1 SUBMITTED 9 MAR 22 1 REVISION REQ. 12 APR & 14 JUN 22; REVISION RECD. 11 MAY & 28 JUN 22 2 ACCEPTED 5 JUL 22 3 ONLINE-FIRST: September 2022 4 DOI: https://doi.org/ 10.18295/squmj.9.2022.053 5 6 Penile Girth Enhancement using Amniotic Membrane in a Rabbit Model 7 A stereological study 8 Ali Ariafar,1 *Saied Karbalay-Doust,2,3 Faisal Ahmed,4 Ali Eslahi,1,5 Sona 9 Tayebi1 10 11 1Department of Urology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; 12 2Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, 13 Shiraz, Iran; 3Anatomy Department, School of Medicine, Shiraz University of Medical Sciences, 14 Shiraz, Iran; 4Urology Research Center, Al-Thora Hospital, Department of Urology, Ibb 15 University of Medical Sciences, Ibb, Yemen; 5Shiraz Geriatric Research Center, Shiraz University 16 of Medical Sciences, Shiraz, Iran. 17 Corresponding Author’s e-mail: karbalas@sums.ac.ir 18 19 Abstract 20 Objectives: This study aimed to evaluate the efficacy of Penile Girth Enhancement (PGE) using 21 Amniotic Membrane (AM) as a graft in a rabbit model. Additionally, stereological studies were 22 used to obtain quantitative histological data regarding the structure of the penis. Methods: In this 23 study, 20 adult male rabbits of similar age and weight were allocated to two sham and 24 surgery+AM groups. Both groups underwent surgery by longitudinal Ishape midline incision of 25 the tunica albuginea on the dorsal surface of the penis. The surgery +AM group underwent PGE 26 by AM graft. The penile length and mid circumference were measured using a Vernier caliper 27 before and two months after the surgery. Stereological studies were used to obtain quantitative 28 histological data regarding the structure of the penis. Results: The mean total volume and 29 diameter of the penis increased in the surgery +AM group (p<0.03 and p<0.04, respectively). 30 2 The stereological evaluation showed a significant increase in the mean volumes of the tunica 31 albuginea and corpora cavernosa in the surgery +AM group compared to the sham group 32 (p<0.01, p< 0.03). Additionally, the mean volume density of the collagen bundles, muscle fibers, 33 and cavernous sinuses and the total number of fibroblasts and smooth muscle cells increased in 34 the surgery +AM group compared to the sham group (p<0.01, p<0.01, p<0.03, p<0.01, and 35 p<0.05, respectively). No infections, bleedings, or other complications were seen. Conclusions: 36 AM is a method that has appeared promising for material use in penile enhancement. Thus, it 37 may be used for PGE in the future. 38 Keywords: Amniotic Membrane; Histopathology; Animal; Penile Girth Enhancement. 39 40 Advances in knowledge 41 - Amniotic membrane is a new method, which has appeared helpful for material use in penile girth 42 enhancement. 43 - Amniotic membrane may be used for humman penile girth enhancement in future. 44 Application to patient care 45 - This study aimed to evaluate the efficacy of penile girth enhancement using amniotic membrane 46 as a graft in a rabbit model. 47 48 Introduction 49 The penis has historically been considered a sign of masculinity. Therefore, its size has become a 50 source of worry for numerous men. Today, some men seek for ways to enlarge their penis in order 51 to increase their self-confidence and make their partners more sexually satisfied.1 52 53 Penile Girth Enhancement (PGE) is carried out for cosmetic purposes and psychological causes in 54 some patients, similar to breast enlargement amongst females.2 Men with a small penis and patients 55 with special urological conditions such as micropenis, Peyronie’s disease, and trauma to the penis 56 may benefit from this procedure.3 PGE aims at improving the penile function and appearance. 57 Nonetheless, there are no suggested guidelines and specific techniques for PGE.4 58 59 3 Generally, PGE can be carried out via such methods as grafts, flaps, fillers, and injections.5 Graft 60 procedures are one of the techniques for PGE, in which more fat tissue is used.6 Other tissues such 61 as small intestinal submucosa and temporalis fascia are also used in graft procedures.3, 7 62 63 Many studies have been done regarding the impact of graft fat procedures on the penis and have 64 indicated the effectiveness of graft fat in enhancing the penile girth. For example, Zhang et al. 65 (2020) evaluated the effectiveness and safety of human acellular dermal matrix graft in the 66 augmentation phalloplasty method.8 Xu et al. (2016) also illustrated the effectiveness and safety 67 of dermal fat graft in augmentation phalloplasty amongst men with a small penis.9 Similarly, 68 Leungwattanakij et al. (2006) showed the promising effect of using small intestinal submucosa on 69 penis enlargement in a rat model.3 In addition, Küçükçelebi et al. (2006) reported that the use of 70 microvascular temporalis fascia strengthened the penis in humans.7 71 Considering the social progress, increase in people’s awareness and sexual needs, and increasing 72 demand for surgical treatment to enlarge the penis, researchers have made genuine attempts to 73 develop new and effective methods for this purpose. 74 75 In the last decade, Amniotic Membrane (AM) has been shown to possess many properties that 76 suggest its value in several medical applications. AM has also been used in many genitourinary 77 surgeries. 10-13 In the current study, AM was used for PGE for the first time. AM transplantation 78 has been used in surgical procedures in the fields of medicine, ophthalmology, dermatology, 79 plastic surgery, urogenital system, and ENT. Many researchers have described these applications 80 separately, each having different effects and techniques.14 It is worth mentioning that AM is the 81 deepest semitransparent layer of the embryonic membrane, which contains an avascular stromal 82 matrix, a thick collagen layer, an overlying basement membrane, and a single layer of cuboidal 83 epithelium.14 84 85 Rabbit has a vascular penis that contains two corpora cavernosa and a corpus spongiosum that 86 encloses the urethra. In addition to the lack of a penile bone, this vascular penis has certain 87 characteristics that make it more similar to human’s penis. Therefore, it is a good animal model 88 for studying the structure of the penis.15 Stereology techniques have been increasingly applied for 89 determining a variety of morphometric variables of three-dimensional structures.16 To the best of 90 4 our knowledge, no study has evaluated the efficiency of the application of AM in PGE in a rabbit 91 model using stereological methods in order to obtain quantitative histological data. The chief 92 advantage of stereological methods is the provision of unbiased and precise assessments. Thus, 93 the present study aims to investigate whether PGE using AM accelerates the regeneration of 94 various parts of the penile tissue and leads to an increase in its size. 95 96 Methods 97 Experimental design 98 A total of 20 adult male New Zealand White (Oryctolagus cuniculus) rabbits (weight: 1600–2500 99 grams; age: 18 weeks) were obtained from the University’s Center of Comparative and 100 Experimental Medicine. The rabbits were kept individually in cages with a 12/12-h light-dark 101 cycle at room temperature of 22–24 °C and humidity of 50% and had access to water and food ad 102 libitum. All animals were kept according to the Animal Care and Ethics Committee of the 103 University. The rabbits were divided two sham and surgery +AM groups using simple random 104 sampling (n=10). In the both groups, the surgery was done by a longitudinal I-shape midline 105 incision of the tunica albuginea on the dorsal surface of the penis .The second group (surgery +AM 106 group) underwent PGE using AM. 107 108 . All animals underwent the surgical procedure, but only six rabbits in the sham group and seven 109 rabbits in the surgery + AM group were included in stereological studies. 110 111 Human amniotic membrane preparation 112 Human AM, provided by Burn and Wound Healing Research Center, were kept in alcohol (95%) 113 until application. (In this center, AM are provided from delivery rooms and are employed as a 114 biological dressing in burn patients).AM were gained from women delivery no history of 115 premature rupture of membrane, endometritis, or meconium ileus. All women were seronegative 116 tests for human immunodeficiency virus, hepatitis types B and C, and syphilis.17 117 118 Surgical procedure 119 All rabbits were anesthetized using the intramuscular injection of ketamine (10–15 mg/kg) and 120 xylazine (6–10 mg/kg). Supplemented doses of ketamine were administered as needed to maintain 121 5 a uniform level of anesthesia. All animals were well shaved and prepared with a povidone iodine 122 topical antiseptic solution and were then draped with sterile sheets. After that, the penis was 123 exposed under aseptic conditions and then, the glans was sutured with 4/0 nylon held with a 124 mosquito clamp under gravity to stretch the penis downward. 125 126 In the both groups, the surgery was done by a longitudinal I-shape midline incision of the tunica 127 albuginea on the dorsal surface of the penis. In the surgery +AM group, the AM graft (3*15 mm2 128 piece) was placed on the dorsal surface of the penis between the edges of tunica albuginea and 129 over the covernosal tissue in both sides of penis and was sutured with a 6-0 PDS (polydioxanone) 130 [Figure 1]. 131 132 All rabbits were housed individually and were fed with standard feed throughout the experiment. 133 Antibiotics were also administered intramuscularly to all groups for three days. After the operation, 134 the rabbits were observed for bleeding, hematoma, swelling, penile deviation, and other 135 complications. 136 137 The penile length and mid circumference were measured using a digital Vernier caliper (accuracy: 138 0.5 mm). The girth of the penis was measured at the mid-penile body in the flaccid state. The 139 penile length during the flaccid state was measured from the palpable lower border of the pubic 140 symphysis to the tip of the glans. The mean length and girth of each rabbit category were 141 determined and compared to those of other rabbit categories.18 142 143 Penile tissue preparation 144 After two months, all the rabbits were sacrificed with deep anesthesia. The penis and skin sutures 145 were removed in its entirety by dissecting along the shaft to the crura and separating each cru from 146 its point of attachment at the ischial tuberosity. The penis was divided to 8-12 sections based on 147 length with equal distances between the sections “T” [Figure 2 a]. The sections of each penis were 148 processed, embedded, sectioned (4 and 25 µm), and stained (hematoxylin-eosin) [Figure 2 b].19 149 150 Estimation of the volumes of the penis and its components 151 6 The sections with a 4-µm thickness were used in order to estimate the volume of the penis and the 152 volume density of the penile components. The penis is composed of skin, penile fascias (superficial 153 fascia or dartos fascia and deep fascia or buck’s fascia), tunica albuginea, paired corpora 154 cavernosa, and a single corpus spongiosum that contains a spongy tissue and the urethra. In each 155 penile section, the borders between the regions were identified and characterized [Figure 3 a]. The 156 corpora cavernosa contains fibrous tissues (collagen bundles), smooth muscle cells, cavernous 157 sinuses, and vessels.19 The volumes of the fascia (superficial and deep fascia), tunica albuginea, 158 and corpora cavernosa were estimated using a video microscopy system and the software designed 159 at the University’s Histomorphometry and Stereology Research Center. The volumes of the penis 160 and its components were estimated by using the “Cavalieri method” at 12X magnification [Figure 161 3 b]: 162 163 V(penile component) = ∑p × A(p) × T 164 165 Where ∑p was the total number of points hitting the structure of interest, A(p) was the area related 166 to every grid point, and “T” was the distance between the sections.19 167 168 Estimation of the volume density of the collagen bundles, smooth muscle cells, cavernous sinuses, 169 and vessels of the corpora cavernosa 170 The volume density “Vv” of collagen bundles, smooth muscle cells, cavernous sinuses, and vessels 171 was calculated by the “point-counting method” and the following formula19 [Figure 4 a]: 172 173 Vv(structure / corpora cavernosa) = P(structure) / P(corpora cavernosa) 174 175 Where “P(structure)” showed the number of points placed on the mentioned structures and 176 “P(corpora cavernosa)” indicated the number of points superimposed on the corpora cavernosa. 177 The total volume of each structure was calculated by the following formula: 178 179 V(structure) = Vv(structure/ corpora cavernosa) × V(corpora cavernosa) 180 181 7 Estimation of the numerical density of the fibroblasts and smooth muscle cells in the corpora 182 cavernosa 183 The numerical density “Nv(fibroblasts or myocyte / cavernous bodies)” and the total number of 184 fibroblasts and smooth muscle cells were estimated using the “optical disector” technique utilized 185 on 25 µm sections. The optical disector contained an Eclipse microscope with a high Numerical 186 Aperture (NA=1.30) ×40 oil-immersion objective lens connected to a video camera that 187 transmitted microscopic live images to a computer monitor and an electronic microcator with 188 digital readout for estimating the number of fibroblasts by moving in the Z-direction. The 189 numerical density (NV) of the fibroblasts and smooth muscle cells was estimated using the 190 following formula: 191 192 Nv (fibroblasts or myocyte / cavernous bodies) = ΣQ–/ (Σp× (a/f) × h) × (t/BA) 193 194 Where “∑Q” was the number of sampled fibroblasts or myocytes, ”∑P” was the number of 195 disectors, a(f) was the area of the frame, “h” was the height of the disector, and “t” was the mean 196 section thickness. The upper and lower borders of each section were considered guard zones. The 197 total number of fibroblasts or myocytes was estimated by multiplying the numerical density by 198 V(cavernous bodies) [Figure 4 b].19 199 200 Fibroblasts were recognized by their specific criteria (having plentiful and irregularly branched 201 cytoplasms, a large ovoid euchromatic nucleus, and a prominent nucleolus). Smooth muscle cells 202 were also recognized by their spindle shape and single central nucleus.20 203 204 Statistics and data analysis 205 GraphPad Prism software, version 8.0.0 for Windows (GraphPad Software, San Diego, California, 206 USA) was applied to analyze the data. The data were compared using Mann-Whitney U test and 207 were presented as dot plots. P<0.05 was considered statistically significant. 208 209 Results 210 The total volume, diameter, and length of the penis 211 8 The total volume, length, and diameter of the penis increased by respectively 26%, 8%, and 4% in 212 the surgery +AM group in comparison to the sham group. There was also a significant increase in 213 the mean volume and diameter of the penis in the surgery +AM group compared to the sham group 214 (p<0.03 and p<0.04, respectively) [Figure 5 a and b]. However, there was no significant difference 215 between the surgery +AM and sham groups regarding the mean length of the penis [Figure 5 c]. 216 217 The volumes of the fascia, tunica albuginea, and corpora cavernosa of the penis 218 The mean volumes of the fascia, tunica albuginea, and corpora cavernosa increased by respectively 219 15%, 29%, and 40% in the surgery +AM group in comparison to the sham group. The results also 220 revealed a significant increase in the mean volumes of tunica albuginea and corpora cavernosa in 221 the surgery +AM group compared to the sham group (p<0.01 and p<0.03, respectively) [Figure 5 222 e and f]. However, there was no significant difference between the surgery +AM and sham groups 223 concerning the mean volume of the fascia [Figure 5 d]. 224 225 The volume density of the collagen bundles, smooth muscle cells, cavernous sinuses, and vessels 226 of the corpora cavernosa 227 The mean volume density of the collagen bundles, smooth muscle cells, and cavernous sinuses 228 increased by respectively 24%, 33%, and 32% in the surgery + AM group in comparison to the 229 sham group. The results indicated a significant increase in the mean volume density of the collagen 230 bundles, smooth muscle cells, and cavernous sinuses in the surgery +AM group compared to the 231 sham group (p<0.01, p<0.01, and p<0.03, respectively) [Figure 6 a, b, and c]. However, there was 232 no significant difference between the surgery +AM and sham groups in terms of the mean volume 233 of the vessels (Figure 6 d). 234 235 The number of fibroblasts and smooth muscle cells of the corpora cavernosa 236 The mean number of fibroblasts and smooth muscle cells increased by 41% and 36%, respectively 237 in the surgery +AM group in comparison to the sham group. There was also a significant increase 238 in the mean number of fibroblasts and smooth muscle cells in the surgery +AM group compared 239 to the sham group (p<0.01 and p<0.05, respectively) [Figure 6 e and f]. 240 241 Discussion 242 9 This study aimed to determine the effectiveness of AM as a graft in PGE in a rabbit model. The 243 results revealed a significant increase in the diameter and volume of the penile corpora cavernosa 244 and the number of fibroblasts and smooth muscle cells in the corpora cavernosa in the animals that 245 had undergone PGE surgical procedures. 246 Penile enlargement is usually done by auto tissue transplantation, cell injection, or implantation of 247 artificial or natural materials.8 Autologous tissue transplantation from the adjacent tissues is one 248 of the most common surgeries performed for PGE. Autologous fat tissue has also been recently 249 used for PGE.8, 21 The utilization of an AM graft for PGE was first introduced in the present 250 research. 251 252 In the previous studies, different techniques were described for PGE and a variety of exogenic 253 materials were utilized in the procedures. However, no standard guidelines are available. 254 Moreover, the employed exogenic materials have shown different degrees of success. For example, 255 autologous fat, silicone, and hyaluronic acid gel were injected to the subcutaneous space of the 256 penile body. Additionally, dermal fat grafts as well as a cellular dermal matrix derived from a 257 donated human skin tissue (allograft) were used for PGE procedures.21,22 In a prior research, 258 dermal cellular porcine grafts were used in 69 participants and the results revealed a promising 259 long-term outcome. After one year of follow-up, the penial circumference increased by 3.1 and 260 2.4 cm during flaccidity and erection, respectively.23 However, the use of pelvicol acellular matrix 261 for PGE was not suitable due to the high rate of complications.24 Overall, these injectable materials 262 carried a risk of foreign body response, swelling, and penile deviation.25 However, autologous fat 263 grafting reduced the risk of foreign body response and was found to improve PGE.26 On the other 264 hand, evidence demonstrated that autologous fat transplantation would lose a large amount of its 265 volume over time and, consequently, needed several procedures to bring about a favorable 266 outcome.25 In the present study, swelling and penile deviation were not observed in the 267 experimental groups. 268 269 AM is composed of connective tissue with a significant collagen and extracellular matrix structure. 270 The inner surface is enclosed by a single-layer cubical epithelium, which is avascular, has anti-271 scarring, anti-inflammatory, and antiangiogenic properties, and contains several growth factors. 272 Moreover, it has been reported to possess the exclusive quality of avoiding graft versus host disease 273 10 and to facilitate wound healing.27 The mechanism of action of AM has been thought to be related 274 to the rich biological construct of the amnion and chorion membranes, which include layers of 275 basement membranes and a variety of intrinsic factors that play a vital role in cell proliferation and 276 differentiation. It has also been reported that the AM epithelial cells secrete angiogenic factors.28 277 These properties make human AM an ideal tissue graft for reconstruction in different tissues. 278 Additionally, AM is resistant to rejection and is easy to obtain, derive, and store.27 Leungwattanakij 279 et al. studied penile reconstruction using small intestinal submucosa in 20 rats. In that study, PGE 280 was performed via the bilateral incision of tunica albuginea and the plane of dissection was 281 between the tunica albuginea and the cavernous tissue. The tunica defect was covered with a piece 282 of small intestinal submucosa. The histological study showed moderate amounts of fibrosis under 283 the graft and the elastic fibers of the graft were oriented in a circular direction.3 In the present 284 study, the same procedure was used and the histological study revealed a significant increase in 285 the mean volumes of the tunica albuginea and corpora cavernosa in the surgery +AM group. 286 Additionally, the mean volume density of the collagen bundles, smooth muscle cells, cavernous 287 sinuses, and vessels (indicating neovascularization into the graft) and the mean number of 288 fibroblasts and smooth muscle cells increased in the surgery +AM group, which represented good 289 tissue acceptance. 290 291 Shakeri et al. reported the proper re-epithelialization of the urethra reconstructed with AM by 292 transitional epithelium with cytokeratin expression in a rabbit model. However, the fistula was 293 detected in one case (5%) and urethral strictures were seen in two cases (10%).29 In another study, 294 Salehipour et al. evaluated the use of human AM in the reconstruction of long ureteral defects in 295 a dog model and concluded that AM was not useful for long urethral defects (3 cm). They 296 mentioned that the use of AM might be studied for shorter defects or as a patch graft.30 Salehipour 297 et al. also assessed the efficacy of human AM grafting in the canine penile tunica albuginea defect. 298 The results of histopathological examinations showed complete re-epithelialization with squamous 299 epithelium and collagen fiber deposition. Besides, no dysplasia was detected.8 300 301 This study had some limitations. Firstly, the operation performed in the sham group might induce 302 scaring, which could have affected the final PGE and make the comparison more difficult. 303 Therefore, a group without surgical procedure (control) had to be added to the group design. 304 11 Secondly, the effects of the surgical operation on ejaculation and erection were not evaluated after 305 PGE. The third study limitation was the increase in collagen in the penis, which could have affected 306 the function of the penis. Therefore, anti-fibrotic drugs can be used to reduce collagen in future 307 studies. 308 309 Conclusions 310 AM is a new method, which has appeared helpful for material use in PGE. Hence, it may be used 311 for human PGE in future. 312 313 Authors’ Contribution 314 AA conceptualized and designed the study. FA and AE were involved in the visualization and 315 investigation. ST collected the data. SK-D and ST drafted the manuscript. SK-D was involved in 316 the validation, review and editing of the manuscript. AA supervised the work. All authors approved 317 the final version of the manuscript. 318 319 Acknowledgement 320 The work was performed at the Histomorphometry and Stereology Research Center, Shiraz 321 University of Medical Sciences, Shiraz, Iran. Hereby, the authors would like to thank Ms. A. 322 Keivanshekouh at the Research Consultation Center (RCC) of Shiraz University of Medical 323 Sciences for improving the use of English in the manuscript. 324 325 Conflicts of Interest 326 The authors declare no conflicts of interest. 327 328 Funding 329 This work was financially supported by grant No. 16032-01-01-1396 from Shiraz University of 330 Medical Sciences, Shiraz, Iran. 331 332 References 333 12 1. Al-Ansari AA, Shamsodini A, Talib RA, Gul T, Shokeir AA. Subcutaneous cod liver oil 334 injection for penile augmentation: review of literature and report of eight cases. 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Is amniotic membrane a suitable biomaterial for reconstruction of long ureteral defects? 425 https://pubmed.ncbi.nlm.nih.gov/14671667/ https://pubmed.ncbi.nlm.nih.gov/14671667/ https://pubmed.ncbi.nlm.nih.gov/16547638/ https://pubmed.ncbi.nlm.nih.gov/23354211/ 15 Saudi J Kidney Dis Transpl 2013 ;24(1):135-8. https://doi: 10.4103/1319-2442.106311. 426 427 428 Figure 1: Surgical procedure was done by the longitudinal I-shaped midline incision of the tunica 429 albuginea on the dorsal surface of the penis and the placement of the AM graft between the tunica 430 albuginea and the corpus cavernosum in both right and left sides of penis. 431 432 433 Figure 2: Processing technique. The penis was cut into 8-12 sections according to its length (a). 434 The sections were embedded in paraffin blocks, sectioned, mounted on a slide, and stained (b). 435 436 16 437 Figure 3: Assessment of the rabbit penile tissue. The penile components were indicated on the 438 histological section by arrows (a). The volumes of the penis and penile components were assessed 439 by Cavalieri’s technique and point-counting method (b). 440 441 442 Figure 4: Point-counting method was employed to estimate the volume density of the collagen 443 bundles, smooth muscle cells, cavernous sinuses, and vessels of the corpora cavernosa (a). Optical 444 disector technique was used to estimate the numerical density of the fibroblasts and smooth muscle 445 cells. The fibroblasts’ or smooth muscle cells’ nuclei coming into focus through scanning of the 446 height of the disector were recorded (the arrow) (b). 447 448 17 449 Figure 5: The aligned dot plots of the total volume (a), diameter (b), and length (c) of the fascia 450 (d), tunica albuginea (e), and corpora cavernosa (f) of the penis in the sham and surgery+AM 451 groups. Each dot shows an animal and the horizontal bars represent the means of the parameters. 452 The p-values and significant differences have been shown on each dot plot by stars. Statistical 453 significance was determined by Mann-Whitney U test. *P=0.03, **P=0.04, ***P=0.01. 454 18 455 Figure 6: The aligned dot plot of the volume density of the collagen bundles (a), smooth muscle 456 cells (b), cavernous sinuses (c), and vessels (d) and number of fibroblasts (e) and smooth muscle 457 cells (f) of the corpora cavernosa in the sham and surgery +AM groups. Each dot represents an 458 animal and the horizontal bars show the means of the mentioned parameters. The significant 459 differences and p-values have been presented on each dot plot by stars. Statistical significance was 460 determined by Mann-Whitney U test. *P=0.01, **P=0.03, ***P=0.05. 461