Abstract
Purpose
To investigate whether personalized embryo transfer (pET) predicted by a modified RNA-sequencing-based endometrial receptivity test (rsERT) model can improve intrauterine pregnancy rate (IPR) in patients with a receptive window of implantation (WOI).
Design
A retrospective pilot study was conducted in the Center for Reproductive Medicine, Central South University, from January 2018 to December 2023. A total of 524 patients with receptive WOI results from rsERT were assigned into two groups based on whether they underwent conventional embryo transfer (conventional ET) or pET. Patients in the conventional ET were matched with those in the pET group at a 1:1 ratio using propensity score matching (PSM).
Results
Before PSM, the IPR (55.73% vs. 46.19%, P = 0.032) and implantation rate (IR) (47.51% vs. 34.03%, P = 0.000) in the pET group were significantly higher than that in the conventional ET group. However, the number and types of transferred embryos differed significantly between the two groups. After adjusting for confounding factors, IPR (57.38% vs. 44.81, P = 0.016) and IR (46.81% vs. 33.10%, P = 0.001) remained significantly higher in the pET group compared to the conventional ET group. The implantation failure rate was significantly lower in the pET group compared to controls (42.62% vs. 55.19%, P = 0.016). Additionally, the multiple-pregnancy rate was significantly higher in the pET group compared to the conventional ET group (10.29% vs. 1.68%, P = 0.001).
Conclusions
Women with receptive WOI results could benefit from the receptivity-timed pET predicted by the newly refined rsERT. These findings provide a basis for future research in precision medicine for embryo transfer.
Supplementary Information
The online version contains supplementary material available at 10.1007/s10815-024-03246-y.
Keywords: Window of implantation, Endometrial receptivity, RNA sequencing, Personalized embryo transfer, Frozen embryo transfer
Introduction
Even though embryo selection provided by time-lapse microscopic photography or preimplantation genetic testing ensures a competent euploid embryo, there is still a risk of implantation failure or early miscarriage [1, 2]. It has been estimated that approximately two-thirds of implantation failures can be attributed to endometrial factors [3]. In these cases, a displaced temporal window of implantation (embryo-endometrial asynchrony) and/or disrupted endometrial receptivity (molecular pathologies) are often blamed for the failure [4].
The window of implantation (WOI) is a self-limited period when the uterine milieu is favorable to blastocyst acceptance and implantation. It begins in the mid-secretory phase coinciding with the 7th day after the luteinizing hormone (LH) surge and lasts for 4–5 days [5]. However, the optimal duration of the WOI is relatively short and is approximately 24–48 h in humans [6]. When the WOI is advanced or delayed, asynchrony between the embryo and endometrium may occur, resulting in implantation failure.
In today’s transcriptomic era, with the development of sophisticated bioinformatic information technologies, commercially available diagnostic tools for endometrial dating have been introduced in the clinical practice of assisted reproductive technology (ART) [7–9]. These tools are applied to individuals, including infertile women who have experienced at least one failed euploid embryo transfer [10, 11] or have a history of recurrent implantation failure (RIF) [9, 12–15]. Infertile patients without previous failed cycles were also eligible for these tests [16, 17], and some of them were tested due to a scarcity of viable frozen embryos or atrophic endometrium on sonographic assessment [11]. The rate of displaced WOI ranges from 17.7% [14] to 64.2% [18] in previously reported studies. Moreover, incongruent or even contradictory results have been reported regarding the clinical efficacy of personalized embryo transfer (pET) based on the results from endometrial receptivity analysis. Some studies demonstrated that pET improved reproductive outcomes among infertile women in their first IVF attempts [17] or women who had experienced RIF [9]. Others challenged the benefits of the endometrial receptivity analysis, as no significant difference was found between pET indicated by putative WOI results and those who underwent conventional embryo transfer (ET) procedures [10–12, 16, 18].
Previously, we developed a novel RNA-sequencing-based endometrial receptivity test (rsERT) containing 175 genetic biomarkers to accurately determine the endometrial WOI [9]. The three-time-point sampling method from the same subject at 48-h intervals during the same menstrual cycle (LH + 5/LH + 7/LH + 9) allows a more precise identification of the optimal WOI. Subsequently, we proposed the clinical application of this tool in patients who experienced multiple implantation failures. The rsERT can personalize the timing of ET, which considerably improves the pregnancy outcomes of RIF patients, particularly in the context of a displaced WOI [9]. To avoid repeated sampling and provide a less invasive alternative to identify the optimal time for embryo transfer, we recruited participants to modify the rsERT and successfully established an estimation method by single-time-point sampling [19].
To our knowledge, there is currently no study specifically addressing the efficacy of endometrial receptivity tests for patients with receptive WOI. The current study aimed to determine whether infertile patients without a problem of displaced WOI could benefit from individualized ET with hourly precision guided by the modified rsERT. Therefore, we performed a pilot study by retrospectively comparing pregnancy outcomes between the pET and conventional ET among women with receptive WOI results provided by rsERT.
Materials and methods
Study design and participants
The current pilot study was retrospectively carried out from January 2018 to December 2023 at the reproductive medicine center of a tertiary hospital, in compliance with the ethical principles of the Declaration of Helsinki. Participants who were referred to rsERT and had a receptive WOI result were eligible for this study.
The inclusion criteria were as follows: 1. Age 20–40 years old; 2. Body mass index (BMI) 18.5–28 kg/m2; 3. The result of WOI predicted by rsERT was receptive; 4. Patients underwent frozen embryo transfer according to the predictive result of WOI by adopting hormone replacement therapy (HRT-FET). The exclusion criteria were as follows: (1) endometrial abnormalities (e.g., intrauterine adhesion, endometrial polyps, endometrial hyperplasia, and chronic endometritis); (2) submucous myoma distorting the uterine cavity confirmed by hysteroscopy or ultrasonography; (3) genital tuberculosis; and (4) severe comorbidities (e.g., hypertension, diabetes, or malignant tumors).
The study was approved by the ethical committee of Xiangya Hospital, Central South University, Changsha, China (reference number 2017002). Written informed consent was obtained from all patients prior to sample collection.
Endometrial sampling and processing
The sampling cycle was conducted in the cycle prior to ET. All participants performed three-time-point rsERT or the modified rsERT by receiving hormonal endometrial preparation. Estradiol administration was started at 4 mg daily for 6 days from the third day of the menstrual cycle, and the dosage could be increased to 6 mg or more when the endometrial thickness was suboptimal. Progesterone (P4) supplementation (vaginal progesterone suppository, 200 mg, tid) was started after at least 12 days of estrogen usage if the endometrium was > 7 mm and the endogenous P4 serum level was close to zero. The day of starting P4 supplementation was considered P + 0. For the three-time-point rsERT, endometrial tissue was collected on days 4th, 6th, and 8th after P4 supplementation (i.e., P + 3, P + 5, and P + 7, respectively). For the modified rsERT, the endometrium was exclusively collected on P + 5. Specifically, all participants in this cohort were asked to detail the exact time when the vaginal progesterone suppository was first administered. The operator also needed to accurately record the timing of endometrial biopsy to obtain the estimated optimal WOI from the modified rsERT prediction model.
Before sampling, the cervix was cleansed with saline. The tip of the endometrial sampler was placed into the uterine fundus, and 5–10 mm3 of endometrial tissue was aspirated into the sampler. The collected endometrial tissues were immediately placed into 1.5 ml of RNAlater buffer (AM7020; Thermo Fisher Scientific, Waltham, MA, USA) for RNA stabilization, sealed, and cryopreserved at − 20 °C. Sequencing analysis was carried out within 7 days after sampling.
RNA extraction, library construction, and sequencing
RNA sequencing of endometrial biopsy samples was conducted following the protocol described in a previous study [9]. In brief, total RNA extraction, RNA quality control, reverse transcription and amplification, and next-generation sequencing (NGS) library construction were carried out by using commercial reagent kits. Subsequently, single-end sequencing was performed on the HiSeq 2500 platform (Illumina, San Diego, CA, USA) under relevant parameters. The read length was set to 140 bp, and the volume of raw data was approximately 5 million reads.
Conventional ET and modified rsERT-guided pET with optimal hour recommendation
As depicted in Supplementary Fig. 1, three endometrial samples from an individual who had a receptive WOI result were reported as pre-receptive (P + 3), receptive (P + 5), and post-receptive (P + 7). In this case, conventional ET was performed according to our routine working schedule. The initial P4 was given between 10:00 a.m. and 1:00 p.m. on P + 0. Frozen-thawed cleavage-stage embryos or blastocysts were transferred between 10:00 a.m. and 12:00 p.m. on P + 3 or P + 5 respectively. For blastocysts transfer, the duration of P4 exposure ranged from 117 to 122 h.
The modified rsERT not only reports whether the timing of endometrial biopsy was receptive but also recommends the optimal WOI timing for ET. On the basis of the modified rsERT result, pET would be scheduled as close as possible to the recommended optimal WOI, corresponding to the timing of blastocyst transfer. Day 3 cleavage-stage embryos were transferred two days earlier accordingly. For example, an endometrial sample aspirated at 10:00 a.m. on P + 5 was receptive (with the optimal WOI being + 6 h from the timing of endometrial biopsy) by the modified rsERT, the blastocyst transfer would be conducted at 4:00 p.m. on P + 5 in the subsequent programmed mock cycle (with initial P4 administered at the same time as in the sampling cycle, i.e., 9:00 a.m. on P + 0). It is worth noting that for patients whose displacement of optimal WOI is within 3 h (i.e., P4 exposure ranging from 117 to 123 h for blastocyst transfer), our center also adopts conventional ET.
Pregnancy outcome measures
All patients were followed up to assess pregnancy outcomes as follows. Plasma β-human chorionic gonadotropin (β-hCG) was measured 12 days after ET. The intrauterine pregnancy and number of gestational sacs were assessed by ultrasound 28 days after ET among patients with β-hCG positivity. The primary outcome measure was the intrauterine pregnancy rate (IPR). The secondary outcome was the implantation rate (IR). IPR refers to the number of patients with intrauterine gestation per ET cycle. IR refers to the number of intrauterine gestational sacs observed divided by the number of embryos transferred. Biochemical pregnancy loss was defined as an early spontaneous abortion after the identification of a β-hCG positivity followed by a consecutive fall in plasma β-hCG concentration before the ultrasonic detection of the gestational sac. Ongoing pregnancy was defined as a sustained pregnancy at 12 weeks gestation. Early spontaneous abortion refers to spontaneous abortion before 12 weeks gestation. Late spontaneous abortion refers to spontaneous abortion between 12 and 28 weeks gestation. Implantation failure rate (IFR) was defined as the ratio of cycles not resulting in an intrauterine pregnancy to the total number of transferred cycles.
Statistical analysis
Statistical analysis was performed using IBM SPSS software (version 25.0, IBM Corp.). Continuous variables were described as the mean ± standard deviation (SD) or median and interquartile range (IQR) according to the distribution. Categorical variables were presented as frequencies and percentages. Between-group differences among variables were analyzed by Student’s t-test or Mann–Whitney test and Pearson’s chi-squared test or Fisher’s exact test for continuous and categorical variables, respectively. Propensity score matching (PSM) was performed to adjust for the parity and the number and types (cleavage-stage embryo or blastocyst) of transferred embryos. Women who underwent pET indicated by modified-rsERT with hourly precision were nearest-neighbor-matched in a 1:1 ratio to their control counterparts (conventional ET after indication of a receptive endometrium by rsERT) with a caliper width of 0.02. Values of P < 0.05 for two-sided tests were considered statistically significant.
Results
Participants and baseline characteristics before PSM
From January 2018 to December 2023, there were 524 eligible participants with a receptive endometrial WOI result provided by either the three-time-point rsERT or the modified rsERT. 210 participants were routinely given initial P4 between 10:00 a.m. and 1:00 p.m. on P + 0, and underwent conventional ET in the morning between 10:00 a.m. to 12:00 p.m. on P + 5 for blastocysts or on P + 3 for cleavage-stage embryos (conventional ET group). Of these, 116 patients (55.24%) underwent three-time-point rsERT followed by conventional ET, while 94 patients (44.76%) performed conventional ET because their displacement of WOI was within 3 h predicted by the modified rsERT. There were 314 eligible patients who had receptive results with the optimal hourly timing recommendation and underwent pET according to the guidance of modified rsERT (pET group). The baseline clinical characteristics of the two groups are shown in Table 1.
Table 1.
Baseline clinical characteristics of the patients in the conventional ET group and pET group
| Characteristics | Conventional ET group N = 210 | pET group N = 314 | P value |
|---|---|---|---|
| Age (y) | 31.66 ± 3.79 | 31.93 ± 3.78 | 0.501 |
| BMI (kg/m2) | 21.74 ± 2.36 | 21.57 ± 2.62 | 0.182 |
| Infertility duration (y) | 4.95 ± 3.12 | 4.74 ± 3.46 | 0.154 |
| Gravidity n (%) | 0.174 | ||
|
0 ≥ 1 |
103 (49.05) 107 (50.95) |
173 (55.10) 141 (44.90) |
|
| Parity n (%) | 0.080 | ||
|
0 ≥ 1 |
170 (80.95) 40 (19.05) |
272 (86.62) 42 (13.38) |
|
| Types of infertility n (%) | 0.280 | ||
|
Primary infertility Secondary infertility |
111 (52.86) 99 (47.14) |
181 (57.64) 133 (42.36) |
|
| No. of previous failed cycles n (%) | 0.190 | ||
|
0–1 ≥ 2 |
40 (19.05) 170 (80.95) |
75 (23.89) 239 (76.11) |
|
| Prevalence of RIF n (%) | 131 (62.38) | 168 (53.50) | 0.044 |
| Main etiology of infertility n (%) | 0.426 | ||
|
Tubal Ovulation disorder Endometriosis Diminished ovarian reserve Male aOthers |
132 (62.86) 12 (5.71) 13 (6.19) 16 (7.62) 20 (9.52) 17 (8.10) |
189 (60.19) 18 (5.73) 10 (3.18) 23 (7.32) 42 (13.38) 32 (10.20) |
|
| PGS or PGD n (%) | 19 (9.05) | 34 (10.83) | 0.508 |
| FSH (mIU/ml) | 6.52 (5.78, 7.80) | 6.50 (5.62, 7.60) | 0.789 |
| LH (mIU/ml) | 5.17 (3.74, 7.00) | 5.40 (4.00, 7.30) | 0.188 |
| E2 (pg/ml) | 36.54 (27.70, 46.64) | 41.40 (30.50, 53.10) | 0.004 |
| AMH (ng/ml) | 2.62 (1.55, 4.18) | 2.70 (1.69, 4.77) | 0.123 |
ET, embryo transfer; pET, personalized embryo transfer; BMI, body mass index; RIF, recurrent implantation failure; PGS, preimplantation genetic screening; PGD, preimplantation genetic diagnosis; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; AMH, anti-Mullerian hormone
aOthers included recurrent spontaneous abortion, chromosomal or genetic abnormalities, and unexplained infertility
Compared with women in the conventional ET group, the pET group had a significantly lower prevalence of RIF (53.50% vs. 62.38%, P = 0.044) and a significantly higher level of serum basal E2 [41.40 (30.50, 53.10) vs. 36.54 (27.70, 46.64) pg/ml, P = 0.004]. Other baseline clinical parameters, including age, BMI, infertility duration, the prevalence of gravidity and parity, types of infertility, number of previous failed cycles, main etiology of infertility, the prevalence of preimplantation genetic screening/diagnosis (PGS/PGD), basal serum follicular stimulating hormone (FSH), basal luteinizing hormone (LH) and anti-Mullerian hormone (AMH), were all comparable between the two groups (P > 0.05).
Cycle characteristics and pregnancy outcomes before PSM
The conventional ET group and pET group were similar in endometrial thickness (9.66 ± 1.93 vs. 9.61 ± 1.61, P = 0.767) and the proportion of endometrial patterns (P = 0.680) (Table 2). However, higher rates of D5 or D6 blastocysts were transferred in women in the pET group compared to those in the conventional ET group (73.53% vs. 53.43%, P = 0.000) (Table 2). There was no significant difference in the transfer rates of good-quality D3 embryos (conventional ET vs. pET: 93.59% vs. 95.73%, P = 0.043) and blastocysts (conventional ET vs. pET: 39.11% vs. 32.62%, P = 0.144) between the two groups. Additionally, more embryos were transferred in the conventional ET group than in the pET group (1.60 ± 0.50 vs. 1.41 ± 0.49, P = 0.000).
Table 2.
HRT-FET cycle characteristics and pregnancy outcomes in the conventional ET group and pET group
| Characteristics | Conventional ET group N = 210 | pET group N = 314 | P value |
|---|---|---|---|
| Endometrial thickness (mm) | 9.66 ± 1.93 | 9.61 ± 1.61 | 0.767 |
| Endometrial types n (%) | 0.680 | ||
|
A B C |
39 (18.57) 162 (77.14) 9 (4.29) |
68 (21.66) 232 (73.89) 14 (4.45) |
|
| Transferred embryo stage n/N (%) | 0.000 | ||
|
D3 cleavage-stage embryos D5 or D6 blastocysts |
156/335 (46.57) 179/335 (53.43) |
117/442 (26.47) 325/442 (73.53) |
|
| Good-quality D3 embryos transferred rate n/N (%) | 146/156 (93.59) | 112/117 (95.73) | 0.443 |
| Good-quality blastocysts transferred rate n/N (%) | 70/179 (39.11) | 106/325 (32.62) | 0.144 |
| No. of transferred embryos n | 1.60 ± 0.50 | 1.41 ± 0.49 | 0.000 |
| β-hCG positive rate n (%) | 123 (58.57) | 201 (64.01) | 0.209 |
| Biochemical pregnancy loss n (%) | 22 (10.48) | 23 (7.32) | 0.207 |
|
Intrauterine pregnancy rate n (%) Implantation failure rate n (%) |
97 (46.19) 113 (53.81) |
175 (55.73) 139 (44.27) |
0.032 0.032 |
| Implantation rate n/N (%) | 114/335 (34.03) | 210/442 (47.51) | 0.000 |
|
Ectopic pregnancy rate n (%) Early spontaneous abortion rate n/N (%) Ongoing pregnancy rate n (%) aLate spontaneous abortion rate n/N (%) aPre-term birth rate n/N (%) aTerm birth rate n/N (%) aLive birth rate n/N (%) aMultiple pregnancy rate n/N (%) |
4 (1.90) 13/97 (13.40)b 87 (41.43) 6/92 (6.52)c 8/205 (3.90) 68/205 (33.17) 78/205 (38.05) 6/205 (2.93) |
3 (0.96) 33/175 (18.86)d 146 (46.50) 6/162 (3.70)e 23/301 (7.64) 104/301 (34.55) 128/301 (42.52) 21/301 (6.98) |
0.446 0.250 0.253 0.478 0.085 0.748 0.314 0.047 |
HRT-FET, hormone replacement therapy-frozen embryo transfer; ET, embryo transfer; pET, personalized embryo transfer; β-hCG, β-human chorionic gonadotropin
aFive and thirteen subjects are in ongoing pregnancy but have not yet delivered live births in the conventional ET group and pET group, respectively
bThree subjects experienced twin pregnancies, with one experiencing an early miscarriage and the other having a full-term live birth
cAmong the 6 cycles of late miscarriage, 2 had live births
dFour subjects experienced twin pregnancies, with one experiencing an early miscarriage and the other having a full-term live birth
eAmong the 6 cycles of late miscarriage, 1 had live births
In the pET group, the IPR (55.73% vs. 46.19%, P = 0.032) and IR (47.51% vs. 34.03%, P = 0.000) were significantly higher than that in the conventional ET group (Table 2). Additionally, the multiple pregnancy rate was significantly higher in the pET group than in the conventional ET group (6.98% vs. 2.93%, P = 0.047). With respect to implantation failure, a significantly higher IFR was observed in the conventional ET group (53.81% vs. 44.27%, P = 0.032).
Baseline characteristics after PSM
In the propensity score-matched cohort, there were 183 infertile women in the conventional ET group and 183 women in the pET group. Table 3 illustrates the detailed information of the baseline characteristics. Patient profiles were broadly similar between the two groups after PSM (Table 3). Serum E2 levels were also comparable between the two groups [conventional ET vs. pET: 35.60 (26.20, 44.71) vs. 32.60 (25.00, 40.90) pg/ml, P = 0.129]. Moreover, the prevalence of RIF was comparable between the two groups (conventional ET vs. pET: 60.11% vs. 54.64%, P = 0.291).
Table 3.
Baseline clinical characteristics of the patients in the conventional ET group and pET group after PSM
| Characteristics | Conventional ET group N = 183 | pET group N = 183 | P value |
|---|---|---|---|
| Age (y) | 31.69 ± 3.72 | 31.90 ± 3.94 | 0.775 |
| BMI (kg/m2) | 21.80 ± 2.38 | 21.82 ± 2.78 | 0.615 |
| Infertility duration (y) | 4.87 ± 2.93 | 4.67 ± 3.30 | 0.166 |
| Gravidity n (%) | 0.530 | ||
|
0 ≥ 1 |
94 (51.37) 89 (48.63) |
100 (54.64) 83 (45.36) |
|
| Parity n (%) | 0.888 | ||
|
0 ≥ 1 |
153 (83.61) 30 (16.39) |
152 (83.06) 31 (16.94) |
|
| Types of infertility n (%) | 0.527 | ||
|
Primary infertility Secondary infertility |
100 (54.64) 83 (45.36) |
106 (57.92) 77 (42.08) |
|
| No. of previous failed cycles n (%) | 0.898 | ||
|
0–1 ≥ 2 |
38 (20.77) 145 (79.23) |
39 (21.31) 144 (78.69) |
|
| Prevalence of RIF n (%) | 110 (60.11) | 100 (54.64) | 0.291 |
| Main etiology of infertility n (%) | 0.666 | ||
|
Tubal Ovulation disorder Endometriosis Diminished ovarian reserve Male aOthers |
116 (63.39) 10 (5.46) 11 (6.01) 11 (6.01) 18 (9.84) 17 (9.29) |
109 (59.56) 8 (4.37) 7 (3.83) 16 (8.74) 24 (13.12) 19 (10.38) |
|
| PGS or PGD n (%) | 19 (10.38) | 20 (10.93) | 0.865 |
| FSH (mIU/ml) | 6.50 (5.50, 7.70) | 6.40 (5.40, 7.30) | 0.749 |
| LH (mIU/ml) | 5.05 (3.51, 6.92) | 5.00 (3.90, 6.75) | 0.767 |
| E2 (pg/ml) | 35.60 (26.20, 44.71) | 32.60 (25.00, 40.90) | 0.129 |
| AMH (ng/ml) | 2.70 (1.61, 4.22) | 2.94 (1.78, 5.00) | 0.153 |
ET, embryo transfer; pET, personalized embryo transfer; PSM, propensity score matching; BMI, body mass index; RIF, recurrent implantation failure; PGS, preimplantation genetic screening; PGD, preimplantation genetic diagnosis; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; AMH, anti-Mullerian hormone
aOthers included recurrent spontaneous abortion, chromosomal or genetic abnormalities, and unexplained infertility
Cycle characteristics and pregnancy outcomes after PSM
After adjusting for parity, the number and types of transferred embryos, the rates of transferred blastocysts (conventional ET vs. pET: 60.14% vs. 60.64%, P = 0.904), the transfer rates of good-quality cleavage-stage embryos (conventional ET vs. pET: 92.86% vs. 97.30%, P = 0.126) and blastocysts (conventional ET vs. pET: 40.83% vs. 30.99%, P = 0.059), and the number of transferred embryos (conventional ET vs. pET: 1.54 ± 0.51 vs. 1.54 ± 0.50, P = 0.879) did not exhibit significant between-group differences (Table 4). Endometrial thickness and endometrial patterns were still comparable between the two groups (P > 0.05).
Table 4.
HRT-FET cycle characteristics and pregnancy outcomes in the conventional ET group and pET group after PSM
| Characteristics | Conventional ET group N = 183 | pET group N = 183 | P value |
|---|---|---|---|
| Endometrial thickness (mm) | 9.60 ± 1.92 | 9.68 ± 1.60 | 0.231 |
| Endometrial patterns n (%) | 0.250 | ||
|
A B C |
33 (18.03) 142 (77.60) 8 (4.37) |
44 (24.04) 128 (69.95) 11 (6.01) |
|
| Transferred embryo stage n/N (%) | 0.904 | ||
|
D3 cleavage-stage embryos D5 or D6 blastocysts |
112/281 (39.86) 169/281 (60.14) |
111/282 (39.36) 171/282 (60.64) |
|
| Good-quality D3 embryos transferred rate n/N (%) | 104/112 (92.86) | 108/111 (97.30) | 0.126 |
| Good-quality blastocysts transferred rate n/N (%) | 69/169 (40.83) | 53/171 (30.99) | 0.059 |
| No. of transferred embryos n | 1.54 ± 0.51 | 1.54 ± 0.50 | 0.879 |
| β-hCG positive rate n (%) | 107 (58.47) | 119 (65.03) | 0.197 |
| Biochemical pregnancy loss n (%) | 21 (11.48) | 12 (6.56) | 0.100 |
|
Intrauterine pregnancy rate n (%) Implantation failure rate n (%) |
82 (44.81) 101 (55.19) |
105 (57.38) 78 (42.62) |
0.016 0.016 |
| Implantation rate n (%) | 93/281 (33.10) | 132/282 (46.81) | 0.001 |
|
Ectopic pregnancy rate n (%) Early spontaneous abortion rate n/N (%) Ongoing pregnancy rate n (%) aLate spontaneous abortion rate n/N (%) aPre-term birth rate n/N (%) aTerm birth rate n/N (%) aLive birth rate n/N (%) aMultiple pregnancy rate n/N (%) |
4 (2.19) 11/82 (13.41)b 74 (40.44) 6/78 (7.69)c 7/179 (3.91) 57/179 (31.84) 66/179 (36.87) 3/179 (1.68) |
2 (1.09) 19/105 (18.10)d 89 (48.63) 3/97 (3.09) 15/175 (8.57) 63/175 (36.00) 78/175 (44.57) 18/175 (10.29) |
0.685 0.387 0.115 0.190 0.069 0.409 0.140 0.001 |
HRT-FET, hormone replacement therapy-frozen embryo transfer; ET, embryo transfer; pET, personalized embryo transfer; PSM, propensity score matching; β-hCG, β-human chorionic gonadotropin; IPR, intrauterine pregnancy rate; IR, implantation rate
aFour and eight subjects are in ongoing pregnancy but have not yet delivered live births in the conventional ET group and the pET group, respectively
bThree subjects experienced twin pregnancies, with one experiencing an early miscarriage and the other having a full-term live birth
cAmong the 6 cycles of late miscarriage, 2 had live births
dThree subjects experienced twin pregnancies, with one experiencing an early miscarriage and the other having a full-term live birth
Compared to the conventional ET group, the IPR (57.38% vs. 44.81%, P = 0.016) and IR (46.81% vs. 33.10%, P = 0.001) were significantly higher in the pET group (Table 4). The IFR was significantly lower in patients who underwent pET compared to those who underwent conventional ET (42.62% vs. 55.19%, P = 0.016). In addition, patients in the pET group had a significantly higher rate of multiple pregnancies than those in the conventional ET group (10.29% vs. 1.68%, P = 0.001). Higher rates of β-hCG positivity (65.03% vs. 58.47%, P = 0.197), ongoing pregnancy (48.63% vs. 40.44%, P = 0.115), preterm birth (8.57% vs. 3.91%, P = 0.069), and live birth (44.57% vs. 36.87%, P = 0.140) were observed in the pET group, although the difference was not significant. A trend toward a lower rate of biochemical pregnancy loss was observed in patients with pET (6.56% vs. 11.48%, P = 0.100). Ectopic pregnancy, early and late spontaneous abortion, and term birth rate were not significantly different between the two groups (P > 0.05).
Discussion
This pilot study aimed to investigate the clinical efficacy of endometrial receptivity tests for infertile women with receptive endometria. We propose that the opening of the endometrial implantation window strengthens gradually before it reaches its plateau and then tapers. It is presumed that the putative timing for ET recommended by the modified rsERT is nearest to the most favorable WOI, which may enable some less competent embryos to implant. Consequently, the IPR and IR were significantly higher in the modified rsERT-guided pET group. This encouraging result might pave the way for precision medicine in ET.
Before the emergence of molecular diagnostic tools for WOI, histological evaluation was considered the gold standard for endometrial phase assignment to identify the receptive status. Under the regulation of ovarian steroids and paracrine-secreted molecules from neighboring cells, the genetic profiles of the endometrium undergo cyclical alterations across phases of the menstrual cycle [20]. On this basis, in 2011, Diaz-Gimeno et al. developed the endometrial receptivity analysis (ERA), which includes 238 genes differentially expressed during the menstrual cycle to identify the receptivity status of an endometrial sample [7]. A higher prevalence of a displaced WOI was detected in RIF patients compared to their non-RIF counterparts [15, 21]. By synchronizing ET to a receptive maternal endometrium, RIF patients with endometrial origin would benefit from this receptivity-timed transfer under the guidance of ERA [15, 22]. Furthermore, among patients at their first appointment, the pET group showed a significantly higher cumulative pregnancy rate compared to the FET and fresh ET groups, suggesting that ERA may have clinical benefits for these individuals [17]. Conversely, the most recent randomized controlled trial (RCT) reported similar results between the receptivity-timed FET and the standard FET group regardless of the ERA result among good prognosis populations [23]. To date, no consensus has been reached, and accumulating evidence has questioned the application of ERA since the embryonic factor is a major cause of most implantation failures [24].
However, no studies have specifically evaluated whether patients with a non-displaced WOI would benefit from endometrial receptivity testing. In the earlier version of rsERT (i.e., three-time-point rsERT), women with receptive results only conducted conventional ET because it could not provide hour-level precision [9]. For the purposes of this study, the three-time-point rsERT serves as a control. Each individual underwent rsERT and followed by conventional ET due to technological limitations, thus avoiding ethical issues arising from intentionally not performing pET. In the recent modified rsERT [19], we identified the optimal timing for embryo implantation by precisely manipulating the duration of P4 exposure (from the initiation of P4 supplementation to ET), based on the notion that optimal WOI was driven under the influence of the P4 exposure duration rather than its serum concentration. In this case, patients whose optimal WOI displacement is within 3 h also underwent conventional ET in our center due to the similar P4 exposure duration as in conventional ET.
In the original cohort, significantly higher IPR and IR were observed in the pET group compared to the conventional ET group. The conventional ET group had a lower blastocyst transfer rate, while fewer embryos were transferred in the pET group. These factors may, in turn, bias the actual results of the present study. We then adjusted for the parity, and the number and types of transferred embryos to reduce confounders to a lack of comparability in cycle characteristics between the two groups. After PSM, IPR and IR remained significantly higher among patients who underwent pET, as indicated by the modified rsERT. This result suggests that even patients who were not bothered with a displaced WOI could benefit from the receptivity-timed ET according to the newly refined rsERT. Compared to the conventional ET group, pET group had significantly lower IFR, indicating that the receptivity-timed pET could improve pregnancy outcomes in the early gestational period by decreasing IF. Rincon et al. reported that the average WOI length varied from 29 to 36 h, and a few patients had extraordinarily narrow WOIs (< 24 h) [25]. As such, even displacement within a few hours would influence reproductive outcomes.
We noticed that patients in the pET group also achieved numerically higher rates of ongoing pregnancy and live births. However, due to the limited sample size, the between-group differences were not significant. Notably, a higher multiple pregnancy rate was observed in the pET group, suggesting that the strategy of single embryo transfer should be adhered to when initiating pET. These results indicate that even for women without significant displacement of WOI, the modified rsERT may have a more comprehensive clinical application by carefully manipulating P4 administration and transferring embryos at the recommended timing.
Our results should be interpreted with caution, considering the retrospective nature of the study. There are potential heterogeneity and possible confounding factors among the study populations. Although we applied PSM to balance some covariates that might be associated with outcomes, the sample sizes in both groups were relatively small after being adjusted for confounders. Based on our sample size (i.e., 210 in conventional ET and 314 in pET), we achieved 57.25% power to detect a difference between the two groups with respect to the IPR and 86.96% power for the IR at a significance level of 0.05. As such, our results were preliminary and the present study should be considered as a pilot study. Ideally, a well-designed RCT with a control arm that does not perform pET after receiving the modified rsERT would be required to fully validate the final results. However, the majority of patients who have been advised to rsERT in our center were RIF patients. We were concerned that ethical issues might be raised if we did not offer pET when an optimal timing was recommended by the modified rsERT. Specifically, patients with a receptive result from the three-time-point rsERT, or those with a WOI displacement within 3 h predicted by the modified rsERT, were treated with conventional ET in actual clinical practice and were set as controls.
Conclusion
The current study reports the clinical efficacy of an endometrial receptivity test among women without a disturbance of displaced WOI. The newly modified rsERT allowed WOI prediction and provided optimal timing for embryo transfer via single time point biopsy. Infertile women with receptive WOI results could benefit from the modified rsERT. It offers an alternative in terms of counseling and tailoring the appropriate timing of embryo transfer in women undergoing ART.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant number 82371682) and Postdoctoral Fellowship Program of CPSF (GZC20233157). The funders did not take part in any of the process of this manuscript.
Author contribution
Y. P. L. and T. Y. were involved in the study concept and design. Q. Z., J. Z., N. L., D. L, and Y. M. L. took part in the acquisition of data. Y. W., B. X., and Z. H. were responsible for the analysis and interpretation of data. Y. Z. and C. W. participated in the model optimization. T. Y. and Z. H. contributed to manuscript drafting. All authors read and approved the final version of this article.
Data availability
The primary data for this study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval
The study was approved by the ethical committee of Xiangya Hospital, Central South University, Changsha, China (reference number 2017002). Written informed consent was obtained from all the patients before sample collection.
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.
Tianli Yang and Zhaojuan Hou contributed equally to this work and should be considered co-first authors.
Contributor Information
Tianli Yang, Email: xinzifan@aliyun.com.
Yanping Li, Email: liyanp@csu.edu.cn.
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Supplementary Materials
Data Availability Statement
The primary data for this study are available from the corresponding author upon reasonable request.