Quantity of Relaxin Receptors in the Ligament of Head of Femur in Infants with Developmental Hip Dysplasia

Background Developmental dysplasia of hip (DDH) etiology is still a controversial issue. While many factors involved in the etiology, one of which is the hormone relaxin. Relaxin concentrations in patients with DDH may lead to pathodynamic changes during hip development by altering the physiological nature of the ligament, as well as by long-term exposure to relaxin during pregnancy. Our objective in this study was to determine the quantity of relaxin receptors in the ligamentum teres and their part in causing DDH. Methods We identied infants between birth and 3 years of age who had undergone open reduction for DDH between 2010 and 2012. 12 hips of 12 miss abortus fetus between 20 to 35 weeks of gestation were used as control group. Specimens obtained from two groups were stained with Relaxin-2 antibody and the amount of staining for relaxin receptors was determined using an ordinal H score.


Introduction
The incidence of developmental dysplasia of the hip (DDH) is 2.7 to 17 per 1000 births worldwide [1].
Hormonal changes, as well as genetic diseases such as Ehler Danlos syndrome, may be involved in DDH associated with joint laxity [2]. Relaxin, a peptide hormone, is one of most common hormones associated with DDH [3]. Relaxin has collagenolytic effects on ligamentous tissue in both pregnant and non-pregnant women. It may also be associated with laxity of fetal ligaments [4].
Two hypotheses address the importance of relaxin in joint laxity. One hypothesis is that it exerts a direct effect on fetal ligament laxity by mediating progesterone and estrogen concentrations in connective tissue [5]. Relaxin also stimulates collagen turnover by increasing the collagenase expression and downregulates collagen synthesis [6]. The second hypothesis is that it exerts an indirect effect that reduces relaxin concentrations in maternal blood. This reduction can impair relaxation of the pelvic ligaments and thus lead to malpositioning the fetus [7]. However, relaxin's effect on joints as mediated through weakened ligaments remains unclear. Although prolonged exposure to relaxin during pregnancy may increase joint laxity during late pregnancy, the acute effect of short-term exposure on ligaments has not been determined [8].
The ligament of head of femur is a cone-shaped ligament extending between the femoral head and the acetabulum. It is a collagenous structure more robust than the capsule in fetuses and may be important in stabilizing the fetal hip joint [9]. The ligament may prevent or slow the progression of an unstable hip to a dislocated hip, particularly at term, when the discrepancy between the size of the femoral head and socket depth is greatest [10]. In fact, Weinstein and Ponseti reported that the ligamentum teres in patients with DDH is longer and thicker than normal, as a result of laxity and the abnormal position of the femoral head [11].
We believe that higher relaxin concentrations in patients with DDH may lead to pathodynamic changes during hip development by altering the physiological nature of the ligament, as well as by long-term exposure to relaxin during pregnancy. We also believe that the number of relaxin receptors is an important clue in explaining the female:male ratio and clinical prognosis of early dysplastic hips. Accordingly, our objective in this study was to determine the quantity of relaxin receptors in the ligamentum teres and their part in causing DDH.

Patients And Methods
This prospective study was approved by the institutional review board of authors' a liated institutions.
Parents and guardians provided written informed consent to have their children enroll in the study and to have samples taken for analysis We identi ed infants between birth and 3 years of age who had undergone open reduction for DDH between 2010 and 2012. 12 hips of 12 miss abortus fetus between 20 to 35 weeks of gestation were used as control group. Infants with teratologic hip dislocations, any accompanying syndrome, or a history of intra-articular intervention of the hip, such as hip surgery or arthrography, were excluded.

Immunohistochemical Analysis
The ligamentum teres ligaments were harvested from infants who had undergone open reduction through a medial or anterolateral incision, xed in 10% formaldehyde for 24 hours, and embedded in para n blocks. Sections, 3-µm thick, were stained with hematoxylin & eosin. Sections were mounted on lysinecoated slides and depara nized for immunohistochemical analysis (Fig. 1).
Antigen retrieval was performed in a citrate buffer solution, and relaxin-2 antibody (GPR106 antibody, Biorbyt, US) was used at 1:50 dilution. Placental tissue was used as a positive control (Fig. 2). We stained the nucleus of cells with relaxin antibody. Preparations were analyzed under light microscopy at a magni cation of 400⊆ at the most intense staining area. The intensity of staining for relaxin was categorized as low, medium, and high, as indicated in Fig. 3. The proportion of cells in each staining category was determined from 100 cells in the densest area of staining by a single pathologist with experience in immunologic staining. Pathologist was blinded for cases and control groups.
The amount of staining for relaxin receptors was determined using an ordinal H score [12]: H score = 3x(percent of cells stained at high intensity ) + 2x(percent of cells stained at medium intensity) + 1x(percentage of cells stained at mild intensity).
The H scores ranges from zero (no relaxin receptors) to 300 (all cells are stained with relaxin at high intensity). They are signi cantly correlated with the PCR data and are also sensitive to small changes in receptor expression [13].

Statistical Methods
The H scores were compared between groups with two-tailed Student's t-test. Alpha was set at 0.05. Data were analyzed with the SPSS software package.

Results
We analyzed data from 26 infants (20 girls) who underwent open reduction for DDH and 12 hips of 12 fetus (9 female) used as control group. The 26 infants ranged in age from 10 to 36 months (mean [SD] age, 22 [6] months. The mean (SD) H scores of infants with DDH were signi cantly higher than those of controls: 215 (59) vs. 52 (48); P = 0.00; 95% CI, (Table 1). Statistically signi cant difference between the two groups in terms of gender was not found.

Discussion
The cause of developmental dysplasia of the hip is still debated, although ligamentous laxity is a contributory factor [1]. Few studies have found evidence for a hormonal effect on DDH. Only a few studies have found an association between DDH and serum or cord blood relaxin concentrations, and many others have found no association. However, our study appears to be the only one that measured relaxin receptors in the ligament of head of femur [14].
Relaxin acts on the extracellular matrix through metalloproteinases by enhancing collagen degredation [6]. Relaxin is associated with both local and generalized laxity [15]. In patients with laxity-associated arthrosis of the rst carpometacarpal joint, the concentration of relaxin inhibitors was higher in the volar oblique and anterior oblique ligaments, indicating that relaxin was involved as a cause of laxity [16,17].
Another study in healthy humans found that trapezial-metacarpal joint laxity was proportional to serum relaxin concentrations [15]. A study in rats found that recombinant human relaxin 30 ng/mL weakened the joint capsule and decreased collagen accumulation in a dose-dependent way [18].
In women athletes, the "monthly window of potential injury" has been associated with hormonal changes, as de ned by Möller-Nielsen [19]. This window coincided with peak concentrations of relaxin during the menstrual cycle, and generalized joint laxity was increased during these peak times [19]. Relaxin also increased ligamentous laxity during pregnancy [20]. Relaxin receptors are up-regulated by estrogen therapy, and joint laxity occurring after the rst trimester of pregnancy is synchronous with peak relaxin contrations [20].
In one of two separate studies by Dragoo, tibial translation in guinea pigs was increased by 12.8% (absolute increase, 1.09 mm) 21 days after relaxin administration. The maximum load of the anterior cruciate ligament was also weaker by 36%, dropping from 64.1 N to 40.4 N, and relaxin altered the structure of the ligament by collagenolytic effects [21]. In the second study, relaxin increased the synthesis of collagenase and many matrix metalloproteinases in anterior cruciate ligament rupture and decreased local collagen synthesis in women orthopedic patients [22]. Increases in estrogen and relaxin during the normal menstrual cycle last only a very short time and are insu cient to explain any collagenolytic effect. It appears that long-term uctuations are required to remodel the collagen [22].
Prolidase activity is increased in patients with DDH [23]. High prolidase activity indicates increased collagen metabolism, which is consistent with relaxin's mechanism of action. In the present study, we found that the number of relaxin receptors was twice as high in infants with DDH than in the elderly control group. We believe that this higher number of receptors and long-term exposure to relaxin from the mother during pregnancy may alter the nature of the ligamentum teres, resulting in hyperlaxity, possibly predisposing the fetus to DDH. However, in this case, it appears that the higher number of fetal receptors, rather than the concentration of relaxin from the mother, might be the cause because Forst et al. found that relaxin concentrations in specimens from cord blood were lower in neonates with DDH than in those of a healthy control group [4]. In a similar study, Vogel et al. also found no relationship between relaxin concentrations and DDH in 15 infants with DDH [24].
The ligamentum teres is important in stabilizing the pathophysiology of DDH [25]. Wenger et al. suggested that the strength of the ligamentum teres was crucial in the biomechanics of the hip and recommended surgical reconstruction for patients with DDH [25]. Another study on rats reported that greater strength of the ligamentum teres helps proper remodelling of the acetabulum against the femoral head [26].
The risk of DDH is four times as high in females as in males [27]. Desteli et al. found a negative relationship between the number of estrogen receptors and collagen metabolism in the ligamentum teres harvested from infants with DDH [28]. Furthermore, relaxin receptor isoforms are down-regulated with progesterone and high-dose estrogen, increasing the laxity, whereas progesterone and estrogen are downregulated with testosterone [29]. Testosterone concentrations are 8 times as high in men as they are in women, and testosterone reduces the expression of the relaxin receptor, as well as decreasing the passive range of motion in joints [30].
The following questions arise from these ndings: because relaxin is a maternal hormone, is it likely that attenuated relaxin activity improves a hip from Graf type IIa to type I in neonates? In addition, in hips with more relaxin receptors, although relaxin concentrations are reduced in time, does the long-term action of relaxin cause permanent dysplasia? Finally, can the down-regulation of relaxin receptors caused by testosterone in men be the reason for their lower rate of DDH? All these questions may guide future studies.
Our study had some limitations. We used missabortus fetuses as control group. As the hip development nishes before 20 week, we used to prefer fetuses over 20 weeks but we cannot be sure that these fetuses has a genetic abnormality or would develop a DDH in future. In this preliminary study, we used a single evaluator to score the intensity of staining. Given that we found an association between relaxin receptors and DDH, however, future studies should reduce the chances of bias by using two or more evaluators.

Conclusions
Our data support the conclusion that the number of relaxin receptors in the ligamentum teres is signi cantly higher in infants with DDH than in healthy control patients and increased number of relaxin receptors in the ligamentum teres could be a risk factor for DDH. This prospective study was approved by the institutional review board of authors' a liated institutions.
Parents and guardians provided written informed consent to have their children enroll in the study and to have samples taken for analysis.

Conset for Publication
Parents and guardians provided written informed consent to related informations to be published.

Availability of Data and Materials
Datasets are available in the form of an excel table.

Competing Interests
All authors declare that they have no con ict of interest.

Funding
No funding has been received.

Author's Contributions
Semih Ayanoğlu M.D. is responsible for design of the work Haluk Çabuk M.D. is responsible for design of the work and interpretation of data for the work Fatmagül Kuşku Çabuk is responsible for examination of pathologic materials Kubilay Beng is responsible for study desing and data analysis Timur Yıldırım is responsible for medical writing and ethical considerations Süheyla Uyar Bozkurt is responsible for examination of pathologic materials and data collection