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REPRODUCTIVE BIOLOGY
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L-carnitine supplementation reduces oocytecytoskeleton damage and embryo apoptosis inducedby incubation in peritoneal fluid from patients withendometriosisGihan Mansour, M.D.,a,b,c Hussein Abdelrazik, M.D.,a,d Rakesh K. Sharma, Ph.D.,a,b,d

Emad Radwan, M.D., Ph.D.,c Tommaso Falcone, M.D.,d and Ashok Agarwal, Ph.D.a,b,d

a Reproductive Research Center; b Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio; c Suez Canal

University Hospital, Ismailia, Egypt; and d Glickman Urological Institute and Kidney Institute, Cleveland Clinic, Cleveland, Ohio

Objective: To investigate the protective effect of L-carnitine (LC) against deleterious substances present in theperitoneal fluid (PF) of patients with endometriosis, which may affect the oocyte cytoskeleton and embryogenesis.Design: Experimental study.Setting: Research embryology laboratory at an academic hospital.Patient(s): Frozen metaphase II mouse oocytes and embryos.Intervention(s): One hundred metaphase II mouse oocytes were divided into five groups and incubated: PF fromendometriosis patients; PF from endometriosis patientsþ LC; PF from tubal ligation patients (patient control); LConly; and human tubal fluid (HTF) alone. A total of 180 eight-cell mouse embryos were divided into: endometriosisonly; tubal ligation only; endometriosis þ LC; LC alone; and HTF alone.Main Outcome Measure(s): Protective effect of LC on oocytes and embryos.Result(s): Incubation of the oocytes and the embryos with PF from patients with endometriosis statisticallysignificantly damaged the oocyte microtubules and chromosomes and increased embryo apoptosis comparedwith controls. Incubation with LC (0.6mg/mL) statistically significantly improved microtubule and chromosomestructure and decreased the level of embryo apoptosis.Conclusion(s): We propose the use of LC as a supplement in patients with endometriosis, a novel approach thatmay help improve in vitro fertilization outcome in these patients. (Fertil Steril� 2008;-:-–-. �2008 by Amer-ican Society for Reproductive Medicine.)

Key Words: L-carnitine, antioxidant, endometriosis, microtubules, chromosomes, oocytes, embryo

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COREndometriosis is diagnosed whenever endometrial tissue is

found outside the uterus. It is a common gynecologic disorderthat affects approximately 14% of all women, 30% to 50% ofinfertile women, and 84% of women with infertility and pel-vic pain (1, 2). Surgically induced endometriosis in rats resultsin decreased fecundity (3), and intraperitoneal injections ofperitoneal fluid (PF) from women with endometriosis intomice lowers the implantation rate (4). Moderate or severe en-

UJanuary 10, 2008; revised and accepted February 7, 2008.

at the 63rd annual meeting of the American Society of Repro-

Medicine, Washington, DC, October 13–17, 2007.

support provided by the Cleveland Clinic Research Programs

ttee (RPC 2006-1135).

nothing to disclose. H.A. has nothing to disclose. R.K.S.has

to disclose. E.R. has nothing to disclose. T.F. has nothing to

. A.A. has nothing to disclose.

quests: Ashok Agarwal, Ph.D., HCLD, Professor and Director,

ctive Research Center, Staff, Department of Obstetrics and

logy and Glickman Urological and Kidney Institute, Cleveland

500 Euclid Avenue, Desk A19.1, Cleveland, Ohio (FAX:

-3118/216-445-6049; E-mail: [email protected]).

2/08/$34.0016/j.fertnstert.2008.02.097 Copyright ª2008 American S

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dometriosis causes infertility due to mechanical disruption ofovulation or efficient gamete transport (5). Even in its mildestforms, endometriosis may contribute to infertility (6).

The PF microenvironment has an important role in thepathogenesis and progression of endometriosis; the increasednumber and activation of peritoneal macrophages and in-creased concentrations of inflammatory cytokines such as in-terleukin-6 (IL-6), IL-1, and tumor necrosis factor-a (TNF-a)lead to increased vascularization and mitotic activity andstimulate the growth of ectopic endometrium (7–10).

Another pathophysiologic cause of endometriosis-associ-ated infertility is the presence of high levels of reactive oxygenspecies (ROS) in the PF. Oxidative stress caused by ROS mayplay a role in the development and progression of endometri-osis (11). Increased generation of ROS by PF macrophages,with increased lipid peroxidation in patients with endometri-osis, has been demonstrated, although other researchers havereported contrary findings (12). Diminished PF antioxidants

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(13), elevated oxidized lipoproteins (12), lysophosphatidylcholine (13), and other markers of lipid peroxidation providefurther evidence of oxidative stress in the peritoneal microen-vironment of patients with endometriosis (14).

The oocyte spindle is a dynamic structure composed ofmicrotubule bundles that are polar polymers of a-tubulinand b-tubulin heterodimers. The spindle is responsible foroocyte meiotic division. During the second meiotic division,the chromosomes are equatorially localized in the meioticspindle of the oocytes (15).

In a study done by our group, exogenous exposure of meta-phase II mouse oocytes to hydrogen peroxide (H2O2) causeddamage in spindle structure, as evident by changes in microtu-bule morphology and alterations in chromosomal alignment.Significantly increased damage was seen with increased dura-tion of incubation. Higher damage also was seen after expo-sure to both TNF-a alone and in combination with H2O2

compared with controls. Moreover, oocytes incubated withH2O2 and vitamin C as an antioxidant demonstrated less dam-age compared with those incubated with H2O2 alone (16).

L-carnitine (LC) is a small, water-soluble molecule thatplays a very important role in fat metabolism. It is essentialfor the normal mitochondrial oxidation of fatty acids and ex-cretion of acyl-CoA esters and affects adenosine triphosphate(ATP) levels (17). L-carnitine can stabilize mitochondrialmembranes, increase the supply of energy to the organelle,and protect the cell from apoptotic death (18). Reduction ofapoptosis through the mitochondrial pathway by the adminis-tration of LC to mouse fibroblasts in culture media has beendemonstrated (18). In a recent study done by our group, we re-ported that supplementation of the culture media with LC (0.6mg/mL) statistically significantly reduced the apoptosis levelin mouse embryos treated with actinomycin-D (apoptosis-in-ducing factor). Supplementation of the culture media with LCat 0.6mg/mL antagonized the oxidative effect of a very highdose of H2O2 (500 mM). In addition, LC was able to neutralizethe antiproliferative effect of TNF-a and significantly reducedthe level of DNA damage in embryos (19).

Our study objective was to investigate the protective effectof LC against the toxic effects (cytokines or oxidative stress)of PF in patients with endometriosis and subsequently to ex-amine oocyte cytoskeleton changes using metaphase II mouseoocytes or early embryo development using eight-cell mouseembryos. Antagonizing these deleterious factors may help im-prove oocyte and embryo quality, thereby improving in vitrofertilization (IVF) outcomes in patients with endometriosis.

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MATERIALS AND METHODS

The study was approved by the Cleveland Clinic institutionalreview board.

Patients

The study included a cohort of 38 female patients who under-went laparoscopy at the Cleveland Clinic from March 2006 toMarch 2007. Patients were classified into two groups, those

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with endometriosis (n¼ 23) and a control group consisting ofpatients with tubal ligation or tubal ligation reversal (n¼ 15).The indications for laparoscopy were chronic pelvic pain,infertility or both, tubal ligation, or sterilization reversal. Allpatients included in the study had no significant comorbiditiesexcept for the primary indication of surgery. After obtaininginformed consent, intraoperative peritoneal samples werecollected. Of the 43 women who contributed their PF, fivewere excluded because of blood-contaminated PF. All patientsincluded had general anesthesia using the same approach.

EDPROOFPeritoneal Fluid Preparation

During laparoscopy, PF was collected from the posterior cul-de-sac. Peritoneal fluid cellular constituents were removed bycentrifugation at 600� g for 5 minutes. The supernatant wasthen collected and stored at �70�C. All samples were col-lected under identical conditions.

Mouse Oocyte Preparation

Cryopreserved metaphase II mouse oocytes were obtainedfrom Embryotech Laboratories, Inc. (Wilmington, MA).For thawing, each straw was removed from its liquid nitrogencontainer and placed at room temperature for 2 minutes. Thestraw was cut, and the oocytes were released into a Petri dishcontaining 500 mL of Dulbecco’s phosphate buffer saline(PBS; Irvine Scientific, Santa Ana, CA) and kept for 5 min-utes. Subsequently, they were transferred into another Petridish containing 500 mL of PBS to equilibrate for 10 minutesat room temperature. Oocytes were then incubated in 500 mLof human tubal fluid (HTF; Irvine Scientific) for 1 hour in 5%CO2 at 37�C to ensure complete repolymerization of themicrotubules before transfer into the PF (16).

L-Carnitine Preparation

In our recent study conducted on mouse embryos (19), wedemonstrated that LC at 0.6 mg/mL was able to reduce apo-ptosis, act as an antioxidant, and antagonize the antiprolifer-ative effect of TNF-a. The LC was diluted 1:1 with either PFfrom patients with endometriosis or with HTF to give an LCconcentration of 0.6 mg/mL.

Experiment 1: Effect of Incubation of Oocytes with thePeritoneal Fluid on Spindle Structure

A total of 100 metaphase II oocytes were randomly dividedinto five groups: PF from endometriosis patients; PF from en-dometriosis patients þ LC; PF from tubal ligation patients(patient control); LC only; and HTF only. Oocytes were eval-uated under the microscope for maturity (presence or absenceof the polar body) and incubated in HTF for 1 hour for com-plete repolymerization. Peritoneal fluid was diluted with HTFmedia (1:1).

Microtubules and chromosomal staining Microtubules weredetected by modified indirect immunocytochemical techni-ques as reported in our earlier study. For microtubule staining,oocytes were placed in fixation solution (2% formaldehyde,

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0.2% Triton X-100) in 500 mL of PBS for 30 minutes and thenincubated in anti-a-tubulin monoclonal antibody (Sigma-Aldrich, St. Louis, MO) (1:300) for 60 minutes, followed byincubation in fluorescein isothiocyanate (FITC)–labeledanti-mouse antibody (Sigma-Aldrich) (1:50) for 30 minutes.For chromosome staining, oocytes were incubated in 10 mg/mLof propidium iodide (Sigma-Aldrich) for 15 minutes (16, 20).

Each staining step was followed by a minimum of threerinses in PBS. Five oocytes were loaded on a slide in a micro-drop (2 mL) of Antifade (Slow Fade Light Antifade; Molec-ular Probes, Inc., Eugene, OR) as anti-bleach and werecovered with a cover slip. Alterations in the microtubulestructure were observed both by epifluorescence microscopyusing the blue filter (excitation 450–490, suppression 515) formicrotubules and green filter (excitation 546, suppression580) for chromosomal alignment. Slides were stored at�20�C in the dark until they were evaluated for more detailsusing confocal microscopy.

Confocal microscopic analysis and scoring of microtubulesand chromosomes Slides were examined using a laser-scanning confocal microscope Leica TCSSP2 (Leica Laser-technik, GmbH, Heidelberg, Germany) equipped with anargon ion laser for the excitation of FITC for microtubulesand propidium iodide for chromosome (excitation 488 nm,barrier 500–555 nm for FITC; excitation 568 nm, barrier575–675 nm for propidium iodide). Microtubule distri-bution and chromosome alignment for each oocyte wereexamined.

Scoring of microtubules and chromosomes according to themorphologic evaluation was done by the method describedearlier elsewhere by our group (16). In brief, spindle morphol-ogy was classified as normal (scores 1, 2) when a barrel-shaped structure with slightly pointed poles was formed byorganized microtubules; score 3 when we found a reductionin the longitudinal dimension of the spindle; or score 4when there was partial or total disorganization, complete ab-sence, or remnant of dispersing spindle (16). Chromosomalconfiguration was regarded as normal (scores 1, 2) when chro-mosomes were arranged in a compact metaphase plate at theequator of the spindle. Chromosomal organization was re-garded as abnormal (scores 3, 4) when chromosomes weredisplaced from the plane of the metaphase plate or when thechromosomes were dispersed or appeared condensed.

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Experiment 2: Effect of Incubation of Mouse Embryos withPeritoneal Fluid and L-Carnitine on Apoptosis

A total of 180 eight-cell embryos were divided into endome-triosis only; tubal ligation only; endometriosis þ LC; LCalone; and HTF alone. They were incubated in a 37�C and5% CO2 incubator for 24 hours. Embryos were fixed informaldehyde 3.7%. The DNA damage of the embryos wasestimated by the terminal deoxynucleotidyl transferase(TdT) -mediated dUTP nick-end labeling (TUNEL) assay.

Measurement of apoptosis in embryos Individual embryoswere stained with the TUNEL technique (in situ cell death

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detection system; Roche Diagnostic Corporation, Indianapo-lis, IN). After we had washed the embryos in PBS, they werefixed in 3.7% paraformaldehyde in PBS (pH 7.4) for 1 hour atroom temperature. Embryos were washed at least three timesin PBS containing 0.3% polyvinylpyrrolidone (PBS/PVP)and permeabilized in 0.5% Triton X-100 on ice for 2 minutes.The embryos were then washed three times in PBS/PVP andincubated in TUNEL reaction cocktail at 37�C for 1 hour inthe dark. Negative controls consisted of embryos incubatedwithout terminal transferase enzyme. The embryos werewashed extensively and mounted with slight cover slip com-pression in Vectashield with 40,60-diamidino-2-phenylindolehydrochloride (DAPI) antibleaching solution (Vector Labs,Burlingame, CA). The slides were sealed with clear nailpolish and stored at�20�C in the dark for analysis by confocalmicroscope. The TUNEL positive (apoptotic) nuclei (cell)appeared as green, and the normal chromatin appeared asblue.

Confocal microscopy for apoptosis Images were collectedwith a Leica TCS-SP2 laser scanning spectral confocalmicroscope (Leica Lasertechnik, GmbH, Heidelberg, Ger-many). The specimen was excited at 364 nm (ultraviolet)for DAPI and at 488 nm for visualization of TUNEL staining.Images were collected sequentially at each level of thespecimen to prevent crosstalk of the fluorophores and thenwere collected along the z-axis of the sample with a stepsize of 1 to 3 mm. Each optical section of the blastocystwas analyzed for TUNEL-negative nuclei stained withDAPI and TUNEL-positive nuclei stained with DAPI andTUNEL.

The percentage of apoptotic cells in each embryo was cal-culated by using the projection of the three-dimensional stackof images that was created with the Leica software. The orig-inal stack of embryo images was transferred to Velocity soft-ware (Improvision, Lexington, MA) for analysis and forcounting of both damaged and intact individual blastomereDNA. This software allows penetration of the scanned em-bryo to observe the TUNEL staining and verify whethereach blastomere is independently stained. The apoptoticblastomeres in each embryo were counted, and the level ofapoptosis in each group was calculated.

Differences between samples and controls were assessedusing the Kruskal-Wallis test for overall group comparisonsand the Wilcoxon rank sum test for pairwise comparisons.Summary statistics are presented as frequency and percentfor categorical data and as median and interquartile range(25th and 75th percentile) for quantitative data. All hypothe-sis testing was two-tailed. P<.05 was considered statisticallysignificant. Statistical analyses were performed using R ver-sion 2.3.1 (http://www.R-project.org).

RESULTS

The results on the effect of PF and LC on oocyte spindle andembryo apoptosis are shown in Tables 1 to 3 and Figures 1and 2.

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TABLE 1Microtubule scores after incubation of oocytes with peritoneal fluid (PF) from endometriosis, tuballigation (control), L-carnitine (LC) alone, carnitine supplementation, and the human tubal fluid (HTF)control group.

Microtubules

Treatment Score 1 Score 2 Score 3 Score 4 P value

Endometriosis PF, group 1a,b,c,d 2 (10%) 3 (15%) 6 (30%) 9 (45%) < .001Tubal ligation PF (patient control), group 2e,f,g 6 (30%) 12 (60%) 1 (5%) 1 (5%)Endometriosis PF þ LC (0.6 mg/mL), group 3h,i 4 (20%) 9 (45%) 5 (25%) 2 (10%)LC alone (0.6mg/mL), group 4j 6 (30%) 10 (50%) 3 (15%) 1 (5%)HTF alone, group 5 7 (35%) 11 (55%) 1 (5%) 1 (5%)

Notes: Each group consisted of 20 oocytes. Overall P¼ .001, using Kruskal-Wallis pairwise test. P< .05 was consideredstatistically significant.

a P< .001 for group 1 vs. 2.b P¼ .009 for group 1 vs. 3.c P¼ .001 for group 1 vs. 4.d P< .001 for group 1 vs. 5.e P¼ .13 for group 2 vs. 3.f P¼ .70 for group 2 vs. 4.g P¼ .78 for group 2 vs. 5.h P¼ .28 for group 3 vs. 4.i P¼ .09 for group 3 vs. 5.j P¼ 0.53 for group 4 vs. 5.

Mansour. Effect of L-carnitine on oocytes and embryos. Fertil Steril 2008.

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Experiment 1: Effect of Incubation of Oocytes with thePeritoneal Fluid on Spindle Structure

Our study showed that incubation of the oocytes in LC ata concentration of 0.6 mg/mL did not cause a statistically sig-nificant effect on the oocyte microtubules or chromosomescores, and these were similar to tubal ligation controls.Both microtubule and chromosome scores were statisticallysignificantly decreased (>2) in oocytes incubated with PFfrom patients with endometriosis compared with the oocytesincubated in PF from patients with tubal ligation alone(P<.001 and P¼.006) (see Tables 1 and 2).

Statistically significant improvement in the microtubulescore was seen when 0.6 mg/mL of LC was added to thePF from endometriosis patients (P¼.009). Oocytes incubatedin PF from patients with endometriosis plus LC demonstrateda decrease in chromosomal changes (P¼.06). Generally,there was a decrease in chromosomal changes between pa-tients with endometriosis and other patient groups (overallP¼.032; .003%P%.06 for endometriosis vs. each of theother groups) (see Tables 1 and 2; Fig. 1).

Experiment 2: Effect of Incubation of Mouse Embryos withPeritoneal Fluid and L-Carnitine on Apoptosis

Our study showed that incubation of the embryos with PFfrom patients with endometriosis statistically significantlyincreased the level of apoptosis compared with the tuballigation group control (P<.001). The extent of apoptosiswas comparable in embryos incubated in LC (0.6 mg/mL)


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and tubal ligation controls. Statistically significant improve-ment in the level of apoptosis was seen when LC (0.6 mg/mL)was added to the PF from endometriosis patients (P<.001)(see Table 3; Fig. 2).

DISCUSSION

Reactive oxygen species are involved in the etiopathogenesisof defective embryo development (21) and retardation ofembryo growth (5). An increase in ROS production leads toarrest of embryo development. Reactive oxygen speciesmay originate in the embryo or from extraneous factors. Inthe in vitro fertilization (IVF) setting, strategies to reduceROS production, such as addition of free radical scavengersand lowering the oxygen tension, are important for improvingthe fertility potential in assisted reproduction (22). Reactiveoxygen species induce cell membrane damage, DNA dam-age, and apoptosis. Apoptosis results in fragmented embryos,which have limited potential to implant and hence result inpoor fertility outcomes (23).

In a study evaluating the possible beneficial effects of LCon tissue injury and oxidative stress in acetic acid-inducedcolitis in rats, results showed that acetic acid administrationsignificantly decreased reduced glutathione, superoxide dis-mutase, and catalase levels in colonic homogenate. Supple-mentation of LC prevented the depletion of free radicalscavengers such as reduced glutathione levels and causeda statistically significant increase in superoxide dismutaselevels (24).

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TABLE 2Chromosome scores after incubation of oocytes with peritoneal fluid (PF) from endometriosis, tuballigation (control), L-carnitine (LC) alone, carnitine supplementation, and the human tubal fluid (HTF)control group.

Chromosomes

Treatment Score 1 Score 2 Score 3 Score 4 P value

Endometriosis PF, group 1a,b,c,d 2 (10%) 4 (20%) 8 (40%) 6 (30%)Tubal ligation PF, (patient control), group 2e,f,g 5 (25%) 11 (55%) 2 (10%) 2 (10%) .032Endometriosis PF þ LC (0.6 mg/mL), group 3h,i 6 (30%) 7 (35%) 3 (15%) 4 (20%)LC alone (0.6mg/mL), group 4j 4 (20%) 9 (45%) 4 (20%) 3 (15%)HTF group, group 5 5 (25%) 11 (55%) 3 (15%) 1 (5%)

Notes: Each group consisted of 20 oocytes. Overall P¼ .032, using Kruskal-Wallis pairwise test. P< .05 was consideredstatistically significant.

a P¼ .006 for group 1 vs. 2.b P¼ .06 for group 1 vs. 3.c P¼ .052 for group 1 vs. 4.d P¼ .003 for group 1 vs. 5.e P¼ .65 for group 2 vs. 3.f P¼ .39 for group 2 vs. 4.g P¼ .95 for group 2 vs. 5.h P¼ .79 for group 3 vs. 4.i P¼ .58 for group 3 vs. 5.j P¼ .34 for group 4 vs. 5.


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L-carnitine was reported to down-regulate cytokines suchas IL-1, IL-6, and TNF-a and/or increase clearance of thesecytokines in rats implanted subcutaneously with sarcoma tu-mor. A statistically significant reduction in the concentrationsof the cytokines (IL-1b: 423 � 33 vs. 221 � 60; IL-6: 222 �18 vs. 139 � 38; TNF-a: 617 � 69 vs. 280 � 77 pg/mL;P%.05) was seen in the sarcomas groups that were untreatedversus those treated with LC (25).

UNCORTABLE 3

Level of apoptosis after incubation of mouse embryosfrom endometriosis, tubal ligation (control), L-carnit

TreatmentAp

(25th

Endometriosis PF (n ¼ 40)Tubal ligation (patient control) (n ¼ 40)Endometriosis PF þ LC (0.6 mg/mL) (n ¼ 40)LC alone (0.6 mg/mL) (n ¼ 40)HTF (n ¼ 20)

a P< .05 was considered statistically significant for apoptoscompared with the control group.

b P< .05 was considered statistically significant for differencrank-sum test compared with the endometriosis group.



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Recently, we demonstrated the possible role of LC inimproving the outcome of IVF. We reported that incubationof two-cell mouse embryos with 500 ng/mL of TNF-a statis-tically significantly decreased the blastocyst developmentrate (%BDR) compared with the control group (P<.001).L-carnitine at 0.6 mg/mL was able to neutralize the anti-proliferative effect of TNF-a and improve the %BDR. Incu-bation of mouse embryos in 500 mmol/L H2O2 alone

with human tubal fluid (HTF), peritoneal fluid (PF)ine (LC) alone, and carnitine supplementation.

Apoptosis (%)

optosis (%) Medianand 75th percentiles) P valuea P valueb

39.1 (30.3, 46) < .001 —6.7 (3, 10) — < .0016.2 (3.3, 10) .80 < .0013.5 (0, 8.2) .13 < .0013.9 (0, 6.9) .036 < .001

is level using Kruskal-Wallis or Wilcoxon rank-sum test

es in apoptosis level using Kruskal-Wallis or Wilcoxon

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FIGURE 1

Effect of incubating metaphase II mouse oocytes with peritoneal fluid from tubal ligation patients as patientcontrol, L-carnitine (LC) alone, and peritoneal fluid from endometriosis patients with and without LC. (A) Mouseoocyte incubated with tubal ligation patients. (B) Mouse oocytes incubated in LC alone. (C) Mouse oocyteincubated in peritoneal fluid from endometriosis patients. Both microtubule and chromosome scores werestatistically significantly decreased (>2) in oocytes incubated with peritoneal fluid from patients withendometriosis compared with the oocytes incubated in peritoneal fluid from patients with tubal ligation alone(P< .001 and P¼ .006, respectively). (D) Mouse oocyte incubated in peritoneal fluid from endometriosis patientsand supplemented with 0.6 mg/mL of LC. Statistically significant improvement in the microtubule score was seenwhen 0.6 mg/mL of LC was added to the peritoneal fluid from endometriosis patients (P¼ .009). Oocytesincubated in peritoneal fluid from patients with endometriosis and LC demonstrated a decrease in chromosomalchanges (P¼ .06). Scale of magnification bar ¼ 10 mm.

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statistically significantly decreased the %BDR and incre-ased the level of apoptosis (P<.001 and P<.01) comparedwith the control group. L-carnitine exhibited strong anti-oxidant effects as it was able to antagonize a very highconcentration of H2O2 (up to 500 mmol/L). At 0.6 mg/mL,LC statistically significantly improved the %BDR (P<.001)and decreased the level of apoptosis (P<.007) com-pared with the group treated with H2O2 alone (500 mmol/L)(19).


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Endometriosis is characterized by changes in the intrafol-licular and PF environment and increased levels of certaincytokines such as TNF-a and ROS, which affect oocytesand embryo development. In our earlier study, we investi-gated the effect of oxidative stress on metaphase II mouseoocyte spindle structure. Significant alterations in microtu-bule chromosomal alignment were reported after exposureto high concentration of H2O2. Exposure of the oocytes toboth TNF-a alone and in combination with H2O2 resulted

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FIGURE 2

Effect of incubating eight-cell mouse embryos with peritoneal fluid from tubal ligation patients as patient control,L-carnitine (LC) alone, and peritoneal fluid from endometriosis patients with and without LC. (A) Mouse embryosincubated in peritoneal fluid from tubal ligation patients (control group). (B) Mouse embryos incubated in LCalone. (C) Mouse embryos incubated in peritoneal fluid from endometriosis patients. Incubation of the embryoswith peritoneal fluid from patients with endometriosis statistically significantly increased the level of apoptosiscompared with the tubal ligation group control (P¼ .036). (D) Mouse embryos incubated in peritoneal fluid fromendometriosis patients and supplemented with 0.6 mg/mL of LC. Statistically significant improvement in the levelof apoptosis was seen when LC (0.6 mg/mL) was added to the peritoneal fluid from endometriosis patients(P< .001). Scale of magnification bar ¼ 10 mm.

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in a concentration-dependent and time-dependent increase inspindle compared with controls (16).

These results are in agreement with our present findingswhere we have demonstrated that incubation of oocyteswith PF from patients with endometriosis statistically signif-icantly increased the microtubule and chromosome scorescompared with the oocyte group incubated in PF from pa-tients with tubal ligation (P<.001 and P<.006, respective-ly).We attribute this to the high concentration of ROS (26)and TNF-a or other cytokines that have been reported to behigher in patients with endometriosis (27). Statistically


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significant improvement in the microtubule score was seenwhen 0.6 mg/mL of LC was added to the endometriosis PF

(P<.001). Oocytes incubated in PF from patients with endo-

metriosis and LC demonstrated a decrease in chromosomal

alignment (P¼.06). This decrease was statistically significant

for the overall endometriosis group versus all other groups

(P¼.032). The improvement in microtubule and chromo-

some alignment after addition of LC may be due to the strong

antioxidant properties of LC. Additionally, it may be the

result of the down-regulation of the cytokines that are known

to be present in the PF of endometriosis patients.

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The oocyte spindle is responsible for oocyte meiotic divi-sion (15). Our results may explain the significant reductionin the oocyte cleavage rate in women with endometriosiscompared with controls (28). This could be explained by del-eterious effects on the oocyte spindle in patients with endo-metriosis.

The embryotoxicity of PF from patients with and withoutendometriosis has been studied before, but the results havebeen conflicting. Although some investigators have demon-strated that the PF from individuals with endometriosis isnot embryotoxic when studied in an in vitro mouse embryomodel (29, 30), others have shown that embryotoxicity is in-creased in women with endometriosis (31). These conflictingresults may be due to differences in the PF concentration,severity of the disease, incubation time, or type of culturemedia (32).

Our results showed that incubation of the preimplantationmouse embryos with PF from patients with endometriosisstatistically significantly increased the level of apoptosiscompared with the controls (P<.001). Statistically significantimprovement in the apoptosis level was seen after adding 0.6mg/mL of LC to the PF from endometriosis patients(P<.001). We also reported earlier that PF from patientswith endometriosis decreases the development of early em-bryogenesis in mouse embryos but does not increase the levelof apoptosis (22). Again, this may be due to the difference inconcentration of PF in these studies.

Oxygen-controlled incubators have been introducedrecently in clinical embryology procedures, and these mayhelp reduce the harmful effects of free radicals and therebyimprove blastocyst rates. However, LC works with multiplemechanisms; one of them is by antagonizing ROS formationthrough its potent antioxidant effect. In addition, LC also candecrease the level of apoptosis in the presence of an apoptoticinducer and decrease the antiproliferative effect induced bythe presence of cytokines such as TNF-a (19).

In conclusion, improvement in oocyte scores and embry-onic developmental competence may be accomplished byaddition of LC to the PF from patients with endometriosis.This effect of LC may be due to reduction in the extent ofDNA damage or potent antioxidant and anti-TNF-a effects.It would be interesting to examine the association betweenthe severity of endometriosis and the protective effect ofsupplementing the IVF media with LC. This may resultin an improvement in the oocyte spindle structure and,subsequently, improvement in embryo quality. The use ofLC as a supplement is a novel approach that can have im-portant clinical applications in the assisted reproductionsetting, and its use may improve the fertility outcomes ina category of patients in which the IVF outcome is still un-satisfactory.

899900901902

Acknowledgment: The authors thank Dr. Judith Drazba; director of the Imag-

ing Core Laboratory, Lerner Research Institute, Cleveland Clinic, for her

valuable support. Jeff Hammel, M.S., provided statistical assistance.


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1 L-carnitine supplementation reduces oocytecytoskeleton damage and embryo apoptosisinduced by incubation in peritoneal fluid frompatients with endometriosis

G. Mansour, H. Abdelrazik, R. K. Sharma, E. Radwan,T. Falcone, and A. AgarwalCleveland, Ohio; and Ismailia, Egypt

L-carnitine supplementation protects the oocyte spin-dle structure and reduces apoptotic effects on em-bryos exposed to peritoneal fluid from endometriosispatients.

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Mansour et al. Effect of L-carnitine on oocytes and embryos Vol. -, No. -, - 2008

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article in press - cleveland clinicuncorrected proof reproductive biology l-carnitine...

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