Effect of endometrial thickness on ectopic pregnancy in frozen embryo transfer cycles: an analysis including 17,244 pregnancy cycles

ORIGINAL ARTICLE: EARLY PREGNANCY

Effect of endometrial thickness on ectopic pregnancy in frozen embryo transfer cycles: an analysis including 17,244 pregnancy cycles Hongfang Liu, M.D., Jie Zhang, M.D., Bian Wang, M.D., and Yanping Kuang, M.D. Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

Objective: To investigate whether endometrial thickness (EMT) influences the incidence of ectopic pregnancy (EP) in frozen embryo transfer (FET) cycles. Design: Retrospective cohort study. Setting: Academic tertiary-care medical center. Patient(s): A total of 16,556 patients were enrolled between January 2010 and December 2017, comprising 16,701 intrauterine, 488 ectopic, and 45 heterotopic pregnancy cycles after FET. Intervention(s): None. Main Outcome Measure(s): EP was the primary outcome. EMT was the main measured variable. Data were analyzed with the binary logistic general estimating equations model to calculate the adjusted odds ratio (aOR) for EP. Result(s): After adjusting for confounders, EMT remained statistically significant as an independent risk factor for EP. Compared with women with an EMT of R14 mm, the aORs for women with EMT in the ranges 7–7.9, 8–9.9, and 10–11.9 mm were 2.70 (95% confidence interval [CI], 1.65–4.40), 2.06 (95% CI, 1.33–3.20), and 1.66 (95% CI, 1.07–2.58), respectively. Hormone replacement treatment for endometrial preparation during FET increased the risk of EP after adjustment for confounding variables. Conclusion(s): EMT is inversely proportional to EP rate in FET cycles and is therefore a potential quantitative marker of endometrial receptivity and uterine contractibility in an FET cycle. The predictive validity of EMT value must be evaluated in further studies. (Fertil SterilÒ 2019;-:-–-. Ó2019 by American Society for Reproductive Medicine.) Key Words: Ectopic pregnancy, endometrial thickness, frozen embryo transfer, assisted reproduction technology, in vitro fertilization Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertilityand-sterility/posts/53046-28008

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espite significant improvements in IVF techniques over the last four decades, ectopic pregnancy (EP) remains a significant problem, occurring in 1%–2% of spontaneous conceptions (1) and 2%–5% of IVF cases (2, 3). EP is not only lifethreatening but also increases the emotional and financial burden of an

already expensive and stressful IVF process. The etiology of EP after IVF is not well understood. Tobacco use and tubal disease (2) as well as a history of EP and surgery (4) are known risk factors. IVF procedures including ovarian stimulation and embryo culture may also increase the risk of EP (5, 6).

Received March 24, 2019; revised August 3, 2019; accepted September 3, 2019. H.L. has nothing to disclose. J.Z. has nothing to disclose. B.W. has nothing to disclose. Y.K. has nothing to disclose. H.L. and J.Z. should be considered similar in author order. Supported by the National Natural Science Foundation of China (grant nos. 81571397 and 81771533) and the China Postdoctoral Science Foundation (grant no. 2018M630456). Reprint requests: Yanping Kuang, M.D., Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Jiao Tong University School of Medicine, Shanghai, People’s Republic of China (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2019 0015-0282/$36.00 Copyright ©2019 American Society for Reproductive Medicine, Published by Elsevier Inc. https://doi.org/10.1016/j.fertnstert.2019.09.003 VOL. - NO. - / - 2019

Endometrial thickness (EMT), an indirect indicator for endometrial receptivity, is easily measured by transvaginal ultrasound (7). A thin endometrial lining, both in fresh and frozen ET (FET) cycles, is associated with lower IVF success rates (8). However, pregnancy rates may even decline at a thickness >14 mm (9). Increased EMT has been shown to be an independent risk factor for placenta previa (10). Little is known, however, regarding the effect of EMT on EP incidence. To date, only one study has investigated the relationship between EMT and risk of EP (11), but it mainly focused on fresh cycles. A number of investigators observed a lower incidence of EP in FET compared with fresh 1

ORIGINAL ARTICLE: EARLY PREGNANCY transfer in stimulated cycles, suggesting that a difference in the tubal/uterine environment contributes to abnormal implantation after IVF (12). Moreover, some researchers claimed that there was a negative effect of ovarian stimulation on the endometrium, as reflected by increased EP rates (13, 14). Hence, the exact role of EMT in the development of EP remains obscure. In the era of a freeze-all policy, further exploration of the influence of EMT on the incidence of EP originating from FET is of utmost importance. Therefore, the aim of the present study was to survey the relationship between EMT and EP occurrence in FET cycles.

MATERIALS AND METHODS Study Design We conducted a retrospective study of all FET cycles for which the outcome was a pregnancy between January 2010 and December 2017 at the Department of Assisted Reproduction of the Ninth People’s Hospital of Shanghai Jiao Tong University School of Medicine. The study was approved by the Independent Ethics Committee of Shanghai Ninth People’s Hospital. FET cycles resulting in an intrauterine, ectopic, or heterotopic pregnancy were included. The exclusion criteria were as follows: an endometrial preparation regime other than a modified natural, mild stimulation, or hormone replacement therapy (HRT) cycle; ET performed on days other than day 3 or 5; and uterine abnormalities or endometrial polyp.

Definition of EP, Intrauterine Pregnancy (IUP), and Thin Endometrium The dependent variable in our study was an ectopic or heterotopic pregnancy as opposed to an intrauterine pregnancy as in previous studies (11, 12). Additionally, our study excluded pregnancy at an unknown location and suspicious adnexal mass to remove any uncertainty from the outcome. An IUP was defined by the implantation of at least one embryo in the uterus (15), whereas EP was defined as the presence of at least one gestational sac outside the uterine cavity. Heterotopic pregnancy, which was defined as a coexistence of an intra- and extrauterine gestation sac, was also classified as EP (n ¼ 45). The incidence of EP was calculated based on the number of EP cycles per 100 clinical pregnancy tests after FET. The definition of a thin endometrium was variable in previous studies. Normally, an EMT <7 or 8 mm on hCG trigger day or before progestin administration is defined as a thin endometrium (8, 16, 17). In our study, a thin endometrium was defined as <8 mm as in previous studies (18, 19).

ART Protocols and Embryo Assessment Details on ovarian stimulation, oocyte retrieval, and IVF/intracytoplasmic sperm injection (ICSI) procedures have been described elsewhere (20). Briefly, cleavage-stage embryos (day 3) were graded according to the Cummins’s criteria (21). Only good-quality embryos (including grade I and II) were classified as high quality and selected for vitrification. Suboptimal day 3 embryos were placed in extended culture to the blastocyst stage (day 5). Blastocyst quality was assessed 2

according to the scoring system of Gardner and Schoolcraft (22), and embryos graded R 3BB were considered as good blastocysts. All embryos were thawed on the day of transfer, and post-thawed embryos with R50% blastomeres were considered as surviving.

Embryo Vitrification The vitrification and thawing procedure has been described elsewhere (23). Briefly, embryo vitrification was carried out via the Cryotop carrier system with dimethyl sulfoxide, ethylene glycol, and sucrose as cryoprotectants. For thawing, embryos were sequentially transferred through a series of diluted sucrose solutions (1 M / 0.5 M / 0 M sucrose). Notably, during the study period, a complete change in the type of culture medium was made. Specifically, before January 2013, the commercial sequential available culture medium was used. From January 2013 onward, continuous single-culture media were introduced. Except for this switch, the laboratory procedures and conditions remained constant throughout the study.

Endometrial Preparation Regime Endometrial preparation for FET was carried out by modified natural, mild stimulation, or HRT cycles. A modified natural cycle was used for patients with regular ovulatory cycles, whereas in patients with an irregular menstrual cycle, a mild stimulation cycle or HRT cycle were used according to the physicians’ or patients’ preference. For a mild stimulation cycle, letrozole was used to induce follicular growth. A daily dose of 2.5 mg letrozole was administered starting on cycle day 3 for 5 days. Serum estrogen level was monitored, and transvaginal ultrasound was performed from cycle day 10 onward. If the diameter of the dominant follicle was <10 mm, a bolus of 75 IU hMG was supplemented until the diameter exceeded 17 mm. If the main follicle was >14 mm, no more hMG was given. In both the modified natural and mild stimulation cycles, when the mean diameter of the dominant follicle was R17 mm and EMT was R7 mm, with E2 >150 pg/mL and P <1 ng/mL, one of two procedures was performed depending on serum LH value: for LH <20 mIU/mL, a bolus of 5,000 IU hCG was administered on the same night, P was given 3 days later, and the cleavagestage embryo was transferred 5 days later after hCG injection, whereas the blastocyst was transferred 7 days later. If serum LH concentration was R20 mIU/mL, hCG was administered on the same afternoon, and P was given 2 days later. The cleavage-stage embryo and blastocyst were transferred 4 and 6 days later, respectively. A daily dose of 6 mg E2 was administered in an HRT cycle starting from day 3 of the menstrual cycle. A transvaginal ultrasound was performed 12–14 days later. If the EMT reached R7 mm and there was no dominant follicle or signs of ovulation, a daily dose of 400 mg natural micronized P was vaginally administered. The duration of E2 treatment did not exceed 21 days since a previous study found that prolonged E2 exposure decreased the clinical pregnancy and live birth rates (24). ET was scheduled depending on the timing of the embryo freezing. VOL. - NO. - / - 2019

Fertility and Sterility® Specifically, cleavage-stage ET was arranged to be performed 3 days later and blastocyst transfer was performed on the fifth day. Once pregnancy was achieved, luteal support was continued until 10 weeks of gestation.

Embryo Transfer ET was performed with a Cook catheter under the guidance of ultrasound following the standard procedures of our department. One or two embryos were transferred into the middle of the uterine cavity according to Chinese legislation. All embryos were thawed on the day of transfer, and post-thawed embryos with R50% blastomeres were considered as surviving.

Assessment of Primary Exposure EMT was measured by transvaginal ultrasound scan on the day of hCG administration in modified natural cycles or mild stimulation cycles. In HRT cycles, EMT was recorded from the last ultrasound scan before starting P administration. The maximum distance between two outer edges in the endometrial image of a longitudinal section of the uterus observed by vaginal ultrasound was used to measure EMT, which was the main exposure. For clinical application of these findings, EMT was classified into the following groups representing regular 2 mm intervals: 7–7.9, 8–9.9, 10–11.9, 12– 13.9, and R14 mm. The grouping interval was established based on the 10th (8.2 mm), 50th (10.3 mm), and 90th (13.6 mm) percentiles of the study population and previous studies (18, 25, 26). All ultrasound measurements were performed by highly trained sonographers via a Voluson E8 (GE Healthcare) with a 6 MHz intracavity probe.

Statistical Analysis and Analyzed Variables Variables associated with EP were selected based on the availability of data and previous literature (3, 27, 28). Data on each subject including age, body mass index (BMI), gravidity, parity, cause of infertility, fertilization method, protocol, stage and number of embryos transferred, and year of treatment were collected. Tubal factor infertility (TFI) was caused by hydrosalpinx, obstructions, damage, salpingectomy, scarring, congenital malformations, or other factors that impeded the normal function of the fallopian tubes such as prior tube surgery or history of EP. The primary outcome measure for the study was EP versus IUP as in previous studies. Data were analyzed by SPSS version 23 (IBM). Normally distributed data were recorded as mean  SD, and nonnormally distributed data as mean and range. Continuous variables were compared using the Student’s t test. Frequencies were compared using with the c2 test or Fisher’s exact test. Some patients with multiple cycles were recruited; thus the observations within the same patients may be correlated and nonindependent. Therefore, we use the generalized estimated equations model to calculate the adjusted odds ratios (aOR) and 95% confidence intervals (CIs) for the association between EP and significant characteristics selected from the univariate analysis. P < .05 was considered statistically significant.

RESULTS Figure 1 shows the process of cycle selection to achieve the final study sample. The first exclusion was 2,835 biochemical pregnancies and 431 cycles that were lost to follow-up, as in previous studies (5, 12). We further excluded 1,428 cycles of

FIGURE 1

Flowchart of patients. Liu. Effect of EMT on ectopic pregnancy. Fertil Steril 2019.

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ORIGINAL ARTICLE: EARLY PREGNANCY women with uterine abnormalities or endometrial polys, 457 cycles with embryos transferred on days other than day 3 and day 5, and 354 cycles in which EMT data were not recorded. Finally, 17,244 FET cycles resulting in a clinical pregnancy were included: 16,701 intrauterine pregnancy cycles, 498 EP cycles, and 45 heterotopic pregnancy cycles; of these, 543 (3.15%) were diagnosed with EP and 16,701 with IUP. The basic characteristics of all patients and cycles are displayed in Table 1. The EP rate did not change over the study period. No difference was observed in terms of maternal age, BMI, endometriosis, polycystic ovarian syndrome, and diminished ovarian reserve. EP rates were higher in female patients with TFI as compared with those without TFI and were higher in cases of double ET, cleavage ET, nonnatural endometrial preparation, and thin endometrium. Univariate analysis is detailed in Table 2; an EMT between 7 and 7.9 mm was associated with a threefold increased risk of EP compared with EMT >14 mm (odds ratio [OR] ¼ 3.11; 95% CI, 1.95–5.03). Moreover, an EMT between 8 and 9.9 mm was associated with a twofold increased risk of EP (OR ¼ 2.32; 95% CI, 1.51–3.58), whereas an EMT between 10 and 11.9 mm was associated with a nearly twofold increased risk of EP (OR ¼ 1.78; 95% CI, 1.15–2.76) compared with an EMT between 7 and 7.9 mm. Patients with TFI or endometriosis also showed an increased risk for EP (87% and 17%, respectively), although the latter was not statistically significant. In contrast, male factor infertility was a protective factor for EP (OR ¼ 0.37; 95% CI, 0.25–0.55). FET after mild stimulation cycles or HRT cycles increased EP risk by 40% and twofold, respectively, compared with modified natural cycles; and cleavage-stage transfer increased the risk threefold compared with blastocyst transfer (OR ¼ 2.82; 95% CI, 1.97–4.04). In addition, the EP rate of double ET was 77% greater than that of single ET (OR ¼ 1.77; 95% CI, 1.31–2.41). All variates with P < .1 in the univariate analysis were included in the multivariate regression analysis; the results are shown in Table 3. After adjusting for confounding factors, EMT between 7 and 7.9 mm remained associated with a nearly threefold increased risk of EP compared with EMT >14 mm (aOR ¼ 2.70; 95% CI, 1.65–4.40), whereas the risk was twofold higher for EMT between 8 and 9.9 mm (aOR ¼ 2.06; 95% CI, 1.33–3.20) and for EMT between 10 and 11.9 mm (aOR ¼ 1.66; 95% CI, 1.07–2.58). FET after HRT remained associated with a twofold increased risk of EP compared with modified natural cycles (aOR ¼ 2.25; 95% CI, 1.76–2.87). Mild stimulation cycles remained associated with a 47% increased risk (aOR ¼ 1.47; 95% CI. 1.16–1.86) compared with modified natural cycles. Cleavage-stage transfer remained associated with a nearly threefold increased risk compared with blastocyst transfer (aOR ¼ 2.87; 95% CI, 2.07– 4.11), and the risk was 36% higher for double ET compared with single ET (aOR ¼ 1.36; 95% CI, 1.00–1.84).

DISCUSSION To date, this is the first large retrospective cohort study to investigate whether EMT affects EP rates after FET cycles.

4

TABLE 1 Basic characteristics.

Variable Total Age <30 30–34 35–39 R40 Body mass index Tubal factor infertility Yes No Male factor infertility Yes No Endometriosis Yes No Polycystic ovary syndrome Yes No Diminished ovarian reserve Yes No Gravidity 0 R1 Parity 0 R1 Intracytoplasmic sperm injection fertilization Yes No Protocol Modified natural cycle Mild stimulation cycle Hormone replacement therapy Endometrial thickness, mm <8 8–9.9 10–11.9 12–13.9 R14 No. of embryos transferred 1 2 Stage of embryo transferred Cleavage Blastocyst Year of treatment 2010–12 2013–15 2016–17

Intrauterine pregnancy, n

Ectopic pregnancy, n (%)

16,701

543 (3.2)

P value NS

5,702 7,048 3,151 800 21.84.6

195 (3.3) 224 (3.1) 108 (3.3) 16 (2.0) 21.63.0

7,068 9,633

314 (4.3) 229 (2.3)

1,932 14,769

25 (1.3) 518 (3.4)

1,241 15,460

33 (2.4) 510 (3.2)

NS .00 .00 NS NS

1,383 15,318

47 (3.3) 496 (3.1) NS

563 16,138

20 (3.4) 523 (3.1)

9,113 7,588

231 (2.5) 312 (3.9)

15,220 1,481

494 (3.1) 49 (3.2)

.00 NS NS 4,320 12,364

147 (3.3) 396 (3.1)

4,836

105 (2.1)

7,773

236 (2.9)

4,092

202 (4.7)

.00

.00 1,220 5,696 5,651 2,811 1,323

66 (5.1) 230 (3.9) 175 (3.0) 49 (1.7) 23 (1.7) .00

2,355 14,346

46 (1.9) 497 (3.3) .00

14,181 2,520

507 (3.5) 36 (1.4)

1,685 8,175 6,841

55 (3.2) 267 (3.2) 221 (3.1)

NS

Note: Data are reported as n or n (%), except body mass index, which is reported as mean  standard deviation. NS ¼ no statistically significant differences between groups. Liu. Effect of EMT on ectopic pregnancy. Fertil Steril 2019.

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TABLE 2

TABLE 3

Univariate analysis of factor associated with ectopic pregnancy.

Predictor variable Age Body mass index Gravidity 0 R1 Parity 0 R1 Tubal factor infertility Yes No Endometriosis Yes No Polycystic ovary syndrome Yes No Diminished ovarian reserve Yes No Male factor infertility Yes No Protocol Modified natural cycle Mild stimulation cycle Hormone replacement therapy Endometrial thickness, mm <8 8–9.9 10–11.9 12–13.9 R14 No. of embryos transferred 1 2 Embryo stage Cleavage Blastocyst Year of treatment

Odds ratio

P value

Predictor variable

0.99 0.99

0.97, 1.01 0.96, 1.02

NS NS

1 1.62

— 1.37, 1.93

— .00

1 1.02

— 0.76, 1.37

— NS

1.87 1

1.57, 2.22 —

.00 —

1.17 1

0.82, 1.65 —

NS —

1.05 1

0.77, 1.42 —

NS —

1.10 1

0.70, 1.73 —

NS —

0.37 1

0.25, 0.55 —

.00

1 1.40 2.27

— 1.11, 1.77 1.79, 2.89

— .01 .00

Gravidity 0 R1 Tubal factor infertility Yes Others Male factor infertility Yes No Protocol Modified natural cycle Mild stimulation cycle Hormone replacement therapy No. of embryos transferred 1 2 Embryo stage Cleavage Blastocyst Endometrial thickness, mm <8 8–9.9 10–11.9 12–13.9 R14

3.11 2.32 1.78 1.00 1

1.92, 5.03 1.51, 3.58 1.15, 2.76 0.61, 1.65 —

.00 .00 .01 NS —

1 1.77

— 1.31, 2.41

— .00

2.82 1 1.02

1.97, 4.04 — 0.97, 1.07

.00 — NS

Note: Variables entered in regression model listed. NS ¼ no statistically significant differences between groups. Liu. Effect of EMT on ectopic pregnancy. Fertil Steril 2019.

Our findings showed that a thin endometrium is an independent risk factor for EP after FET. Moreover, TFI, nonnatural endometrial preparation regime, stage of ET, and the number of embryos transferred were also associated with an increased risk for EP after FET.

Comparison with Other Studies As expected, TFI, double ET, and cleavage-stage ET were associated with increased risk of EP, whereas male factor infertility decreased the risk of EP after FET in both univariate and multivariate analyses in our study, which is consistent with previous studies (27). Additionally, BMI and polycystic ovarian syndrome were not significantly related to EP in our study, which coincides with previous studies (29, 30). Endometriosis and diminished ovarian reserve, which have been shown to be associated with EP rate (27, 31), were not VOL. - NO. - / - 2019

Multiple analysis of factors associated with ectopic pregnancy.

95% confidence interval

Adjusted odds ratio

95% confidence interval

P value

1 1.32

— 1.10, 1.58

— .02

1.88 1

1.58, 2.25 —

.00 —

0.36 1

0.24, 0.54 —

.00 —

1 1.47 2.25

— 1.16, 1.86 1.76, 2.87

— .00 .00

1 1.36

— 1.00, 1.84

— .04

2.87 1

2.01, 4.11 —

.00 —

2.70 2.06 1.66 0.95 1

1.65, 4.40 1.33, 3.20 1.07, 2.58 0.57, 1.56 —

.00 .00 .03 NS —

Note: Variables entered in regression model listed. NS ¼ no statistically significant differences between groups. Liu. Effect of EMT on ectopic pregnancy. Fertil Steril 2019.

risk factors for EP in the present work, although this could be due to the very small proportion of patients in the study population with endometriosis or diminished ovarian reserve. Few studies have evaluated whether EMT can be used as an independent factor to identify patients with EP and IUP, and the findings have been controversial. Most of them investigated whether EMT measurements after a positive hCG test could predict pregnancy of unknown locations. It was suggested that EMT lacks sensitivity in predicting the occurrence of EP in women with a normal ultrasound and a positive pregnancy test (32). In contrast, EMT after a positive pregnancy test tended to be thinner in patients with EP (33), which is consistent with our findings. The reason why a thin endometrium results in higher EP rates is unclear and likely complex. It is speculated that differences in oxygen tension may play a role. A thin EMT means the implanting embryo would be much closer to the spiral arteries in the basal endometrium layer, so the embryos would be exposed to higher oxygen concentrations due to the production of reactive oxygen species. It is well known that higher oxygen concentrations inhibit the growth of embryos (34). Compared with the higher oxygen tension in the thin endometrium, the low oxygen concentration in the fallopian tube could explain the higher EP risk. Another possible mechanism by which a thin endometrial lining affects the incidence of EP is uterine peristalsis. It was previously demonstrated that the uterine peristaltic wave frequency was higher in women with EP group than 5

ORIGINAL ARTICLE: EARLY PREGNANCY in those with IUP, although no statistical significance was found due to the uneven distribution of the sample size (35). On the contrary, a thicker endometrium was shown to be an independent risk factor for placenta previa (10). The authors of that study (10) hypothesized that increased EMT is proportional with uterine peristalsis and thus affects the site of embryo implantation. This contradicts our observation that a thin endometrium is an independent risk factor for EP. Uterine peristaltic wave frequency increases with serum estrogen levels and reach a peak in the preovulatory phase; then the uterine wave-like activity decreases with serum P concentrations to provide a quiet environment for embryo implanting. Previous studies hypothesized that higher wave activity can push the implanting embryos into the fallopian tubes (36, 37), resulting in an EP. Uterine peristaltic wave frequency was higher in controlled ovarian hyperstimulation cycles than in natural cycles (38), which could explain the higher EP rate in mild stimulation cycles and HRT cycles compared with modified natural cycles after FET. So the thin endometrium was thought to be closely associated with uterine peristaltic frequencies, which is in contrast to Rombauts’s study (10). The study mentioned above (38) also found that uterine waves from the cervix to fundus increased, whereas those from the fundus to cervix decreased in controlled ovarian hyperstimulation cycles compared with natural cycles, suggesting that the direction of uterine peristalsis influences the risk of EP, although additional studies are needed to confirm this possibility. Although many studies have shown that all of the current endometrial preparation regimens are equally successful in terms of ongoing pregnancy and live birth rates (39–45), there have been no studies comparing the effect of different endometrial preparation regimens on EP. A fresh ET study found that antagonist protocols increased EP risk twofold compared with agonist protocols (11), indicating that the protocol influences the EP rate. It was also reported that a different type of GnRH suppression protocol used in fresh autologous cycles was associated with EP rate (5). That study suggested that lower serum E2 levels in GnRH antagonist cycles compared with luteal GnRH agonist cycles were associated with decreased EP rates. Compared with modified natural cycles, simulation cycles or HRT cycles showed an increased risk of EP rates after FET in our study, which could be due to higher hormone levels in the former since an altered hormone environment has been shown to affect embryo-tube transport (12, 46).

Strengths and Limitations of Our Study The main strength of our study is that EMT was divided into more detailed groups, which has provided insight into EP risk after FET over a different range of EMTs. In addition, most studies investigated whether EMT measured after a positive pregnancy test could be a single predictor for pregnancy of unknown location (47). Our study investigated whether EMT recorded before P application would affect embryo implantation, which could determine whether the ET would proceed if EMT is an independent risk factor of EP. 6

Moreover, our study had rigid exclusion criteria and a robust analysis to minimize the potential effects of confounding factors. Furthermore, this was a large cohort single-center study in which embryos were evaluated by the same trained embryologists, and EMT was assessed by the same trained sonographers, thus reducing the intercenter and interobserver variability, respectively. We must also highlight the limitations of our study. Due to its retrospective design, there is some intrinsic defect that cannot be avoided. In addition, confounding factors known to influence pregnancy outcome, such as smoking, were not recorded in the database. According to published statistics, only 2.7% of women in China smoke (http://www. xinhuanet.com//video/sjxw/2016-05/31/c_129029080.htm). IVF patients consciously reduce risks such as smoking that may negatively influence pregnancy outcome and child health. Thus, like endometriosis or diminished ovarian reserve, smoking is not supposed to play a role in the outcome due to a very small number of smokers within the study population.

Conclusion EMT evaluation continues to plays an important role in assisted reproduction despite significant technological advances. A thin endometrium is associated not only with a higher EP rate but also with a lower birth rate and higher risk of obstetric complications (25, 48). Thus, EMT represents a promising quantitative marker of endometrial receptivity and uterine contractibility in an FET cycle. One study suggested that women with a history of EP have a 40% higher recurrence risk of EP after IVF in comparison with women with no history of EP (49). ET in patients with higher risks of EP, such as a hormone replacement cycle with a thin EMT or a patient with a history EP with a thin EMT, should be seriously evaluated to determine whether it should proceed. Furthermore, another hypothetical mechanism of EP is the conflicting signals to the embryo from the uterine and fallopian epithelia (50). Studies have found that thin endometrium showed a different number of cytokines compared with normal-thickness endometrium (51). Our study considers whether EP has some endometrial mechanism and whether the risk of EP can be ameliorated through mitigating uterine peristalsis. The predictive validity of the EMT value needs to be tested in a further study. Acknowledgments: The authors thank the doctors, nurses, laboratory staff, and study participants for their contributions.

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