follicular fluid

Follicular fluid

FF provides a very important microenvironment for the development of oocytes. FF is a product of both the transfer of blood plasma constituents that cross the blood follicular barrier and of the secretory activity of granulosa and thecal cells [4]. It is reasonable to think that some biochemical characteristics of the FF surrounding the oocyte may play a critical role in determining oocyte quality and the subsequent potential to achieve fertilization and embryo development. The analysis of FF components may also provide information on proteomics changes in blood serum, as the circulating biochemical milieu may be reflected in the composition of FF.

Follicular fluid has an important role in both follicle and oocyte development, from antral to pre-ovulatory follicles (Figure 4-1). This fluid fills the follicular antrum and surrounds the ovum in an ovarian follicle. In human, a single preovulatory follicle can contain up to 15 ml of fluid and provides a conducive microenvironment for the developing follicle and it’s oocyte. Follicular fluid is produced from secreted plasma of blood vessels and capillaries, which are usually found within the thecal layer [183]. It begins with a simple capillary network surrounding the follicle at the antral stage, and then turns to a more complicated network as the follicle grows. Follicular fluid production is also facilitated by hydrostatic forces, generated by contraction of cilia on individual cells. Moreover, osmotic pressure created by the differences in the solute concentrations between the two areas facilitates the mechanism [182]. Lastly, secretions from the granulosa and theca cells contribute to the production of follicular fluid [182] The formation of the follicular cavity is unique. It starts with development of multiple foci of fluid within the granulosa layers. These foci will expand and coalesce, forming a larger, centrally located antrum. The lumen is also formed by cell death or/and apoptosis. To contain and accommodate more fluid in a growing follicle, the surrounding theca and granulosa cells will become oedematous and swell to form a tight seal. Increasing fluid volume within the antral cavity results in differentiation of granulosa cell layers into mural or cumulus cells

1. Fortune JE. Ovarian follicular growth and development in mammals. Biol Reprod. 1994;50:225–232. doi: 10.1095/biolreprod50.2.225. [PubMed] [CrossRef] [Google Scholar]
2. Rodgers, R.J. and H.F. Irving-Rodgers, Formation of the ovarian follicular antrum and follicular fluid. Biol Reprod, 2010. 82(6): p. 1021-9.
3. Dumesic, D.A., et al., Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril, 2015. 103(2): p. 303-16.

Follicular fluid is the medium by which signaling mediators are transported in and out of the follicle, as well as within the follicle between various cell types. Given that intrafollicular communication is critical for normal oocyte development and reproduction, much effort has been directed at better understanding intrafollicular signaling. It is evident that there is communication from the mural granulosa cells to the cumulus complex [4–6]; from the cumulus complex to the oocyte; and from the oocyte back to the somatic compartment [7–10]

Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science. 2004;303(5658):682–4. doi: 10.1126/science.1092463. [PubMed] [CrossRef] [Google Scholar]

5. Russell DL, Robker RL. Molecular mechanisms of ovulation: co-ordination through the cumulus complex. Hum Reprod Update. 2007;13(3):289–312. doi: 10.1093/humupd/dml062. [PubMed] [CrossRef] [Google Scholar]
6. Zhang M, Su YQ, Sugiura K, Xia G, Eppig JJ. Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science. 2010;330(6002):366–9. doi: 10.1126/science.1193573. [PMC free article][PubMed] [CrossRef] [Google Scholar]
7. Chang CL, Wang HS, Soong YK, Huang SY, Pai SY, Hsu SY. Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2. J Biol Chem. 2011;286(50):43193–203. doi: 10.1074/jbc.M111.297358. [PMC free article][PubMed] [CrossRef] [Google Scholar]
8. Gilchrist RB, Lane M, Thompson JG. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update. 2008;14(2):159–77. doi: 10.1093/humupd/dmm040. [PubMed] [CrossRef] [Google Scholar]
9. Gui LM, Joyce IM. RNA interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice. Biol Reprod. 2005;72(1):195–9. doi: 10.1095/biolreprod.104.033357. [PubMed] [CrossRef] [Google Scholar]
10. Balasch J, Guimera M, Martinez-Pasarell O, Ros J, Vanrell JA, Jimenez W. Adrenomedullin and vascular endothelial growth factor production by follicular fluid macrophages and granulosa cells. Hum Reprod. 2004;19(4):808–14. doi: 10.1093/humrep/deh204.

The importance of FF

physiologically, FF is important for steroidogenesis, follicular growth, oocyte maturation, the ovulation process, and transportation into the oviduct. This viscous, straw-coloured fluid mainly contains sodium and potassium, which plays an important role for osmotic balance. Apart from that, hormones, interleukins, protein, Glutathione (GSH), reactive oxygen species (ROS), sugar and anti-apoptotic factors are also found in follicular fluid [188]. Anti-oxidant such as GSH is required to ensure the redox balance within the follicular fluid and is fundamental for oocyte growth. However imbalance of oxidant and antioxidant can be pathological and is known to affect meiosis negatively, for instance by causing DNA damage [189-191].

Hormones in FF such as FSH, hCG and LH are essential to promote oocyte maturation by interaction between somatic and endocrine cells [183]. Gonadotropins within FF also play an important role in the secretion of several substances by granulosa cells. Other hormones such as growth hormone, prolactin, oestradiol and progesterone are also found in normal follicular fluid. When compared to women who were unstimulated, FF from those who undergone stimulated cycle has significantly lower level of oestradiol, luteinising hormone and androstendione [196]. These hormones are not routinely measured except for research purposes to find suitable biomarkers for oocyte quality. Since both maturation and ovulation are inflammatory process, pro inflammatory markers such as interleukins and cytokines are commonly found in FF [197]. The majority of proteins in FF were discovered from proteomic analysis done to find markers for oocyte quality assessment [188]. Additionally reactive oxygen species, which have the ability to react with and damage proteins, lipids and nucleic acid, can be found in the FF. It is known that supra physiological ROS has a detrimental effect on oocytes. insulin-like growth factors I and II (IGF-I and -II) are polypeptides that promote cell proliferation and differentiation in several tissues; their biological availability is regulated by a family of IGF-binding proteins (from IGFBP-1 to IGFBP-6). The intrafollicular levels of IGF-II, IGFBP-3 and IGFBP-4 were found to be significantly correlated with oocyte fertilization, cleavage, embryo development and embryo morphological score on day 3 [58]. In the same study, multiple regression analysis showed that the combination of high FF levels of IGFBP-3 and 4 with low FF levels of pregnancy-associated plasma protein-A (PAPP-A) was significantly correlated with fertilization and embryo development. In other studies, FF levels of both IGF-I an d IGFBP-1 were positively correlated with oocyte quality and maturity [28,59,60], and the ratio IGF-1/IGFBP-1 was shown to be significantly higher both in serum and in FF in women whose IVF was successful [50]. In a recent study, however, IGF-I FF levels were not found to reflect embryo quality and IVF outcome [61]. Overall, further studies are needed to estabilish if IGFs and IGF-BPs have the potential to become useful non-invasive biomarkers of oocyte quality in the clinical practice. Pro-inflammatory cytokines can be found in FF as a result of ovarian local synthesis and release during follicular maturation and ovulation; for example, FF IL-1beta derives both from the plasma ultrafiltrate and from the local synthesis by luteinizing granulosa cells. A positive correlation was observed between the serum concentrations of IL-1beta and E2 on the day of hCG injection [63]. Higher FF IL-1beta levels were associated with normal fertilization [64], but surprisingly they were lower in FFs whose oocytes were able to generate better embryos and successful IVF attempts [10]. It is possible that IL-1beta leads to cytoplasmic maturation and normal fertilization, but does not play a role in post-fertilization embryo development. In some studies, IL-2 and IL-10 were found to correlate with specific hormonal milieus within the follicle, but not with IVF outcome [65–67]. Ledee [68] recently detected significantly higher levels of IL-2 and interferon (IFN-gamma) in FFs whose oocytes generated early cleaving embryos. In the same study, IL-12 levels were higher in the FF of follicles corresponding to highly fragmented embryos, and granulocyte colony-stimulating factor (G-CSF) was particularly elevated in the fluid of follicles corresponding to embryos with high implantation potential. Bili [69] studied the FF levels of IL-1alpha, IL-2, TNF-alpha and leukotriene B4 (LTB4) and reported that there was no significant relationship between their individual FF concentration and oocyte maturity, fertilization and likelihood of pregnancy. On the other hand, IL-1alpha/TNF-alpha, IL-1alpha/LTB4, TNF-alpha/LTB4 ratios were significantly different in the FF of the women who became pregnant versus the ones who did not. He suggested that IL-1alpha, TNF-alpha and LTB4 may take part in the process of follicle wall degradation, and their concentration in optimal proportion may reflect a better intrafollicular milieu able to optimize oocyte development and maturation.

188. Revelli, A., et al., Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol, 2009 7: p189. Agarwal, A., S. Gupta, and R.K. Sharma, Role of oxidative stress in female reproduction. Reprod Biol Endocrinol, 2005. 3: p. 28. 190. Jozwik, M., et al., Oxidative stress markers in preovulatory follicular fluid in humans. Mol Hum Reprod, 1999. 5(5): p. 409-13.
189. de Bruin, J.P., et al., Ultrastructure of the resting ovarian follicle pool in healthy young women. Biol Reprod, 2002. 66(4): p. 1151-60.

de los Santos, M.J., et al., Hormonal and molecular characterization of follicular fluid, cumulus cells and oocytes from pre-ovulatory follicles in stimulated and unstimulated cycles. Hum Reprod, 2012. 27(6): p. 1596-605.

197. Choi, Y.S., et al., Alteration in the intrafollicular thiol-redox system in infertile women with endometriosis. Reproduction, 2015. 149(2): p. 155-62.
198. Singh, A.K., et al., Markers of oxidative stress in follicular fluid of women with endometriosis and tubal infertility undergoing IVF. Reprod Toxicol, 2013. 42: p. 1
199. Revelli, A., et al., Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol, 2009. 7: p. 40.

• Wang TH, Chang CL, Wu HM, Chiu YM, Chen CK, Wang HS. Insulin-like growth factor-II (IGF-II), IGF-binding protein-3 (IGFBP-3), and IGFBP-4 in follicular fluid are associated with oocyte maturation and embryo development. Fertil Steril. 2006;86:1392–1401. doi: 10.1016/j.fertnstert.2006.03.064. [PubMed] [CrossRef] [Google Scholar]
• Artini PG, Battaglia C, D’Ambrogio G, Barreca A, Droghini F, Volpe A, Genazzani AR. Relationship between human oocyte maturity, fertilization and follicular fluid growth factors. Hum Reprod. 1994;9:902–906

50 Fried G, Remaeus K, Harlin J, Krog E, Csemiczky G, Aanesen A, Tally M. Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization. J Assist Reprod Genet. 2003;20:167–176. doi: 10.1023/A:1023656225053

59 Jimena P, Castilla JA, Peran F, Molina R, Ramirez JP, Acebal M, Vergara F, Herruzo A. Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization. J Reprod Fertil. 1992;96:641–647.

60 Oosterhuis GJ, Vermes I, Lambalk CB, Michgelsen HW, Schoemaker J. Insulin-like growth factor (IGF)-I and IGF binding protein-3 concentrations in fluid from human stimulated follicles. Hum Reprod. 1998;13:285–289. doi: 10.1093/humrep/13.2.285.

61-Asimakopoulos B, Abu-Hassan D, Metzen E, Al-Hasani S, Diedrich K, Nikolettos N. The levels of steroid hormones and cytokines in individual follicles are not associated with the fertilization outcome after intracytoplasmic sperm injection. Fertil Steril. 2008;90:60–64. doi: 10.1016/j.fertnstert.2007.05.054.

62- Wang XF, Xing FQ, Chen SL. Interleukin-1beta expression on ovarian granulosa cells and its clinicalimplication in women undergoing in vitro fertilization. Di Yi Jun Yi Da Xue Xue Bao. 2002;22:934–936.

• Mendoza C, Cremades N, Ruiz-Requena E, Martinez F, Ortega E, Bernabeu S, Tesarik J. Relationship between fertilization results after intracytoplasmatic sperm injection, and intrafollicular steroid, pituitary hormone and cytokine concentrations. Hum Reprod. 1999;93:863–868.
• Barak Y, Amit A, Lessing JB, Paz G, Homonnai ZT, Yogev L. Improved fertilization rate in an in vitro fertilization program by egg yolk-treated sperm. Fertil Steril. 1992;58:197–198.
• Geva E, Lessing JB, Lerner-Geva L, Azem F, Yovel I, Ben-Yosef D, Barkai U, Amit A. Interleukin-10 in preovulatory follicular fluid of patients undergoing in-vitro fertilization and embryo transfer. Am J Reprod Immunol. 1997;37:187–190.
• Cerkiene Z, Eidukaite A, Usoniene A. Follicular fluid levels of interleukin-10 and interferon-gamma do not predict outcome of assisted reproductive technologies. Am J Reprod Immunol. 2008;59:118–126.
• Lédée N, Lombroso R, Lombardelli L, Selva J, Dubanchet S, Chaouat G, Frankenne F, Foidart JM, Maggi E, Romagnani S, Ville Y, Piccinni MP. Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor. Hum Reprod. 2008;23:2001–2009. doi: 10.1093/humrep/den192.
• Bili H, Tarlatzis BC, Daniilidis M, Fleva A, Bontis J, Tourkantonis A, Mantalenakis S. Cytokines in the human ovary: presence in follicular fluid and correlation with leukotriene B4. J Assist Reprod Genet. 1998;15:93–98. doi: 10.1007/BF02766833

10 Mendoza C, Ruiz-Requena E, Ortega E, Cremades N, Martinez F, Bernabeu R, Greco E, Tesarik J. Follicular fluid markers of oocyte developmental potential. Hum Reprod. 2002;17:1017–1022. doi: 10.1093/humrep/17.4.1017.

Endometriosis protmics and different samples

Proteomics is a new and challenging perspective in the field of noninvasive biomarkers for early detection of endometriosis, which includes all the protein “fingerprints” used for endometriosis diagnosis. Despite promising results, these technologies need better standardization and are cost and time-intensive [12].

Blood

The chronic inflammatory nature of endometriosis further challenges the specificity of tests based on mediators of inflammation.

In endometriosis, proteomic analysis has been performed on several tissue types including the serum, plasma, urine, endometrium, peritoneum, and follicular fluid, however most of these biomarker panels have not been verified or clinically validated [62]. Despite that, the authors of several published reviews on proteomic analysis [253, 324] concluded that proteomic analysis might be used in infertile women with or without pelvic pain to predict the presence of the disease [325].

Serum and Blood

there are 14 available publications that have examined serum and plasma using proteomic techniques. Less than half [241, 327-330] of the proteomic analysis used gel-based methods whereas 9 publications [75, 248, 267, 324, 331-333] used protein chip analytic techniques. Faserl et al. 2011 [327], by using 2D-DIGE MALDI-MS/MS, discovered 25 protein spots with significantly (P0.02) higher vitamin-D binding protein in women with compared with those without endometriosis. They also found a higher GC*2 allele product in endometriosis and speculated that this gene was unable to activate the phagocytic function to facilitate the implantation of endometrial tissue within the peritoneal cavity.

By using 2D-MALDI-TOF MS, verified by Western Blot (using different set of serum samples n=60), Gajbhiye et al [328] have identified three endometrial antigens, Topomyosis 3 (TPM3), Stomatin-like protein 2 (SLP2) and Tropmodulin 3 (TMOD 3), and they proposed using these markers for early diagnosis of endometriosis. Using the same technique, Nabeta et al in 2011 [329], found high anti-syntaxin 5 (STX5), which have 53.6% sensitivity and 72.2% specificity for the diagnosis of endometriosis, which was better than using CA-125 alone. However, when STX5 and CA-125 were combined, they reported that the sensitivity was increased to 69.6%. The authors acknowledged that their discoveries require further clinical validation.

Gajbhiye et al. [106] included 40 endometriosis patients in their study and noted higher serum levels of autoantibodies compared to tropomodulin 3 (TMOD3), tropomyosin 3 (TPM3), and stomatin-like protein 2 (SLP2) in contrast to control’s serum samples. Elevated values were associated with both minimal to mild and moderate to severe disease. Additionally, in women with endometriomas, autoantibodies against IGF-2 mRNA-binding protein 1 (IMP1) were identified by Yi et al. to be significantly elevated compared to healthy controls [107].

After employing these methods, we found differential proteins at 4210, 5264, 2660, 5635, 5904 Da to distinguish between patients with EM and healthy controls. These proteins have high diagnostic accuracy, either singly or combined. We also found that the 5635 Da peak is associated with early-stage or minimal EM and could help monitor early disease. LC-MS/MS sequencing showed the 4210 Da peptide to correspond uniquely to ATP1B4, and the 5904 Da protein to FGA isoform 1/2 subunit (Yang Zhao 2015)

Zhang et al 2006 [241], the study that has combined serum and endometrium in their analysis using 2D-MalDI-TOF-MS, have identified 13 protein spots, which were differentially expressed, out of which 11 were known proteins. Some of the proteins were involved in the regulation of cell cycle, signal transduction or immunological function

The most commonly used biomarker for preoperative assessment is CA-125 (CA-125). This is a glycoprotein found within the cells lining the female genital tract and is raised in both epithelial ovarian cancer and other gynaecological diseases (115–117). This was systematically reviewed with a meta-analysis finding insignificant sensitivities and specificities to justify its use as a predictive marker (116) though serum levels appear to rise with increasing disease severity (118). CA-125 along with other glycoproteins has been analysed by research teams in Leuven who have kept a bank of frozen blood samples from patients since 1999. The team were able to demonstrate the accuracy of CA-125 with sensitivity and specificity of 78 and 51% (119).

The rest of serum proteomic studies used gel free mass spectrometry, and these studies reported a variable sensitivity and specificity for endometriosis. For instance, Fassbender et al in 2011 [73] reported a sensitivity of 75% and specificity of 86% for the diagnosis of mild endometriosis using a model based on five peptides and protein peaks (range 4.898-14.698 m/z). However, an earlier study done by Seeber et al 2008 [267], found six differentially expressed proteins and by combining monocyte chemoattractant protein-1, migration inhibitory factor leptin and CA-125, the author reported the diagnostic capability could be up to 73% with a 94% specificity.

Long et al. [113] collected serum samples from several affected women and compared them to controls, in order to detect different protein fingerprints of this disease, by using MALDI-TOF–MS. Their results indicated that 13 protein peaks were over-expressed, and five protein peaks were down-regulated in the affected group compared to healthy subjects.

62 May, K.E., et al., Peripheral biomarkers of endometriosis: a systematic review. Hum Reprod Update, 2010. 16(6): p. 651-74.

253. Fassbender, A., et al., Biomarkers of endometriosis. Fertil Steril, 2013. 99(4): p. 1135- 45.
254. Fassbender, A., et al., Proteomics analysis of plasma for early diagnosis of endometriosis. Obstet Gynecol, 2012. 119(2 Pt 1): p. 276-85
255. Siva, A.B., P. Srivastava, and S. Shivaji, Understanding the pathogenesis of endometriosis through proteomics: recent advances and future prospects. Proteomics Clin Appl, 2014. 8(1-2): p. 86-98.
256. Thambisetty, M., et al., Plasma biomarkers of brain atrophy in Alzheimer’s disease. PLoS One, 2011. 6(12): p. e28527.
257. Zhang, H., et al., Use of proteomic analysis of endometriosis to identify different protein expression in patients with endometriosis versus normal controls. Fertil Steril, 2006. 86(2): p. 274-82.
258. Faserl, K., et al., Polymorphism in vitamin D-binding protein as a genetic risk factor in the pathogenesis of endometriosis. J Clin Endocrinol Metab, 2011. 96(1): p. E233-41.
259. Gajbhiye, R., et al., Identification and validation of novel serum markers for early diagnosis of endometriosis. Hum Reprod, 2012. 27(2): p. 408-17.
260. Fassbender, A., et al., TRIzol treatment of secretory phase endometrium allows combined proteomic and mRNA microarray analysis of the same sample in women with and without endometriosis. Reprod Biol Endocrinol, 2010. 8: p. 123.
261. Seeber, B., et al., Panel of markers can accurately predict endometriosis in a subset of patients. Fertil Steril, 2008. 89(5): p. 1073-81.
262. Fassbender, A., et al., Proteomics analysis of plasma for early diagnosis of endometriosis. Obstet Gynecol, 2012. 119(2 Pt 1): p. 276-85
263. Liu, H., et al., Detection of endometriosis with the use of plasma protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Fertil Steril, 2007. 87(4): p. 988-90.
264. Jing, J., et al., Two novel serum biomarkers for endometriosis screened by surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry and their change after laparoscopic removal of endometriosis. Fertil Steril, 2009. 92(4): p. 1221- 7. 333. Wolfler, M.M., et al., Mass spectrometry and serum pattern profiling for analyzing the individual risk for endometriosis: promising insights? Fertil Steril, 2009. 91(6): p. 2331- 7.
265. Zheng, N., C. Pan, and W. Liu, New serum biomarkers for detection of endometriosis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Int Med Res, 2011. 39(4): p. 1184-92.
266. Gajbhiye R., Sonawani A., Khan S., Suryawanshi A., Kadam S., Warty N., Raut V., Khole V. Identification and validation of novel serum markers for early diagnosis of endometriosis. Hum. Reprod. 2012;27:408–417. doi: 10.1093/humrep/der410.
267. Yi Y.-C., Wang S.-C., Chao C.-C., Su C.-L., Lee Y.-L., Chen L.-Y. Evaluation of serum autoantibody levels in the diagnosis of ovarian endometrioma. J. Clin. Lab Anal. 2010;24:357–362. doi: 10.1002/jcla.20415.

Zhao, Y., Liu, Y.N., Li, Y., Tian, L., Ye, X., Cui, H. and Chang, X.H., 2015. Identification of biomarkers for endometriosis using clinical proteomics. Chinese medical journal, 128(04), pp.520-527.

241. Zhang, H., et al., Use of proteomic analysis of endometriosis to identify different protein expression in patients with endometriosis versus normal controls. Fertil Steril, 2006. 86(2): p. 274-82.
242. Omland, A.K., et al., Pregnancy outcome after IVF and ICSI in unexplained, endometriosis-associated and tubal factor infertility. Human Reproduction, 2005. 20(3): p. 722-7. 116. Omland, A.K., et al., Intracytoplasmic sperm injection (ICSI) in unexplained and stage I endometriosis-associated infertility after fertilization failure with in vitro fertilization (IVF). Journal of Assisted Reproduction & Genetics, 2006. 23(7-8): p. 351-7. 117. Omland, A.K., et al., Natural cycle IVF in unexplained, endometriosis-associated and tubal factor infertility. Human Reproduction, 2001. 16(12): p. 2587-92.
243. Kuroda, K., The impact of endometriosis, endometrioma and ovarian cystectomy on assisted reproductive technology. Reprod Med Online, 2009. 8: p. 113-118
244. Olivennes, F., et al., Endometriosis: a stage by stage analysis–the role of in vitro fertilization. Fertil Steril, 1995. 64(2): p. 392-8.
245. Fassbender, A., et al., TRIzol treatment of secretory phase endometrium allows combined proteomic and mRNA microarray analysis of the same sample in women with and without endometriosis. Reprod Biol Endocrinol, 2011. 9(1): p. 44

A further study analyzed distinct patterns of serum proteins in 90 endometriotic women, using SELDI-TOF MS. Following surgical intervention, 51 out of 90 patients were diagnosed with endometriosis, while 39 were unaffected. The researchers concluded that a unique combination of proteins, with molecular weights ranging between 2000 and 20,000 Da made a difference between affected women and controls. The sensitivity of this technique was 81.3%, with a specificity of 60.3% [114]. According to Zheng et al. [115], a proteomic fingerprint model including three peptide peaks showed a sensitivity and specificity of 91.4% and 95%, respectively, for the detection of endometriosis, when compared to controls. In an independent cohort, this combination of peptides revealed a sensitivity of 89.3% and a specificity of 90%.

Wang and collab. [116] illustrated a panel of five protein peaks with a specificity of 90% and a sensitivity of 91.7% for endometriosis, and Jing et al. [117] highlighted two protein peaks with specificity and sensitivity of 97% and 87%, respectively.

Given the inflammatory nature of the disease, inflammation-related proteins have also been investigated for their utility as noninvasive biomarkers. Plasma levels of AXIN1 and ST1A1 were analyzed using ELISA in both affected subjects and healthy controls. AXIN1 and ST1A1 had higher values in endometriosis when compared to healthy controls, regardless of the anatomical location of the lesions [118]. AXIN1 is a promising protein that should be further investigated as a biomarker for endometriosis diagnosis.

Signorile and coworkers [119] used 2D gel analysis in order to describe the potential of two proteins (serum albumin and complement C3 precursor) as diagnostic markers for endometriosis. Their technique was easily reproducible, and their results indicated a sensitivity/specificity for albumin and the complement C3 of 83.3%/83.3% and 58.1%/100%, respectively. In conclusion, this study confirmed the statistical significance of the differential expression for these two proteins in endometriotic women with respect to unaffected individuals. The same authors conducted, in the year 2014, a study comprising 120 women with endometriosis and 20 healthy controls, in order to highlight their serum levels of Zn-alpha2-glycoprotein. After performing ELISA, they observed that the serum levels of this protein were significantly increased in the endometriosis group than in healthy women [120]. In the issue of this observation, the analysis of Zn-alpha2-glycoprotein levels in the serum could become an innovative noninvasive diagnostic test for endometriosis.

115. Zheng N., Pan C., Liu W. New serum biomarkers for detection of endometriosis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Int. Med. Res. 2011;39:1184–1192. doi: 10.1177/147323001103900406.
116. Wang L., Zheng W., Mu L., Zhang S.-Z. Identifying biomarkers of endometriosis using serum protein fingerprinting and artificial neural networks. Int. J. Gynaecol. Obstet. 2008;101:253–258. doi: 10.1016/j.ijgo.2008.01.018.
117. Jing J., Qiao Y., Suginami H., Taniguchi F., Shi H., Wang S. Two novel serum biomarkers for endometriosis screened by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and their change after laparoscopic removal of endometriosis. Fertil. Steril. 2008;92:1221–1227. doi: 10.1016/j.fertnstert.2008.08.078.
118. Malin E., Bodil R., Gunnar E., Bodil O. AXIN1 in Plasma or Serum Is a Potential New Biomarker for Endometriosis. Int. J. Mol. Sci. 2019;20:189. doi: 10.3390/ijms20010189.
119. Signorile P.G., Baldi A. Supporting evidences for potential biomarkers of endometriosis detected in peripheral blood. Data Brief. 2015;5:971–974. doi: 10.1016/j.dib.2015.10.047.
120. Signorile P.G., Baldi A. Serum Biomarker of Endometriosis. J. Cell Physiol. 2014;229:1731–1735. doi: 10.1002/jcp.24620.

Biomarkers

The increased incidence of this pathology in women with early menarche compels the development of novel noninvasive diagnostic biomarkers for faster diagnosis, appropriate treatment, and for triaging potential patients for surgery [9,10]. A biomarker is a biological molecule that can be “objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention” [11]. Therefore, a biomarker or a panel of biomarkers found in the biological fluids of the affected women could be an expedient diagnostic tool for endometriosis as well as an objective assessment of the effectiveness of the treatment [12].

Recently, a wide range of papers revealed the importance and effectiveness of various putative biomarkers from the biological fluids of affected patients. Despite considerable research on this topic, noninvasive biomarkers of endometriosis have eluded the transition from bench to bedside. Some of the limitations of biomarker studies consist of reduced datasets, methodological flaws (the variability of biomarkers under physiological conditions such as menstrual phases), the lack of reproducibility across multiple studies, and, last but not least, high costs of the complex assays.

• Ahn, S.H.; Singh, V.; Tayade, C. Biomarkers in endometriosis: Challenges and opportunities.  Steril.2017, 107, 523–532.
• Irungu, S.; Mavrelos, D.; Worthington, J.; Blyuss, O.; Saridogan, E.; Timms, J.F. Discovery of non-invasive biomarkers for the diagnosis of endometriosis.  Proteom.2019, 16, 14.
• Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework.  Pharmacol. Ther.2001, 69, 89–95
• May, K.E.; Conduit-Hulbert, S.A.; Villar, J.; Kirtley, S.; Kennedy, S.H.; Becker, C.M. Peripheral biomarkers of endometriosis: A systematic review.  Reprod. Update2010, 16, 651–674.

Endometrial tissue

The hormonal variation in ovulatory women throughout their menstrual cycle results in endometrial molecular signature change depending on the stage in the cycle. This presents a significant challenge with regard to endometrial-based biomarker development. Although a cycle phase specific test may be acceptable to optimise sensitivities and specificities, this may not be practical with women having irregular menstrual cycles. This is particularly relevant in studies analysing eutopic mRNA expression (142). Recent studies have found that aberrant neuronal growth may contribute to abnormal fertility and uterine disorders including endometriosis

The hypothesis that increased 53. neuronal innervation to endometrial cells (eutopic and ectopic) could be reflected in an endometrial biopsy to detect a neuronal protein called protein gene product 9.5. The association between protein gene product 9.5 in the functional layer of the endometrium and the presence of endometriosis in the pelvis has, like many markers, shown promise (142–148). This C-terminal hydrolase dissociates ubiquitin peptide bonds and thus regulates proteolysis (149). The use of this semi-invasive biomarker has sensitivities ranging from 80 to 81% with specificity 92–100% and did not appear to vary by phase of the menstrual cycle (148,150).

To date, 19 publications have analysed eutopic endometrium of women with endometriosis for proteomic research. More than half of the studies (n=9) used gel base techniques whereas the rest used the protein chip technique (n=6). One study combined gel and liquid chromatography techniques (Zhang 2010). Only one study used isobaric labelling for the analysis [245]. The rest of the studies used liquid chromatography with tandem mass spectrometry. All studies, except Xue Y. 2018 [241] had at least one control group for comparison. The majority of the studies have performed immunohistochemistry Xue Y. 2018 [72, 243, 250] and/or Western Blotting [243, 250, 338]. None of the study used ELISA for verification/validation and none has used more than 1000 samples, which is the suggested benchmark for biomarker validation [318

The studies using gel-based proteomic studies are usually combined with MALDI-TOF-MS [242, 243, 247, 250] or DIGE-TOF-MS [72, 241]. By using the gel-based technique, protein were first identified and then isolated before being quantified using mass spectrometry.

Stephens et al in 2009 [72] also discovered vimentin was differentially expressed, alongside with peroxiredoxin 6, and ribonuclease/ angiogenin inhibitor 1 (RNH1). However these two studies with similar methodology did not reach consensus on a large body of proteins discovered. Apart from that, four studies [242, 243, 247, 250] used 2D MALDI-TOF-MS technique. Rai et al in 2010 [243] analysed the eutopic endometrium according to the different stages of endometriosis. They identified upregulated proteins (MVP, HSP90-beta, GRP78, HSP70, HSP60, HSP27, and DJ-1) and downregulated protein (ERp57), which later were verified by Western blotting and immunohistochemistry. An earlier study by Fowler et al. 2007 [247] identified dysregulated proteins in women with endometriosis which involved molecular chaperones (Heat Shock Protein 90 and Annexin A2), proteins involved in cellular redox state (peroxiredoxin 2), proteins involved in protein and DNA formation/breakdown (Ribonucleoside-diphosphate Reductase, Prohibitin and Prolyl 4-hydroxylase), and secreted proteins (Apolipoprotein A1). Chehna-Patel et al. 2010 [250] discovered 11 spots, out of which 4 (Haptoglobin, Rho-GDIa, SM-22a, and Rab37) have been verified (n=6) by both immunohistochemistry and western blotting. Ten Have and colleagues [242] discovered 21 up-regulated proteins in the endometriosis and pathway analysis that showed the proteins that were involved in apoptosis, immune reaction, glycolytic pathway, cell structure and transcription factors. The most recent publication [245], which is also the only study that used isobaric labelling technique, included the combination of the eutopic endometrium and the endometrioma cyst wall in their analysis.

This study interestingly found that Vimentin was upregulated, which is in keeping with one of the previous publications [72, 241]. In the proteomic analysis of eutopic endometrium, a gel-free technique with protein chip has been used in Surface Enhance Laser Desorption IonisationTime of Flight (SELDI-TOF). This technique provides distinctive proteomic profiles in the form of mass/charge (m/s). Published studies on proteomic analysis of endometrial tissue using SELDI-TOF [73-75, 246, 248, 339] have started as early as 2009 but now have been superseded by newer techniques. The number of samples used in the studies utilising this technique varies from 9 to 53 and some studies did not indicate the respective menstrual cycle phases where the samples were collected from [73, 74, 246, 248]. Multiple surfaces (see Separation and fractionation of complex protein mixture, of the protein chip [73, 74, 246, 248] have been used in this technique to maximise protein detection. None of the studies using this technique has been verified or validated.

142. Aghajanova L, Giudice LC. Molecular evidence for differences in endometrium in

severe versus mild endometriosis. Reprod Sci. SAGE Publications; 2011

Mar;18(3):229–51.

143. Tokushige N, Markham R, Russell P, Fraser IS. High density of small nerve fibres

in the functional layer of the endometrium in women with endometriosis. Hum

Reprod. 2006 Mar;21(3):782–7.

144. Tokushige N, Markham R, Russell P, Fraser IS. Nerve fibres in peritoneal

endometriosis. Hum Reprod. Oxford University Press; 2006 Nov;21(11):3001–7.

145. Tokushige N, Markham R, Russell P, Fraser IS. Different types of small nerve

fibers in eutopic endometrium and myometrium in women with endometriosis.

Fertil Steril. 2007;88:795–803.

146. Al-Jefout M, Andreadis N, Tokushige N, Markham R, Fraser I, Tokushige N, et al.

A pilot study to evaluate the relative efficacy of endometrial biopsy and full

curettage in making a diagnosis of endometriosis by the detection of endometrial

nerve fibers. Am J Obstet Gynecol. Elsevier; 2007 Dec;197(6):578.e1-578.e4.

147. Al-Jefout M, Dezarnaulds G, Cooper M, Tokushige N, Luscombe GM, Markham

R, et al. Diagnosis of endometriosis by detection of nerve fibres in an endometrial

biopsy: a double blind study. Hum Reprod. 2009 Dec 1;24(12):3019–24.

148. Elgafor el Sharkwy IA. Combination of non-invasive and semi-invasive tests for

diagnosis of minimal to mild endometriosis. Arch Gynecol Obstet. Springer Berlin

Heidelberg; 2013 Oct 3;288(4):793–7.

149. Boudreaux DA, Chaney J, Maiti TK, Das C. Contribution of active site glutamine to

rate enhancement in ubiquitin C-terminal hydrolases. FEBS J. Blackwell

Publishing Ltd; 2012 Mar;279(6):1106–18.

150. Zevallos HB-V, McKinnon B, Tokushige N, Mueller MD, Fraser IS, Bersinger NA.

Detection of the pan neuronal marker PGP9.5 by immuno-histochemistry and quantitative PCR in eutopic endometrium from women with and without endometriosis. Arch Gynecol Obstet. 2015 Jan 22;291(1):85–91.

245. Marianowski, P., et al., Proteomic analysis of eutopic and ectopic endometriotic tissues based on isobaric peptide tags for relative and absolute quantification (iTRAQ) method. Neuro Endocrinol Lett, 2013. 34(7): p. 717-21
246. Zhang, H., et al., Use of proteomic analysis of endometriosis to identify different protein expression in patients with endometriosis versus normal controls. Fertil Steril, 2006. 86(2): p. 274-82.

Xue, Y., Xu, P., Xu, S., Xue, K., Xu, L., Chen, J., Xu, J., Shi, X., Li, Q. and Gu, L., 2018. Peptidomic analysis of endometrial tissue from patients with ovarian endometriosis. Cellular Physiology and Biochemistry, 47(1), pp.107-118.

243. Rai, P., et al., Differential proteome profiling of eutopic endometrium from women with endometriosis to understand etiology of endometriosis. J Proteome Res, 2010. 9(9): p. 4407-19.
244. Chehna-Patel, N., et al., “Spot”-ting differences between the ectopic and eutopic endometrium of endometriosis patients. Fertil Steril, 2010. 94(6): p. 1964-71, 1971 e1.
245. Zhang, Y., et al., Protein analysis by shotgun/bottom-up proteomics. Chem Rev, 2013. 113(4): p. 2343-94
246. Zhang, H., et al., 17betaE2 promotes cell proliferation in endometriosis by decreasing PTEN via NFkappaB-dependent pathway. Mol Cell Endocrinol, 2010. 317(1-2): p. 31-43.
247. Zhang, H., et al., Use of proteomic analysis of endometriosis to identify different protein expression in patients with endometriosis versus normal controls. Fertil Steril, 2006. 86(2): p. 274-82. 242. Ten Have, S., et al., Proteomic analysis of protein expression in the eutopic endometrium of women with endometriosis. Proteomics Clin Appl, 2007. 1(10): p. 1243-51. 243. Rai, P., et al., Differential proteome profiling of eutopic endometrium from women with endometriosis to understand etiology of endometriosis. J Proteome Res, 2010. 9(9): p. 4407-19
248. Marianowski, P., et al., Proteomic analysis of eutopic and ectopic endometriotic tissues based on isobaric peptide tags for relative and absolute quantification (iTRAQ) method. Neuro Endocrinol Lett, 2013. 34(7): p. 717-21.
249. Kyama, C.M., et al., ProteinChip technology is a useful method in the pathogenesis and diagnosis of endometriosis: a preliminary study. Fertil Steril, 2006. 86(1): p. 203-9.
250. Fowler, P.A., et al., An investigation of the effects of endometriosis on the proteome of human eutopic endometrium: a heterogeneous tissue with a complex disease. Proteomics, 2007. 7(1): p. 130-42.
251. Fassbender, A., et al., TRIzol treatment of secretory phase endometrium allows combined proteomic and mRNA microarray analysis of the same sample in women with and without endometriosis. Reprod Biol Endocrinol, 2010. 8: p. 123
252. Chehna-Patel, N., et al., “Spot”-ting differences between the ectopic and eutopic endometrium of endometriosis patients. Fertil Steril, 2010. 94(6): p. 1964-71, 1971 e1.
253. Stephens, A.N., et al., Post-translational modifications and protein-specific isoforms in endometriosis revealed by 2D DIGE. J Proteome Res, 2010. 9(5): p. 2438-49.
254. Fassbender, A., et al., TRIzol treatment of secretory phase endometrium allows combined proteomic and mRNA microarray analysis of the same sample in women with and without endometriosis. Reprod Biol Endocrinol, 2011. 9(1): p. 44.
255. Kyama, C.M., et al., Evaluation of endometrial biomarkers for semi-invasive diagnosis of endometriosis. Fertil Steril, 2011. 95(4): p. 1338-43 e1-3.
256. Wang, L., et al., Identification biomarkers of eutopic endometrium in endometriosis using artificial neural networks and protein fingerprinting. Fertil Steril, 2010. 93(7): p. 2460-2
257. Ding, X., et al., Differences in mitochondrial proteins in the eutopic endometrium of patients with adenomyosis and endometriosis identified using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. J Int Med Res, 2010. 38(3): p. 987-93

Follicular fluid of women with endometriosis

Whilst the majority of publications have examined serum, peritoneal fluid and endometrium of women with endometriosis, limited studies have analysed FF and its effect on the oocyte maturation process. Available studies on FF however, were using samples collected during a stimulated cycle of IVF treatment. This may not be representative, and cannot be generalised to unstimulated women.

Overall, a clear correspondence between specific FF biochemical characteristics and measurable oocyte quality-linked, embryo-related variables has not been established to date. In the last years, the research in this area has progressed toward a more complex type of molecular analysis, metabolomics, that is the analysis of all substances contained in a biological fluid.

INFLAMMATION

Inflammation is believed to play a fundamental role in the development and progression of endometriosis(44,45). Endometriotic tissue, unlike endometrial tissue, is associated with the overproduction of inflammatory markers; prostaglandins, metalloproteinases, cytokines, and chemokines (46–49). Overexpression of prostaglandin E2 in endometriotic tissue is sustained by overexpression of cyclooxygenase (COX) 2 and CYP19A1. Prostaglandin E2 released following the inflammatory response stimulates the expression of all steroidogenic genes necessary to enable the endometriotic stromal cell to synthesise estradiol from cholesterol (50). Oestrogen enhances the survival or persistence of endometriotic tissue, prostaglandins and cytokines mediate pain, inflammation, and infertility (51,52). Pro-inflammatory cytokines such as tumour necrosis factor α (TNFα) and IL-1b initiate the development and progression of endometriosis via: pleiotropic, cytostatic, chemoattractant, or angiogenic effects (53). The presence of peritoneal TNFα and serum levels of Inter-Leukin-1b have been associated with endometriosis suffers (54), dysmenorrhoea (55), and severe endometriosis (45). The inflammatory process of oxidative stress occurs when there is an imbalance between reactive oxygen species (ROS) production and the antioxidant defence (56). Oxidative stress has been associated with several chronic inflammatory diseases including endometriosis (44). As a result, ROS promote the growth and adhesion of 40. endometrial cells within the peritoneal cavity, leading to disease establishment and symptoms of pain and infertility (57,58)

44. Santulli P, Chouzenoux S, Fiorese M, Marcellin L, Lemarechal H, Millischer A-E, 422. et al. Protein oxidative stress markers in peritoneal fluids of women with deep infiltrating endometriosis are increased. Hum Reprod. 2015 Jan 1;30(1):49–60.
45. Lambert S, Santulli P, Chouzenoux S, Marcellin L, Borghese B, de Ziegler D, et al. Endometriosis: increasing concentrations of serum interleukin-1β and interleukin1sRII is associated with the deep form of this pathology. J Gynecol Obstet Biol Reprod (Paris). 2014 Nov;43(9):735–43.
46. Wu Y, Kajdacsy-Balla A, Strawn E, Basir Z, Halverson G, Jailwala P, et al. Transcriptional characterizations of differences between eutopic and ectopic endometrium. Endocrinology. 2006 Jan;147(1):232–46.
47. Sharpe-Timms KL, Cox KE. Paracrine regulation of matrix metalloproteinase expression in endometriosis. Ann N Y Acad Sci. 2002 Mar;955:147-56; discussion 157-8, 396–406.
48. Noble LS, Simpson ER, Johns A, Bulun SE. Aromatase expression in endometriosis. J Clin Endocrinol Metab. 1996 Jan;81(1):174–9
49. Tseng JF, Ryan IP, Milam TD, Murai JT, Schriock ED, Landers D V, et al. Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab. 1996 Mar;81(3):1118–22.
50. Bulun SE, Lin Z, Imir G, Amin S, Demura M, Yilmaz B, et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol Rev. 2005 Sep;57(3):359–83.
51. Bruner KL, Matrisian LM, Rodgers WH, Gorstein F, Osteen KG. Suppression of matrix metalloproteinases inhibits establishment of ectopic lesions by human endometrium in nude mice. J Clin Invest. 1997 Jun 15;99(12):2851–7. 423.
52. Ryan IP, Taylor RN. Endometriosis and infertility: new concepts. Obstet Gynecol Surv. 1997 Jun;52(6):365–71.
53. Reis FM, Petraglia F, Taylor RN. Endometriosis: hormone regulation and clinical consequences of chemotaxis and apoptosis. Hum Reprod Update. 2013 Jul 1;19(4):406–18.
54. Eisermann J, Gast MJ, Pineda J, Odem RR, Collins JL. Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil Steril. 1988 Oct;50(4):573–9.
55. Scholl B, Bersinger NA, Kuhn A, Mueller MD. Correlation between symptoms of pain and peritoneal fluid inflammatory cytokine concentrations in endometriosis. Gynecol Endocrinol. 2009 Nov 23;25(11):701–6.
56. Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005 Jul 14;3:28. 57. Jackson LW, Schisterman EF, Dey-Rao R, Browne R, Armstrong D. Oxidative stress and endometriosis.
57. Carvalho LFP, Samadder AN, Agarwal A, Fernandes LFC, Abrão MS. Oxidative stress biomarkers in patients with endometriosis: systematic review. Arch Gynecol Obstet. 2012 Oct 12;286(4):1033–40

follicular fluid