Abstract
Purpose
Multiple factors have been shown to influence the rate of clinical pregnancy after FET in IVF treatment, including embryo quality, synchronization of embryo and endometrium, and endometrial receptivity (ER). The subendometrial blood flow conditions could also contribute potentially major effects toward the establishment and maintenance of pregnancy. We conducted a retrospective cohort study to examine the correlation between subendometrial blood flow, as determined by Doppler ultrasound, and pregnancy outcomes in IVF patients with a thin endometrium (endometrium thickness [EMT] ≤ 0.7 cm).
Methods
This was a retrospective cohort study conducted at a university-affiliated reproductive hospital from January 2017 to April 2023. The EMT and subendometrial blood flows were assessed using transvaginal color Doppler ultrasound and evaluated by experienced clinical ultrasound physicians on the endometrial transformation day. The pregnancy outcomes were followed up and documented in clinical medical records through the IVF cohort study at our center.
Results
In the patients with 0.5 cm ≤ EMT ≤ 0.7 cm, the embryo implantation rate was statistically significant increased in the patients with the presence of subendometrial blood flow (OR 1.484; 95% CI, 1.001–2.200; P = 0.049; aOR 1.425; 95% CI, 1.030–2.123; P = 0.003). Patients with discernible subendometrial blood flow have superior live birth (P = 0.028), clinical pregnancy (P = 0.049), and embryo implantation (P = 0.027) compared to the patients without subendometrial blood flow when the EMT is ≤ 0.7 cm.
Conclusions
The presence of subendometrial blood flow detected by ultrasound was positively associated with successful embryo implantation and favorable pregnancy outcomes in patients with thin endometrium undergoing FET.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-024-03245-z.
Keywords: Embryo transfer, Pregnancy outcome, Subendometrial blood flow, Thin endometrium, Doppler ultrasound
Introduction
In the process of in vitro fertilization or intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET), factors such as high-quality embryos and a well-functioning endometrium are indispensable for successful embryo implantation and favorable pregnancy outcomes [1]. Among these factors, endometrial receptivity (ER)—the capacity of the endometrium to facilitate blastocyst localization, adherence, and successful implantation—plays a pivotal role in assisted reproductive technology (ART) outcomes [2].
Ideally, methods for ER assessment should be easy to implement in clinical practice and preferably noninvasive. Color Doppler ultrasound has the advantages of non-invasions, repeatability, and real-time monitoring, which makes it suitable for evaluating ER [3]. To date, ultrasound has evaluated ER using a variety of parameters, notably morphological parameters such as endometrial thickness (EMT), echogenicity, volume, and peristalsis, supplemented by hemodynamic measures including uterine artery, endometrial, and subendometrial blood flow [4–6]. Endometrial blood flow assessment offers an added physiological dimension to conventional morphological ultrasound parameters [7]. Notably, angiogenesis plays a critical role in endometrium growth, embryo implantation, and other female reproductive processes.
In ART, thin endometrium is typically defined by an EMT of < 0.7 cm, although some practitioners set this benchmark as < 0.8 cm [8, 9]. This description stems from evidence that both clinical pregnancy and live birth rates tend to decline when EMT falls below the threshold of 0.8 cm for fresh embryo transfer cycles and 0.7 cm for frozen embryo transfer (FET) cycles [10]. Therefore, more attention should be given to ER in patients with thin endometrium to guide such patients to improve the success rate of ET.
Previous studies have largely focused on elucidating the predictive value of endometrial and subendometrial blood flow in the clinical pregnancy success rates [11, 12]. However, it remains unknown whether endometrial and subendometrial blood flow can be used as predictors of FET success and favorable pregnancy outcomes, particularly in patients with a thin endometrium. In this work, we analyzed data from a relatively large number (2505) of FET cycles at our center to identify potential correlations between subendometrial blood flow and pregnancy outcomes. We especially focused on analyzing the relationship between subendometrial blood flow and pregnancy outcomes of FET in patients with thin endometrium.
Materials and methods
Participants
From January 2017 to April 2023, a total of 2505 cycles of FET from 2505 patients conducted at the Reproductive Hospital Affiliated with Shandong University were included in this retrospective cohort study. A single embryo was transferred at the blastocyst stage for every cycle, and every cycle was the first FET cycle for the patients. The inclusion criteria were as follows: age 20–44 years old, normal ovarian reserve (basal follicle-stimulating hormone [FSH] < 12 IU/L and anti-Mullerian hormone [AMH] ≥ 1.1 ng/mL), normal thyroid function (thyroid-stimulating hormone [TSH] = 0.277–4.2 mIU/L), and normal chromosome karyotype for both husband and wife (chromosome polymorphisms were also considered normal). The exclusion criteria were as follows: abnormal anatomy of the genital tract, malignant diseases such as thyroid cancer and breast cancer, and no subendometrial blood flow result on the day of endometrial transformation. Briefly, the EMT and the subendometrial blood flow of these patients were examined by transvaginal color Doppler ultrasound on the day of endometrial transformation. This study was conducted according to the ethical guidelines of the World Medical Association (Declaration of Helsinki) and was approved on 24 December 2021 by the Ethics Committee of Reproductive Hospital Affiliated to Shandong University ([2021] IRB no. 140). Written informed consent was obtained from all patients before treatment, and the patients consented to the use of their retrospective data in scientific publications.
IVF procedure
According to the patient’s age, preference, and ovarian reserve indicators (antral follicle count, AMH, FSH, estrogen [E2], basal FSH/luteinizing hormone [LH]), a variety of conventional controlled ovarian hyperstimulation (COH) protocols were used, including ultra-long gonadotropin-releasing hormone (GnRH) agonist protocol, long GnRH agonist protocol, short GnRH agonist protocol, minimal stimulation protocol, or GnRH antagonist protocol [13]. When at least two dominant follicles were 18 mm or greater in mean diameter, 5000 to 10,000 IU human chorionic gonadotropin (hCG) was administered to induce ovulation. Oocyte retrieval was performed 34–36 h after hCG administration. On day 5 of embryo culture, the embryo score was assessed according to Gardner morphological criteria [14] on the basis of the degree of expansion and the development of the inner cell mass and trophectoderm. All blastocysts were then vitrified on day 5 or day 6 according to embryo development.
In the embryo transplantation cycle, endometrial preparation was performed and monitored by ultrasound. Endometrium was prepared by natural cycles or other artificial cycles, depending on the individual condition. The natural cycle program was mainly applied to women with regular menstrual cycles and normal ovulation. The artificial cycle program was the first choice for women with oligomenorrhea or irregular menstruation, such as patients with polycystic ovary syndrome, endometriosis, or endometrial dysplasia in the natural cycle program. If patients did not ovulate in the natural cycle or exhibited poor endometrial response in the artificial cycle, the ovulation induction cycle program or artificial cycle with GnRH agonist pretreatment was then used. Patients undergoing embryo transfer in natural cycles or ovulation induction cycles initiated luteal support from the day of ovulation, while for patients undergoing endometrial preparation through artificial cycles, luteal support was initiated when the endometrial thickness reached 7 mm until 12 weeks of gestation. On the day of endometrial transformation, which is comparable to the day of ovulation, EMT and subendometrial blood flow were measured and recorded by ultrasound. FET was performed 5 days after the day of endometrial transformation. FET has a significantly lower risk of ovarian hyperstimulation syndrome than fresh ET [15]; thus, our study only included single FET cycles. Luteal-phase support continued until 12 weeks of gestation.
Ultrasound measurement and assessment
Endometrial features, including EMT, endometrial blood flow, and subendometrial blood flow, were measured by transvaginal 5–10 MHz ultrasonography with a four-dimensional ultrasound instrument (GE Voluson E8; USA). The settings for this study were as follows: low frequency; pulse repetition frequency, 0.7 kHz. To assess blood vessels and the location of the myometrium and the endometrial relationship, the sector of interest was adjusted to cover the endometrial cavity in the longitudinal plane of the uterus. The color gain was adjusted to 2.0 to optimize blood flow detection in the small vessels of the endometrial and subendometrial areas. The ultrasound measurements were performed by two well-trained physicians independently, followed by further assessment and verification by one experienced clinical ultrasound physician before the approval of the official ultrasound report. We performed inter-observer and intra-observer consistency tests for EMT and subendometrial blood flow. Satisfactory of inter- and intra-observer agreement was documented for the EMT and subendometrial blood flow (Table S1). Clinical assessment of uterine artery (UtA) hemodynamics is currently limited to ultrasound (US) Doppler velocimetry [16]. Color Doppler flow mapping is the real-time display of two-dimensional (2D) flow patterns superimposed on cross‐sectional pulse-echo images of anatomical structures [17]. As shown by transvaginal color Doppler ultrasound, the endometrium is a hypoechogenic zone, the uterine cavity and the junction of the endometrium with the myometrium are hyperechoic zones, and the myometrium is a hypoechogenic zone. A triple-line pattern was defined as a hypoechogenic endometrium surrounded by a hyperechogenic zone.
The zones of vascular penetration into the subendometrial and endometrial regions were defined as Type I, Type II, and Type III on the basis of the Applebaum classification [18], as shown in Fig. 1. In Type I, the blood vessel passes through the hypoechoic zone (myometrium) on the outside of the endometrium but does not reach the hyperechoic zone (the junction of the endometrium with the myometrium) on the outer edge of the endometrium (the absence of endometrial blood flow or subendometrial blood flow). In Type II, the blood vessel passes through the outer edge of the endometrial hyperechoic zone (the junction of the endometrium with the myometrium) but does not enter the hypoechoic zone (the endometrium) (the absence of endometrial blood flow with the presence of subendometrial blood flow). In Type III, the blood vessel passes through the inner hypoechogenic zone (the endometrium) (the presence of both endometrial and subendometrial blood flow). According to the Applebaum classification, patients were then divided into Category 1 (Type I) or Category 2 (Type II and Type III). In other words, subendometrial blood flow was absent in Category 1, whereas Category 2 implies the presence of subendometrial blood flow. In addition, patients were divided into five groups according to EMT: 0.5 cm ≤ EMT ≤ 0.7 cm; 0.7 cm < EMT ≤ 0.8 cm; 0.8 cm < EMT ≤ 0.9 cm; 0.9 cm < EMT ≤ 1.0 cm; or EMT > 1.0 cm. The setting of the 0.5 cm < EMT ≤ 0.7 cm group was defined as thin endometrium.
Fig. 1.
Ultrasonographic measurement and assessment of endometrial blood flow. A Representative ultrasound image of Type I blood flow in the subendometrial and endometrial regions. B Representative ultrasound image of Type II blood flow in the subendometrial and endometrial regions. C Representative ultrasound image of Type III blood flow in the subendometrial and endometrial regions. Asterisks are located in the myometrium region, circles are located in the endometrium region, and dotted lines refer to the junction of the endometrium with the myometrium
Pregnancy outcome assessment
The pregnancy outcome assessment and statistical analysis were performed according to the collected pregnancy outcome data from all participants. Biochemical pregnancy, which is usually used to evaluate the success of embryo implantation to the endometrium, was determined by measuring blood β-hCG levels higher than 50 IU/L at 14 days after FET. Clinical pregnancy was confirmed by a vaginal ultrasound examination showing the amniotic sac inside and outside of the uterus at 4 weeks following transplantation. Abortions occurring before 12 weeks of gestation were defined as early miscarriage, while abortions after 12 weeks of gestation were defined as middle/late miscarriage. Blood β-hCG ≥ 50 IU/L measured at 14 days after FET, but not persisting to the clinical pregnancy stage, was defined as biochemical pregnancy miscarriage. Term delivery refers to delivery between 37 and 42 weeks of gestation. Delivery that occurred at or after 42 weeks of gestation was defined as a postterm delivery. Preterm delivery refers to deliveries that occurred between 28 and 37 weeks of gestation. Pregnancy outcomes were divided into two groups based on whether embryo implantation was successful. Successful embryo implantation included term birth, preterm birth, postterm birth, clinical pregnancy, early abortion, and middle or late abortion.
Statistical analysis
The required sample size for this study was estimated using PASS software version 2021 (NCSS, USA). The study power and α-value were set at 95% and 0.05, respectively. Power analysis indicated that a total of 680 participants (number of groups = 5) were needed. Logistic binary regression was used to analyze the effects of subendometrial blood flow on successful embryo implantation in different endometrial thickness groups. Odds ratios and 95% CIs were calculated in each group of EMT. The Kolmogorov–Smirnov test was performed to evaluate the distribution of continuous variables. Continuous data with a normal distribution are expressed as the mean ± standard deviation (SD) and were analyzed with a two-tailed Student’s t test. The chi-squared test was used to assess the differences in frequencies with percentages. A P value of < 0.05 was considered statistically significant. SPSS 25.0 software was used for statistical analysis.
Results
We reviewed 34,797 cycles of FET performed at our center from January 2017 to April 2023, of which 2615 cycles had subendometrial blood flow measured on the day of endometrial transformation, 76 cycles were excluded due to transfer of more than 1 embryo, 1 cycle was excluded due to cancellation for patient reasons, and 22 cycles were excluded due to female age > 44 years. Six cycles were excluded for the transfer of cleavage embryos and 5 cycles for donor eggs, resulting in 2505 cycles from 2505 patients being included in the study.
Baseline data
To further exclude possible interference of baseline characteristics on the interpretation of our findings, we divided the patients with thin endometrium (i.e., 0.5 cm < EMT ≤ 0.7 cm) into Category 1 or Category 2 based on the Applebaum classification method and compared age, duration of infertility, body mass index (BMI), type of infertility, AMH, basal FSH, basal LH, TSH, number of retrieved oocytes, number of blastulas, type of fertilization method, endometrial preparation protocols, and EMT between the two groups. No significant differences in these characteristics were observed between the two groups (P > 0.05) (Table 1). The characteristics of the patients with endometria exceeding 0.7 cm are presented in Tables S2-5. In the patients with 0.7 cm < EMT ≤ 0.8 cm, Category 1 patients were older than Category 2 patients (37.49 ± 5.14 vs 36.32 ± 4.63 years, P = 0.000) and had a higher BMI (24.10 ± 3.68 vs 23.26 ± 3.55 kg/m2, P = 0.001), lower rates of primary infertility (26.16% vs 35.69%, P = 0.003), lower AMH (3.79 ± 3.02 vs 4.64 ± 3.49 ng/mL, P = 0.000), and fewer retrieved oocytes (11.35 ± 7.06 vs 13.01 ± 7.20, P = 0.001) and blastulas (5.13 ± 3.94 vs 5.81 ± 3.69, P = 0.009). In the patients with 0.8 cm < EMT ≤ 0.9 cm, Category 1 patients were older than Category 2 patients (37.69 ± 4.96 vs 36.01 ± 4.70 years, P = 0.000) and had a lower AMH (4.20 ± 3.51 vs 4.95 ± 3.58 ng/mL, P = 0.019). In patients with 0.9 cm < EMT ≤ 1.0 cm, Category 1 patients were older than Category 2 patients (37.78 ± 5.20 vs 35.92 ± 4.30 years, P = 0.001) and had a higher BMI (24.64 ± 4.08 vs 23.77 ± 3.48 kg/m2, P = 0.048) and lower AMH (3.96 ± 2.77 vs 4.86 ± 3.30 ng/mL, P = 0.021). In patients with EMT > 1.0 cm, Category 1 patients were older than Category 2 patients (37.57 ± 5.09 vs 36.30 ± 4.68 years, P = 0.039) and had a higher BMI (24.84 ± 3.96 vs 23.93 ± 3.40 kg/m2, P = 0.049), lower AMH (3.66 ± 3.58 vs 4.70 ± 3.26 ng/mL, P = 0.022), lower basal FSH (7.40 ± 3.16 vs 6.57 ± 2.15 IU/L, P = 0.027), and fewer retrieved oocytes (10.20 ± 4.92 vs 12.14 ± 6.07, P = 0.005) and blastulas (4.29 ± 2.78 vs 5.37 ± 3.21, P = 0.006).
Table 1.
Baseline characteristics of the patients with thin endometrium (0.5 cm ≤ endometrial thickness ≤ 0.7 cm)
| Variables | Category 1 (Type I) N = 186 |
Category 2 (Type II & III) N = 217 |
P value |
|---|---|---|---|
| Age (years) | 37.66 ± 4.67 | 36.85 ± 4.70 | 0.085 |
| Duration of infertility (years) | 3.04 ± 3.18 | 2.99 ± 2.71 | 0.858 |
| BMI (kg/m2) | 23.51 ± 3.30 | 22.94 ± 3.15 | 0.078 |
| Primary infertility (%) | 27 (14.52) | 48 (22.12) | 0.051 |
| AMH (ng/ml) | 3.62 ± 3.08 | 4.04 ± 3.08 | 0.193 |
| Basal FSH(IU/L) | 7.38 ± 3.30 | 6.89 ± 2.66 | 0.119 |
| Basal LH(IU/L) | 6.17 ± 6.86 | 6.10 ± 3.80 | 0.903 |
| TSH (uIU/mL) | 2.36 ± 1.52 | 2.18 ± 0.97 | 0.187 |
| No. of retrieved oocytes | 11.02 ± 7.05 | 12.34 ± 7.02 | 0.061 |
| No. of blastulas | 5.39 ± 3.81 | 6.06 ± 3.67 | 0.074 |
| Fertilization method, no. (%) | 0.939 | ||
| IVF | 107 (57.53) | 128 (58.99) | |
| ICSI | 74 (39.78) | 84 (38.71) | |
| PGT | 5 (2.69) | 5 (2.30) | |
| Endometrial preparation protocols (number of cycles) | 0.313 | ||
| HRT cycle | 98 (52.69) | 129 (59.45) | |
| Natural cycle | 37 (19.89) | 42 (19.35) | |
| Ovulation stimulation cycle | 51 (27.42) | 45 (20.74) | |
| Others | 0 | 1 (0.46) | |
| Endometrial thickness (cm) | 0.66 ± 0.05 | 0.67 ± 0.04 | 0.343 |
Data are presented as the mean ± standard deviation or n (%). P < 0.05 was considered statistically significant. Independent sample t tests, Mann–Whitney U tests, or chi-square tests were used as appropriate
Abbreviations: BMI body mass index, AMH anti-Müllerian hormone; FSH follicle-stimulating hormone, LH luteinizing hormone, TSH thyroid-stimulating hormone, IVF in vitro fertilization, ICSI intracytoplasmic sperm injection, PGT preimplantation genetic testing, HRT hormonal replacement treatment
Embryo implantation
The patients were divided into five groups according to EMT, and the effect of the presence of subendometrial blood flow on embryo implantation was analyzed for each group via logistic regression (Table 2). In the patients with 0.5 cm ≤ EMT ≤ 0.7 cm, the embryo implantation rate was higher in the patients with the presence of subendometrial blood flow (Category 2) than Category 1 (53.92% vs 44.08%, P = 0.049; OR 1.484; 95% CI, 1.001–2.200; P = 0.049). However, when the thickness of the endometrium exceeded 0.7 cm, there was no significant correlation between successful embryo implantation and subendometrial blood flow (P > 0.05). Maternal age, type of infertility, BMI, AMH, number of oocytes retrieved, and number of blastocysts were also potential influence factors for embryo implantation. A multivariate logistic regression was subsequently conducted, and subendometrial blood flow was still significantly associated with successful embryo implantation in patients with thin endometrium after adjusting for confounding variables (aOR 1.425; 95% CI, 1.030–2.123; P = 0.003) (Table 2).
Table 2.
Univariate and multivariate logistic regression analyses of the association between the presence of subendometrial blood flow and embryo implantation in different endometrial thickness groups
| Group | Implantation rate | OR (95% CI) | P value | aOR (95% CI) | P value |
|---|---|---|---|---|---|
| 0.5 cm ≤ EMT ≤ 0.7 cm | |||||
| Category 1 N = 186 | 82 (44.08) | 1.0 (Reference) | 1.0 (Reference) | ||
| Category 2 N = 217 | 117 (53.92) | 1.484 (1.001–2.200) | 0.049 | 1.425 (1.020–2.123) | 0.003 |
| 0.7 cm < EMT ≤ 0.8 cm | |||||
| Category 1 N = 407 | 252 (61.92) | 1.0 (Reference) | 1.0 (Reference) | ||
| Category 2 N = 489 | 300 (61.35) | 0.976 (0.745–1.280) | 0.862 | 0.848 (0.628–1.144) | 0.280 |
| 0.8 cm < EMT ≤ 0.9 cm | |||||
| Category 1 N = 236 | 147 (62.29) | 1.0 (Reference) | 1.0 (Reference) | ||
| Category 2 N = 355 | 224 (63.10) | 1.035 (0.737–1.455) | 0.842 | 0.922 (0.628–1.354) | 0.679 |
| 0.9 cm < EMT ≤ 1.0 cm | |||||
| Category 1 N = 128 | 83 (64.84) | 1.0 (Reference) | 1.0 (Reference) | ||
| Category 2 N = 216 | 159 (73.61) | 1.512 (0.943–2.426) | 0.086 | 1.464 (0.866–2.475) | 0.155 |
| EMT > 1.0 cm | |||||
| Category 1 N = 108 | 71 (65.74) | 1.0 (Reference) | 1.0 (Reference) | ||
| Category 2 N = 163 | 104 (63.80) | 0.919 (0.552–1.530) | 0.744 | 0.946 (0.539–1.662) | 0.847 |
Adjusted for maternal age, type of infertility, BMI, AMH, number of retrieved oocytes and blastulas. Data are presented as n (%). P < 0.05 was considered statistically significant and indicated in bold. Logistic regression analyses were performed
Abbreviations: EMT endometrial thickness, OR odds ratios, aOR adjusted odds ratios, CI credible intervals
Pregnancy outcomes
To investigate whether the presence of subendometrial blood flow was associated with successful pregnancy outcomes in IVF patients with thin endometrium, we next examined the pregnancy outcomes in the thin endometrium patient group (0.5 cm ≤ EMT ≤ 0.7 cm) (Table 3). Category 1 patients had a lower biochemical pregnancy rate (49.46% vs 60.37%, P = 0.028), clinical pregnancy rate (44.09% vs 53.92%, P = 0.049), and live birth rate (29.57% vs 40.09%, P = 0.027) than Category 2 patients. We also explored the effects of different endometrial preparation protocols on pregnancy outcomes in patients with thin endometrium. Different endometrial preparation protocols did not affect pregnancy outcomes, even when classified as Category 1 or Category 2 (Table S6).
Table 3.
Pregnancy outcomes of patients with thin endometrium (0.5 cm ≤ endometrial thickness ≤ 0.7 cm)
| Pregnancy outcome | Category 1 (Type I) N = 186 |
Category 2 (Type II & III) N = 217 |
P value |
|---|---|---|---|
| Biochemical pregnancy rate | 92 (49.46) | 131 (60.37) | 0.028 |
| Clinical pregnancy rate | 82 (44.09) | 117 (53.92) | 0.049 |
| Live birth rate | 55 (29.57) | 87 (40.09) | 0.027 |
Data are presented as n (%). P < 0.05 was considered statistically significant and indicated in bold. The chi-square test was used
However, no significant difference in the biochemical pregnancy rate, clinical pregnancy rate, or live birth rate existed between patients in Category 1 and Category 2 when the thickness of the endometrium exceeded 0.7 cm (Tables S7-10). As shown in Table S11, maternal age, type of infertility, BMI, AMH, and number of retrieved oocytes and blastulas were included in the logistic regression analysis to eliminate the potential effects of these confounding factors. The final results were consistent with those of the abovementioned analyses.
Discussion
Main findings
In the patients with 0.5 cm ≤ EMT ≤ 0.7 cm, the embryo implantation rate was statistically significant increased in the patients with the presence of subendometrial blood flow (OR 1.484; 95% CI, 1.001–2.200; P = 0.049; aOR 1.425; 95% CI, 1.030–2.123; P = 0.003). Patients with discernible subendometrial blood flow have superior live birth (P = 0.028), clinical pregnancy (P = 0.049), and embryo implantation (P = 0.027) compared to the patients without subendometrial blood flow when the EMT is ≤ 0.7 cm.
Strengths and limitations
Although previous studies have described the application of transvaginal color Doppler sonography for determining subendometrial blood flow to predict pregnancy outcome, this is, to the best of our knowledge, the first in-depth study investigating the specific impacts of subendometrial blood flow on pregnancy outcomes in patients with thin endometrium. Our findings thus provide an effective predictor of pregnancy outcomes following FET in patients with a thin endometrium, underscoring the necessity for improved subendometrial blood flow in IVF patients. However, further studies are warranted to examine the potential implications of our findings for clinical practice, particularly for naturally conceived women and IVF patients without thin endometrium.
One limitation of our study was that endometrial echo, uterine cavity volume, and other factors related to ER that could influence pregnancy outcomes were not included in our analysis due to incomplete data. In addition, the possible impacts of differences in embryo quality could not be entirely excluded. The retrospective study design and limited data for other factors contributing to pregnancy outcomes such as the variation in E2 and other hormones during endometrial preparation should be considered when interpreting the results. All patients undergoing FET can undergo the assessment of blood flow, among them, primary infertility patients, patients with thin endometrium, and patients with a limited number of embryos available for transfer are particularly recommended, which may lead to some selection bias. The different effects of regimens for improving subendometrial blood flow, including mesenchymal stem cells, growth hormone (GH), sildenafil, aspirin, and low molecular weight heparin (LMWH), are also worthy of being evaluated.
Interpretation
Multiple factors have been shown to influence the rate of clinical pregnancy after FET, including embryo quality, synchronization of embryo and endometrium, and ER [19]. Notably, the uterine arteries travel from the muscle layer to the endometrial surface, eventually forming capillary networks and sinus-shaped capillaries [20, 21]. Thus, the endometrial and subendometrial blood flow conditions could also contribute potentially major effects toward the establishment and maintenance of pregnancy. Based on this strong influence of the endometrium on the success of IVF treatments, increasing numbers of studies use noninvasive Doppler ultrasound to measure the morphological and hemodynamic parameters related to ER to characterize the relationship between subendometrial blood flow and pregnancy outcome [6].
One previous meta-analysis analyzed ten articles, including 895 pregnant women and 882 nonpregnant women, and demonstrated that subendometrial blood flow, particularly on the day of ET, was correlated with pregnancy outcomes and could therefore help identify the most suitable time for ET [22]. Similarly, a prospective study investigating patients who underwent ET cycles with endometrium ≥ 0.7 cm on day 14 revealed that several Doppler ultrasound indices, including endometrial vascularization flow, were significantly higher in the pregnant group than in the nonpregnant group. These results indicated that ultrasound measurements of subendometrial blood flow could serve as a useful parameter in predicting pregnancy during ET cycles [11]. However, the statistical power of that study was limited by its small sample size and advanced age of patients. Moreover, that study only investigated patients with EMT ≥ 0.7 cm and did not include patients with a thin endometrium. In addition, several studies have shown that uterine artery conditions, such as the presence of endometrial and subendometrial blood flow, are associated with successful pregnancy outcomes in IVF patients [12, 23, 24], which highlights the predictive value of hemodynamics in the endometrial area.
However, these results remain controversial, and other studies have reported contradictory conclusions. For example, one study found that the endometrial and subendometrial vascular parameters measured by color Doppler ultrasound during IVF cycles could not be used to predict pregnancy outcomes [25]. Notably, this study only included patients in their first IVF cycle following a standard, long ovarian stimulation regimen of GnRH agonist who had the two highest-scoring embryos transferred on day 3 after fertilization. Although that study considered embryo quality and excluded interference from the endometrial preparation method, this research did not include patients with thin endometrium, which was the focus of our study.
The reason for the failure of FET associated with a thin endometrium may be related to the partial pressure of oxygen. The thin or missing functional layer of the endometrium indicates a closer distance between the implanted embryo and the spiral artery, leading to a higher oxygen concentration of the basal endometrium. Compared with the normal low oxygen tension of the superficial endometrium, it has been suggested that the hyperoxia that occurs near the basal endometrium may be harmful [26]. High blood flow resistance, downregulation of VEGF expression, insufficient epithelial growth, and insufficient vascularization are all considered pathophysiological characteristics of a thin endometrium [27]. The high blood flow resistance of the uterine radial artery may be a trigger that can impair the growth of the glandular epithelium and cause a decrease in the level of VEGF in the endometrium. Low VEGF levels can lead to vascular dysplasia, reducing blood flow in the endometrium. This vicious cycle leads to a thin endometrium [28]. The uterine artery blood flow reflects the blood flow of the entire uterine cavity, while the subendometrial blood flow is mainly determined by the terminal artery of the uterine artery. Our study confirms that subendometrial blood flow has no significant effect on pregnancy outcomes in patients with medium or thick endometrium. The reason may be that even if the terminal arterial perfusion is poor, good blood flow of the uterine artery and, thus, good blood perfusion throughout the uterine cavity can still be enough to achieve better pregnancy outcomes. In patients with thin endometrium, blood perfusion in the uterine cavity is inherently poor; however, if the terminal artery has more branches, it may relatively increase the pregnancy rate. Admittedly, further research is needed to identify the actual underlying pathological mechanisms of our clinical findings.
Potential avenues to improve the ER of patients with a thin endometrium have been gradually revealed. Cell therapy has been proposed in recent years as an alternative treatment for thin endometrium, including the use of stem cells, PRP, and growth factors as treatments [29]. A clinical case study reported that the use of autologous mesenchymal stem cells increased ER for pregnancy in women with a thin endometrium. Intrauterine autologous platelet-rich plasma (PRP) infusion has been shown to improve EMT, implantation rate, and clinical pregnancy rate in patients with thin endometrium [30, 31]. Some drugs have also been reported to improve the ER, such as sildenafil, aspirin, GH, and LMWH [32–34].
Conclusion
The presence of subendometrial blood flow detected by ultrasound was positively associated with successful embryo implantation and favorable pregnancy outcomes in patients with thin endometrium undergoing FET.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors would like to thank all patients who participated in this study and give special thanks to colleagues of the IVF laboratory, PGT laboratory, and nurses and doctors at the Reproductive Hospital Affiliated to Shandong University.
Author contribution
JH.Y., Y.L., and GY.Z. conceived and designed the project; ZW.Z., JN.L., YC. Y., and MW. Z. analyzed the data and wrote the manuscript; JH.Y., Y.L., GY.Z., and Q.Z. critically revised the manuscript. All authors were involved in interpreting the data and had final approval of the submitted and published versions.
Funding
This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604, 2022YFC2702400), the National Natural Science Foundation of China (82101784, 82171648), the Key Research and Development Program of Shandong Province (2021LCZX02), the Natural Science Foundation of Shandong Province (ZR2020QH051), the Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154), the Shandong Provincial University Young Innovators Team Initiative Plan, and the Young Scholars Program of Shandong University.
Data availability
The datasets used and analyzed during the current study are available from the corresponding authors on reasonable request.
Code availability
Not applicable.
Declarations
Ethics approval
This study was conducted according to the ethical guidelines of the World Medical Association (Declaration of Helsinki) and was approved on 24 December 2021 by the Ethics Committee of Reproductive Hospital Affiliated to Shandong University (IRB no. 140).
Consent to participate
Written informed consent was obtained from all patients before treatment, and the patients consented to the use of their retrospective data in scientific publications.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Zhaowen Zang and Jianan Lyu contributed equally to this work.
Contributor Information
Guangyong Zhang, Email: guangyongzhang@hotmail.com.
Yan Li, Email: yanli.sdu@gmail.com.
Junhao Yan, Email: yyy306@126.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding authors on reasonable request.
Not applicable.