Success of oocyte retrieval in modified natural cycle assisted reproductive techniques: a retrospective cohort study

Background This study investigates how clinical factors and hormonal levels affect oocyte retrieval in modified natural cycles, aiming to improve understanding and enhance treatment protocols.

A retrospective analysis of data from 306 women undergoing in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) at a fertility clinic (2018–2023) was conducted. Clinical factors and hormonal levels were analysed. Follicular development was monitored via ultrasound.

Baseline comparisons between the oocyte retrieval and non-retrieval groups showed no significant differences in age, body mass index, infertility types, duration, associated conditions (e.g. tubal issues, ovulation disorders and endometriosis) or assisted reproductive methods (p > 0.05). After human chorionic gonadotropin (HCG) injection, both groups showed significant increases in progesterone, oestradiol and luteinising hormone levels (p < 0.0001). Before HCG injection, the retrieval group had significantly higher levels of progesterone, oestradiol and LH (p < 0.05), with oestradiol remaining higher afterward (p < 0.01). There were significant differences between the two groups on anti-Müllerian hormone levels (p ˂ 0.05). No significant differences were found in follicle-stimulating hormone, total testosterone, thyroid hormone levels and antral follicle count (p > 0.05).

In conclusion, the findings suggest that clinical factors do not impact oocyte retrieval in modified natural cycle assisted reproductive techniques. However, high hormone levels on the day of HCG injection in the study subjects may indicate a greater possibility of successful oocyte retrieval. PLAIN LANGUAGE SUMMARY Nowadays, the global demand for assisted reproductive technologies (ARTs) has grown significantly. For some patients, natural cycle egg retrieval is more suitable for their clinical condition, meaning that no ovulation-inducing drugs are used or only ovulation-inducing low-dose medicines are used to mature 1–2 follicles. However, some patients are unable to obtain eggs during the egg retrieval procedure. Understanding what affects egg development in these cycles can help improve treatment outcomes and guide personalised fertility care. We evaluated data from 306 women undergoing ART in China to find the role of related factors on egg acquisition in modified natural cycle assisted reproductive techniques. We found that women who had higher levels of some sex hormones have a greater possibility of egg acquisition. These findings suggest that hormone levels may play a more important role in successful egg retrieval than other clinical factors. Monitoring these indicators during treatment could help doctors better predict outcomes and tailor fertility treatments to individual patients using modified natural cycles.

Assisted reproductive technologies (ARTs), including in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI), have revolutionised the treatment of infertility, offering hope to millions of couples worldwide (Palermo et al. Citation2017). The global demand for ART has grown exponentially, with over 13 million children born through these technologies to date (Adamson et al. Citation2025). Despite significant advancements, the success rates of ART remain variable, with live birth rates per cycle ranging from 20% to 40%, depending on patient demographics and clinical protocols (Chambers et al. Citation2016). A critical determinant of ART success is follicular development, which is influenced by a complex interplay of hormonal dynamics, ovarian reserve and patient-specific factors, including age, body mass index (BMI) and underlying causes of infertility (Moslehi et al. Citation2018). Follicular development is a hormonally regulated process that depends on the coordinated secretion of follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol (E2) and progesterone (P4) (Li and Chian Citation2017). During controlled ovarian stimulation (COS), exogenous gonadotrophins are administered to promote multifollicular growth and to retrieve multiple mature oocytes for fertilisation (Racca et al. Citation2020). Oocyte retrieval during a natural cycle is a commonly used strategy for patients with decreased ovarian reserve (Chen et al. Citation2015). Natural cycle oocyte retrieval optimises the use of the body’s inherent hormonal environment, allowing for the collection of mature oocytes without the need for hormonal stimulation (Rose Citation2014). As fertility treatments continue to evolve, understanding the implications of ovarian reserve and tailoring strategies like natural cycle retrieval can significantly impact patient outcomes (Di Guardo et al. Citation2022), providing hope for individuals with infertility. Age is one of the most well-established factors affecting ART success, with declining oocyte quality and quantity leading to reduced pregnancy rates in women over 35 (Balasch Citation2010). Additionally, obesity has been linked to altered folliculogenesis, impaired oocyte maturation and decreased implantation rates (Aird and Graham Citation2017). Other factors, such as endometriosis, tubal factor infertility and ovulatory disorders, may further complicate ART outcomes by affecting endometrial receptivity and follicular microenvironment (Benlioglu et al. Citation2025). Hormonal fluctuations during follicular development play a crucial role in determining follicular growth and oocyte competence. Elevated progesterone levels at the end of the follicular phase have been linked to premature luteinisation and reduced pregnancy rates (Mesen and Young Citation2015). Similarly, E2 levels are critical for endometrial preparation and oocyte maturation, with both excessively high and low levels associated with poorer outcomes (Huang et al. Citation2019). The LH surge, whether endogenous or triggered by human chorionic gonadotropin (hCG), is essential for final oocyte maturation; however, premature LH rises can disrupt follicular development (Kol and Humaidan Citation2010). Thyroid hormones and androgens also modulate ovarian function, with hypothyroidism and hyperandrogenism (e.g. in polycystic ovary syndrome) influencing folliculogenesis and ART success (Taieb and Feryel Citation2024). Given the multifactorial nature of ART outcomes, identifying reliable predictors of follicular response is crucial for individualised treatment planning. Previous studies have explored the role of antral follicle count (AFC), AMH and baseline FSH in predicting ovarian response (Tsakos et al. Citation2014). However, less attention has been paid to the dynamic changes in hormonal levels during natural cycle follicle development. Additionally, while some studies have examined the impact of infertility aetiology on ART outcomes, few have comprehensively evaluated how different causes of infertility interact with hormonal profiles to influence natural cycles oocyte retrieval success. Understanding these interactions could lead to more tailored approaches to natural cycle egg retrieval and oocyte retrieval based on both the underlying cause of infertility and individual hormonal responses. This study aims to investigate the influence of (1) clinical factors (age, BMI and infertility causes) and (2) hormonal profiles (progesterone, oestradiol, LH, FSH, thyroid hormones and androgens) in women with modified natural cycle oocyte retrieval process IVF/ICSI. Our comprehensive approach includes a detailed assessment of both static and dynamic hormonal parameters throughout the follicular development cycle, along with careful evaluation of follicular development patterns.

Study design and population This retrospective cohort study analysed data from 306 women undergoing modified natural cycle oocyte retrieval IVF, or ICSI at a single fertility clinic between 2018 and 2023, dividing participants into oocyte retrieval (n = 242) and non-oocyte retrieval (n = 64) groups based on whether oocyte was obtained through oocyte egg retrieval. Inclusion criteria: In this study, women aged 20–45 years with infertility combined with diminished ovarian reserve (DOR) who were treated with modified natural cycle oocyte retrieval were included. Among them, DOR diagnosis meets the standards of the ‘Chinese Expert Consensus on Clinical Diagnosis and Treatment of Ovarian Reserve Hypofunction (2023 Edition)’: age ≥35 years old and AMH <1.2 ng/mL, or age <35 years old and AMH <0.5 ng/mL, or basal AFC <5; the following conditions are met: the World Health Organization (WHO) diagnosis criteria for infertility (couples live regularly, have not contraceptive for 1 year but are not pregnant, or the woman is not contraceptive for 6 months and is not pregnant when she is ≥35 years old). Moreover, all participants should have had complete hormonal and ultrasonographic data. Exclusion criteria: (1) Combined with other types of ovarian diseases: such as ovarian malignant tumours or borderline tumours; (2) severe endocrine and metabolic diseases: such as uncontrolled hyperthyroidism/hypoxia (TSH > 4.5 mIU/L or 7.0 mmol/L), Cushing syndrome; (3) orthologic lesions of the uterine cavity: such as adenomyosis (ultrasound suggests diffuse thickening of the myometrium with fence-like echo), endometrial polyps (diameter >1 cm), uterine adhesion (confirmed by hysteroscopy); (4) severe systemic diseases: such as cardiac insufficiency (NYHA grade ≥ III), chronic renal failure (creatinine >178 μmol/L), liver failure (ALT >3 times normal upper limit); (5) those with incomplete data: ≥2 missing key indicators (such as basal hormones, ultrasound AFC and oocyte retrieval outcome); (6) those with donor gametes; (7) patients with premature ovulation. In this study, patients did not use a stimulation protocol; all underwent modified natural cycle IVF. All patients underwent oocyte retrieval. For the oocyte retrieval group, all oocytes retrieved without oocytes were included in the study, regardless of whether they were mature metaphase II oocytes. Therefore, a small number of oocytes retrieved from patients may be in metaphase I oocytes or germinal vesicle (GV) stage. The cut-off used to determine the timing of HCG triggering was E2 level of ≥100 pg/mL. Data collection Clinical parameters including age, BMI, infertility type/duration and causes (tubal, ovulatory, endometriosis, male factor) were recorded alongside cycle-specific data (ART type, gonadotrophin dosage) and hormonal levels (P4, E2, LH, FSH, AMH, total testosterone, free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH)) were measured before and after hCG trigger, with follicular development tracked via serial transvaginal ultrasounds. In detail, all patients were fasted in the early morning (fasting for 8–12 hours), and the trained nurse used vacuum blood collection vessels (BD Company, Franklin Lakes, NJ, containing EDTA-K2 anticoagulant or coagulant) for cubital vein collection. Basic hormones: Collected on the 2nd to 4th day of the menstrual cycle (the 1st day of natural menstrual cycle is the 1st day of the cycle), used to detect FSH, LH, E2, P4, testosterone, AMH, TSH, FT3 and FT4. Oocyte retrieval hormone: Tests LH, E2 and P4 at 10 am on the day of HCG injection and again at 10 am on the 1st day after the injection of HCG were measured. Each sample was sent to the laboratory within two hours after blood collection, and the procoagulant tube sample is left to stand at room temperature (20–25 °C) for 30 minutes to coagulate, and the EDTA-K2 anticoagulant tube sample is processed immediately; separate serum (procoagulant tube) or plasma (anticoagulant tube) at 3000 r/min for 10 minutes (centrifugal radius 15 cm); serum/plasma was aliquoted into EP tube (1.5 mL), mark the patient ID and sampling time, store in a −80 °C refrigerator, and re-temperature uniformly before detection (re-temperature for 30 minutes at room temperature to avoid repeated freeze–thaw). Hormonal levels were measured using standardised electrochemiluminescence immunoassays (ECLIAs) with the Roche Cobas e411 Analyzer (Roche Diagnostics, Basel, Switzerland) for accurate quantification of P4, E2, LH, FSH, testosterone and thyroid markers (T3, T4, TSH), as well as AMH. Regarding the sensitivity, precision and calibration procedures: The ECLIAs were performed using Roche cobas® electrochemiluminescence immunoassay kits (Basel, Switzerland) according to the manufacturer’s standard protocols. The sensitivity of the assays for each sex hormone was as follows: (oestradiol: 5 pg/mL; testosterone: 0.02 ng/mL; FSH: 0.1 IU/L; LH: 0.1 IU/L; P: 0.1 nmol/L; TSH: 0.001 mIU/L; FT3: 0.2 pmol/L; FT4: 0.1 pmol/L; AMH: 0.01 pmol/L), which meets the requirements for clinical and research applications. For precision, the intra-assay coefficient of variation (CV) was <5%, and the inter-assay CV was <8%, as verified by quality control samples included in each run. Calibration was performed prior to each batch of measurements using the manufacturer-provided calibrators, covering the full analytical range of the assays. Calibration curves were validated to ensure linearity and accuracy within the measuring range. All measurements were performed in duplicate to minimise random errors. Both intra-assay and inter-assay variabilities were assessed. Intra-assay variability was evaluated by repeated measurements of the same sample within a single run, and inter-assay variability was determined by analysing quality control samples across multiple runs on different days. The results of these variability assessments confirmed the reliability and reproducibility of the measurements. Follicular development was tracked using serial transvaginal ultrasounds, conducted on the GE Voluson E10 ultrasound system (GE Healthcare, Chicago, IL). This system, equipped with a 4–8 MHz transvaginal probe, allowed for high-resolution imaging and precise monitoring of follicular size and number throughout the follicular development stage. In this study, all patients underwent the first transvaginal ultrasound monitoring starting on day 8 of ovulation induction treatment. The monitoring frequency was then adjusted based on follicular growth: When the dominant follicle reached a diameter of 10–12 mm, the monitoring frequency was every two days. When the dominant follicle reached a diameter of 14 mm or greater, the monitoring frequency was adjusted to daily until the day of HCG injection. Criteria for determining follicular maturity: The timing of egg retrieval is based on follicular maturity. Specific criteria include: Follicular size: at least one dominant follicle must be ≥16 mm in diameter; Hormone level support: a comprehensive assessment is performed based on the daily serum E2 level (target range: ≥100 pg/mL per mature follicle) and LH peak (no premature LH surge) to ensure that the follicles meet both morphological and functional maturity standards. Some older patients may have follicles with a diameter ≥14 mm but <16 mm, an E2 level ≥100 pg/mL, elevated LH levels, premature ovulation in previous cycles, or a (P) level ≥1.2 ng/mL. Follicle size measurement method: This study used transvaginal two-dimensional ultrasound to measure follicles. The specific procedure was as follows: For each identifiable follicle, two perpendicular diameters (i.e. the long diameter and the transverse diameter) were measured on its largest cross-section (longitudinal section); The average of these two diameters was used as the final follicle diameter (rather than a single maximum diameter or volume); During measurement, intrafollicular septa and areas of increased echogenicity were avoided, and the measurement line was parallel to the follicle wall to reduce systematic error. Considerations for operator and interobserver variability in ultrasound assessment: Interobserver agreement: All follicular ultrasound assessments in this study were performed by two attending physicians with at least five years of experience in reproductive ultrasound. The same operator performed the entire monitoring process for each patient (from the initial assessment to the final assessment before oocyte retrieval) to minimise measurement errors due to differences in operator habits. Interobserver variability control: To verify the reliability of measurement results, we implemented the following measures: A random sample of 20% of patients had follicle diameters measured independently by two operators. The interobserver correlation coefficient (ICC) was 0.94, indicating high agreement between the two measurements. All ultrasound images were stored in the hospital’s PACS system and were reviewed monthly by the department’s ultrasound quality control team during the study period to ensure consistent measurement standards. Ethical approval This study received approval from the Medical Ethics Committee of General Hospital, Tianjin Medical University, with Approval No. IRB2025-YX-327-01. Informed consent was waived because of the retrospective nature of the study, and the analysis used anonymous clinical data. Statistical analysis In this study, we considered α = 0.05 and β = 0.2. The post hoc power analysis using the two-group sample sizes showed that this study has 80% power to detect standardised mean differences of about d ≥ 0.39. The Kolmogorov–Smirnov test was used to test the normality of variables. Data were analysed using independent/paired t-tests or non-parametric equivalents (for non-normal variables) for continuous variables, Chi-square tests for categorical comparisons, and logistic regression to identify oocyte retrieval predictors, with all analyses performed in SPSS 26.0 (Chicago, IL) (p < 0.05 significance threshold). Multicollinearity among independent variables in the logistic regression model was assessed using the variance inflation factor.

presents flow diagram for study participants. Baseline characteristics In the present study, the demographic characteristics of the participants are presented in , which includes a total of 306 individuals. Among them, 67 participants were in the non-oocyte retrieval group, while 239 were in the oocyte retrieval group. The number of cycles in the oocyte retrieval group (2.5 ± 2.0) and the non-oocyte retrieval group (2.7 ± 2.3) showed no statistically significant difference (p > 0.05). No significant differences were observed in age and BMI between the two groups (p > 0.05). The results showed that AFC was not significantly different between the two groups (p > 0.05). Analysis of infertility causes between the oocyte retrieval group and the non-oocyte retrieval group Next, we analysed the types of infertility, including primary infertility, secondary infertility and other types, as well as infertility duration and causes between the two groups. As shown in , there was no significant difference in infertility types between the oocyte retrieval group and the non-oocyte retrieval group (χ2 = 2.36, p = 0.31). We also found that the most common infertility type was secondary infertility in both the oocyte retrieval group (65.30%) and the non-oocyte retrieval group (56.70%). No significant difference was observed in infertility duration between the oocyte retrieval group (3.6 ± 3.7 years) and the non-oocyte retrieval group (5.0 ± 14.7 years) (p > 0.05). Furthermore, we compared the occurrence of fallopian tube issues, ovulation disorders and endometriosis between the two groups. No statistically significant differences were found (χ2 = 0.02, p = 0.90; χ2 = 0.05, p = 0.83; χ2 = 0.70, p = 0.40, respectively), suggesting that these infertility causes might not significantly influence the outcome of oocyte retrieval. Analysis of hormone levels before and after HCG administration between the oocyte retrieval group and the non-oocyte retrieval group Furthermore, we analysed the changes in progesterone, oestradiol and LH levels before HCG injection in both groups. As shown in , in both the oocyte retrieval group and the non-oocyte retrieval group, the levels of progesterone, oestradiol and LH were significantly higher after HCG injection compared to before, with statistical significance (p < 0.0001). When comparing between the two groups, we found that before HCG injection, the oocyte retrieval group had significantly higher levels of progesterone, oestradiol and LH than the non-oocyte retrieval group (p < 0.05). After HCG injection, the oestradiol level in the oocyte retrieval group remained significantly higher than that in the non-oocyte retrieval group (p = 0.001). These results suggest that higher levels of progesterone, oestradiol and LH before oocyte retrieval may contribute to successful oocyte retrieval in individuals undergoing ART treatment. Analysis of sex hormones and thyroid hormones levels between the oocyte retrieval group and the non-oocyte retrieval group As shown in , we conducted a retrospective analysis of the levels of FSH, LH, total testosterone, FT3, FT4, AMH and TSH in both the oocyte retrieval group and the non-oocyte retrieval group. There were significant differences between the two groups on AMH levels (p ˂ 0.05); however, the results showed no statistically significant differences in these hormone levels between the two groups (p > 0.05). In the logistic regression analysis, higher AMH levels were associated with increased odds of successful egg retrieval (OR = 1.836, 95% CI: 0.987–3.414, p = 0.055). However, this association was not significantly different. All other variables were also not statistically significant ().

In this study, we aimed to analyse the baseline characteristics, infertility causes, clinical information and hormone levels between two groups: an oocyte retrieval group and a non-oocyte retrieval group. Our findings provide important insights into factors that may influence oocyte retrieval outcomes, and offer comparisons with previous studies in the field of ART. The demographic characteristics of participants in the oocyte retrieval group and the non-oocyte retrieval group revealed no significant differences in age or BMI. This finding aligns with previous studies that suggest BMI and age may not be primary factors in determining the success of oocyte retrieval in ART treatments (Rafael et al. Citation2023). However, while BMI and age may not be significant determinants in this study, other factors, such as ovarian reserve and hormonal profiles, have been shown to play a more critical role in determining the success rates of oocyte retrieval (Chen et al. Citation2015). In this study, we are unable to investigate the impact of other lifestyle and environmental factors; however, evidence supports the role of these factors in the outcomes of ART (Kumar et al. Citation2018, Mínguez-Alarcón et al. Citation2018). Regarding the causes of infertility, our study did not find significant differences between the two groups, which is consistent with previous literature that reports the causes of infertility, such as fallopian tube issues, ovulation disorders and endometriosis, do not always correlate strongly with retrieval success (Wang et al. Citation2022). Interestingly, we found that secondary infertility was the most common infertility type in both groups. This finding mirrors trends observed in other studies, where secondary infertility is more common than primary infertility in patients undergoing ART (Jabeen et al. Citation2022). Additionally, the lack of significant differences in infertility duration between the two groups aligns with the findings of a previous study, which found that infertility duration did not significantly impact ART outcomes (Matthiesen et al. Citation2011). In the clinical information analysis, we found that the number of cycles did not differ significantly between the two groups. This result is consistent with a previous study (Saunders and Saunders Citation2000). We found that basal hormone levels (referring to the hormone index on the third day of menstrual bleeding from the first day of menstrual bleeding), including FSH, LH, TT, FT3, FT4 and TSH levels, were not significantly different between the two groups. However, women in the oocyte retrieval group had significantly higher AMH levels. Our hormone analysis on the day of HCG injection demonstrated significantly higher levels of progesterone, oestradiol and LH in the oocyte retrieval group. Previous findings from studies show that higher baseline hormone levels contribute to better ART outcomes (Tulic et al. Citation2020). Another study also reported that higher progesterone levels are positively correlated with oocyte retrieval success (Zhu et al. Citation2014), which corroborates our findings. A possible reason for lower hormone levels in the group with non-oocyte retrieval is empty follicle syndrome (EFS) (Singh et al. Citation2018). Also, a recent study highlighted the predictive value of the AMH as a determining factor in the selection of the oocyte donor candidates; they showed that a cut-off of AMH = 3.2 ng/mL is predictive of the retrieval <12 oocytes (Vale-Fernandes et al. Citation2023). Another study reported the predictive cut-off for poor ovarian response was AMH = 0.72 ng/mL (Reis et al. Citation2024). A pilot study in the Indian population revealed that day-2 serum AMH concentration significantly correlated with the oocytes’ retrieval count in women undergoing ovulation induction for IVF (Singh et al. Citation2013). A multi-centre study also demonstrated that in the NC-IVF protocol, AMH acts as an effective predictor of the number of oocytes (Magaton et al. Citation2025). Analysis of Public Gamete Bank between 2011 and 2021 also suggested that AMH = 1.12 ng/mL was considered as a threshold level for successful donation (Oliveira et al. Citation2023). Furthermore, the analysis of thyroid hormone levels showed no significant differences between the two groups. This outcome is consistent with a recent study (Wijs et al. Citation2022), which concluded that thyroid function, although important, does not always exhibit a direct link to ART outcomes, at least not in the general population. The potential biological mechanisms linking higher baseline hormones with improved retrieval include regulating follicle growth via the PI3K/AKT and MAPK signalling pathways, as well as increasing vascular endothelial growth factor (VEGF) in human granulosa cells (Doldi et al. Citation1997, Long et al. Citation2021). The results showed that the AFC was not significantly different between the two groups. Previous studies have shown (Nakagawa et al. Citation2008, Zheng et al. Citation2018), that a higher number and better size of follicles are directly associated with improved ART outcomes (Ortega et al. Citation2018). The results emphasise the importance of follicular health and the role of ovarian reserve in oocyte retrieval success. Reasons for oocyte non-retrieval include: (1) Empty follicle syndrome (Revelli et al. Citation2017): Ultrasound shows a ‘morphologically normal dominant follicle’ (e.g. diameter ≥18 mm, clear follicular wall), but after puncture, only follicular fluid is aspirated, and no oocytes are retrieved. (2) Follicular maturity is determined solely by ultrasound and hormonal parameters. Misjudgment can lead to either ‘too early’ oocyte retrieval (when oocytes are immature and difficult to identify) or ‘too late’ oocyte retrieval (when oocytes are aged and degenerated). Despite the valuable insights this study provides, several limitations should be acknowledged. First, the study is observational in nature, and while it can reveal associations, it does not establish causality. Future studies utilising randomised controlled trials (RCTs) would help provide more robust evidence regarding the factors influencing oocyte retrieval outcomes. Additionally, while we analysed several hormones and clinical factors, other potential variables such as genetic factors, lifestyle choices (e.g. smoking, alcohol use, lifestyle and environmental factors) and environmental factors were not included in this analysis. Incorporating these variables could further enhance the understanding of the factors that influence oocyte retrieval success. Another limitation of the study is the retrospective design, which inherently introduces the possibility of selection bias and inaccurate data reporting, particularly with respect to infertility duration and clinical history. Moreover, the sample size in the non-oocyte retrieval group was relatively small compared to the oocyte retrieval group. Imbalanced group sample sizes can reduce statistical power due to the limitations of precision in the comparison for the smaller group. While we found no significant differences in the majority of parameters between the two groups, a larger sample size in the non-oocyte retrieval group could potentially reveal subtler differences that might have been undetected in this study. Also, potential inter-operator variability in ultrasound assessments may have influenced measurements. To validate findings, future prospective, multicentre studies with large sample size are warranted. By examining the impact of various factors on oocyte retrieval, researchers can provide valuable insights for enhancing infertility treatment protocols. Integrating hormonal profiling into NC-ART offers the potential for more personalised treatment, optimised ART processes and improved support for patients throughout their fertility journey.

In conclusion, the findings suggest that clinical factors do not impact oocyte retrieval in modified natural cycle assisted reproductive techniques. However, for women who had higher levels of LH, E2 and P on the day of HCG injection, the possibility of egg acquisition is higher. Future studies in other settings should be performed to support these findings. Supplemental material STROBE checklist.doc Download MS Word (87 KB)STROBE checklist.docAcknowledgements Clinical trial registration. Not applicable. All authors have read and agreed to the published version of the manuscript. Disclosure statement No potential conflict of interest was reported by the author(s). Data availability statement The data are available from the corresponding author on reasonable request. Additional information Funding

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