italian journal of gynaecology & obstetrics · 1 gynaecology italian journal of the official...

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GynaecologyItalian Journal

of

The Official Journal of theSocietà Italiana di Ginecologia e Ostetricia

(SIGO)

Quarterly

& Obstetrics

Partner-Graf

Dec

embe

r 201

6 - V

ol. 2

8 - N

. 5 -

Qua

rter

ly -

ISSN

238

5 - 0

868

Gynaecology

Italian Journalof

The Official Journal of theSocietà Italiana di Ginecologia e Ostetricia

(SIGO)

Quarterly

& Obstetrics

Partner-Graf

Editor in Chief

Paolo Scollo, Catania

Editors

Herbert Valensise, Roma Enrico Vizza, Roma

Editorial Board

Cervigni Mauro, RomaChiantera Vito, NapoliCosta Mauro, GenovaDe Stefano Cristofaro, AvellinoDe Vita Davide, SalernoLa Sala Giovanni Battista, Reggio EmiliaLocci Maria Vittoria, NapoliMarci Roberto, RomaMonni Giovanni, CagliariRagusa Antonio Franco, MilanoSirimarco Fabio, NapoliTrojano Vito, BariViora Elsa, Torino

Editorial Staff

Roberto Zerbinati Serena Zerbinati

Management, Administrative officePartner-Graf Srl - Via F. Ferrucci, 73 - 59100 PratoTel 0574 527949 - Fax 0574 636250E-mail: [email protected]

The Italian Journal of Gynaecology & Obstetrics is a digital magazine.You can download it freely fromwww.italianjournalofgynaecologyandobstetrics.com orwww.italianjog.com

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Table of contents

Editorial

Haptoglobin gene polymorphism and oxidative stress effects on ovarian reservein Beta-Thalassemia major womenSally S. El Tawab, Nadia A. Sadek, Maher A. Kamel, Khaled S. Salem

Ovarian reserve biomarkers usefulness for optimization of counselling in a public network for fertility preservation in oncological patientsMarta Manno, Francesco Tomei, Fabio Puglisi, Francesco Zaja, Paolo Metus, Renato Tozzoli, Maurizio Mascarin, Massimo Manno

Laparoscopic Indocyanine green sentinel lymph node mapping in early ovarian cancer. A pilot study and review of the literatureMichela Angelucci, Giacomo Corrado, Emanuela Mancini, Ermelinda Baiocco, Benito Chiofalo, Ashanti Zampa, Arabella Bufalo, Enrico Vizza

It. J. Gynaecol. Obstet.2016, 28: N.2

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It. J. Gynaecol. Obstet.2016, 28: N. 5

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Editorial

Dear SIGO members,

As I send my final editoral as SIGO President, I would first like to express my sincere gratitude to all the members of the past Council and all the staff that have collabotated with me during these three years. It was a great honor for me to have served SIGO and its members over the past three years rappresenting the Italian Gyneacologists in the Istitution both national as well international, improving the standards of care in Ostetrics and gynacology through education, training and public awareness.

The past SIGO Congress of Rome-October 2016, was a great succes both from the scientific point of view as well as for the numbers we had: over 1,700 attendees, 515 members of faculty, 352 exposition stands. More than 80% of interviewed defined the event from interesting to relevant from scientific point of view. A selection of abstracts are published as supplement of present issue.

In Rome-2016, during the SIGO general assembly, I had already the opportunity to report in detail the SIGO work in the last three years during my Presidential mandate, but here I would like to remember what has been done for the new course of Italian Journal of Ostetrics & Gynaecology that has been trasformed from printed version to on line magazine. Another very important iniziative that take place during the last three years as been the renovation and restiling of SIGO web-site making it a more flexible instrument of information for our society. Please visit www.SIGO.IT now to explore the features and become acquainted with the new SIGO-site.

In closing, I wish to the new President Prof. Giovanni Scambia and the new Counsil a good, and satisfactory work in the process of renovation of our Society making SIGO more and more intergrate with the international societies as well as more rapresentative in the Italian institution.

I am sure that an important instrument in this process is and will be The Italian Journal of Ostetrics & Gynaecology.

All the best for 2017 and for SIGO!

Prof. Paolo ScolloS.I.G.O. President

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Haptoglobin gene polymorphism and oxidative stress effects on ovarian reserve in Beta-Thalassemia major womenDr. Sally S. El Tawab1, Dr. Nadia A. Sadek2, Dr. Maher A. Kamel3, Dr. Khaled S. Salem4

1 Lecturer of Obstetrics and Gynecology, Faculty of Medicine, Alexandria University, Shatby MaternityUniversity Hospital, Alexandria, Egypt

2 Professor of Hematology, Medical Research Institute, Alexandria University, Alexandria, Egypt3 Assistant Professor of Biochemistry, Medical Research Institute, Alexandria University, Alexandria,

Egypt4 Consultant of Haematology, Gamal Abel Naser Hospital, Health Insurance Organization, Alexandria,

Egypt

ABSTRACTObjective: to study the impact of haptoglobin gene polymorphism on iron overload, oxidative stress and anti-mullerian hormone in BTM women in Egypt.Methods: case-control study. 47 BTM women, aged between 16-26 years and 47 age-matched regularly menstruating women as control. Haptoglobin Hp1/2 gene polymorphism by PCR, Hemoglobin electrophoresis, serum haptoglobin, ferritin, malondialdehyde MDA, FSH, LH, estrogen and ant-mullerian hormone levels were all analyzed. Results: serum ferritin level and Serum MDA, showed a significant higher level in thalassemic group, p<0.001. Significant lower levels of LH, E2 and AMH were found in thalassemic group compared to the control p<0.001. A significant difference (p<0.05) between both groups as regarding distribution of haptoglobin gene polymorphism was detected. In thalassemic group, Hp2-2 was the most frequent. (odds ratio 2.616 at 95% CI, 1.134-6.033). A significantly lower AMH level in patients with Hp2-2 gene polymorphism. Conclusion: beta thalassemic patients are at great risk of oxidative stress. Haptoglobin gene polymorphism has a great impact on the extent of oxidative stress in these patients. BTM patients have lower serum AMH levels than the age -matched control. AMH significantly negatively correlated with serum ferritin and MDA. This was more evident in patients with HP2-2 genotype.

Keywords: thalassemia, haptoglobin gene polymorphism, oxidative stress, ovarian reserve, MDA, anti-mullerian hormone.

Corresponding to: [email protected] 2016, Partner-Graf srl, PratoDOI: 10.14660/2385-0868-54

Gynaecology & ObstetricsItalian Journal of

December 2016 - Vol. 28 - N. 5 - Quarterly - ISSN 2385 - 0868

SOMMARIOObiettivo: studiare l’impatto di aptoglobina polimorfismo del gene sul sovraccarico di ferro, lo stress ossidativo e anti-mullerian ormone in beta-thalassemia major de donne in Egitto.Metodi: caso-controllo in studio terziario centro medico. 46 beta-major di donne di eta compresa tra i 16-26 anni sono stati inclusi nello studio. 47 pari età sani durante il periodo mestruale regolarmente le donne e servitor come controllo. Aptoglobina Hp1/2 polimorfismo del gene mediante PCR, emoglobina elettroforesi, siero aptoglobina, ferritina,la malondialdeide MDA livello, FSH, LH, estrogeni e anti-mullerian i livelli ormonali: sono stati analizzati tutti. Risultati: livelli siericidi di ferritina sierica e MDA, ha monstrato un livello significantivamente superior nel gruppo thalassemia, p<0.001. Abbassamento significativo dei livelli di LH, E2 e AMH sono stati trovati in thalassemia gruppo rispetto al controllo p<0.001. Una differenza significativa p<0.05 fra i due gruppi per quanto riguarda la distribuzione di aptoglobina polimorfismo del gene e stata rilevata. In thalassemic grappo, Hp 2-2 e stato il piu frequente e seguita da Hp 2-1 quindi Hp 1-1 (odds ratio 2.616 al 95% CI 1.134-6.033). Un significativamente inferior livello di AMH in pazienti con Hp 2-2 poliorfismo del gene. Conclusione: o pazienti Beta thalassemi sono a grande rischio di stress ossidativo. Aptoglobina poliformismo del gene ha un grande impatto sulla misura di stress ossidative in questi pazienti. BTM pazienti hanno inferiore AMH nel siero livelli rispetto a quelli dell’eta-adattato per il controllo. AMH significativamente correlate negativament con la ferritina sierica e MDA. Questo e stato pui evidente nei pazienti con Hp2-2 genotipo.


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Ovarian reserve in Beta-Thalassemia

INTRODUCTIONBeta thalassemia major β-TM is the most

common genetically-determined chronic haemolytic anaemia in Egypt, as in many other Mediterranean countries. It has been estimated that 1000 children out of 1.5 million live births are born annually with β-TM, with a carrier rate of 9-10%(1). β -thalassemia occurs when there is a quantitative reduction of β globin chains that are usually structurally normal. They are caused by mutations that affect the β globin locus and are extremely heterogeneous. These genetic defects lead to a variable reduction in β globin output ranging from a minimal deficit (mild β+ thalassemia alleles) to complete absence (β0 thalassemia)(2).

In β-thalassemia, the synthesis of normal α globin chains from the unaffected α globin gene continues as normal, resulting in the accumulation within the erythroid precursors of excess unmatched α globin. The free α globin chains are not able to form viable tetramers and instead precipitate in the red cell precursors in the bone marrow forming inclusion bodies. They are responsible for the extensive intramedullary destruction of the erythroid precursors and hence the ineffective erythropoiesis that underlies all β-thalassemia. Anemia in β-thalassemia thus results from a combination of ineffective erythropoiesis, peripheral hemolysis, and an overall reduction in hemoglobin synthesis(3). The life expectancy of patients with TM has significantly increased in recent years with subsequent increase in the reproductive potential and desire to have children. However, complications are still frequent and affect the patient’s quality of life(4). β-TM female patients usually suffer from hypogonadotropic hypogonadism associated with amenorrhea, anovulation and infertility. Infertility is assumed to be mainly from iron deposition in the hypothalamic-pituitary-ovarian axis, but recent evidence attribute it to iron-induced oxidative stress(5).

Oxidative stress occurs when reactive oxygen species ROS exceeds anti-oxidant capacity. These oxygen free radicals are unstable, highly reactive and capable of initiating uncontrolled chain reactions, resulting in cellular damage(6). In the cell membrane, lipid peroxidation begins when electrons from lipids reacts with unstable free radicals promoting a chain reaction with successive oxidation that results in lipid instability and formation of byproducts such as malondialdehyde MDA, a highly toxic molecule. Thus, serves as a reliable marker of oxidative stress(7).

Haptoglobin, Hp is an abundant acute phase plasma glycoprotein of the α 2-globulin fraction that binds free hemoglobin Hb forming a stable Hp-Hb complex cleared through endocytosis by the macrophage scavenger receptors CD 163 and inhibits the dissociation of ferric heam from globin, thus preventing oxidative damage(8). Several Hp-genotyping methods based on conventional polymerase chain reaction PCR have been developed. Hp genotypes have different biophysical characteristics and functional efficacies that account for their distinict anti-oxidant capacities(9).

Previous research showed that Hp 1-1 is more effective than Hp 2-2 in preventing Hb-induced oxidation of plasma components. It was found that Hp 2-2-Hb complexes are cleared more slowly than Hp 1-1-Hb complexes, which may allow the Hb which is complexed with Hp 2-2 more opportunity to react with oxidizing agents (10).

Ovarian reserve refers to residual follicles that are available for procreation, recent studies have shown that anti-mullerian hormone AMH and antral follicle count are equally effective in testing ovarian reserve(11). Moreover, it has been demonstrated that performing both together doesn’t increase the predictive power(12). Early follicle development, before secondary follicle recruitment, is largely gonadotrophin independent. AMH serum levels are not affected by dominant follicle growth during the late follicular phase of the normal menstrual cycle. This renders AMH easy to use clinically as opposed to other currently available markers of ovarian aging, such as inhibin B, estradiol (E2) and FSH, which are all menstrual cycle dependent and constitute relatively late markers of the ongoing process of primordial follicle pool depletion(13).

OBJECTIVESTo study the impact of haptoglobin gene

polymorphism on iron overload and oxidative stress in beta-thalassemia major women in Egypt, and their subsequent influence on AMH level as a marker of ovarian reserve.

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Dr. Sally S. El Tawab et al..Ovarian reserve in Beta-Thalassemia

MATERIAL AND METHODSAfter obtaining the approval of both Medical

Research Institute Ethics Committee and Alexandria faculty of medicine Ethics Commitee on the study protocol, and an informed consent from all participants or their parents, 94 women were included in the study.

47 females with established β-TM followed up in the Haematology Clinic of Student Sporting Hospital, as well as the Haematology department, Medical Research Institute from September 2015-June 2016. They were previously diagnosed as having β-TM by clinical signs, hemoglobin electrophoresis and were followed up regularly in haematology clinic. All were on regular transfusion of 350-500 ml of packed RBCs at 2-4 weeks interval for more than 10years. All were adherent to iron chelation therapy using desferrioxamine DFO at a dose 20-50 mg/kg/day. Patients with apparent acute infection were excluded. The presence of an acute-phase reaction was excluded by measurement of C-reactive protein CRP. If CRP >6mg/L, they were excluded from the study. 47 healthy regularly menstruating females, of matched age served as control. None of the control were thalassemia minor and none had a history of previous blood transfusion, anemia, liver diseases, or active inflammatory conditions, previous pelvic surgery and were not taking any medication.

The menstrual pattern, age at menarch, presence of amenorrhea either primary or secondary were obtained by self -reporting. Blood samples from all regularly menstruating women were taken in the early follicular phase to obtain basal levels of E2, FSH, AMH. For those having with amenorrhea or irregular mensis, blood samples were collected randomly. A cut-off value of AMH ≤ 1ng/ml was used to predict poor ovarian reserve(14).

Haptoglobin Hp level was quantitatively determined by means of immunonephelometry automated chemistry analyzer (BN ProSpec system, Siemens). Using anti sera (NAS HAPT), a liquid animal sera which produced by immunization of rabbits with highly purified human Hp. Hp in serum form immune complexes in an immunochemical reaction with specific antibodies. These complexes scatter a beam of light passed through the sample; the intensity of the scattered light is proportion of the Hp in the serum.

Serum malondialdehyde MDA, determined as thiobarbituric acid reactive substances (TBARS) according to the method of Draper and Hadley.

MDA has been identified as the product of lipid peroxidation that reacts with TBA to give pink species absorbing at 532nm. The unknown MDA containing samples are first reacted with TBA at 95° C and at low PH. After a brief incubation, the samples and standards can be read spectrophotometrically. The concentration (nmol/ml) of MDA in sample was obtained from a standard curve made by preparing serial dilutions of tetramethoxypropane TMP 1,2,4,6.8.12 nmol/ml, in ethanol treating them like the sample.

Genotyping of Hp polymorphism by conventional PCR: Venous EDTA blood samples were obtained and nuclear DNA was isolated from blood using Fermentas whole blood genomic DNA isolation kit (Fermentas, EU) according to the manufacturer instructions. Genotyping of Hp was assayed by PCR based DNA amplification of a 1757-bp Hp 1 allele-specific sequence and 3481-bp Hp2 allele specific sequence using four primers set.

Primer A: 5-GAGGGGAGCTTGCCTTTCCATTG-3Primer B: 5-GAGATTTTTGAGCCCTGGCTGGT-3Primer C: 5-CCTGCCTCGTATTAACTGCACCAT-3Primer D: 5-CCGAGTGCTCCACATAGCCATGT-3

Two sets of PCR reactions were performed. After electrophoresis of the reaction products in 1% agarose gel, Hp genotyping-specific banding patterns were obtained. With primers A and B, Hp1-1 and Hp 2-2 genotypes were characterized by single bands representing the 1757 and 3481 bp products. Primers C and D were used for detecting 349 bp Hp-2 allele specific product. Serum concentrations of FSH were measured using Chemiluminescent Microparticle Immunoassay (CMIA), (ARCHITECT system, Abbott Laboratories, Abbott Park, IL, USA) expressed as mIU/ml. Serum AMH was measured by enzyme-linked immunosorbent assay (ELISA) kit (Immunotech-Beckman Coulter, Webster, TX, USA) and expressed in ng/ml.

STATISTICAL ANALYSISData were fed to the computer and analyzed

using IBM SPSS software package version 20.0. Qualitative data were described using number and percent. Quantitative data were described using median, range (minimum and maximum) or mean, standard deviation and significance of the obtained results was judged at the 5% level. Chi-square test, for categorical variables, to


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compare between different groups. Student t-test, for normally quantitative variables, to compare between two studied groups. Mann Whitney test, for abnormally quantitative variables, to compare between two studied groups. Odd ratio (OR):used to calculate the ratio of the odds and 95% Confidence Interval of an event occurring in one risk group to the odds of it occurring in the non-risk group. Kruskall–Wallis test was performed where appropriate. Multivariate linear regression analysis for serum AMH, ferritin and MDA after log transformation to correct the heterogeneity of variance, using Wilcoxon signed rank test.

RESULTSThe study was conducted on 47 β-TM women,

aged between 16-26 years, mean 21.4±2.7, a positive family history was found in 24%, a positive consanguinity between parents was found in 32%. As regards frequency of blood transfusion, 52% had regular transfusion every 2 weeks, 16% had transfusion every 3 weeks and 32% had less frequent transfusion every 4 weeks. The age at first transfusion was given ranged from 6 to 36 months of age, with mean of 12.9±7.58 months.

All beta-TM patients were receiving DFO chelation therapy regularly, 32% by subcutaneous infusion using electronic infusion pump over 8-12 hours, 3-5 days per week, 44% via intramuscular route and 24% via intravenous route. 47 age matched with mean 20.5±3.3, regularly menstruating females served as control. Using the definition of hypo-gonadotrophic hypogonadism as FSH and LH levels ≤ 2 mIU /ml with accompanying E2 level ≤ 20pg/ml, 21 of 47 β-TM (44.6%) suffered from it. Primary amenorrhea was found in 10/47 (21%) and secondary amenorrhea in 11/47 (23.4%). Poor ovarian reserve taking ≤1ng/ml as a cut-off level(14) was found in 6/47 patients (12.5%). None of the patients was on hormone replacement therapy and all women were virgin, eliminating the possibility of transvaginal ultrasound for antral follicular count AFC, leaving AMH as the ideal choice for ovarian reserve assessment in these patients.

Both groups were compared against several laboratory items (table 1), hemoglobin level Hb was significantly lower in thalassemic group p<0.001, with mean 6.7±1.2 reflecting a range far away from the optimum target. At the same time, iron overload represented by serum ferritin level

showed a significant higher level p<0.001. Serum MDA, the chosen marker of oxidative stress showed a significant higher level in thalassemic group, p<0.001. Moreover, significant lower levels of LH, E2 and AMH were found in thalassemic group compared to the control p<0.001. The data in table 2, shows significant difference (p<0.05) between both groups as regarding distribution of haptoglobin gene polymorphism.

In thalassemic group, Hp2-2 was the most frequent followed by Hp2-1 then Hp1-1 (odds ratio 2.616 at 95% CI, 1.134-6.033), with allele frequency Hp-2 72.3%. In contrast to the control group, in which Hp2-1 was more frequent than Hp2-2 followed by Hp1-1, with allele frequency Hp-2 57.4%. Table 3 illustrates a correlation between the three haptoglobin genotypes with serum haptoglobin, serum ferritin, MDA and AMH in thalassemic group.

Table 1.Comparison between the two studied groups according to different parameters

Table 2.Comparison between the two studied groups according to HP poly

Table 3.Relation between HP gene polymorphism with different parameters

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Ovarian reserve in Beta-Thalassemia Dr. Sally S. El Tawab et al..

Hp was lowest with Hp2-2 and serum ferritin was highest in the same Hp genotype but with no statistic significant difference. A significant higher MDA p<0.05 and a significant lower AMH p<0.001 (figure 1) was in patients with Hp2-2 genotype.

Multi-variant regression analyses were done to determine the factors that correlate with AMH, serum ferritin and MDA in the thalassemic group (table 4).

When AMH was used as the dependent variable, only serum ferritin of all the explanatory variables was found to correlate significantly and inversely with serum AMH (B= -0.532, SE=0.166, p<0.001).

When serum ferritin was used as the dependent variable; haptoglobin, LH, and AMH of the explanatory variables were found to correlate significantly and inversely with serum ferritin (B= -0.238, SE=0.0.089, p<0.001), (B=-0.323, SE=0.2, p<0.001) (B= -0.268, SE=0.06, p<0.001) respectively. While MDA correlated significantly positive with serum ferritin (B= 0.242, SE=0.144, p<0.001). When MDA was used as the dependent variable, only serum ferritin of all the explanatory factors correlated significantly positively with

Table 4.Multivariate linear regression analysis for serum AMH, ferritin & MDA

DISCUSSIONIn the past; reproductive and sexual health were

considered irrelevant to β-TM patients as they rarely survived beyond adolescence. Improved medical treatment for these patients introduced in the late 1970s, involving regular optimum red blood cell transfusion and daily subcutaneous iron chelation therapy, has significantly decreased patient morbidity and increased survival beyond adolescence into middle age. As a result, patient care has expanded to include encouraging these patients to aspire to the same sexual, and reproductive goals as their peers(15). According to the current evidence-based knowledge, a significant prooxidants/antioxidants imbalance with subsequent increased oxidative stress exists in patients with β-TM, which is mainly caused by tissue injury due to overproduction of free radicals by secondary iron overload, alteration in serum trace elements, and alteration in antioxidant enzymes level(16).

In the present study, a discrepancy in the prevalence of Hp genotypes in β-TM patients versus control was found. While Hp2-2 was more common in thalassemic patients, Hp2-1 predominated in the control group. The variable capacity of the three Hp phenotypes to prevent

Figure 1.Correlation between serum AMH, ferritin and MDA in cases group

Figure 2.Relation between HP gene polymorphism with AMH

MDA (B= 0.59, SE=0.069, p<0.001).Finally, figure 2, demonstrate the significantly lower AMH level in patients with Hp2-2 gene polymorphism.


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oxidative stress induced by free Hb could be related to differences in their affinity for Hb. The protective effect is more marked for Hp1-1 than Hp2-1 and Hp2-2. Studies have demonstrated that internalization of Hb-Hp complex by CD 163 receptors is more potent for Hb-Hp 2-2 complex than for that bound to Hp1-1 or Hp 2-1. Hence, it was proposed that individuals with Hp2-2 phenotype are under greater oxidative stress(17). We also demonstrated, very high ferritin levels in the β-TM. Furthermore, the study showed that HP2-2 was associated with the highest serum ferritin.

In this study, a significant higher level of MDA, a marker of lipid peroxidation and oxidative stress was found in BTM group versus control (p<0.001) reflecting a state of significant oxidative stress in patient group. More importantly, is the highest level of MDA significantly found in patients with Hp2-2 phenotype as compared with the other phenotypes (p=0.005). Our results support the finding of Blum et al(18) who reported that the Hp-2 protein is associated with increased generation of oxidative active iron, while Hp-1 protein is associated with increased production of antioxidant cytokine interleukin IL-10 in diabetic mice with myocardial infarction. They stated that Hp-1 phenotype has an antioxidant and anti-inflammatory properties.

In a similar study evaluating the reproductive capacity in thalassemic women, the study concluded that AMH served as such a better marker than AFC, which in many cases is lower despite a normal AMH. Thalassemia women had a considerably lower ovarian volume compared with reported normal controls (1.25 ± 1.2 cm3 vs 6-6.6 cm3)(19) also representing impaired ovarian reserve, probably a result of a halt in follicle maturation because of lack of gonadotropin stimulation and direct iron toxicity to ovarian tissue(20).

Meanwhile, the previous studies concluded that labile, potentially toxic, iron plays a major role in causing reproductive tissue damage,(21,22)

suggesting a role for “free” iron in the pathogenesis of impaired fertility in β-TM women, our study demonstrated a significant correlation between the high ferritin level, with high MDA oxidative stress marker and low AMH levels. Moreover, β-TM women with Hp2-2 genotype found to be the most affected from the resulting higher oxidative stress. Accurate ovarian reserve testing may motivate some women to start a family at an earlier age (or alternatively apply fertility preservation by means of oocyte freezing) or alternatively reassure others that postponing childbearing will not interfere with her chances to achieve a pregnancy later on (13). This matches a recent study which concluded that patients receiving chronic transfusion and heavy metal overload are at high risk of being associated with impaired gonadal function. Furthermore, elective cryopreservation of ovarian tissue or oocytes should possibly be discussed to preserve future fertility(23).

Beta thalassemic major patients are at great risk of oxidative stress. Haptoglobin gene polymorphism has a great impact on the extent of oxidative stress in these patients. β-TM patients have lower serum AMH levels than the age –matched healthy regularly menstruating control. AMH significantly negatively correlated with serum ferritin and MDA, marker of oxidative stress. This affection was more evident in patients with HP2-2 genotype. Thus, concluding that the higher the oxidative stress is, the more impaired ovarian function might be existed in β-TM women.

ACKNOWLEDGEMENTNone

DISCLOSUREThe results of this manuscript have not been

distorted by research funding or conflicts of interest.

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LA NATURA CHE AIUTA

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Ovarian reserve biomarkers usefulness for optimization of counselling in a public network for fertility preservation in oncological patientsMarta Manno1, Francesco Tomei1, Fabio Puglisi3, Francesco Zaja4, Paolo Metus2, Renato Tozzoli2,Maurizio Mascarin5, Massimo Manno1

1 Pathophysiology of Human Reproduction, Semen&Oocyte Bank, Maternal Paediatric Dpt, Pordenone2 Laboratory Dpt, Pordenone3 Dpt of Oncology, University of Udine4 Clinical Hematology, University of Udine5 CRO (Oncological Reference Center), Aviano

ABSTRACTObjective: the aim of the present study was to investigate the strength and reliability of biomarkers to optimize counselling in cancer patients.Methods: retrospective observational study of fertility preservation cycles, performed in cancer patients referred to Pathophysiology of Human Reproduction, Semen and Oocyte Bank (Pordenone, Italy).Ovarian reserve was evaluated with antral follicle count by 2D ultrasound and AMH determination by Beckman Coulter Generation II ELISA and also Roche Elecsys fully automated method, when available.All patients were stimulated with antagonist protocol and modified ovulation triggering and when indicated according to random start protocol, to reduce the lag between referral and fertility preservation. Aromatase inhibitors were added to gonadotropin in oestrogen receptor positive patients.Results: numbers of retrieved/vitrified oocytes were used to evaluate fertility preservation efficiency. Fertility preservation was completed in 17 breast cancer oestrogen receptor positive patients, 6 breast cancer oestrogen receptor negative patients, 10 lymphomas patients and one gastrointestinal stromal tumour, one colon adenocarcinoma, one thyroid sarcoma, one medulloblastoma and one Ewing’s sarcoma. The mean number of vitrified oocytes was 8.18 ± 5.22 in breast cancer oestrogen receptor positive, 11 ± 6.26 in breast cancer oestrogen receptor negative and 11.1 ± 8.81 in lymphomas. AMH was the most effective biomarker as predictor for fertility preservation outcomes and correlation was comparable with the two methods.Conclusions: AMH seems to be the best biomarker to predict FP efficiency in cancer patients. Aromatase inhibitor introduction for ovarian stimulation seems to reduce its reliability.

SOMMARIOObiettivo: lo scopo dello studio è stato quello di indagare l’affidabilità dei biomarkers per ottimizzare il counselling in pazienti affetti da patologie neoplastiche.Metodi: studio retrospettivo dei cicli di preservazione della fertilità eseguiti presso la “Fisiopatologia della Riproduzione Umana e Banca del Seme e degli Ovociti” dell’ospedale di Pordenone.La riserva ovarica è stata valutata tramite conta dei follicoli antrali e determinazione dell’AMH.I pazienti sono stati stimolati con protocollo antagonista e triggering ovulatorio modificato e, quando indicato, secondo il protocollo “random start stimulation” per ridurre il periodo tra riferimento e pick up. Gli inibitori dell’aromatasi sono stati aggiunti alle gonadotropine nei pazienti affetti da carcinoma mammario recettore positivo.Risultati: il numero di ovociti recuperati/vitrificati è stato utilizzato per valutare l’efficienza dei cicli di preservazione della fertilità. Questa è stata portata a termine in 17 pazienti affetti da carcinoma mammario recettore positivo, 6 da carcinoma mammario recettore negativo, 10 da linfoma, uno da tumore stromale gastrointestinale, uno da adenocarcinoma del colon, uno da sarcoma della tiroide, uno da medulloblastoma ed uno da sarcoma di Ewing.Il numero medio di ovociti vitrificati è risultato 8,18 ± 5,22 nei carcinomi mammari recettore positivi, 11 ± 6.26 nei carcinomi mammari recettore negativi e 11,1 ± 8,81 nei linfomi. Conclusioni: l’AMH è risultato il migliore biomarker per predire l’efficienza della Fertility Preservation in pazienti affetti da patologie neoplastiche.L’introduzione di inibitori dell’aromatasi per la stimolazione ovarica sembra ridurre la sua affidabilità.

Correspondence to: [email protected] Copyright 2015, Partner-Graf srl, PratoDOI: 10.14660/2385-0868-55


Keywords: oocytes, vitrification, fertility preservation, AMH, breast cancer, lymphomas, aromatase inhibitor.


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Biomarkers in fertility preservation

positive (17 cases), while only a minority (6 cases) were oestrogen receptor negative.

Multiple follicular growth was induced with different protocols according to oestrogen receptor status. In fact, in patients with oestrogen receptor positive neoplasm, the ovarian stimulation was carried out by co-administration of aromatase inhibitors (AI) (letrozole 5 mg daily) starting two days before gonadotropins introduction, in order to keep oestrogen levels as low as possible during multifollicular growth. Notwithstanding the lack of prospective randomized trials comparing aromatase inhibitors plus gonadotropins with the standard protocol with gonadotropins alone evaluating cancer recurrence as primary endpoint, this approach is generally adopted according to a precautionary principle suggesting to minimize the oestrogen exposure of the potentially residual neoplastic cells in oestrogen receptor positive tumours. Moreover, the safety of this protocol seems to be preliminarily confirmed by retrospective studies showing no higher recurrence rates of neoplastic diseases in patients undergoing fertility preservation, than those who do not(5-6).

In all our patients, a protocol with antagonist was adopted to minimize the risk of ovarian hyperstimulation syndrome (OHSS)(7) by inducing final oocyte maturation by GnRH agonists instead of human chorionic gonadotropin administration. This complication could be particularly undesirable in patients who need to start chemotherapy (CT) as soon as possible, due to the risk of worsening prognosis with its deferral. Moreover, in order to minimize the lag between the start of fertility preservation program and the beginning of the chemotherapy, an approach with random start stimulation (RSS) has been adopted when necessary. With this approach, the stimulation can start in any phase of the ovarian cycle, in accordance with the most recent evidences on the existence of multiple waves of folliculogenesis in each ovarian cycle. Oocytes were vitrified on open devices (Cryotop) according to Kuwayama protocol(8).

Antral follicle count (AFC) was performed immediately before the start of ovarian stimulation and therefore not always in the early follicular phase, considering follicles of 2-9 mm in diameter observed with 2D transvaginal ultrasound. AMH values were determined using random sampling and each sample was stored at -20 °C before assay with Beckman Coulter Generation II ELISA (BC Gen II) system and, after its recent implementation, Elecsys Roche automated method.

INTRODUCTIONThe combined effect of reproductive

program deferral, increased incidence of cancers in childbearing age women and the huge improvement of their quod vitam prognosis, induce a growing demand for fertility preservation (FP) through oocytes vitrification. This trend was highlighted in a recent statement of the main Italian scientific societies in this field(1). Moreover, a recent international statement highlighted also the need for early counselling about fertility preservation opportunities for all women affected by cancer(2). Fertility Preservation substantially contributes to improve the quality of life for patients whose survival is now guaranteed, in the large majority of cases, by new cancer therapies. The introduction of oocyte vitrification into our daily laboratory practice, in the light of its non-experimental nature(3), has substantially contributed to the implementation of fertility preservation programs, finally ensuring gender equity in reproductive potential preservation in cancer patients. Even if artificial gametogenesis from both embryos and induced pluripotent stem cells has been recently validated in mouse(4), we think that a long time is needed until it could be implemented in human reproduction. So, at present an effective FP can be applied only with gametes cryopreservation before gonadotoxic therapies.

MATERIALS AND METHODSWith the aim of optimizing fertility preservation

counselling, we have retrospectively analysed all the cycles performed at Pathophysiology of Human Reproduction, Semen and Oocyte Bank at Pordenone Hospital, from October 2012 until now. The correlations between FP cycles outcomes, humoral and biophysical markers, and methods of ovarian stimulation were evaluated.

The 38 patients referred by neighbouring centers (Clinical Hematology University of Udine, Oncology Department of Udine and CRO of Aviano) mostly belong to two main groups: 23 breast cancer patients stimulated before or after surgery, according to oestrogen receptor status and 10 patients with Hodgkin’s (HL) or non-Hodgkin’s lymphomas (NHL). In addition to these two main groups, a gastrointestinal stromal tumour (GIST), a colon adenocarcinoma, a thyroid sarcoma, a medulloblastoma and an Ewing’s sarcoma were referred for FP. Patients affected by breast cancers were mainly oestrogen receptor

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M. Manno et al.Biomarkers in fertility preservation

RESULTSWe evaluated FP cycles outcomes (oocytes

retrieved and vitrified), humoral and biophysical markers and methods for ovarian stimulation (table 1).

Table 1.Overview of all Fertility Preservation cycles

Overall, the mean number of oocytes retrieved at pick-up was 12.95 ± 8.35, 80% of which was mature (10.37 ± 7.34 oocytes). The mean value of AMH was 4.6 ± 3.85 ng/ml. The parameters for which we assessed the correlation with retrieved


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and vitrified oocytes were: AMH measured with the Beckman Coulter Generation II ELISA and, when available, Roche Elecsys method, AFC, starting-dose, total dose of gonadotropins and peak oestradiol. The values which best correlated with the number of vitrified oocytes were oestradiol peak and AMH, although overall correlations were low (table 2).

Both, the starting-dose and gonadotropins total dose, negatively correlated with the number of vitrified oocytes. In table 3 are reported the mean values of retrieved and vitrified oocytes in receptor positive breast cancers, receptor negative breast cancers and lymphomas (table 3).

The differences observed in retrieved and vitrified oocytes in the three subgroups were not significant. Correlation values reported in table 2 were calculated also in the different sub-groups: receptor positive breast cancers (table 4), receptor negative breast cancers (table 5) and lymphomas (table 6). This group includes the patients treated with the aromatase inhibitor, due to receptor positive status. In these patients with mean AMH value 4.44 ± 3.63 ng/ml, a mean of 11.12 ± 7.23 oocytes was retrieved, 8.18 ± 5.22 of which vitrified (74%). In this sub-group, the biomarker which best correlated with vitrified oocytes was the AMH level, even if the correlation value was low (table 4).

In these patients, with mean AMH value 3.8 ± 2.91 ng/ml, a mean of 12.67 ± 5.79 and 11 ± 6.26 (87%) were respectively retrieved and vitrified. In this subgroup, as expected, peak oestradiol highly correlated with the number of both retrieved and vitrified oocytes, due to the well-known relationship between the number of growing follicles and oestradiol output, in absence of aromatase inhibitors administration (table 5).

However, in this group, the statistical evaluation is only preliminary and not conclusive, due to the small number of observations presently available. The second largest group of patients was that of lymphomas. Even for this group the parameter with the best correlation was the AMH value, with very high Pearson index (0.87). In these patients with mean AMH value 3.2 ± 2.35 ng/ml, a mean of 13.5 ± 10.22 oocytes were retrieved, of which an average of 11.1 ± 8.81 vitrified (82%) (table 6).

Moreover, means of patients’ age, AMH, AFC, starting dose, peak oestradiol, total units of gonadotropin, retrieved and vitrified oocytes between different sub-groups were compared by Student’s t-test.

A significant difference of peak oestradiol was observed between the patients with oestrogen receptor positive breast cancers and patients with both lymphomas (433.71 ± 431.40 vs 1967.2 ± 1387.13; p= 0.7x10-2) and oestrogen receptors negative breast cancers (433.71± 431.40 vs 1289.5±586.92; p= 0.01). In addition, a significant difference between lymphomas and breast cancers patients’ ages was observed (28.9 ± 4.68 vs 32.94 ± 4.34; p= 0.04).

Recently, in our laboratory a fully automated method for AMH determination (Elecsys Roche)


Table 6.Correlation r Pearson indexes between retrieved/vitrified oocytes and different parameters in lymphoma patients

Table 2.Correlation r Pearson indexes between retrieved/vitrified oocytes and the different parameters studied

Table 3.Retrieved and vitrified oocytes in the different subgroups

Table 4.Correlation r Pearson indexes between retrieved/vitrified oocytes and different parameters in receptor positive breast cancers patients

Table 5.Correlation r Pearson indexes between retrieved/vitrified oocytes and different parameters in receptor negative breast cancer patients

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has been implemented. After this implementation, we tried to compare AMH reliability for the number of retrieved and vitrified oocytes prediction with both non-automated and automated method.

Due to the observation that AI introduction for ovarian stimulation could reduce AMH reliability, we arbitrarily choose to compare AMH performance with both methods, only in patients stimulated without AI co-treatment. The correlation between oocytes retrieved/vitrified and AMH value was comparable with BC Gen II and Elecsys methods (table 7).

We also determined the delay from diagnosis and start of chemotherapy. Time to chemotherapy was determined adding times between diagnosis and surgery, time between surgery and FP referral and between referral and pick-up.

For our breast cancer patients, the mean delay was 67.67 days ± 21.10 (table 8).

In lymphomas patients, due to the lack of surgical therapy, the delay consists of only two periods (table 9).

For lymphomas patients the mean delay was 34.25 days ± 11.23.

The length of controlled ovarian stimulation was comparable in RSS and conventional protocol (9 days ± 0.82 with RSS vs 9.11 ± 2.27). Moreover in patients treated with random start stimulation protocol, the average number of vitrified oocytes was 15.9 ± 9.87 vs 9.43 ± 7.13 in conventional protocol in front of comparable ovarian reserves as evaluated with mean AMH level (3.78 ± 2.49 vs

4.69 ± 3.36; p=n.s.) with comparable starting and cumulative dose of gonadotropins (table 10).

Three patients were not included in the statistical evaluation, due to previous treatments with gonadotoxic drugs (for sarcoma, neuroblastoma and NHL). In these patients, six MII oocytes, four oocytes all lysated during decoronization and zero oocytes were respectively retrieved. These numbers are significantly lower than those observed in other patients and considered as needed for an acceptable probability of pregnancy(9). Previous chemotherapy with alkylating agents therefore substantially impaired fertility preservation cycle efficacy.

On the contrary, in patient 9 (table 1) previously treated with six cycles of ABVD for HL, 16 oocytes were retrieved, of which 12 were mature. This patient was included in the statistical evaluation of the group of lymphomas, as the therapy with ABVD, considered less gonadotoxic than protocols carried out in the other three patients(10), allowed an oocyte recovery compatible with a good chance of pregnancy.

DISCUSSIONWe tried to find the most predictive parameters

of FP programs effectiveness and to compare results with those of previous studies reporting AMH as the best biomarker for this purpose(11-12).

AMH predictability for FP outcomes in terms of retrieved and vitrified oocytes was excellent in oestrogen receptor negative breast cancers and lymphomas, but markedly lower in oestrogen receptor positive breast tumours. As expected, due to aromatase inhibitors co-administration, a significant difference of peak oestradiol was observed between the patients with oestrogen receptor positive breast cancers and patients with both lymphomas and oestrogen receptors negative

Biomarkers in fertility preservation M. Manno et al.

Table 10.Overview of our 10 RSS Fertility Preservation cycles

Table 8.Delays to chemotherapy for breast cancer patients

Table 9.Delays to chemotherapy for lymphomas patients

Table 7.Correlation r Pearson indexes between retrieved/vitrified oocytes and AMH levels with BC Gen II and Elecsys Roche methods in AI-cycles


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breast. In the small subgroup of the oestrogen receptor negative breast cancers, even peak oestradiol showed a good predictive power even if its usefulness is limited by its post hoc availability. Surprisingly, peak oestradiol correlated very well with retrieved and vitrified oocyte in oestrogen receptor negative breast cancers, but not in the subgroup of lymphomas, even if both subgroups were stimulated without aromatase inhibitors co-treatment.

In addition, lymphomas patients were significantly younger than breast cancers one. AMH as FP efficiency biomarker predictor was more reliable than AFC in each subgroup.

Moreover, both the starting dose, estimated on the basis of individual patient ovarian reserve and the total dose of gonadotropins were negatively correlated with the number of oocyte retrieved. This seems to suggest that the major determinant of oocyte recovery is ovarian responsiveness, in comparison with starting or cumulative dose of gonadotropins.

Concerning the efficacy of different protocols for ovarian stimulation the number of vitrified oocytes, with and without aromatase inhibitors, was not significantly different (p=0.08). However we cannot exclude that this trend will become statistically significant increasing the number of observations. So at present, a type β statistical error due to the small number of observations cannot be excluded at all. So a definitive costs/benefits evaluation of letrozole co-treatment for ovarian stimulation in oestrogen dependent tumours is not presently possible. Indeed, different opinions regarding this protocol exist in the literature with both positive(13) and negative effects reported as well(14). Moreover, recent evidences from other authors show that the timing of letrozole introduction also seems to influence ovarian response and mature oocytes number, more than its use per se(15). The advantage in terms of prognosis of the underlying disease, without adequately statistically powered studies evaluating the frequency of relapses with traditional stimulation or with AI addition, is not exactly quantifiable. Presently, the prescription of AI in oestrogen sensitive cancers is suggested only in accordance with a general principle of precaution but a detrimental effect on both number and percentage of mature oocytes cannot be ruled out. Moreover, our observations suggest that peak oestradiol does not correctly predict the number of retrieved oocytes if ovarian stimulation is performed with aromatase inhibitor use.

Regarding the overall interval before


chemotherapy, the delay introduced by fertility preservation per se was extremely shorter than that before referral. However, overall delay in breast cancer patients was longer than that recently suggested as acceptable(16).

Our preliminary observations seem to suggest that ovarian stimulation with random start approach allows to retrieve at least the same or even an higher number of mature oocytes, if compared with standard protocol. These data are in agreement with previous observations by other authors(17-18). Considering a possible detrimental effect of AI use on the number of retrieved/vitrified oocytes a realistic explanation of this observation could be that the percentage of patients treated with RSS and AI was lower (20%) than that of patients treated with gonadotropin alone (80%). Anyway even in AI+ protocol the number of vitrified oocytes was suitable for a good chance of pregnancy, according to what suggested in recent elective fertility preservation programs(9).

However, even using RSS to minimize the delay of CT, we urgently need to sensitize both oncologists and haematologists to a fast patient referral for fertility preservation, even before surgery, if indicated. The possibility that clinical prognosis of receptor negative cancers could be improved by pre-surgery fertility preservation due to shortened time to chemotherapy should be addressed by future studies.

In our experience, 12 patients refused fertility preservation due to the advanced stage of breast cancers or lymphomas, or due to the perceived physical burden of the procedure.

With antagonist protocol and modified triggering, no cases of ovarian hyperstimulation syndrome were observed even in patients with an high number of retrieved oocytes. Thus, ovarian stimulation with antagonist protocol and modified triggering seems to be safe for our daily clinical practice of FP.

Moreover our preliminary data show that fertility preservation can be done with a limited further delay of chemotherapy if compared with that between diagnosis and patient referral for FP.

Anyway, several burning questions still remain unanswered in FP programs. Today how many patients are presently referred for FP? The ASCO guidelines suggest that in the past only a small percentage of cancer patients were referred to the centers performing fertility preservation(19). Which is the best protocol for ovarian stimulation? Gonadotropins alone or gonadotropin plus AI in oestrogen receptor positive cancers? Which is the best drug for ovarian stimulation in such patients:

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Biomarkers in fertility preservation M. Manno et al.

classic gonadotropins or the more user friendly corifollitropin needing only one injection for the first seven days of stimulation? Moreover, the problem of how FP can be matched with ovarian protection by pharmacological treatment by GnRH analogue administration is still an open question. To avoid OHSS we need to do ovarian stimulation using antagonist protocol without GnRH receptors down regulation, allowing ovulation triggering by GnRH agonist. Therefore, GnRH analogue administration before ovarian stimulation for FP is not possible. However, with GnRH analogue administration after oocytes retrieval, immediately before chemotherapy a flare effect, due to gonadotropins mediated angiogenic effect, could theoretically increase post chemotherapy ovarian damage. At present, we don’t know neither the percentage of utilization of vitrified oocytes, nor the ART outcome in cancer patients with vitrified oocytes. Presently data regarding ART outcomes with vitrified oocytes in patients with history of cancer are not completely consistent. Some studies show comparable results with that of infertile couples(20), while others show that the number of retrieved oocytes is related to the type of cancer. Anyway, ART outcome also in such case are clearly related to the mean age of the patients(21) which is variable in different types of cancers. It could be that in older patients, emergency IVF with embryo cryopreservation could be a more effective approach if compared with oocyte vitrification notwithstanding ethical qualms. However, such an approach is presently not allowed by the Italian law on reproduction, due to limitations for embryo cryopreservation.

FP cost effectiveness, acceptability and clinical outcomes in comparison with post hoc ovodonation should also be addressed by future studies.

The last but not least problem is the presently limited affordability of FP in our public health insurance system due to shortage of centres offering such practices.

In conclusion, our preliminary data seem to confirm that antagonist protocol with modified agonist triggering of ovulation is a good approach to avoid OHSS. RSS seems equally effective than conventional protocol. AI introduction for receptor positive breast cancer stimulation, reduced the number of retrieved/vitrified oocytes, and AMH level correlation with the number of retrieved/vitrified oocytes was lower in cycles with AI if compared with both oestrogen receptor negative cancers and lymphomas stimulated with gonadotropins alone. Aromatase inhibitors co-treatment is able to significantly reduce peak oestradiol level but presently the impact of this protocol on cancers recurrence is not clear and a detrimental effect on FP effectiveness cannot be excluded.

Both automated AMH and BC Gen II AMH correlated well with both oocytes retrieved and vitrified in cycles without AI co-treatment. AMH BC Gen II correlation with both retrieved and vitrified oocytes in FP cycles with ovarian stimulation without AI was lower. Our data confirm previous observations of lower AMH values with the new automated assay(22).

The mean delay to chemotherapy in our patients was different according to the kind of neoplasm: this lag was shorter in lymphomas if

REFERENCES1) Pinto C, Lenzi A, Scollo P. Raccomandazioni AIOM- SIE- SIGO su oncofertilità 2016.2) Loren AW, Mangu PB, Beck LN, Brennan L, Magdalinski AJ, Partridge AH et al. Fertility Preservation for Patients With Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update. J Clin Oncol 2013; 31: 2500-2510.3) The Practice Committees of the American Society for Reproductive Medicine and Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril 2013; 99: 37-43.

4) Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N et al. Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature 2016; 539: 299-303.5) Kim J, Turan V, Oktay K. Long-Term Safety of Letrozole and Gonadotropin Stimulation for Fertility Preservation in Women With Breast Cancer. J Clin Endocrinol Metab 2016; 101: 1364-1371.6) Kim J, Turan V, Oktay K. Safety of fertility preservation by ovarian stimulation with letrozole and gonadotropin in patients with breast cancer: a prospective controlled study with subgroup analysis. ASRM Abstract 2014; 102: e32.


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7) Oktay K, Türkçüoğlu I, Rodriguez-Wallberg KA. GnRH agonist trigger for women with breast cancer undergoing fertility preservation by aromatase inhibitor/FSH stimulation. Reprod Biomed Online 2010; 20: 783-788.8) Kuwayama M. Highly efficient vitrification method for cryopreservation of human oocytes. RBM Online 2005; 11: 300-308.9) Cobo A, Garcia-velasco JA, Coello A, Domingo J, Pellicer A, Remohi J. Ooocyte vitrification as an efficient option for elective fertility preservation. Fertil Steril 2016; 105: 755-764.10) Boltežar L, Pintarić K, Jezeršek Novaković B. Fertility in young patients following treatment for Hodgkin’s lymphoma: a single center survey. J Assist Reprod Genet 2016; 33: 325-333.11) Emirdar V, Turan V, Moy F, Bedoschi G, Oktay KH. Value of Antimullerian hormone and antral follicle count in predicting fertility preservation cycle outcomes. ASRM Abstracts 2015; 104: e264.12) Iliodromiti S, Anderson RA, Nelson SM. Technical and performance characteristics of anti-Müllerian hormone and antral follicle count as biomarkers of ovarian response. Hum Reprod Update 2015; 21: 698-710.13) Pereira N, Hancock K, Cordeiro CN, Lekovich JP, Schattman GL, Rosenwaks Z. Comparison of ovarian stimulation response in patients with breast cancer undergoing ovarian stimulation with letrozole and gonadotropins to patients undergoing ovarian stimulation with gonadotropins alone for elective cryopreservation of oocytes. Gynecol Endocrinol 2016; 26: 1-4.14) Revelli A, Porcu E, Levi Setti PE, Delle Piane L, Merlo DF, Anserini P. Is letrozole needed for controlled ovarian stimulation in patients with estrogen receptor-positive breast cancer? Gynecol Endocrinol 2013; 29: 993-996.

15) Diaz-Garcia C, Domingo J, Romero A, Martinez M, Rubio JM, Garcia-Velasco JA et al. The timing of administration of letrozole significantly affects the oocyte rate in breast cancer patients undergoing controlled ovarian stimulation for fertility preservation. Fertil Steril 2015; 104: e327.16) de Melo Gagliato D, Gonzalez-Angulo AM, Lei X, Theriault RL, Giordano SH, Valero, V et al. Clinical impact of delaying initiation of adjuvant chemotherapy in patients with breast cancer. J Clin Oncol 2014; 32: 735-744.17) Cakmak H, Katz A, Cedars M, Rosen MP. Effective method for emergency fertility preservation: random-start controlled ovarian stimulation. Fertil Steril 2013; 100: 1673-1680.18) von Wolff M, Capp E, Jauckus J, Strowitzki T, Germeyer A. Timing of ovarian stimulation in patients prior to gonadotoxic therapy: an analysis of 684 stimulations. Eur J Obstet Gynecol Reprod Biol 2016; 199: 146-149.19) Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K et al. American Society of Clinical Oncology Recommendations on Fertility Preservation in Cancer Patients. J Clin Oncol 2006; 24: 2917-2931. 20) Oktay K, Turan V, Bedoschi G, Pecheco FS, Moy F. Fertility preservation success subsequent to concurrent aromatase inhibitor treatment and ovarian stimulation in women with breast cancer. J Clin Oncol 2015; 33: 2424-2429.21) Alvarez RM, Ramanathan P. Fertility preservation in female oncology patients: the influence of the type of cancer on ovarian stimulation response. Hum Reprod 2016 (dew158).22) Nelson SM, Pastuszek E, Kloss G, Malinowska I, Liss J, Lukaszuk A et al. Two new automated, compared with two enzyme-linked immunosorbent, antimüllerian hormone assays. Fertil Steril 2015; 104: 1016-1021.


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Laparoscopic Indocyanine green sentinel lymph node mapping in early ovarian cancer. A pilot study and review of the literatureMichela Angelucci1, Giacomo Corrado1, Emanuela Mancini1, Ermelinda Baiocco1,Benito Chiofalo2, Ashanti Zampa1, Arabella Bufalo1, Enrico Vizza1

1 Department of Experimental Clinical Oncology, Gynecologic Oncology Unit, “Regina Elena”National Cancer Institute, Rome, Italy.

2 Department of Human Pathology in Adulthood and Childhood “G. Barresi”, Unit of Gynecology andObstetrics, University of Messina, Italy

ABSTRACTObjective: there are still few studies concerning the Sentinel lymph node (SLN) in ovarian cancer. In this pilot study we described the feasibility of SLN mapping with Indocyanine Green (ICG) in five patients affected by early stage ovarian cancer, during laparoscopic surgery. Material and Methods: the tracer has been injected into the hilum of the ovary through a spinal needle (22-gauge size) connected to 20 cm-long infusion tubing for intravenous fluid delivery advanced inside the lateral trocar (10 mm) through a Johann clamp. Results: a total of 6 SNs was detected in all patients. The median interval from the tracer injection to the detection of all SNs was 2 minutes (range, 1 - 3 minutes). In three patients, the first detected SN was found in the area of common iliac artery, in two patients at paracaval region. The median number of lymph nodes removed per patient was 2 (range, 0-2). All the detected SLNs were identified ipsilateral to the site of injection. Only in two patients we detected a second SLN, respectively in preaortic region and in under mesenteric region. No allergic reaction has occurred. Conclusions: ICG laparoscopic SLN mapping in early stage ovarian cancer is a procedure feasible and promising. Additional prospective multicenter studies are needed to evaluate the best technique for staging early stage ovarian cancer patients.

Keywords: sentinel lymph node biopsy, ovarian cancer, Indocyanine Green.

SOMMARIOObiettivo: ci sono ancora pochi studi riguardanti il linfonodo sentinella (SLN) nel carcinoma ovarico. In questo studio pilota abbiamo descritto la fattibilità di mappatura SLN con verde di Indocianina (ICG) in cinque pazienti affetti da cancro ovarico fase iniziale, durante la chirurgia laparoscopica.Materiali e metodi: il tracciante è stato iniettato nell’ilo dell’ovaio attraverso un ago spinale (formato 22-gauge) collegato a 20 cm di lunghezza di tubo di infusione per la somministrazione di liquidi per via endovenosa avanzato all’interno del trocar laterale (10 mm) attraverso una Johann laparoscopica.Risultati: un totale di 6 SN è stato rilevato in tutti i pazienti. L’intervallo mediano di tempo dalla iniezione tracciante per l’individuazione di tutti i SN era di 2 minuti (range, 1 - 3 minuti). In tre pazienti, il primo rilevato SN è stato trovato nella zona dell’arteria iliaca comune, in due pazienti nella regione paracavale. Il numero mediano di linfonodi rimossi per paziente è stato di 2 (range 0-2). Tutti i linfonodi sentinella rilevati sono stati identificati omolateralmente al sito di iniezione. Solo in due pazienti abbiamo rilevato un secondo SLN, rispettivamente, nella regione preaortica e in regione sotto mesenterica. Nessuna reazione allergica si è verificata.Conclusioni: il SLN laparoscopico con IDG nel carcinoma ovarico fase precoce è una procedura fattibile e promettente. Ulteriori studi multicentrici prospettici sono necessari per valutare la tecnica migliore da poter eseguire in pazienti affetti da cancro ovarico in stadio iniziale.

Correspondence to: [email protected] 2015, Partner-Graf srl, PratoDOI: 10.14660/2385-0868-56



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Sentinel node biopsy in ovarian cancer

INTRODUCTIONOvarian cancer is the most unfavorable

prognosis gynecological neoplasia. Lymph node involvement is an asymptomatic early phenomenon, able to modify the course of disease. In patients with clinical early-stage disease, an occurrence of occult lymph node metastasis upgrades the disease to FIGO stage IIIC(1). The incidence of positive lymph node in patients with early stage ovarian cancer is between 5.1-15%(2-3). Therefore, an accurate surgical staging with systematic pelvic and para-aortic lymphadenectomy, has a significant therapeutic value in early stage ovarian cancer. A lymph node sampling, represent an insufficient procedure because some metastatic lymph nodes could remain undetected(4). On the other hand, the systematic lymphadenectomy is the gold standard, but the radical procedure implicates too many morbidities such as lymphocists (13%) nerve and vessel injury (4%) increased blood loss and increased surgery times(5).

With the advent of laparoscopic surgery, the concept of sentinel lymph node (SN) biopsy for assessing the regional lymph node status has been studied in all gynecological malignancies. The SN technique is effective in vulvar and breast cancer, whereas in cervical and endometrial cancers are currently ongoing(6). In ovarian cancer, SN data availability is exiguous, mainly because the tracers dissemination after injection through the cortex is unpredictable and because the risk of tumor spread is very high(7-8). Another unsolved question concerns the tracer injected during the SN procedure. Usually in other pathologies, node localization is achieved by the combination of injected dye and radioactive tracer. The commonly used dyes are the isomers isosulphan blue and Patent blue V (PBV). Concerning the early-stage ovarian cancer, the radioisotope is considered an inconvenient target to the tracer injections, because require a too long time to be absorbed from the lymphatic system and because a lymphoscintigram is always needed for localizing the SNs(7). Fluorescent indocyanine green (ICG) dye has been used and tested by several groups in SLN mapping in various solid tumors(9) Near-infrared (NIR) fluorescent dye with ICG has a higher signal-to-background ratio, is cheaper, has fewer adverse effects and less toxicity, and has infrequent allergic reactions(10).

The aim of our paper is to describe the SN procedure in early stage ovarian cancer with tracer injection into the ovarian ligaments during laparoscopic surgery.

MATERIALS AND METHODSPatients with clinical International Federation

of Gynecology and Obstetrics (FIGO) stage I - II ovarian carcinoma, good performance status (PS=0-1), no previous tumors, no previous chemotherapy or radiotherapy, who underwent laparoscopic primary debulking surgery were enrolled in this study. Preoperatively, all patients underwent CT and PET/CT scans to evaluated suspicious nodal involvement that could exclude their case from the nodal mapping.

The SPIES Full HD Image 1S H3-Z FI camera with Karl Storz Near Infrared (NIR/ICG) System (Karl Storz Endoscopy, GmbH, Mittelstrasse, Tuttlingen, Germany) was used and all patients signed an informed consent and the Local Ethic Committees approved the study.

After the induction of pneumoperitoneum, the operative trocars were positioned in the standard manner. The injection procedure was carried out after ascertaining the absence of suspicious gross peritoneal lesions or enlarged lymph nodes.

The ICG (Indocyanine green, PULSION Medical Systems SE, Feldkirchen, Germany) concentration used was 1.25 mg/mL. A 25 mg vial with ICG powder was diluted in 20 cc of aqueous sterile water, and 0.5 to 1 mL of ICG solution was injected near the hilum of the ovary, respectively into the right ovarian pedicle (3 patients), into the right broad ligament (1 patient), into left ovarian parenchyma (1 patient). A percutaneous introduction of the needle was performed with low intra-abdominal pressure (4-5 mmHg) in order to have the ovarian surface close to the abdominal wall during the injection of the tracer. A typical spinal needle (22-gauge size) is made up of an obturator having an end cap, a cannula having a needle bevel at one extremity and a hub at the opposite end. We have disengaged the obturator from the cannula and we have connected the 20 cm-long hub to infusion tubing for intravenous fluid delivery. The infusion tubing is instead connected to indocyanine green infusor. The needle is advanced inside the lateral trocar (10 mm) through a Johann clamp and in this way we can inject the indocyanine in the sites described above (Figure 1 A-B). Subsequently, by using the real time fluorescent infrared light of the SPIES camera, the entire retroperitoneal area was explored to find the fluorescent tracer in the lymphatic channels and to identify the anatomical location of the SLNs.

25

Sentinel node biopsy in ovarian cancer M. Angelucci et al.

RESULTSBetween April 2016 and May 2016, 5 patients

(age range 43-67 years; mean 54,7 years) with clinical FIGO stage I - II ovarian carcinoma underwent laparoscopic abdominal hysterectomy (LAH) and bilateral salpingo-oophorectomy (BSO) with radical omentectomy and with pelvic and para aortic lymph node dissection, at the Department of Gynecologic Oncology, Regina Elena National Cancer Institute. (Table 1).The distribution of histology and stage is shown in Table 2.

Two epithelial histologies were represented: serous (3 patients) and endometrioid (2 patients).For serous types the grading was 3 in both patients and for endometrioid types grading was 2 in both patiens. A variety of stages were represented, in particular patients with endometrioid ovarian cancer showed stage IA and patients with serous ovarian cancer were affected by stage IIB and IC respectively. LVSI was negative in all of patients. Median time of surgery was 196 minutes (range 180 to 215). Median total nodes removed were 21 (range 18 to 28), in two patient ipsilaterally to the injection site and in two patients bilaterally. A total of 6 SNs was detected in all patients. The median interval from the tracer injection to the detection of all SNs was 2 minutes (range, 1 - 3 minutes). In three patients, the first detected SN was found in the area of common iliac artery, in two patients at paracaval region. The median number of lymph nodes removed per patient was 2 (range, 0-2). All the detected SLNs were identified ipsilateral to the site of injection. Only in two patients we detected a second LSN, respectively in preaortic region and in under mesenteric region. The biopsy of all of 6 SNs was negative: the remaining removed 22 not-sentinel node were negative too. (Table 3).

No intra o post-operative complications were registered and no allergic reaction occurred. In absence of positive nodes sensitivity could not be calculated. Specificity and negative predictive value (NPV) were 100%.

Figure 1.Inoculation of IDG in the Right ovarian pedicle.A) Before and B) After inoculation of IDG

Table 1.Caracteristics of patients

Table 3.Site of SLN locatio

Table 2.Hystology of patients DISCUSSION

Although the sentinel node biopsy has been applied successfully for breast cancer, vulvar cancer melanoma and other gynecological cancers(6), the role of SLN mapping in ovarian cancer is still unclear. The most common


26


causes are the different mapping techniques (tracers, injection sites) and the risk of tumor dissemination. Lymph node involvement in early stage ovarian cancer plays a crucial prognostic role. Literature shows that, the prevalence of pelvic and para-aortic node metastases in 689 early stage ovarian cancer is 20%(11). Systematic pelvic and para-aortic lymphadenectomy (LAE) is an important prognostic factor but, compared with a lymph node sampling, LAE takes prolonged hospitalization, more blood loss and longer surgical time(12).

Kleppe et al. in the 2015, realized a 3D reconstruction of a lymphatic drainage pathways of the ovaries in human fetal pelvis aged 15 weeks, previously sectioned and stained with hematoxylin and eosin or with azan. In addition, series of selected sections were stained with antibodies against Lyve-1 (ReliaTech), S100 (DAKO), and >-smooth muscle actin (SMA; Sigma-Aldrich). Micrographs were made with a microscope and camera and a particular software was used to assemble the 3D volume and to reconstruct the 3D surface meshes. Authors identified 2 major and 1 minor lymphatic drainage pathways of the ovaries: an abdominal pathway, running via the infundibulopelvic ligament to the para-aortic (left side)/paracaval (right side) regions; a pelvic pathway, draining via the lateral parametrium and supraureteral pathway to the internal iliac artery and obturator fossa; and thirdly an inguinal pathway, draining via the round ligament of the uterus to the inguinal regions. Sentinel nodes can be found in any of these regions(13).

Subsequently, the authors determined the feasibility and the safety of the SN procedure performed with tracer injection into the ovarian ligament. Injection of radioactive tracers resulted in the identification of SNs in all patients, suggesting that this procedure could potentially be incorporated into routine clinical practice in patients with early-stage ovarian cancer(4). Therefore same authors proposed a phase 1 study protocol which provides a blue dye and radioactive colloid injection into the ligamentum ovarii proprium and the ligamentum infundibulo-pelvicum before starting the surgical staging procedure. In the analysis this protocol calculates the percentage of patients in whom is feasible to identify sentinel nodes. Other study parameters are the anatomical localization of the sentinel nodes and the incidence of false negative lymph nodes(14).

Up to the present, a small amount of studies on SNB in ovarian cancer have been published.

Table 4 summarizes the data of these studies in addition to the current one.

Three previously published series explored the feasibility of SLN mapping of the ovary performed during traditional open surgery and overall recruited 59 patients. In a pilot study, Negishi et al(7) found SLNs in 11 patients by the injection of a mixture of activated carbon CH40 and polyvinylpyrrolidone into the ovarian cortex with an overall detection rate of 100%. Another two studies injected near the hilum a solution of technetium radiocolloid and blue dye(8-4). A total of 30 blue SLNs were located in 15 of 16 patients (94%). With great interest, the authors found a statistically significance difference in the level of the right- and left-side SLNs in relation to Inferior Mesenteric Artery (IMA). The right-side injections drained below the IMA, whereas left-side injections located SLNs above the IMA. Kleppe et al(4) in their series of 22 patients injected the tracer close to the ovary on the dorsal and the ventral side of the ovarian ligament and the suspensor ligament. They successfully completed the SLN mapping in 96% of cases. In most patients (67%) the hot spot nodes were detected in the paracaval or in the para-aortic region exclusively, only 9% in the pelvic region, and 24% in both the paracaval/para-aortic and pelvic region. In 19 of 21 patients, SLNs were found ipsilateral to the injection site. The same authors(13) confirmed their preliminary hypotheses with the study of the hystologic lymphatic pathway of the ovary. They

Table 4.Review of literature on SLN in early ovarian cancer

27

Sentinel node biopsy in ovarian cancer M. Angelucci et al.

found 3 main lymphatic drainage pathways from the ovaries the abdominal pathway, the pelvic pathway and the inguinal pathway. The first 2 seem to be the major route for lymphatic drainage from the ovary. The inguinal pathway seeems to disappear during embryologic development, but in a limited pecentage of women it may persist and can explain the occurence of isolated inguinal metastases.

The U.S. Food and Drug Administration approved ICG for use in humans since 1959 for cardiac and liver functions, but for tissue ICG, its use is off label. Moreover, ICG is widely used particularly for cholangiography and as a tool to delineate the extrahepatic biliary tree for NIR fluorescence(15). More recently, the clinical effectiveness of NIR fluorescence using ICG has been evaluated for SLN mapping in gynecologic malignancies, particularly in laparoscopic or robotic approach(16-19). Buda et al(20) recently published their experience about the feasibility of ovarian SLN mapping during laparoscopy using the fluorescent dye ICG and near-infrared real-time SPIES ICG (Karl Storz). In these patients the same protocol as Kleppe et al(4) has been adopted and the injection has been carried out through a percutaneous approach with a 22-G spinal needle (12 cm). Their results are similar to the previous series published concerning the incidence and the location of nodal involvement in early stage ovarian cancer. 100% of the SLNs were located ipsilaterally to the side of injection. and in 30% the paracaval/para-aortic location was associated with pelvic migration on the common iliac nodes. Most of the right ovarian SLNs were located below the IMA (inferior mesenteric artery), whereas SLNs were located above the IMA when the left ovary was injected.

In our small series, four patients with clinical FIGO stage I and II ovarian carcinoma underwent to laparoscopic SLN mapping using Fluorescent Indocyanine Green injected near the hilum of the ovary, respectively into the right ovarian pedicle (3 patients), into the right broad ligament (1

patient), into left ovarian parenchyma (1 patient). A spinal needle (22-gauge size) was connected to 20 cm-long hub to infusion tubing for intravenous fluid delivery. The needle was advanced inside the lateral trocar (10 mm) through a Johann clamp and the indocyanine was injected into the right ovarian pedicle (3 patients), into the right broad ligament (1 patient), into left ovarian parenchyma (1 patient). Median total nodes removed were 21 (range 18 to 28), in two patient ipsilaterally to the injection site and in two patients bilaterally. A total of 6 SNs was detected in all patients. In three patients, the first detected SN was found in the area of common iliac artery, in one patient at paracaval region.

Only in two patients a second LSN was detected, respectively in preaortic region and in under mesenteric region. The biopsy of all of 6 SNs was negative: the remaining removed 22 not-sentinel node were negative too. Despite the very small series in our experience, we want highlight the innovative laparoscopic injection technique, through a spinal needle connected to infusion tubing. Moreover we used ICG tracer, a new injection agent that relies on near-infrared imaging. Early reports concerning its use in cervical and endometrial cancer suggest very high SLN detection rates(21).

Our paper shows that the laparoscopic Indocyanine Green Sentinel lymph node mapping in ovarian cancer is a feasible, safe and inexpensive technique, suggesting that technique could potentially be incorporated into routine clinical practice. Unfortunately the sample size is very small. Additional prospective multicenter studies are needed to determine the best SN procedure to reduce the morbidity and rate of complications associated with complete pelvic and paraortic lymphadenectomy.

CONFLICT OF INTERESTThe authors have no conflict of interest.


28


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13) Kleppe M, Kraima AC, Kruitwagen RF, et al. Understanding Lymphatic Drainage Pathways of the Ovaries to Predict Sites for Sentinel Nodes in Ovarian Cancer. Int J Gynecol Cancer. 2015;25(8):1405-14.14) Kleppe M, Van Gorp T, Slangen BF, et al. Sentinel node in ovarian cancer: study protocol for a phase 1 study. Trials. 2013;14:47.15) Kaneko J, Ishizawa T, Masuda K, et al. Indocyanine green reinjection technique for use in fluorescent angiography concomitant with cholangiography during laparoscopic cholecystectomy. Surg Laparosc Endosc Percutan Tech. 2012;22(4):341-4.16) Jewell EL, Huang JJ, Abu-Rustum NR, et al. Detection of sentinel lymph nodes in minimally invasive surgery using indocyanine green and near-infrared fluorescence imaging for uterine and cervical malignancies. Gynecol Oncol. 2014;288(133):274–7.17) Sinno AK, Fader AN, Roche KL, et al. A comparison of colorimetric versus fluorometric sentinel lymph node mapping during robotic surgery for endometrial cancer. Gynecol Oncol. 2014;134:281–6.18) Plante M, Touhami O, Trinh XB, et al. Sentinel node mapping with indocyanine green and endoscopic near-infrared fluorescence imaging in endometrial cancer. A pilot study and review of the literature. Gynecol Oncol. 2015;137:443–7.19) Handgraaf HJ, Verbeek FP, Tummers QR, et al. Real-time nearinfrared fluorescence guided surgery in gynecologic oncology: a review of the current state of the art. Gynecol Oncol. 2014;135:606–1320) Buda A, Passoni P, Corrado G, et al. Near-infrared Fluorescence-guided Sentinel Node Mapping of the Ovary With Indocyanine Green in a Minimally Invasive Setting: A Feasible Study. J Minim Invasive Gynecol. 2016; in press21) E.L. Jewell, J.J. Huang, N.R. Abu-Rustum, et al. Detection of sentinel lymph nodes in minimally invasive surgery using indocyanine green and near-infrared fluorescence imaging for uterine and cervical malignancies. Gynecol Oncol. 2014; 133 (2): 274–277.

1. DENOMINAZIONE DEL MEDICINALE: MECLON® “20% + 4% crema vaginale” MECLON® “200 mg/10 ml + 1 g/130 ml soluzione vaginale”. 2. COMPOSIZIONE QUA-LITATIVA E QUANTITATIVA: Crema vaginale. 100 g contengono: Principi attivi: Metro-nidazolo 20 g; Clotrimazolo 4 g. Eccipienti: contiene sodio metil p-idrossibenzoato e sodio propil p-idrossibenzoato. Per l’elenco completo degli eccipienti, vedere paragrafo 6.1. Soluzione vaginale. Flacone da 10 ml. 10 ml contengono: Principio attivo: Clotrimazolo 200 mg. Flacone da 130 ml. 130 ml contengono: Principio attivo: Metronidazolo 1 g. Eccipienti: contiene sodio metil p-idrossibenzoato e sodio propil p-idrossibenzoato. Per l’elenco completo degli eccipienti, vedere paragrafo 6.1. 3. FORMA FARMACEUTICA: Crema vaginale. Soluzione vaginale. 4. INFORMAZIONI CLINICHE: 4.1 Indicazioni terapeutiche: Crema vaginale. Cervico-vaginiti e vulvo-vaginiti causate da Trichomonas vaginalis anche se associato a Candida albicans, Gardnerella vaginalis ed altra � ora batterica sensibile. MECLON® crema vaginale può essere impiegato anche nel partner a scopo pro� lattico. Soluzione vaginale. Coadiuvante nella terapia di cervico-vaginiti, vulvo-vaginiti causate da Trichomonas vaginalis anche se associato a Candida albicans, Gardnerella vaginalis ed altra � ora batterica sensibile. MECLON® soluzione vaginale può essere impiegato anche dopo altra terapia topica od orale, allo scopo di ridurre il rischio di recidive. 4.2 Posologia e modo di somministrazione: Crema vaginale. Somministrare profondamente in vagina il contenuto di un applicatore una volta al giorno per almeno sei giorni consecutivi, preferibilmente alla sera prima di coricarsi, oppure secondo pre-scrizione medica. Nelle trichomoniasi, maggior sicurezza di risultato terapeutico si veri� ca con il contemporaneo uso di Metronidazolo per via orale sia nella donna non gestante che nel partner maschile. Per un’ottimale somministrazione si consiglia una posizione supina, con le gambe leggermente piegate ad angolo. Per ottenere una migliore steriliz-zazione è preferibile spalmare un po’ di MECLON® crema vaginale anche esternamente, a livello perivulvare e perianale. Se il medico prescrive il trattamento del partner a scopo pro� lattico, la crema deve essere applicata sul glande e sul prepuzio per almeno sei giorni. Istruzioni per l’uso: Dopo aver riempito di crema un applicatore, somministrare la crema in vagina mediante pressione sul pistone, � no a completo svuotamento. Soluzione vaginale. Somministrare la soluzione vaginale pronta una volta al giorno, preferibilmente al mattino, oppure secondo prescrizione medica. Nella fase di attacco l’uso della soluzione vaginale deve essere associato ad adeguata terapia topica e/o orale. L’irrigazione va eseguita preferibilmente in posizione supina. Un lento svuotamento del � acone favorirà una più prolungata permanenza in vagina dei principi attivi e quindi una più ef� cace azione antimicrobica e detergente. Istruzioni per l’uso: Dopo aver versato il contenuto del � aconcino nel � acone, inserire la cannula vaginale sul collo del � acone stesso. Introdurre la cannula in vagina e somministrare l’intero contenuto. 4.3 Controindicazioni: Iper-sensibilità verso i principi attivi od uno qualsiasi degli eccipienti. 4.4 Avvertenze speciali e opportune precauzioni d’impiego: Evitare il contatto con gli occhi. Il consigliato im-piego contemporaneo di Metronidazolo per via orale è soggetto alle controindicazioni, effetti collaterali ed avvertenze descritte per il prodotto summenzionato. Evitare il trat-tamento durante il periodo mestruale. Tenere il medicinale fuori dalla portata e dalla vista dei bambini. 4.5 Interazioni con altri medicinali e altre forme di interazione: Nessuna. 4.6 Gravidanza e allattamento: In gravidanza il prodotto deve essere impie-gato solo in caso di effettiva necessità e sotto il diretto controllo del medico. 4.7 Effetti sulla capacità di guidare veicoli e sull’uso di macchinari: MECLON® non altera la capacità di guidare veicoli o di usare macchinari. 4.8 Effetti indesiderati: Dato lo scarso assorbimento per applicazione locale dei principi attivi Metronidazolo e Clotrimazolo, le reazioni avverse riscontrate con le formulazioni topiche sono limitate a: Disturbi del sistema immunitario: Non nota (la frequenza non può essere de� nita sulla base dei dati disponibili): reazioni di ipersensibilità. Patologie della cute e del tessuto sottocutaneo: Molto rari (frequenza <1/10.000): fenomeni irritativi locali quale prurito, dermatite allergica da contatto, eruzioni cutanee. L’eventuale manifestarsi di effetti in-desiderati comporta l’interruzione del trattamento. 4.9 Sovradosaggio: Non sono stati descritti sintomi di sovradosaggio. 5. PROPRIETÀ FARMACOLOGICHE: 5.1 Proprietà farmacodinamiche: Categoria farmacoterapeutica: Antinfettivi ed antisettici ginecolo-gici/Associazioni di derivati imidazolici - Codice ATC: G01AF20. Meccanismo d’azione/effetti farmacodinamici: Il MECLON® è una associazione tra Metronidazolo (M) e

Clotrimazolo (C). Il (M) è un derivato nitroimidazolico ad ampio spettro di azione antipro-tozoaria e antimicrobica. Ha effetto trichomonicida diretto ed è attivo su cocchi Gram-positivi anaerobi, bacilli sporigeni, anaerobi Gram-negativi. Presenta attività spiccata sulla Gardnerella vaginalis. Non è attivo sulla � ora acido� la vaginale. Il (C) è un imidazolico con spettro antifungino molto ampio (Candida, etc.). È attivo anche su Trichomonas vaginalis, cocchi Gram-positivi, Toxoplasmi, etc. È stato documentato che l’associazione Clotrimazolo-Metronidazolo dà luogo ad effetti di tipo additivo, pertanto essa è in grado di conseguire tre vantaggi terapeutici principali: 1) Ampliamento dello spettro d’azione antimicrobica, per sommazione degli effetti dei due principi attivi; 2) Potenziamento dell’attività antimicotica, antiprotozoaria ed antibatterica; 3) Abolizione o ritardo della comparsa dei fenomeni di resistenza. Studi microbiologici in vitro hanno dimostrato che l’attività trichomonicida e antimicotica risulta potenziata quando il (M) e il (C) sono as-sociati nelle stesse proporzioni che sono presenti nel MECLON®. Anche l’attività anti-batterica esaminata su diversi ceppi di microorganismi è risultata elevata ed è emerso un potenziamento di essa quando i due principi attivi del MECLON® vengono associati. 5.2 Proprietà farmacocinetiche: Dalle indagini farmacocinetiche sui conigli, cani e ratti risulta che dopo ripetute applicazioni topiche di MECLON® non si rilevano concentrazioni apprezzabili di Clotrimazolo e Metronidazolo nel sangue. Per applicazione vaginale nella donna il (M) e il (C) vengono assorbiti in una percentuale che varia tra il 10% e il 20% circa. 5.3 Dati preclinici di sicurezza: La tossicità acuta del MECLON® nel topo e nel ratto (os) è risultata molto bassa, con una mortalità di appena il 20% dopo 7 giorni, a dosi molto elevate (600 mg/Kg di (C) e 3000 mg/Kg di (M), sia da soli che associati). Nelle prove di tossicità subacuta (30 giorni) il MECLON®, somministrato per via locale (genitale) nel cane e nel coniglio, non ha determinato alcun tipo di lesione nè locale nè sistemica anche per dosi molte volte superiori a quelle comunemente impiegate in terapia umana (3-10 Dtd nel cane e 100-200 Dtd nel coniglio; 1 Dtd = dose terapeutica/die per l’uomo = ca. 3,33 mg/Kg di (C) e ca. 16,66 mg/Kg di (M)). Il MECLON® somministrato durante il periodo di gravidanza per via topica vaginale nel coniglio e nel ratto non ha fatto evidenziare alcun segno di sofferenza fetale per dosi die di 100 Dtd, nè in� ussi negativi sullo stato gestazionale. 6. INFORMAZIONI FARMACEUTICHE: 6.1 Elenco degli eccipienti: Crema vaginale. Eccipienti: Stearato di glicole e polietilenglicole; Pa-raf� na liquida; Sodio metile p-idrossibenzoato; Sodio propile p-idrossibenzoato; Acqua depurata. Soluzione vaginale. Flacone da 10 ml. Eccipienti: Alcool ricinoleilico; Etanolo; Acqua depurata. Flacone da 130 ml. Eccipienti: Sodio metile p-idrossibenzoato; Sodio propile p-idrossibenzoato; Acqua depurata. 6.2 Incompatibilità: Non sono note incom-patibilità con altri farmaci. 6.3 Periodo di validità: Crema vaginale: 3 anni. Soluzione vaginale: 3 anni. 6.4 Precauzioni particolari per la conservazione: Questo medicinale non richiede alcuna particolare condizione per la conservazione. 6.5 Natura e contenuto del contenitore: MECLON® crema vaginale. Tubo in alluminio verniciato internamente con resine epossidiche e fenoliche. Gli applicatori monouso sono di polietilene. Tubo da 30 g + 6 applicatori monouso. MECLON® soluzione vaginale. Flaconi di polietilene a bassa densità; � aconcini di polietilene; cannule vaginali di polietilene. 5 � aconi da 10 ml + 5 � aconi da 130 ml + 5 cannule vaginali monouso. 6.6 Precauzioni particolari per lo smaltimento e la manipolazione: Nessuna istruzione particolare. 7. TITOLARE DELL’AUTORIZZAZIONE ALL’IMMISSIONE IN COMMERCIO: ALFA WASSERMANN S.p.A. - Sede legale: Via E. Fermi, n. 1 - Alanno (PE). Sede amministrativa: Via Ragazzi del ‘99, n. 5 - Bologna. 8. NUMERI DELL’AUTORIZZAZIONE ALL’IMMISSIONE IN COMMERCIO: MECLON® crema vaginale: A.I.C. n. 023703046. MECLON® soluzione vaginale: A.I.C. n. 023703059. 9. DATA DELLA PRIMA AUTORIZZAZIONE/RINNOVO DELL’AUTORIZZAZIONE: 11.05.1991 (GU 07.10.1991) / 01.06.2010. 10. DATA DI REVISIONE DEL TESTO: Determinazione AIFA del 27 Ottobre 2010.

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Medicinale non soggetto a prescrizione medica (SOP) . CLASSE C.

Riassunto delle Caratteristiche del Prodotto

1. DENOMINAZIONE DEL MEDICINALE: MECLON® “100 mg + 500 mg ovuli”. 2. COMPOSIZIONE QUALITATIVA E QUANTITATIVA: Un ovulo da 2,4 g contiene: Principi attivi: Metronidazolo 500 mg; Clotrimazolo 100 mg. Per l’elenco completo degli ecci-pienti, vedere paragrafo 6.1. 3. FORMA FARMACEUTICA: Ovuli. 4. INFORMAZIONI CLINICHE: 4.1 Indicazioni terapeutiche: Cerviciti, cervico-vaginiti, vaginiti e vulvo-va-giniti da Trichomonas vaginalis anche se associato a Candida o con componente batteri-ca. 4.2 Posologia e modo di somministrazione: Lo schema terapeutico ottimale risul-ta il seguente: 1 ovulo di MECLON® in vagina, 1 volta al dì. 4.3 Controindicazioni: Ipersensibilità verso i principi attivi od uno qualsiasi degli eccipienti. 4.4 Avvertenze speciali e opportune precauzioni d’impiego: Evitare il contatto con gli occhi. Il consi-gliato impiego contemporaneo di Metronidazolo per via orale è soggetto alle controindi-cazioni, effetti collaterali ed avvertenze descritte per il prodotto summenzionato. MECLON® ovuli va impiegato nella prima infanzia sotto il diretto controllo del medico e solo nei casi di effettiva necessità. Tenere il medicinale fuori dalla portata e dalla vista dei bambini. 4.5 Interazioni con altri medicinali e altre forme di interazione: Nessuna. 4.6 Gravidanza e allattamento: In gravidanza il prodotto deve essere impie-gato solo in caso di effettiva necessità e sotto il diretto controllo del medico. 4.7 Effetti sulla capacità di guidare veicoli e sull’uso di macchinari: MECLON® non altera la capacità di guidare veicoli o di usare macchinari. 4.8 Effetti indesiderati: Dato lo scar-so assorbimento per applicazione locale dei principi attivi Metronidazolo e Clotrimazolo, le reazioni avverse riscontrate con le formulazioni topiche sono limitate a: Disturbi del sistema immunitario: Non nota (la frequenza non può essere de� nita sulla base dei dati disponibili): reazioni di ipersensibilità. Patologie della cute e del tessuto sottocutaneo: Molto rari (frequenza <1/10.000): fenomeni irritativi locali quale prurito, dermatite aller-gica da contatto, eruzioni cutanee. L’eventuale manifestarsi di effetti indesiderati com-porta l’interruzione del trattamento. 4.9 Sovradosaggio: Non sono stati descritti sintomi di sovradosaggio. 5. PROPRIETÀ FARMACOLOGICHE: 5.1 Proprietà farmacodinami-che: Categoria farmacoterapeutica: Antinfettivi ed antisettici ginecologici/Associazioni di derivati imidazolici - Codice ATC: G01AF20. Meccanismo d’azione/effetti farmacodi-namici: Il MECLON® è una associazione tra metronidazolo (M) e clotrimazolo (C). Il (M) è un derivato nitroimidazolico ad ampio spettro di azione antiprotozoaria e antimicrobica. Ha effetto trichomonicida diretto ed è attivo su cocchi Gram-positivi anaerobi, bacilli sporigeni, anaerobi Gram-negativi. Presenta attività spiccata sulla Gardnerella vaginalis. Non è attivo sulla � ora acido� la vaginale. Il (C) è un imidazolico con spettro antifungino molto ampio (Candida, etc.). È attivo anche su Trichomonas vaginalis, cocchi Gram-positivi, Toxoplasmi, etc. È stato documentato che l’associazione Clotrimazolo-Metronidazolo dà luogo ad effetti di tipo additivo, pertanto essa è in grado di conseguire tre vantaggi terapeutici principali: 1) Ampliamento dello spettro d’azione antimicrobica, per sommazione degli effetti dei due principi attivi; 2) Potenziamento dell’attività antimi-

cotica, antiprotozoaria ed antibatterica; 3) Abolizione o ritardo della comparsa dei feno-meni di resistenza. Studi microbiologici in vitro hanno dimostrato che l’attività trichomo-nicida e antimicotica risulta potenziata quando il (M) e il (C) sono associati nelle stesse proporzioni che sono presenti nel MECLON®. Anche l’attività antibatterica esaminata su diversi ceppi di microorganismi è risultata elevata ed è emerso un potenziamento di essa quando i due principi attivi del MECLON® vengono associati. 5.2 Proprietà farmacoci-netiche: Dalle indagini farmacocinetiche sui conigli, cani e ratti risulta che dopo ripetute applicazioni topiche di MECLON® non si rilevano concentrazioni apprezzabili di Clotrimazolo e Metronidazolo nel sangue. Per applicazione vaginale nella donna il (M) e il (C) vengono assorbiti in una percentuale che varia tra il 10% e il 20% circa. 5.3 Dati preclinici di sicurezza: La tossicità acuta del MECLON® nel topo e nel ratto (os) è risul-tata molto bassa, con una mortalità di appena il 20% dopo 7 giorni, a dosi molto elevate (600 mg/Kg di (C) e 3000 mg/Kg di (M), sia da soli che associati). Nelle prove di tossi-cità subacuta (30 giorni) il MECLON®, somministrato per via locale (genitale) nel cane e nel coniglio, non ha determinato alcun tipo di lesione nè locale nè sistemica anche per dosi molte volte superiori a quelle comunemente impiegate in terapia umana (3-10 Dtd nel cane e 100-200 Dtd nel coniglio; 1 Dtd = dose terapeutica/die per l’uomo = ca. 3,33 mg/Kg di (C) e ca. 16,66 mg/Kg di (M)). Il MECLON® somministrato durante il periodo di gravidanza per via topica vaginale nel coniglio e nel ratto non ha fatto evidenziare alcun segno di sofferenza fetale per dosi die di 100 Dtd, nè in� ussi negativi sullo stato gesta-zionale. 6. INFORMAZIONI FARMACEUTICHE: 6.1 Elenco degli eccipienti: Eccipienti: Miscela idro� la di mono, di, tri-gliceridi di acidi grassi saturi. 6.2 Incompatibilità: Non sono note incompatibilità con altri farmaci. 6.3 Periodo di validità: 3 anni. 6.4 Precauzioni particolari per la conservazione: Questo medicinale non richiede alcuna particolare condizione per la conservazione. 6.5 Natura e contenuto del contenitore: 10 ovuli in valve in PVC, racchiusi in scatola di cartone. 6.6 Precauzioni particolari per lo smaltimento e la manipolazione: Nessuna istruzione particolare. 7. TITOLARE DELL’AUTORIZZAZIONE ALL’IMMISSIONE IN COMMERCIO: ALFA WASSERMANN S.p.A. - Sede legale: Via E. Fermi, n. 1 - Alanno (PE). Sede amministrativa: Via Ragazzi del ‘99, n. 5 - Bologna. 8. NUMERO DELL’AUTORIZZAZIONE ALL’IMMISSIONE IN COMMERCIO: A.I.C. n. 023703010. 9. DATA DELLA PRIMA AUTORIZZAZIONE/RINNOVO DELL’AUTORIZZAZIONE: 27.11.1978 (GU 16.01.1979) / 01.06.2010. 10. DATA DI REVISIONE DEL TESTO: Determinazione AIFA del 27 Ottobre 2010.

100 mg + 500 mg ovuli, 10 ovuli. Prezzo: € 12,50.

Medicinale non soggetto a prescrizione medica (SOP) . CLASSE C.

italian journal of gynaecology & obstetrics · 1 gynaecology italian journal of the official...

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