Endometriyozis-oosit kalitesi

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1 Endometriyozis-oosit kalitesi
Pınar ÖZCAN, MD, PhD Bezmialem Universitesi Tıp Fakültesi Kadın Hastalıkları ve Doğum ABD

2 ENDOMETRİYOZİS İLİŞKİLİ İNFERTİLİTENİN PATOJENİK MEKANİZMASI
Endometriyoma cerrahisi over rezervini azaltabilir Neden ve etki ilişkisi Endometriosis, one of the most common gynaecologic diseases, affects about 10–15% of reproductive-age women and is associated with subfertility (Donnez et al., 2002; Holoch and Lessey, 2010). The prevalence of this condition increases to 40% in women with subfertility, and about 30– 50% of affected women are estimated to be infertile (Holoch and Lessey, 2010). The mechanisms involved in the aetiopathogenesis of infertility in patients with endometriosis are likely to be multifactorial, although this complexity has not yet been fully elucidated (De Ziegler et al., 2010). independently from its pathogenesis, clinical treatment of the ovarian endometriotic cyst is presently the focus of much interest and controversy due to the overwhelming evidence demonstrating that ovarian reserve is negatively affected following surgical excision of these cysts a 2% rate of premature menopause following surgery has been reported after surgery Cerrahi öncesi endometriyomanın varlığının fertilite üzerindeki olumsuz etkisi

3 Endometriyozis fekunditeyi azaltır
MİLD MODERATE SEVERE 7.4 2.8 Most studies report that pregnancy rates are lower in women with endometriosis than in normal controls, but the specific mechanisms that may account for this difference arepoorly understood. The monthly fecundity rate (MFR) for normal couples of reproductive ages typically range around 30% for the first three cycles and decline to 4% when the couples have been trying to conceive for >1 year. The couples conceiving within the earlier months are the most fertile, and 18% of the couples did not conceive at the end of 1- year in the study. However, the MFR in couples diagnosed with both endometriosis and infertility is between 2% and 10% per month. Even minimal endometriosis may be associated with marked subfertility. Olive, Fertil Steril 1985 Dmowski, Fertil Steril 2002

4 ENDOMETRİYOZİS İLİŞKİLİ İNFERTİLİTENİN PATOJENİK MEKANİZMASI
Follikülogeneziste değişiklikler Ovulatuar disfonksiyon Kötü oosit kalitesi Luteal faz defekti Azalmış fertilizasyon Anormal embriyogenezis The controversy regarding whether endometriosis is a cause of subfertility or an incidental finding is ongoing. An association between endometriosis and infertility has repeatedly been reported in the literature, but an absolute cause-an deffect relationship has yet to be confirmed Further, the follicular fluid from patients has been shown to contain factors such as cytokines and growth factors that might promote the maintenance of endometriotic lesions and lead to a suboptimum follicular environment During normal ovulation, the LH surge starts a cascade of events in the follicle that leads to the expulsion of the oocyte-cumulus complexChanges in proteolytic enzymes cytokines inflammatory and the vasculature, all of which are required for normal ovulation, can also be found in the follicles of women with endometriosis. Collectively, these data provide evidence of mechanisms that could cause ovulatory dysfunction in endometriosis. A phenomenon exists whereby oocytes become trapped in a luteinizing corpus hemorrhagicum. This failure of ovulation, defined as luteinized unruptured follicle syndrome (LUFs), has been associatedwith endometriosis and infertility in women

5 Granuloza hücre fonksiyonu, hormon reseptivitesi, hormon biosentezi
Folliküler mikroçevre kalitesi Kumulus hücreleri-oosit arasındaki çift yönlü iletişimin yeterli olması Oocyte competence depends on the quality of the follicular microenvironment, and the presence of adequate bidirectional cumulus cell-to-oocyte signalling is a prerequisite for the acquisition of both oocytes and cumulus cell competence. Granulosa cells play an essential role in follicular differentiation, leading to optimal conditions for oocyte development, ovulation, fertilization and subsequent implantation (Adashi, 1994). Moreover, the bi-directional communication between the oocyte and these cells occurs throughout follicular development and is essential for the acquisition of developmental competence in mammalian oocytes Oosit kalitesi

6 ENDOMETRİYOZİS İLİŞKİLİ İNFERTİLİTENİN PATOJENİK MEKANİZMASI
Uzamış foliküler faz Azalmış foliküler büyüme hızı Azalmış dominant folikül büyüklüğü Azalmış foliküler östradiol konsantrasyonu Bozulmuş LH surge Folliküler LH reseptörlerinde yetersizlik Mural ve oositi çevreleyen kumulus granuloza hücrelerinde apopitozis Others have implicated impaired luteinizing hormone (LH) production as the primary pathophysiology causing impaired ovulation (43). It was suggested that gonadotropin-surge attenuating factor (GnSAF), which is a small polypeptide in the FF primarily produced by small follicles, plays a role in the decreased levels of LH in endometriosis patients. Gonad- otropin-surge attenuating factor decreases the ability of E2 to sensitize the pituitary to gonadotropin-releasing hormone, thereby decreasing the pituitary’s potential to produce LH. Because estrogen levels are lower in the FF of endometriosis patients, the antagonistic actions of GnSAF against LH production are likely to result in suboptimal LH levels and impaired ovulation (44).

7 Inflamatuar response-oxidative stress
Our knowledge of the pathophysiology of endometriomas (or ‘chocolate cysts’) is still limited as it is supported by controversial theories proposed for its pathogenesis. The term ‘chocolate cyst’ was applied to describe an ovarian cyst lined with endometrial tissue histologically and functionally similar to eutopic endometrium and in which the internal fluid is generally thought to arise from the accumulation of menstrual debris deriving from the shedding of the active implants inside the cystThe cyst contains non-resorbed blood derived from repeated hemorrhages of ectopic endometrial cells lining the cyst wall during menstrual cycles. The cyst fluid contains cellular damagemediating factors, proteolytic enzymes and inflammatory molecules in concentrations from tens to hundreds of times higher than those present in serum or in non-endometriotic cysts Among the different cellular damage-mediating factors present in the cyst fluid, iron has attracted much attention due to its role in the pathogenesis of endometriosis and as a potential cause of carcinogenesis of the cyst An endometrioma contains a chocolate colored fluid which is thought to contain non-resorbed blood derived fromrepeated hemorrhages of the endometriotic cells in the cyst during menstrual cycles. As a result of this assumption, molecular analysis of the fluid has been limited. An issue to be clarified is whether the content of an endometriotic cyst could be a potential source of toxicity for the surrounding healthy tissue To clarify a potential detrimental effect of the cyst on the surrounding ovarian tissue, the issue here is: ‘Is this wall an impermeable barrier or are the endometrioma contents able, at least in part, to leak out from the cyst?’ This makes this type of cyst an unusual entity from a morphological, structural and also surgical point of view. Based on its nature, this barrier between the fluid of the cyst and the normal ovarian tissue might be incompletely impermeable

8 FSH LH E2 A FSH receptors on granulosa cells
Theca externa cells FSH receptors on granulosa cells Theca interna cells LH receptors on theca cells Granulosa cells FSH Follicular antrum Zona pellucida Oocyte Cumulus Oophorus cells Two possible mechanisms can be proposed to explain this vascular dysfunction: (i) the imbalance between ROS production and the ability of biological systems to detoxify the reactive intermediates leads to impaired vascular relaxation, enhanced endothelial transcytosis, up-regulation of proinflammatory molecules and alteration of endothelial cell fibrinolytic activity. The loss of cortex-specific stroma should be considered in the context of its potential harmful effect on folliculogenesis. The ovarian stroma not only provides a blood supply though its capillaries to the primordial follicles but also acts in a co-ordinated and synergistic way with other compartments to induce the transition from primordial to primary follicles (Motta et al., 2003; Skinner, 2005; Oktem and Urman, 2010). Ovarian cortical stromal cells are the source of theca cells. In summary, most authors agree that follicular density is lower in the ovarian cortex adjacent to the endometriotic cyst. This phenomenon is associated with tissue alterations, such as formation of fibrosis and vascular deficiency inhibition of ovarian angiogenesis and capillary loss are consequently mediated directly by increased ROS levels and indirectly by the cellular injury that in turn triggers over-expression of factors affecting the vascular system, such as TSP-1 (Csanyi et al., 201 Apoptosis in ovarian cells is a good indicator of poor oocyte quality (Nakahara et al. 1997). Death of cumulus cells probably leads to reduced oocyte quality and maturation attributable to the loss of the essential support that the cumulus cells give to the oocyte (e.g., pyruvate, hormones, growth factors; Russell and Robker 2007). A LH E2 Capillary network Basement membrane

9 Comprehensive overview of the signaling effect of ROS in tumor progression. Growth factor (HGF), cytokine (TGFβ), the tumor promoter (TPA) and integrin engagement may induce ROS generation via NADPH oxidase or derived from mitochondria. Oxidative activation and/or inactivation of PKC and PTP, respectively, by ROS result in MAPK activation followed by activation of various transcriptional factors including SMAD, AP-1, Ets-1 and Snail. Each transcriptional factor then regulate its target genes such as E-cahedrin, MMP, integrin and biglycan leading to EMT, migration and invasion The local factors in the cysts claimed to be responsible for the induction of genetic alterations in endometriotic cells lining the inside of ovarian endometriomas are as follows: (i) inflammatory factors and cytokines that, via NF-kB activation, have been found to promote angiogenesis, cell proliferation, inhibition of apoptosis and production ofROS thatmay, in turn, induceDNAdamage and mutations; (ii) an altered steroid hormone balance or responsiveness as estrogens have been linked to the pathogenesis of gynecological cancers (iii) iron-associated oxidative stress, currently considered the most important trigger, being able to attack ‘fragile’ sites in the genome, including the susceptible p53 pathway Apoptosis or programmed cell death of the embryo can occur through several mechanisms associated with endometriotic lesions such as increased concentrations of inflammatory cytokines or reactive oxygen species (ROS; Agic et al. 2006; Jana et al. 2010; Zeller et al. 1987; Fig. 2). Inflammatory cytokines such as tumor necrosis factor-α can activate caspase-dependent signaling pathways to increase apoptosis (Hu 2003). ROS can cause mitochondrial damage and DNA strand breaks (Lao et al. 2009). This might also encourage the cell to undergo programmed cell death or apoptosis. Probably the most significant effect of iron and other redox-active metals-induced formation of ROS on signalling pathways has been observed in the mitogen-activated protein kinase (MAPK) pathways. This involves activation of nuclear transcription factors which control the expression of genes responsible for the repair of damaged DNA, genes that power the immune system, arrest the proliferation of damaged cells, and induce apoptosis. The nuclear transcription factor NF-kappaB, is involved in inflammatory responses and AP-1 is important for cell growth and differentiation. P53 is a gene whose disruption is associated with more than half of all human cancers

10 Several triggers of apoptosis in human ovaries have been extensively studied and some of them are also implicated in chronic inflammation caused by endometriosis, such as members of the tumor necrosis factor (TNF) superfamily (20). It is possible that local inflammation may have a direct impact on follicular apoptosis and atresia in ovaries affected by endometriosis. The cyst contains non-resorbed blood derived from repeated hemorrhages of ectopic endometrial cells lining the cyst wall during menstrual cycles. The cyst fluid contains cellular damagemediating factors, proteolytic enzymes and inflammatory molecules in concentrations from tens to hundreds of times higher than those present in serum or in non-endometriotic cysts Among the different cellular damage-mediating factors present in the cyst fluid, iron has attracted much attention due to its role in the pathogenesis of endometriosis and as a potential cause of carcinogenesis of the cyst Changes in the ovarian steroid enzyme pathways may be modulated by cytokines secreted by ovarian and white blood cells (16, 38). Tedeschi et al. (39) reported that endothelin-1 was a potent inhibitor of rat granulosa cell steroidogenesis in vitro. According to Abae et al. (40), immunoreactive endothelin-1was elevated in the FF of patientswith endometriosis-associated infertility. It was also shown that increased levels of IL-6 in the preovulatory follicles of endometriosis patients result in decreased aromatase activity via the MAPK signal pathway. The decreased aromatase activity causes a decrease in intrafollicular conversion of androstenedione to estrone and then a diminished conversion ofAndrostenedione to testosterone,which is aromatized toE2 (41, 42).The resulting decrease in follicular levels of E2 can then give rise to fertility problems including decreased fertilizing capacity (41).Altered levels of progesterone have also been found in the FF of endometriosis patients, indicating that altered steroidogenesis most likely plays a significant role in endometriosis-associated infertility. However, a direct relationship between infertility and modified progesterone levels has yet to be established (16) Gupta S, Fertil Steril 2008

11 Aromatase is present in granulosa cells and plays a fundamental role in follicle maturation and the establishment of oocyte quality decreased aromatase activity in the granulosa cells of women with endometriosis, which might lead to defects in granulosa cell steroidogenesis and abnormal oocyte functioning 40 with endometriosis vs 41 patients without endometriosis A possible explanation for this finding is the absence of reduced CYP19A1 expression in mural granulosa cells from infertile women with endometriosis submitted to ovarian stimulation for IVF, as Abreu et al. (2011) previously observed. As these cells are primarily responsible for the production of follicular oestradiol (Hillier et al., 1994; Whitelaw et al., 1992), the production of this hormone would be preserved even in the presence of reduced CYP19A1 expression in cumulus cells, as observed in the present study. Cumulus cells express lower levels of CYP19A1 than mural granulosa cells, and are therefore less active in steroidogenesis (Whitelaw et al., 1992). Studies comparing the expression of CYP19A1 in mural granulosa cells and the cumulus cells from infertile women with endometriosis are necessary to confirm this hypothesis. we demonstrated a lower expression of the CYP19A1 in cumulus cells of infertile patients with endometriosis undergoing ovarian stimulation for ICSI compared with infertile women without endometriosis. We also observed a lower number of fertilized oocytes in these women. On the basis of these results, we hypothesize that the reduced expression of the CYP19A1 gene in cumulus cells might be involved in the impairment of oocyte quality associated with endometriosis, indicating a new perspective for understanding the pathogenesis of endometriosisrelated infertility. Barcelos, RBM online 2015

12 The mechanism underlying the anti-apoptotic effects of testosterone on granulosa cells, and found that a low-testosterone status is a potentially important step in the development of premature ovarian insufficiency in patients with endometriosis. In AR knockout mice, intensive granulosa apoptosis occurs during the periovulatory period [16], and these mice develop the POI phenotype with a loss of follicles we therefore analyzed the serum testosterone levels in endometriosis patients, and evaluated whether a low serum testosterone level correlates with the apoptosis of granulosa cells. Furthermore, we assessed whether a favorable testosterone level prevents granulosa cell apoptosis and clarified the underlying mechanisms using a basic approach employing human immortalized granulosa cells derived from a primary human granulosa cell tumor, the COV434 cell line Serum samples were collected from 46 patients with endometriosis and from 62 patients without endometriosis who received assisted reproductive therapy. In order to evaluate the role of the serum testosterone level in the apoptosis of granulosa cells in the endometriosis patients, 10 subjects with ovarian endometrioma who underwent unilateral salpingo-oophorectomy were also recruited for the analysis. flutamide (an androgen receptor inhibitor) The mean serum testosterone concentration in the patients with endometriosis was significantly lower than that observed in the patients without endometriosis. Furthermore, high expression of a pro-apoptotic Bcl-2 member, BimEL, in the follicles was found to be associated with a low serum testosterone level. We clarified the underlying mechanisms using a basic approach employing human immortalized granulosa cells derived from a primary human granulosa cell tumor, the COV434 cell line. The in vitro examination demonstrated that testosterone inhibited apoptosis induced by sex steroids depletion via the PI3K/Akt-FoxO3a pathway in the COV434 cells. In conclusion, we elucidated the mechanism underlying the anti-apoptotic effects of testosterone on granulosa cells, and found that a low-testosterone status is a potentially important step in the development of premature ovarian insufficiency in patients with endometriosis. testosterone has been shown to enhance follicular recruitment [38], promote follicular growth and development [39], increase the insulin-like growth factor 1 (IGF-1) expression in the primate ovary, which plays an essential role in regulating follicular development [40], and increase follicular sensitivity to FSH stimulation via androgen receptors [41, 42]. The major question remaining is why there is a low testosterone status in endometriosis patients. There has been no evidence that endometriosis attenuates the function of the theca cells, which are the major source of androgens in the ovaries. To examine the pathology associated with the low testosterone induced by endometriosis in greater detail, measurement of androstenedione, which is a precursor of testosterone, in the serum and follicle fluid will be required. In addition, to evaluate the adrenal function, the measurement of dehydroepiandrosterone may also be necessary. Ono YJ, PLOS one, 2014

13 Endometriosis patients showed the highest incidence of apoptotic granulosa cells in both cumulus and mural regions [12] when compared to other infertile patients (fig. 1). Among these endometriosis patients, individuals with more advanced stages of endometriosis presented higher incidences of apoptosis in the granulosa cells Saito H, Gynecol Obstet Invest 2002

14 Saito H, Gynecol Obstet Invest 2002
The cell cycle of granulosa cells was determined by flow cytometric analysis. Usually, the LH surge causes mitosis arrest in the granulosa cells by concurrent inhibition of cycline D2 and upregulation of cycline-dependent kinase inhibitors, p27 and p21. Thus, changes in cell-cycle kinetics, assessed by flow cytometric measurements of DNA in individual granulosa cells aspirated from the follicles of infertile patients, may be related to the state of follicle growth and oocyte maturation. The cell cycle state of granulosa cells was divided into 4 phases: apoptosis, G0/G1 phase, S phase and G2/M phase. The results of the flow cytometric analysis showed that the endometriosis patients had a higher incidence of apoptotic granulosa cells when compared with the other infertility factor patients. Among the endometriosis patients, the incidence of granulosa cells in the S phase was higher than in other patients and lower in the G2/M phase than in the other phases (table 1) [16]. Thus, flow cytometry analysis showed that endometriosis caused alterations in the cell cycle of granulosa cells It has been determined that various cytokines, including IL-1·, IL-1ß, IL-6, IL-8, and IL-10, are increased in the serum, peritoneal fluid, and follicular fluid of patients with endometriosis [17–19]. It has also been reported that these cytokines can activate several cyclin-dependent kinase inhibitors in various cells [20–22]. However, the precise mechanisms involved, and the reason for why cytokines adversely affect the cell cycle in the granulosa cells of patients with endometriosis remain unclear. The higher cytokine production in these patients may be responsible for the disturbance of the cell cycle in the granulosa cells, as in other cells, and consequently have pathogenic effects on folliculogenesis. Saito H, Gynecol Obstet Invest 2002

15 Changes reflected ovarian interstitial microvascular injury of OEC, pathologically supported the findings of blood flow changes within ovarian interstitial arteries, and prospectively predicted OEC-induced ovarian interstitial vessel injury. (10 ovarian benign teratomas,10 ovarian serous cystadenomas and 10 ovarian mucinous cystadenomas) preoperative TV-CDS; (2) postoperatively test for changes in TSP-1 protein and mRNA and CD34-MVD in ovarian interstitial specimens; and (3) evaluate the relationship between ultrasonic flow indices [colour Doppler flow classification and resistance indices (RI) values] and pathological indices (TSP-1 and CD34-MVD) grade 0 if the colour signal was absent, grade I if the colour signal was star shaped, grade II if the colour signal was strip shaped, and grade III if the colour signal was reticular shaped. The expressions of CD34-MVD and TSP-1 were examined via immunohistochemical assay. The expression of TSP-1 was categorised into three grades: negative or sparsely positive (-), scored from 0 to 4; focally positive (?), scored from 5 to 8; and strongly positive (??), scored from 9 to 12. Microvessel density was defined as the number of blood vessels that were characterised as CD34-positive tube-like structures. Western blot of TSP-1 was performed Results Blood flow, most of star-shaped, within ovarian interstitial arteries in the OEC group was diminished; however, arterial spectra exhibited a high-resistance flow manifesting a significantly higher RI compared with that of the control group (P\0.01). In ovarian interstitial specimens, there were significantly (P\0.01) lower CD34- MVD and higher TSP-1 protein and mRNA in the OEC group than in the controls. CD34-MVD and TSP-1 showed remarkably negative correlation (rs = -0.76, P\0.01). RI values correlated negatively with MVD values (rs = -0.91, P\0.01), but positively with TSP-1 (rs = 0.81, P\0.01), while flow classification correlated positively with MVD values (rs = 0.66, P\0.01), but negatively with TSP-1 (rs = -0.54, P\0.01). Conclusions Changes in CD34-MVD and TSP-1 reflected ovarian interstitial microvascular injury of OEC, pathologically supported the findings of blood flow changes within ovarian interstitial arteries, and prospectively predicted OEC-induced ovarian interstitial vessel injury. This has important clinical value: early treatment, instead of allowing the cyst to become bigger, is of great importance for OEC patients, because a greater number of functional tissue blood vessels would be destroyed as the disease progresses. Qui JJ, Arch Gynecol Obstet 2012

16 This prospective cohort study included 117 infertile women undergoing IVF in a tertiary infertility
There were 47 patients with unilateral endometrioma and 17 patients with bilateral endometrioma, while the 53 control patients had unexplained or male factor infertility. Concentrations of IL-1b, IL-6, IL-8, IL-10, IL-12 and TNF-a were measured in serum and in the fluid of the largest pre-ovulatory follicles from each ovary of each participant Cytokine levels in the follicular fluid from the two ovaries in women with unilateral endometriomas were comparable, and were not significantly altered compared with that of control groups with male factor infertility, unexplained infertility or bilateral endometriomas. Compared with serum levels, the follicular fluid levels of IL-8 and IL-6 were higher, suggesting a local production or recruitment. The follicular fluid IL-8 level varied considerably and showed an inverse relationship with IL-12, IL-10 and TNF-/, suggesting a complex interaction between various immune cells. A small group of patients (n ¼ 3) had increased levels of all follicular fluid cytokines combined with moderately to slightly elevated serum levels and these patients had a significantly lower ovarian response. A possible association between endometrioma and local intrafollicular inflammation was examined by comparing cytokine concentrations in the follicular fluid from the two ovaries across infertility diagnoses (Table II). The concentrations of IL-6 and IL-8 in follicular fluid were comparable in the affected and non-affected ovary of women with unilateral endometriomas, in women with bilateral endometriomas and in infertile women without endometriosis. TNF-a was detected in follicular fluid derived from women with bilateral endometriomas only. Follicular fluid concentrations of IL-1b, IL-10 and IL-12 were below the lower limit of quantification. Opeion H, Hum Repr 2013

17 Reduced vascularization Increased oxidative stress
The‘‘burnout’’ hypothesis may explain the mechanism partly responsible for the reduced ovarian reserve in women with endometriomas. Formation of endometriomas may cause focal inflammation in ovarian cortex. This inflammation could result in structural alteration to the ovarian cortex, which manifest as massive fibrosis and loss of cortex-specific stroma that maintains follicular nests. Focal loss of follicular density may be associated with a vicious circle of dysregulated folliculogenesis that eventually results in burnout of the stockpile of dormant follicles ovarian follicle numbers were decreased in women with endometriosis and that this decrease was associated with the presence of endometriosis and fibrosis in cortical tissue. This could be considered a sequela of focal endometriotic inflammation. Early follicular development may be activated and follicular atresia increased in ovaries with endometriomas compared to contralateral ovaries without cysts. These results indicate that upregulated recruitment and, at the same time, demise of early follicles may occur in ovaries with endometriomas, resulting in focal exhaustion of primordial follicles that constitute the ovarian reserve. Dolmans et al. (13) and David et al. (14) reported activation of follicular recruitment after xenografting of human ovarian cortex to immunodeficient mice, and this accelerated growth ‘‘without brakes’’ led to loss of primordial follicles due to hypoxic stress and reduced vascularization. This ‘‘burnout’’ hypothesis was already suggested by Dolmans et al. in similar upregulation of follicular recruitment could occur in ovaries with endometriomas. this inflammation and fibrosis, with associated reduced vascularization and increased oxidative stress. may be responsible for follicle activation with subsequent follicle loss, as proven by the increased numbers of follicles. It is very important tounderline thatROS, together with the potent profibrotic PAI-1 and members of the TGF-b family are all involved in tissue fibrosis. The pathological manifestations of fibrosis are the expansion of mesenchymal elements, includingmyofibroblasts, excessive accumulation of ECM proteins and tissue contraction. Myofibroblasts, as the cells most responsible for the development of fibrosis, derive fromactivated resident fibroblasts or from fibroblasts deriving from epithelial-to-mesenchymal transition or endothelial-to-mesenchymal transition. They synthesize mostly collagen and fibronectin . The demonstration of increased oxidative stress affecting the normal ovarian cortex surrounding an endometrioma strongly supports the possibility that a ROS-induced fibrogenic response may be induced at this level. Reduced vascularization Increased oxidative stress Dysregulated folliculogenesis Oocyte apoptosis Follicular atresia Kitajima M, Fertil Steril 2014

18 Ovaries with endometriomas, which may be more prone to local pelvic inflammation, showed activated follicular recruitment and atresia of early follicles. The potential contribution of inflammation to follicle ‘‘burnout’’ in case of endometriomas is discussed 13 women <40 years of age monolocular unilateral endometriotic cysts In ovaries with endometriomas, biopsy was taken at a distance of approximately 1.5 cm from the endometrioma bulk, at the antimesenteric level if possible. In control ovaries, biopsy was taken from normal-looking tissue at the antimesenteric level. To evaluate early-stage endometriomas and avoid the confounding effects of enlarged endometriomas on the histologic features of healthy ovarian cortex, only cortical samples from endometriomas measuring %4 cm were included Kitajima M, Fertil Steril 2014

19 Oxıdative stress Eritrositler Apopitotik endometrioma hücreleri
aktive olmuş makrofajlar ROS damage to lipids, proteins, telomerase activity, telomeres, DNA and mitochondria Another way in which cells are damaged through OS is via lipid peroxidation, which is the oxidative destruction of polyunsaturated fatty acids in the plasma membrane (27). It leads to ‘‘increased membrane permeability, degraded membrane integrity, inactivated enzymes and structural damage of the DNA; cell death rapidly follows’’ 4-hydroxy-2-nonenal (4HNE), one of the products of lipid peroxidation excessive ROS generation, depolarization of mitochon-drial membrane potential and significant increase in DNA damagein granulosa cells of women with endometriosis as compared withcontrols ROS-induced oxidative stress alters cellular function by regulating gene expression and protein activity of pro-inflammatory cytokines, adhesion molecules, growth and angiogenic factors as well as by affecting the normal action of important signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathways, the AP-1 transcription factor, the NF-kB pathway and hypoxia-inducible transcription factors (Michiels et al., 2002;Wu, 2006; Defrere et al., 2011). Ovarian tissue frompatientswho underwent laparoscopic cystectomyfor endometriotic cysts and other benign ovarian cysts (dermoid and serous)was immunostained for 8-OHdG(Matsuzaki and Schubert, 2010). The immunostaining for 8-OHdG was .10-fold greater in the ovarian cortex of patients with endometrioma compared with patients with other cysts. Explanations for this finding can be various; the presence of the cyst as an entity inducing a structural distortion of the ovarian architecture may initiate a local ovarian inflammatory reaction causing ROS production; alternatively, factors present in the cyst, for example iron, might diffuse into the surrounding tissue causing ROS generation; finally, several ROS aremembrane permeable,making them excellent candidate paracrinemolecules for the surrounding tissues (Bryan et al., 2012). It has been reported that ROS have the potential to cause catastrophic damage to healthy tissue if left unharnessed (Bryan et al., 2012). Granuloza hücre disfonksiyonu, mitokondrial defektler ve oosit telomer kısalması

20 Uncomplexed iron together with superoxide, which reduces Fe(III) and hydrogen peroxide, which is decomposed by the Fenton reaction, provides a lethal mixture containing hydroxyl radicals that can directly damage DNA, lipids and proteins (Jonova and Valko, 2011). One of the proteins that plays an important role in iron balance is ferritin that captures and buffers the intracellular labile iron pool as a main function (Picard et al., 1998). Ferritin levels were found to be higher in the peritoneal fluid of women with endometriosis compared with control women (Van Langendonckt et al., 2002). Ferritin is a heteropolymer that contains two different subunits, the H- and L-chains which are similarly regulated by iron, although they have distinctive functional activities: the H-chain has ferroxidase activity and readily sequesters the excess cytosolic iron, while the L-chain, with a more efficient iron nucleation activity, assists the H-chain in the functionality of the hybrid molecules (Cozzi et al., 2003; Arosio and Levi, 2010). Also essential to iron homeostasis is the transport of iron by the protein transferrin (Tf). Iron-bearing Tf binds tightly to its specific transferrin receptor (TfR), a homodimeric transmembrane protein. Efficient delivery of iron is critically dependent on Tf/TfR interactions. In pelvic endometriosis, peritoneal macrophages were found to express higher levels of TfR1 and transferrin levels in the peritoneal fluid were also increased (Martinez-Roman et al., 1997). The presence of iron-related compounds that are potentially toxic to developing ovarian follicles adjacent to the endometrioma during IVF procedures has been poorly investigated (Sanchez et al, 2013). the average concentration of free iron in endometriotic cysts iscomparable with the iron concentration reported in hepatic carcinoma tissue, which is a sufficient amount of iron to induce a malignant transformation (iii) iron-associated oxidative stress, currently considered the most important trigger, being able to attack ‘fragile’ sites in the genome, including the susceptible p53 pathway Jomova K, Current Pharmaceutical Design, 2011

21 Iron and its major storage proteins represent molecular signs of the gonadotoxic insult to individual follicles developing adjacent to the cyst during IVF cycles. This is reflected by the low rate of oocyte retrieval from endometrioma-proximal follicles The cyst contains non-resorbed blood derived from repeated hemorrhages of ectopic endometrial cells lining the cyst wall during menstrual cycles. The cyst fluid contains cellular damagemediating factors, proteolytic enzymes and inflammatory molecules in concentrations from tens to hundreds of times higher than those present in serum or in non-endometriotic cysts Among the different cellular damage-mediating factors present in the cyst fluid, iron has attracted much attention due to its role in the pathogenesis of endometriosis and as a potential cause of carcinogenesis of the cyst Total iron levels were higher in endometrioma-proximal follicles compared with endometrioma-distal ones (P ¼ 0.009) and to follicles in the healthy ovary (P ¼ 0.02). L ferritin was higher in proximal versus distal follicles (P ¼ 0.044) or follicles from the healthy ovary (P ¼ 0.027).Hferritin was higher in the proximal and distal follicles compared with follicles in the healthy ovary (P ¼ and P ¼ , respectively).Hferritin transcript levels in granulosa cellswere higher in proximal follicles versus follicles fromhealthy ovary (P ¼ 0.02). TfR1 transcript levels were higher in proximal versus distal follicles (P ¼ 0.03) and versus follicles from the healthy ovary (P ¼ 0.04). The oocyte retrieval rate was lower in proximal and distal follicles than in follicles from the healthy ovary (P ¼ and P ¼ 0.04,respectively). an up-regulation of TfR1 has been reported under inflammatory conditions (presence of IL-6 and TNF-a) (Wang et al., 2013). Therefore, the observed TfR1 up-regulation on luteinized granulosa cells of follicles adjacent to the endometrioma might be induced by the inflammatory microenviroment, masking the expected decrease due to ironoverload 4 cm in diameter 13 infertile women with unilateral endometrioma undergoing controlled ovarian stimulation and IVF-ICSI procedure standard long protocol for ovulation induction Sanchez AM Hum Rep 2014

22 Fertilization rate and percentage of Grades I and II embryo formation were observed to be significantly lower and DNA fragmented embryo significantly higher in women with endometriosis Though the role of reactive oxygen species (ROS) in female infertility has been a subject of rigorous research worldwide, there is inadequate information on the cut-off value of ROS in the oocyte microenvironment beyond which ART outcome may be adversely affected. An upper ROS level in follicular fluid (FF) samples ofwomenundergoing IVF beyond which good quality embryo formation is unlikely, is established. ROS, lipid peroxidation and total antioxidant capacity were estimated. The upper cut-off ROS level beyond which viable embryo formation is not favorable was found to be ∼107 cps/400l FF. This level, determined in women with tubal factor infertility, was further validated in women with endometriosis and PCOS and correlated with fertilization and pregnancy rate and embryo quality. Summarizing, a threshold level in FF has been established for the first time beyond which ROS may be considered toxic for viable embryo formation and pregnancy outcome. with a GnRH agonist Janaa SK, Reproductive Toxicology 2010

23 Increased ROS and NO in endometriosis and infertility associated with poor oocytes and embryo quality tOxidative stress and trace elements in the oocytes environment is explored in endometriosis and impacton in vitro fertilization (IVF) outcome assessed. Follicular fluid was aspirated at the time of oocyte retrievalfrom endometriosis (n = 200) and tubal infertility (n = 140) and the analytes measured using spectroscopyand HPLC. Increased concentration of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation(LPO), iron, lead, cadmium and reduced levels of total antioxidant capacity (TAC), superoxide dismutase(SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), vitamins A, C, E, copper, zincand selenium was observed compared to tubal infertility. Increased ROS and NO in endometriosis andtubal infertility associated with poor oocytes and embryo quality. Increased levels of ROS, NO, LPO, cad-mium and lead were observed in women who did not become pregnant compared to women who did.Intrafollicular zinc levels were higher in women with endometriosis who subsequently became pregnantfollowing IVF. The direct relationship between intrafollicular oxidative andnitrosative stress and immature oocytes and poor quality embryosmotivated us to plot the ROC curve to establish the cut-off levelof intrafollicular ROS within which the chances of good qual-ity oocytes formation is significanT The threshold level of ROSin endometriosis cases was estimated to be cps. ROS lev-els beyond this limit appear to be toxic and are associated withthe formation of immature oocytes Singh A, Reproductive Toxicology 2013

24 patients with infertility and endometriosis exhibit more signs of
The normal ovarian cortex surrounding endometriotic tissues was affected more severely by oxidative stress, granulosa cells from patients with infertility and endometriosis exhibit more signs of severe oxidative DNA damage These findings prompt the question of whether removal of endometriotic tissues may improve the microenvironment of the ovary in patients with ovarian endometriosis in terms of oxidative stress A possible explanation might be that oxidative damage may persist in the normal ovarian cortex that remains after the removal of endometriosis tissue. Matsuzaki S, Fertil Steril 2010

25 Altered follicular microenvironment with
increased oxidative stress and NO insufficiency, and their secondary impact on GCs and oocytes, is the likely mechanism to explain poor oocyte quality in women with endometriosis that fail to conceive after ART. Nitric oxide forms a vital component of the oocyte microenvironment during folliculogenesis, ovulation, and oviductal journey, and plays a positive role during oocyte maturation, fertilization, and beginning of embryo developmentNitric oxide is a free radical and a bioregulator of apoptosis (65). Low levels of NO are important in ovarian function and implantation. However, higher amounts of NO and nitric oxide synthase (NOS) are seen in the endometrium of women with endometriosis (66). This is partly because of the fact that peritoneal macrophages express higher levels of NOS, have higher NOS enzyme activity, and have been shown to produce more NO in response to immune stimulation in vitro (67) Patient(s): Women with and without endometriosis undergoing ART (n = 28). Result(s): Clinical characteristics and ART live birth outcomes were no different between groups A and B. Women from group A had significantly lower peak serum E2 (2, pg/mL vs. 2, pg/mL) and higher apoptosis (80.0% vs. 22.2%) and NT staining (70.0% vs. 22.2%) in GCs compared with group B. Fewer IOs underwent IVM to MII ( ) in group A compared with group B ( ). IVM oocytes had significantly higher incidence of cortical granule loss (83.3% vs. 24.0%) and spindle disruption (66.7% vs. 16.0%) and higher zona pellucida dissolution timing ( s vs s) in group A compared with group B. FF nitrate levels were significantly higher in women who failed to conceive in group A ( nmol/L) compared with those that did conceive ( nmol/L). Conclusion(s): Increased protein nitration, GC apoptosis, resistance to IVM, and oocyte aging indicate the involvement of oxidative dysregulation of NO in the pathophysiology of altered follicular milieu and poor oocyte quality in women with endometriosis Nitric oxide is a ubiquitous free radical in the oocyte microenvironment that plays a vital role in virtually every step of oocyte development, including meiotic maturation, fertilization, embryonic cleavage, and implantation . Furthermore, we have demonstrated a significant role of NO in delaying oocyte aging and maintaining the integrity of the spindle apparatus (39, 40). Decreased bioavailability of NO under certain pathologic conditions could therefore result in abnormalities in oocyte viability and developmental capacity (40, 41). Although endometriosis could affect NO production as well as metabolism, one of the main mechanisms affecting NO bioavailability could be its consumption by superoxide (42), with resultant formation of highly reactive peroxynitrite (ONOO). Peroxynitrite promotes nitration of tyrosine residues, depletes lipid soluble antioxidants, and initiates lipid peroxidation (42–45). reactive nitrogen species, in particular nitric oxide (NO), are present in endometrioma fluid in the 20–40 mM/l concentration range (Shawky et al., 2001), which is similar to the concentration detected in peripheral blood ( mM/l) (Manau et al., 2000). The analysis of NO has been, however, limited to a small number of patients and no comparison with other types of cysts has been performed. An excess of NO is also toxic since it can damage proteins, carbohydrates, nucleotides and lipids. Goud PT, Fertil Steril 2014

26 Endometriosis is associated with inflammatory changes in the follicular fluid (FF) and peritoneal fluid (PF) environments. An increased percentage of B lymphocytes, natural killer cells, and monocyte-macrophages in the FF have been noted. This suggests the possibility of altered immunologic function in the FF of patients with endometriosis The methylation level of the oocyte genome remains low until the oocyte is activated during folliculogenesisEndometriotic lesion secretory products or inflammatory mediators from elevated numbers of peritoneal macrophages and other immune cells present in the peritoneal fluid might affect the methylation status of the genome of the embryo or fetus (Hill et al. 1988). This can occur by changing the gene expression of enzymes such as DNA methyltransferases (DNMTs) and histonemodifying enzymes such as histone deacetylases (Haaf 2006). One suggestion is that, in ectopic endometrium of women with endometriosis, DNMT1, DNMT3A and DNMT3B are over-expressed when compared with control levels (Wu et al. 2007). Inflammatory mediators might cause increased DNA methylation by a secondary mechanism (Ushijima and Okochi-Takada 2005). ROS associated with inflammation cause DNA damage such as halogenated pyrimidines, which mimic methylated cytosines (Lao et al. 2009; Valinluck and Sowers 2007). These halogenated pyrimidines cause DNMT1 to recognize the hemi-methylation of the DNA leading to the methylation of the opposite strand of DNA (Lao et al. 2009; Valinluck and Sowers 2007).

27 Telomeraz- dna hasarı

28 Alteration of oocyte cytoskeleton might be one of the causes of poor oocyte quality in patients with
endometriosis. Mansour G, Fertil Steril 2009

29 FF of infertile women with mild endometriosis can impair nuclear maturation and promote meiotic anomalies in bovine oocytes matured in vitro. These findings provide evidence about the mechanisms associated with infertility in women with early stage endometriosis Because of the ethical limitations of working with human embryos and experimentation in women, animal models of endometriosis are frequently used to study the anomalies associated with endometriosis These models have many advantages such as decreased cost and ethical limitations compared with working on primates Oxidative stress plays a large role in infertility, and its effect is exerted through multiple mechanisms. In addition, OS has been shown to induce oocyte degeneration and apoptosis through disturbing themeiotic spindle. Oocyte quality depends on the appropriate acquisition of cytoplasmic and nuclear maturation, where the latter depends on the presence of a normal cell spindle This microtubular structure functions primarily in assisting chromatid segregation, concomitant with the extrusion of the second polar body, ensuring the end of the meiotic process (Coticchio et al., 2010). The meiotic spindle of the oocyte is extremely sensitive to factors such as oxidative stress (Hu et al., 2001; Eichenlaub-Ritter et al., 2002; Mullen et al., 2004), which, in turn, promotes meiotic anomalies and chromosome instability, and is associated with increased apoptosis and impairments of preimplantation embryo development (Liu et al., 2003; Navarro et al., 2004, 2006) Da Broi MG, Hum Reprod 2014

30 Induced peritoneal endometriosis in a mouse model is associated with a decrease in oocyte quality and embryo number Cohen J, J Assist Reprod Genet 2015

31 Eve götürülecek mesaj Endometriyozis oosit kalitesi üzerinde detrimental etkiye sahip Kortex-spesifik stroma kaybı Ovarian angiogenezisin inhibisyonu Artmış granuloza hücre apoptozisi Ovarian steroid enzime pathwaylerinde değişiklikler Hücre siklusu kinetiklerinde değişimler DNA hasarı, kromozomal instabilite Gene ekspresyonlarında değişiklikler (i) ROS permeate inside the surrounding cells or are formed in the otherwise healthy tissue as a response to the cyst presence, as shown by the finding that a marker of DNA damage is 10-fold higher in the tissue surrounding the endometrioma than other types of cyst (Matsuzaki and Schubert, 2010); (ii) ROS and TGF-b induce tissue fibrosis, also involving the actions of proteolytic enzymes (Radisky et al., 2007; Bryan et al., 2012; Samarakoon et al., 2013); (iii) an increased fibrosis causes a reduction of cortex-specific stromal cells. Stromal cells are essential for the structure of the follicle and act as mediators of nutrients and molecular signals (Skinner 2005; Oktem and Urman 2010; Kitajima et al., 2011); (iv) fibrosis formation in cortical tissue is also a common pathogenic feature of follicular loss (Meirow et al., 2007) as most authors agree that the follicular density in healthy tissue is at least 2-fold higher than in tissue surrounding the endometriotic cyst (Kitajima et al., 2011; Kuroda et al., 2012); (v) smooth muscle metaplasia also occurs, mostly in the rim of endometriotic cysts and probablydisrupting the organization of the physiological smooth muscle network present in the cortical stroma and periovulatory follicle (Fukunaga, 2000); (vi) inhibition of ovarian angiogenesis and capillary loss are consequently mediated directly by increased ROS levels and indirectly by the cellular injury that in turn triggers over-expression of factors affecting the vascular system, such as TSP-1 (Csanyi et al., 2012). Infertility associated with endometriosis can be even more puzzling, research into therapeutic modalities for subfertility associated with endometriosis needs to be continued, particularly with regard to targeting the molecular mechanisms

32 Teşekkürler

33

34

35 Filippi F, Fertil Steril 2014

36 Benaglia L, Fertil Steril 2013

37 Wallach EE, Fertil Steril 2008

38 Wallach EE, Fertil Steril 2008

39 The presence of ovarian endometriomas is associated with a reduced responsiveness to gonadotropins
Somigliana E, Fertil Steril 2006

40 A lower expression of the CYP19A1 in cumulus cells of infertile patients with endometriosis undergoing ovarian stimulation for ICSI compared with infertile women without endometriosis. A lower number of fertilized oocytes in these women. On the basis of these results, the reduced expression of the CYP19A1 gene in cumulus cells might be involved in the impairment of oocyte quality associated with endometriosis, indicating a new perspective for understanding the pathogenesis of endometriosisrelated infertility. Barcelos IDES, RBM online 2015