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Decreased ovarian reserve in female Sprague–Dawley rats induced by isotretinoin (retinoic acid) exposure

      Abstract

      Isotretinoin is a retinoid widely used for the treatment of severe nodulocystic acne. Although it has broad side effects, there is no well-designed study about its effects on the ovary. This study investigated possible toxic effects of isotretinoin on female gonads. A total of 30 female rats were randomly divided into three equal groups according to the dose of isotretinoin they were administered: 0 mg/kg/day (group 1), 7.5 mg/kg/day (group 2) or 15 mg/kg/day (group 3). Thirty days after the treatment, the effects of isotretinoin on the ovaries were evaluated with serum anti-Müllerian hormone (AMH) concentrations, apoptosis by TUNEL assay and immunohistochemical observations by proliferating cell nuclear antigen (PCNA). The percentage of atretic follicles was calculated for each stage of folliculogenesis. The serum AMH concentrations were found to be lower in both isotretinoin groups. The percentage of atretic follicles in both isotretinoin groups was higher than the control. The number of PCNA-positive granulosa cells was decreased in the isotretinoin groups. The number of ovarian follicles with apoptotic granulosa cells was increased in the experimental groups. These data are the first to identify that exposure of isotretinoin may be responsible for decreased ovarian reserve and toxic effects on rat ovaries.
      Isotretinoin (13-cis-retinoic acid) is a retinoid that is widely used for the treatment of severe acne. Although it has a broad side effects profile, no well-designed study about its effects on the ovary has been published. This study was designed to investigate possible toxic effects of isotretinoin on female gonads. A total of 30 female rats were divided into three equal groups according to the dose of isotretinoin to be administered: 0 mg/kg/day (group 1), 7.5 mg/kg/day (group 2) or 15 mg/kg/day (group 3). Thirty days after the treatment, ovarian effects of isotretinoin were evaluated with serum anti-Müllerian hormone (AMH) concentrations and immunohistochemical examination. The percentage of atretic follicles was calculated for each stage of folliculogenesis. The serum AMH concentrations were lower in both isotretinoin groups. The percentage of atretic follicles in both isotretinoin groups was higher than the control. The number of proliferating cell nuclear antigen-positive granulosa cells was decreased in the isotretinoin groups. The number of ovarian follicles with apoptotic granulosa cells was increased in the experimental groups. These data are the first to identify that exposure of isotretinoin may be responsible for decreased ovarian reserve and toxic effect on rat ovaries.

      Keywords

      Introduction

      Isotretinoin (13-cis-retinoic acid) is a retinoid that has been widely used for the treatment of severe recalcitrant nodulocystic acne since 1982 (
      • Jones H.
      • Blanc D.
      • Cunliffe W.J.
      13-cis Retinoic acid and acne.
      ). The exact mechanism of retinoid action is not clear. There are numerous studies indicating the modes of action of isotretinoin, which include the induction of apoptosis and cell cycle arrest in human sebaceous gland cells (SEB-1 sebocytes) near increased TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) staining of treated cells (SEB-1 sebocytes) and increased concentrations of cleaved caspase 3 (
      • Nelson A.M.
      • Gilliland K.L.
      • Cong Z.
      • Thiboutot D.M.
      13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes.
      ).
      The physiological functions of retinoids include the control of proliferation, apoptosis and differentiation in normal cells during growth and development; they also have tumour-suppressive capacity (
      • Altucci L.
      • Gronemeyer H.
      The promise of retinoids to fight against cancer.
      ).
      Isotretinoin for acne treatment is used for approximately 6 months at a dose of 0.5–1 mg/kg/day to a cumulative dose of 120–150 mg/kg. However, there is emerging evidence that much lower dosages (as low as 5 mg/day) are just as effective but have significantly fewer adverse effects (
      • Sardana K.
      • Garg V.K.
      Efficacy of low-dose isotretinoin in acne vulgaris.
      ). It has been used not only for severe acne vulgaris but also for the management of other dermatological conditions, such as rosacea, folliculitis, sarcoidosis, granuloma annulare, seborrhoeic dermatitis, myelodysplastic syndromes, chemoprevention of skin neoplasms, periorificial dermatitis and a variety of disorders of keratinization (
      • Akyol M.
      • Ozcelik S.
      Non-acne dermatologic indications for systemic isotretinoin.
      ,
      • Brelsford M.
      • Beute T.C.
      Preventing and managing the side effects of isotretinoin.
      ).
      Isotretinoin has a broad spectrum of side effects. The most common side effects occur in the mucocutaneous and ocular regions. Yet the most notable side effect is the induction of birth defects due to fetal exposure to isotretinoin, which is pregnancy category X (
      • Tzimas G.
      • Nau H.
      The role of metabolism and toxicokinetics in retinoid teratogenesis.
      ). Therefore, contraception is necessary during isotretinoin treatment in women of childbearing age, beginning 1 month before, during and 3 months after treatment.
      Acne vulgaris often affects young people between 12 and 18 years of age, some of whom are treated with isotretinoin. While this drug is known to have many side effects, there are only a few studies describing the effects on reproductive organs (
      • Comitato R.
      • Esposito T.
      • Cerbo G.
      • Angelini F.
      • Varriale B.
      • Cardone A.
      Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula.
      ,
      • Ferguson S.A.
      • Cisneros F.J.
      • Gough B.J.
      • Ali S.F.
      Four weeks of oral isotretinoin treatment causes few signs of general toxicity in male and female Sprague–Dawley rats.
      ,
      • Gencoglan G.
      • Tosun M.
      Effects of isotretinoin on spermatogenesis of rats.
      ).
      Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance, is a dimeric ovarian glycoprotein produced by the granulosa cells of healthy, small, growing follicles (
      • Lee M.M.
      • Donahoe P.K.
      Müllerian inhibiting substance: a gonadal hormone with multiple functions.
      ,

      Themmen, A.P., 2005. Anti-Müllerian hormone: its role in follicular growth initiation and survival and as an ovarian reserve marker. J. Natl. Cancer. Inst. Monogr. 18–21.

      ). AMH expression disappears when a follicle becomes atretic, and it is also a reliable marker of ovarian reserve, with decreasing concentrations correlated with reduced response potential (
      • Fleming R.
      • Broekmans F.
      • Calhaz-Jorge C.
      • Dracea L.
      • Alexander H.
      • Andersen A.N.
      • Blockeel C.
      • Jenkins J.
      • Lunenfeld B.
      • Platteau P.
      • Smitz J.
      • de Ziegler D.
      Can anti-Müllerian hormone concentrations be used to determine gonadotrophin dose and treatment protocol for ovarian stimulation?.
      ,
      • Nelson S.M.
      • La Marca A.
      The journey from the old to the new AMH assay: how to avoid getting lost in the values.
      ). Recent studies suggest that serum AMH may be useful as a biomarker for ovarian reserve following chemotherapy and exposure to other gonadotoxic agents (
      • Anders C.
      • Marcom P.K.
      • Peterson B.
      • Gu L.
      • Unruhe S.
      • Welch R.
      • Lyons P.
      • Behera M.
      • Copland S.
      • Kimmick G.
      • Shaw H.
      • Snyder S.
      • Antenos M.
      • Woodruff T.
      • Blackwell K.
      A pilot study of predictive markers of chemotherapy-related amenorrhea among premenopausal women with early stage breast cancer.
      ,
      • Anderson R.A.
      • Cameron D.A.
      Pretreatment serum anti-mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer.
      ,
      • Lie Fong S.
      • Lugtenburg P.J.
      • Schipper I.
      • Themmen A.P.
      • de Jong F.H.
      • Sonneveld P.
      • Laven J.S.
      Anti-mullerian hormone as a marker of ovarian function in women after chemotherapy and radiotherapy for haematological malignancies.
      ,
      • van Beek R.D.
      • van den Heuvel-Eibrink M.M.
      • Laven J.S.
      • de Jong F.H.
      • Themmen A.P.
      • Hakvoort-Cammel F.G.
      • van den Bos C.
      • van den Berg H.
      • Pieters R.
      • de Muinck Keizer-Schrama S.M.
      Anti-Müllerian hormone is a sensitive serum marker for gonadal function in women treated for Hodgkin‘s lymphoma during childhood.
      ). AMH is an earlier predictor of ovarian reserve in the ageing process compared with other identified ovarian hormones, such as FSH, inhibin B or oestradiol and does not vary significantly across the menstrual cycle (
      • Anderson R.A.
      What does anti-Müllerian hormone tell you about ovarian function?.
      ).
      This study postulated that, in isotretinoin-exposed female rats, decreasing concentrations of serum AMH could be a sign of ovarian damage and that isotretinoin causes damage to the ovary by increasing the percentage of atretic follicles. As far as is known, there is no study in the literature investigating the effects of isotretinoin treatment on female rat ovaries.

      Materials and methods

      Animals

      Female, 7–8-week-old, Sprague–Dawley rats were obtained from the Institute of Experimental Medicine, Istanbul University. The animals were maintained on a 12/12 light/dark cycle with ad-libitum access to food and water. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Istanbul University.

      Experimental design

      A total of 30 female rats were randomly divided into three experimental groups according to the dose of isotretinoin to be administered: 0 mg/kg/day (group 1), 7.5 mg/kg/day (group 2) or 15 mg/kg/day (group 3). Each group was comprised of 10 animals.

      Drug administration

      Capsules of isotretinoin (Zoretanin; Actavis Laboratories) were opened and transferred to class A volumetric flasks and diluted with soybean oil (cat. no. 8001-22-7; MP Biomedicals) to obtain suspension at the desired concentrations. All drug preparations were conducted in a darkened room and amber bottles were used for storage. Rats were gavaged once daily with either 0 (soybean oil only), 7.5 or 15 mg/kg/day in a volume of 1.42 ml/kg. Daily gavages continued for 30 consecutive days. The doses of 7.5 or 15 mg/kg/day were given to produce serum isotretinoin concentrations comparable to those of humans treated with 1 mg/kg/day based on the results of a previously published study (
      • Ferguson S.A.
      • Siitonen P.H.
      • Cisneros F.J.
      • Gough B.
      • Young J.F.
      Steady state pharmacokinetics of oral treatment with 13-cis-retinoic acid or all-trans-retinoic acid in male and female adult rats.
      ). Serum isotretinion concentrations were not measured in the current study.

      Histopathological evaluation

      At the end of the experiment, the ovaries were individually immersed in Bouin’s fixative, dehydrated in alcohol and embedded in paraffin. Serial sections of 5 μm were obtained, deparaffinized and stained with haematoxylin–eosin (H and E). Analysis of normal and atretic primordial, primary and preantral follicles was performed in the largest cross-section of each ovary. The total number of normal and atretic follicles per ovary was calculated, and the percentage of atretic follicles was calculated as 100 × atretic follicles total follicles (
      • Zhang H.
      • Zhang X.
      • Yuan Z.
      • Li X.
      • Li W.
      • Zhou Q.
      • Min F.
      • Xie Y.
      • Liu B.
      • Duan X.
      Germ cell loss induced by 12C6+ ion irradiation in young female mice.
      ).

      Classification of follicles

      Follicles were histologically classified as normal or atretic. Normal follicles had an intact granulosa layer with a compact and well-organized arrangement. Atretic follicles were classified based on the previously described criteria (
      • Hay M.R.
      • Cran D.G.
      • Moor R.M.
      Structural changes occurring during atresia in sheep ovarian follicles.
      ). Early atretic follicles contained a minimal number of pyknotic cells, degenerated cells and/or apoptotic bodies distributed along the antral border of the granulosa layer. Advanced atretic follicles contained numerous pyknotic cells, degenerated cells and/or apoptotic bodies in the granulosa layer, while the granulosa layer had already disintegrated in the late atretic follicles.

      PCNA immunohistochemistry

      For immunohistochemical observations, the ovaries were fixed in Bouin‘s, embedded in paraffin and sectioned at 5 μm thickness. Immunocytochemical reactions were performed according to the ABC technique (
      • Hsu S.M.
      • Raine L.
      • Fanger H.
      Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures.
      ). The procedure involved the following steps: (i) endogenous peroxidase activity was inhibited by 3% H2O2 in distilled water for 30 min; (ii) the sections were washed in distilled water for 10 min; (iii) non-specific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907; Carpinteria, CA, USA) with PBS, diluted 1:4; (iv) the sections were incubated with specific mouse monoclonal anti-PCNA antibody (cat. no. MS-106-B; Thermo LabVision, USA) diluted 1:50 for 1 h at room temperature; (v) the sections were washed in PBS 3 × 3 min; (vi) the sections were incubated with biotinylated anti-mouse IgG (DAKO LSAB 2 Kit); (vii) the sections were washed in PBS 3 × 3 min; (viii) the sections were incubated with ABC complex (DAKO LSAB 2 Kit); (ix) the sections were washed in PBS 3 × 3 min; (x) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories); (xi) the sections were washed in tap water for 10 min and then dehydrated; (xii) the nuclei were stained with haematoxylin; and (xiii) the sections were mounted in DAKO paramount. All dilutions and thorough washes between steps were performed using PBS, unless otherwise specified. All steps were carried out at room temperature unless otherwise specified. As a negative control, a primary antibody was replaced with PBS.
      Immunohistochemical staining was scored in a semi-quantitative manner, in order to determine the differences between the control group and the experimental groups. The numbers of the positive staining were recorded as absence; 0 (−), a few; 1 (±), few; 2 (+), medium; 3 (++), high; 4 (+++), and very high; 5 (++++). This analysis was performed in serial sections from each animal at ×400 magnification.

      Ovarian apoptotic activity

      Apoptosis was evaluated by the TUNEL assay. The TUNEL method, which detects fragmentation of DNA in the nucleus during apoptotic cell death in situ, was employed using an apoptosis detection kit (TdT-Fragel DNA Fragmentation Detection Kit, cat. no. QIA33; Calbiochem, USA). All reagents listed below are from this kit and were prepared following the manufacturer‘s instructions. The ovaries were fixed in Bouin’s and embedded in parafin. Serial sections of 5 μm were obtained and deparaffinized. They were then incubated with 20 mg/ml proteinase K for 20 min and rinsed in TBS. Endogenous peroxidase activity was inhibited by incubation with 3% hydrogen peroxide. Sections were then incubated with equilibration buffer for 10–30 s and then with TdT-enzyme in a humidified atmosphere at 37°C, for 90 min. They were subsequently put into pre-warmed working strength stop/wash buffer at room temperature for 10 min and incubated with blocking buffer for 30 min. Each step was separated by thorough washes in TBS. Labelling was revealed using DAB, counter staining was performed using methyl green, and sections were dehydrated, cleared and mounted.
      The positive staining of TUNEL cell numbers was scored in a semi-quantitative manner in order to determine the differences between the control group and the experimental groups. The numbers of the positive staining were recorded as absence; 0 (−), a few; 1 (±), few; 2 (+), medium; 3 (++), high; 4 (+++), and very high; 5 (++++). This analysis was performed in serial sections from each animal at ×400 magnification.

      Serum AMH concentrations

      Anti-Müllerian hormone concentrations in the serum were measured by USCN Life Science enzyme-linked immunosorbent assays (ELISA). A monoclonal antibody specific for AMH was pre-coated onto a microplate; any AMH present was bound by the immobilized antibody. Standards or samples were then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for AMH. Next, avidin conjugated to horseradish peroxidase was added to each microplate well and incubated. A substrate solution was added to the wells and colour developed in proportion to the amount of AMH bound in the initial step. The colour development was stopped and the intensity of the colour was measured. The detection limit of this assay was less than 0.053 ng/ml.
      The investigators were blinded to the groups for both the histopathological evaluations and AMH assays.

      Statistical analysis

      All data were analysed using Statistical Package for the Social Sciences for Windows version 16.0 (SPSS, Chicago, IL, USA). All data were presented as mean ± standard error (SE). Differences in measured parameters between the three groups were analysed with a nonparametric test (Kruskal–Wallis). Dual comparisons between groups exhibiting significant values were evaluated with a Mann–Whitney U test. Kruskal–Wallis and Mann–Whitney tests, which are both rank-sum tests, were used due to the small sample size. Statistical significance was defined as P < 0.05.

      Results

      Serum AMH concentrations

      The serum concentrations of AMH were measured. The mean serum AMH concentrations were 2.41 ± 0.17, 1.80 ± 0.18, 1.47 ± 0.10 ng/ml in groups 1, 2 and 3, respectively (P = 0.003). Serum AMH concentrations were observed as being lower in groups 2 and 3 compared with group 1 (P = 0.026, P = 0.001, respectively). However, no significant difference was found between group 2 and group 3 (Figure 1).
      Figure thumbnail gr1
      Figure 1Serum concentrations of anti-Müllerian hormone (AMH; mean ± SE). The serum AMH concentrations in the control animals (soybean oil only) were higher than in group 2 (7.5 mg/kg/day isotretinoin) and group 3 (15 mg/kg/day isotretinoin) (P < 0.05, P < 0.01, respectively). There was no significant differences between group 2 and group 3. Ten animals per group.

      Isotretinoin exposure increased the percentage of atretic follicles

      The percentage of atretic follicles was calculated at each stage of folliculogenesis. The percentage of atretic follicles in both experimental groups was higher than in the control group. Primordial, primary, preantral and antral follicles in group 2 (7.5 mg/kg/day isotretinoin) and group 3 (15 mg/kg/day isotretinoin) showed significant degeneration. The mean atretic primordial follicle percentages were 34.9 ± 1.14, 38.3 ± 1.15 and 51.3 ± 1.04% in groups 1, 2 and 3, respectively (P < 0.001). The mean atretic primary follicle percentages were 17.0 ± 0.57, 20.9 ± 0.67, 35.2 ± 0.68% in groups 1, 2 and 3, respectively (P < 0.001). The mean atretic preantral follicle percentages were 20.9 ± 0.45, 24.9 ± 0.62, 45.7 ± 0.71% in groups 1, 2 and 3, respectively (P < 0.001). The percentages of mean atretic antral follicles were 56.6 ± 0.84 in group 1, 61.2 ± 0.98 in group 2 and 87.6 ± 1.03% in group 3 (P < 0.001). The data showed that there was a dose response increase in the percentages of all stages of atretic follicles in conformity with increased doses of isotretinoin (P < 0.001) (Figure 2). The percentage of healthy follicles was calculated at each stage of folliculogenesis and, in contrast to atretic follicles, the mean percentage of healthy follicles was lower in both experimental groups (Figure 3).
      Figure thumbnail gr2
      Figure 2Proportions (mean ± SE) of atretic primordial, primary, preantral and antral follicles in the ovary. The proportions of atretic follicles in group 2 (7.5 mg/kg/day isotretinoin) and group 3 (15 mg/kg/day isotretinoin) were higher than in group 1 (soybean oil only) (P < 0.001). Ten animals per group, largest cross-section of each ovary analysed.
      Figure thumbnail gr3
      Figure 3Proportions (mean ± SE) of healthy primordial, primary, preantral and antral follicles in the ovary. The proportions of healthy follicles in group 2 (7.5 mg/kg/day isotretinoin) and group 3 (15 mg/kg/day isotretinoin) were lower than group 1 (soybean oil only) (P < 0.001). Ten animals per group, largest cross-section of each ovary analysed.

      Immunohistochemical findings

      PCNA-positive cells were strongly detected in the granulosa cells of the control group (Figure 4a and Table 1). However, the signal intensity of positive cells was significantly lower in the granulosa cells of the experimental groups (P = 0.001) (Figure 4b and Table 1). The number of PCNA-positive granulosa cells decreased in association with follicular atresia. Analysis of ovaries in group 2 showed that the number of follicles containing PCNA-positive cells with advanced atretic features was significantly higher than in group 1 (P = 0.029) (Figure 4c and Table 1).
      Figure thumbnail gr4
      Figure 4Immunohistochemical detection of PCNA in groups 1, 2 and 3. (a) Group 1 (soybean oil only); (b) group 2 (7.5 mg/kg/day isotretinoin), (c) group 3 (15 mg/kg/day isotretinoin). Arrows indicate PCNA-positive granulosa cells, arrowhead indicates nuclei of oocytes and asterisk indicates atretic follicle. Immunoperoxidase, hematoxylin counterstain, Bar = 50 μm.
      Table 1Semi-quantitative comparison of the number of PCNA- and TUNEL-positively stained cells in the follicles of ovaries from each group.
      Group 1Group 2Group 3
      PCNA+++++++++
      TUNEL++++++
      The numbers of the positive staining were recorded as a few; 1 (±), few; 2 (+), medium; 3 (++), high; 4 (+++), and very high; 5 (++++). (n = 10 animals for each group, 10 sections were analysed for each ovary). Group 1 = soybean oil only (control); group 2 = 7.5 mg/kg/day isotretinoin; and group 3 = 15 mg/kg/day isotretinoin.

      Evaluation of apoptosis

      The number of ovarian follicles containing apoptotic granulosa cells was higher in the experimental groups (P = 0.001) (Figure 5ac and Table 1). Analysis of rat ovaries by TUNEL showed that there were apoptotic cells in both the follicular wall and the antrum (Figure 5b and Table 1). On the other hand, analysis of ovaries in group 3 showed that the number of follicles containing apoptotic cells with advanced atretic features was significantly higher than group 2 (P = 0.012) (Figure 5c and Table 1).
      Figure thumbnail gr5
      Figure 5In-situ end labelling of DNA fragmentation on ovary sections. (a) Group 1 (soybean oil only); (b) group 2 (7.5 mg/kg/day isotretinoin), (c) group 3 (15 mg/kg/day isotretinoin). Arrows indicate TUNEL-positive granulosa cells. Bar = 50 μm.

      Discussion

      Isotretinoin is used effectively for treatment of acne vulgaris, a condition that often affects young people between 12 and 18 years of age. While it is known to have broad side effects, there is no well-designed study about its effects on the ovaries. In this study, exposure to isotretinoin in female rats induced ovarian damage that was detected by decreased serum AMH concentrations and increased apoptotic granulose cells in ovarian follicles. An experimental study investigated the side effects of isotretinoin on the ovaries by calculating gonads/bodyweight ratio and reported no adverse effect on the ovaries (
      • Ferguson S.A.
      • Cisneros F.J.
      • Gough B.J.
      • Ali S.F.
      Four weeks of oral isotretinoin treatment causes few signs of general toxicity in male and female Sprague–Dawley rats.
      ). Gonads/bodyweight ratio is not a proper method to investigate ovarian function. Thus, it is not possible to conclude that isotretinoin has no adverse effect on the gonads. Several histomorphological and biochemical methods have been described to demonstrate ovarian function, such as antral follicle count, detection of apoptotic activity in ovaries and serum hormone concentrations (
      • Anderson R.A.
      • Cameron D.A.
      Pretreatment serum anti-mullerian hormone predicts long-term ovarian function and bone mass after chemotherapy for early breast cancer.
      ,
      • Lee C.J.
      • Park H.H.
      • Do B.R.
      • Yoon Y.
      • Kim J.K.
      Natural and radiation-induced degeneration of primordial and primary follicles in mouse ovary.
      ). These methods are more reliable and have been used in numerous studies related with ovarian toxicity.
      Therefore, this study used hormonal, histopathological and immunohistochemical methods to detect the effects of isotretinoin on ovaries. As a hormonal method, AMH was used as it is a reliable marker of ovarian reserve independent of the menstrual cycle (
      • La Marca A.
      • Sighinolfi G.
      • Giulini S.
      • Traglia M.
      • Argento C.
      • Sala C.
      • Masciullo C.
      • Volpe A.
      • Toniolo D.
      Normal serum concentrations of anti-Müllerian hormone in women with regular menstrual cycles.
      ,
      • Overbeek A.
      • Broekmans F.J.
      • Hehenkamp W.J.
      • Wijdeveld M.E.
      • van Disseldorp J.
      • van Dulmen-den Broeder E.
      • Lambalk C.B.
      Intra-cycle fluctuations of anti-Müllerian hormone in normal women with a regular cycle: a re-analysis.
      ). In this study, AMH concentrations were significantly lower in the groups treated with 7.5 and 15 mg/kg/day isotretinoin compared with the control group. AMH concentrations in the group treated with 15 mg/kg/day isotretinoin showed an insignificant decrease compared with the group treated with 7.5 mg/kg/day isotretinoin. According to the concentrations of AMH, it may be concluded that isotretinoin has an adverse effect on the ovaries. However, this study cannot postulate that this effect is dose-dependent by measuring the concentrations of AMH.
      Few studies have investigated the effects of retinoids on pituitary, adrenal or gonadal hormone metabolism. Recently, a study investigated the effects of retinoids on pituitary hormone concentrations (
      • Karadag A.S.
      • Ertugrul D.T.
      • Tutal E.
      • Akin K.O.
      Isotretinoin influences pituitary hormone levels in acne patients.
      ). They found significant decreases in TSH, free triiodothyronine, thyroid-stimulating hormone, adrenocorticotropic hormone, LH, total testosterone and prolactin concentrations after 3 months of treatment. The mechanism behind the decrease in these hormone concentrations is not fully understood. However, recent evidence derived from translational studies of therapeutic and adverse effects of isotretinoin in cell systems, animal studies and human subjects indicates that isotretinoin’s primary mode of action is the enhancement of the nuclear activity of the transcription factor Forkhead box O1 (FoxO1;
      • Melnik B.C.
      Isotretinoin and FoxO1: a scientific hypothesis.
      ). At the promoter level, the genes for proopiomelanocortin (POMC) and carboxypeptidase E, which processes POMC to alpha-melanocyte-stimulating hormone, are suppressed by FoxO1 (
      • Sasaki T.
      • Kitamura T.
      Roles of FoxO1 and Sirt1 in the central regulation of food intake.
      ). This explains impaired down-stream pituitary hormone signalling observed during isotretinoin treatment of acne patients (
      • Karadag A.S.
      • Ertugrul D.T.
      • Tutal E.
      • Akin K.O.
      Isotretinoin influences pituitary hormone levels in acne patients.
      ). Thus, isotretinoin/FoxO1-mediated POMC gene regulation is likely the reason for decreased pituitary hormones derived from POMC during isotretinoin treatment. Moreover, FoxO, especially FoxO1, at the promoter level up-regulate the expression of pivotal genes involved in the induction of apoptosis, like Bcl2 interacting mediator of cell death (Bim), Fas-ligand (FasL), tumour necrosis factor receptor-associated death domain and tumour necrosis factor-related apoptosis inducing ligand (TRAIL), which were recently reviewed (
      • van der Vos K.E.
      • Coffer P.J.
      The extending network of FOXO transcriptional target genes.
      ). Isotretinoin-mediated activation of caspase 3 has been observed in Dalton’s lymphoma ascites cells (
      • Guruvayoorappan C.
      • Pradeep C.R.
      • Kuttan G.
      13 Cis-retinoic acid mediates apoptosis in Dalton‘s lymphoma ascites cells by regulating gene expression.
      ) and B16F-10 melanoma cells (
      • Guruvayoorappan C.
      • Pradeep C.R.
      • Kuttan G.
      13-cis-Retinoic acid induces apoptosis by modulating caspase-3, bcl-2, and p53 gene expression and regulates the activation of transcription factors in B16F–10 melanoma cells.
      ). Thus, substantial evidence has been provided in the literature that isotretinoin induces apoptosis in various cell types, such as sebocytes, lymphoma and melanoma cells. Furthermore, FoxO, especially FoxO1, control apoptotic signalling pathways, eventually activating caspase 3. Most recent evidence underlines the role of up-regulated FoxO1 expression in mouse follicular granulosa cell apoptosis induced by oxidative stress (
      • Shen M.
      • Lin F.
      • Zhang J.
      • Tang Y.
      • Chen W.K.
      • Liu H.
      Involvement of the up-regulated FoxO1 expression in follicular granulosa cell apoptosis induced by oxidative stress.
      ). The presented data of this study, the proposed isotretinoin-FoxO1 hypothesis of
      • Melnik B.C.
      Isotretinoin and FoxO1: a scientific hypothesis.
      and the observed involvement of FoxO1 in mouse granulosa cell apoptosis observed by
      • Shen M.
      • Lin F.
      • Zhang J.
      • Tang Y.
      • Chen W.K.
      • Liu H.
      Involvement of the up-regulated FoxO1 expression in follicular granulosa cell apoptosis induced by oxidative stress.
      fit very well together and imply that isotretinoin via up-regulation of FoxO1 stimulates sebocyte apoptosis (isotretinoin’s therapeutic effect in the treatment of acne) and cancer cell apoptosis (isotretinoin’s chemopreventive mode of action as an anti-tumour agent). Unfortunately, it also promotes follicular granulosa cell apoptosis (isotretinoin’s adverse effect, promoting follicular atresia). Future studies using the employed experimental methodology may be most useful to unravel isotretinoin-induced FoxO1-mediated apoptosis of granulosa cells by investigating granulosa cell FoxO1 expression at the mRNA and protein level with and without exposure to isotretinoin.
      These data also may be the explanation of menstrual irregularities, although there have been several reports which could not explain its mechanism clearly (
      • Christmas T.
      Roaccutane and menorrhagia.
      ,
      • Cox N.H.
      Amenorrhoea during treatment with isotretinoin.
      ,
      • Kwon H.J.
      • Lee J.Y.
      • Cho B.K.
      • Park H.J.
      Menstrual irregularity during isotretinoin treatment.
      ).
      The studies mentioned above have shown the effects of isotretinoin on the reproductive system indirectly. The present study preferred to use the histopathological analysis of the ovaries in order to investigate the effect of isotretinoin more clearly.
      The changes of morphological characteristics of primordial, primary, preantral and antral follicles due to isotretinoin treatment were investigated.
      At the largest cross section of the ovarian slices, the percentage of atretic follicles was calculated at each stage of oogenesis. The percentage of atretic primordial, primary, preantral and antral follicles was higher in the experimental groups in comparison to the control group. Histopathological analysis of rat ovaries after administration of 7.5 mg/kg/day and 15 mg/kg/day isotretinoin showed that the number of follicles containing apoptotic cells with advanced atretic features was significantly higher than the 0 mg/kg/day group (soybean oil). Dual comparisons of the percentages of atretic follicles were conducted between the groups. The percentage of atretic primordial follicles in 7.5 mg/kg/day isotretinoin group were higher than the control group, but this increase was not significant. On the other hand, the percentage of atretic primordial follicles was significantly higher in 15 mg/kg/day isotretinoin group compared with the control group. Also degenerated primordial follicles were considerably increased with increasing doses of isotretinoin at all stages of follicles. These findings can indicate that isotretinoin has a dose-dependent effect on the ovaries.
      Apoptosis is an essential component of ovarian function and development and a key event in the process of atresia. During fetal life, apoptosis mainly involves the oocyte. Alternatively, it involves the granulosa cells of the growing follicle during the adult life (
      • Hussein M.R.
      Apoptosis in the ovary: molecular mechanisms.
      ,
      • Johnson A.L.
      Intracellular mechanisms regulating cell survival in ovarian follicles.
      ). There is no study available investigating the effect of isotretinoin on the process of apoptosis in the ovaries. There are studies examining its effects on male gonads and spermatogenesis only. Some of these studies showed that retinoic acid can decrease the number of germinal epithelial cells in rat testis by increasing apoptosis (
      • Comitato R.
      • Esposito T.
      • Cerbo G.
      • Angelini F.
      • Varriale B.
      • Cardone A.
      Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula.
      ). Previous studies indicated that retinoic acid reduces the proliferation of fetal and neonatal gonocytes in dispersed testicular cell cultures by acting on both apoptosis and mitosis (
      • Boulogne B.
      • Levacher C.
      • Durand P.
      • Habert R.
      Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes.
      ). Therefore, the present study investigated the apoptotic and PCNA-positive granulosa cells; the number of PCNA-positive granulosa cells decreased in association with follicular atresia. Additionally, apoptotic granulosa cells in the ovarian follicles increased in the experimental groups compared with the control group.
      A potential weakness of this study is that the histopathologist did not classify the stages of cycles since they may affect the survival and death of granulosa cell, which are under the control of some intraovarian factors (
      • Matsuda F.
      • Inoue N.
      • Manabe N.
      • Ohkura S.
      Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells.
      ).
      In conclusion, according to histopathological, immunohistochemical and hormonal investigations, isotretinoin has a detrimental effect on the ovaries. Other experimental studies should be designed to determine whether or not this effect is irreversible. Also, future studies investigating the effect of isotretinoin on human ovaries should be re-evaluated.

      Acknowledgements

      This study was supported by Namik Kemal University Research Funding.

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