N-acetylcysteine improves morphologic and functional aspects of ovarian grafts in rats1 1 Research performed at Microsurgical Laboratory, Operative Technique and Experimental Surgery, Department of Surgery, Federal University of São Paulo, São Paulo, Brazil. Master thesis at the Interdisciplinary Surgical Science Post Graduate Program.

Elivânia Marques Gomes de Amorim Luciana Lamarão Damous Maria Clara Silva Durando Márcia Viviane Alves Saraiva Marcia Kiyomi Koike Edna Frasson de Souza Montero About the authors

Abstract

PURPOSE:

To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC.

METHODS:

Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test.

RESULTS:

At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation.

CONCLUSION:

In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.

Transplantation; Autologous; Acetylcysteine; Histology; Estrogen; Ovarium; Rats


INTRODUCTION

Life expectation of adult women, who survived adolescence or infancy cancer treatment has increased, thanks to early diagnostic and treatment in oncologic centers1Callejo J, Salvador C, Miralles A,Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab.2001;86:4489-94. , 2Akar M, Oktay K. Restoration of ovarian endocrine function by ovarian transplantation. Trends Endocrinol Metab. 2005;16(8):374-80.. Early menopause induced by cancer treatment is often followed by osteoporosis, early cardiovascular diseases and infertility, besides a prolonged hormonal reposition treatment requirement (TRH)1.

There are several alternatives aiming to reduce the adverse effects of chemotherapy and radiotherapy on ovaries, such as the transposition (temporary removal) of ovaries, also known as oophorectomy, the use of antagonistic drugs to Gonadotropin Releasing Hormones (GnRH) together with chemotherapy and new apoptosis antagonistic drugs3Bedaiwy MA, Shahin AY, Falcone T. Reproductive organ transplantation: advances and controversies. Fertil Steril. 2008;90(6):2031-55.. For those patients, who need immediate oncologic treatment, a promising option is the oophorectomy, the cryopreservation of ovaries and its posterior re-implantation, after the end of treatment1Callejo J, Salvador C, Miralles A,Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab.2001;86:4489-94. . For the preservation of treated women fertility, several techniques may be used: some already widely used, such as cryopreservation of embryos, mature or premature follicles, others are still experimental, such as the transplantation of ovarian tissue3Bedaiwy MA, Shahin AY, Falcone T. Reproductive organ transplantation: advances and controversies. Fertil Steril. 2008;90(6):2031-55. , 4Israely T, Dafni H, Granot D, Nevo N, Tsafriri A, Neeman M. Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts. Biol Reprod. 2003;68(6):2055-64..

Ovarian transplantation has been studied in several experiments, both on animals and humans, and has been described as a promising alternative for those patients, who will be submitted to oncologic treatment5Liu L, Wood GA, Morikawa L, Ayearst R, Fleming C, McKerlie C. Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries. Human Reprod. 2008;23(1):122-8. , 6Schmidt KLT, Andersen CY, Loft A, Byskov AG, Ernst E, Andersen NA. Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantatio. Human Reprod. 2005;20(12):3539-46.. Even thought, until now, it has been described a short survival time of the transplanted ovarian, with loss of more than 50% of the ovarian follicles after transplantation5Liu L, Wood GA, Morikawa L, Ayearst R, Fleming C, McKerlie C. Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries. Human Reprod. 2008;23(1):122-8.. Several factors are involved in this problem, but ischemia seems to play an important role in this process.1Callejo J, Salvador C, Miralles A,Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab.2001;86:4489-94. , 5Liu L, Wood GA, Morikawa L, Ayearst R, Fleming C, McKerlie C. Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries. Human Reprod. 2008;23(1):122-8.

Some studies have been performed, in search for strategies for the attenuation of the ischemia and reperfusion lesion (I/R): angiogenic factors, which stimulate new vessels formation, antioxidating substances, such as E vitamin, GnRH analogous and ischemic preconditioning7Damous LL, Silva SM, Carbonel APF, Simões RS, Simões MJ, Montero EFS. Effect of Remote Ischemic Preconditioning on Rat Estradiol Serum Levels and Follicular Development After Ovarian Transplantation 2009. Transpl Proc. 41:830-3.

Yang HY, Cox SL, Jenkin G, Findlay J, Trounson A, Shaw J. Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts. Reproduction. 2006;131(5):851-9.

Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil.1998; 114(2):341-6.
- 1010 Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril. 2004; 82(3):679-85..

Some previous works have shown the positive effect of N-Acetylcysteine (NAC) at 150 mg/kg dose on various organs. NAC is a low molecular weight thiol, it is derived from cysteine and acts removing reacting species of oxygen, besides improving the action of nitric oxide, acting on the regulation of microcirculation1111 Montero EF, Quireze C Jr., D'Oliveira DM. Bile duct exclusion from selective vascular inflow occlusion in rat liver: role of ischemic preconditioning and N-acetylcysteine on hepatic reperfusion injury. Transplant Proc. 2005;37:425-7.

12 Franco RK, Zumioth AV, Kume MH, Seyboth CD, Marcolini DR. Study on the effect of N-acetylcystein on fasciocutaneous flaps submitted to ischemia and reperfusion: experimental model in rats. Rev Bras Ortop. 2006;41(11/12):474-82.
- 1313 Galhardo MA, Júnior CQ, Riboli Navarro PG, Morello RJ, De Jesus Simões M, De Souza Montero EF. Liver and lung late alterations following hepatic reperfusion associated to ischemic preconditioning or N-acetylcysteine. Microsurgery. 2007;27(4):295-9.. Usta et al.14 14 Usta U, Inan M, Erbas H, Aydogdu N, Puyan OF, Altaner S. Tissue damage in rat ovaries subjected to torsion and detorsion: effects of L-carnitine and N-acetyl cysteine. Pediatr Surg Int. 2008; 24:567-73.showed NAC improved tissue recovering in ovaries, which had been submitted to torsion and distortion. Some other studies use NAC in polycystic ovarian syndrome patients, resulting in improved sensibility to insuline1515 Masha A, Manieri C, Dinatale S, Bruno GA, Ghigo E, Martina V. Prolonged treatment with N-acetylcysteine and L-arginine restores gonadal function in patients with polycystic ovary syndrome. J Endocrinol Invest. 2009;32(11):870-2. , 1616 Nasr A. Effect of N-acetyl-cysteine after ovarian drilling in clomiphene citrate-resistant PCOS women: a pilot study. Reproductive BioMedicine Online. 2010; 20,403-9..

Considering the positive effects of NAC in clinical or experimental studies, this work aims to evaluate the post transplant effect of NAC on ovarian graft in rats treated with N-Acetylcysteine.

METHODS

This project was approved by the board of the Research Ethics Committee, UNIFESP, under protocol 0907/2010. Procedures have been performed at the laboratory of experimental microsurgery of UNIFESP.

Female Wistar rats, virgins, age 3 to 4 months, weight 200 to 250 grams have been used. All animals came from the Center for Experimental Models Developing for Biology and Medicine, UNIFESP. Animals were kept in proper sanitary conditions, having their environmental temperature and humidity checked, water and food always available along all experiment.

Daily, vaginal smears were performed during 8 days before of transplantation procedures to evaluate the estral cycle. Females with regular cycle were selected, and ovarian transplantations were performed during diestrum. After transplantation, vaginal smears were performed daily and euthanasia was performed during estrum or proestrum stages. During follow-up of four or 15 days, vaginal smears were performed to determine cycle regularity return.

One hour before ovarian transplantation, animals were randomly allocated into five groups, with 10 animals each, according to the treatment. Four groups were treated subcutaneously - along the linea alba - with NAC, at doses of 150mg/kg, 300mg/kg, 600mg/kg or 1200mg/kg - NAC150, NAC300, NAC600 and NAC1200 groups, respectively. One group was treated with 0.9% Sodium chloride solution and was considered control, named GTx.

The autologous left ovarian transplantation was performed in anesthetized rat (Xilazine, 15 mg/kg and Ketamine, 60 mg/kg). The anaesthesia was applied on the lateral side of the left hind leg, intramuscularly.

After medial laparotomy, the left ovarian was carefully removed, through a ligation of the pedicle, and on the junction of the left horn, using 6-0 size nylon. The left ovarian was washed using 0.9% Sodium chloride solution and uterine tubes and periovarian fat were removed. To ovarian transplantation, the left ovarian was fixed in retroperitoneal position, close to the large vessels and, without vascular anastomosis. The right ovarian was removed and anatomical-pathological evaluation was conducted to exclude previous existing disorders.

At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment, respectively.

The ovarian graft was fixed in 10% formalin and embedded to paraffin to histological procedures. Five micra slides were stained with haematoxylin-eosin and assessed by blinded investigator.

For the evaluation of follicular development, ovarian follicles were counted and classified in developing follicles, independently of their stages and atretic follicles, which were subdivided according to the degree of maturity into immature follicles (including primordial, preantral follicles) and antral follicles (with only one bulky antrum) besides the corpora lutea1717 D'Acampora AJ, Tramonte R, Manoel FS, Schmidt RR, Muller CT, Silva HT, Oliveira A, Raymundi SD. Histological analysis of the viability of half ovary autologous transplantation in retroperitoneal of rats. Acta Cir Bras. 2004; 19(4):318-26. . Measurements were obtained with the image analysis program AxionVision REL 4.6, Karl Zeiss. Counting was always performed in 4 fields per animal at 10x magnification.

Data are presented as mean±SED or median (interquartile range). The comparison among groups in the same time was analyzed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks, when appropriated, complemented by Student-Newman-Keuls test. Comparison between studied times was analyzed by Mann-Whitney test. Differences were considered for p<0.05.

RESULTS

After 4 or 14-16 days of follow-up, serological levels of estradiol were similar among groups (p=0.051 and p=0.51, respectively).

At 4th day after transplantation, ovarian graft morphology indicated that NAC treatment promoted high numbers of atretic follicles in NAC300 and NAC600 groups (Figure 1). Primordial follicle, preantral follicle, antral follicle and functioning and degenerated corpora lutea were similar among groups.

Figure 1
Number of atretic follicle on 4th-day of autologous ovarian transplantation in rats. Rats previously treated with NAC at dosis of 300 and 600 mg/kg (NAC300 and NAC600, respectively) augmented the number of atretic follicle in the graft. One way analysis of variance complemented by Student-Newman-Keuls test; *, p

At 14th day after transplantation, ovarian graft morphology indicated that NAC treatment promoted high numbers of primordial follicles in NAC600 and NAC1200 groups (Figure 2). Preantral follicle, antral follicle, atretic follicle, functioning and degenerated corpora lutea were similar among groups.

Figure 2
Number of primordial follicle on 14th-day of autologous ovarian transplantation in rats. Rats previously treated with NAC at dosis of 600 and 1200mg/kg (NAC600 and NAC1200, respectively) showed elevation in number of primordial follicle in graft. One way analysis of variance complemented by Student-Newman-Keuls test; *, p

The comparison between two intervals showed that high number of atretic follicle and antral follicle in GTx group in 14th day. NAC promoted a better preservation of viable follicles on latest time (14th day after surgery), showing significant results on those groups NAC300 and NAC600 (Table 1; Figure 3).

Table 1
Follicles and Corpora Lutea after ovarian transplantation in groups treated with NAC in different doses.

Figure 3
Photomicrographs of ovarian grafts of the groups at 14 days : GTx (A), NAC150 (B), NAC300 (C), NAC600(D), NAC1200 (E). HE - 10 X. F=Follicle CL=Corpus luteum

Comparing the response of the animals at 4 and 14 days after surgery, the animals, which had been treated with NAC1200 showed a better tissue preservation on later stage, with a significant higher number of premature follicles (p=0.018). In all treatment groups at day 14 after transplantation there was an increase in the number of functional corpora lutea, but this was significant just in the group treated with NAC150 (p=0.02). NAC 600 group showed a tendency instead (p=0.074). For what concerns to the degradation of the transplants, NAC treatment promoted a better preservation of the corpus luteum, reducing the degenerative process at the later analysis stage, on day 14.

DISCUSSION

Experimental ovarian transplantation has been performed for some years: autologous, heterologous, with or without vascular pedicle, both in humans and laboratory animals. This operation has been performed in different sites of implantation, and the implants have been functional, both functionally and morphologically1Callejo J, Salvador C, Miralles A,Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab.2001;86:4489-94. , 3Bedaiwy MA, Shahin AY, Falcone T. Reproductive organ transplantation: advances and controversies. Fertil Steril. 2008;90(6):2031-55. , 1818 Fabbri R, Pasquinelli G, Montanaro L, Mozzanega B, Magnani V, Tamburini F, Venturoli S, Keane D. Healthy early preantral follicle can be obtained in a culture of frozenthawed human ovarian tissue of 32 weeks. Ultrastruct Pathol. 2007;31(4):257-62..

At transplantation, half of the primordial follicles are lost, persisting until the stage of neoangiogenesis5Liu L, Wood GA, Morikawa L, Ayearst R, Fleming C, McKerlie C. Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries. Human Reprod. 2008;23(1):122-8.. Many studies have tried to preserve the ovarian tissue from I/R lesion, using substances such as E vitamin, ascorbic acid, and GnRH analogous, or physical strategies including the ischemic preconditioning2Akar M, Oktay K. Restoration of ovarian endocrine function by ovarian transplantation. Trends Endocrinol Metab. 2005;16(8):374-80. , 8Yang HY, Cox SL, Jenkin G, Findlay J, Trounson A, Shaw J. Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts. Reproduction. 2006;131(5):851-9.

Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil.1998; 114(2):341-6.
- 1010 Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril. 2004; 82(3):679-85..

Some work have already used NAC at different doses on several organs, such as kidney, liver, lungs, fasciocutaneous flap, intestine and ovarian, showing good results on I/R lesions1212 Franco RK, Zumioth AV, Kume MH, Seyboth CD, Marcolini DR. Study on the effect of N-acetylcystein on fasciocutaneous flaps submitted to ischemia and reperfusion: experimental model in rats. Rev Bras Ortop. 2006;41(11/12):474-82.

13 Galhardo MA, Júnior CQ, Riboli Navarro PG, Morello RJ, De Jesus Simões M, De Souza Montero EF. Liver and lung late alterations following hepatic reperfusion associated to ischemic preconditioning or N-acetylcysteine. Microsurgery. 2007;27(4):295-9.
- 1414 Usta U, Inan M, Erbas H, Aydogdu N, Puyan OF, Altaner S. Tissue damage in rat ovaries subjected to torsion and detorsion: effects of L-carnitine and N-acetyl cysteine. Pediatr Surg Int. 2008; 24:567-73. , 1818 Fabbri R, Pasquinelli G, Montanaro L, Mozzanega B, Magnani V, Tamburini F, Venturoli S, Keane D. Healthy early preantral follicle can be obtained in a culture of frozenthawed human ovarian tissue of 32 weeks. Ultrastruct Pathol. 2007;31(4):257-62.

19 Montero EFS, Abrahão MS, Koike MK, Manna MCB, Ramalho CEB. Intestinal ischemia and reperfusion injury in growing rats: hypothermia and N-acetylcysteine modulation. Microsurgery. 2003;23:517-21.

20 Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci. 2003;60(1):6-20.

21 Danilovic A, Lucon AM, Srougi M, Shimizu MHM, Lanhez LE, Nahas WC, Seguro AC. Protective effect of N-acetylcysteine on early outcomes of deceased renal transplantation. Transplant Proc. 2011;43: 1443-9.
- 2222 Millea PJ. N-Acetylcysteine: Multiple clinical applications. Am Fam Physician. 2009;80:265-9..

So in order to mitigate the damage and I/R as the first study in ovarian transplantation, it was decided to give NAC subcutaneously, in order to have a better control over the given volumes and at different doses to determine the relation between the doses and the effect on the ovarian tissue.

There are further applications of NAC, besides its mucolytic activity, in the prevention of obstructive chronic lung disease, in the prevention of kidney complications during contrast procedures, in the reduction of viral flu and pulmonary fibrosis. Furthermore, NAC gave good results on fertility of patients with clomifen-resisting polycystic ovarian2323 Badawy A, State O, Abdelgawad S. N-Acetyl cysteine and clomiphene citrate for induction of ovulation in polycystic ovary syndrome: A crossover trial. Acta Obstet Gynecol Scand. 2007;86:218-22.. Recent studies have also suggested the use of NAC as chemiopreservative against cancer, auxiliary in the eradication of Helicobacter pylori, in the hearing loss caused by gentamycin in kidney dialysis patients, HIV infection, cardiac diseases, smoke and epileptic2222 Millea PJ. N-Acetylcysteine: Multiple clinical applications. Am Fam Physician. 2009;80:265-9.. NAC used for long time (5-6 weeks) improved the sensibility to insulin, testosterone levels and lipidic profile in women affected by polycystic ovarian syndrome2424 Löhrke B, Xu J, Weitzel JM, Krüger B, Goldammer T, Viergutz T. Acetylcysteine impairs survival of luteal cells through mitochondrial dysfunction cytometry. 2010;77(4):310-20..

A study realized on polycystic ovarian syndrome patients, which compared the effect of metformin to those of the association metformin + NAC for short times (5 days), showed NAC is effective in inducing ovulation, even if the number of mature follicles was not significant, as it was confirmed in our experience2323 Badawy A, State O, Abdelgawad S. N-Acetyl cysteine and clomiphene citrate for induction of ovulation in polycystic ovary syndrome: A crossover trial. Acta Obstet Gynecol Scand. 2007;86:218-22. , 2525 Elnashar A, Fahmy M, Mansour A, Ibrahim K. N-Acetyl cysteine vs. metformin in treatment of clomiphene citrateresistant polycystic ovary syndrome: A prospective randomized controlled study. Fertil Steril. 2007;88:406-9..

Oktay et al.2626 Oktay K, Newton H, Gosden RG. Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril. 2000;73(3):599-603. observed the levels of estradiol in the xenotransplantation and found signals of a estrogenic effect in the genitalia, even if there was a lower number or absence of growth of antral follicles. This observation confirms, genital organs of rodents are sensitive to low levels of sexual steroids2Akar M, Oktay K. Restoration of ovarian endocrine function by ovarian transplantation. Trends Endocrinol Metab. 2005;16(8):374-80. , 2626 Oktay K, Newton H, Gosden RG. Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril. 2000;73(3):599-603., data corroborates our results, as we detected low levels of estrogen in all transplanted groups, despite the histology showed graft functionality in the later phase. Callejo et al.2727 Callejo J, Vilaseca S, Ordi J, Cabré S, Lailla JM, Balasch J. Heterotopic ovarian transplantation without vascular pedicle in syngeneic Lewis rats: long-term evaluation of effects on ovarian structure and function. Fertil Steril. 2002; 77(2):396-402., analyzing the longevity of the grafts and estradiol levels, suggest, estradiol and FSH are reliable markers of the transplant functionality. The same authors, analyzing intraperitoneal and subcutaneous implantation of ovarian transplantations, found high estradiol levels and low FSH levels in the intraperitoneal transplantation of ovarians after 30 days2727 Callejo J, Vilaseca S, Ordi J, Cabré S, Lailla JM, Balasch J. Heterotopic ovarian transplantation without vascular pedicle in syngeneic Lewis rats: long-term evaluation of effects on ovarian structure and function. Fertil Steril. 2002; 77(2):396-402.. Li et al.2828 Li F, Tao Y, Zhang Y, Li Y, Fang F, Liu Y, Cao H, Zhang X, Zhou S. Follicle growth and oocyte development back muscle of immune-intact adult castrated male mice. Reproduction. 2010;140(3):465-76. also describe an elevation of estradiol levels studying follicular growth and oocyte development after transplantation in muscle of spayed male mice.

D'Acampora et al.1717 D'Acampora AJ, Tramonte R, Manoel FS, Schmidt RR, Muller CT, Silva HT, Oliveira A, Raymundi SD. Histological analysis of the viability of half ovary autologous transplantation in retroperitoneal of rats. Acta Cir Bras. 2004; 19(4):318-26. analysed the transplantation in female rats and perceived, on the 7th day the recovery of the follicular production, which persisted until the 14th day, with the presence of follicles in 75% of the ovaries. Israely et al.4Israely T, Dafni H, Granot D, Nevo N, Tsafriri A, Neeman M. Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts. Biol Reprod. 2003;68(6):2055-64. implanted grafted ovarian transplants in granulation tissues and showed better vascularization and an higher number of viable follicles, which may serve as a model to reduce the ischemic period and to extended the functionality of the transplant. In this same study NAC was important on the latest stage, protecting the follicles from being atretic, with an anti-apoptosis effect.

Damous et al.7Damous LL, Silva SM, Carbonel APF, Simões RS, Simões MJ, Montero EFS. Effect of Remote Ischemic Preconditioning on Rat Estradiol Serum Levels and Follicular Development After Ovarian Transplantation 2009. Transpl Proc. 41:830-3. showed a tendency to the increase of the follicles number at 48 hours using PCI-R. It is important to stress the direct correlation existing between follicle size and its susceptibility to damages caused by ischemia: antral follicle has an higher tendency to degeneration than primordial follicle. According to Damous et al.7Damous LL, Silva SM, Carbonel APF, Simões RS, Simões MJ, Montero EFS. Effect of Remote Ischemic Preconditioning on Rat Estradiol Serum Levels and Follicular Development After Ovarian Transplantation 2009. Transpl Proc. 41:830-3., PCI-R showed positive effects at the 5th day after surgery, with the presence of ovarian follicles at different development stages and corpus luteum, even if an intense inflammation infiltrate was present. D'Acampora et al.1717 D'Acampora AJ, Tramonte R, Manoel FS, Schmidt RR, Muller CT, Silva HT, Oliveira A, Raymundi SD. Histological analysis of the viability of half ovary autologous transplantation in retroperitoneal of rats. Acta Cir Bras. 2004; 19(4):318-26. showed similar findings.

Fabbri et al.1818 Fabbri R, Pasquinelli G, Montanaro L, Mozzanega B, Magnani V, Tamburini F, Venturoli S, Keane D. Healthy early preantral follicle can be obtained in a culture of frozenthawed human ovarian tissue of 32 weeks. Ultrastruct Pathol. 2007;31(4):257-62. studied ovarian tissue from a woman, who suffered breast cancer at the age of 26: after freezing, defrosting and 32 weeks growth, the authors could observe the synergy between NAC and FSH, with improvement on growth and preservation of the pre-antral follicle. On follicular growth, after some days of development, the follicle shows some signals of atresy. In vitro studies have produced the complete development from primordial follicle to ovulatory mature follicle just in mice. In humans, the same studies have only produced the maturation from pre-antral to antral stage and from primordial to primary and secondary follicles2525 Elnashar A, Fahmy M, Mansour A, Ibrahim K. N-Acetyl cysteine vs. metformin in treatment of clomiphene citrateresistant polycystic ovary syndrome: A prospective randomized controlled study. Fertil Steril. 2007;88:406-9.. The return to gonad functionality on long term ovarian transplantation depends on the survival and growth of the follicles, which itself depends, from the capacity to ovulate2929 Fulghesu AM, Ciampelli M, Muzj G, Belosi C, Selvaggi L, Ayala GF. N-Acetyl cysteine treatment improves insulin sensitivity in women with polycystic ovary syndrome. Fertil Steril. 2002;77:1128-35..

CONCLUSION

In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.

REFERENCES

  • 1
    Callejo J, Salvador C, Miralles A,Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab.2001;86:4489-94.
  • 2
    Akar M, Oktay K. Restoration of ovarian endocrine function by ovarian transplantation. Trends Endocrinol Metab. 2005;16(8):374-80.
  • 3
    Bedaiwy MA, Shahin AY, Falcone T. Reproductive organ transplantation: advances and controversies. Fertil Steril. 2008;90(6):2031-55.
  • 4
    Israely T, Dafni H, Granot D, Nevo N, Tsafriri A, Neeman M. Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts. Biol Reprod. 2003;68(6):2055-64.
  • 5
    Liu L, Wood GA, Morikawa L, Ayearst R, Fleming C, McKerlie C. Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries. Human Reprod. 2008;23(1):122-8.
  • 6
    Schmidt KLT, Andersen CY, Loft A, Byskov AG, Ernst E, Andersen NA. Follow-up of ovarian function post-chemotherapy following ovarian cryopreservation and transplantatio. Human Reprod. 2005;20(12):3539-46.
  • 7
    Damous LL, Silva SM, Carbonel APF, Simões RS, Simões MJ, Montero EFS. Effect of Remote Ischemic Preconditioning on Rat Estradiol Serum Levels and Follicular Development After Ovarian Transplantation 2009. Transpl Proc. 41:830-3.
  • 8
    Yang HY, Cox SL, Jenkin G, Findlay J, Trounson A, Shaw J. Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts. Reproduction. 2006;131(5):851-9.
  • 9
    Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil.1998; 114(2):341-6.
  • 10
    Kim SS, Yang HW, Kang HG, Lee HH, Lee HC, Ko DS. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Steril. 2004; 82(3):679-85.
  • 11
    Montero EF, Quireze C Jr., D'Oliveira DM. Bile duct exclusion from selective vascular inflow occlusion in rat liver: role of ischemic preconditioning and N-acetylcysteine on hepatic reperfusion injury. Transplant Proc. 2005;37:425-7.
  • 12
    Franco RK, Zumioth AV, Kume MH, Seyboth CD, Marcolini DR. Study on the effect of N-acetylcystein on fasciocutaneous flaps submitted to ischemia and reperfusion: experimental model in rats. Rev Bras Ortop. 2006;41(11/12):474-82.
  • 13
    Galhardo MA, Júnior CQ, Riboli Navarro PG, Morello RJ, De Jesus Simões M, De Souza Montero EF. Liver and lung late alterations following hepatic reperfusion associated to ischemic preconditioning or N-acetylcysteine. Microsurgery. 2007;27(4):295-9.
  • 14
    Usta U, Inan M, Erbas H, Aydogdu N, Puyan OF, Altaner S. Tissue damage in rat ovaries subjected to torsion and detorsion: effects of L-carnitine and N-acetyl cysteine. Pediatr Surg Int. 2008; 24:567-73.
  • 15
    Masha A, Manieri C, Dinatale S, Bruno GA, Ghigo E, Martina V. Prolonged treatment with N-acetylcysteine and L-arginine restores gonadal function in patients with polycystic ovary syndrome. J Endocrinol Invest. 2009;32(11):870-2.
  • 16
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  • 1
    Research performed at Microsurgical Laboratory, Operative Technique and Experimental Surgery, Department of Surgery, Federal University of São Paulo, São Paulo, Brazil. Master thesis at the Interdisciplinary Surgical Science Post Graduate Program.

Publication Dates

  • Publication in this collection
    2014
Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia Al. Rio Claro, 179/141, 01332-010 São Paulo SP Brazil, Tel./Fax: +55 11 3287-8814 - São Paulo - SP - Brazil
E-mail: sgolden@terra.com.br