Introduction
Chronic pelvic pain (CPP) is one of the most prominent manifestations of pelvic venous disease (PeVD) [1]. V. Hansrani et al. [2] in their work demonstrated association between CPP and pelvic varicose veins in women. 10% of all women worldwide are diagnosed as having dilated pelvic veins and reflux therein, with 60% of these found to develop pelvic congestion syndrome (PCS) accompanied by CPP [3-5]. The causes of CPP in patients with PeVD are still poorly studied. In some patients, dilation of pelvic veins is an incidental finding during examination and is not accompanied by any clinical manifestations [6, 7], whereas others on the background of analogous morphological changes of pelvic veins are found to develop pelvic congestion syndrome (PCS) accompanied by CPP [6, 8]. It was determined that the degree of pelvic vein dilation does not influence the occurrence of PCS [9, 10]. A series of studies demonstrated the effect of reflux in pelvic varicose veins on the development of clinical manifestations of PCS [11, 12]. The results of several studies suggest possible association between neurogenic inflammation, hyperproduction of vasoactive neuropeptides and formation of CPP in patients with PeVD [13-15]. Stones R.W et al. revealed increased content of calcitonin-gene-related peptide (CGRP) and substance P (SP) in the wall of gonadal veins and in ovaries in patients with PCS [16, 17].
Objective. The study was undertaken to investigate the relationship between pelvic venous reflux, CGRP and SP levels in blood plasma and the presence of CPP in patients with PeVD using ultrasonographic, radionuclide and immunoenzymatic methods of examination.
Patients and methods
Our prospective non-randomized cohort study enrolled one hundred and ten 21-to-42-year-old women examined at the University Surgical Clinic of the Pirogov Russian National Research Medical University between 2019 and 2022. It was approved by the Local Ethics Committee (protocol N 206) and registered at clinicaltrials.gov (NCT03921788). All participants (of the study) were familiarized with the aim and methods of the study and signed the informed consent.
The inclusion criteria were as follows: reproductive age of patients, presence of pelvic varicose veins with reflux confirmed on duplex ultrasound scanning (DUS) and no concomitant diseases accompanied by chronic pain syndrome, The exclusion criteria were menopause, pregnancy, presence of nutcracker syndrome, May-Thurner syndrome, post-thrombotic disease and oncological diseases, absence of reflux in pelvic veins according to DUS, virgo intacta, thus ruling out the performance of transvaginal DUS.
Besides patients with PeVD, the study included twenty 21-to-26-year-old female volunteers with no clinical manifestations of disease of the pelvic veins or lower limbs chronic pain syndrome, dilation and reflux in pelvic and lower-limb veins according to the data of DUS. Inclusion of volunteers into the study was necessary to determine the reference values of CGRP and SP in apparently healthy people used in this study as the norm.
Study design
The clinical and ultrasonographic examination was performed in 387 women suspected as having PeVD. Of these, 243 patients complained of CPP and 154 were referred to our clinic from other medical facilities due to DUS-revealed pelvic vein dilation with no complaints. All patients were examined by the gynecologist, urologist and neuropathologist to exclude/confirm their pathology, with additional studies performed if necessary.
According to the inclusion criteria, the study enrolled a total of 110 patients with PeDV. Of these, 70 patients diagnosed as having symptomatic form of PeVD characterized by CPP, dyspareunia, dysuric disorders, vulvar varicosis were assigned to Group 1 (symptomatic) and 40 patients with no clinical manifestations of PeDV to Group 2 (asymptomatic). Physical examination and DUS of pelvic and lower-limb veins in order to assess pelvic congestion syndrome was followed by emission computed tomography of pelvic veins with in vivo labeled red blood cells. Group III (n=20) was composed of healthy volunteers undergoing only clinical examination and DUS, with no symptoms and sings of pelvic or lower-limb vein disease, or pelvic vein dilatation with reflux therein revealed. In order to determine the levels of vasoactive neuropeptides (CGRP, SP) in blood plasma, all patients underwent enzyme immunoassay. The flowchart of the study is shown in Fig. 1.
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Duplex ultrasound scanning
DUS of lower-limb and pelvic veins was performed using the unit Esaote MyLab Class C (Esaote, Italy). The study protocol included sequential assessment of the condition of superficial and deep veins of lower limbs, perineal veins, inferior vena cava, renal iliac and pelvic (parametrial, uterine, gonadal) veins. During DUS, we used the Valsalva maneuver and compression tests in order to detect blood reflux in pelvic, perineal and lower-limb veins. A decrease in the diameter of pelvic veins (gonadal, parametrial, uterine) by more than 5 mm was regarded as their varicose dilatation [18]. Reflux in pelvic and deep veins of lower limbs was regarded as pathological if its duration exceeded 1 s [18, 19]. Reflux in lower-limb superficial veins was considered pathological if lasing more than 0.5 s [20].
DUS of lower limb and perineal veins
The study was performed using a 7.5-13 MHz transducer according to the standard technique, with the patient in the supine and standing position, assessing patency, diameter of veins, the presence of pathological reflux in the saphenofemoral and saphenopopliteal junctions, trunks of the great saphenous (GSV) and small saphenous (SSV) veins, veins of the labia majora (vulvar veins), determining the presence/absence of retrograde blood flow through ostial tributaries of the GSV.
Transabdominal DUS of iliac, inferior saphenous, renal and gonadal veins
The study was performed by means of convex (3-5 MHz) and linear (3-18 MHz) transducers with the patient in the supine and semi-sitting (45°) position, assessing patency and diameters of external, common, internal iliac, inferior vena cava and renal veins, measuring blood flow velocity in the iliac and left renal veins, assessing the presence/absence of reflux in internal iliac (IIV) and gonadal veins, measuring the reflux duration if present. May-Thurner syndrome was diagnosed we assessed the anterior-posterior size of the left common iliac vein (CIV) in its distal portion immediately above the ostium of the iliac vein, as well as registering linear blood flow velocity in the regions of interest. The values of the gradient between the proximal and distal portions of the common iliac vein exceeding 2.5 were considered as a sign of hemodynamically significant stenosis of the left CIV [21].
We studied the state of the left renal vein (LCV) using transversal and longitudinal scanning of the vessel, measuring its diameters in projection of its compression of the superior mesenteric artery (Dstenosis), in the area of the renal hilum (Dhilum), determining the peak flow velocity in the LRV in the compression zone (PFVstenosis) and in the area of the hilum renalis (PFVhilum). Increased blood velocity more than 100 cm/s in the area of narrowing of the LRV (at the level of SMA) and more than 12 cm/s in the area of hilum renalis, increased ratios of PFVstenosis/PFVhilum >4 and Dstenosis/Dhilum >5 were regarded as ultrasonographic criteria for hemodynamically significant stenosis of the LRV, the presence of nutcracker syndrome [22, 23].
Transvaginal DUS of pelvic veins
It was the main method of determining pelvic venous reflux (PVR). The study was carried out with the patient in the supine, semi-sitting (torso angle 45 °) and semi-standing position using endovaginal microconvex transducers with 3-9 MHz and 3.5-10 MHz frequency [11, 19], assessing patency and diameters of gonadal veins (GV), parametrial veins (PV), uterine veins (UV), IIV, the presence and duration of reflux in the mentioned vessels. IIV tributaries and retrograde blood flow through them were not studied.
PVR was assessed in accordance with the previously worked out classification [11]. Pelvic vein reflux up to 1 s was regarded as a physiological phenomenon. Type I PVR (mild) was characterized by the duration of 1-2 s, type II PVR (moderate) - 2.1-5 s, and type III PVR (severe) - >5 s of spontaneous reflux with no loading tests.
SPECT of pelvic veins with in vivo labeled RBCs
This study was performed in order to determine the degree of pelvic congestion syndrome (PCS), using the gamma-chamber Discovery NM/CT 670 (GE, USA). The Perfotech solution for the in vivo labeling of RBCs was injected in the cubital vein in a dose of 2 mL, followed by 99mTc-pertechnetate injection 20 min later. SPECT was performed at 20 min post-injection using a 360° circular orbit rotation of gamma detector for obtaining images of the distribution of labeled RBCs in the pelvic veins. In a healthy individual, no or only negligible accumulation of labeled RBCs in pelvic venous plexuses is identified. Venous congestion syndrome (PCS) was characterized by deposition of labeled RBCs in parametrial and uterine veins, visualization of GVs (more often left). Activity of labeled RBCs in pelvic veins was registered by means of computer equipment in pulses per second (PPS). Pelvic venous congestion (PVC) of the uterine and parametrium was objectively assessed calculating the pelvic venous congestion coefficient (CPVC) as the ratio of pulses registered from the region “parametrial veins” to the pulses calculated in the region “common iliac vein”, since the activity of labeled RBCs in the common iliac vein is most stable. The activity of erythrocyte-phosphate-pertechnetate complexes in pelvic veins depends on the degree of their dilation, presence of reflux and, consequently, the degree of blood accumulation therein. In health, the ratio of PPS in parametrial veins to the PPS in the common iliac artery does not exceed 0.5. Since dilation of pelvic veins and development of reflux therein are followed by accumulation of labeled RBCs, the values of CPVC also increase. An increase of CPVC to 1 was regarded as grade I PVC, from 1.1 to 1.5 as grade II, > 1.5 - grade III [24]. Grade I corresponded to mild PVC, grade II - to moderate, and grade III - to severe PVC.
Effective dose of radiation per patient during the study amounted to 2.1 mSv.
Enzyme immunoassay
Venous blood was procured from the cubital vein at the same time, 8.00-8.30 a.m., on an empty stomach, in a sitting position, 7 days after the last menstruation into 4.0-ml vacuum tubes (vacutainers) containing K2-EDTA. Blood sampling was followed by centrifugation for 10 minutes at 3000 rpm. The obtained blood plasma was aliquoted by 1.0 ml in 2 Eppendorf tubes. The biological material was immediately frozen and stored at -80 °C for further analysis. Blood plasma CGRP and SP was determined by means of the competitive enzyme-linked immunosorbent assay (ELISA), using commercial kits Peninsula Laboratories, LLC of the BachemGroup (USA). Reference standards and specimens were analyzed in duplicates, using the manufacturer recommended protocol № 5 with incubation at 4° С for 14-16 hours (overnight). Optic density was measured on immunoenzymatic analyzer Stat Fax 2100 (microplate photometer Awareness Technology Inc., USA) in standard 96-well plates at wavelength of 450 nm. The concentration of neuropeptides was calculated using the computer program Cobas EIA Recalibration Software (F. Hoffmann - La Rochet Ltd. Switzerland).
Assessment of results
We evaluated the presence and degree of CPP, duration of PVR and its incidence in pelvic veins, degree of deposition of labeled RBCs in pelvic veins (CPVC) and blood plasma CGRP and SP in patients and volunteers, followed by analysis of interrelations of these parameters.
Statistical methods
The statistical analysis was performed using the Microsoft Excel (Microsoft Corp, USA) Statistical 10 (StatSoft, TIBCO, USA) and on-line calculator VassarStats (open Internet resource: http://vassarstats.net/). The results are presented as quantitative and categorical variables. For qualitative variables we used the Mann-Whitney test, for categorical variables - the Chi-square test. We calculated the mean and standard deviation (M±SD), odds ratio with 95% confidence interval (CD), Student’s t criterion. Results were regarded as statistically significant if p<0.05. To determine correlations between different parameters we calculated the Pearson’s criterion. Assessment of interrelation of various factors with probability of CPP development was performed with the help of logistic regression, with its results presented as odds ratio with a 95% CI and p-values for the Wald test.
Results
Clinical data
Symptomatic PeVD (n=70) was characterized by CPP (100%), dyspareunia (84.3%), heaviness in the epigastric region (70%), vulvar varicosis (24%), dysuric disorders (20%). Amongst patients with asymptomatic PeVD (n=40) we observed a statistically significant increase in the body mass index (BMI) compared with symptomatic patients (25.3±1.2 kg/m2 vs 21.3±2.2 kg/m2, respectively, p=0.004) and increased frequency of detecting CEAP class 3 CVD (57.5 % vs 25%, respectively, p=0.01). The characteristics of the patients and volunteers are shown in Table 1.
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There were no differences in age, number of pregnancies and deliveries, accompanying pathology between group 1 and 2 patients. The volunteers had no symptoms of PeDV, accompanying pathology, pregnancy and labor, thus suggesting they had no factors influencing the production of CGRP and PS.
Ultrasonographic data
Frequency of detecting reflux in GVs in symptomatic patients amounted to 45.7%, in asymptomatic to 10%, with no GV refluxes in volunteers revealed (Table 2). Duration of reflux in the GV in symptomatic and asymptomatic patients amounted to 4.1±1.7 and 1.4±0.3 s, respectively (p=0.002). No statistically significant differences in diameters of GVs in patients with symptomatic and asymptomatic PeVD were revealed. Similar data were obtained when comparing the duration of reflux in PVs and their diameters for symptomatic and asymptomatic patients. The UV diameters did not statistically differ in Group 1 and 2 patients and volunteers, with reflux of these veins detected in 24.3% of symptomatic patients. No UV reflex in symptomatic patients and volunteers was revealed.
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Duration of PVR turned out to be longer in symptomatic patients (Table 3). Type II and III PVR in GV was found in 41.4% of symptomatic patients and in only 5% of patients with asymptomatic PVR (Fig. 2). Type II and III PVR was registered in 80% of symptomatic patients and in 10% asymptomatic patients. PVR in UVs was more often in patients with type I PVR (12.9%), type II less frequently (10%) and type III (1.4%) in symptomatic patients.
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DUS results suggested a larger number of pelvic veins involved into pathological process (i. e., having reflux) and prevailing of prolonged (more than 2 s) PVR in symptomatic patients. On the contrary, the overwhelming majority of asymptomatic patients had type I reflux only in the PV. In the volunteers, dilation of pelvic veins and reflux therein was not registered.
Radionuclide data
100% of patients with symptomatic and asymptomatic PeVD demonstrated the presence of deposition of labeled RBCs in parametrial and uterine veins but its degree was different (Table 4).
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95.7% of symptomatic patients were found to have grade II and III PVC, with grade II PVC and grade I PVC revealed in 15% and 85% of asymptomatic patients, respectively. We noted significant difference of CPVC in patients with symptomatic and asymptomatic PeVD (1.9±04 vs 0.7±0.2, respectively, p=0.008). Visualisation of GV was noted in a third of patients. In asymptomatic patients, GVs were not visualized (Fig. 3).
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Findings of enzyme immunoassay
Concentration of vasoactive neuropeptides of blood plasma in volunteers was regarded as the normal parameter, proceeding from the fact that they had no conditions accompanied by pain syndrome or inflammation. The CGSP and SP levels in symptomatic patients significantly exceeded those in asymptomatic patients (CGSP - 0.48±0.06 ng/ml vs 0.19±0.02 ng/ml, p=0.001; SP - 0.38±0.08 ng/ml vs 0.13±0.03, respectively, p=0.001) (Table 5).
It was noted that in asymptomatic patients, the concentration of vasoactive neuropeptides was also higher than in volunteers, despite the absence of pain syndrome (CGRP - 0.19±0.02 ng/ml vs 0.06±0.003 ng/ml, p=003; SP - 0.13±0.03 ng/ml vs 0.03±0.002 ng/ml, respectively, p=0.03).
The multivariate regression analysis demonstrated that significant factors for the development of CPP in patients with PeVD were as follows: reflux in pelvic veins for more than 2 s (i. e., type II and III PVR) determined in 2 and more venous collectors of the pelvis, the coefficient of pelvic vein congestion, and increased concentration of CGRP and SP (Table 6).
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Discussion
CPP complicates the course of PeVD in 59-75% of patients [3, 6, 11]. The real clinical practice and research results suggest that the degree of pelvic vein dilation was not associated with clinical manifestations of PeVD. Many studies have demonstrated that in patients with PeVD it is namely reflux in pelvic veins rather than their diameter that is accompanied by the development of CPP [9-12]. Along with it, pelvic venous reflux is revealed during DUS in all patients with PeVD, but the degree of CPP varies significantly and in part of patients is completely absent.
The literature contains sporadic studies devoted to examining causes of CPP in patients with PeVD, with the data presented therein incapable of fully explaining the mechanisms of formation of CPP. It is known that varicose dilation of veins and reflux therein are accompanied by the development of vein-specific inflammation in which hypoxia of venous wall occurs [25]. Hypoxic state of tissues is accompanied by increased synthesis of cytokines, neurokines and vasoactive neuropeptides [24]. The latter, particularly CGRP, serve as the key factor in pathogenesis of migraine, and blockers of this protein have successfully been used for treatment of this pathology [26, 27].
CGRP and SP are regarded by many authors as neurotransmitters-algogens preconditioning the formation of pain syndrome in various diseases. Intravenous administration of CGRP is accompanied by increased CPP in patients with PeVD but does not influence healthy volunteers [21]. According to R.W. Stones [13], the development of pelvic pain in patients with PeVD is caused not so much by dilation of pelvic veins as by formation of neurogenic inflammation. The author compared migraine in which headache appears on the background of vascular disorders, with the development of pain syndrome in impairment of venous outflow from pelvic organs. The role of CGRP in the development of neurogenic inflammation was reported by L.Y. Qiao et al., X.Q. Pan et al., Z. Kee et al., according to whose opinion this type of inflammation is accompanied by edema, vasodilation and destruction of mast cells, and elevation of CGRP expression results in the development of pain syndrome [15, 28-30]. R.W. Stones et al. [1] supposed that impairment of blood outflow from pelvic organs in women with PeVD leads to increased synthesis of SP and that hypersensitivity of receptors to this neuropeptide predisposes to formation of CPP.
The mentioned data served an incentive for studying interconnections between CPP, reflux in pelvic veins and blood plasma CGRP and SP in patients with PeVD. Our previous studies showed that treatment using modern venoactive drugs in a series of patients with PeVD did not exert significant influence on CPP [31].
Our study demonstrated that the duration of PVR and its frequency in pelvic veins predetermined formation of CPP. It was found that the duration of CPP longer than 2 s was characteristic of patients with PeVD and CPP, and shorter reflux was not accompanied by any clinical manifestations. Involvement into the pathological process of all pelvic veins (gonadal, parametrial, uterine) is a significant factor for the development of CPP in patients with PeVD, Chances of formation of CPP in patients with diffused lesions of pelvic veins exceed those in patients with isolated dilation of only PVs 7.57-fold (95% CI 2.43-23.58). Along with it, 10% of patients with asymptomatic course of PeVD had a combination of reflux of GV and PV with the duration longer than 2 s, but despite this, CPP and other symptoms of PeVD were absent, which, probably was associated with neurobiological causes.
SPECT of pelvic veins demonstrated pronounced (grade II and III PVC) deposition of labeled RBCs in parametrial and uterine varicose veins of in 95.7% of patients with symptomatic PeVD and in only 15% of patients with an asymptomatic form of the disease (p=0.001, r=0.89, very strong correlation).
The obtained data strongly suggest that long PVR combined with involvement into the pathological process of several pelvic vein collectors results in deposition of blood in pelvic varicose veins and formation of CPP. These findings may be considered as hemodynamic factors for the development of chronic pain syndrome in patients with PeVD.
Taking into consideration that a small number of patients with asymptomatic course of PeVD were found to have multimodal lesions of pelvic veins with reflux of more than 2 s, to explain the formation of pain syndrome by only hemodynamic factors seemed insufficient.
We studied the content of CGRP and SP in patients with PeVD and healthy volunteers. The values of CGRP and SP found in volunteers were used as the norm. It was determined that in patients with PeVD and CPP, the levels of CGRP and SP significantly exceeded the normal parameters (CGRP - 0.19±0.02 ng/ml vs 0.06±0.003 ng/ml, р=0.03; SР - 0.13±0.03 ng/ml vs 0.03±0.002ng/ml, respectively, р=0.03). This made it possible to suppose that dilation of pelvic veins with blood reflux therein resulted in production of CGRP and SP, thus indirectly confirming the theory of venospecific inflammation in varicose vein dilation [32]. Probably, the concentration of these neuropeptides in blood plasma exceeding 0.2 mg/ml is accompanied by the development of CPP in patients with PeVD. Hence, increased levels of CGRP and SP may be regarded as a neurobiological factor of formation of CPP in patients with PeVD.
The regression analysis results confirmed the proposed suggestions. The chances for developing CPP in patients with PeVD and TVR of more than 2 s amounted to 6.36 (95% CI 2.04-1984) in patients with reflux in the GV and 31.5 (95% CI 9.74-101.79) in patients with reflux in PV. Pronounced deposition of blood in pelvic veins (grade II and III) according to SPECT findings was accompanied by a 126.55 - fold increase in chances for developing CPP in patients with PeVD (95% CI 29.80-537.406).
Limitations. This study was based on the results of examining a small number of patients, which was conditioned by strict inclusion/exclusion criteria. Blood to determine the content of CGRP and SP was sampled from the peripheral, cubital vein. It is most likely that in case of blood sampling for EIA immediately from the gonadal vein, the levels of the studied neuropeptides would have been higher. In this study, we assessed concentrations of only CGRP and SP, not taking into consideration possible changes in activity of cytokines and neuropeptides, thus warranting further research of neurobiological factors of the development of pain syndrome in patients with PeVD.
Conclusion
The underlying the occurrence of CPP in patients with PeVD is reflux in pelvic veins. This hemodynamic factor becomes a trigger for the development of a cascade of molecular and cellular processes leading to overproduction of CGRP and SP and formation of pain syndrome in PeVD. Reflux in pelvic veins longer than 2 s, involvement into the pathological process of more than 1 pelvic venous collector in combination with increased blood plasma CGRP and SP may be regarded as hemodynamic and neurobiological factors for the development of CPP in patients with PeVD.