Resectoscopic Metroplasty with Uterine Septum Excision: A Histologic Analysis of the Uterine Septum

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RESECTOSCOPIC METROPLASTY WITH UTERINE SEPTUM EXCISION. AN HISTOLOGIC ANALYSIS OF THE UTERINE SEPTUM Fabiana Divina Fascilla MD , Leonardo Resta MD , Rossella Cannone MD , Domenico De Palma MD , Oronzo Ruggiero Ceci MD , Vera Loizzi MD , Attilio Di Spiezio Sardo MD , Rudi Campo MD , Ettore Cicinelli MD , Stefano Bettocchi MD PII: DOI: Reference:

S1553-4650(19)31334-2 https://doi.org/10.1016/j.jmig.2019.11.019 JMIG 4018

To appear in:

The Journal of Minimally Invasive Gynecology

Received date: Revised date: Accepted date:

21 May 2019 31 October 2019 9 November 2019

Please cite this article as: Fabiana Divina Fascilla MD , Leonardo Resta MD , Rossella Cannone MD , Domenico De Palma MD , Oronzo Ruggiero Ceci MD , Vera Loizzi MD , Attilio Di Spiezio Sardo MD , Rudi Campo MD , Ettore Cicinelli MD , Stefano Bettocchi MD , RESECTOSCOPIC METROPLASTY WITH UTERINE SEPTUM EXCISION. AN HISTOLOGIC ANALYSIS OF THE UTERINE SEPTUM, The Journal of Minimally Invasive Gynecology (2019), doi: https://doi.org/10.1016/j.jmig.2019.11.019

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RESECTOSCOPIC METROPLASTY WITH UTERINE SEPTUM EXCISION. AN HISTOLOGIC ANALYSIS OF THE UTERINE SEPTUM. Fascilla Fabiana Divina MD1, Resta Leonardo MD2, Cannone Rossella MD1, De Palma Domenico MD1, Ceci Oronzo Ruggiero MD1, Loizzi Vera MD1, Di Spiezio Sardo Attilio MD3, Campo Rudi MD4, Cicinelli Ettore MD1, Bettocchi Stefano MD5 1

2

3

4 5

II Unit of Obstetrics and Gynecology, Department D.I.M.O., University “Aldo Moro”, Policlinico of Bari, Italy Department of Emergency and Organ Transplantation (DETO) Section of Anatomic Pathology, University “Aldo Moro”, Policlinico of Bari, Italy Department of Public Health, School of Medicine, University of Naples Federico II, Naples, Italy Leuven Institute for Fertility and Embryology (LIFE), Louvain, Belgium Inter-Departmental Project Unit of “Minimal-Invasive Gynecological Surgery”, University “Aldo Moro”, Policlinico of Bari, Italy

KeyWords: Uterine Septum; Office Hysteroscopy; Mini-Resectoscope; Histology Corresponding Author: Prof. Stefano Bettocchi Head of the “Minimal-Invasive Gynecological Surgery” Unit University “Aldo Moro” of Bari Azienda Ospedaliero-Universitaria “Policlinico” Piazza Giulio Cesare 11, 70124 Bari, Italy [email protected] Author’s conflict of interest Prof. Stefano Bettocchi: consultant for Karl Storz Co. Prof. Attilio Di Spiezio Sardo: consultant for Karl Storz Co. Prof. Rudi Campo: consultant for Karl Storz Co.

PRECIS The aim of this study is to evaluate the histology of the uterine septum and to define its macroscopic anatomical structure

ABSTRACT Study Objective: To evaluate the histology of the uterine septum after its complete hysteroscopic excision. Design: Case series. Setting: II Gynecological and Obstetric Unit of the University of Bari, Italy and Pathological Anatomy Department of the University of Bari, Italy. Patients: 35 patients between 25 and 41 years old diagnosed with uterine septum by 3D ultrasound as per ESHRE/ESGE 2013/Salim 2003 criteria. Office hysteroscopy was also performed to define the anatomy of the uterine cavity and to exclude the presence of other endometrial pathology. Interventions: Operative hysteroscopic septum resection was performed. The septum was initially incised with a “L shape” bipolar electrode with the 5 mm Bipolar Mini-Resectoscope (Karl Storz, Germany). Then, using the bipolar loop, two triangles of the septum were excised in parallel, obtaining uninterrupted entire septum-long strips, from the fundus to the apex of the septum. The strips were immediately removed from the uterus and reassembled “in vitro” in order to reconstruct the macroscopic 3D structure of the septum for a complete morphological and histological evaluation. Measurements and Main Results: Patients presented an average BMI of 24,8 and were all nulliparous. Histologic evaluation of the uterine septa showed a different conformation of the muscle bundles along the septum. In the apex and edges of the septum, the muscle cells were arranged in nodules circumscribed by a thin area of collagen fibers. Medium size vessels were distributed in the collagen fibers around the muscle cells. Only few capillary vessels are present in the muscle nodules. This pattern is very similar to the cell arrangement of leiomyomas. In the core of the septa, near the base, the muscle bundles show a linear course, with concurrent collagen fibers and vessels. All the above-mentioned characteristics were consistently present in every patient. On high-power histological fields (200x), the muscle portion ranged from 48,3 ± 1,8% (mean 6,0) in the apex and borders, to 48,5 ± 1,3% (mean 6,0) in the core. The collagen fibers ranged from 27.1 ± 1.1% (mean 4,0) in the apex and borders, to 26.7 ± 1.3% (mean 5,0) in the core. Conclusion: This study allowed us, by removing the entire septum as a whole structure, to redefine the concept of the septum as a complex structure based on islands of muscle fibers irregularly arranged in vertex, in a context of collagen tissue, similar to the structure of myomas. It appears to deeply involve the anterior and posterior uterine wall, resembling a “reverse letter H”.

INTRODUCTION The uterine septum is a well-known uterine malformation with serious implications in the field of reproduction.1-7 Little information is available in the current literature about the histology of the septum, although some papers showed limited data due, in our opinion, poor methodology adopted when collecting the biopsy samples.8-12 Prior studies failed to sample the septum in its entirely, only limiting the histologic evaluation to random collected biopsies. Surgical treatment of the uterine septum has been performed during the last thirty years as resectoscopic metroplasty, 13- 18 replacing the surgical laparotomy approach19-21 with proven better results in terms of pregnancy rate, patients discomfort, surgical complications and cosmetics results.22, 23 While positive results in terms of reproductive outcome are widely reported, 24-27 there is no evidence in the literature of the risks of persistence/recurrence of an intracavitary spur after hysteroscopic metroplasty, despite the common evidence of a variable presence this spur at followup, even when the surgery has been performed by expert surgeons. Understanding the histology of the septum is mandatory to establish the best surgical approach for hysteroscopic metroplasty. Our group decided to study the different aspects that could influence the outcome of metroplasty, in terms of both recurrence of the disease and fertility outcome. The goal of this study is primarily to define the histology of the uterine septum (U2.C0.V0 class of the ESGE/ESHRE 2013 CONUTA Classification28), studying the whole structure, up to the fundus, instead of collecting small superficial samples of the septum. 8,10

MATERIAL & METHODS This case series study was performed at the II Obstetrics and gynecology Unit at the University Medical School “Policlinico” of Bari, after approval of the Hospital’s Ethics Research Committee (Ref. 5746) from November 2016 to June 2018. Written informed written consent was obtained from each participant. Thirty-five reproductive age patients diagnosed with uterine septum, classified as U2.C0.V0 as per ESGE/ESHER 2013 CONUTA classification by using 3D Transvaginal Ultrasound,28 met inclusion criteria and were invited to participate. Thirteen patients outside the reproductive age and/or with septum involving the cervix and/or the vagina (C1, C2 and/or V1, V2) were intentionally excluded from the study in order to standardize the histological results. All the patients presented to Universitary OB/GYN office setting. All the included patient had initial diagnostic in-office hysteroscopy (OH) using a 4 mm hysteroscope (B.I.O.H. or “Bettocchi Size 4”, Karl Storz Co, Tuttlingen, Germany) with a stable intrauterine pressure at 45 mm/Hg maintained by a fluid management pump (Hysteromat E.A.S.I., Karl Storz Co, Tuttlingen, Germany) performed during the early proliferative phase of the menstrual cycle (from day 4 to day 9). All the procedures were performed in an office setting, without analgesia/sedation or anesthesia. An endometrial biopsy under direct hysteroscopic visualization was collected in all patients. The patients also underwent, before or after OH, 3D Trans-Vaginal (or Trans-Rectal) Ultrasound (3D TV/TR US) with Color/Power Doppler (Voluson E10, General Electrics, USA, and WS80A Elite, Samsung, South Corea) during the secretory phase of the menstrual cycle (from day 16 to day 22) in order to enhance the 3D contrast. The diagnosis of uterine septum was performed following the criteria of the ESGE/ESHRE 2013 CONUTA Classification. 28

Surgical hysteroscopic metroplasty was then performed in the operating room under general anesthesia using a bipolar 15 Fr. Mini-Resectoscope (Karl Storz Co., Tuttlingen, Germany) connected to an advanced bipolar generator (Autocon III 400, Karl Storz Co., Tuttlingen, Germany). Intrauterine pressure was kept stable around 45 mm/Hg using a dedicated pump (Hysteromat E.A.S.I., Karl Storz Co, Tuttlingen, Germany). The surgical procedures were performed during the early proliferative phase of the menstrual cycle (from day 4 to day 9). The septum was initially incised using an “L shape” bipolar electrode (Karl Storz Co., Tuttlingen, Germany) up to the fundal area. The septum was then longitudinally transected into two parts, forming two triangles on the anterior and posterior uterine wall, with the base on the fundus and the apex facing the Internal Cervical Os (I.C.O.). Then, using the bipolar loop (Karl Storz Co., Tuttlingen, Germany), we resected the two triangles in parallel, uninterrupted, long strips, from the fundus to the apex. The strips were removed from the uterus immediately after the resection and reassembled in vitro in order to reconstruct the macroscopic 3D structure of the septum for a complete morphological and histological evaluation (Fig. 1). Shortly after removal, tissues were fixed in neutral buffered formalin (pH 7.0), dehydrated, and embedded in paraffin using standard histological techniques. Sections from Formalin-Fixed Paraffin-Embedded (F.F.P.E.) blocks were stained with hematoxylin-eosin and periodic acid-Schiff and trichromic stain according to Masson for routine histologic analysis. Serial sections (5-micron) were processed for immunohistochemistry on Leica BondMax instruments (Leica Biosystem, Germany) using Refine HRP-kits (Leica DS9800, Leica Biosystem, Germany), including all buffer solutions (Leica Microsystems, Germany). Samples were treated in order to identify the different histological components of the tissue (CD31, Smooth Muscle Actin -SMA-, Desmin-Clone -DC-) with Clone 1A10 diluted 1/50 (Novocastra, Leica Biosystem, Germany), Clone 1A4 diluted 1/100 (Dako, USA) and DE-R-11 diluted 1/100 (Novocastra, Leica Biosystem, Germany), respectively. A Reichert microscope (Buffalo, New York, USA) with a digital camera (JTV Polyvar 2, Zeiss, Germany) and Sony Trinitron monitor (Sony, Japan) were used for quantitative analysis of immunostained cells. The areas occupied by muscle cells or by collagen bundles were counted in 10 microscopic fields (each measuring 140 x 110 microns, total amplitude: 15,400 square microns) at 400X magnification. Statistical analysis was performed using Student’s T-test. Descriptive statistic was used to analyze the data.

RESULTS Our study was based on thirty-five patients, aged between 25 and 41 years old, with an average BMI of 24,8 (Tab. 1). All the patients underwent OH at our University Centre. Nine patients had “incidental finding of uterine septum on ultrasound performed for other gynecologic complaints (3 for pelvic pain, 6 for abnormal uterine bleeding). These patients were the youngest in the group, not wishing, at that time, to become pregnant. Our policy, in this case, is to inform the patients about the “fertility risks” related to the septum, proposing an early treatment. The remaining 26 patients (74,29%) were trying to conceive, but with negative results, including IVF failure: 15 (42.86%) had a history of repetitive spontaneous abortion during the first trimester, while 11 (31.43%) were sterile. All patients were nulliparous.

All selected patients had negative endometrial hysteroscopic biopsies and there were no additional anomalies present inside the uterine cavity. The procedure was successfully completed in all the patients and no complication was encountered. Differential samplings of the uterine septa show a different conformation of the muscle bundles: in the apex and in the borders of the septum the muscle cells are arranged in different-sized nodules circumscribed by thin septa of collagen fibers. Medium size vessels are distributed in the collagen fibers around the muscle cells. Only few capillary vessels are present in the muscle nodules. This pattern is very similar to the cell arrangement of leiomyomas (Fig. 2). In the core of the septa, near the base, the muscle bundles show a linear course, with concurrent collagen fibers and vessels (Fig. 3). All the above-mentioned characteristics were consistently present in all the patients. On high-power histological fields (200x), the muscle portion ranged from 48,3 ± 1,8% (mean 6,0) in the apex and borders, to 48,5 ± 1,3% (mean 6,0) in the core. The collagen fibers ranged from 27.1 ± 1.1% (mean 4,0) in the apex and borders, to 26.7 ± 1.3% (mean 5,0) in the core. The differences are not statistically significant (Tab. 2).

DISCUSSION By removing the entire septum as a whole structure, we were able to define not only the histology but also the macroscopic anatomical structure of the uterine septum. Understanding the histology of the septum is mandatory to determine the best surgical approach. Past studies have differed in their conclusions regarding the histologic differences in different components of the septum. Dabirashrafi et all.12 in 1995 on 16 patients treated with Tompkins metroplasty was based on the histological analysis of 4 biopsy tissue samples obtained 1) from the septum near the serosal layer, 2) from the midpoint of the septum, 3) from the tip of the septum and 4) from the left uterine wall opposite the septum. A variation in the structural components of the septum is described, with a cranio-caudal reduction of fibrous components and increase of muscular components towards the apex, where the vessels, defined as "…with muscular component", are reduced. Our data contrast with these previously reported findings, showing a variation in the organization, rather than in the relative composition of the different components. In our opinion, the study published by Dabirashrafi el all.12 has limited scientific validity, due to the small size of the samples analyzed (3 to 5 mm) lacking a complete, three-dimensional analysis of the septum. In a recent study conducted by Moety et al.8, 41 patients with primary infertility or recurrent abortion diagnosed with uterine septum underwent hysteroscopic metroplasty using a 26 Fr. Resectoscope. During hysteroscopy, with the use of 5 Fr. micro-scissors, isolated superficial tissue fragments (0.51mm) were collected at the apex, the medial portion and the base of the septum. From a histological point of view, a variability in the muscular and fibrous components of the septum is reported. Interestingly, there was an increase in fibrotic tissue, compared to controls, in those patients with primary infertility and an increased muscular component in patients with a history of repeated abortions. In our opinion, the study validity is limited by the biopsy sampling method, that allow only to evaluate the superficial layers and not the internal organization of the septum. Sparc et al.10 reported the septum as a fibromuscular structure with slightly more pronounced connective tissue components in 72.3% of the cases, while in 27.6% the histology resembled normal myometrium. They also found numerous blood vessels in the septum, with medium-wide

muscular arteries and enlarged sinusoidal and capillary spaces. Again in this study the methodology used to collect the samples was not designed to study the entire structure of the septum: only the distal part of the septum was included, using a monopolar loop, collecting one or two specimens measuring 5-8 mm in length and 4-5 mm wide, while the rest of the septum was resected with a monopolar needle. In contrast, thanks to the technique that we used to excise the septum, we were able to analyze the entire uterine septum, providing a complete evaluation of the structure from both a histological and a structural point of view. Moreover, by combining the histological results with our findings during the resection of the septum, we are the first to study in vitro a three-dimensional structural of the septum. Accordingly, we believe it should no longer be considered as a spur in the middle of the uterine cavity, but as a real three-dimensional structure located in the myometrium. The septum has a "myoma-like" component with variable thickening, characterized by muscle cells arranged in nodules of different sizes circumscribed by a thin septum of collagen fibers. Medium-sized vessels are distributed in the collagen fibers around the muscle cells and only a few capillary vessels are present in the muscular nodules. This component is mostly present at the uterine fundus, where it forms two large circular structures that we have denominated "Mickey Mouse ears", then reduces while caudalizing, and thickens once again at the level of the free distal margin (Fig. 4). Furthermore, we demonstrated the presence, at the cranial portion of the septum, between the "Mickey Mouse ears", of a notch of “good” muscle near the normal myometrium, which is differentiated by the arrangement of the muscle fibers (Fig. 4). In the normal myometrium, the muscle fibers appear as a crossed section while in this area the muscle fibers have a parallel distribution. These findings explain why the septum presents a variable and irregular vascularization at ultrasound, due to the presence of lowflow vessels which correlates whit our now described histological structure.

CONCLUSIONS This study allowed us, by removing the entire septum as a whole structure, to redefine the concept of the septum as a complex structure based on islands of muscle fibers irregularly arranged in vertex, in a context of collagen tissue, similar to the structure of myomas. It appears to deeply involve the anterior and posterior uterine wall, resembling a “reverse letter H” (Fig. 5). We consider this study only as the first step in more complex research devoted to the uterine septum. These data should pave the way for the development of new surgical techniques, as well as promoting a re-evaluation of the endometrial function and its impact on fertility.

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Zreik TG, Troiano RN, Ghoussoub RA, Olive DL, Arici A, McCarthy SM. Myometrial tissue in uterine septa. J Am Assoc Gynecol Laparosc. 1998 May;5(2):155-60. PubMed PMID: 9564063.

10. Sparac V, Kupesic S, Ilijas M, Zodan T, Kurjak A. Histologic architecture and vascularization of hysteroscopically excised intrauterine septa. J Am Assoc Gynecol Laparosc. 2001 Feb;8 (1):111-6. PubMed PMID: 11172125. 11. Gouhar GK, Siam S: Uterine septum structure and reproductive performances: role of 3D TV Ultrasound and MRI. The Egyptian Journal of Radiology and Nuclear Medicine, 2013, 44, 357-365. 12. Dabirashrafi H, Bahadori M, Mohammad K, Alavi M, Moghadami-Tabrizi N, Zandinejad K, Ghafari V. Septate uterus: new idea on the histologic features of the septum in this abnormal uterus. Am J Obstet Gynecol. 1995 Jan;172(1 Pt 1):105-7. PubMed PMID: 7847514. 13. Daly DC, Maier D, Soto-Albors C. Hysteroscopic metroplasty: six years’ experience. Obstet Gynecol. 1989;73:201–205. 14. Bettocchi S, Ceci O, Nappi L, Pontrelli G, Pinto L, Vicino M. Office hysteroscopic metroplasty: three "diagnostic criteria" to differentiate between septate and bicornuate uteri. J Minim Invasive Gynecol. 2007 May-Jun;14(3):324-8. PubMed PMID: 17478363. 15. Valle RF. Hysteroscopic treatment of partial and complete uterine septum. Int J Fertil Menopausal Stud. 1996 May-Jun;41(3):310-5. PubMed PMID: 8799762. 16. Vilos GA. Intrauterine surgery using a new coaxial bipolar electrode in normal saline solution (Versapoint): a pilot study. Fertil Steril. 1999 Oct;72(4):740-3. PubMed PMID: 10521122. 17. Paradisi R, Barzanti R, Fabbri R. The techniques and outcomes of hysteroscopic metroplasty. Curr Opin Obstet Gynecol. 2014 Aug;26(4):295-301. doi: 10.1097/GCO.0000000000000077. Review. PubMed PMID: 24978851. 18. DeCherney AH, Russell JB, Graebe RA, Polan ML. Resectoscopic management of

müllerian fusion defects. Fertil Steril. 1986 May;45(5):726-8. PubMed PMID: 3699175. 19. Rock JA, Jones HW Jr. The clinical management of the double uterus. Fertil Steril. 1977 Aug;28(8):798-806. PubMed PMID: 885269. 20. Bret AJ, Guillet B. [Reconstructive hysteroplasty without muscular resection in uterine malformations; a cause of repeated abortion]. Presse Med. 1959 Feb 25;67(10):394-7. French. PubMed PMID: 13633897. 21. Tompkins P. Comments on the bicornuate uterus and twinning. Surg Clin North Am. 1962 Aug;42:1049-62. PubMed PMID: 13921824. 22. Fayez JA. Comparison between abdominal and hysteroscopic metroplasty. Obstet Gynecol. 1986 Sep;68(3):399-403. PubMed PMID: 2942813. 23. McShane PM, Reilly RJ, Schiff I. Pregnancy outcomes following Tompkins metroplasty. Fertil Steril. 1983 Aug;40(2):190-4. PubMed PMID: 6873316. 24. Shokeir T, Abdelshaheed M, El-Shafie M, Sherif L, Badawy A. Determinants of fertility and reproductive success after hysteroscopic septoplasty for women with unexplained primary infertility: a prospective analysis of 88 cases. Eur J Obstet Gynecol Reprod Biol. 2011 Mar;155(1):54-7. doi: 10.1016/j.ejogrb.2010.11.015. Epub 2010 Dec 23. PubMed PMID: 21185112. 25. Pabuçcu R, Gomel V. Reproductive outcome after hysteroscopic metroplasty in women with septate uterus and otherwise unexplained infertility. Fertil Steril. 2004 Jun;81(6):1675-8. PubMed PMID: 15193494. 26. Tonguc EA, Var T, Batioglu S. Hysteroscopic metroplasty in patients with a uterine septum and otherwise unexplained infertility. Int J Gynaecol Obstet. 2011 May;113(2):128-30. doi: 10.1016/j.ijgo.2010.11.023. Epub 2011 Mar 9. PubMed PMID: 21392763. 27. Paradisi R, Barzanti R, Natali F, Battaglia C, Venturoli S. Metroplasty in a large population of women with septate uterus. J Minim Invasive Gynecol. 2011 Jul-Aug;18(4):449-54. doi: 10.1016/j.jmig.2011.03.017. Epub 2011 May 28. PubMed PMID: 21621483. 28. Grimbizis GF, Gordts S, Di Spiezio Sardo A, Brucker S, De Angelis C, Gergolet M, Li TC, Tanos V, Brölmann H, Gianaroli L, Campo R. The ESHRE/ESGE consensus on the classification of female genital tract congenital anomalies. Hum Reprod. 2013 Aug;28(8):2032-44. doi: 10.1093/humrep/det098. Epub 2013 Jun 14. PubMed PMID: 23771171; PubMed Central PMCID: PMC3712660.

Fig. 1: Scheme explaining the surgical steps: #1 incision/resection of the “Mickey Mouse ears” until, #2, the area is completely resected; #3 incision/resection of the apex of the septum until the central muscular core is reached (during this step the fundal area change its aspect becoming flat); #4 the central muscular core is finally resected

Fig. 2: Histologic images of the Apex and Lateral Wall of the septum, using different preparations A. Masson’s Trichrome Stain (TRI): muscle bundles in a nodular arrangement with a thin cover of collagen fibers B. Smooth Muscle Actin (SMA) preparation: the immunohistochemical reaction for smooth muscle antigen reveals the real nodular arrangement of the muscle cells C. CD 31. Medium-sized vessels are arranged among the collagen fibers around the muscular nodules, where only few small vessels are present

Fig. 3: Histologic images of the Core of the septum, obtained with different preparations A. Masson’s Trichrome Stain (TRI): Linear arrangement of the smooth muscle cells, similar to the normal myometrium

B. Smooth Muscle Actin (SMA) preparation: the arrangement of muscle cells is very linear

C. CD 31. Linear arrangement of the vessels among the collagen fibers

Fig. 4: a 2D structure of the uterine septum

Fig. 5: “H” shape of the Septum

Tab.1: Patients’ biodata Patient Sterility 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Recurrent Occasional abortion diagnosis

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

Age

BMI

30 34 28 40 25 27 29 36 41 38 37 28 36 40 38 25 29 33 37 27 34 37 29 35 26 25 30 33 38 39 29 32 33 40 27

22 25 22 28 24 25 21 26 29 29 26 26 25 26 28 29 25 26 21 22 28 26 28 19 24 24 25 23 19 29 24 25 22 29 20

Tab.2: Comparison between presence of Muscle and Fibrotic tissue in the apex and the core of the uterine septum Patient Sterility 1

Recurrent Occasional abortion diagnosis

X

Age

Apex (%M)

Core %Apex(%M) %Core

30

46

48

Apex (%F)

Core (%F)

%Apex%Core

-2

28

26

2

2

X

34

49

51

-2

28

27

1

3

X

28

51

50

1

28

25

3

4

X

40

51

49

2

26

27

-1

25

46

49

-3

26

27

-1

27

50

50

0

27

26

2

29

48

50

-2

28

25

3

36

46

48

-3

28

28

-1

41

48

49

-1

28

28

0

38

49

51

-2

28

26

3

5 6

X X

7 8

X X

9 10

X X

11

X

37

47

49

-2

26

27

-1

12

X

28

46

48

-2

28

28

-1

36

51

53

-3

27

25

2

40

47

49

28

-2

13

X

14

X

-2

26

15

X

38

46

49

-3

27

25

3

16

X

25

50

51

-1

28

26

2

29

48

50

-2

26

27

-1

33

48

51

-3

25

24

1

37

48

50

-2

28

26

2

27

46

48

-2

27

29

-2

34

49

49

0

27

27

0

37

47

48

-1

29

26

3

29

49

47

2

28

25

3

35

49

50

-1

28

27

1

26

49

48

1

25

26

-1

25

47

48

-1

28

27

1

0

28

29

0

-2

28

26

2

-4

27

29

-2

25

2

17

X

18 19

X X

20

X

21 22

X X

23

X

24

X

25 26

X X

27

X

30

50

50

28

X

33

49

51

38

46

49

39

52

51

1

27

29

47

50

-3

25

27

-2

29

X

30 31

X X

32

X

32

50

49

2

26

28

-2

33

X

33

48

49

-1

28

28

0

40

49

49

0

26

28

-2

27

48

50

-2

26

27

-1

-1,2

27,1

26,7

0,5

34

X

35

X

35

11

15

9

Mean

48,3

49,5

100,0%

31,4%

42,9%

25,7%

S.D.

1,8

1,3

1,1

1,3

Range

6

6

4

5

%M Muscular tissue’s percentage (Apex & Core) %F

Fibrous tissue's percentage (Apex & Core)