Surgical Management of Vesicoureteral Junction Obstruction in Children: A Comparative Study Between Transvesicoscopic Cohen Reimplantation and Transumbilical Laparoendoscopic Single-Site Lich

Abstract

Background:

There are many reports on the application of minimally invasive technology in correction of children's vesicoureteral junction obstruction (VUJO), but there is no report on the treatment of children's VUJO with the transumbilical laparoendoscopic single-site surgery (TU-LESS) Lich–Gregoir method. We aimed to comparatively analyze the therapeutic outcomes of transvesicoscopic ureteral reimplantation Cohen (TUR-C) procedure and TU-LESS Lich–Gregoir (TU-LESS-LG) procedure in pediatric VUJO.

Materials and Methods:

The data of 49 children with VUJO, admitted from January 2016 to January 2020, were retrospectively analyzed. Based on different surgical methods, they were divided into the TUR-C group (23 cases) and the TU-LESS-LG group (26 cases). Demographic characteristics, perioperative characteristics, postoperative complications, recovery of renal function, and improvement of hydronephrosis were compared between the two groups.

Results:

There were no statistical differences in demographic characteristics and preoperative data between the two groups. The TU-LESS-LG group was superior to the TUR-C group in terms of average operation time and postoperative hospital stay. There was no statistical difference between the two groups in terms of postoperative complications, postoperative recovery of renal function, and improvement of hydronephrosis.

Conclusions:

The two surgical methods can achieve a similar curative effect in the treatment of VUJO. The TU-LESS-LG procedure has more advantages of operation time, postoperative hospital stay, wider age range for selection of cases, megaureter tapering, and cosmetic incision, but the operation is more difficult.

Clinical Trial Registration number: 2021(KY-E-048).

Introduction

Vesicoureteral junction obstruction (VUJO) is a common congenital urinary malformation in children. Ureterovesical reimplantation is necessary when obstructive hydronephrosis, ureteral dilatation, consequence urinary tract infection (UTI), and sequels of renal malfunction occur. The surgical approach for reimplantation can be intravesical or extravesical. Cohen's surgical technique and Lich–Gregoir (LG) operation are the two classic approaches.

With the advancement of minimally invasive surgical techniques, the most commonly reported techniques are transvesicoscopic ureteral reimplantation Cohen (TUR-C) and conventional laparoscopic LG procedures, while the TU-LESS Lich–Gregoir (TU-LESS-LG) method is rarely reported. This retrospective study aimed to comparatively analyze the differences and surgical efficacies between TUC-C and TU-LESS-LG procedures.

Clinical data of 49 children with VUJO from our hospital were retrieved and divided into two groups according to different surgical methods, the TUR-C group and TU-LESS-LG group. A comparative analysis of the differences between the two surgical procedures and surgical efficacy was performed.

Materials and Methods

Study design and data evaluation

The study was approved by the institutional review board. A total of 60 children with ureterovesical junction stenosis, who were treated in our institute from January 2016 to January 2020, were analyzed retrospectively. The inclusion criteria for this study were (1) differential renal function (DFR) that was evaluated with dimercaptosuccinic acid (DMSA) as less than 35% and the presence of hydronephrosis that tended to worsen on ultrasound during follow-up; (2) pain or UTI related to hydronephrosis; and (3) voiding cystourethrography (VCUG) performed to exclude vesicoureteral reflux (VUR). Patients with bilateral VUJO were excluded.

As a result, we excluded four cases of bilateral VUJO, two cases with other diseases and two cases with incomplete data. Forty-nine cases were followed up with complete data and they were divided into two groups according to different surgical methods used to correct VUJO: the TUR-C group (23 cases) and TU-LESS-LG group (26 cases).

Age, sex, weight, preoperative UTI, and pain data were collected. The DFR was measured by DMSA, and the anterior-posterior renal and pelvic diameter (APD) and the maximal ureteral diameter (MUD) were measured by ultrasound before surgery. In addition, the operation time, intraoperative blood loss, switch to open surgery, the number of ureteral tapering attempts, and postoperative hospital stay were recorded.

Postoperative complications included hematuria, bladder spasms, and urinary retention. Hematuria bladder spasms were defined as dark red hematuria generally accompanied by bladder spasms and blood clot formation. Children had obvious pain and discomfort in the bladder area. Postoperative care included intravenous antibiotics and hemostatic drugs, oral tolterodine to relieve bladder spasms, and oral sodium bicarbonate tablets to alkalize urine.

Appropriate infusion was administered to maintain urine volume, the urinary catheter was removed when there was no obvious gross hematuria after surgery, and the indwelling time of the urinary catheter was 5–7 days. If the child complained of pain or had pain manifestations, oral acetaminophen at 5–10 mg/kg/time was given based on age and weight. Discharge criteria were as follows: urination was smooth; there was no discomfort such as fever, urinary pain, and hematuria; and the incision healed completely.

Patients were followed up for 6–12 months as outpatients to record whether there were UTIs or other abnormal manifestations. Urinary ultrasonography was performed 3 months after the operation to measure the APD and MUD. VCUG and DMSA scans were performed 6 months after the operation to check whether there was VUR and change in DFR.

Surgical procedure

Transvesicoscopic ureteral reimplantation Cohen (Fig. 1)

The patient was placed in a flat decubitus position using high hip pads and 80°–90° abduction of both lower extremities. A cystoscope was placed to establish the air bladder through which pressure at ∼8–10 mm Hg was applied until the full bladder reached the infraumbilical level. While applying pressure on the skin above the top of the bladder under cystoscopic observation, a suture needle was passed percutaneously through the full thickness of the bladder, leaving the traction line in place.

FIG. 1.

Schematic diagram of the TUR-C procedure. (a) Microscopic release of the right megaureter; (b) the left ureter was fixed to the opposite side and then tapered; (c) suture of the tapered ureter; and (d) established submucosal tunnel and completed ureteral reimplantation. TUR-C = transvesicoscopic ureteral reimplantation Cohen.

The traction line was followed to leave a 5-mm puncture in the bladder and it was secured with the traction line. A 3-mm puncture channel was created in each of the left and right sides below the level of the viewing cytoscope and a trocar was placed and properly secured. Then, the cystoscope was removed, and the Cohen procedure was started, freeing the diseased ureter to an appropriate length outside the bladder wall so that it could be smoothly dragged over the contralateral ureteral opening. If the ureter with the lesion was thick, it was tapered and shaped.

The bladder mucosa was incised longitudinally above the contralateral ureteral opening, about 0.8–1.0 cm. Scissors or separation forceps were used to establish a tunnel under the bladder mucosa. The ureter was cropped and shaped without tension and directed to the new ureteral opening. Sutures were placed intermittently with the bladder mucosa.

The primary ureteral opening in the muscularis was fixed with the ureter using sutures, and the primary ureteral opening was closed as in those with bilateral ureteral lesions using the same method, with reimplantation. The other ureter was reimplanted into the contralateral primary ureteral opening or more lateral to the opening by routine indwelling of the ureteral stent tube and ureter.

TU-LESS Lich–Gregoir method (Supplementary Video S1 and Fig. 2)

The patient was positioned lying flat with a low and high hip to make a curved incision at the lower edge of the umbilicus. The skin, subcutaneous tissue, rectus abdominis, and peritoneum were incised gradually to enter the abdominal cavity. The incision expansion protector was inserted using disposable gloves. Three fingertips of the glove were cut to establish one 5-mm channel and two 3-mm channels or three 5-mm channels to create pneumoperitoneum with pressure of 8–10 mm Hg.

FIG. 2.

Schematic diagram of the TU-LESS-LG procedure. (a) Handheld single-hole multichannel surgical instruments; (b) microscopic release of the ureter; (c) ureteral tapering was performed outside the abdominal cavity; (d) the muscularis layer of the bladder was incised until the bulge in the bladder mucosa; (e) the tapered ureter was anastomosed to the bladder; (f) the bladder muscularis was closed by suturing and the ureter was embedded; (g) postoperative microscopic appearance; and (h) appearance of the abdominal wall incision. TU-LESS-LG = transumbilical laparoendoscopic single-site surgery Lich–Gregoir.

The dilated ureter was searched at the level of the common iliac artery. The posterior peritoneum was opened, freeing the diseased ureter until the ureterovesical junction. The stump was disconnected and sutured while protecting the vas deferens and round ligament of uterus. The pneumoperitoneum was withdrawn, the diseased ureter was dragged from the umbilical incision, and the ureter was cropped and shaped in vitro, making the ureteral orifice into an everted papilla.

The papillary opening allowed more patency for urine drainage and less chance of anastomotic stricture.¹ The ureteral stent tube was placed and pneumoperitoneum was reestablished by intraperitoneal manipulation. The bladder was appropriately filled with saline using a urinary catheter and lifted by the left and right suspension placement (percutaneously) with needle sutures, and the muscularis layer of the bladder was carefully incised (superiorly) with an electrocoagulation hook at the original ureterovesical junction until a mucosal bulge was seen. The length was five times the caliber of the reimplanted ureter.²

The mucosa was opened into the bladder at the original ureteral opening position. The clipped ureter was intermittently anastomosed to the bladder with a 5-0 absorbable suture . The ureter tightly adhered to the bladder mucosa from the bottom up, the bladder muscularis was closed by suturing, the ureter was embedded, the bladder was refilled again and examined for urine leakage, the posterior peritoneum was closed, and a drainage tube was left in place in the pelvic cavity.

Statistical analysis

SPSS, v20.0, software was used for statistical analysis. The count data are expressed as mean ± standard deviation; a two-sample Student's t-test was used to compare sample means between the two groups, and paired Student's t-test was used to compare sample means within groups.

The comparison of sample rates between the two groups was performed using the chi-square test, differences of DFR, APD, and MUD between the two groups after the operation were compared using the rank-sum test, and p < 0.05 was considered statistically significant.

Results

Preoperative characteristics

In the TUR-C group (n = 23), there were 13 boys and 10 girls, age was 8–58 months, mean age was 17.91 ± 12.16 months, weight was 7.7–21.6 kg, mean weight was 11.43 ± 3.85 kg, nine cases (39.1%) had left VUJO, 14 cases (60.9%) had right VUJO, 14 cases (60.9%) had UTIs and/or pain, and preoperative DFR of <35% was seen in 19 cases (82.6%).

In the TU-LESS-LG group (n = 26), there were 14 boys and 12 girls, age was 6–42 months, the average age was 15.54 ± 10.65 months, weight was 6.4–19.5 kg, the average weight was 10.97 ± 4.11 kg, left VUJO was found in nine cases (39.1%), right VUJO was seen in 14 cases (60.9%), UTI or pain was seen in 14 cases (60.9%), and preoperative DFR of <35% was found in 19 cases (82.6%). There was no statistical difference in the preoperative data between the two groups (see Table 1).

Table 1.

Patients' Demographics and Surgical Outcomes

Patients	TUR-C	TU-LESS-LG
No. of patients	23	26
Mean age, months (mean ± SD)	17.91 ± 12.16	15.54 ± 10.65	p = 0.524
Mean weight, kg (mean ± SD)	11.43 ± 3.85	10.97 ± 4.11	p = 0.696
Left VUJO, n (%)	9 (39.1)	10 (38.5)	p = 0.962
Right VUJO, n (%)	14 (60.9.0)	16 (61.5)	p = 0.962
Indications for surgery
UTIs or/and pain, n (%)	14 (60.9)	15 (57.7)	p = 0.821
Preoperative DFR<35%, n (%)	19 (82.6)	19 (73.1)	p = 0.649
Conversion to open surgery, n (%)	2 (8.7)	0^a (0)	p = 0.077
Mean operation time, minutes (mean ± SD)	136.26 ± 11.30	126.85 ± 12.18	p = 0.008
Hospitalization, days (mean ± SD)	6.87 ± 0.97	6.04 ± 1.28	p = 0.015
No. of ureteral tapering attempts, n (%)	12 (52.2)	18 (69.2)	p = 0.221
Postoperative DFR improvement, n (%)	15 (65.2)	15 (57.7)	p = 0.590
Bladder spasms, n (%)	2 (8.7)	0 (0)	p = 0.077
Postoperative urinary retention, n (%)	0 (0)	2 (7.7)	p = 0.106
Early UTI, n (%)	1 (4.3)	1 (3.8)	p = 1.000
Postoperative VUR, n (%)	1 (4)	2 (7.7)	p = 1.000

An additional 3-mm trocar in the right abdomen was used in one patient.

DRF = differential renal function; SD = standard deviation; TUR-C = transvesicoscopic ureteral reimplantation Cohen; TU-LESS-LG = transumbilical laparoendoscopic single-site surgery Lich–Gregoir; UTI = urinary tract infection; VUJO = vesicoureteral junction obstruction; VUR = vesicoureteral reflux.

Intraoperative variables and complications

In the TUR-C group, two patients were switched to open surgery, one of whom was 8 months old, weighed 7.7 kg, had a small bladder capacity, and the ureter was large and difficult to taper, therefore the patient was switched to open surgery. The other was an obese child, aged 11 months and weighing 14.5 kg; the trocar had slipped during the operation, causing air leakage; and it was difficult to maintain the surgical field of vision, so the patient was switched to open surgery.

The average operation time was 136.26 ± 11.30 minutes and average hospital stay was 6.87 ± 0.97 days. Ureteral tapering was performed in 12 cases (52.2%). There were two cases of postoperative hematuria due to bladder spasms, of which one case had a right VUJO, the MUD before the operation was 23 mm, the ureter was tapered during the operation, the operation time was 172 minutes, and the duration of hematuria was 3 days.

The other case with left VUJO was switched to open surgery and the MUD before the operation was 12 mm. The ureter was tapered during the operation, the operation time was 125 minutes, and hematuria lasted for 4 days. After treatment such as saline bladder irrigation, the hematuria disappeared.

In the TU-LESS-LG group, one obese child was 17 months old and weighed 16.9 kg. Due to difficulty in suturing the ureter to the bladder, an auxiliary hole of 3 mm was created. The average operation time was 126.85 ± 12.18 minutes and average hospital stay was 6.04 ± 1.28 days. Ureteral tapering was performed in 18 cases (69.2%). Urinary retention occurred in two cases after the operation, all of which were left VUJO cases.

One case had an MUD of 18 mm before the operation and underwent ureteral tapering during the operation. The other case had an MUD of 8 mm before the operation and the ureter was not tapered during the operation. The condition improved after recatheterization for one week. In terms of average operation time and postoperative hospital stay, the results of the TU-LESS-LG group were better than the TUR-C group. There were no statistical differences in other observation indicators between the two groups (see Table 1).

Follow-up data

All cases were followed up in the outpatient department for more than 6 months. In the TUR-C group, one case of UTI improved with oral antibiotics. VCUG was performed after the operation, and one case had mild VUR without symptoms. In the TU-LESS-LG group, one case of UTI improved with oral antibiotics, and VCUG was performed after the operation. Two cases had mild VUR and were asymptomatic.

Postoperative urinary ultrasound showed that the APD of the renal pelvis and the MUD of the two groups were better than those before the operation. There was no difference between the two groups. DMSA scans were examined after the operation. DFR improved in 15 cases (65.2%) in the TUR-C group and 15 cases (57.7%) in the TU-LESS-LG group. There was no deterioration of DFR in both groups. The postoperative DFR of the two groups was better than that before the operation. There was no statistical difference between the two groups (see Tables 1 and 2).

Table 2.

Postoperative Follow-up for Each Group

	Group	Preoperative	Postoperative	p
DRF, % (mean ± SD)	TUR-C	24.50 ± 6.18	36.22 ± 5.50	0.275
DRF, % (mean ± SD)	TU-LESS-LG	20.96 ± 5.36	34.53 ± 7.46	0.275
APD, mm (mean ± SD)	TUR-C	16.13 ± 5.18	8.26 ± 3.44	0.532
APD, mm (mean ± SD)	TU-LESS-LG	19.31 ± 4.70	10.70 ± 3.77	0.532
MUD, mm (mean ± SD)	TUR-C	11.78 ± 5.23	9.52 ± 4.20	0.732
MUD, mm (mean ± SD)	TU-LESS-LG	11.58 ± 3.73	9.23 ± 2.89	0.732

APD = anterior–posterior renal and pelvic diameter; MUD = maximal ureteral diameter.

Discussion

The surgical procedure of vesicoureteral reimplantation is complex and its overall therapeutic principle is to relieve ureteral end obstruction and maintain patency of the ureteral opening after reimplantation in the bladder, which has an antireflux effect and does not affect the physiological function of urinary storage and voiding of the bladder.

In the Cohen method for intravesical surgery, the ureter is tapered and shaped in the form of a transverse submucosal tunnel that opens on the contralateral side. The buried ureter has a certain length, good antireflux effect, and is not prone to distortion or deformation. The success rate of the operation is high, but in the traditional open surgery, the abdominal wall incision is large, needs to open the bladder, and there are postoperative complications such as hematuria and bladder spasms.³

In 2005, Yeung et al.⁴ reported the laparoscopic ureterovesical bladder reimplantation technique with gas insufflation of the bladder. The principle of reimplantation was to fill the bladder with carbon dioxide gas to establish a certain degree of operating space for surgery. Since the bladder was not opened, the surgical trauma was less and the method was quickly popularized and developed. Since then, multiple units reported the procedure, and various modified procedures based on the initial method have been used with satisfactory surgical outcomes.^5

–8

The LG procedure is an extravesical approach, in which the bladder is not incised, but entraps the reimplanted ureter at the muscular layer of the outer wall of the bladder, thereby acting as an antireflux structure. In 1994, Ehrlich et al.⁹ reported on two patients with VUR treated using the laparoscopic LG method, but this technique was not adopted and recognized by surgeons until 2004.¹⁰

With the advancement and development of surgical technology, endoscopic surgery is less traumatic compared with open surgery: the surgical field is clear and magnified and the surgical effect is comparable with that of open surgery, with the advantage of a more esthetic abdominal wall.^10,11

Some scholars reported that transumbilical single-hole laparoscopic ureteral reimplantation was used to treat various adult ureteral pathologies with satisfactory results.^12,13 However, no formal case report has been found on the use of TU-LESS in pediatric ureterovesical reimplantation.

Comparison of indicators and technical characteristics of the two surgery approaches

The Cohen procedure is a classic intravesical approach and the surgical operation is performed on the bladder. Therefore, the TUR-C procedure has certain conditions for selection of cases. The child needs to have a certain bladder capacity; the larger the bladder capacity, the more convenient the surgical operation, and the smaller the bladder capacity, the more difficult the operation and the more prone to complications. It has been reported in the literature that TUR-C is easy to perform when the child's age is 14 months and bladder capacity is above 130 mL, which reduce the incidence of complications.¹⁴

In the TUR-C group in this study, two patients who were switched to open surgery were less than 12 months old. The reason for the transition to open surgery was that the bladder capacity was too small, which was not conducive to the tapering of the megaureter and leakage of the puncture channel. In addition, it was difficult to establish a gas bladder. The LG operation is performed outside the bladder without opening the bladder, the method has little dependence on bladder volume, and it is suitable for cases of ectopic ureteral opening.

In this study, in the TU-LESS-LG group, the youngest child was 6 months old and weighed 6.4 kg, and the operation outcome was good. For children over 3 years old, due to the long distance of laparoscopic operation, 5-mm instruments need to be used. The single hole diameter is small, and the operation interference is obvious, especially when suturing and knotting.

Compared with VUR, patients with ureteral end obstruction are prone to complications with the megaureter, and the probability of tapering is higher. In this study, ureteral tapering was performed in 12 cases (52.2%) in the TUR-C group and 18 cases (69.2%) in the TU-LESS-LG group. When tapering the ureter, the TU-LESS-LG procedure can drag the ureter out of the umbilical puncture hole for accurate cutting,^15,16 while the TUR-C procedure can only taper the ureter under the microscope, which is more difficult and time-consuming.

In this study, the average operation time in the TU-LESS-LG group was better than that of the TUR-C group. The most time-consuming part in the former was the anastomosis of the ureter and bladder and closure and embedding of the ureter by suture of the bladder muscle layer, while in the latter, it was establishment of the bladder puncture channel and tapering of the ureter.

Regarding surgical operation, both minimally invasive surgical methods require fine and stable operation under endoscopy. The disadvantages of the TUR-C method are that establishment of the puncture channel is cumbersome; the trocar can easily prolapse, causing air leakage; the pressure cannot be maintained; and the operation in the bladder is limited with restricted operation space. The advantages are that there is a triangular operating plane on the observation mirror, operating forceps conform to the operating habits of conventional laparoscopy surgeons, and the learning curve is relatively short.

The disadvantages of the TU-LESS-LG method are that the light source and instruments are coaxial, the mutual interference is large, the operation range is small, the surgeon needs to be familiar with the reverse operation, and the learning curve is relatively long. The advantage is that establishment of pneumoperitoneum is simple and stable. The differences in indications and technical details of the two procedures are shown in Table 3.

Table 3.

Comparison of Indications and Techniques of Two Surgical Methods

	TUR-C	TU-LESS-LG
Age	≥12 Months	≥6 Months
Diseases	VUJO, VUR	VUJO, VUR, ectopic ureter
Establishment of surgical puncture channel	Difficulty, slippage of the trocar, and air leakage	Easy and stable
Surgical visual field	Clear and stable	Susceptible to interference
Megaureter tapering	Difficulty	Easy
Difficulty of laparoscopic operation	Relatively easy, there is a triangular operation plane	Relatively difficult, the “single hole” effect, and the instruments interfere with each other

Comparison of the safety and efficacy of surgery between the two groups

In this study, the two groups of patients were able to achieve good results after surgery and there was no case of aggravated DFR damage. Comparing the improvement rate of renal function and the improvement rate of hydronephrosis between the two groups, there was no difference in the curative effect between the two groups. The mean operative time and postoperative hospital stay in the TU-LESS-LG group were better than those in the TUR-C group.

In terms of surgical complications, bladder spasms occurred in two cases (8.7%) in the TUR-C group with the main manifestations of postoperative bladder pain and continuous hematuria. It was considered that the incision of the bladder mucosa and establishment of a submucosal tunnel may have caused bladder spasms to a certain extent, but this phenomenon did not occur in the TU-LESS-LG group.

Postoperative urinary retention occurred in two cases (7.7%) in the TU-LESS-LG group, which was eliminated after 1–2 weeks of catheterization. The LG procedures have been reported in the literature to have the risk of damage to the detrusor nerve branches of the bladder from the pelvic floor plexus^17,18; however, no urinary retention occurred in the TUR-C group.

Other complications, such as postoperative UTI and VUR, showed no difference between the two groups. In terms of the cosmetic effect of the incision, the incision with the TU-LESS-LG method was concealed in the umbilicus, which was better than the TUR-C method.

Conclusions

In conclusion, the two types of minimally invasive surgery methods for treatment of VUJO had similar surgical outcomes, and both operations had certain operational difficulties. In terms of selection of cases, TU-LESS-LG has a wider range of options, is less affected by bladder capacity, and is more conducive to the tapering of the giant ureter. The surgical incision is hidden, but the method needs to overcome the single-port operation limitation and long learning curve.

The number of cases in this study was still small, and there may have been bias in evaluation of perioperative and long-term complication rates and renal function recovery, which needs to be improved by increasing the number of cases at a later date with extension of the follow-up period.

Footnotes

Authors' Contributions

Study conception and design were performed by Y.L. and J.C. Material preparation and data collection and analysis were performed by W.L., H.D.., P.C., C.S., C.W., Y.L., and Y.L. The first draft of the manuscript was written by W.L. All authors read and approved the final manuscript.

Ethical Approval

This project fully considered and protected the rights and interests of the study subjects. It meets the criteria of the Ethics Review Committee. The Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University has approved the protocol.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was financed by grants from the Science and Technology Project of Guangxi Health Committee (No. S2019104).

Supplementary Material

Supplementary Video S1

Abbreviations Used

References

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Supplementary Material

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Surgical Management of Vesicoureteral Junction Obstruction in Children: A Comparative Study Between Transvesicoscopic Cohen Reimplantation and Transumbilical Laparoendoscopic Single-Site Lich–Gregoir Techniques

Abstract

Background:

Materials and Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Study design and data evaluation

Surgical procedure

Transvesicoscopic ureteral reimplantation Cohen (Fig. 1)

TU-LESS Lich–Gregoir method (Supplementary Video S1 and Fig. 2)

Statistical analysis

Results

Preoperative characteristics

Intraoperative variables and complications

Follow-up data

Discussion

Comparison of indicators and technical characteristics of the two surgery approaches

Comparison of the safety and efficacy of surgery between the two groups

Conclusions

Footnotes

Authors' Contributions

Ethical Approval

Author Disclosure Statement

Funding Information

Supplementary Material

Abbreviations Used

References

Supplementary Material