. L.X. participated in the acquisition and analysis of data. Y.P.L. participated in the study conception and design as well as the embryo transfer procedure. All authors contributed to the production of the manuscript.
1243
