{"paper_id":"3c8ede85-55ba-4d8e-9d96-a2e74a8dde0a","body_text":"FINAL REPORT ON A SHORT-TERM \nGRANT RESEARCH PROJECT \n·.. ... 304/PPSP/6131183 \nEFFECT OF PERITONEAL FLUID \nON EARLY EMBRYONIC \nDEVELOPMENT \nDR. LIZA BINTI NOORDIN \n\n. . \n.. . '· \n. .. \n.· \n. . \nRESEARCHERS \n1. Dr. Liza Noordin \n(Department of Physiology , PPSP) \n2. Prof. Gregory Tan Jin San \n(International Medical University , Kuala Lumpur) \n3. Assoc. Prof. Mohd. Shu kri Othman \n(Department of Obstetric & Gynecology, PPSP) \n.. \n----\nr~' . . ' . . I n I ' . ' \n· ·· · '!\" '\"·'\\' P··ll' · It'!· ' 'l ' (\"•/\\Ti f \\Jor\\l\"iVrl~'-' l.. ,_.....,,\\ '\\ .. t . J , ~ \\ l. • ' \"- .. \n<'. ~ .' • J : \n[,/f . : \".... , .' •. L:!.:.<KK \n1_- 1 l' \nl \nr . \" .., \n--\nf • \n\nTABLE OF CONTENTS \nTable of contents \nGlossary \nAbstract \nAbstrak \n1. BACKGROUND \n2. GENERAL OBJECTI VES \n3. GENERAL METHODS AND MATERIALS \n4. EMBRYOTOXICITY OF PERITONEAL FLUI D WITH \nENDOMETRIOSIS (PF-E) \n4.1 Introduction \n4.2 Methods and materials \n4.3 Results \n4.4 Discussi on \n5. PYRUVATE IN EMBRYO GROWTH AND POTENTIAL SUPPORT \nAGAINST THE EMBRYOTOXICITY OF PF-E \n5.1 Introduction \n5.2 Methods and materia ls \n5.3 Results \n5.4 Discussion \nPage \nIll \nv \nvii \n1 \n2 \n3 \n9 \n10 \n11 \n19 \n23 \n25 \n26 \n30 \n\n6. INTERLEUKINS AS POSSIBLE MEDIATORS OF THE \nEMBRYOTOXICITY OF PERITONEAL FLUID \n6.1 Introduction \n6.2 Methods and materials \n6.3 Results \n6.4 Discussion \n7. SUMMARY AND CONCLUSION \nREFERENCES \nAPPENDICES \nPUBLICATIONS AND PRESENTATIONS \n11 \n34 \n36 \n48 \n58 \n64 \n67 \n73 \n78 \n\nGLOSSARY \nDefinition of terms \nEmbryotoxic: Substance(s) that can cause harmful to the embryo \nEmbryotoxicity: The ability of a substance to cause harm to the embryo which \nmay result in death or abnormal development. \nEndometriosis: A condition in which the endometrial tissue that composed of \nendometrial glands, stroma or both, outside the endometrial cavity (Tabibzadeh \neta/., 2003). \nAbbreviation \nAFS American Fertility Society \nATP adenosine triphosphate \nDNA deoxyribonucleic acid \nhCG human chorionic gonadotrophin \nHWM modified Whitten's medium (with Hepes) \nICR Institute Cancer of Research \nIL interleukin \nIVF in vitro fertilization \nmWM modified Whitten's medium \nPF-E peritoneal fluid with endometriosis \niii \n\nPF-NE \nPMSG \nr-AFS \nTNF \nWM \nzp \nperitoneal fluid without endometriosis \npregnant mare serum gonadotrophin \nrevised American Fertility Society \ntumor necrosis factor \nWhitten's medium \nzona pellucida \nlV \n\nABSTRACT \nThe aetiology of endometriosis associated with infertility remains poorly \nunderstood. In recent years, the potential influence of peritoneal fluid (PF) or its \ncellular components have been proposed as possible mediators of infertility in \nendometriosis through it toxic effects on pre-implantation embryo. Several \nfactors have been identified as embryotoxic factors, however, the mechanism \nof embryotoxicity have not well clarified. Studies were therefore undertaken to \nclarify the possible mechanisms of embryotoxicity in endometriosis and to \ndetermine the possible embryotoxic factor(s) that involved in the process. The \nrole of exogenous nutrient (pyruvate) has also been examined in reducing or \neliminating the embryotoxicity. Peritoneal fluid was collected from infertile \nwomen at reproductive age with 21 endometriosis (PF-E) (7 minimal or mild, 7 \nmoderate, 7 severe) and 7 without endometriosis (PF-NE). Addition of PF-E to \nthe culture medium from all stages of endometriosis significantly suppressed \nthe mouse embryo growth at all stages of development, at 24, 48 and 72 hours, \nwhich correlated with the severity of the disease, as compared to control (no \nperitoneal fluid). Excessive pyruvate was able to reduce the embryotoxicity at \nall stages of development in minimal or mild and moderate PF-E, and only \nduring the first 24 hours in severe PF-E. The level of IL-6 concentration was \nsignificantly higher in PF-E as compared to PF-NE and correlated with the \nseverity of the disease. However, no significant difference was noted in the \nlevel of IL-8 between the two groups. Both interleukins were found to be \nembryotoxic with IL-6 is more potent. A positive correlation between the levels \nof IL-6 and its embryotoxicity was noted. These findings propose embryotoxicity \nto be a possible mechanism of infertility in endometriosis and the growth \nv \n\npromoting effects of pyruvate indicated the mechanism of embryotoxicity might \ninvolve functional disruption of the intermediary metabolism such that \nexcessive pyruvate is required to maintain embryo growth. \nVl \n\nKESAN CECAIR PERITONEUM KE ATAS PERKEMBANGAN AWAL \nEMBRIO (ABSTRAK) \nEtiologi penyakit endometriosis berkaitan dengan masalah kesuburan masih \nkurang difahami. Sejak kebelakangan ini, cecair peritoneum (CP) atau \nkomponen sel-sel di dalamnya telah dicadangkan sebagai perantara yang \nberupaya mempengaruhi masalah kesuburan di dalam penyakit endometriosis \nmelalui kesan toksiknya ke atas embrio di peringkat pra-implantasi. Beberapa \nfaktor telah dikenalpasti sebagai embriotoksin, walaubagaimanapun, \nmekanisme ketoksikan embrio ini masih belum dijelaskan dengan sempurna. \nKajian ini telah dikendalikan untuk menjelaskan mekanisme ketoksikan embrio \npada pesakit endometriosis dan mengenalpasti faktor-faktor embriotoksin yang \nmungkin terlibat. Peranan nutrien eksogenus (piruvat) didalam mengurangkan \natau menghilangkan ketoksikan embrio juga telah dijalankan. Cecair \nperitoneum diperolehi daripada wanita diperingkat umur reproduktif yang \nmempunyai masalah kesuburan, iaitu 21 Endometriosis (CP-E) ( 7 ringan, 7 \nsederhana, 7 teruk) dan 7 tiada endometriosis (CP-TE). Tambahan CP-E ke \ndalam media kultur, dari semua peringkat endometriosis, didapati merencat \nperkembangan embrio pada setiap peringkat perkembangan, iaitu pada 24, 48 \ndan 72 jam selepas kultur, dan didapati kolerasi terhadap keterukan penyakit \nendometriosis, berbanding dengan kumpulan kawalan (tiada cecair \nperitoneum). Tambahan piruvat yang berlebihan didapati mampu \nmengurangkan ketoksikan .em brio di setiap peringkat perkembangan, pada CP­\nE ringan dan CP-E sederhana, dan bagi CP-E teruk, kesan ini hanya dilihat \npada 24 jam yang pertama sahaja. Paras IL-6 pada CP-E adalah tinggi secara \nsignifikan berbanding dengan CP-TE, dan didapati kolerasi terhadap keterukan \nVll \n\npenyakit. Tiada perbezaan yang signifikan pada paras IL-8 dari kedua-dua \nkumpulan. Kesan ketoksikan embrio dapat dilihat pada kedua-dua interleukin \ndengan IL-6 mempunyai keupayaan yang lebih tinggi. Didapati juga kolerasi \npositif yang signifikan di antara paras IL-6 dan kesan ketoksikan embrio. \nPenemuan ini mencadangkan ketoksikan embrio sebagai mekanisme terhadap \nmasalah kesuburan pada pesakit endometriosis dan peranan piruvat didalam \nmenggalakkan pertumbuhan embrio menunjukkan mekanisme ketoksikan \nembrio ini mungkin melibatkan gangguan terhadap fungsi metabolisme \nperantara yang dibuktikan dengan kehadiran piruvat yang berlebihan \ndiperlukan untuk mengekalkan pertumbuhan embrio. \nVlll \n\n1. BACKGROUND \nThe peritoneal fluid is the physiologic environment of the fallopian tube and the \noocyte after ovulation, and believed to be an exudation product of the ovary, at \nleast in part (Bouckaert eta/., 1986). This fluid has been shown to contain a \nplethora of substances, including macrophages, cytokines, growth factors, \nenzymes, proteins and prostaglandins, which subject to change with \npathological conditions (Harada et a/., 2001 ). The direct communication of the \nperitoneal fluid with the lumen of the fallopian tube (Seli & Arici, 2000), suggests \nthat it may serve as a medium for fertilization and early embryonic development. \nbut whether it exerts a regulatory role on them is unknown. \nIn recent years, many investigators have focused on this fluid to study various \naspects of endometriosis, one of the most frequently encountered gynecologic \ndiseases (AI-Fozan & Tulandi, 2003; Tabibzadeh et a/., 2003). To date, the \naetiology of endometriosis associated with infertility remains poorly understood. \nA number of postulates have been proposed to explain the possible aetiology of \nendometriosis associated with infertility ranging from alterations in the \nperitoneal fluid (Ryan & Taylor, 1997; Gomez-Torres et a/., 2002), ovulatory \ndysfunction (Dmowski et a/., 1986; Ronnberg, 1990), sperm phagocytosis \n(Soldati et al. I 1989; Jha et a/., 1996)~ impaired fertilization (Cahill et a/., 1997; \nAzem et a/., 1998) and implantation defects (Pellicer et a/., 1998; lllera et a/.. \n2000). \nMany authors (Harada et a/. I 2001; Gomez-Torres et a/., 2002) suggested that \nthe factors that cause the infertility in endometriosis might be hidden in the \n\nperitoneal fluid especially because this fluid is a major controlling the peritoneal \nmicroenvironment (Taketani eta/., 1992; Tabibzadeh eta/., 2003) where most \nof the reproductive organs located. The fluid is in contact with peritoneal \nendometrial implants as well as the tubal microenvironment in which fertilization \noccurs, hence subtle alterations of this fluid and /or cellular constituents might \nadversely influence reproduction. Despite continuously bathes the pelvic cavity, \nuterus, fallopian tubes and ovaries (Seli & Arici, 2000) and host the processes \nof ovulation, gamete transportation, fertilization and early embryonic \ndevelopment (Syrop & Halme, 1987), this fluid is away from routine site of \ninvestigations. \nA recent focus of attention concerning this fluid is to determine the factor(s) \nwithin it that is believed can cause harmful to the embryo or known as \nembryotoxic factors, as a mediator of infertility in endometriosis and to \nd~termine whether this factor(s) can be reduced or e~iminated from the fluid. \nThere is thus a need to re-define the role of peritoneal fluid especially in \nendometriosis, on early embryonic development. \n2. GENERAL OBJECTIVES \n1. To determine the effects of peritoneal fluid (embryotoxicity) from women \nwith endometriosis (PF-E) on in vitro development of early mouse \nembryos and to relate with the severity of the disease. \n2 \n\n2. To determine whether the embryotoxicity can be reduced or eliminated \nfrom the PF-E. \n3. To determine the embryotoxic factor(s) in the PF-E as a possible \nmediator of infertility in endometriosis. \n4. To determine the embryotoxicity of the embryotoxic factor(s) determined \non in vitro development of early mouse embryos. \n3. GENERAL METHODS AND MATERIALS \n3.1 Subject selection \nThe diagnosis of endometriosis was based solely upon direct laparoscopic \nvisualization of endometriotic implants or during laparotomy. Disease was \nstag·ed as minimal or mild, moderate and severe, according to the classification \nof revised American Fertility Society (r-AFS), 1985. Despite using the same \nclassification, many ~esearchers (Damewood et a/., 1990; Rier et a/:, 1994; \nPolak eta/., 2003) combined the minimal and mild endometriosis as one group. \nPeritoneal fluid samples were obtained from 21 infertile women of reproductive \nage who underwent laparoscopy examination or laparotomy. They were \nincluding 7 women with minimal or mild, 7 with moderate and 7 with severe \nendometriosis. The subjects were from Hospital Universiti Sains Malaysia and \nHospital Kota Bharu, Kelantan. The mean age of these patients is 30.88 :t 0.87 \n(mean _± S.E.M), (range: 26 - 36) with the duration of infertility was at least 18 \nmonths. They received general infertility work ups and non-received hormonal \ntreatment or had intrauterine device insertion within 6 months before the \n\nprocedure. The· inclusion criteria included: infertile, either primary or secondary \nwhereas the exclusion criteria included: age above 40 years and presence of \nother pathological condition which observed during laparoscopy or laparotomy, \nsuch as uterine fibroid or pelvic tumor. The study was approved by the \nUniversiti Sains Malaysia Ethical Committee. \n3.2 Peritoneal fluid collection and preparation \nPeritoneal fluid samples were collected by the gynaecologic surgeon from the \nanterior and posterior cui-de-sacs by Veress needle during laparoscopy either \nfor evaluation for infertility or for treatment of infertility, or during laparotomy. \nThe fluid was obtained before any manipulative procedures done. All patients \nwere placed in a supine position to standardize the method of collection. The \nfluid was placed in a sterile heparinzed tube; the volume was recorded and \ntransported to the laboratory immediately. Those samples with heavily blood \nstained were discarded. The collected peritoneal fluid volume varied from 2 - 10 \nmls, (4.94:!: 0.61, mean+ S.E.M). \nSamples were prepared as described by Tan et a/., (1989). The fluid was \ncentrifuged at 600-x g for 1 0 minutes at 4 oc. The cell-free supernatants were \nheated inactivation in a water bath (56°C) for 30 minutes to inactivate the \ncomplement protein. Then, it was separated into aliquots and stored at -8ooc \ntill the time of assay. \n3.3 Animal preparation \nIn the present study, female and male mice of ICR strain, 8 - 1 o weeks of age \nand weighing 15 - 20 gram were used. The mice were provided by Laboratory \n\nAnimal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia, \nKuala Lumpur. Animals had free access to food pellets and drinking water. \nFemales were superovulated by injected intraperitoneally with 5 IU of pregnant \nmare serum gonadotrophin {PMSG: Folligon, lntervet International B. V, Holand) \n(Appendix A) to stimulate the follicular growth and 46 hours later with 5 IU of \nhuman chorionic gonadotrophin {hCG: Chorulon, lntervet International B.V, \nHoland) {Appendix B) to trigger ovulation. Immediately after hCG injection, the \nfemale mice were caged at a 1: 1 ratio with males for overnight. Mating was \nconfirmed after 18 to 19 hours by the presence of vaginal plug or sperm-positive \nsmear. The protocol was used as described by Tan eta/., (1989). \n3.4 Mouse embryo collection \nTwenty-four hours after mating confirmed, which was about 42 to 44 hours after \nhCG injection, the female mice were sacrificed by cervical dislocation and the \noviducts were dissected and transferred to a Petri dish (NUNC, Denmark) \ncontained few drops of flushing solution, modified Whitten's medium with Hepes \n(HWM) (Appendix C). With one ml syringe attached to a blunt 32-gauge needle, \nthe oviduct was flushed with HWM to separate the 2-cell mouse embryos under \nthe dissecting microscope at room temperature. Only morphologically normal \nembryos were used in the experiment and they were pooled in a Petri dish \ncontained HWM. Therefore, the medium used for oviduct flushing, embryo \ncollection and holding before culture was HWM. \n3.5 Mouse embryo culture \nBefore culture. each sample of the peritoneal fluid supernatant from all stages \nof endometriosis {study group) was thawed and filtered (0.2 f..lm filter, Whatman) \n5 \n\nand added to the culture well (culture well plate: NUNC, Denmark) whereas in \ncontrol group, the culture well only contained the 2-cell embryos in culture \nmedium, without present of peritoneal fluid. \nThe collected normal 2-cell embryos were rinsed with HWM to remove any \nforeign materials. Then, they were distributed randomly between the study and \ncontrol groups, which have been designed according to the respective \nexperiments. Embryos were cultured in groups of 10-12 per well, which \ncontained modified Whitten's medium (mWM) (Appendix D). \nThe embryos were cultured at different time of experiments based on the \nnumber of 2-cell mouse embryos collected. All the experiments were done in \nthe same culture environment. Embryos were cultured in an incubator at 37°C, \nwhich was gassed with 5o/oC02 in air. The pH of culture medium was maintained \nat 7.3-7.4. \n3.6 Embryo evaluation \nViability assessment is an essential part of embryo culture study. Different \nterms have been proposed such as 'embryo quality, 'embryo viability' and \n'developmental competence'. Overstrom, (1996) has proposed five approaches \nto evaluate embryo viability including: \n1. morphology \n2. development in vitro (culture) \n3. differential (live/dead) cell staining \n4. fluorescent metabolic probes and \n5. micro assays of embryo metabolism \n6 \n\nIn this study, the first two approaches were used. Several characteristics \nincluding embryo color/darkness, homogeneity of blastomere size, cytoplasmic \ngranulation and degree of blastomere fragmentation were observed. The \nembryonic stages at 24, 48 and 72 hours were observed under Inverted \nmicroscope (Axiovert S100: Carl Zeiss Company, Germany). \nThe control range for normal growth was defined as described by Tan eta/., \n(1989). \nAt 24 hours: 4 cells or greater \nAt 48 hours: morulae or greater \nAt 72 hours: blastocysts or greater \nFor the degenerated embryos, they were defined as described by Marcos et a/., \n(1985) including dark granular cytoplasm, fragmentation and cell mass retracted \nfrom the zona pellucida. \nThe flow chart of mice preparation and embryo culture is outline in Figure 3. 1. \n3.7 Statistical analysis \nAll analysis was done using the Instal Programme. To evaluate the \ndevelopment of mouse embryos, results from individual culture well were \npooled according to their groups. Data were analyzed by chi-square test and \nexpressed in percentage. p values< 0.05, were considered significant. \n7 \n\nFemale mice (ICR strain: 8 to 10 weeks) \nOvarian hyperstimulation \ni. Injection of PMSG (5 IU) · \nii. Injection of hCG (5 IU), 46 hours later \nMating with male mice \n(ICR strain: 8 to 10 weeks) \nMating confirmed by presence of vaginal plug/sperm-positive smear \n(18-19 hours after mating) \nEmbrvo collection \no. \nFlushing of oviducts with HWM \n(42-44 hours after hCG injection) \n~ \nEmbryo culture \n2-cell mouse embryos with \ni. Control group (no PF-E) \nii. Study group (with PF-E) \nMedium : modified Whitten's medium \nAtmosphere : C02 incubator, with 5o/o C02 in air \npH : 7.3-7.4 \nTemperature : 37°C \nFigure 3.1: Flow chart showing mouse preparation and embryo culture \n8 \n\n4. EMBRYOTOXICITY OF PERITONEAL FLUID WITH ENDOMETRIOSIS \n(PF-E) . \n4.1 Introduction \nEmbryotoxicity is defined as the ability of a substance to cause harm to an \nembryo, which may result in death or abnormal development. There is \nincreasing evidence that macrophages, cytokines and other local products \npresent in PF-E may be the mediators for infertility by causing alterations in the \nperitoneal environment, subsequently generate embryotoxic activity (Gomez­\nTorres et a/., 2002). This is supported by the facts that peritoneal fluid \nsurrounds the ovary and proximal part of the fallopian tubes and passes easily \ninto the tubal lumen in which fertilization takes place (Seli & Arici, 2000). Early \nembryonic growth is therefore exposed to this fluid with its cellular and soluble \ncomponents. \nThe embryotoxic factors can be an antibody, a cytokine or may be nutritional \n(Fein eta/., 1998). There is a large array of factors that have been reported to \nhave a potential in the inhibition of early embryo growth such as IL-1 (Fakih et \na/., 1987; Taketani eta/., 1992), TNF-a (Taketani eta/., 1992), IL-6 (Gomez­\nTorrez et a/., 2002), interferon (IFN)-cr (Seli & Arici, 2000), ovum capture \ninhibitor (OCI) (Suginami & Yano, 1988) and various types of autoantibodies \nsuch as phospholipid, ribonucleoprotein and double stranded DNA (Gieicher et \na/., 1987). \n9 \n\nQ \nPeritoneal fluid embryotoxicity has been studied in women with and without \nendometriosis. Although several investigators (Marcos eta/., 1985; Tan eta/., \n1989; Ill era et a/., 2000; Gomez-Torres et a/., 2002) report that peritoneal fluid \nobtained from infertile patients with endometriosis adve~sely affect mouse \nembryo cleavage and viability in vitro, others (Dodds et a/., 1992; Awadalla et \na/., 1997) have found no harmful effects of this fluid on embryo growth, when \ncompared to peritoneal fluid from infertile women without endometriosis (PF­\nNE). \nTo this end, the relationship between infertility and embryotoxicity in \nendometriosis is still not clear. The reason for these equivocal studies is unclear, \nalthough PF-E has frequently been shown to be toxic to the pre-implantation \nembryo (Harada eta/., 2001). \nSpecific objectives: \n1. To determine the embryotoxicity of PF-E on the development of early \nmouse embryos. \n2. To determine the effects of different stages of PF-E on the development \nof early mouse embryos. \n4.2 Methods and materials \nMouse embryo culture: \n• mWM was added to each culture well. \n10 \n\nII \nStudy group: \n50 f..ll of each sample of PF-E (prepared as previously described in the \nGeneral Methodology: Chapter 3) was added to each culture well \ncontaining mWM. \nControl group: \nEach culture well was prepared with 1 ml mWM (control without the \nperitoneal fluid) \n• The collected 2-cell mouse embryos were distributed randomly \n• The total volume in each culture well was 1 mi. \n• There were 7 wells used for each stage of PF-E and the control group. \n• The embryos were incubated and observed at 24, 48 and 72 hours. \n4.3 Results \nAddition of 5o/o (50J.tl) heat-inactivated PF-E to the culture medium from all \nstages of endometriosis significantly {p<O. oo 1 versus control) suppressed the \nembryo growth at all stages of embryo development, which are shown in Figure \n4.1 (minimal or mild PF-E), Figure 4.2 (moderate PF-E) and Figure 4.3 (severe­\nPFE). A positive relationship between embryotoxicity and the severity of \nendometriosis was also observed, as shown in Figure 4.4. \nThe morphology of the embryos is shown in Figure 4.5 (normal embryos) and \nFigures 4.6 and 4. 7 (degenerated embryos). Normal embryos consist of \nblastomeres of even in size and shape and no cytoplasmic fragments whereas \nthe degenerated embryos either contain dark granular cytoplasm, are \nfragmented or have cell mass retracted from the zona pellucida. \n11 \n\n100% l \n90% j \n80% j \nt/) 70% 1 \n0 \ni!' I .0 60% i E \nG) \nI C) \n50% I \nc \na. \n0 \n(1) \n> G) \n\"C 40% l -0 \n~ \n30% i \n0 \nI \n20% ! \nI \nI \n10% i \nI . \nI \nI \n! I 0% -1 \n!._ -· -·--· \n24 H 48 H 72 H \nhours \n• Control (n=76) l \n'I 0 Minimal or mild I \nL PF-E (~=80) I \n! \nI \ni \n- .I \nFigure 4.1: Effects of minimal or mild PF-E on 2-cell mouse embryo \ndevelopment. a p<0.001 versus control. () refers to number of embryos used in \nthe experiment. \n12 \n\n., \n! \nI \nI \nI \nI \ntn \n0 \n~ .c \nE \nQ) \n100% \n90% \n80% \n70% \n60% \nC) \nc: \n·a. 50% \n0 \n-~ \nQ) \n\"'C \n-0 \n~ \n40% ~ \n! \n30% ~ \nI I \n20% i \n10% \n0% \n! •control (n=76) II \nII \nj DModerate PF-E II I (n=78) \ni 24 H 48 H 72 H 1 \ni hours i \nL_ ____ ·-------- -----·-----·----------- - ---- --------- -- _j \nFigure 4.2: Effects of moderate PF-E on 2-cell mouse embryo development \n3 p<0.001 versus controL ()refers to number of embryos used in the experiment \n13 \n\n100% \n90% \n80% \ntn 70% \n0 \n~ .c 60% E \nC1) \nOl \nc \nc. 50% \n0 \nC1) \n> C1) \n40% \"0 \n-0 \n~ 0 \n30% \n20% \n10% \n0% --, \n24 H 48 H 72 H \n~ • Control (n=76) l \nI I I 0 Severe PF-E I' \n! (n=82) 'I L . -------- - . -- ·- - I \nI \nI \n' ' I \n' I \nI \n! \nI hours . I \nI \nL __ ------ --- ----------------- --· -·· -· ------ -· __ j \nFigure 4.3: Effects of severe PF-E on 2-cell mouse embryo development. \nap<0.00 1 versus control. ()refers to number of embryos used in the experiment. \n14 \n\n100% -\n90% -\ntJ) \n80% .J ~ \n:J \n0 \n.t::. \nN 70% .. \nt--\n-C'O \ntJ) 60% -· 0 \n~ \n..0 \nE 50% -\nQ) \nCl \nr:: 40% \n-C'O \n~ \nQ) \nr:: 30% Q) \nCl \nQ) \n'0 \n- 20% 0 \n~ 0 \n10% \n0% . . \na \n\"0 \nE \n'-\n0 \nro \nE \nc \n~ \na,b \nQ) ...... ro '-Q) \n\"0 \n0 \n~ \na,b,c \nQ) \n'-Q) \n> Q) \nU) \n1 Control \n0 PF-E \nStages of \nendome triosis \nFig 4.4: Percentage of degenerating embryos at 72 hours in different stages of \nPFE. a p<0.001 vs. control. b p<0.001 vs. minimal or mild PF-E. Cp<O.OS vs. \nmoderate PF-E. \n15 \n\n2-cell embryo Morula \n4-cell embryo Blastocyst \n8-cell embryo Hatching embryo \nFigure 4.5: Photographs showing normal mouse embryos. \na: zona pellucida b: blastomere \nlo \n\n'.-\nFigure 4.6: Photographs showing degenerated mouse embryos. \na: dark granular cytoplasm \nb: fragmentations \nc: cell mass retracted from the zona pellucida \n17 \n\n.•, \nFigure 4. 7: Photographs showing degenerated mouse embryos. \na: dark granular cytoplasm \nb: fragmentations \nc: cell mass retracted from the zona pellucida \n1R \n\n4.4 Discussion \nAlthough it is generally agreed that endometriosis is associated with infertility, \ncausality is not well defined. The potential influence of peritoneal fluid or its \ncellular components as mediators of infertility has only been examined in the \nlast decade. Experimental data seem· to indicate that there is a direct \nembryotoxic effect of peritoneal fluid as one of the underlying causes of \ninfertility. The presence study examines the possibility of embryotoxicity as a \nmechanism of infertility in vvomen with endometriosis. \nHeat-inactivated of PF-E is found to be toxic to early mouse embryos as \ncompared to the control. Embryotoxicity appears to increase with increased \nseverity of endometriosis. By 72 hours, the highest percentage of \ndegenerated embryos is noted in severe PF-E, followed by moderate and \nminimal or mild PF-E (88%, 74% and 47% respectively). The percentage of \ndegenerated embryos in the control group is 20%. Embryotoxicity is therefore \nmost mark in severe endometriosis, indicating the presence of perhaps more \ntoxic factors with increased severity of endometriosis. \nThe presence finding supports the hypothesis that embryotoxicity may be a \nmechanism of infertility in endometriosis. The embryotoxicity as observed is \ncongruent with the findings of several studies (Morcos et a/., 1985; Tan et a/., \n1989; lllera et a!., 2000; Gomez-Torres et a/., 2002). The relationship with \nseverity of endometriosis however appears to contradict t he finding of Dodds \net a/., 1992 and Awadalla et a!. , 1997. \n19 \n\nGenerally, the percentage of degenerated embryos was observed to increase \nafter 24 hours of culture. By 72 hours, the majority of the degenerated embryos \nthat do not reach the blastocyst stage or greater are noted to contain mainly \nfragments or celt mass retracted from the zona pellucida. \nHowever, it is uncertain from the present study whether the embryos underwent \nthe process of necrosis or apoptosis as a result of peritoneal fluid \nembryotoxicity. It could be a mix of both processes. Future studies could focus \non the ultrastructural changes associated with embryotoxicity to differentiate the \nmechanism of cell degeneration. According to Betts and King (2000), most of \nthe mechanism in cell death is not restricted to a single mode of death \n(apoptosis or necrosis); there is some overlap in the initial cellular responses \nand triggers of each pathway of cell death. \nIt is acknowledged that there are essential cellular components for cell survival \nsuch as the plasma membrane, mitochondria, lysosomes, the nucleus or other \ncellular organelles that have their own functions (Kanduc et a/., 2002). Plasma \nmembrane, mitochondria and the nucleus and its DNA, have been predominant \nareas of study as 'vulnerable site' whose destruction or malfunction threatens \nthe functioning of the cellular unit. \nIt can be hypothesized that the embryotoxic factor may disrupt the essential \ncellular components organization, especially the mitochondria, an essential \ncomponent for intermediary metabolism and the most sensitive to changes in \nmedia composition. Mitochondria are important sites of aerobic respiration, \n20 \n\nwhich produce over 85% of all ATP production (Benes & Balaban, 1983). They \nhave also reported that the major source of ATP in the in vitro mammalian pre­\nimplantation embryo is aerobic respiration, which is presumed similar to in vivo. \nIn addition, an intact plasma membrane is also important for embryo survival as \nit provides both a physical and a selective physiological barrier with the \nextracellular environment (Overstrom, 1996). This membrane is essential for \nmaintenance of ionic gradient, intracellular pH, solute-specific exchange and co­\ntransport mechanism (Overstrom, 1996). If membrane integrity is compromised \nprobably by the embryotoxic factors, there will be a disruption in the ability to \nregulate cellular homeostasis such as pH and osmotic pressure that may result \nin developmental failure (Hansen, 2002). \nNucleus is important for DNA synthesis, which is required for protein synthesis. \nIn conjunction with the activation of the embryonic genome, there are major \nchanges occur in protein synthesis as the embryo growth. These changes are \nnecessary for cell division, compaction, blastocyst formation an9 hatching. \nThe presence study confirm the embryotoxicity of PF-E, however the \nmechanism of actions still remains unclear. The possible mechanisms of \nembryotoxicity is outline in Figure 4.8. The effects of intermediary metabolism \nand interleukins will be examined to elucidate further the mechanism of \nembryotoxicity in endometriosis. \n21 \n\nNucleus \n~ \nDisrupt protein \nsynthesis \nPF-E \n~ \nMacro phages \nl \nEmbryotoxic factor( s) \n~ \nMitochondria Plasma membrane \nl \nDisrupt intermediary \nmetabolism \nl \nJ ATP production \n~ \nDegenerated \nembryos \n~ \nDisrupt cellular \nhomeostasis \nFigure 4.8: Possible mechanisms of embryotoxicity in endometriosis \n22 \n\n5. PYRUVATE IN EMBRYO GROWTH AND POTENTIAL SUPPORT \nAGAINST THE EMBRYOTOXICITY OF PF-E \n5.1 Introduction \nCarbohydrates and amino acids are the most significant nutrients to the \ndeveloping human pre-implantation embryo (Devreker & Englert, 2000). The \npatterns of uptake and utilization of energy substrates such as pyruvate, lactate, \nglucose and glutamine have been described in pre-implantation embryos of \nmany mammalian species (Overstorm, 1996). \nIn order to have successful development of embryos in culture, it is important to \nconsider their nutrient requirements. According to Conaghan et a/., ( 1993), the \nnutrient that supplied in embryo culture media mainly at concentrations that are \nwell above those presence in vivo, however, excessive amounts of substrates \nmay be detrimental to embryo health or produce abnormal patterns of. \ndevelopment. Nutrient requirements for preimplantation embryo have been \nstudied mainly in experimental animals (Devreker & Englert, 2000). \nThe development competence of embryos in vitro is significantly reduced \ncompared to in vivo-produced embryos (Thompson, 2000), however, providing \nappropriate nutrients in the culture media can enhance the in vitro development. \nIn recent years, the focus of studies on nutrient requirement for energy sources \nin early embryos has been on consumption and utilization of exogenous \nsubstrates. However, the contribution of exogenous nutrients to the energy \nneeds is poorly understood. One of the nutrients that have been studied \n23 \n\n• \nextensively is pyruvate, an essential substrate for the early preimplantation \nmouse and human (Hardy eta/., 1989) . \nPyruvate is a three-carbon (triose) ketoacid, produced at the end stages of \nglycolysis. It acts as an energy source by being oxidized in the mitochondrion \n(Butcher eta/., 1998). The ATP production from this substrate involved the acid \ncitric cycle and oxidative phosphorylation (Thompson, 2000). Though pyruvate \nis an essential component of all the media used for human in vitro fertilization \n(Conaghan et a/., 1993), little is known regarding its role on early embryo \ngrowth and the mechanism by which it enters the embryo. Studies have shown \nthat pyruvate uptake by human embryos has been correlated with embryo \nviability and the ability to develop to the blastocyst stage (Hardy et a/., 1989). \nSeveral authors agreed that human embryo in the early stage (pre-compaction) \nhas an initial preference for pyruvate over glucose. as a nutrient (Hardy et a/., \n1989; Conaghan et a/., 1993). As development proceeds, the embryonic \nmetabolism shifts to utilize glucose as the primary source of ATP (Thompson, \n2000; Gardner eta/., 2002). In the routine culture of preimplantation embryos, a \nsingle culture media is used (Sakkas et a/., 1993), however, due to a difference \nin nutrient requirements, it is plausible that optimal development of the \nmammalian embryo in culture requires the two or more media since culture \nconditions that support excellent development of the blastocyst. probably \ndetrimental to the zygote (Gardner, 1998). Furthermore, a static environment \ndoes not allow for the metabolic and developmental changes as they have \nduring normal development (Sakkas eta/., 1993). \n24","source_license":"CC0","license_restricted":false}