With the hope to provide suggestions for EP prevention, this matched case-control study was designed to identify risk factors related to EP in women undergoing ART working backwards from the outcome to exposure. To our knowledge, this is the first study to analyze only endometrial and embryonic developmental factors associated with ectopic pregnancy after precise matching of other risk factors.
In this study, transferring cleavage stage embryo was found to be a risk factor of EP. This is consistent with some previous studies, which have shown that blastocyst transfer may reduce EP in IVF/intracytoplasmic sperm injection (ICSI) cycles[39,43,44], and day-3 embryo transfers was an ectopic pregnancy risk factor in IVF[45]. In the case of cleavage-stage ETs, the embryos are not temporally prepared for immediate implantation regardless of whether the endometrium is in a receptive state and can migrate within the upper female reproductive tract before implantation. Selecting blastocysts has the advantage of physiological synchronization with the uterine endometrium, therefore, it may lead to better pregnancy outcomes[46]. Meanwhile, it has been reported that uterine contractile decreased on day 5, which may be another reason for the decreased EP rate after blastocyst transfer[30]. But in contrast, many studies [30,47-50] considered the stage of embryos did not affect the EP rate. In addition, other researches have indicated that blastocyst transfer may increase the risk of EP compared with the cleavage stage embryo transfer on account of the potentially higher implantation rate of each blastocyst [31,51]. The reason why they did not come to the same conclusion may be related to different research sample sizes, patients’ age, experimental designs and analysis methods among studies, even blastocyst culture techniques in reproductive centers. In our center, almost all embryos transferred in the fresh cycle are cleavage stage, blastocysts can only be transferred in the frozen cycle, which leads to the lack of data of blastocyst transfer in the fresh cycle, which may be one of the reasons why our results are different from some previous studies. Although EP risk was lower when the blastocyst was transferred, all-blastocyst-incubation strategy is likely to lead to zero blastocyst formation and canceling of transfer for the elderly women, this will be a huge blow to the elderly couples hoping to have a baby.
The success of in vitro fertilization and embryo transfer (IVF-ET) cycles depends primarily on embryo quality and uterine receptivity, both are indispensable. It’s speculated that mismatch between the embryo stage transferred and the receptive state of the endometrium can lead to increased incidence of EP after ART treatment, as it will lead to inadequate embryonal-endometrial crosstalk, the blastocyst travels in the uterine cavity for a longer time to wait for the endometrial implantation window, it is also possible that the endometrium is not capable of receiving embryo implantation, increasing the likelihood that the embryo migrating outside the uterus, eventually leading to an EP.
A variety of studies have suggested that ultrasound can be used as a noninvasive and simple method to assess the endometrial receptivity[52]. Several sonographic parameters have been evaluated, including EMT, endometrial pattern, endometrial volume and endometrial and subendometrial blood flow[53-57]. Many studies have proposed that a correlation exists between EMT and uterine receptivity[58-63]. Few studies use EMT during ART therapy to predict future EP, the cutoff value for EP associated EMT is also debated. One study showed that the EMT > 12 mm (OR 0.27; 95% CI 0.13–0.56) prior to embryo transfer was a protective factor against EP[64]. Our study showed that, for fresh cycles, the EMT of the EP group on transformation day was significantly lower than that of the IUP group, the best cutoff point was 9.35mm, the thinner the endometrial was, the more likely EP would occur. However, for frozen cycles, we came to the opposite conclusion from Hongfang Liu's study[65], no significant difference was found in EMT between the two groups.
It’s known the endometrium can be divided into basal layer and functional layer. The functional layer is a highly dynamic tissue that changes periodically under the action of steroid hormones, creating an endometrial implantation window[66]. The ultrasonic appearance of the endometrium reflects these periodic changes. It can be considered that the endometrium measured on the first time point was the basal layer, and endometrial growth amplitude represents the functional layer thickness at the late proliferative stage (EMT on transformation day minus EMT on the first time point). Take into account the different length of stimulation in each patient, we included the average daily growth rate of the endometrium in our analysis. The results showed that there was no difference in EMT on the first time point between the two groups, suggesting that the thickness of the basal layer is always constant in most patients. In fresh cycles, the endometrial growth amplitude and daily growth rate of EP patients were significantly smaller/slower than that of IUP group, the cutoff value were 4.9mm and 0.491mm/d, respectively. However, for the frozen embryo cycles, these indicators had no difference between two groups. In general, all the studies showed that thinner EMT may be associated with worse endometrial receptivity in fresh cycles. A previous study confirmed that an increased EMT was positively correlated with an increased risk of placenta praevia [67], hypothesized that increased EMT is a sign of the frequency and/or amplitude of uterine peristalsis wave, which may increase the risk of the embryos being dislodged from their initial transfer location. Combined with our results, it can be speculated that EMT may be related to the direction of endometrial peristaltic waves, thicker endometrium may represent fundus-to-cervix uterine peristalsis, leading to a higher incidence of placenta praevia [67], as well as a lower EP rate in the present. In frozen embryos cycle, endometrium growth depends on exogenous estrogen stimulation, rather than the hyperphysiological levels of endogenous estrogen caused by multiple follicles develop simultaneously, it can be assumed that only very high levels of hormones affect the direction of the endometrial peristaltic waves. A prospective study to determine the actual endometrial implant window by combining molecular biology with pregnancy outcome tracking and to measure the endometrial peristaltic wave, and to analyze their association with different endometrial parameters should be interesting. Further larger studies should confirm a more accurate cutoff value, to guide clinical embryo transfer so as to avoid the occurrence of EP as much as possible.
Our study indicated that in fresh cycles, when endometrial pattern on transformation day was C, EP was more likely to occur. It has been reported that women with lower implantation rates and pregnancy rates show homogenous patterns, although there are conflicting results[68-70]. It is generally believed that the hyperechoic middle line represents the uterine cavity, and the other two hyperechogenic line are related to the endometrium-myometrium interface, but the main reason for the hyperechoic structure of secretory endometrium is still controversial. Fleischer et al.[71] suggested that the homogeneous hyperechoic endometrium during the late secretory phase might indicate the stromal edema by comparing the endometrial chronological date with glandular histology and stromal histology respectively[72]. The transformation process of endometrial from proliferative phase to secretory phase under the influence of hormones is called endometrial decidualization. The disability of decidualization is related to recurrent spontaneous abortion (RSA), infertility and so on.
Progesterone is the hormone responsible for the secretory changes in the endometrium, it is expected that high progesterone levels in responsible to the hyperechogenic endometrium. Investigations have shown a relationship between the serum progesterone level and secretory changes in the endometrium in controlled ovarian hyperstimulation (COH) cycles[73]. Some study suggested that a premature secretory endometrial pattern is caused by the advanced rise of P[74], and this premature conversion is bad for pregnancy[75]. Moreover, another team[76] found that ovarian stimulation would increase PRB expression and lead to the proliferative endometrium persistence. Therefore, delayed endometrial maturation may not be synchronized with embryonic developmental stages. Our study did not analyze the correlation between progesterone levels and endometrial patterns, but in either situation(earlier or later), endometrial development does not match embryo, consequently lead to the increasing EP rate. But there was no difference in frozen embryo cycles, this may be related to the less frequently early P rise in the frozen embryo period. Different unknown mechanisms produced hyperechogenicity, although the exact mechanism is unknown, it is believed that other hormones, such as androgens and exogenous gonadotropins, cause premature echogenicity of the endometrium by direct effects on the endometrium[77,78]. Further studies should be done to explore these mechanisms.
Clinically, combining endometrial and embryo information are often considered together. However, in the present study, a suggestion of combining EMT and embryo stage to predict ectopic pregnancy could not be made since the AUC of combining the two factors was as low as 0.604. Many factors known to affect EP such as the sequelae stage of pelvic inflammatory disease and maternal age cannot be cured before ET. In this study, our initial aim was to analyze only the effects of endometrial and embryonic parameters on EP after matching other well-known influencing factors, hoping that EP can be prevented by changing the parameters of endometrium and embryo to some extent in the IVF-ET cycle, in further study, when we use more of the risk parameters such as number of embryos transferred to set up a model, it would be likely to get a better prediction effect.
In addition, it is necessary to note that the correlation between EMT and pattern and embryo stage and pregnancy outcome shown in our study does not imply a causal relationship, we must acknowledge its function more than its mere thickness and pattern. The relationship may also result from some other factors that are responsible for endometrial receptivity (such as blood flow or some other underlying physiological machinery responsible for periodic endometrial changes). Despite a higher pattern C rate in EP group, we disagree with the idea that embryo cryopreservation and subsequent ET in a frozen cycle. We agree with Friedler[79] that endometrial pattern offers important predictive information but should not be used as an absolute predictor of conception. We believe that such patients should be adequately counseled and given the most adaptive advice, routine endometrial peristaltic wave examination in patients with endometrium pattern C or thickness less than 9.35mm on transformation day may be of certain significance.
This study has some limitation, the most important of which is that it is retrospective in nature, moreover, lifestyle factors such as alcohol consumption, smoking, risky sexual behaviors and some other factors which were previously reported to be associated with higher EP risks[80] were not evaluated. However, we believe the results are of interest because similar but not the same studies have published with debate results, there is no consensus about the optimal cutoff value of EMT and the prediction or prevention strategy of EP after ART. A well-designed and powered randomized clinical trial will be needed to achieve these ends.