{"paper_id":"33e2b2af-d655-4682-8a05-0b1c5b3dc07a","body_text":"How Can We Optimize the Granulose Cell \nFunction of Oocytes from an Endometriosis \nPatient to Improve the Reproductive Outcomes-A \nShort Communication?\nKulvinder Kochar Kaur*1, Gautam Allahbadia2 and Mandeep Singh3\n 1,2Centre for Human Reproduction, India\n3Department of Neurologist, India\nIntroduction\nEndometriosis, results in infertility in 50% infertile women [1]. Explanations given are \npoor oocyte quality as well as low grade embryos associated with abnormal folliculogenesis \n[2], with reduced chances of fertilization correlated with enhanced oxidative stress, high \nreactive oxygen species (ROS) levels/local inflammation [3]. Particularly escalated amounts \nof cytokines are found in follicular fluid (FF) of patients with Endometriosis might result in \novulatory impairment [4]. Further Akila et al. reviewed how in the pathogenesis of ovarian \npathologies like Endometriosis and polycystic ovarian syndrome (PCOS) are affected by \nenhanced ROS, enhanced oxidative stress in the ovary by concentrating on mural granulosa-\nlutein cells of in vitro  Fertilization (IVF) subjects. Synthesis of anti-oxidant enzymes like \nperoxiredoxin 4, superoxide dismutase and catalase and oxidative stress damage response \nproteins like aldehyde dehydrogenase 3, member A2 reduces with aging in human granulosa \nlutein cells, that helps in an unbalance in ROS/anti-oxidants which modulate molecular injury \nas well as changes in cellular functioning .Increased oxidative stress in the granulosa cells \nassociates with reduced expression of follicular stimulating hormone( FSH) receptor (FSHR) \nas well as a dysregulated FSHR signalling pathway and thus might have a role in impaired \nsteroidogenic function as well as bad ovarian response to FSH in women who are aging. In case \nof women having Endometriosis along with those with PCOS have<antioxidants producing \nability which may result in abnormal follicle formation as well as infertility. More investigations \nof the signalling pathways in relation to cellular responses to oxidative stress might throw \nlight into molecular properties of these diseases and allow formation of new treating \nstrategies for increasing reproductive potential in these women. Kunitomi et al. [5] posited \nthat endoplasmic reticulum (ER) Stress is stimulated by great oxidative stress in granulose \ncells in ovaries from Endometrioma and this modulates oxidative stress-stimulated apoptosis. \nCrimson Publishers\nWings to the Research Short Communication\n*Corresponding author: Kulvinder Kochar \nKaur, Centre for Human Reproduction, \nIndia\nSubmission: \n  January 17, 2020\nPublished: \n  January 22, 2020\nVolume 3 - Issue 4\nHow to cite this article: Kulvinder Kochar \nK, Gautam A, Mandeep S. How Can We Op-\ntimize the Granulose Cell Function of Oo -\ncytes from an Endometriosis Patient to Im-\nprove the Reproductive Outcomes-A Short \nCommunication?. Perception in Reproduc-\ntive Medicine.3(4). PRM.000570.2020.\nDOI: 10.31031/PRM.2020.03.000570\nCopyright@ Kulvinder Kochar Kaur, This \narticle is distributed under the terms of \nthe Creative Commons Attribution 4.0 \nInternational License , which permits \nunrestricted use and redistribution \nprovided that the original author and \nsource are credited.\nISSN: 2640-9666\n245Perception in Reproductive Medicine\nAbstract\nEndometriosis, results in infertility in 50% infertile women. Explanations given are poor oocyte quality \nas well as low grade embryos associated with abnormal folliculogenesis, with reduced chances of \nfertilization correlated with enhanced oxidative stress, high reactive oxygen species (ROS) levels/local \ninflammation. Particularly escalated amounts of cytokines are found in follicular fluid of follicles of \nEndometriosis subjects. Here we further detail on the work going on in finding the role of granulosa cells \nin Endometriosis subjects with regards to increased inflammation, alteration in cytokines in follicular \nfluid that associates with follicular fluid in causing the oxidative stress, and how the upstream pathway \ninvolving tumour necrosis factor alpha (TNFα) and nuclear factor kappa B( NFκB) that influences both \ntelomere length as well as telomerase activity, with shorter telomeres as well as reduced telomerase \nactivity associated with aging follicles as well as those observed in patients with premature ovarian \ninsufficiency (POI). Thus, this enhanced granulosa cell NFκB corresponds with inflammation found in FF \nalong with effect on telomeres. Ways of targeting it seems to be one answer in improving reproductive \npotential of Endometriosis patients.\nKeywords: Endometriosis; Poor oocyte quality; Oxidative stress; High reactive oxygen species (ROS); \nNFκB; TNFα; Telomere length; Telomerase activity; POI\n\n246\nPerceptions Reprod Med\n      Copyright © Kulvinder Kochar Kaur\nPRM.MS.ID.000570. 3(4).2020\nHuman granulosa-lutein cells (GLC’s) from Endometrioma patients \nexpressed great mRNA amounts correlated with unfolded protein \nresponse (UPR). Additionally, amounts of phosphorylated ER Stress \nsensor proteins, inositol-requiring enzyme (IRE1) and double \nstranded RNA-activated protein kinase-like ER kinase (PERK), were \nenhanced in granulosa cells from Endometrioma patients. Knowing \nthat ER Stress causes phosphorylation of ER Stress sensor proteins \nand stimulates UPR, these observations point that these cells were \nunder ER Stress. H 2O2, that stimulates oxidative stress, enhanced \nexpression of UPR-related mRNA in cultured human GLC’s \nwith this action getting ameliorated by pre-treatment utilizing \ntauroursodeoxycholic acid (TUDCA), that is an ER Stress inhibitor \nutilized clinically. Therapy with H2O2 enhanced apoptosis as well as \naction of pro- apoptotic factors caspase 8 as well as caspase 3 both \nof which got ameliorated by TUDCA. This pointed that stimulated \nER Stress stimulated by oxidative stress in GCs in ovaries having \nEndometrioma modulates apoptosis of these cells causing ovarian \ndysfunction in Endometriosis patients [6].\nReviewing the quality of oocytes in relation to endometriosis, \nSanchez et al. [7] tried to concentrate on direct oocytes studied \ninstead of most studies concentrating on cumulus cells or FF \ncontent. In summary It has been posited that morphological defects \nseen that include i)cytoplasmic granularity as well as/or presence of \nvacuoles might affect fertilization although limited predictive value \nof these findings in view of restrictions of non-invasive techniques \nwith simple transmitted light microscopy along with subjectivity in \nmorphological analysis as well as morphology might be influenced \nby other factors like ovarian stimulation or hormonal milieu. Goud \net al did functional studies examining immature oocytes obtained \nfrom Endometriosis women as compared to control. They observed \nthat oocytes from Endometriosis women showed enhanced cortical \ngranule loss as well as ii) zona pellucida (ZP) hardening, probably \ninterfering with fertilization dissolution of the ZP and the ability \nof embryo to undergo hatching and implantation (Figure1); [8,9].\nFigure 1:   Courtesy ref no.-7-Representative \nmorphological changes in oocytes from women \naffected by endometriosis.\n Further capacity of oocytes to undergo in vitro  maturation \n(IVM) to metaphase II stage was checked, observing a significant \nreduction of number of germinal vesicles (GV) as well as metaphase \nI (M1) oocytes could reach metaphase II stage in Endometriosis \ngroup as compared to controls. iii)Since spindle structure needs \nto coordinate the alignment as well as normal segregation of \nhomologous chromosomes and sister chromatids in 2 subsequent \nmeiotic division, disruption of meiotic spindle causes abnormal \nchromosomes alignment and fertilization. Of the 2 methods by \nwhich spindle morphology is studied polarized light microscopy \novercomes fixing problem of confocal microscopy. Barcelos et al. \nchecked spindle morphology following IVM comparing oocytes \nobtained from Endometriosis patients (n=35) with oocytes from \ncontrol group with tubal factor infertility (n=19). Till recently \nno significant differences were observed. Recently Goud et al. \nusing same IVM, observed a>percentage of abnormal spindles \nin oocytes obtained from Endometriosis patients as compared to \nART secondary to male factor infertility (66.7% vs 16%, p<0.05). \nOnly study that observed spindle in mature oocytes unlike all \nearlier ones on IVM ,failed to see any significant changes in \nEndometriosis patients iv)Cytoplasm of mature oocytes have a very \nhigh mitochondrial content as compared to other cell types, since \nit can contain up to 105 mitochondria. The 1 st and only study that \nexamined correlation between cytoplasm ultrastructure of oocytes \nand Endometriosis presence was conducted by Xu et al regarding \nintra cytoplasmic sperm injection (ICSI). Using transmission \nelectron microscopy (TEM) 50MII oocytes from patients with \nlaparoscopically diagnosed minimal/mild Endometriosis \ncontrol found a >number of Endometriosis patients contained \ndecentralized chromatin with a voluminous nucleolus as compared \nto control. Further oocytes from patients with Endometriosis had \nboth a > percentage of abnormal mitochondria (containing small/\nswollen as well as blurred vacuoles) and in total lower quantity of \nmitochondrial DNA (mtDNA) copies seen using quantitative PCR. \nThey concluded low mtDNA amount particularly shows reduced \noocyte quality with minimal/mild Endometriosis.\nSirtuins are a family of deacetylases which modify structural \nproteins, metabolic enzymes, and histones for altering cellular \nprotein placement as well as function. In mammals, 7 Sirtuins, \nthat are related to functions like oxidative stress or metabolic \nhomeostasis correlated with increasing age, degeneration/\ncarcinoma. Gonzalez Fernandez et al. [10] explored gene expression \nof Sirtuins by qRT-PCR in human mural granulosa-lutein cells (hGL)\nfrom IVF subjects in various infertility diagnostic groups and in \noocyte donors (OD; Control grp). In Study 1 gene expression levels \nof Sirtuins as well as association with age as well as IVF parameters \nin women having no ovarian factor was done. They observed that \nsignificantly>expression levels of SIRT1, SIRT2 as well as SIRT5 \nin patients ≥40yrs old as compared to OD and in women among \n27-39yrs age with tubal/male factor and no ovarian factor (NOF). \nOnly SIRT2, SIRT5 and SIRT7 expression was associated with \nage. In Study 2 gene expression of Sirtuins in poor responders \n(PR), Endometriosis (EM)and PCOS was done. In contrast to NOF \nControls, they observed >, SIRT2 gene expression in all diagnostic \ngroups although SIRT3, SIRT5, SIRT6 and SIRT7 expression was > \nonly in PR. Associated with clinical parameters , SIRT1, SIRT6 and \nSIRT7 correlated in a positive manner with FSH as well as LH doses \n\n247\nPerceptions Reprod Med\n      Copyright © Kulvinder Kochar Kaur\nPRM.MS.ID.000570. 3(4).2020\ngiven to EM patients. The number of mature oocytes obtained in PR \ncorrelated in a positive manner with expression amounts of SIRT3, \nSIRT4, SIRT5. Hence emphasizing on that cellular physiopathology \nin PR’s follicles might be correlated with cumulative DNA damage \nthat points that future studies are required (Figure2); [8]. Since FF \nsurrounds the granulosa cell- oocyte complex and represents one of \nthe modulating factors in the inter cell conversation among the cells \nwithin the follicle the substances present in FF are cytokines and \nimmune cells ,that includes IL-6.IL12, sHLA-G, macrophages, NK \ncells and lymphocytes. These cells as well as cytokines may impact \nthe granulosa cell-oocyte complex, change in immune component \ncontents might be involved in alteration in folliculogenesis, \noocyte maturation, oocyte quality as well as ovulation. Moreover, \nchanges in these balances are probably responsible for immune \nmediated conditions like Endometriosis. Prins et al., gave a \ndetailed evaluation on FF immune function, as well as FF substance \nalterations in Endometriosis patients. Escalation of macrophages \nin FF of Endometriosis patients as well as various cytokines have \nbeen documented. The part played by particular immune cells \nin FF and insight about the biological mode in healthy women as \nwell as Endometriosis patients is unknown. More studies in this \nfield will give us greater understanding in the role of FF immune \ncells and the influence of change in these balances in patients with \nEndometriosis [9].\nFigure 2:  Courtesy ref no.-8-Scheme of alterations in NOF and PR groups and possible Sirtuins’ roles. According \nto our studies, NOF women between 18 to 38 years old may avoid OS damage by OSR, triggering a normal \nsignaling response and maintaining an equilibrated OS/OSR status (A). It is possible that this equilibrium \nfavors OS during pre-menopausal aging (B). In that case, OSR are not sufficient to protect cells from OS actions \nand SIRT1, SIRT2 and SIRT5 gene expression increase would be required to recover homeostasis. Failing this \nresponse, women poor responders show a different sirtuin pattern (C). In this group, women between 25 to \n38 years old have an imbalance between OS and OSR similar to older NOF women. Cellular attempts to reach \nhomeostasis by increasing SIRT1, SIRT2 and SIRT5 gene expression are insufficient and it is necessary activate \nprotein, lipid and DNA repair mechanisms and others sirtuins’ expression. Despite the fact that SIRT6 and SIRT7 \ngene expression increase, cells cannot response to signaling and homeostasis cannot be recovered, leading to a \nclinically poor response to follicle stimulation.\nIn follicular formation there is key role of granulosa cell [10]. \nIn case of endometriotic subjects, abnormalities of granulosa cell \nmight interfere with oocyte maturation and result in poor oocyte \nquality [11]. Telomerase was found as a biomarker of the potential \nthat germ cells possess [12]. Premature aging and decreased \nfecundity were seen in mice having Telomerase deficiency [13]. \nEarlier Li et al. [14] revealed that telomerase activity (TA) in \ngranulosa cell (GTA), had a positive association with IVF treatment \nresults [14]. Healthy follicles showed > amounts of TA, as compared \nto decreased GTA =>enhancement of atretic follicles [15]. Subjects \nhaving subtle and biochemical premature ovarian insufficiency \n(POI) further demonstrated reduced TA in their granulosa cells \n[16,17]. Overall these observations point that GTA might work as \nan ovarian function biomarker.\nThe effect of ovarian endometriosis on granulose cells \nwas recently studied by Li et al. [18], that correlates molecular \nphysiology with results in fertility. An efficient oocyte as well as \ngranulosa cell crosstalk taking place the ovarian follicles sees to it \nthat formation of a mature oocyte occurs for obtaining an embryo \nhaving full competency resulting in a pregnancy that is viable. Li \net al. [18] described how internally endometriotic lesions present \non the ovarian tissue resulted in intrinsic injury by evaluating the \ninflammatory paths as well as telomerase action within granulosa \ncell. 80 women got enrolled prospectively having stage II-III \nEndometriomas as well as 104 controls who were having oocyte \nrecovery utilizing artificial reproductive therapy (ART) therapy. \nThe 2 groups developed pregnancy (clinical)with equal chances, \nthat agrees with most of current literature and helps in giving \nus assurance with regard to ART overcoming the problems in \nendometriotic subjects. But emphasis has been paid by them on the \nimpaired results of folliculogenesis which takes place when lesions \nare existing. \n\n248\nPerceptions Reprod Med\n      Copyright © Kulvinder Kochar Kaur\nPRM.MS.ID.000570. 3(4).2020\nActually, the antral follicle count (AFC), luteinizing hormone \n(LH) amounts as well as the number of oocytes recovered, and \nmature oocytes all were < in their Endometriomas group. Stimulated \nby these observations they tried to evaluate the molecular reasons \nbehind this effect on follicle formation via a number of repeated in \nvitro studies where they separated granulosa cell from the follicular \nfluid ,evaluated them for any basal inflammatory changes as well \nas culturing them in the exogenous inflammatory stimulants added \nfor getting a response. Their findings were similar to the common \nfactors in such settings like the nuclear factor (NF) κB as well \nas tumour necrosis factor alpha (TNF α) paths that seem to be \ncontrolled separately in granulosa cell in endometriotic subjects \nas compared to controls. Particularly action of NF κB in addition to \nits 2 helpers IK κβ as well as IKB α was > in the granulosa cell from \nthe follicular fluid of endometriotic subjects as compared to healthy \ncells. To find the upstream regulators of NF κB parts of granulosa \ncell of endometriotic subjects. Their observation was that TNF α \nfound within the follicular fluid (FF) had a positive association \nwith the expression of NFκB in granulosa cell, a finding which was \nlater confirmed by crucial cell culture studies ,where TNFα therapy \ndirectly escalated the excess of NF κB as well as IKB α. The biggest \nimportance of these experiments was the innovative understanding \nof the telomerase enzyme action when endometriosis was existent \nalong with its associated inflammation. Recently trying to assess \ntelomere length as well as telomerase activity in granulosa cell of \npatients with infertility has been on the rise that has turned to be a \ngood marker of oocyte competence. Telomeres represent conserved \nareas at the distal part of chromosomes which aid in conferring \nprotection to genomic integrity.\nEscalated mitotic action of granulosa cell as well as follicular \nhormones lead to the shortened length of telomeres that has to \nbe restored by telomerase activity. Minimal but essential proof \nhas aided in the understanding that the activity of telomerase is \n>in healthy as well as small follicles as compared to atretic larger \nfollicles and this activity of enzymes can be enhanced by estrogens \n[18]. This telomeres length as well as telomerase enzyme activity \nas far as theoretical explanation  is that they represent divergence \nbut separate dynamics got detailed in particular situations ,Like \ngranulosa cell of women having a diagnosis of premature ovarian \nfailure(POF) had shorter telomeres as well as < telomerase activity \nas compared to control ladies, while absence of normal telomerase \nactivity enhances the chances for women to have POF 11 times [19]. \nThough experiments of cellular modes with regards to telomere \nstructures within oocytes as well as granulosa cell might aid in \nunfolding the underlying causes related to infertility, mostly little \nwork has been done in this field.\nIn 2017 Li et al. [18] utilized the niche, detailing the telomeres \nlength as well as telomerase activity in polycystic ovarian syndrome \n(PCOS). Their observations were shorter telomeres but the \ngranulosa cell telomerase activity (GTA) was same in PCOS subjects \nas compared to controls [19,20]. In this study, they found that GTA \nin ovarian endometriotic subjects associated positively with the \nnumber of mature oocytes, negatively with granulosa cell NF κB \nactivity and reduced following TNF α therapy. Greater information \nregarding GTA dynamics was added by studies on (human \ntelomerase reverse transcriptase (hTERT), the catalytic subunit \nof the telomerase enzyme was reduced with TNF α therapy and \nenhanced by NFκB inhibitor. An essential observation of this study \nalthough negative is that both GTA as well as hTERT were only little \nlower in endometriotic subjects as compared to controls (p=0.16 \nand p=0.55 respectively) while NF κB inhibitor did not escalate \nGTA in granulosa cell culture. It needs to be seen if lowering TNF α \nactivity or use of NFκB inhibitor might be of help in re-establishing \nthe hTERT activity needs to be studied further.\nOn the basis of in vitro findings, they posited a possible mode \nwhere lesion led to escalation of FF TNFα stimulates NFκB cascade \nactivation in granulosa cell that in turn decreases telomerase \nactivity and effects physiologic increase in telomeres that is key \nfor folliculogenesis as well as oocyte quality. Important is follicular \nTNFα levels was not separate in case as well as controls but the \nin vivo  findings here correlated with limited size sample size or \ninclusion of earlier operated endometriotic subjects that might \nhave contribute to the complicated nature regarding this. There \nis requirement for experiments detailing pathophysiology of \ngranulosa cell in endometriotic subjects and time to unveil why \nsubfertility in this disease both for patients as well as professionals \n[21].\nConclusion\nThus trying to understand why infertility results in \nEndometriosis patients lot of efforts have been put in originally \nmost studies concentrated on studying follicular fluid for finding \nthe reason of poor quality of oocytes among Endometriosis patients \nwhere researchers observed enhanced oxidative stress as well as \nER Stress in cases of Endometriosis subjects. \nLater deciding to study oocytes directly researchers observed \n1. Altered mitochondrial numbers as well as abnormal \nmitochondria along with reduced mitochondrial DNA in oocytes of \nEndometriosis patients \n2. Spindle abnormalities \n3. ZP hardening of oocytes of Endometriosis patients \n4. Abnormal spindle patterns and \n5. Altered morphology in form of dark central granulation \nby carrying out study on oocytes during IVM mostly.\nFurther the role of Sirtuins in effecting the alteration in cytokines \nin FF associated with Endometriosis patients was emphasized as \nreflecting which Sirtuins are associated with poor oocyte quality \nwith aging, PR as well as Endometriosis patients and PCOS subjects. \nLi et al. [18] utilized the niche,detailin g the telomeres length as well \nas telomerase activity in PCOS subjects where they found shorter \ntelomeres but the granulosa cell telomerase activity (GTA) was same \nin PCOS subjects found that GTA in ovarian endometriotic subjects \nassociated positively with the number of mature oocytes, negatively \nwith granulosa cell NF κB activity and reduced following TNF α \ntherapy. Greater information regarding GTA dynamics was added \n\n249\nPerceptions Reprod Med\n      Copyright © Kulvinder Kochar Kaur\nPRM.MS.ID.000570. 3(4).2020\nby studies on hTERT , the catalytic subunit of the telomerase enzyme \nwas reduced with TNF α therapy and enhanced by NF κB inhibitor \n.An essential observation of this study although negative ,is that \nboth GTA as well as hTERT were only little lower in endometriotic \nsubjects as compared to controls. On the basis of in vitro findings, \nthey posited a possible mode where lesion led to escalation of FF \nTNFα stimulates NF κB cascade activation in granulosa cell that in \nturn decreases telomerase activity and effects physiologic increase \nin telomeres that is key for folliculogenesis as well as oocyte quality. \nImportant is follicular TNFα levels was not separate in case as well \nas controls But the in vivo  findings here correlated with limited \nsize sample size or inclusion of earlier operated endometriotic \nsubjects Further work is being needed to find why granulosa cells \nare involved in Endometriosis patients ,studying a larger number \nof cohort to try to get better possible therapies to reduce oxidative \nstress and see to it that abnormal cytokines are prevented so that \nwe get optimum oocytes of good quality.\nReferences\n1. Eskenazi B, Warner ML (1997) Epidemiology of endometriosis. Obstet \nGynaecol Clin North Am 24(2): 235-258.\n2. 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(2009) \nCorrelation of telomere length and telomerase activity with occult \novarian insufficiency. J Clin Endocrinol Metab 94(12): 4835-4843.\n21. Vigano P , Makieva S (2019) Lifting the Endometrioma veil on granulosa \ncells. Fertil Steril 112(5): 813-814.\nFor possible submissions Click below: \nSubmit Article","source_license":"CC0","license_restricted":false}