Abstract
We aimed to investigate the effects of the position of the transferred air bubble with the clinical pregnancy rate (PR) in frozen-thawed embryo transfer (FET) cycles. A prospective clinical study was carried out at Reproductive Medicine Center of West China Second University Hospital between June 2020 and May 2021. A total of 1159 women who underwent FET were included in this study. Transabdominal ultrasonographic guidance was used during the transfer procedure. The distance from the air bubble to endometrial cavity fundus (DAF) was measured in the freeze-frame ultrasound immediately after ET. In DAF ≤3 mm, 3–15 mm, and ≥15 mm group, the clinical PR in women transferred with cleavage embryos was 33.3% (7/21), 55.0% (153/280), and 31.3% (5/16), respectively, and the difference was statistically significant (P < 0.05). Among women transferred with blastocysts, the clinical PR was 63.0% (34/54), 68.5% (485/708), and 55.0% (44/80), respectively, and the difference was statistically significant (P < 0.05). In multivariate logistic regression model for clinical PR, the clinical PR was associated with age, embryo quality, number of embryo transferred, and endometrial thickness. DAF was an independent risk factor influencing clinical PR in blastocyst FET cycles rather than in cleavage embryo FET cycles. In conclusion, our results suggested that DAF was associated with the clinical PR and DAF between 3 mm and 15 mm is the optimal position in blastocyst FET cycles.
Lay summary
Embryo transfer is the last step in the IVF process. The position of the transferred embryo in the uterine cavity may affect the clinical pregnancy rate. The relationship between the position of the embryo in the uterine cavity at the time of transfer and the clinical pregnancy rate is disputed. In this study, the distance from the air bubble to endometrial cavity fundus measured using the freeze-frame ultrasound immediately after embryo transfer was used to indicate the position of the embryo in the uterine cavity. This study demonstrates that the position of the transferred air bubble in the uterine cavity is an independent factor on clinical pregnancy rate in blastocysts (embryos on the fifth day of fertilization) frozen-thawed embryo transfer cycles but not in cleavage embryos (embryos on the third day of fertilization) frozen-thawed embryo transfer cycles. The distance from the air bubble to endometrial cavity fundus between 3 mm and 15 mm is the optimal position in blastocyst frozen-thawed embryo transfer cycles.
Introduction
Infertility is a prevalent issue in various countries worldwide; according to the 2023 WHO report, 1 in 6 people were globally affected by infertility (https://www.who.int/zh/news/item/04-04-2023-1-in-6-people-globally-affected-by-infertility). Assisted reproductive technology (ART) is considered as an effective treatment for infertility around the world (Inhorn & Patrizio 2015, Sharma et al. 2018). In vitro fertilization embryo transfer (IVF-ET) is a crucial method used to address infertility. Embryo transfer is the procedure of transferring embryos cultured in vitro into the uterine cavity and is the final and crucial step of IVF-ET process. Studies have shown that ultrasound guidance during the transfer procedure can improve the implantation rate and clinical pregnancy rate (PR) of the IVF-ET cycle (Brown et al. 2000, Coroleu et al. 2000, Wood et al. 2000, Tang et al. 2001, Teixeira et al. 2015, Penzias et al. 2016).
However, the optimal position for transferred embryos in the uterine cavity remains a topic of controversy. Previous studies investigated optimal embryo position through catheter tip indicated that catheter tip in the lower part of the uterine cavity results in higher PRs because of avoiding bleeding caused by touching the uterine fundus (Coroleu et al. 2002, Oliveira et al. 2004, Cavagna et al. 2006, Pacchiarotti et al. 2007, Tiras et al. 2010, Kwon et al. 2015). Recent studies preferred to use the transferred air bubbles as indicators but got different conclusions (Marieke et al. 2007, Friedman et al. 2011, Cenksoy et al. 2014, Hayashi et al. 2020, Bayram et al. 2021). The effect of the position of transferred embryos on clinical PR has been an interesting topic in recent years.
Nevertheless, it is worth to note that the catheter tip does not accurately represent the embryo, for the actual position of the transferred embryo is some distance from the tip of the catheter. The position of air bubble seems to be a more exact indication of embryo placement because the embryos are sandwiched between the air bubbles and the air bubbles are deposited along with the embryos at the time of transfer (Gergely et al. 2005, Marieke et al. 2007, Friedman et al. 2011, Cenksoy et al. 2014, Hayashi et al. 2020, Bayram et al. 2021). However, there is still no consensus on the optimal position of the transferred air bubble.
It has previously been observed that the air bubble closer to the fundus was associated with higher PR (Marieke et al. 2007, Friedman et al. 2011, Cenksoy et al. 2014, Hayashi et al. 2020, Bayram et al. 2021). In contrast, some studies even showed that the bubbles away from the fundus of the uterus increased clinical PR (Waterstone et al. 1991, Frankfurter et al. 2003, 2004, Pope et al. 2004). However, other studies have found that the position of the embryo air bubble at the time of transfer had no impact on clinical PR (Saravelos et al. 2016, Fıçıcıoğlu et al. 2018). These contrasting conclusions may be related to the type of study, the population included, and the statistical method. Also, some studies were retrospective, the included population did not distinguish fresh from frozen cycles, and no multivariate regression analysis was performed. As we know, besides the position of the transferred air bubble, various factors affect the pregnancy outcomes of ET, including female age, embryo stages, quality and quantity, fresh or frozen cycles, endometrial thickness, and ovarian stimulation protocol (Hayashi et al. 2020). However, some studies did not strictly control these potential confounders when analyzing the effect of the position of air bubbles on clinical PR.
These conflicting results suggest that further research is needed to analyze the effect of transferred air bubble position on clinical PR. To clarify the relationship between air bubble position and clinical PR, we conducted a prospective study; only women undergoing frozen-thawed embryo transfer (FET) were included, and multiple logistic regression was used to adjust for the aforementioned variables. It was aimed to determine whether the position of the transferred air bubble affects clinical PR in FET cycles; the results were expected to guide clinical practice.
Materials and methods
Study design, sample selection, and data collection
This is a prospective study. The study obtained the ethical approval and consent from the Ethics Committee, Sichuan University. All participants provided informed consent to take part in our study; informed consent was obtained from participants before any study procedures were initiated. Women undergoing FET at Reproductive Medicine Center of West China Second University Hospital, Sichuan University, between June 2020 and May 2021 were included in the study. Because they might conceal their previous pregnancy history, there were no requirements for primary and secondary infertility. To reduce bias, we excluded a number of factors that might affect pregnancy.
Inclusion criteria: Women aged 20–45 years old.
Exclusion criteria: (i) uterine malformations (e.g., monohorn uterus, mediastinal uterus, etc.) and uterine organic diseases (uterinemyoma, adenomyosis, etc.); (ii) endometrial abnormalities including intrauterine adhesions and endometrial polyps; (iii) untreated hydrosalpinx; (iv) cycle of egg donation or freezing, cycle of sperm extraction; (v) systemic diseases (thyroid disease, hyperprolactinemia, etc.); (vi) incomplete or missing follow-up data; (vii) blood stained on the catheter tip after transplantation; (viii) obesity with body mass index (BMI) >28; (ix) preimplantation genetic diagnosis (PGD) cases; and (x) posterior uteri.
Ovarian stimulation protocol
Standard controlled ovarian stimulation was performed, including gonadotropin-releasing hormone (GnRH) antagonist protocol, follicular phase long-acting long protocol, luteal phase short-acting long protocol, mild stimulation protocol, and progestin-primed ovarian stimulation (PPOS) with recombinant FSH. GnRH agonist and long protocol were the usual options. Patients with poor ovarian reserve function were treated with mild stimulation protocol and PPOS. Gonadotropin dosage varied according to patients’ medical history, BMI, ovarian reserve and response as determined by serial E2 levels on the second to fourth day of the menstruation, AMH, and transvaginal US assessment of follicular development.
Embryo freezing and thawing
Oocytes were inseminated 4–6 h after retrieval either by conventional IVF or by ICSI and cultured (the culture medium was Vitralife G-1/2 PLUS medium; Vitrolife, Sweden) for 3 or 6 days according to standard laboratory procedures. Embryos at cleavage or blastocyst stage which were not fresh transferred were vitrified following the manufacturer’s protocol (Kitazato, Japan) and using an open support for vitrification (Cryotop, Kitazato). On the day of FET, embryos were warmed following manufacturer’s instructions (Kitazato) and cultured for at least 2 h before FET.
Embryo grading
Embryos were graded according to the Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting (Balaban et al. 2011). Cleavage embryos were classified as good-quality embryos (grade I and II embryos) if they had six to eight cells on day 3, with less than 20% anucleate fragments. Good-quality blastocysts were defined as 4-5AA, 4-5AB, 4-5BA, and 4-5BB.
Endometrial preparation
Endometrial preparation protocol was performed with either a hormone replacement therapy (HRT) cycle or natural cycle. All patients underwent endometrial preparation with HRT using a similar method. Oral E2 administration (Progynova 2 mg; Bayer Holding Ltd.) was started with a dose of 2–8 mg/day from day 3 of the menstrual cycle. When the endometrial thickness was still less than 3 mm after 10 days of estrogen use, the dose of estrogen was increased by 2 mg. The progesterone (8% progesterone vaginal gel 90 mg/day or progesterone 40–60 mg/day) was supplemented if the endometrial thickness was ≥6 mm and progesterone concentration <1.5 ng/mL.
For cleavage embryo FET cycles, embryo transfer was scheduled on the third full day after progesterone administration or ovulation. For blastocyst FET cycles, embryo transfer was scheduled on the fifth full day after progesterone administration or ovulation.
Number of embryos transferred
In patients transferred with cleavage embryos, two embryos were transferred usually unless only one embryo remains. In patients transferred with blastocysts, one embryo was transferred usually and two embryos were transferred if the patient demanded.
Embryo transfer procedure
Embryo transfer was performed according to the instructions for ET provided by the manufacturer of ET catheter (Kitazato, Japan). The fertility doctor, embryologist, and ultrasound doctor did the procedure together. Patients were placed in a lithotomy position with a moderately full bladder. The embryos were loaded into the catheter according to the manufacturer’s instructions by the embryologist. The fertility doctor removed the mucus in the cervical canal with a sterile cotton swab and then placed the catheter sheath in the cervical canal under US guidance. The embryologist pressed the syringe and delivered the embryo to the middle of the uterine cavity under ultrasound guidance. The catheter was then returned to the embryologist to confirm that there were no retained embryos. Two embryologists alternate once a week. Three fertility doctors alternate once a week. The positioning of the air bubble was not intentional for each patient. The distance from the air bubble to endometrial cavity fundus (DAF) was measured in the freeze-frame ultrasound immediately after ET. The measurement of DAF was performed by the same operator. The patient was then brought to another bed and kept supine for approximately 30 min. Luteal phase support was sustained until the tenth week of gestation.
Outcomes
The primary outcome of this study was clinical pregnancy defined by β-HCG positive and the pregnancy sac found by ultrasound.
Statistical analysis
Statistical analysis was performed using SPSS version 22.0. Continuous variables were expressed with mean ± s.d. and compared by Student’s t-test. Categorical variables were expressed as percentages (%) and compared using chi-square test or Fisher’s exact tests. The categorical variables included DAF, age, endometrial thickness on the day of ET, quality of transferred embryos, single (SET) or double embryo transfer (DET), and ovarian stimulation protocol. Multivariate binary logistic regression model was used to identify the regulators of clinical pregnancy from categorical variables by calculating odds ratios (ORs) and 95% CIs. For all statistical tests, P < 0.05 was considered statistically significant.
Results
A total of 1159 women were included in this study, of which 317 were transferred with cleavage embryos and 842 with blastocysts; the clinical PR was 52.1% and 66.9%, respectively. There were no difference in terms of age, number of embryos transferred, and endometrial thickness in different DAF groups. The characteristics of women according to DAF are shown in Table 1.
Characteristics of women included in this study.
| ≤3 mm | 3-15 mm | ≥15 mm | P | |
|---|---|---|---|---|
| Age (year) | ||||
| Day 3 | 32.52 ± 5.50 | 32.86 ± 5.16 | 34.00 ± 5.45 | 0.656 |
| Day 5 | 30.48 ± 4.54 | 30.69 ± 4.02 | 30.90 ± 4.67 | 0.841 |
| Embryos transferred, n | ||||
| Day 3 | 1.86 ± 0.36 | 1.85 ± 0.36 | 1.75 ± 0.48 | 0.556 |
| Day 5 | 1.04 ± 0.19 | 1.13 ± 0.34 | 1.15 ± 0.36 | 0.106 |
| Endometrial thickness (mm) | ||||
| Day 3 | 0.48 ± 0.10 | 0.51 ± 0.09 | 0.53 ± 0.09 | 0.168 |
| Day 5 | 0.48 ± 0.06 | 0.50 ± 0.08 | 0.53 ± 0.11 | 0.161 |
DAF, distance from the air bubble to endometrial cavity fundus.
The most optimal DAF was 3–15 mm, which presented an inverted U shape. The clinical PRs with different DAF were shown in Table 2.
The clinical PRs with different DAF in cleavage embryos/blastocysts FET cycles.
| DAF | Clinical PRs with | |||
|---|---|---|---|---|
| Cleavage embryos | Blastocysts | |||
| n/total n | % | n/total n | % | |
| ≤3 mm | 7/21 | 33.3 | 34/54 | 62.9 |
| 4 mm | 9/17 | 52.9 | 36/52 | 69.2 |
| 5 mm | 20/42 | 47.6 | 79/124 | 63.7 |
| 6 mm | 26/43 | 60.4 | 71/104 | 68.2 |
| 7 mm | 13/31 | 41.9 | 72/102 | 70.5 |
| 8 mm | 21/32 | 65.6 | 69/93 | 74.1 |
| 9 mm | 18/26 | 69.2 | 47/66 | 71.2 |
| 10 mm | 18/36 | 50 | 30/51 | 58.8 |
| 11 mm | 8/19 | 42.1 | 28/39 | 71.7 |
| 12 mm | 6/11 | 54.5 | 23/32 | 71.8 |
| 13 mm | 12/17 | 70.5 | 23/31 | 74.1 |
| 14 mm | 1/3 | 33.3 | 7/10 | 70.0 |
| ≥15 mm | 5/16 | 31.2 | 26/48 | 55.0 |
DAF, distance from the air bubble to endometrial cavity fundus.
Univariate analyses
The data were stratified according to DAF, age, embryo quality and quantity, stimulation protocol and endometrial thickness, and the results of univariate analyses are summarized in Table 3.
Subgroup analysis of clinical PR according to age, DAF, embryo quality and quantity, ovarian stimulation protocol, and endometrial thickness in cleavage embryos/blastocysts FET cycles.
| Variables | Day 3 embryos | Day 5 embryos | ||||
|---|---|---|---|---|---|---|
| n/total n | % | P | n/total n | % | P | |
| Age, years | 0.002 | 0.000 | ||||
| ≤30 | 58/104 | 55.7 | 309/420 | 73.6 | ||
| 31–37 | 101/184 | 54.8 | 223/369 | 60.4 | ||
| >37 | 6/29 | 20.6 | 31/53 | 58.5 | ||
| DAF, mm | 0.039 | 0.043 | ||||
| ≤3 | 7/21 | 33.3 | 34/54 | 63.0 | ||
| 3–15 | 153/280 | 55.0 | 485/708 | 68.5 | ||
| ≥15 | 5/16 | 31.3 | 44/80 | 55.0 | ||
| Embryo quality and quantity | 0.000 | − | ||||
| SET day 3 (A/B) | 16/49 | 32.7 | ||||
| DET day 3 (B + B) | 25/64 | 39.0 | ||||
| DET day 3 (A + B) | 53/99 | 53.5 | ||||
| DET day 3 (A + A) | 71/105 | 67.6 | ||||
| SET day 5 (B) | − | − | 42/87 | 48.2 | 0.000 | |
| SET day 5 (A) | 437/647 | 67.5 | ||||
| DET day 5 (A/B+ A/B) | − | − | 84/108 | 77.8 | ||
| Ovarian stimulation protocol | 0.152 | 0.342 | ||||
| GnRH antagonist protocol | 78/154 | 50.6 | 318/463 | 68.6 | ||
| Luteal phase short-acting long protocol | 59/104 | 56.7 | 173/276 | 62.6 | ||
| Follicular phase long-acting long protocol | 18/30 | 60.0 | 64/92 | 69.5 | ||
| Others | 10/29 | 34.5 | 8/11 | 72.7 | ||
| Endometrial thickness, mm | 0.010 | 0.021 | ||||
| <4 | 3/15 | 20 | 26/50 | 52.0 | ||
| ≥4 | 162/302 | 53.6 | 537/792 | 67.8 | ||
‘A’ presents high-quality embryo; ‘B’ presents non-high-quality embryo.
DAF, distance from the air bubble to endometrial cavity fundus; DET, double embryos transfer; PR, pregnancy rate; SET, single embryo transfer.
Among women transferred with cleavage embryos, the clinical PR were 33.3% (7/21) with DAF ≤3 mm, 55.0% (153/280) with DAF between 3 and 15 mm, and 31.3% (5/16) with DAF ≥15 mm, which was statistically significant between different DAF groups (P < 0.05). There were two ectopic pregnancies in the DAF between 3 and 15 mm group.
Among women transferred with blastocysts, the clinical PR for those with DAF ≤3 mm, 3–15 mm, and ≥15 mm was 63.0% (34/54), 68.5% (485/708), and 55.0% (44/80), respectively, and the difference was statistically significant (P < 0.05). Additionally, one ectopic pregnancy was observed in the DAF 3–15 mm group.
In both cleavage embryos and blastocysts FET cycles, the clinical PR of women aged >37 years was significantly higher than women aged >37 years (P < 0.05, P < 0.05). The quantity and quality of the embryo had a positive effect on the clinical PR (P < 0.05, P < 0.05). The clinical PR in women with endometrial thickness ≥4 mm was significantly higher than in those with <4 mm (P < 0.05, P < 0.05).
The clinical PR in women with different ovarian stimulation protocol was not statistically significant different (P > 0.05, P > 0.05).
Multivariate logistic regression analysis of clinical PR
Multivariate logistic regression analysis was performed for clinical PR (yes vs no) against relevant variables, such as age, quantity and quality of transferred embryos, endometrial thickness, and DAF. The results indicated that age ≤30 years, higher quality embryo, DET, endometrial thickness ≥4 mm increased the clinical PR. After adjusting for potential confounding factors, the clinical PR was 1.810 times higher in DAF 3–15 mm group compared to DAF ≥15 mm groups (OR: 1.810, 95% CI: 1.118–2.931) in blastocyst FET cycles. But DAF was not related to clinical PR in cleavage embryo FET cycles. The OR and the 95% CI from the multivariate models were shown in Table 4.
Multivariate logistic regression models of clinical PR according to age, embryo quality and quantity, endometrial thickness, and DAF in cleavage embryo/blastocyst FET cycles. Data are presented as OR (95% CI).
| Variables | Day 3 embryos | Day 5 embryos |
|---|---|---|
| Age, years | ||
| ≤30 | 4.651 (1.659–13.039) | 1.788 (0.979–3.268) |
| 31–37 | 4.691 (1.753–12.553) | 0.996 (0.546–1.817) |
| >37 | 1.00 (reference) | 1.00 (reference) |
| DAF, mm | ||
| ≤3 | 0.899 (0.206–3.924) | 1.519 (0.736–3.134) |
| 3–15 | 2.379 (0.764–7.406) | 1.810 (1.118–2.931) |
| ≥15 | 1.00 (reference) | 1.00 (reference) |
| Embryo quality and quantity | ||
| SET day 3 (A/B) | 1.00 (reference) | |
| DET day 3 (B+B) | 0.996 (0.435–2.281) | |
| DET day 3 (A+B) | 1.824 (0.854–3.896) | |
| DET day 3 (A+A) | 3.618 (1.672–7.829) | |
| SET day 5 (B) | − | 1.00 (reference) |
| SET day 5 (A) | − | 2.078 (1.310–3.295) |
| DET day 5 (A/B+ A/B) | − | 3.415 (1.818–6.415) |
| Endometrial thickness, mm | ||
| <4 | 1.00 (reference) | 1.00 (reference) |
| ≥4 | 4.386 (1.155–16.660) | 1.945 (1.076–3.516) |
Discussion
Embryo transfer is the final step in the IVF treatment. Whether the position of the transferred air bubble in the uterine cavity has an impact on clinical PR is still not very clear. The evidence available to date is inconsistent. In this study, we identified that the most optimal DAF was 3–15 mm and that DAF was an independent risk factor influencing clinical PR in women transferred with blastocysts but not in women transferred with cleavage embryos.
According to our findings, the air bubble being too far (≥15 mm) or too close (≤3 mm) to endometrial cavity fundus would decrease the clinical PR. This result was similar to some previous studies. Cenksoy et al. reported that the ideal position of air bubbles would be at distance <10 mm from the endometrial fundus (Cenksoy et al. 2014). A retrospective cohort study by Friedman et al. suggested that DAF <10 mm transferred with either fresh or frozen blastocyst was associated with higher PR (Friedman et al. 2011). A prospective study in Japan in which only single frozen and thawed good-quality blastocysts was transferred indicated that the clinical PR was higher when the air bubble position was at a distance between 6 mm and 10 mm (Hayashi et al. 2020). A retrospective study by Asina et al., in which only euploid blastocysts were included, revealed that the probability of clinical PR decreases as DAF increases (Bayram et al. 2021). A study performed by Lambers and colleagues indicated that the higher clinical PRs were found when the day 2/day 3 embryo air bubbles were closer to the fundus in fresh cycles (Marieke et al. 2007).
However, our result conflicted with that of the following studies, which reported that the clinical PR was higher when the embryo was located away from the fundus of the uterus. Research indicated that for every additional millimeter embryos are deposited away from the fundus, with the odds of clinical pregnancy increasing by 11% (Pope et al. 2004). Another research indicated that the PR of fresh cleavage embryo or blastocyst ET was favorably affected by directing embryo placement to the lower to middle uterine segment (Frankfurter et al. 2004). A study by Coroleu et al. in which catheter tip was used as an embryo indicator indicated that embryos should be replaced 15–20 mm from the fundus in order to improve implantation rate (Coroleu et al. 2002). A similar finding was also reported in other studies using catheter tips as embryo indicators (Waterstone et al. 1991).
The reason of the opposite conclusion might be these studies were conducted about 10–20 years ago, when the transferred catheters may not have been as soft as they are now; that is, the catheters were too hard, causing endometrial bleeding and affecting the clinical pregnancy rate. Secondly, the catheter tip did not represent the position of the embryo itself. Because of the thrust of the injection, the embryo was actually located closer to the fundus than the catheter tip. Thirdly, some studies did not separate blastocysts from cleavage embryos, and the number of embryos transferred was not the same. However, both the stage and number of transferred embryos affect the clinical pregnancy rate.
In this study, multivariate logistic regression analysis indicated that DAF was an independent risk factor influencing clinical PR in women transferred with blastocysts but not in women transferred with cleavage embryos. However, some previous studies did not conduct a multivariate logistic regression analysis, which maybe another reason explained the contradictory conclusions. The finding in this study is consistent with that of Sun et al., who observed that only blastocyst transfer showed a significant interaction with transfer depth (Sun et al. 2022). As embryo implantation occurs 6–7 days after fertilization, and previous studies have found that the transferred embryo swims in the uterine cavity, though it was only observed for 60 min after transfer (Saravelos et al. 2016, Fıçıcıoğlu et al. 2018), we hypothesized that implantation will occur within 24–36 h after blastocyst transfer, while the cleavage embryo will still whirl around and relocate for 72–96 h before implantation. Our study did not dynamically look at embryo migration in the uterine cavity, which is something we will investigate further in the future.
In this study, multivariate regression analyses indicated that younger age (only in cleavage embryo FET cycles), higher quality embryo, DET, and endometrial thickness ≥4 mm contribute to higher clinical PR.
Age was known to be negatively correlated with IVF clinical PR. However, in this study, the clinical PR between women aged ≤30 years and women aged 31–40 years was not statistically significant, it may be that FET transferred embryos was obtained when they were younger. Interestingly, in this study, age was an independent predictor influencing the clinical PR only in women who were transferred with cleavage embryos. However, in women who were transferred with blastocysts, age was not an independent risk factor. The reason may be that maternal age at retrieval influences the number of euploid blastocysts, but it does not impair the implantation potential of euploid embryos (Irani et al. 2018, Hayashi et al. 2020).
We also identified that embryo quality and quantity are independent predictors influencing the clinical PR in both cleavage embryo and blastocyst FET cycles. Higher quality embryo or DET increased clinical PRs (Bayram et al. 2021).
Endometrial thickness was significantly associated with the clinical PR in this study. The clinical PR was higher in women with a thicker endometrium than in those with a thinner endometrium in both cleavage embryos and blastocysts FET cycles, which is consistent with previous studies (Hayashi et al. 2020, Bayram et al. 2021).
There are several limitations to this study. First, the direction of air bubble movement may not be consistent between the anterior and retroverted uteri. In addition, the poor retroverted uterus image may cause measurement errors, so women with retroverted uteri were not included in this study. Second, this study was a prospective but non-randomized study due to the influence of previous research conclusions (Marieke et al. 2007, Friedman et al. 2011, Hayashi et al. 2020, Bayram et al. 2021). Clinicians favored placing embryos in the upper uterine cavity, resulting in the sample of embryos being placed in the lower uterine cavity small after grouping; a larger study is needed to optimize ET strategy. Third, it is difficult to precisely control the position and migration of the air bubbles in real practice since the final position depends on the pressure of push on syringe which may be different in different patient. Therefore, we need a more standardized method of embryo transfer that allows the surplus value of exact positioning at embryo transfer to be analyzed.
In conclusion, the position of the transferred air bubble in the uterine cavity at the time of transfer has a significant effect on the clinical pregnancy rate in blastocyst FET cycles but not in cleavage embryo FET cycles. A higher pregnancy rate can be achieved when transferred air bubble at a distance of 3–15 mm from the fundus. These findings suggest that the position of the blastocyst at the time of transfer should be taken into consideration.
Declaration of interest
The authors declare that the study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding
This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Data sharing statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Author contribution statement
LH designed the study, wrote the first draft and did the statistical analyses. JH contributed to data collection and statistical analyses. QM and SJ contributed to data collection and corrected the draft. YL implemented the study, provided supervision and corrected the draft. DZ provided discussion and corrected the draft. XL contributed to data collection.
Acknowledgements
The authors thank our patients and all participants in the data collection. We would like to thank the medical staff and patients in the Reproductive Medicine Center of West China Second University Hospital, Sichuan University, for recording the data and cooperating with the treatment.
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