Understanding endometriosis: a threat to fertility

Endometriosis is a gynaecological disease that affects female reproductive organs by the growth of endo - metrium-like tissues inside and outside the pelvic cavity. This paper explored how endometriosis can lead to increased chances of infertility through a dysregulated immune system and impaired oocyte quality. Running title: Understanding endometriosis

endometriosis, inflammatory cascade, interleukin, oocyte, granulosa cells, cytotrophoblasts Jeya Verschuren1 Verschuren et al. Medical Journal of Cell Biology 2024 DOI: 10.2478/acb-2024-0004 Received: 26.02.2024 Accepted: 29.03.2024 1STN (Student Scientific Society) Anatomia-Klinika-Nauka, Division of Anatomy, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland *Correspondence: [email protected] Full list of author information is available at the end of article Verschuren et al. Medical Journal of Cell Biology (2024) 31

Endometriosis is a common chronic gynecologi - cal disease that affects approximately 1 in 10 wo - men [1]. It is characterised by the growth of endo - metrium-like tissues inside and outside the pelvic cavity, mainly affecting female reproductive organs such as the uterus and ovaries. It can cause symp - toms such as pelvic pain and heavy menstrual ble - eding. Since these symptoms are non-specific and often associated with a typical menstrual cycle, this condition is often misdiagnosed or not diagnosed at all. In fact, in patients between 18 and 45 years of age, the average delay of diagnosis is 6.7 years [2]. According to the WHO, endometriosis affects aro - und 10% (190 million) of reproductive age women and girls globally and specifically 14 million women affected across the EU [3]. In Asian countries, preva - lence is high and affects approximately 6.8% to 15.7% [4]. If endometriosis is left untreated, symp - toms will worsen and can lead to infertility. Studies have shown that 30 to 50% of women affected with this disorder are infertile and endometriosis acco - unts for up to 25% of general infertility cases [5]. This increase chance in infertility can be connected to how endometriosis affects the patients’ immune system through generation of reactive oxygen spe - cies and recruitment of immunocompetent cells, as well as how it impairs oocyte quality by inhibiting follicular granulosa cells. Immune system dysregulation In patients with endometriosis, increased per - centages of immunocompetent cells such as macro - phages and neutrophils are observed. This points to the involvement of the immune system in endo - metrial lesions. In the pelvic cavity of those affected with endometriosis, macrophages are accumulated and can lead to the dysfunction of the immune re - sponse [6]. When macrophages are activated, they recruit neutrophils and stimulate an inflammatory response through the release of cytokines which promote the angiogenesis of endometrial cells. Such cytokines include IL-1, IL-6, and IL-8. IL-1 IL-1 is an interleukin typically responsible for regulation of the inflammatory cascade. Through binding to receptors on target cells, they can illicit inflammatory responses by recruiting immune cells such as macrophages and neutrophils [7]. However, when there is over-secretion such as in endometrio - sis, IL-1 can cause tissue damage. IL-1β is a particular subtype of this cytokine involved in pathogenesis by forming new blood vessels in “tissues surrounding endometriotic lesions by producing vascular endo - thelial growth factor (VEGF)” [8]. In physiology, IL-1 Ra (receptor antagonist) binds to IL-1 receptors and acts against these cytokines, diminishing their amounts before inflammation occurs [9]. However, in patients with endometriosis, decreased concen - tration of IL-1 Ra is apparent and has even been shown to cause dysmenorrhea [10]. IL-6 Similar to IL-1, secretion of IL-6 is also intensi - fied in the immune dysfunction found in endome - trial pathology. IL-6 helps to develop and sustain endometrial foci and implants by up-regulating secretion of a glycoprotein similar to haptoglo - bin called endometriosis protein-I (ENDO-1) [11]. Similar to how haptoglobin binds to free haemo - globin in the blood, ENDO-1 adheres itself to ma - crophages and creates a feed-forward loop that protects the endometrial implants by blocking the phagocytic function of macrophages [8]. This dimi - nished phagocytosis allows for the survival of these implants as well as an increase in secretion of IL-6, which will thereby increase ENDO-1 production, re - sulting in the feed-forward loop. IL-8 IL-8 is a “chemotactic factor” which recruits im - mune cells such as neutrophils and macrophages to the site of inflammation [8]. It is found that when IL-8 concentrations are increased, there is a high proliferation of “ovarian endometrioma-derived stromal cells” which can be linked to the growth of these cells in the peritoneum [12]. It has been speculated that increased concentrations of these immune cells in the peritoneal fluid are “toxic to embryo survival and sperm function” due to the “alterations of decidual microenvironment” [12]. Specifically, through the interaction between ma - crophages and cytotrophoblasts. Cytotrophoblasts are integral cells making up the outer layer of a bla - stocyst (the early stage of an embryo), providing nutrients to the growing embryo and later develo - ping into the placenta [13]. Activated macrophages in patients with endometriosis disrupt these impor - tant cells by inhibiting their invasiveness, thereby being detrimental to embryo growth [14]. ROS and OS Another effect of an unregulated immune sys - tem is the overproduction of reactive oxygen spe - cies (ROS) through stimulation by cytokines and macrophages during inflammation. Although these species are present in regular physiological states and are even associated with supporting female fer - tility processes such as “folliculogenesis (the deve - lopment of a follicle needed for release of a mature oocyte), oocyte maturation, and hormone signal - ling” [12], they must be held in a careful balance with antioxidants or else oxidative stress will occur. When this balance is lost, oxidative stress (OS) can lead to infertility through damaging oocyte DNA and driving post-ovulatory oocytes to apop - tosis [12]. This pathomechanism is driven by the Verschuren et al. Medical Journal of Cell Biology (2024) 32 reduction of glutathione levels by OS. Glutathione is a key antioxidant regulating apoptosis, therefore its decrease leads to morphological changes in the cell and eventually cell death [15]. Oxidative spe - cies also cause oocyte ageing and cell cycle arrest in follicular oocytes, thus ultimately hindering pre - gnancy. It is clear that in patients with endometrio - sis, the dysregulation of the immune system and the consequent ROS production is not only involved in the pathogenesis of the condition, but can lead to infertility as well. Oocyte impairments Oocyte quality is one of the most important fac - tors in fertility and can be characterised by an oocy - te’s ability to mature and be fertilised [16]. Oocytes are immature eggs or ovum found in the ovaries that mature within a follicle and have not been fertilised. If there is damage to an oocyte’s quality, embryo development may be impaired. In order to describe how endometriosis affects the abilities of an oocyte, we must consider a main factor affecting oocyte growth: granulosa cells. When primordial germ cells become oogonia and later proliferate into oocytes, the oocytes are encapsulated by a lay - er of granulosa cells. Granulosa cells Granulosa cells are somatic cells in the ovaries that surround an oocyte in a follicle [17]. These gra - nulosa cells communicate directly with oocytes in primordial follicles but when the oocytes start to grow, they are separated from the granulosa cells by the zona pellucida. The zona pellucida is a coat of glycoproteinaceous matrix that surrounds the oocyte and is responsible for the “binding of sperm to un - fertilised eggs” [18]. Since it completely encapsulates the oocyte, this disrupts the communication between the granulosa cells and the oocyte. Therefore, hete - rologous gap junctions begin to form on cytoplasmic projections called transzonal projections to restore communication [19,20]. Transzonal projections are follicular cell processes branching from granulosa cells that penetrate the zona pellucida, reaching the oocyte. The connection between the oocyte mem - brane and these projections is called a gap junction [21]. Through these junctions, granulosa cells pro - vide growing oocytes with nutrients like amino acids and glucose substrates, as well as paracrine signals such as cAMP and cGMP to regulate oocyte proliferation by maintaining oocyte meiotic arrest. Given this, it is clear that functional granulosa cells are crucial in healthy oocyte quality. Endometriosis impairs this quality not by attacking the oocytes directly, but instead by modifying and destroying the granulosa cells. As mentioned earlier, there are high levels of ROS in the cellular environment of patients with endometriosis. This stress affects the endoplasmic reticulum (ER), an organelle within the cell that is responsible for synthesising and folding proteins. The oxidative stress stimulates ER stress due to the increase demand for protein folding. When this occurs and there is an accumulation of unfolded or misfolded proteins in the ER, signal transduc - tion cascades called the unfolded protein response (UPR) are activated to cope with this increase [22]. In physiological conditions, the UPR is a mechanism used to restore homeostasis by “eliminating slow - -folding proteins” , allowing the ER to maintain its folding capacity [23]. However, if ER stress persists and is severe, the UPR ’s adaptive measures will no longer be sufficient and thus terminal UPR is activa - ted [24]. Terminal UPR is designed to remove stres - sed cells through signalling cell suicide and apopto - sis. Specifically, by activating protein kinase R-like endoplasmic reticulum kinase, also known as PERK. PERK is a vital protein in terminal UPR because it can induce the “expression of proapoptotic tran - scription factors” such as activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) [25]. ATF4 and CHOP These factors will drive the apoptosis of cells, including the granulosa cells needed for oocyte de - velopment. With granulosa cells being destroyed, oocytes are no longer getting the adequate amounts of nutrients needed for healthy growth. It has also been found that oxidative stress can cause damage to mitochondria in granulosa cells which can lead to the dyssynchronisation of nuclear and cytopla - smic maturation [26]. This impacts fertility because it may result in embryo development failure. Given the importance of oocyte quality in fertility and gra - nulosa cells vital role in maintaining it, it is evident that the ER stress caused by endometriosis negati - vely impacts fertility.

It is clear that there is a multitude of factors caused by endometriosis that can lead to infertili - ty. The primary focus of treatment research should be on eliminating these consequences before it can permanently negatively impact fertility. Drugs that have anti-inflammatory effects or specific inhibitors of driving interleukins should be studied as these

can eliminate the root pathomechanisms of endometriosis. However, these solutions may only be helpful if patients are diagnosed before the disease causes irreparable damage to the reproduc - tive system. To this day, far too many women are li - ving with endometriosis and do not realise. Proper awareness and education of reproductive health will allow women and girls to recognise irregula - rities in their own menstrual cycles and detect this Verschuren et al. Medical Journal of Cell Biology (2024) 33 disease. Healthcare professionals must also not dis - miss symptoms as typical menstrual manifestations as endometriosis can worsen with time. Regarding areas of research, future studies should investigate if there are any external factors that can lead to the pathogenesis of endometriosis so that the public can be aware and avoid harmful agents. For a dise - ase that impacts so many lives, little is known about the exact causes for its pathogenesis. Areas for rese - arch regarding endometriosis is wide and it is with hope that the further we study this disease, the so - oner we can develop more effective treatments and possibly a cure.

In conclusion, endometriosis can induce a pro - -inflammatory state in which the immune system is unregulated and leads to the proliferation of immu - ne cells and reactive oxygen species, creating a toxic environment for embryo development. Endometrio - sis also affects the quality of oocytes by stimulating terminal UPR and destroying the vital granulosa cel - ls needed for oocyte maturation. Through these me - chanisms, it can be concluded that untreated endo - metriosis creates conditions unsuitable for embryo growth and can therefore be linked to infertility. Ethical approval The study was a descriptive one. No humans or animals were a sub- ject of examinations.

Not applicable. Corresponding author Jeya Verschuren , STN (Student Scientfic Society), Division of Anatomy, Department of Human Morphology and Embryology, Wroclaw Medical University, Chalubinskiego 6a, 50-368 Wro - claw, Poland, e-mail: [email protected] . Conflict of interest statement The authors declare no conflict of interest.

1. Sims OT , Gupta J, Missmer SA, Aninye IO. Stigma and endometrio - sis: a brief overview and recommendations to improve psychoso - cial well-being and diagnostic delay. Int J Environ Res Public Health. 2021;18(15):8210; DOI:10.3390/ijerph18158210. 2. Parasar P , Ozcan P , Terry KL. Endometriosis: epidemiology, diagnosis and clinical management. Curr Obstet Gynecol Rep. 2017;6(1):34-41; DOI:10.1007/s13669-017-0187-1. 3. University of Mu� nster. Translational research on endometriosis. TREN- DO Project [Internet]. Brussels: CORDIS European Commission; 2020 [cited 2024 Feb 20]. Available from: https://cordis.europa.eu/project/ id/101008193/. 4. Yamamoto A, Johnstone EB, Bloom MS, Huddleston HG, Fujimoto VY. A higher prevalence of endometriosis among Asian women does not con - tribute to poorer IVF outcomes. J Assist Reprod Genet. 2017;34(6):765- 74; DOI:10.1007/s10815-017-0919-1. 5. Velarde MC, Bucu MEM, Habana MAE. Endometriosis as a highly re - levant yet neglected gynecologic condition in Asian women. Endocr Connect. 2023;12(11):e230169; DOI:10.1530/EC-23-0169. 6. Ramí�rez-Pavez TN, Martí�nez-Esparza M, Ruiz-Alcaraz AJ, Marí�n-Sa � n- chez P , Machado-Linde F, Garcí�a-Peñarrubia P . The role of peritoneal macrophages in endometriosis. Int J Mol Sci. 2021;22(19):10792; DOI:10.3390/ijms221910792. 7. Boraschi D. What Is IL-1 for? The functions of interleukin-1 across evolution. Front Immunol. 2022;13:872155; DOI:10.3389/ fimmu.2022.872155. 8. Abramiuk M, Grywalska E, Małkowska P , Sierawska O, Hrynkiewicz R, Niedźwiedzka-Rystwej P . The role of the immune system in the deve - lopment of endometriosis. Cells. 2022;11(13):2028; DOI:10.3390/ cells11132028. 9. Arend WP , Malyak M, Guthridge CJ, Gabay C. Interleukin-1 recep - tor antagonist: role in biology. Annu Rev Immunol. 1998;16:27-55; DOI:10.1146/annurev.immunol.16.1.27. 10. Toczek J, Jastrzębska-Stojko Ż � , Stojko R, Drosdzol-Cop A. Endometrio - sis: new perspective for the diagnosis of certain cytokines in women and adolescent girls, as well as the progression of disease outgrowth: a systematic review. Int J Environ Res Public Health. 2021;18(9):4726; DOI:10.3390/ijerph18094726. 11. Sharpe-Timms KL, Żimmer RL, Ricke EA, Piva M, Horowitz GM. Endo - metriotic haptoglobin binds to peritoneal macrophages and alters their function in women with endometriosis. Fertil Steril. 2002;78(4):810-9; DOI:10.1016/s0015-0282(02)03317-4. 12. Didziokaite G, Biliute G, Gudaite J, Kvedariene V. Oxidative stress as a potential underlying cause of minimal and mild endometriosis-related infertility. Int J Mol Sci. 2023;24(4):3809; DOI:10.3390/ijms24043809. 13. Wang Y, Żhao S. Cell types of the placenta. In: Wang Y, Żhao S, editors. Vascular biology of the placenta [Internet]. San Rafael (CA): Mor- gan & Claypool Life Sciences ; 2010 [cited 2024 Feb 22]. Chap - ter 4. Available from: https://www.ncbi.nlm.nih.gov/books/ NBK53245/. 14. Renaud SJ, Postovit LM, Macdonald-Goodfellow SK, McDonald GT , Caldwell JD, Graham CH. Activated macrophages inhibit human cyto - trophoblast invasiveness in vitro. Biol Reprod. 2005;73(2):237-43; DOI:10.1095/biolreprod.104.038000. 15. Abbasi B, Dong Y, Rui R. Resveratrol hinders postovulatory aging by modulating oxidative stress in porcine oocytes. molecules. 2021;26(21):6346; DOI:10.3390/molecules26216346. 16. Nikbakht R, Mohammadjafari R, Rajabalipour M, Moghadam MT . Eva - luation of oocyte quality in Polycystic ovary syndrome patients un - dergoing ART cycles. Fertil Res Pract. 2021;7(1):2; DOI:10.1186/ s40738-020-00094-z. 17. Ai A, Tang Ż, Liu Y, Yu S, Li B, Huang H, Wang X, Cao Y, Żhang W. Charac- terization and identification of human immortalized granulosa cells de- rived from ovarian follicular fluid. Exp Ther Med. 2019;18(3):2167-77; DOI:10.3892/etm.2019.7802. 18. Wassarman PM. Żona pellucida glycoproteins. J Biol Chem. 2008;283(36):24285-9; DOI:10.1074/jbc.R800027200. 19. Alam MH, Miyano T . Interaction between growing oocytes and granu - losa cells in vitro. Reprod Med Biol. 2019;19(1):13-23; DOI:10.1002/ rmb2.12292. 20. Clarke HJ. Transzonal projections: essential structures mediating inter- cellular communication in the mammalian ovarian follicle. Mol Reprod Dev. 2022;89(11):509-25; DOI:10.1002/mrd.23645. 21. Clarke HJ. History, origin, and function of transzonal projections: the brid- ges of communication between the oocyte and its environment. Anim Reprod. 2018;15(3):215-23; DOI:10.21451/1984-3143-AR2018-0061. 22. Takahashi N, Harada M, Hirota Y, Nose E, Azhary JM, Koike H, Kunitomi C, Yoshino O, Izumi G, Hirata T , Koga K, Wada-Hiraike O, Chang RJ, Shi - masaki S, Fujii T , Osuga Y. Activation of endoplasmic reticulum stress in granulosa cells from patients with polycystic ovary syndrome con - tributes to ovarian fibrosis. Sci Rep. 2017;7(1):10824; DOI:10.1038/ s41598-017-11252-7. 23. Kim P . Understanding the unfolded protein response (UPR) pa - thway: insights into neuropsychiatric disorders and therapeutic po - tentials. Biomol Ther (Seoul). 2024;32(2):183-91; DOI:10.4062/ biomolther.2023.181. 24. Al-Hetty HRAK, Jabbar AD, Eremin VF, Jabbar AM, Jalil AT , Al-Dulimi AG, Gharban HAJ, Khan MUF, Saleh MM. The role of endoplasmic reticulum stress in endometriosis. Cell Stress Chaperones. 2023;28(2):145-50; DOI:10.1007/s12192-023-01323-2. 25. Fribley A, Żhang K, Kaufman RJ. Regulation of apoptosis by the un - folded protein response. Methods Mol Biol. 2009;559:191-204; DOI:10.1007/978-1-60327-017-5_14. 26. Fan W, Yuan Ż, Li M, Żhang Y, Nan F. Decreased oocyte quality in pa - tients with endometriosis is closely related to abnormal granulosa cells. Front Endocrinol (Lausanne). 2023;14:1226687; DOI:10.3389/ fendo.2023.1226687.