Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium of pregnant rats after local administration of hyaluronidase

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(1)P R A C E. Ginekol Pol. 2014, 85, 365-370. O R Y G I N A L N E po ł o ż n i c t wo. Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium of pregnant rats after local administration of hyaluronidase Histomorfometria i wykrywanie glikozaminoglikanów w nabłonku szyjkowym ciężarnych szczurów po miejscowym podaniu hialuronidazy 0RQLFD5HJLQD/RXUHQoR/X]1(GZDUG$UDXMR-~QLRU1/XL]&DPDQR1 0DQXHOGH-HVXV6LP}HV(GXDUGRGH6RX]D1 1 2. Department of Obstetrics, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil. Department of Morphology, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil.. Abstract Objective: The aim of the study was to detect the presence of glycosaminoglycans and to investigate histomorphometric aspects of the endocervical epithelium in pregnant rats after local administration of hyaluronidase. Materials and methods: Ten pregnant rats were randomly distributed into two groups. On day 18 of pregnancy, 1mL of distilled water and 0.02 mL of hyaluronidase were administered to the control group (CG) and the study group (SG), respectively. On day 20 the rats were sacrificed, followed by dissection and removal of the uterine cervix, which was prepared for histomorphometry (endocervical epithelium thickness and leucocyte infiltration) and for immunohistochemistry with alcian blue reaction and its respective blockers. The paired Student t test was used to compare the groups. Results: The SG was characterized by reduced epithelial thickness (mean: 291.01±71.1 vs. 764.30±50.94; p<0.0001) and a larger number of eosinophils (mean: 3.72±1.60 vs. 0.54±0.70; p<0.0001). Alcian blue staining (pH 0.5) indicated a very strong reaction (3+) for the CG. With pH 2.5, the staining was also very intense (4+) in the CG. With methylation, both groups showed negative reactions after alcian blue staining (pH 2.5). With the methylation reaction followed by saponification and with enzymatic digestion of the lamina, staining showed a weak reaction (1+) in both groups. Conclusion: The SG presented with significant alterations related to the reduction of epithelial thickness and an increase in leucocyte infiltration. Furthermore, the use of hyaluronidase resulted in a significant decrease of the sulfated glycosaminoglycans.. Key words: cervical epithelium / glycosaminoglycans / hyaluronidase / pregnant rats / Streszczenie Cel: Celem pracy było zbadanie histomorfometrycznych aspektów i wykrycie glikozaminoglikanów w nabłonku szyjki macicy u ciężarnych szczurów po miejscowym podaniu hialuronidazy.. Corresponding author: Edward Araujo Júnior Department of Obstetrics, Federal University of São Paulo (UNIFESP) Rua Carlos Weber, 956 apt. 113 Visage Vila Leopoldina, Sao Paulo – SP, Brazil, CEP 05303-000 tel./ fax: +55 11 37965944 e-mail: araujojred@terra.com.br. Nr 5/2014. © Polskie Towarzystwo Ginekologiczne. Otrzymano: 12.06.2013 Zaakceptowano do druku: 30.10.2013. 365.

(2) P R A C E O R Y G I N A L N E poł ożn i ct wo. Ginekol Pol. 2014, 85, 365-370. Monica Regina Lourenço Luz et al. Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium.... Materiały i metody: Dziesięć ciężarnych szczurów podzielono losowo na dwie grupy: 1 ml wody destylowanej podawano w grupie kontrolnej (CG) w 18 dniu ciąży, a 0,02 ml hialuronidazy podawano w grupie doświadczalnej (SG) w tym samym czasie. W 20 dniu ciąży szczury uśmiercano, następnie dokonywano wycięcia szyjki macicy, która została przygotowana do badania histomorfometrycznego (grubość nabłonka i infiltracja leukocytów), a także do histochemicznego badania reakcji alcianem niebieskim i jego odpowiednich blokerów. Test t-Studenta wykorzystano dla porównania obu grup. Wyniki: Zwężenie nabłonka szyjki i większą liczbę eozynofili zaobserwowano w SG. SG scharakteryzowała się zmniejszoną grubością nabłonka (średnia 291,01 ± 71,1 vs 764,30 ± 50,94, p <0,0001) i większą liczbą eozynofili (średnia 3,72 ± 1,60 vs 0,54 ± 0,70, p <0,0001). Bardzo silną reakcję (3+) zaobserwowano w CG przy barwieniu alcianem niebieskim (pH 0,5). Przy pH 2,5 zabarwienie było także bardzo silne (4+) w CG. W przypadku metylacji obie grupy wykazały ujemną reakcję po zabarwieniu alcianem niebieskim (pH 2,5). W reakcji metylacji, po której nastąpiła saponifikacja, oraz enzymatyczne trawienie w blaszce, barwienie wykazało słabą reakcję (1+) w obu grupach. Wnioski: W SG odnotowano istotne różnice odnoszące się do redukcji grubości nabłonka i zwiększoną infiltrację leukocytów. Ponadto, istnieje wyraźna redukcja w ilości glikozaminoglikanów związana ze stosowaniem hialuronidazy.. Słowa kluczowe: naEáoneN s]yMNi macicy / glikozaminoglikany / hialuronidaza / / ciĊĪarne szczury /. Introduction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(34) . Figure 1. Photomicrography showing endocervical epithelium of a rat from the study group. Note the presence of a cyst containing leukocytes in its interior (400 X).. © Polskie Towarzystwo Ginekologiczne. 367.

(35) P R A C E O R Y G I N A L N E poł ożn i ct wo. Ginekol Pol. 2014, 85, 365-370. Monica Regina Lourenço Luz et al. Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium.... Tabl e I . Measurement values of thickness of the endocervical epithelium (μM) of the rats on day 20 of pregnancy in controls and the study group. Groups. Groups. Epithelial thickness. Control group. Study group. Number oI ¿elds. Control group. Study group. 1. 759.10. 356.50. 1. 0. 5. 2. 610.24. 234.57. 2. 2. 3. 3. 850.30. 258.00. 3. 1. 2. 4. 750.10. 319.00. 4. 0. 3. 5. 680.20. 322.00. 5. 0. 2. 6. 780.20. 300.50. 6. 0. 1. 7. 810.24. 245.67. 7. 1. 6. 8. 785.35. 234.50. 8. 2. 4. 9. 679.20. 278.00. 9. 1. 5. 10. 750.20. 358.90. 10. 0. 6. 11. 820.30. 245.67. 11. 0. 4. 12. 789.00. 235.90. 12. 0. 5. 13. 658.24. 450.23. 13. 0. 6. 14. 756.12. 245.00. 14. 1. 3. 15. 768.80. 349.60. 15. 1. 4. 16. 712.35. 254.89. 16. 0. 1. 17. 812.45. 253.00. 17. 1. 2. 18. 780.00. 450.00. 18. 0. 4. 19. 760.12. 359.00. 19. 0. 2. 20. 789.30. 238.00. 20. 0. 4. 21. 810.24. 240.00. 21. 0. 5. 22. 760.12. 317.00. 22. 2. 3. 23. 756.12. 320.00. 23. 1. 2. 24. 679.20. 305.50. 24. 0. 3. 25. 759.10. 240.67. 25. 0. 2. 26. 812.45. 230.50. 26. 0. 1. 27. 750.20. 270.00. 27. 1. 6. 28. 759.10. 340.90. 28. 2. 4. 29. 780.00. 230.67. 29. 1. 5. 30. 610.24. 220.90. 30. 0. 6. 31. 812.45. 400.23. 31. 0. 4. 32. 759.10. 201.50. 32. 0. 5. 33. 780.00. 343.60. 33. 0. 6. 34. 789.00. 230.89. 34. 1. 3. 35. 768.80. 241.50. 35. 1. 4. 36. 750.20. 450.00. 36. 0. 1. 37. 820.30. 359.00. 37. 1. 2. 38. 759.10. 232.50. 38. 0. 4. 39. 750.10. 222.90. 39. 0. 2. 40. 770.00. 402.23. 40. 0. 4. 41. 822.30. 201.50. 41. 0. 2. 42. 783.00. 343.60. 42. 2. 1. 43. 785.00. 230.89. 43. 1. 6. 44. 817.00. 241.50. 44. 0. 4. 45. 805.00. 450.00. 45. 0. 5. 46. 798.00. 359.00. 46. 0. 6. 47. 783.00. 232.50. 47. 1. 4. 48. 692.20. 233.25. 48. 2. 5. 49. 795.00. 234.15. 49. 1. 6. 50. 797.24. 235.10. 50. 0. 3. Mean. 764.30. 291.01*/†. Mean. 0.4. 3.72*/†. Standard deviation. 0.94. 71.11. Standard deviation. 0.7. 1.6. *Paired Student’s t test; †p<0.0001. 368. Ta bl e II. Values of eosinophil count present in the endocervical epithelium of rats on day 20 of pregnancy in controls and the study group, of a field of view at 40x.. *Paired Student’s t test; †p<0.0001. © Polskie Towarzystwo Ginekologiczne. Nr 5/2014.

(36) P R A C E. Ginekol Pol. 2014, 85, 365-370. O R Y G I N A L N E po ł o ż n i c t wo. Monica Regina Lourenço Luz et al. Histomorphometry and detection of glycosaminoglycans in the endocervical epithelium.... 2. Histochemical Analysis. Discussion. 2.1 alcian blue reaction at pH 0.5 2QO\ VXOIDWHG *$* VWDLQHG EOXH LQ WKLV UHDFWLRQ ,Q WKH HQGRFHUYLFDO HSLWKHOLXP D VWURQJO\ SRVLWLYH UHDFWLRQ

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(44) . A. B. Figure 2. Staining of the endocervical epithelium of the cervix of a rat from the control group (A) and from the study group (B) with alcian blue reaction at pH 0.5 (400X).. A. B. Figure 3. Staining of the endocervical epithelium of the cervix of a rat from the control group (A) and the study group (B), with alcian blue reaction at pH 2.5 (400X).. Nr 5/2014. © Polskie Towarzystwo Ginekologiczne. 369.

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Conclusion ,QVXPPDU\RXUVWXG\DOORZVXVWRFRQFOXGHWKDWPRGL¿FDWLRQV HYLGHQFHGLQWKHHQGRFHUYLFDOHSLWKHOLXPVXSSRUWWKHFRQFHSWWKDW K\DOXURQLGDVH LQMHFWHG ORFDOO\ SURPRWHG DOWHUDWLRQV FRQVLVWHQW ZLWKFHUYLFDOPDWXUDWLRQ Authors’ Contribution 1. Monica Regina Lourenco Luz – acquisition of data. 2. Edward Araujo Junior – article draft, corresponding author. 3. Luiz Camano – revised article critically. 4. Manuel de Jesus Simoes – analysis and interpretation of data. 5. Eduardo de Souza – concept, study design. Authors’ statement ³ >RS] S] ^Y MO\^SPc ^RK^ ^RO Z_LVSMK^SYX aSVV XY^ `SYVKÔ ^RO MYZc\SQR^] YP K ^RS\N. party, as understood according to the Act in the matter of copyright and related rights of 14 February 1994, Official Journal 2006, No. 90, Clause 63, with respect to the text, data, tables and illustrations (graphs, figures, photographs); ³ ^RO\O S] XY mMYX»SM^ YP SXÔ\O]^]p aRSMR YMM_\] aROX ^RO K_^RY\ \OWKSX] SX. a financial or personal relationship which unjustly affects his/her actions associated with the publication of the manuscript; ³ KXc ZY]]SLVO \OVK^SYX]RSZ] YP ^RO K_^RY\] aS^R ^RO ZK\^cZK\^SO] SXÔ\O]ÔN. in the publication of the manuscript are revealed in the text of the article; ³ ^RO WKX_]M\SZ^ RK] XY^ LOOX Z_LVS]RON SX Y\ ]_LWS^ÔN ^Y KXc Y^RO\ TY_\XKV. ³ =Y_\MO YP ºXKXMSXQ$ >RO K_^RY\] NOMVK\O XY ]Y_\MO]. References 1. Koong D, Evans S, Mayers C, [et al.]. A scoring system for prediction of successful delivery in low risk birthing units. 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Morphological and morphometric changes in the cervix uteri of the rat at term pregnancy induced by hyaluronidase. Clin Exp Obstet Gynecol. 2004, 31, 225-228. 9. Hamilton JB, Wolfe JM. The effect of male hormone substance upon birth and prenatal development in the rat. Anat Rec. 1938, 70, 433-440. 10. Spallicci MD, Chiea MA, Singer JM, [et al.]. Use of hyaluronidase for cervical ripening: a randomized trial. Eur J Obst Gynecol Reprod Biol. 2007, 130, 46-50. 11. Lev R, Spicer SS. Specific staining of sulfate groups with alcian blue at low pH. J Histochem Cytochem. 1964, 12, 309. 12. Spicer SS, Lillie RD. Saponification as a means of selectively reversing the methylation blockade of tissue basophilia. J Histochem Cytochem. 1959, 7,123-125. 13. Winkler M, Rath W. Changes in the cervical extracellular matrix during pregnancy and parturition. J Perinat Med. 1999, 27, 45-61. 14. Ludmir J, Sehdev HM. Anatomy and Physiology of the Uterine Cervix. Clin Obstet Gynecol. 2000, 43, 433-439. 15. 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