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Characteristics of follicular fluid in ovaries with endometriomas
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EURO-9279; No. of Pages 5 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2016) xxx–xxx
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Characteristics of follicular fluid in ovaries with endometriomas Elisa Giacomini a, Ana M. Sanchez a, Veronica Sarais a, Soha Al Beitawi a, Massimo Candiani b, Paola Vigano` a,* a b
Reproductive Sciences Laboratory, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milano, Italy Vita-Salute San Raffaele University and IRCCS San Raffaele Hospital, Milano, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 December 2015 Received in revised form 19 January 2016 Accepted 29 January 2016
The study of follicular fluid (FF) content nearby endometriomas may assist in elucidating pathophysiology, possible biomarkers related to this disease and the effect of endometriomas on ovarian physiology. As the question ‘‘how endometrioma may intrude the physiology of ovarian tissue?’’ is still open, we aimed to summarize the molecular evidence supporting the idea that endometriomas can negatively influence the content of the surrounding ovarian follicles. An alteration of the iron metabolism and an increased ROS (reactive oxygen species) generation characterize the intrafollicular environment adjacent to endometriomas. Other potentially negative effects include decreased testosterone and anti-Mullerian hormone FF levels although these have been only partially clarified. Alterations in lipid and proteomic patterns have been also observed in FF samples nearby endometriomas. The possibility that endometriomas per se may influence IVF clinical results as a consequence of the detrimental impact on the local intrafollicular environment is also discussed. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endometrioma Follicular fluid Iron Reactive oxygen species
Introduction The role of endometriomas on ovarian physiology is quite controversial. It has been repeatedly observed that the ovarian cortex surrounding the endometriomas is altered when compared with that surrounding non-endometriotic benign ovarian cysts [1– 3]. It has been reported that early follicular development may be activated and follicular atresia increased in ovaries with endometriomas compared to contralateral ovaries without cysts. These results would indicate that up-regulated recruitment and, at the same time, demise of early follicles may occur in ovaries with endometriomas, resulting in focal exhaustion of primordial follicles constituting the ovarian reserve. On the other hand, this worrying evidence contrasts with the available clinical evidence suggesting that the damage would be less than initially thought [4–6]. Two studies showed that, in women with unilateral unoperated endometriomas, spontaneous ovulation occurs less frequently in the affected gonad [4,7]. However, in the prospective observational study by Leone Roberti Maggiore et al., no significant difference in the rate of ultrasonographically documented ovulation was observed between the healthy and the affected ovary [8]. Finally, two studies reported on in vitro fertilization (IVF)
* Corresponding author. Tel.: +39 02 26436228; fax: +39 02 26434311. E-mail address: [email protected] (P. Vigano`).
outcomes in women with bilateral endometriomas. Reinblatt et al. failed to document any effect, whereas Benaglia et al. documented a mild reduction in ovarian responsiveness but similar embryo development and pregnancy rates [5,9]. Evidence from the basic science perspective is scanty in this context. Indeed, the basic research has so far been inadequate in elucidating the potential detrimental effect of an endometrioma presence in the context of the ovarian physiology [10,11]. Unlike non-endometriotic cysts, the endometrioma is not surrounded by a capsule as the physical barrier between the cyst contents and the regular ovarian tissue is a thin wall composed of the ovarian cortex itself or fibroreactive tissue [10]. Thus, it is not so surprising that the surrounding healthy tissue could be damaged by the presence of endometrioma per se. To gain insight into this issue, in this review, we have thus performed a literature search in order to clarify whether the follicular fluids (FFs) adjacent to the endometrioma are somehow modified by the presence of the cyst itself. Methods We searched PubMed for articles published in the English language between January 1990 and December 2015 using the following MeSH search terms: ‘endometrioma’ OR ‘endometriotic ovarian cyst’ OR ‘ovarian endometriosis’ combined with ‘follicular fluid’ with restriction to the human species. Data were extracted
http://dx.doi.org/10.1016/j.ejogrb.2016.01.032 0301-2115/ß 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Giacomini E, et al. Characteristics of follicular fluid in ovaries with endometriomas. Eur J Obstet Gynecol (2016), http://dx.doi.org/10.1016/j.ejogrb.2016.01.032
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independently by three investigators (E.G., A.M.S., S.A.) who also performed an initial screening of the title and abstract of all articles to exclude citations deemed irrelevant to all observers. Reference lists of the selected articles and from other reviews were also evaluated. Results Iron It is well known that the endometriotic cyst contains nonresorbed blood derived mainly from the ectopic endometrial cells lining the cyst wall [12]. Thus, high iron concentration has been found within endometriotic cysts [13,14]. In particular, Yamaguchi and colleagues have demonstrated that iron levels in endometriotic cysts are much higher than those in non-endometriotic ovarian cysts. The concentration of iron in serum is 0.013– 0.027 nmol/L while levels reported inside the endometrioma are up to 10,000-fold higher [14]. The potential role of iron in the pathogenesis of endometriosis has been proposed [15]. In addition, iron could have a toxic action, linked to the catalytic decomposition of hydrogen peroxide (Fenton’s reaction) leading to the formation of reactive oxygen species, that can directly damage DNA, lipids and proteins [16]. Still, the presence of iron and iron-related molecules in the endometrioma that could be potentially toxic for the surrounding ovarian follicles has been poorly investigated. Singh et al. have demonstrated that iron level in the FF of women with endometriosis (stages III and IV) was higher compared with women with tubal infertility undergoing IVF [17].
Nonetheless, only two recent studies have evaluated iron concentration in the follicles of ovaries with endometriomas (Table 1) [18,19]. In the study by our group, we have tested iron levels from individual follicular samples that were obtained from 13 women with unilateral endometrioma undergoing IVF-ICSI procedures. In order to evaluate the potential toxic effect exerted by an endometrioma, we have divided the 103 single follicles tested in three groups: 35 follicles proximal to the endometrioma, 28 from the same ovary but distal to the endometrioma and 40 from the contralateral healthy ovary. Total iron levels were found to be significantly increased in the endometrioma-proximal follicles (74.87 5.27 mg/dL) compared to the endometrioma-distal follicles (56.15 1.91 mg/dL) and contralateral follicles (55.83 4.39 mg/dL). In contrast, in the study by Benaglia and collaborators, 39 women with unilateral endometrioma were recruited. Follicular fluid samples were divided between those from ovaries with endometrioma and those from follicles in the contralateral ovary. The median (IQR) of follicular iron concentration in ovaries with [59(44–74) mg/dL] and without [59(47–73) mg/dL] endometriomas was similar [19]. The discrepancies between the two studies in terms of iron concentration could be due to various reasons. A diluting effect might explain the lack of difference found by Benaglia and colleagues as they have evaluated iron concentration in the whole follicular pool. A different explanation could be due to a type I error present in our study considering that we have analyzed data from 13 women only and the use of several follicles from the same women may indeed lead to an over-appreciation of the association. Similar results were conversely obtained by the two groups in relation to H and L ferritin. Ferritins are major iron storage proteins which main role is the regulation of the intracellular iron
Table 1 Characteristics of studies addressing follicular fluid content from ovary with endometrioma versus control. Study year
Population
Ovarian endometrioma group
Control group
Methods
Results
Cordeiro et al., 2015 [44]
Women undergoing ART, 35 years old
Endometriosis grade III or IV, with unilateral endometrioma (n = 10)
Lipidomic analysis with mass spectrometry (ESI-MS/MS)
Regiani et al., 2015 [27]
Women undergoing ART, 18–37 years old
Endometriosis grade III/IV, with unilateral endometrioma (pool of 10 samples)
Lipid subclasses detected: sphingolipids and phosphatidylcholines with specific m/z involved in apoptosis and cell proliferation Specific pathways detected: oxygen transport and hemoglobin complex (peroxiredoxin 2 and ferritin) and response to ROS (prostaglandine reductase 2)
Nakagawa et al., 2015 [26] Benaglia et al., 2015 [19]
Women undergoing ART, 40 years old
Unilateral endometrioma 10 mm (n = 26)
Women undergoing ART, 18–42 years old
Unilateral endometrioma (n = 39)
Tubal factor infertility or no evidence of female infertility (n = 10); Contralateral unaffected ovary of endometrioma group women (n = 10) Tubal factor infertility with a positive pregnancy outcome (pool of 10 samples); Contralateral unaffected ovary of endometrioma group women (pool of 10 samples) No endometrioma (n = 29); Contralateral unaffected ovary (n = 26) Unaffected ovary (n = 39)
Spectrophotometer (free radical elective evaluator F.R.E.E.- and d-ROM, BAP test) Colorimetric (for iron) and immunochemical (for ferritin)
No differences in d-ROM, BAP values and in BAP/d-ROM ratio Higher levels of ferritin in follicles developed in the affected ovary
Sanchez et al., 2014 [18]
Women undergoing ART, 38 years old
Contralateral unaffected ovary (n = 40 follicles from the same 13 women)
FerroZine(e) (for iron) and ELISA (for ferritin)
Increased iron levels in follicles in proximity to endometrioma. Increased ferritin levels in follicles from the affected ovary
Ono et al., 2014 [37]
Women undergoing ART, 38 years old
Male and tubal factor factor infertility (n = 62)
Electrochemiluminescence assay immunoassay
Lower levels of testosterone
Opøien et al., 2013 [34] Garcia-Velasco et al., 2009 [41]
Women undergoing ART; 38 years old
Unilateral endometrioma <4 cm (n = 35 follicles proximal to the endometrioma and n = 28 from the same ovary but distal from a total of 13 women) Unilateral endometrioma (n = 15); bilateral endometriomas (n = 31) Unilateral endometrioma (n = 47); bilateral endometriomas (n = 17) Unilateral endometrioma >2 cm. (n = 28)
Male factor or unexplained infertility (n = 53)
Bio-Plex Pro Human Cytokine Assay
No differences in IL-6, IL-8 levels
Egg donors (n = 28) and contralateral unaffected ovary (n = 28)
ELISA
Lower AMH levels
Women undergoing ART, 35 years old
Proteomic analysis with mass spectrometry (MSE) and functional enrichment analysis
Please cite this article in press as: Giacomini E, et al. Characteristics of follicular fluid in ovaries with endometriomas. Eur J Obstet Gynecol (2016), http://dx.doi.org/10.1016/j.ejogrb.2016.01.032
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distribution [20]. To evaluate the iron balance inside the FFs, H and L ferritins levels were analyzed and we found that in FFs from endometrioma-affected ovary L ferritin levels (mean SEM) were significantly higher in the proximal follicles compared with the distal ones (0.35 0.07 versus 0.19 0.03 ng/mg of total protein respectively; p = 0.044) and compared to the contralateral ones (0.11 0.02 ng/mg of total protein; p = 0.027). Analogous results were found for H ferritin, whose content was significantly higher in the endometrioma-proximal follicles than in those isolated from the contralateral healthy ovary (1.05 0.15 versus 0.48 0.10 ng/mg of total protein respectively; p = 0.042) [18]. Similarly, in the study by Benaglia and colleagues, levels of total ferritin [median (IQR)] measured were higher in the FF from affected ovaries [57(31– 146) mg/dL] compared to contralateral ones [33(23–67) mg/dL; p = 0.026] [19]. The issue to consider is whether the presence of these factors in the FF could disturb the folliculogenesis and oocyte potential affecting IVF outcomes. In our study, we showed that the oocyte retrieval rate was significantly reduced in the affected ovary compared with the healthy contralateral ovary but no changes in day 3 embryo quality was found. In the case of Benaglia et al., no significant correlations were found between follicular ferritin levels from the affected gonad and the number of developing follicles, number of oocytes retrieved, number of suitable oocytes and the number of embryos obtained, concluding that higher ferritin levels did not seem to affect ovarian function [19]. ROS Oxidative stress is a deleterious condition of biochemical disequilibrium caused by an increase of reactive oxygen species (ROS), by a decrease of defense antioxidants or by a combination of both mechanisms. This condition is characterized by an excessive presence of free-radicals species which are formed under normal physiological conditions but become deleterious when not being quenched by the antioxidant systems, independently of the mechanisms which determine it [21]. Oxidative stress has been proposed by many studies as a potential factor involved in the pathophysiology of endometriosis [22,23]. The link between endometriosis, inflammation and infertility is well established, nonetheless little is known about the association between the possibility of an increased in the oxidative stress status in the surrounding follicles and the presence of endometrioma per se [16,24,25]. Nakagawa and colleagues have found that patients with unilateral endometrioma (n = 26) exhibited oxidative stress values similar to those patients without (n = 29) (Table 1). Indeed, the d-ROM (reactive oxygen metabolite) and BAP (biological antioxidant potential) values were similar in FFs from ovaries with endometrioma and in FFs of patients without endometrioma undergoing IVF cycles (d-ROS: 328.7 97.8 U.CARR and 414.9 84.2 U.CARR respectively; BAP: 2474.3 432.0 mmol/L and 2552.83 435.58 mmol/L, respectively). Similarly, the number of patients with modified BAP/d-ROM ratio <1.0, an index for latent antioxidant potential, was similar between the two groups. Finally, no oxidative stress or antioxidant potential differences in FFs from ovaries with or without endometrioma from the same patient were found [26]. Recently, Regiani et al. [27] have performed a proteomic analysis using a mass spectrometry approach to define the protein profile of FF proximal to endometriomas. Different proteomic profiles were defined matching three groups of pooled follicular fluids: a control group (FFs from women with tubal factor or minimal male factor infertility who achieved pregnancy after IVF treatment) (n = 10), an endometrioma group (FFs from the ovary with an endometrioma) (n = 10) and an endometriosis group (FFs of the contralateral non affected ovary from the same patients) (n = 10). Results of the enrichment functional analysis of protein
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profile showed the strong presence of a hydrogen peroxidemediated catabolic process in the FFs of the endometrioma group. Additionally, the authors suggested that the protein profiles identified in the FFs of the endometrioma group could favor a high oxidative stress status [27]. The presence in the endometrioma group of several proteins involved in cell death mechanisms, such as high levels of ubiquitin, protein S100-A8 and annexin A1, could confirm an involvement of oxidative stress in follicular maturation [28–30]. Inflammatory molecules It is well known that endometriosis induces a chronic inflammatory reaction [31]. In particular, different pro-inflammatory cytokines such as interleukin (IL)-8 [32], IL-6, IL-13, IL-8, IL-17 and CA125 [33] have been found in the content of the ovarian endometriotic cyst. However, it is unknown whether the inflammatory components of the endometrioma can adversely impact the surrounding follicular environment. Opøien et al. have compared the FF content of women with unilateral and bilateral endometriomas with two different control groups, unexplained infertility and a male factor infertility group (Table 1). Levels of IL-6 and IL-8 were similar among the groups. Additionally, TNF-a was only detectable in FF from women with bilateral endometriomas. The concentration of IL-1b, IL-10 and IL12 could not be quantified, due to low levels. The authors have also found that women with higher concentration of cytokines in FF (independently from the presence or not of the endometrioma) retrieved a lower number of oocyte (p < 0.05) [34]. More studies need to be done in order to clarify this aspect of the disease. Hormone profile There are several studies providing arguments in favor of an endocrine dysregulation in endometriosis patients [35]. This could be one of the reason for which women with endometriosis show an impairment of the follicular function leading to oocyte damage. The relationship between FF hormone levels and IVF outcomes has been studied in the past with a particular attention to levels of progesterone, androstenedione and estradiol in FF [36]. In the case of endometrioma, only few articles have studied the hormonal environment in the FF of patients affected (Table 1). Recently Ono et al., have found lower levels of testosterone in FFs of patients with an endometrioma (n = 46) compared to control women (male factor or tubal factor infertility, n = 62) (9.16 3.96 ng/mL versus 17.04 4.94 ng/mL respectively; p = 0.0003). Moreover, a correlation between lower serum testosterone levels and higher levels of apoptosis in granulosa cells from these follicles was found [37]. Currently, serum anti-Mullerian hormone (AMH) level is considered a reliable surrogate marker of ovarian reserve also in women with endometriosis. Additionally, FF levels of AMH may correlate with oocyte quality, fertilization rate and reproductive outcomes [38–40]. Unfortunately, only one study has described AMH levels in FF from women with endometrioma [41]. Considering 3 different groups, FFs from healthy women (n = 28), FFs from women with endometrioma from the affected ovary (n = 28) and FFs from the contralateral healthy ovary from the same women (n = 28), Garcia-Velasco et al. found lower AMH levels in FFs from the ovary with the endometrioma (4.1 2.7 ng/ mL) compared with FFs from the contralateral healthy ovary (4.9 2.6 ng/mL) and with FFs of healthy women without endometriosis (6.2 3 ng/mL; p = 0.039). They have concluded that the presence of the endometrioma itself could reduce per se AMH concentration in the surrounding follicles [41].
Please cite this article in press as: Giacomini E, et al. Characteristics of follicular fluid in ovaries with endometriomas. Eur J Obstet Gynecol (2016), http://dx.doi.org/10.1016/j.ejogrb.2016.01.032
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Fig. 1. Follicular fluid content alterations caused by the presence of endometrioma. Endometriomas impact on the nearby follicular environment: representative scheme. ROS, reactive oxygen species; PTGR2, prostaglandin reductase 2; TNF-a, tumor necrosis factor alpha; IL-6, interleukin 6; IL-8, interleukin 8; AMH, anti-Mullerian hormone; OMICS, proteomics and lipidomics alterations.
‘‘Omics’’ The ‘‘omics’’ approach could be an important tool to find new biomarkers of ovarian behavior with diagnostic significance [42]. In line, Regiani et al. have performed a proteomic analysis to define the profile of FFs of women with endometrioma [27]. Analysis of tandem mass spectrometry (MSE) spectra of the three groups of FFs previously described allowed to identify a total of 535 proteins of which 366 expressed in the endometrioma group divided as follows: 9 proteins in common with the control group, 6 with the endometriosis group, 139 common among the three groups and 212 exclusively recognized in the endometrioma group. Functional enrichment analysis was used to define possible specific pathways for each group. The main pathways identified by protein interactions in the control group included vitamin transport (vitamin-D-binding protein), immune response (complement component 2) and inflammation (alpha-1-trypsin). Response to ROS (prostaglandin reductase 2 – PTGR2), oxygen transport and hemoglobin complex (peroxiredoxin 2 and ferritin) were the specific pathways observed in the endometrioma group whereas the endometriosis group showed pathways involving coagulation processes (kallicrein B and prothrombin) and sterol metabolism (sex-hormone-binding globulin). The study highlighted similar protein patterns between the endometriosis and the endometrioma groups suggesting the prevalence of endometriosis features at different stages of the disease. Additionally, alterations in lipid pattern are common in several diseases and are the result of response to toxin exposure and to both environmental and genetic modifications [43]. To identify lipidic subclasses of potential biomarkers with clinical relevance, the same team [44] has performed a lipidomic profile of FFs from the three identical study groups (Table 1). The metabolic profile performed using electrospray tandem mass spectrometry (ESI-MS) analysis has generated a list of 15 ions differentially expressed in each group. The results of lipid fingerprint analysis highlighted a strong presence of sphingolipids and phosphatidylcholines in the endometriosis and endometrioma groups but with different massto-charge ratio (m/z) between the two groups. Conversely, the control group was enriched of phosphatidylglycerol phosphate, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol bisphosphate. The lipid subclasses identified demonstrating differential expression among the groups have been suggested to
be involved in follicular development and embryo cleavage in the control group [45–47], as well as in apoptosis and cell proliferation particularly in the endometriosis and endometrioma group [48–50]. Conclusion The major findings of this review indicate that the ovarian follicles surrounding an endometrioma are characterized by alterations of the iron metabolism and by an increased ROS generation (Fig. 1). However, it should be noted that studies addressing the impact of endometriomas on the nearby follicular environment are limited in number and further investigation is required to clarify this aspect of the disease. The endometrioma presence may impact the ovarian physiology as an increased oxidative stress status and an alteration of iron metabolism has been observed in the follicular environment. References [1] Maneschi F, Marasa´ L, Incandela S, Mazzarese M, Zupi E. Ovarian cortex surrounding benign neoplasms: a histologic study. Am J Obstet Gynecol 1993;169:388–93. [2] Kuroda M, Kuroda K, Arakawa A, et al. Histological assessment of impact of ovarian endometrioma and laparoscopic cystectomy on ovarian reserve. J Obstet Gynaecol Res 2012;38:1187–93. [3] Kitajima M, Dolmans MM, Donnez O, Masuzaki H, Soares M, Donnez J. Enhanced follicular recruitment and atresia in cortex derived from ovaries with endometriomas. Fertil Steril 2014;101:1031–7. [4] Horikawa T, Nakagawa K, Ohgi S, et al. The frequency of ovulation from the affected ovary decreases following laparoscopic cystectomy in infertile women with unilateral endometrioma during a natural cycle. J Assist Reprod Genet 2008;25:239–44. [5] Benaglia L, Bermejo A, Somigliana E, et al. In vitro fertilization outcome in women with unoperated bilateral endometriomas. Fertil Steril 2013;99: 1714–9. [6] Somigliana E, Benaglia L, Paffoni A, Busnelli A, Vigano P, Vercellini P. Risks of conservative management in women with ovarian endometriomas undergoing IVF. Hum Reprod Update 2015;21:486–99. [7] Benaglia L, Somigliana E, Vercellini P, Abbiati A, Ragni G, Fedele L. Endometriotic ovarian cysts negatively affect the rate of spontaneous ovulation. Hum Reprod 2009;24:2183–6. [8] Leone Roberti Maggiore U, Scala C, Venturini PL, Remorgida V, Ferrero S. Endometriotic ovarian cysts do not negatively affect the rate of spontaneous ovulation. Hum Reprod 2015;30:299–307. [9] Reinblatt SL, Ishai L, Shehata F, Son WY, Tulandi T, Almog B. Effects of ovarian endometrioma on embryo quality. Fertil Steril 2011;95:2700–2. [10] Sanchez AM, Vigano` P, Somigliana E, Panina-Bordignon P, Vercellini P, Candiani M. The distinguishing cellular and molecular features of the
Please cite this article in press as: Giacomini E, et al. Characteristics of follicular fluid in ovaries with endometriomas. Eur J Obstet Gynecol (2016), http://dx.doi.org/10.1016/j.ejogrb.2016.01.032
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[31] Vercellini P, Vigano` P, Somigliana E, Fedele L. Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol 2014;10:261–75. [32] Fasciani A, D’Ambrogio G, Bocci G, Monti M, Genazzani AR, Artini PG. High concentrations of the vascular endothelial growth factor and interleukin-8 in ovarian endometriomata. Mol Hum Reprod 2000;6:50–4. [33] Velasco I, Acie´n P, Campos A, Acie´n MI, Ruiz-Macia´ E. Interleukin-6 and other soluble factors in peritoneal fluid and endometriomas and their relation to pain and aromatase expression. J Reprod Immunol 2010;84:199–205. [34] Opøien HK, Fedorcsak P, Polec A, Stensen MH, A˚byholm T, Tanbo T. Do endometriomas induce an inflammatory reaction in nearby follicles. Hum Reprod 2013;28:1837–45. [35] Pellicer A, Albert C, Mercader A, Bonilla-Musoles F, Remohı´ J, Simo´n C. The follicular and endocrine environment in women with endometriosis: local and systemic cytokine production. Fertil Steril 1998;70:425–31. [36] Wen X, Li D, Tozer AJ, Docherty SM, Iles RK. Estradiol, progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles. Reprod Biol Endocrinol 2010;8:117. [37] Ono YJ, Tanabe A, Nakamura Y, et al. A low-testosterone state associated with endometrioma leads to the apoptosis of granulosa cells. PLOS ONE 2014;23:9– 12. [38] Wunder DM, Guibourdenche J, Birkha¨user MH, Bersinger NA. Anti-Mu¨llerian hormone and inhibin B as predictors of pregnancy after treatment by in vitro fertilization/intracytoplasmic sperm injection. Fertil Steril 2008;90:2203–10. [39] Fanchin R, Louafi N, Me´ndez Lozano DH, Frydman N, Frydman R, Taieb J. Perfollicle measurements indicate that anti-Mu¨llerian hormone secretion is modulated by the extent of follicular development and luteinization and may reflect qualitatively the ovarian follicular status. Fertil Steril 2005;84:167–73. [40] Takahashi C, Fujito A, Kazuka M, Sugiyama R, Ito H, Isaka K. Anti-Mu¨llerian hormone substance from follicular fluid is positively associated with success in oocyte fertilization during in vitro fertilization. Fertil Steril 2008;89:586–91. [41] Garcia-Velasco JA, Motta L, Rodriguez S, Toribio M, Martinez-Salazar J, Pacheco A. Decreased concentrations of AMH in follicular fluid of women with endometriosis: a hypothetical new marker of oocyte quality. J Endometr 2009;1:52–6. [42] Hu ZZ, Huang H, Wu CH, et al. Omics-based molecular target and biomarker identification. Methods Mol Biol 2011;719:547–71. [43] Bou Khalil M, Hou W, Zhou H, et al. Lipidomics era: accomplishments and challenges. Mass Spectrom Rev 2010;29:877–929. [44] Cordeiro FB, Cataldi TR, Perkel KJ, et al. Lipidomics analysis of follicular fluid by ESI-MS reveals potential biomarkers for ovarian endometriosis. J Assist Reprod Genet 2015 [Epub ahead of print]. [45] Rojas FJ, Moretti-Rojas I, Balmaceda JP, Asch RH. Regulation of gonadotropinstimulable adenylyl cyclase of the primate corpus luteum. J Steroid Biochem 1989;32:175–82. [46] Houmard BS, Guan Z, Stokes BT, Ottobre JS. The effects of gonadotropin on the phosphatidylinositol pathway in the primate corpus luteum. Mol Cell Endocrinol 1994;104:113–20. [47] O’Neill C. Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development. Reproduction 2008;136:147–56. [48] Cuvillier O, Andrieu-Abadie N, Se´gui B, et al. Sphingolipid-mediated apoptotic signaling pathways. J Soc Biol 2003;197:217–21. [49] Separovic D, Hanada K, Maitah MY, et al. Sphingomyelin synthase 1 suppresses ceramide production and apoptosis post-photodamage. Biochem Biophys Res Commun 2007;358:196–202. [50] Santos CR, Schulze A. Lipid metabolism in cancer. FEBS J 2012;279:2610–23.
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EURO-9279; No. of Pages 5 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Characteristics of follicular fluid in ovaries with endometriomas Elisa Giacomini a, Ana M. Sanchez a, Veronica Sarais a, Soha Al Beitawi a, Massimo Candiani b, Paola Vigano` a,* a b
Reproductive Sciences Laboratory, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milano, Italy Vita-Salute San Raffaele University and IRCCS San Raffaele Hospital, Milano, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 December 2015 Received in revised form 19 January 2016 Accepted 29 January 2016
The study of follicular fluid (FF) content nearby endometriomas may assist in elucidating pathophysiology, possible biomarkers related to this disease and the effect of endometriomas on ovarian physiology. As the question ‘‘how endometrioma may intrude the physiology of ovarian tissue?’’ is still open, we aimed to summarize the molecular evidence supporting the idea that endometriomas can negatively influence the content of the surrounding ovarian follicles. An alteration of the iron metabolism and an increased ROS (reactive oxygen species) generation characterize the intrafollicular environment adjacent to endometriomas. Other potentially negative effects include decreased testosterone and anti-Mullerian hormone FF levels although these have been only partially clarified. Alterations in lipid and proteomic patterns have been also observed in FF samples nearby endometriomas. The possibility that endometriomas per se may influence IVF clinical results as a consequence of the detrimental impact on the local intrafollicular environment is also discussed. ß 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endometrioma Follicular fluid Iron Reactive oxygen species
Introduction The role of endometriomas on ovarian physiology is quite controversial. It has been repeatedly observed that the ovarian cortex surrounding the endometriomas is altered when compared with that surrounding non-endometriotic benign ovarian cysts [1– 3]. It has been reported that early follicular development may be activated and follicular atresia increased in ovaries with endometriomas compared to contralateral ovaries without cysts. These results would indicate that up-regulated recruitment and, at the same time, demise of early follicles may occur in ovaries with endometriomas, resulting in focal exhaustion of primordial follicles constituting the ovarian reserve. On the other hand, this worrying evidence contrasts with the available clinical evidence suggesting that the damage would be less than initially thought [4–6]. Two studies showed that, in women with unilateral unoperated endometriomas, spontaneous ovulation occurs less frequently in the affected gonad [4,7]. However, in the prospective observational study by Leone Roberti Maggiore et al., no significant difference in the rate of ultrasonographically documented ovulation was observed between the healthy and the affected ovary [8]. Finally, two studies reported on in vitro fertilization (IVF)
* Corresponding author. Tel.: +39 02 26436228; fax: +39 02 26434311. E-mail address: [email protected] (P. Vigano`).
outcomes in women with bilateral endometriomas. Reinblatt et al. failed to document any effect, whereas Benaglia et al. documented a mild reduction in ovarian responsiveness but similar embryo development and pregnancy rates [5,9]. Evidence from the basic science perspective is scanty in this context. Indeed, the basic research has so far been inadequate in elucidating the potential detrimental effect of an endometrioma presence in the context of the ovarian physiology [10,11]. Unlike non-endometriotic cysts, the endometrioma is not surrounded by a capsule as the physical barrier between the cyst contents and the regular ovarian tissue is a thin wall composed of the ovarian cortex itself or fibroreactive tissue [10]. Thus, it is not so surprising that the surrounding healthy tissue could be damaged by the presence of endometrioma per se. To gain insight into this issue, in this review, we have thus performed a literature search in order to clarify whether the follicular fluids (FFs) adjacent to the endometrioma are somehow modified by the presence of the cyst itself. Methods We searched PubMed for articles published in the English language between January 1990 and December 2015 using the following MeSH search terms: ‘endometrioma’ OR ‘endometriotic ovarian cyst’ OR ‘ovarian endometriosis’ combined with ‘follicular fluid’ with restriction to the human species. Data were extracted
http://dx.doi.org/10.1016/j.ejogrb.2016.01.032 0301-2115/ß 2016 Elsevier Ireland Ltd. All rights reserved.
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independently by three investigators (E.G., A.M.S., S.A.) who also performed an initial screening of the title and abstract of all articles to exclude citations deemed irrelevant to all observers. Reference lists of the selected articles and from other reviews were also evaluated. Results Iron It is well known that the endometriotic cyst contains nonresorbed blood derived mainly from the ectopic endometrial cells lining the cyst wall [12]. Thus, high iron concentration has been found within endometriotic cysts [13,14]. In particular, Yamaguchi and colleagues have demonstrated that iron levels in endometriotic cysts are much higher than those in non-endometriotic ovarian cysts. The concentration of iron in serum is 0.013– 0.027 nmol/L while levels reported inside the endometrioma are up to 10,000-fold higher [14]. The potential role of iron in the pathogenesis of endometriosis has been proposed [15]. In addition, iron could have a toxic action, linked to the catalytic decomposition of hydrogen peroxide (Fenton’s reaction) leading to the formation of reactive oxygen species, that can directly damage DNA, lipids and proteins [16]. Still, the presence of iron and iron-related molecules in the endometrioma that could be potentially toxic for the surrounding ovarian follicles has been poorly investigated. Singh et al. have demonstrated that iron level in the FF of women with endometriosis (stages III and IV) was higher compared with women with tubal infertility undergoing IVF [17].
Nonetheless, only two recent studies have evaluated iron concentration in the follicles of ovaries with endometriomas (Table 1) [18,19]. In the study by our group, we have tested iron levels from individual follicular samples that were obtained from 13 women with unilateral endometrioma undergoing IVF-ICSI procedures. In order to evaluate the potential toxic effect exerted by an endometrioma, we have divided the 103 single follicles tested in three groups: 35 follicles proximal to the endometrioma, 28 from the same ovary but distal to the endometrioma and 40 from the contralateral healthy ovary. Total iron levels were found to be significantly increased in the endometrioma-proximal follicles (74.87 5.27 mg/dL) compared to the endometrioma-distal follicles (56.15 1.91 mg/dL) and contralateral follicles (55.83 4.39 mg/dL). In contrast, in the study by Benaglia and collaborators, 39 women with unilateral endometrioma were recruited. Follicular fluid samples were divided between those from ovaries with endometrioma and those from follicles in the contralateral ovary. The median (IQR) of follicular iron concentration in ovaries with [59(44–74) mg/dL] and without [59(47–73) mg/dL] endometriomas was similar [19]. The discrepancies between the two studies in terms of iron concentration could be due to various reasons. A diluting effect might explain the lack of difference found by Benaglia and colleagues as they have evaluated iron concentration in the whole follicular pool. A different explanation could be due to a type I error present in our study considering that we have analyzed data from 13 women only and the use of several follicles from the same women may indeed lead to an over-appreciation of the association. Similar results were conversely obtained by the two groups in relation to H and L ferritin. Ferritins are major iron storage proteins which main role is the regulation of the intracellular iron
Table 1 Characteristics of studies addressing follicular fluid content from ovary with endometrioma versus control. Study year
Population
Ovarian endometrioma group
Control group
Methods
Results
Cordeiro et al., 2015 [44]
Women undergoing ART, 35 years old
Endometriosis grade III or IV, with unilateral endometrioma (n = 10)
Lipidomic analysis with mass spectrometry (ESI-MS/MS)
Regiani et al., 2015 [27]
Women undergoing ART, 18–37 years old
Endometriosis grade III/IV, with unilateral endometrioma (pool of 10 samples)
Lipid subclasses detected: sphingolipids and phosphatidylcholines with specific m/z involved in apoptosis and cell proliferation Specific pathways detected: oxygen transport and hemoglobin complex (peroxiredoxin 2 and ferritin) and response to ROS (prostaglandine reductase 2)
Nakagawa et al., 2015 [26] Benaglia et al., 2015 [19]
Women undergoing ART, 40 years old
Unilateral endometrioma 10 mm (n = 26)
Women undergoing ART, 18–42 years old
Unilateral endometrioma (n = 39)
Tubal factor infertility or no evidence of female infertility (n = 10); Contralateral unaffected ovary of endometrioma group women (n = 10) Tubal factor infertility with a positive pregnancy outcome (pool of 10 samples); Contralateral unaffected ovary of endometrioma group women (pool of 10 samples) No endometrioma (n = 29); Contralateral unaffected ovary (n = 26) Unaffected ovary (n = 39)
Spectrophotometer (free radical elective evaluator F.R.E.E.- and d-ROM, BAP test) Colorimetric (for iron) and immunochemical (for ferritin)
No differences in d-ROM, BAP values and in BAP/d-ROM ratio Higher levels of ferritin in follicles developed in the affected ovary
Sanchez et al., 2014 [18]
Women undergoing ART, 38 years old
Contralateral unaffected ovary (n = 40 follicles from the same 13 women)
FerroZine(e) (for iron) and ELISA (for ferritin)
Increased iron levels in follicles in proximity to endometrioma. Increased ferritin levels in follicles from the affected ovary
Ono et al., 2014 [37]
Women undergoing ART, 38 years old
Male and tubal factor factor infertility (n = 62)
Electrochemiluminescence assay immunoassay
Lower levels of testosterone
Opøien et al., 2013 [34] Garcia-Velasco et al., 2009 [41]
Women undergoing ART; 38 years old
Unilateral endometrioma <4 cm (n = 35 follicles proximal to the endometrioma and n = 28 from the same ovary but distal from a total of 13 women) Unilateral endometrioma (n = 15); bilateral endometriomas (n = 31) Unilateral endometrioma (n = 47); bilateral endometriomas (n = 17) Unilateral endometrioma >2 cm. (n = 28)
Male factor or unexplained infertility (n = 53)
Bio-Plex Pro Human Cytokine Assay
No differences in IL-6, IL-8 levels
Egg donors (n = 28) and contralateral unaffected ovary (n = 28)
ELISA
Lower AMH levels
Women undergoing ART, 35 years old
Proteomic analysis with mass spectrometry (MSE) and functional enrichment analysis
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distribution [20]. To evaluate the iron balance inside the FFs, H and L ferritins levels were analyzed and we found that in FFs from endometrioma-affected ovary L ferritin levels (mean SEM) were significantly higher in the proximal follicles compared with the distal ones (0.35 0.07 versus 0.19 0.03 ng/mg of total protein respectively; p = 0.044) and compared to the contralateral ones (0.11 0.02 ng/mg of total protein; p = 0.027). Analogous results were found for H ferritin, whose content was significantly higher in the endometrioma-proximal follicles than in those isolated from the contralateral healthy ovary (1.05 0.15 versus 0.48 0.10 ng/mg of total protein respectively; p = 0.042) [18]. Similarly, in the study by Benaglia and colleagues, levels of total ferritin [median (IQR)] measured were higher in the FF from affected ovaries [57(31– 146) mg/dL] compared to contralateral ones [33(23–67) mg/dL; p = 0.026] [19]. The issue to consider is whether the presence of these factors in the FF could disturb the folliculogenesis and oocyte potential affecting IVF outcomes. In our study, we showed that the oocyte retrieval rate was significantly reduced in the affected ovary compared with the healthy contralateral ovary but no changes in day 3 embryo quality was found. In the case of Benaglia et al., no significant correlations were found between follicular ferritin levels from the affected gonad and the number of developing follicles, number of oocytes retrieved, number of suitable oocytes and the number of embryos obtained, concluding that higher ferritin levels did not seem to affect ovarian function [19]. ROS Oxidative stress is a deleterious condition of biochemical disequilibrium caused by an increase of reactive oxygen species (ROS), by a decrease of defense antioxidants or by a combination of both mechanisms. This condition is characterized by an excessive presence of free-radicals species which are formed under normal physiological conditions but become deleterious when not being quenched by the antioxidant systems, independently of the mechanisms which determine it [21]. Oxidative stress has been proposed by many studies as a potential factor involved in the pathophysiology of endometriosis [22,23]. The link between endometriosis, inflammation and infertility is well established, nonetheless little is known about the association between the possibility of an increased in the oxidative stress status in the surrounding follicles and the presence of endometrioma per se [16,24,25]. Nakagawa and colleagues have found that patients with unilateral endometrioma (n = 26) exhibited oxidative stress values similar to those patients without (n = 29) (Table 1). Indeed, the d-ROM (reactive oxygen metabolite) and BAP (biological antioxidant potential) values were similar in FFs from ovaries with endometrioma and in FFs of patients without endometrioma undergoing IVF cycles (d-ROS: 328.7 97.8 U.CARR and 414.9 84.2 U.CARR respectively; BAP: 2474.3 432.0 mmol/L and 2552.83 435.58 mmol/L, respectively). Similarly, the number of patients with modified BAP/d-ROM ratio <1.0, an index for latent antioxidant potential, was similar between the two groups. Finally, no oxidative stress or antioxidant potential differences in FFs from ovaries with or without endometrioma from the same patient were found [26]. Recently, Regiani et al. [27] have performed a proteomic analysis using a mass spectrometry approach to define the protein profile of FF proximal to endometriomas. Different proteomic profiles were defined matching three groups of pooled follicular fluids: a control group (FFs from women with tubal factor or minimal male factor infertility who achieved pregnancy after IVF treatment) (n = 10), an endometrioma group (FFs from the ovary with an endometrioma) (n = 10) and an endometriosis group (FFs of the contralateral non affected ovary from the same patients) (n = 10). Results of the enrichment functional analysis of protein
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profile showed the strong presence of a hydrogen peroxidemediated catabolic process in the FFs of the endometrioma group. Additionally, the authors suggested that the protein profiles identified in the FFs of the endometrioma group could favor a high oxidative stress status [27]. The presence in the endometrioma group of several proteins involved in cell death mechanisms, such as high levels of ubiquitin, protein S100-A8 and annexin A1, could confirm an involvement of oxidative stress in follicular maturation [28–30]. Inflammatory molecules It is well known that endometriosis induces a chronic inflammatory reaction [31]. In particular, different pro-inflammatory cytokines such as interleukin (IL)-8 [32], IL-6, IL-13, IL-8, IL-17 and CA125 [33] have been found in the content of the ovarian endometriotic cyst. However, it is unknown whether the inflammatory components of the endometrioma can adversely impact the surrounding follicular environment. Opøien et al. have compared the FF content of women with unilateral and bilateral endometriomas with two different control groups, unexplained infertility and a male factor infertility group (Table 1). Levels of IL-6 and IL-8 were similar among the groups. Additionally, TNF-a was only detectable in FF from women with bilateral endometriomas. The concentration of IL-1b, IL-10 and IL12 could not be quantified, due to low levels. The authors have also found that women with higher concentration of cytokines in FF (independently from the presence or not of the endometrioma) retrieved a lower number of oocyte (p < 0.05) [34]. More studies need to be done in order to clarify this aspect of the disease. Hormone profile There are several studies providing arguments in favor of an endocrine dysregulation in endometriosis patients [35]. This could be one of the reason for which women with endometriosis show an impairment of the follicular function leading to oocyte damage. The relationship between FF hormone levels and IVF outcomes has been studied in the past with a particular attention to levels of progesterone, androstenedione and estradiol in FF [36]. In the case of endometrioma, only few articles have studied the hormonal environment in the FF of patients affected (Table 1). Recently Ono et al., have found lower levels of testosterone in FFs of patients with an endometrioma (n = 46) compared to control women (male factor or tubal factor infertility, n = 62) (9.16 3.96 ng/mL versus 17.04 4.94 ng/mL respectively; p = 0.0003). Moreover, a correlation between lower serum testosterone levels and higher levels of apoptosis in granulosa cells from these follicles was found [37]. Currently, serum anti-Mullerian hormone (AMH) level is considered a reliable surrogate marker of ovarian reserve also in women with endometriosis. Additionally, FF levels of AMH may correlate with oocyte quality, fertilization rate and reproductive outcomes [38–40]. Unfortunately, only one study has described AMH levels in FF from women with endometrioma [41]. Considering 3 different groups, FFs from healthy women (n = 28), FFs from women with endometrioma from the affected ovary (n = 28) and FFs from the contralateral healthy ovary from the same women (n = 28), Garcia-Velasco et al. found lower AMH levels in FFs from the ovary with the endometrioma (4.1 2.7 ng/ mL) compared with FFs from the contralateral healthy ovary (4.9 2.6 ng/mL) and with FFs of healthy women without endometriosis (6.2 3 ng/mL; p = 0.039). They have concluded that the presence of the endometrioma itself could reduce per se AMH concentration in the surrounding follicles [41].
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Fig. 1. Follicular fluid content alterations caused by the presence of endometrioma. Endometriomas impact on the nearby follicular environment: representative scheme. ROS, reactive oxygen species; PTGR2, prostaglandin reductase 2; TNF-a, tumor necrosis factor alpha; IL-6, interleukin 6; IL-8, interleukin 8; AMH, anti-Mullerian hormone; OMICS, proteomics and lipidomics alterations.
‘‘Omics’’ The ‘‘omics’’ approach could be an important tool to find new biomarkers of ovarian behavior with diagnostic significance [42]. In line, Regiani et al. have performed a proteomic analysis to define the profile of FFs of women with endometrioma [27]. Analysis of tandem mass spectrometry (MSE) spectra of the three groups of FFs previously described allowed to identify a total of 535 proteins of which 366 expressed in the endometrioma group divided as follows: 9 proteins in common with the control group, 6 with the endometriosis group, 139 common among the three groups and 212 exclusively recognized in the endometrioma group. Functional enrichment analysis was used to define possible specific pathways for each group. The main pathways identified by protein interactions in the control group included vitamin transport (vitamin-D-binding protein), immune response (complement component 2) and inflammation (alpha-1-trypsin). Response to ROS (prostaglandin reductase 2 – PTGR2), oxygen transport and hemoglobin complex (peroxiredoxin 2 and ferritin) were the specific pathways observed in the endometrioma group whereas the endometriosis group showed pathways involving coagulation processes (kallicrein B and prothrombin) and sterol metabolism (sex-hormone-binding globulin). The study highlighted similar protein patterns between the endometriosis and the endometrioma groups suggesting the prevalence of endometriosis features at different stages of the disease. Additionally, alterations in lipid pattern are common in several diseases and are the result of response to toxin exposure and to both environmental and genetic modifications [43]. To identify lipidic subclasses of potential biomarkers with clinical relevance, the same team [44] has performed a lipidomic profile of FFs from the three identical study groups (Table 1). The metabolic profile performed using electrospray tandem mass spectrometry (ESI-MS) analysis has generated a list of 15 ions differentially expressed in each group. The results of lipid fingerprint analysis highlighted a strong presence of sphingolipids and phosphatidylcholines in the endometriosis and endometrioma groups but with different massto-charge ratio (m/z) between the two groups. Conversely, the control group was enriched of phosphatidylglycerol phosphate, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol bisphosphate. The lipid subclasses identified demonstrating differential expression among the groups have been suggested to
be involved in follicular development and embryo cleavage in the control group [45–47], as well as in apoptosis and cell proliferation particularly in the endometriosis and endometrioma group [48–50]. Conclusion The major findings of this review indicate that the ovarian follicles surrounding an endometrioma are characterized by alterations of the iron metabolism and by an increased ROS generation (Fig. 1). However, it should be noted that studies addressing the impact of endometriomas on the nearby follicular environment are limited in number and further investigation is required to clarify this aspect of the disease. The endometrioma presence may impact the ovarian physiology as an increased oxidative stress status and an alteration of iron metabolism has been observed in the follicular environment. References [1] Maneschi F, Marasa´ L, Incandela S, Mazzarese M, Zupi E. Ovarian cortex surrounding benign neoplasms: a histologic study. Am J Obstet Gynecol 1993;169:388–93. [2] Kuroda M, Kuroda K, Arakawa A, et al. Histological assessment of impact of ovarian endometrioma and laparoscopic cystectomy on ovarian reserve. J Obstet Gynaecol Res 2012;38:1187–93. [3] Kitajima M, Dolmans MM, Donnez O, Masuzaki H, Soares M, Donnez J. Enhanced follicular recruitment and atresia in cortex derived from ovaries with endometriomas. Fertil Steril 2014;101:1031–7. [4] Horikawa T, Nakagawa K, Ohgi S, et al. The frequency of ovulation from the affected ovary decreases following laparoscopic cystectomy in infertile women with unilateral endometrioma during a natural cycle. J Assist Reprod Genet 2008;25:239–44. [5] Benaglia L, Bermejo A, Somigliana E, et al. In vitro fertilization outcome in women with unoperated bilateral endometriomas. Fertil Steril 2013;99: 1714–9. [6] Somigliana E, Benaglia L, Paffoni A, Busnelli A, Vigano P, Vercellini P. Risks of conservative management in women with ovarian endometriomas undergoing IVF. Hum Reprod Update 2015;21:486–99. [7] Benaglia L, Somigliana E, Vercellini P, Abbiati A, Ragni G, Fedele L. Endometriotic ovarian cysts negatively affect the rate of spontaneous ovulation. Hum Reprod 2009;24:2183–6. [8] Leone Roberti Maggiore U, Scala C, Venturini PL, Remorgida V, Ferrero S. Endometriotic ovarian cysts do not negatively affect the rate of spontaneous ovulation. Hum Reprod 2015;30:299–307. [9] Reinblatt SL, Ishai L, Shehata F, Son WY, Tulandi T, Almog B. Effects of ovarian endometrioma on embryo quality. Fertil Steril 2011;95:2700–2. [10] Sanchez AM, Vigano` P, Somigliana E, Panina-Bordignon P, Vercellini P, Candiani M. The distinguishing cellular and molecular features of the
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Please cite this article in press as: Giacomini E, et al. Characteristics of follicular fluid in ovaries with endometriomas. Eur J Obstet Gynecol (2016), http://dx.doi.org/10.1016/j.ejogrb.2016.01.032