The association and diagnostic value of maternal serum placental biomarkers for fetal hypospadias and cryptorchidism

Introduction

Background

Hypospadias is a congenital urogenital disease, with common clinical manifestations including abnormal opening of the male urethra in the penis, ventral scrotum, or perineum, penile curvature, and abnormal foreskin (1). The incidence of hypospadias varies between 1/300 and 1/125 male infants and exhibits ethnic and regional differences (2,3).

Cryptorchidism is a congenital disease in which at least one testicle does not descend into the scrotum. The prevalence of this condition varies geographically, ranging from 1% to 4.6% in newborn males at term and 1% to 45% in premature male infants (4). Cryptorchidism may reflect neonatal testicular hypoplasia, and may subsequently manifest as low fertility or testicular cancer (5).

According to a common pathogenesis and potential correlations, hypospadias, cryptorchidism, poor semen quality, and testicular germ cell cancer, are all commonly summarized as testicular dysgenesis syndrome hypothesis. The hypothesis is that severe urogenital disorders cause disruption of embryonic processes and gonadal development. Hypotheses thought that the pathogenic mechanisms and epidemiological features of this syndrome are complex and its etiology involves multiple factors, including genetics, in-utero growth disorders, endocrine disruptors, or the environment (6), and to some extent, hypospadias and cryptorchidism are potential manifestations of testicular dysgenesis syndrome in childhood.

The measurement of placental molecules has been widely used for prenatal screening. Currently, maternal screening is usually performed during the developmental window of the first/second trimester [first-trimester (FT): 10–13⁺⁶ weeks; second-trimester (ST): 15–20⁺⁶ weeks] in order to estimate the risk of Down’s syndrome, trisomy 18, and neural tube defects in the fetus (7,8). The most common maternal serum biomarkers are pregnancy-associated plasma protein-A (PAPP-A), free beta human chorionic gonadotropin (free β-hCG), alpha-fetoprotein (AFP), and unconjugated estriol (uE3) (9,10).

Rationale and knowledge gap

The prevalence of congenital hypospadias and cryptorchidism is increasing on a global basis (3,11-13). The clinical diagnosis of hypospadias and cryptorchidism is predominantly made after the birth of the infant by a clinician who performs a visual examination for an abnormal urethral opening, or by a physical examination to determine whether both of the testes are located normally in the scrotum (1,14-16). The prediction of congenital hypospadias and cryptorchidism by the analysis of maternal serum biomarkers has been less frequently reported (17-19).

The precise association between alterations in the levels of placental markers in the maternal serum during pregnancy and disorders of reproductive development in males remains unclear. In the 1970s, researchers discovered that human chorionic gonadotropin (hCG) binds to the luteinizing hormone chorionic gonadotropin receptor (LHCGR) in the male fetal gonads to stimulate the initiation of steroid release (20). In addition, follow-up studies reported that male newborns with combined LHCGR mutations were born with hypogonadism and pseudohermaphroditism (21,22). In another study, Peycelon, Sharpe and Schneuer et al. reported a correlation between changes in the levels of hCG and the risk of proximal hypospadias (18,19,23). Subsequently, Chen et al. suggested that the combination of maternal serum AFP and ST-free β-hCG exhibited higher sensitivity and specificity for fetal hypospadias (24). Nutman et al. noted that compared with controls, newborns with cryptorchidism had a lower pregnancy uE3 levels (25). And Cripps et al. found that estrogen is required for normal nuclear development at the distal end of the penis and for the location of the mature urethral opening (26).

Objective

Given the clear biological plausibility, recent experimental evidence, and the limited body of epidemiological evidence, we conducted a retrospective and case-control study to investigate the association and diagnostic value of placental markers in the maternal serum (free β-hCG, PAPP-A, AFP, and uE3) for the prediction of hypospadias and cryptorchidism. We present this article in accordance with the STARD reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-83/rc).

Methods

Participants

This was a retrospective and case-control study that analyzed samples of prenatal serum from the first/second trimester of pregnancy and pregnancy outcomes of approximately 671,605 pregnant women with male or female infants between October 2014 and September 2020 at four prenatal screening centers: the Hangzhou Women’s Hospital (Hangzhou Maternity and Child Health Care Hospital), Hangzhou Yuhang District Maternity and Child Health Care Hospital, Hangzhou Fuyang District Maternity and Child Health Care Hospital, and Zhejiang Xiaoshan Hospital. Among 671,605 pregnant women, 51,807 pregnant women had complete follow-up records. All pregnancies underwent maternal serological screening for the appropriate biomarkers in early (FT: 10–13⁺⁶ weeks) and middle (ST: 15–20⁺⁶ weeks) gestations, and all pregnant women were routinely examined for structural abnormalities by ultrasound prior to blood sampling. Following hospital delivery, each infant was routinely examined by a neonatologist or pediatrician to investigate for and diagnose hypospadias or cryptorchidism. After excluding some pregnancies according to the exclusion criteria, 342 pregnant women’s infants were diagnosed as hypospadias or cryptorchidism by neonatologists or pediatricians. There were 222 cases in the hypospadias group and 120 cases in the cryptorchidism group. Based on the total number of cases in the hypospadias group and the cryptorchidism group, a 1:1 random sampling was performed to form the control group from pregnant women with male infants exhibited normal development during the same period; 311 pregnant women with normal male infant development in the same period were selected as the control group. As shown in Figure 1 (27).

Figure 1 Flow chart of the study population.

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Human Research Ethics Committee of the Hangzhou Women’s Hospital Medical Ethics Committee [No. 2023-A-(014)], and individual consent for this retrospective analysis was waived. The other centers were also informed and approved this study.

Diagnostic and exclusion criteria

Diagnostic criteria

Diagnoses were made by a senior neonatologist or pediatrician in accordance with the diagnostic criteria developed by the International Classification of Diseases (ICD-10) and the Chinese Birth Defects Surveillance Network. Hypospadias is a congenital malformation characterized by an abnormal opening of the urethra in the penis, ventral scrotum, or perineum; this condition can be diagnosed by visual observation after birth.

For children suspected of having cryptorchidism, an evaluation conducted by experienced neonatologist or pediatrician remains the most crucial assessment step, enabling the distinction between normally positioned gonads, retractile testes, palpable undescended or ectopic testes, and non-palpable testes (28). Cases diagnosed with hypospadias and cryptorchidism are followed up until the age of 1 year. Spontaneous descent of cryptorchidism is rare after 1 year of age. Meanwhile, the health records of children up to 1 year of age are reviewed on a monthly basis by the Maternal and Child Health Centers in each district and reported on a quarterly basis. Some children require diagnostic assistance by imaging such as ultrasound. The normal group were defined as follows: (I) the pregnancy progressed well; (II) the urethral opening was in a normal position; and (III) the testes were located in the scrotum (27).

Exclusion criteria

We excluded the following cases: pregnant women who had offspring with hypospadias and cryptorchidism and who did not participate in prenatal screening; gender abnormalities of hypospadias combined with cryptorchidism, abnormal adrenal sex characteristics (female pseudohermaphroditism), true hermaphroditism and male pseudohermaphroditism; twin and multiple pregnancies, and women who had been diagnosed with other medical conditions such as insulin-dependent diabetes mellitus and severe pregnancy complications. We also excluded women who smoked, those that had conceived by in vitro fertilization, and newborns that had chromosomal abnormalities, neural tube defects, and other birth defects; elevated testes, spontaneously descending, and retracting testes; other birth defects of the genitourinary tract; and cases for which there was an incomplete set of clinical information (27).

Reagents and instruments

This research utilized a 1235 Auto Automatic Time-resolved Fluorescence Immunoassay Analyzer (PerkinElmer, Shelton, USA), along with PAPP-A, free β-hCG, AFP, uE3 kits, enhancement solution, washing solution, standards (all Wallac Oy, Turku, Finland), and Internal quality control products (Biosan, Hangzhou, China).

Detection method

We collected 3–5 mL of venous blood from each participant and separated the serum samples after 30 min. Then 1–2 mL of serum was aspirated, transferred to centrifuge tubes, and stored temporarily in a refrigerator at 2–8 ℃ to await analysis. Samples were transported at low temperatures and sent for testing within 1 week; following prenatal screening, the remaining serum samples were stored in a refrigerator at −80 ℃. We performed serological PAPP-A, FT-free β-hCG, ST-free β-hCG, AFP, and uE3 tests during the first/second trimester of pregnancy by applying the dissociation enhanced-lanthanide fluoroimmunoassay method, which was performed in accordance with the manufacturer’s instructions.

Representation of PAPP-A, free β-hCG, AFP, and uE3 levels

To reduce the effect of gestational week and maternal weight on each marker, we utilized median multiple of median (MoM) values instead of the original concentrations to indicate the levels of each placental marker. MoM = Original Conj/Median (19,24) in which ‘Original Conj’ represents the original concentrations of PAPP-A, FT-free β-hCG, ST-free β-hCG, AFP, and uE3 and ‘Median’ represents the median of the original concentrations of the corresponding index. Since the median was assessed on the basis of the change in gestational week, dividing the test result by the median yielded an initial MoM value.

Screening models for hypospadias and cryptorchidism

Hypospadias: FT-free β-hCG MoM value model; PAPP-A MoM value model; AFP MoM value model; uE3 MoM value model; ST-free β-hCG MoM value model; AFP + ST-free β-hCG combination model; AFP + ST-free β-hCG + uE3 combination model; PAPP-A + AFP + uE3 combination model.

Cryptorchidism: FT-free β-hCG MoM value model; PAPP-A MoM value model; AFP MoM value model; uE3 MoM value model; ST-free β-hCG MoM value model; AFP + ST-free β-hCG + uE3 combination model; PAPP-A + ST-free β-hCG + uE3 combination model; PAPP-A + uE3 combination model (27).

Statistical analysis

We used Excel 2013 to create the database, and IBM SPSS 21.0 (Chicago, USA) for statistical processing. The normality of data was tested using the Shapiro-Wilk test; if data were not normally distributed, they were expressed as median and percentile [M (P_2.5, P_97.5)]. Comparisons between more than two groups were analyzed by the Kruskal Wallis H test. Receiver operating characteristic (ROC) curves were used to determine the cut-off; then, we calculated the optimal cut-off, the area under the curve (AUC), and the Youden index. We used a range of indicators to evaluate the performance of the models, including sensitivity (Sen), specificity (Spe), false negative rate (FNR), false positive rate (FPR), positive likelihood ratio (+ LR) and negative likelihood ratio (−LR).

Results

Basic clinical information

A total of 653 pregnant women across the three groups were involved in this study, including 222 cases in the hypospadias group, 120 cases in the cryptorchidism group, and 311 cases in the control group. The gestational age of the control group, the hypospadias group and the cryptorchidism group were 118 [109–129], 118 [110–132], and 118 [110–135] days, respectively, the difference was not statistically significant (P>0.05). There were no statistically significant differences between the groups in terms of age and maternal weight (P>0.05; see Table 1) (27).

Placental biomarkers in maternal serum

Maternal serum PAPP-A levels during early pregnancy were significantly lower in both the hypospadias and cryptorchidism groups than in the control group [0.70 (0.10–2.41) vs. 0.92 (0.31–3.01) vs. 1.03 (0.33–2.69) MoM)] (P<0.001). In addition, maternal serum free β-hCG levels during early pregnancy were lower in both the hypospadias and cryptorchidism groups than in the control group [1.08 (0.27–4.88) vs. 1.06 (0.29–3.68) vs. 1.11 (0.33–3.08) MoM]; however, these differences were not significant (P>0.05).

Compared with the control group, maternal serum AFP, ST-free β-hCG, and uE3 MoM levels in the Hypospadias group were higher, higher, and lower, respectively, while maternal serum AFP, ST-free β-hCG and uE3 MoM levels in the cryptorchidism group were lower, higher, and lower, respectively; these differences were all significant (all P<0.001). Maternal serum AFP, ST-free β-hCG, and uE3 MoM levels were then compared in a two-by-two manner between the three groups. The differences in AFP and ST-free β-hCG levels between the hypospadias and control groups were significantly different (P<0.05), while the differences in uE3 MoM levels differed significantly between the cryptorchidism and control groups (P<0.05). All other comparisons between the three groups were not significant (P>0.05). See Table 2 (27) and Figure 2.

Figure 2 Levels of PAPP-A, FT-free β-hCG, AFP, ST-free β-hCG, and uE3 in maternal serum from the three groups. (A) Levels of PAPP-A in maternal serum from the three groups. (B) Levels of FT-free β-hCG in maternal serum from the three groups. (C) Levels of ST-free β-hCG in maternal serum from the three groups. (D) Levels of AFP in maternal serum from the three groups. (E) Levels of uE3 in maternal serum from the three groups. ^ns, P>0.05; ***, P<0.001. AFP, α-fetoprotein; free β-hCG, free β human chorionic gonadotropin; FT, first-trimester; MoM, multiple of median; PAPP-A, pregnancy-associated plasma protein A; ST, second-trimester; uE3, unconjugated estriol.

The screening of neonatal hypospadias and cryptorchidism by PAPP-A, FT-free β-hCG, AFP, ST-free β-hCG, and uE3 levels alone or in combination

Table 3 shows the AUCs for predicting neonatal hypospadias using maternal serum PAPP-A in early pregnancy, AFP, and uE3 levels alone: the AUCs were 0.667, 0.703, and 0.728, respectively (all P<0.05). However, FT-free β-hCG and ST-free β-hCG had no diagnostic value for predicting neonatal hypospadias (AUC =0.561, 0.585; both P>0.05). The AUCs for combined models (AFP + ST-free β-hCG, AFP + ST-free β-hCG + uE3, and PAPP-A + AFP + uE3) were 0.682, 0.745 and 0.795, respectively (all P<0.05).

Of the multiple index models, the Sen, Spe, FPR, and +LR evaluation indexes were ranked as follows: the PAPP-A + AFP + uE3 model was better than the AFP + ST-free β-hCG + uE3 model, which was better than the AFP + ST-free β-hCG model. The PAPP-A + AFP + uE3 model exhibited the best predictive value; Sen, Spe, FNR, FPR, +LR, and −LR were 0.800, 0.788, 0.200, 0.212, 3.774, and 0.254, respectively. See Table 3 and Figure 3A (27).

Figure 3 ROC curve for the diagnosis of PAPP-A, FT-free β-hCG, AFP, ST-free β-hCG, and uE3 levels for neonatal hypospadias and cryptorchidism. (A) ROC curve for the diagnosis of maternal serum biomarkers for neonatal hypospadias; (B) ROC curve for the diagnosis of maternal serum biomarkers for neonatal cryptorchidism. AFP, α-fetoprotein; free β-hCG, free β human chorionic gonadotropin; FT, first-trimester; MoM, multiple of median; PAPP-A, pregnancy-associated plasma protein A; ROC, receiver operating characteristic; ST, second-trimester; uE3, unconjugated estriol.

The models that utilized PAPP-A, FT-free β-hCG, AFP, and ST-free β-hCG alone had no diagnostic value for predicting neonatal cryptorchidism (P>0.05). The AUC for maternal serum uE3 levels alone to predict cryptorchidism was 0.648 [95% confidence interval (CI): 0.540–0.756, P=0.04], and the AUC for the combined PAPP-A + uE3 model was 0.658 (95% CI: 0.531–0.784, P=0.03), as shown in Table 3. According to the ROC curves, the Sen, Spe, FPR, and + LR evaluation indices were ranked as follows: the PAPP-A + uE3 model was better than the PAPP-A + ST-free β-hCG + uE3 model, which was better than the AFP + ST-free β-hCG + uE3 model in the multiple index combination model. The combined PAPP-A + uE3 model exhibited the best predictive value, with a sensitivity of 0.778 and a specificity of 0.614 when the optimal cutoff value was 0.056 (+LR =2.016, −LR =0.362). Overall accuracy of predictive models was not sufficient. See Table 3 and Figure 3B (27).

Discussion

We conducted a retrospective case-control study to analyze the association and diagnostic value of placental biomarkers in the maternal serum with/for hypospadias and cryptorchidism. We detected statistical differences in the maternal serum levels of PAPP-A, ST-free β-hCG, AFP, and uE3 in offspring with hypospadias and cryptorchidism. However, there was no statistical difference in terms of FT-free β-hCG levels between the case and control groups. Specifically, maternal serum levels of PAPP-A and uE3 were lower than normal while the levels of AFP and ST-free β-hCG were higher than normal in offspring with hypospadias. In contrast, the maternal levels of serum PAPP-A, AFP, and uE3 were all lower, while the levels of ST-free β-hCG were higher. than normal in offspring with cryptorchidism. Comparison of the single-indicator models and combined-indicator models, identified the best models as (hypospadias: PAPP-A + AFP + uE3 model; AUC =0.795) and (cryptorchidism: PAPP-A + uE3 model; AUC =0.658) (27).

The precise association between placental biomarkers in the maternal serum during pregnancy and disorders of reproductive organ development in males has yet to be elucidated. Several studies have revealed that placental dysfunction may represent a common cause of both cryptorchidism and hypospadias (29). Moreover, indirect evidence indicates that preterm birth, fetal growth retardation, and low birth weight represent common or separate risk factors for hypospadias and cryptorchidism (30-32). Alterations in the levels of maternal serum biomarkers are one of the manifestations of placental dysfunction. Biologically, PAPP-A expresses protein hydrolase activity and acts on insulin-like growth factors to promote normal placental and fetal growth (33). Thus, low levels of PAPP-A may represent an indicator of impaired placental function and impaired implantation. In addition, low levels of PAPP-A are the strongest predictor for low birth weight and preterm birth and can predict pregnancy loss (34). Few studies have investigated the association between low levels of PAPP-A and congenital genitourinary abnormalities (35,36). The results of our present study suggested that levels of PAPP-A in the maternal serum were reduced in both neonates with hypospadias and cryptorchidism when compared to the normal group. Similarly, Yinon and Proctor et al. described the results of a prospective study of 30 male infants with hypospadias and reported low levels of PAPP-A in the maternal serum (35,36). In another study, Weidner et al. reported that although the testes usually descend into the scrotum shortly before birth, a transabdominal period of testicular descent also occurs between the weeks 10 and 15 of gestation. Thus, cryptorchidism may actually begin during early gestation (37). Therefore, we hypothesized that there is an association between PAPP-A levels in the maternal serum and cryptorchidism. An animal experiment conducted by Kim et al. also indicated that the expression of PAPP-A in supporting cells may regulate the development and differentiation of neonatal porcine testicular cells through the insulin-like growth factor (IGF) axis (38).

Since fetal testosterone secretion during the first 14 weeks of pregnancy is influenced by placental hCG (39), placental dysfunction leads to an inadequate supply of hCG and therefore affects testosterone levels; thus, there is a failure of normal maturation and closure of the male fetal urethra (40). The results of the present study showed that levels of FT-free β-hCG in maternal serum from the hypospadias group were lower than those in the normal group, although this difference was not statistically significant. However, Huhtaniemi et al. previously reported significantly higher hCG levels in fetal gonads than in other tissues in fetal organs collected at the early termination of pregnancy (41). In another study, Peycelon et al. found that alterations in the levels of free β-hCG in the maternal serum were associated with proximal hypospadias by comparing free β-hCG MoM measured during early gestational screening for Down’s syndrome (18). This previous study also showed that levels of ST-free β-hCG in the maternal serum were higher in fetuses with hypospadias than in the normal group; these findings were similar to those of our present study. In our previous study, we found that the combination of maternal serum AFP and ST-free β-hCG levels for fetal hypospadias had a higher sensitivity and specificity (24). Based on this previous study, we added PAPP-A and uE3 markers and performed a combined screening protocol for early/mid-trimester pregnancy. We found that the PAPP-A + AFP + uE3 model could better predict the risk of fetal hypospadias, with an AUC of 0.795. In addition, in the present study, we also found that the PAPP-A + uE3 model could also predict the risk of fetal cryptorchidism (AUC: 0.658), with a lower −LR and a higher +LR (27).

In this study, we found that AFP and ST-free β-hCG levels did not differ statistically between the cryptorchidism and the control groups. In contrast, Chedane et al. found that total hCG and hCG MoM levels were significantly lower in a cryptorchidism group than in a control group (42), while AFP and AFP MoM levels were similar when compared between the two groups. Boyd et al. found that boys with maternal serum AFP levels ≥2.5 MoM had a 63% higher risk of cryptorchidism than boys with maternal serum AFP levels within 25% of the median (43); from this, we can infer that the levels of AFP in the maternal serum may reflect placental dysfunction and contribute to development of cryptorchidism, at least in part.

In this study, we also observed that the levels of uE3 in the maternal serum were significantly lower in the cryptorchidism group when compared with the control group. A previous cohort study by Nutman et al. also showed that the levels of uE3 in the maternal serum of fetuses delivered with cryptorchidism were lower than those in the maternal serum of those with newborn males with a normal testicular position at birth (25). However, these authors concluded that levels of uE3 in the maternal serum during pregnancy (15–19 weeks of gestation) could not be used to predict the occurrence of cryptorchidism at birth (25). Another cohort study found that total estradiol was significantly lower in a cryptorchidism group than in a control group (P=0.03), and that uE3 was also significantly lower in the case group composed of white pregnant women (P=0.05) (44). These findings are identical to those of the present study. Our present results suggested that the AUC of uE3 alone for cryptorchidism at birth was 0.648, while the combination of PAPP-A and uE3 showed a better predictive value. Hughes et al. previously suggested that although estrogen did not play a role in normal testicular descent (45), uE3 was the primary estrogen formed during pregnancy, and its levels might reflect the ability of the placenta to form estrogen during fetal testicular development and migration to the scrotum. This previous study was restricted to boys diagnosed with cryptorchidism at the age of 1 year because spontaneous descent of cryptorchidism was relatively rare at that time.

Our study found the association between maternal serum placental biomarkers and congenital hypospadias and cryptorchidism. However, there were also some limitations that need to be considered. First, this study relied on stored samples, and the diagnosis of newborns was based on the observations of a single doctor. Although our diagnosticians are highly experienced and are regularly tested on their theory and skills, there is still the possibility of some bias. Second, our included sample was only representative of the area of Hangzhou, China. Third, our study did not include stratified risk factor analysis for parental factors [body mass index (BMI), smoking, and occupation] and social determinants of related health (income, education, marital status, and race). Another limitation relates to imprecision in the estimates of means. The confidence intervals are very wide and are affected by several factors, such as the confidence level (e.g., 95%, 99%), and the high variability of the data. The use of different statistical methods or formulas to calculate confidence intervals may result in different ranges. Furthermore, the distribution of the data can also affect the range of the confidence intervals, as well as the methods used for sample collection and experimental design. Finally, the severity of hypospadias and cryptorchidism was not graded in this study; therefore, the correlation between relevant markers and the severity of hypospadias and cryptorchidism could not be judged. It is hoped that a larger long-term prospective validation study will be conducted in the future to elucidate the predictive value and mechanisms of the prediction models (27).

Conclusions

The detection of placental biomarker levels in maternal serum has indicative value and relevance for the screening of neonatal hypospadias and cryptorchidism, in which the combined detection of maternal serum PAPP-A, AFP and uE3 could improve the screening effect compared with the combined detection of other single or multiple markers, and provide a scientific basis for the early diagnosis of fetal urological disorders.

Acknowledgments

We are grateful to all of the participants and contributors in this study. We would like to express our gratitude to Jun He from Zhejiang Xiaoshan Hospital, Haiya He from Hangzhou Fuyang District Maternity and Child Health Care Hospital, and Xuelian Chu from Hangzhou Yuhang District Maternity and Child Health Care Hospital, among others, for their hard work in the detection of maternal serum placental biomarkers and for helping to collect the data. We also thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-83/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-83/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-83/prf

Funding: This study was supported by Hangzhou Medicine and Health Science and Technology Plan Project (No. ZD20250268).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-83/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Human Research Ethics Committee of the Hangzhou Women’s Hospital Medical Ethics Committee [No. 2023-A-(014)], and individual consent for this retrospective analysis was waived. The other centers were also informed and approved this study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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