Comparison of uterine, endometrial and subendometrial blood flows in predicting pregnancy outcomes between fresh and frozen-thawed embryo transfer after GnRH antagonist protocol: a retrospective cohort study

Abstract This retrospective cohort study aimed to compare endometrial receptivity and pregnancy rate between fresh embryo transfer (ET) and frozen-thawed ET after gonadotrophin-releasing hormone (GnRH) antagonist protocol in normal ovarian responders. The patients were divided into two groups: the fresh ET group and the frozen-thawed ET group. Uterine artery resistance index (RI) and endometrial thickness were lower in the frozen-thawed ET group. The proportion of detectable endometrial–subendometrial flow was significantly higher in the frozen-thawed ET group. There was no significant difference in miscarriage rate between the two groups. Frozen-thawed ET group had a significantly higher CPR (56.0% vs. 48.1%), implantation rate (32.2% vs. 26.4%), and LBR (45.4% vs. 36.5%) than the fresh ET group. In GnRH antagonist protocol, elective frozen-thawed ET should be ideally taken, as this could improve embryo implantation rate, clinical pregnancy rate, and live birth rate, thus presenting an effective strategy to enhance the embryo utilization rate. IMPACT STATEMENT What is already known on this subject? The clinical pregnancy rate following fresh embryo transfer (ET) was lower than frozen-thawed ET after GnRH antagonist protocol. IVF success depends on embryo quality, embryo-endometrium interaction and endometrial receptivity. A good blood supply toward the endometrium is generally considered a requirement for implantation. What do the results of this study add? Uterine artery RI and endometrial thickness were significantly lower in the frozen-thawed ET group. The proportion of detectable endometrial–subendometrial flow was significantly higher in the frozen-thawed ET group. Frozen-thawed ET group had a significantly higher clinical pregnancy rate, implantation rate and live birth rate than the fresh ET group after GnRH antagonist protocol. What are the implications of these findings for clinical practice and/or further research? In GnRH antagonist protocol, elective frozen-thawed ET should be ideally taken, as this could improve embryo implantation rate, clinical pregnancy rate and live birth rate, thus presenting an effective strategy to enhance the embryo utilization rate.


Introduction
Gonadotrophin-releasing hormone (GnRH) antagonist protocol has become increasingly popular in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment.The main reason for this advanced shift in clinical practice is the need to minimize the occurrence of the largest drawback in controlled ovarian hyperstimulation (COH), ie ovarian hyperstimulation syndrome (OHSS).However, there is still no consensus on the optimal protocol.IVF success depends on embryo quality, embryo-endometrium interaction and endometrial receptivity (Lessey and Young 2019).Recently, we found that the clinical pregnancy rate following fresh embryo transfer (ET) was lower than frozen-thawed ET after the GnRH antagonist protocol, which poses the question of whether the endometrium in the GnRH antagonist protocol is optimally prepared for implantation.Different methods have been proposed to evaluate endometrial receptivity, such as the histologic dating of an endometrial biopsy (Tilburgs et al. 2020), the evaluation of endometrial cytokines in endometrial tissues (Xu et al. 2018, Li et al. 2021), using endometrial biomarker with immunolocalization (Cope and Monsivais 2022), the genomic study of a timed endometrial biopsy (Silveira et al. 2017) or more commonly a noninvasive ultrasound examination of the endometrium (Polanski and Baumgarten 2021).Transvaginal color Doppler power ultrasound examination of the endometrium is a commonly used noninvasive strategy to assess endometrial receptivity during IVF/ICSI treatment (Silva Martins et al. 2019).A good blood supply toward the endometrium is generally considered a requirement for implantation and therefore assessment of endometrial blood flows in IVF/ICSI treatment has attracted a lot of attention in recent years.
Cryopreservation of embryos with vitrification is considered ever more efficient in IVF/ICSI.Also, frozen-thawed ET is the most commonly used to prevent serious OHSS and increase the cumulative pregnancy rate (Li et al. 2019).However, it remains unclear whether the endometrium would be more optimally prepared for implantation if it is interfered with for at least one menstrual cycle after the COH by GnRH antagonists and whether frozen-thawed ET can replace fresh ET after GnRH antagonist protocol.
The aim of this prospective study was to evaluate the endometrial receptivity through the uterine, endometrial and subendometrial blood flows by transvaginal color Doppler power ultrasound and pregnancy rates between fresh ET and frozen-thawed ET after GnRH antagonist protocol in normal ovarian responders.

Study design and patients
This retrospective cohort study was performed at the Affiliated Hospital of Kunming University of Science and Technology, Kunming, China, with approval from the Ethics Committee of the First People's Hospital of Yunnan Province.Data were collected from patients who underwent their first IVF/ICSI cycles between January 2019 and March 2021.Data were de-identified to ensure patient privacy.

GnRH antagonist protocol
Patients did not undergo any therapeutic interventions except for routine procedures.A total of 721 women underwent, a standardized GnRH antagonist protocol with recombinant follicle stimulating hormone (FSH) (Gonal-f; Merck Serono, Switzerland) starting on day 2 or 3 of their menses, with doses ranging from 150 to 300 international units per day, according to their age and response to stimulation day 6.A GnRH antagonist (Cetrorelix; Cetrotide, Merck Serono, Switzerland) was used for pituitary suppression when a leading follicle achieved 14 mm.Final oocyte maturation was induced by administration of 250 lg of recombinant human chorionic gonadotropin (hCG) (Ovidrel; Merck Serono, Switzerland) as soon as three follicles reached 18 mm in diameter by ultrasound evaluation.Oocyte retrieval was performed 34-36 h after trigger, and conventional insemination or ICSI was performed as clinically appropriate.The good quality embryos included grade I and grade II embryos.

Fresh ET
Fresh ET was performed on the third day after oocyte retrieval.A maximum of two top best cleavage-stage embryos were transferred under ultrasound guidance, while other embryos were used to develop blastocysts.

Frozen-thawed ET
In the freeze-all group, a maximum of two top best cleavagestage embryos were chosen for cryopreserve with vitrification on day 3, awaiting frozen-thawed ET, while other embryos were used to develop blastocyst, after which it was cryopreserved.Vitrification is an ultrarapid method of cryopreservation whereby the embryo is transitioned from 37 � Cto À 196 � C in <1 s, resulting in extremely fast cooling rates.After at least one menstrual cycle, the frozen cleavage-stage embryos were thawed and transferred in a natural or artificial cycle.In the natural cycles, regular ultrasonic evaluation of the endometrium thickness and mean diameter of the dominant follicle was performed from the 10th day of the menses.When the endometrium was �7 mm, and the diameter of the dominant follicle was 18-20 mm, serum LH was tested.If serum LH was <20 IU/l, 5 000 IU hCG (Livzon Pharmaceutical Group, Inc., Guangdong, China) was used to trigger final oocyte maturation.Thawing and transferring were performed three days after ovulation.In the artificial cycle, the endometrial preparation started on day 3 of each patient's menstrual cycle, when estradiol valerate (E 2 V) (Progynova, Bayer, Germany) was orally administered at a dose of 4 mg/d.On day 10 of the cycle, an ultrasound was performed.If the endometrial thickness was< 7 mm, the Progynova dose was raised to 3 mg twice daily for sevendays.After one week, the endometrium was checked once again.The frozen-thawed ET was scheduled when the endometrium thickness was �7mm.The progesterone administration with vaginal micronized progesterone (Utrogestan, Lab Besins Internat S.A., Paris) for 200 mg twice daily or in gel (Crinone 8%; Merck Serono, Switzerland) for a single daily administration starting 3 days before ET.Also, the oral administration of Progynova was continued.If the endometrium was <7 mm, the ET was canceled.

Luteal phase support
Luteal phase support started on the day of oocyte retrieval.All patients received vaginal micronized progesterone (Utrogestan) for 200 mg twice daily or in gel (Crinone 8%) for a single daily administration.If the qualitative hCG urine tests were positive 14 days after ET, the patients were advised to continue the luteal support until the 8-10th gestational week.In the artificial cycle group, the same dose of oral Progynova was continued until the intrauterine gestational sac with a heartbeat was observed, after which the dose was gradually decreased.

Ultrasound investigation
Transvaginal ultrasound measurements of all patients were performed between 8 and 10 AM on the ET day after the patients had emptied their bladder.Endometrial thickness, pulsatility index (PI), resistance index (RI) and systolic/diastolic (S/D) ratio of the uterine artery and the pattern of endometrial-subendometrial blood flow distribution were examined by Philips HD15 (Electronics Inc., the Netherlands).The average values of all the ultrasonography parameters from three measurements were analyzed for each subject.The results of the adjunctive ultrasound assessment did not affect subsequent clinical management procedures.The double thickness of the endometrium was measured (maximum distance between each myometrial/endometrial interface through the longitudinal axis of the uterus).Using color Doppler in the two-dimensional (2D) mode, flow velocity waveforms were obtained from the ascending main branch of the uterine artery on the right and left side of the cervix before it entered the uterus in a longitudinal plane.The gate of the Doppler was positioned when the vessel with good color signals was identified on the screen.The PI, RI and S/D ratio of the uterine artery were electronically calculated when similar consecutive waveforms of good quality were obtained.As there were no differences in uterine artery PI, RI and S/D between the left and the right sides, the averaged results were taken.The area of interest was the endometrium and the subendometrial regions within 10 mm of the echogenic endometrial borders.The pulse repetition frequency was chosen for a color velocity range of 3 cm/s, and the color gain was adjusted to 80%±2% to optimize blood flow detection in the small vessels.The pattern of endometrial-subendometrial blood flow distribution was determined by detected power Doppler signals in the endometrial-subendometrial regions.The mode was turned to power Doppler by using 'CPA' key.For those with vascularization penetrating into the subendometrial and endometrial regions, we adopted the definition from Applebaum (1995), which was summarized as follows: zone 1, vessels penetrating the outer hypoechogenic area surrounding the endometrium but not entering the hyperechogenic outer margin of the endometrium (Figure 1); zone 2, vessels penetrating the hyperechogenic outer margin of the endometrium but not entering the hypoechogenic inner area (Figure 2) and zone 3, vessels entering the hypoechogenic inner area (Figure 3).

Definition of clinical outcomes
A qualitative hCG urine test was performed 14 days after ET.Clinical pregnancy was confirmed by transvaginal ultrasound scanning of the gestational sac four to five weeks after ET.The implantation rate reflected the number of gestational sacs seen per embryo transferred.An ectopic pregnancy was defined by the diagnosis of extra uterine pregnancy confirmed by laparoscopy or ultrasound.Ongoing pregnancy was defined as a viable pregnancy at week 12 of pregnancy.

Statistical methods
Quantitative data were presented as mean ± SD if they conformed to a normal distribution or as medians (interquartile range) if they had non-normal distribution.Qualitative data were presented as numbers or percentages.Normal distribution was identified using the Kolmogorov-Smirnov test.Student's t-test was used for between-group comparisons for normally distributed variables, whereas the Mann-Whitney U  test was used to analyze non-normally distributed data.Chisquared test or Fisher exact test was used for qualitative variables as appropriate.Ranked data's comparison was carried out by the Wilcoxon rank sum test.A logistic regression analysis was performed to determine the variables that could be independently associated with the clinical pregnancy rate.Maternal age, basal FSH level, body mass index (BMI), infertility status (primary vs. secondary), method of insemination (IVF vs. ICSI), number of transferred embryos, treatment allocation (fresh vs. frozen-thawed), endometrial thickness, the PI, RI and S/D ratio of the uterine artery and the pattern of endometrial-subendometrial blood flow distribution were included in the analysis.The backward LR method was used to select variables in the logistic regression model.Variables were removed from the logistic regression model if p > .1.
The two-tailed value of p < .05 was considered statistically significant.All the data were analyzed with SPSS (version 16; SPSS Inc., Chicago, IL).

Results
A total of 721 patients were enrolled.Table 1 shows the baseline characteristics at the cycle level between the fresh ET and frozen-thawed ET groups.Patients' demographic and infertility characteristics of both groups were not significantly different (all p > .05).Clinical characteristics of GnRH antagonist protocol in days of stimulation, number of M II oocytes, the proportion of ICSI cycles, and fertilization rate were similar (all p > .05).
Ultrasound parameters of endometrial receptivity are summarized in Table 1.The endometrial thickness in the frozenthawed ET group was significantly lower than in the fresh ET group (p < .05).Both groups were similar regarding uterine artery PI and S/D ratio (p > .05),whereas the uterine artery RI was higher in the fresh ET group than the frozen-thawed ET (p < .05).The proportion of detectable endometrial-subendometrial blood flow was significantly higher in frozen-thawed ET group (p < .05).
When the outcome was clinical pregnancy, the maternal age, basal FSH level, BMI, infertility status (primary vs. secondary), method of insemination (IVF vs. ICSI), number of transferred embryos, treatment allocation (fresh vs. frozenthawed), endometrial thickness, the PI, RI and S/D ratio of the uterine artery and the pattern of endometrial-subendometrial blood flow distribution were included in the first step of the binary logistic regression model.The independent variables were maternal age, treatment allocation (fresh vs. frozen-thawed), and the pattern of endometrial-subendometrial flow distribution, suggesting these variables to be the factors that significantly related to clinical pregnancy rate in GnRH antagonist protocol (Table 3).

Discussion
This retrospective cohort study found that the frozen-thawed ET group had significantly higher clinical pregnancy rate, ongoing pregnancy rate, implantation rate and live birth rate compared to the fresh ET group after the GnRH antagonist protocol.In addition, the proportion of detected endometrial-subendometrial blood flow was significantly higher in the frozen-thawed ET group.In our analysis, patients in the frozen-thawed ET group were 1.438 times more likely to achieve clinical pregnancy when compared to the fresh ET group (odds ratio [OR] ¼ 1.438; 95% confidence interval (CI): 1.001-2.064;p <.05).The miscarriage rate, which was 20.1% in the fresh ET group, was higher than in the frozen-thawed ET group, possibly due to the lack of endometrial and subendometrial blood flows.As a result, the endometrial receptivity was damaged in the fresh ET cycle after the GnRH antagonist protocol.
In recent years, GnRH antagonist protocol with high potency and fewer side effects has become increasingly popular in IVF/ICSI, having emerged as an alternative for preventing premature LH surges (Kadoura et al. 2022).It has been confirmed that both GnRH and GnRH receptors are present in preimplantation embryos, fallopian tubes, ovary and endometrium (Tzoupis et al. 2020), suggesting that GnRH antagonist appears to exist and have functional roles in multiple and diverse effects (Pousias et al. 2019).GnRH can modulate matrix metalloproteinases and their endogenous inhibitors, tissue-specific inhibitors, which have an integral role in cyclic remodeling events in the endometrium for the implantation process.In addition, GnRH controlled the expression of kisspeptin (KISS1), which is related to the efficiency of assisted reproductive technology (D'Occhio et al.

2020).
There is still some controversy about the influence between the GnRH antagonist and endometrial receptivity.Consistent with our conclusion, most studies suggested that the GnRH antagonist can damage endometrial receptivity.The current study showed that GnRH-ant weakened the activization of c-kit receptor by decreasing its expression, causing the impaired growth ability of human endometrial stromal cells (ESCs) (Xu et al. 2022).Moreover, Qian Chen et al showed that GnRH-ant affects CKB expression in endometrial epithelial cells (EECs), resulting in cytoskeletal damage and migration failure (Chen et al. 2020).On the contrary, a study did not prove the influence on the extent of decidualization of endometrial stromal cells, suggesting that GnRH antagonist affect neither the capacity of the endometrium to support invasion nor the invasive potential of the blastocyst in the early stages of implantation (Klemmt et al. 2009).
Endometrial receptivity in ART cycle has always been a challenge for physicians requiring real-time data to improve treatment options.We found greater implantation and pregnancy rates in frozen-thawed ET than in fresh ET, suggesting COH with GnRH antagonist adversely affects endometrial receptivity for implantation following IVF/ICSI.As confirmed by our study, the shortcoming in fresh ET can be resolved by frozen-thawed ET.There is no evidence to suggest that the freeze-all strategy is inferior to the conventional strategy of fresh transfer when comparing cumulative live birth rates (Johnson et al. 2022, Venetis 2022).A systematic review (Wang and Hu 2021) suggested that IVF outcomes could be improved by frozen-thawed ET compared with fresh ET, which could be due to better embryo-endometrium synchrony achieved with endometrium preparation cycles.The endometrial preparation in frozen-thawed cycles can be controlled more precisely in optimal conditions.The advantage of frozen-thawed ET is that it provides a more physiologic environment for ET to occur.
The administration of GnRH antagonist results in changes in the endometrium, which have been demonstrated at the subcellular level and histologically observed.In recent years, several considerable efforts have been made to discover the evaluation method of endometrial receptivity.However, testing tissue damage on the endometrium to detect mRNA, protein or lipids cannot be used in the ET cycle, even though a receptive endometrium could be identified at that time point.Instead, transvaginal ultrasonography, which is noninvasive, simple and effective, can examine endometrial thickness, morphology and blood flow status to predict endometrial receptivity (Ahmadi et al. 2020).
To the best of our knowledge, the radial artery is the branch of the uterine artery, which divides after passing through the myometrial-endometrial junction to form the basal arteries, supplying the basal portion of the endometrium, and the spiral arterioles that continue up toward the endometrial surface.As the spiral artery is challenging to examine, the spiral artery in the endometrium could be examined instead of the uterine artery.In the present study, uterine artery RI and endometrial thickness were significantly lower in the frozen-thawed ET group; however, in the logistic regression analysis, there was no association between endometrial thickness and uterine arterial RI between pregnancy outcomes.Prasad et al. also argued that uterine artery PSV values, S/D values, and RI could not predict pregnancy outcomes (Prasad et al. 2017).Similarly, we suggest that uterine artery S/D, RI and PI could not be used alone to predict endometrial receptivity.In the present study, patients with the presence zone 1 of endometrial-subendometrial blood flow were estimated to be 0.022 times more likely to become clinical pregnant when compared to zone 3 (OR¼ 0.022; 95% CI: 0.012-0.042;p <.001).Those with zone 2 of endometrial-subendometrial blood flow were estimated to be 0.227 times more likely to become clinical pregnant compared to zone 3 (OR¼ 0.227; 95%CI: 0.135-0.379;p<.001).Koo et al. also found that increased endometrial blood flow during the midluteal phase may correlate with successful COH and IVF-ET (Koo et al. 2018).Yet, there is little consensus on the relationship between endometrial volume, 3D power Doppler indices, including vascularization index (VI), flow index (FI) and vascularization flow index (VFI) of endometrial and subendometrial region with the IVF outcome (Zhang et al. 2016, Wang et al. 2018, Maged et al. 2019, Sini et al. 2022;Wang et al. 2018;Maged et al. 2019).
The main strength of the present study is that it was applicable in daily clinical practice because 2D transvaginal color Doppler power ultrasound is extraordinarily common.Good endometrial-subendometrial blood flows on the day of ET were found to be associated with high pregnancy success in frozen-thawed ET after GnRH antagonist protocol, as this was indicative of endometrial receptivity.
In conclusion, our study proved that the endometrial receptivity was damaged in the fresh ET cycle after the GnRH antagonist protocol, which was caused by the lack of endometrial and subendometrial blood flows.However, many previous studies confirmed that the risk of maternal hypertensive disorders in pregnancy or having a large-forgestational-age baby and a higher birth weight might be increased with frozen embryos (Maheshwari et al. 2018, Zaat et al. 2021).Accordingly, in GnRH antagonist protocol, elective frozen-thawed ET should be ideally taken, as this could improve embryo implantation rate, clinical pregnancy rate and live birth rate, thus presenting an effective strategy to enhance the embryo utilization rate.

Figure 1 .
Figure 1.Zone 1: vessels penetrating the outer hypoechogenic area surrounding the endometrium but not entering the hyperechogenic outer margin of the endometrium.

Figure 2 .
Figure 2. Zone 2: vessels penetrating the hyperechogenic outer margin of the endometrium but not entering the hypoechogenic inner area.

Table 2 .
Reproductive outcome per embryo transferred.

Table 1 .
Characteristics of patients.

Table 3 .
Results of the final logistic regression model.Backward LR statistic test.p value <.05 was considered statistically significant.b OR were adjusted for other variables in the equation. a