Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium

Australian Veterinary Journal

Volume 86, No 8, August 2008 © 2008 CSIROJournal compilation © 2008 Australian Veterinary Association

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Blackwell Publishing Asia

Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium

J HILL

a,b

and R GILBERT

a

Objective

A suboptimal uterine environment contributes tothe bovine repeat breeder syndrome. Subclinical endometritisis a component, so the mechanism by which inflammationaffects embryo survival was investigated by assessing theeffect of non-cellular products of an inflamed endometrium onembryo development to blastocyst.

Design

Endometrial fluid from a lactating dairy cow wascollected by lavage using embryo culture medium. Asepticinflammation was then induced by infusion of glycogen andlavage was repeated 6 h later. The recovered fluid was used toculture Day 5 in-vitro-derived embryos for 2 days. Embryodevelopment and quality were compared for two treatmentgroups (culture media conditioned by inflamed or non-inflamedendometrium) and two controls (control or control + serum).

Results

Development to blastocyst was higher for conditionedmedia or media + serum (inflamed 42.2%; non-inflamed49.3%; control + serum 50.9%; control 16.9%; P < 0.001).Blastocyst cell numbers were lower for the conditioned-inflamedgroup (inflamed, 83.1; non-inflamed, 99.8; control + serum,100.6; control, 110.1; P < 0.001). Trophectoderm cell number,but not inner cell mass number, was reduced in the conditioned-inflamed group and the inner cell mass:trophectoderm ratiowas increased (P < 0.001).

Conclusion

Altering the embryo culture environment with theproducts of endometrial inflammation had a subtle effect onembryo quality. An increased inner cell mass:trophoblast ratiois likely to negatively affect embryonic survival. Altered embryoquality is a mechanism for early embryonic failure in cows withsubclinical endometritis. Culture of embryos with normalendometrial fluid may improve in vitro embryo production.

Key words:

bovine reproduction, endometritis, embryonic loss,in vitro fertilisation

Aust Vet J

2008;86:312–316 doi: 10.1111/j.1751-0813.2008.00326.x

IVF in vitro fertilisationSOF synthetic oviductal fluidFCS fetal calf serumFSH follicle-stimulating hormone

LH Luteinising hormone

N

ormal fertility requires a normal functioning reproductivesystem. In dairy cows, management, disease and geneticsare the broad factors that determine fertility. A suboptimal

uterine environment contributes to infertility through earlyembryonic loss. Clinical endometritis, with the appearance ofpurulent material at the external cervical os, or echogenic fluidon ultrasonography, is an obvious sign that the uterine environ-ment is hostile to a developing embryo and occurs commonlyin the cow (

≈

10% of dairy cows).

1–3

Subclinical, mild endometritisis more difficult to diagnose, requiring more invasive techniquessuch as culture, cytology or biopsy of the endometrium, and itseffect on fertility is less certain.

4

An impaired uterine environment is likely to be the primaryproblem of repeat breeder cows,

3

which show a high incidence ofembryonic death at Day 6 following fertilisation (i.e. shortlyafter entry of the embryo into the uterus) and the increased rateof pregnancy loss continues up until day 19.

5

Embryos derivedfrom normal cows fail to survive when transferred into the uterusof repeat breeders, whereas embryos derived from repeat breedersshowed normal survival rates following transfer into normalcows,

6

a result that indicates poor embryo quality is related to asuboptimal uterine environment.

As a preliminary study of how mild inflammation affects embryoquality, we investigated the effect of the inflamed endometriumon the development of embryos from Days 5 to 7. This periodwas selected because it is the time of entry of the embryo into theuterus and it is also thought to be the critical period for embryonicdeath.

7

The embryo culture media were conditioned by infusioninto either an inflamed or non-inflamed uterus to create mediathat had been exposed to endometrial products likely to be eitherbeneficial or detrimental to embryo development. Inflammationwas induced by intrauterine infusion of glycogen, which hasbeen found to reliably induce a mild, non-septic endometritis incows.

8

In vitro conditioning of embryo culture media is com-monly achieved by co-culture with somatic cells, but we usedthis novel in-vivo method of conditioning of embryo culturemedia. The major goal, as part of understanding the contributionof subclinical inflammation to bovine postpartum subfertility,was to investigate the effect of inflammatory uterine fluid onbovine embryos at the stage they would normally be entering theuterus (Day 5). A secondary objective was to investigate ifexposure of embryo culture medium to normal endometrial fluidresulted in a significant beneficial effect.

a

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, New York 14853-6401, USA

b

Present Address: University of Queensland, School of Veterinary Science, Brisbane, QLD 4072; [email protected]

avj_326.fm Page 312 Tuesday, July 22, 2008 11:29 AM

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Material and Methods

Conditioning of embryo culture medium

One pluriparous lactating Holstein cow was used in this study.Oestrus was detected visually and 7 days later in vivoconditioning of the embryo culture medium was performed.The horn ipsilateral to the corpus luteum was infused with30 mL of modified synthetic oviductal fluid (SOF) embryoculture medium

9

through an embryo transfer catheter, then 10to 20 s later recovery of the media began, with approximately70% of the solution recovered into a sterile syringe (i.e. non-inflamed lavage). Immediately after, glycogen (Sigma, St Louis,MO, USA; 20 mL of 1% solution) was infused intrauterinethrough the same catheter, which was then removed. Six hourslater, a second catheter was placed in the same horn and 30 mLof fresh embryo culture medium was infused, the horn wasmassaged and the media recovered (i.e. inflamed lavage).

Cytology was performed for both the non-inflamed andinflamed lavage to confirm normal cytology of the initial sampleand the presence of acute inflammation following glycogeninfusion. Samples of both the non-inflamed and inflamed lavage(200

µ

L) were placed into a cyto-tek chamber (Sakura) andcentrifuged at 15,000 rpm for 10 min in a cyto-tek centrifuge(Miles Scientific). Slides were stained with Diff-Quick (DadeDiagnostic) and observed at

×

200 magnification for neutrophils.

To remove the cellular inflammatory products, the non-inflamedand inflamed media were passed through a 0.5-

µ

m filter and theresulting fluid was diluted 1:2 with fresh embryo culturemedium to increase the volume to that required for subsequentculture experiments, before being passed through 0.2-

µ

m filterfor storage as aliquots at –20

°

C until use in the IVF experiments.The control SOF media were treated similarly.

In vitro embryo production

Ovaries from predominantly Holstein cattle were collected froma nearby abattoir. The fluid from follicles between 2 and 8 mmin diameter was aspirated into a 50-mL centrifuge tube thatcontained HEPES-buffered Medium 199 (Gibco LaboratoriesInc., Grand Island, NY, USA). Oocytes with multiple layers ofcumulus cells and homogeneous cytoplasm were selected formaturation for 22 h in Medium 199 supplemented with 10%v:v fetal calf serum (FCS; Gibco), 0.1 units/mL follicle-stimulatinghormone (FSH; Sioux Biochem), 0.1 units/mL luteinisinghormone (LH; Sioux Biochem), 1 mg/mL oestradiol (Sigma),0.1 mmol/L cysteamine (Sigma) and 1% penicillin–streptomycin.Maturation and fertilisation occurred in a 5% CO

2

in airatmosphere. Matured oocytes were fertilised with frozen–thawedbovine semen prepared by Percoll gradient separation: 50 oocyteswere incubated with 1.0

×

10

6

/mL spermatozoa in 500

µ

Lfertilisation medium, consisting of bicarbonate-buffered modifiedTyrode’s solution supplemented with heparin, penicillamine,hypotaurine and adrenaline (Sigma). After 20 h of culture, anyremaining cumulus cells were removed from fertilised oocytes byvortexing for 3 min in a HEPES-buffered version of the culturemedium and were then washed three times prior to culture.

Embryos were cultured for 5 days in SOF culture medium

9

ingroups of 5 to 7 per 50-

µ

L drop of culture medium overlaid withmineral oil, at 39

°

C in a 5% CO

2

, 7% O

2

, 88% N

2

atmosphere.

Treatments

On Day 5 after fertilisation (100 h in culture), embryos that hadreached at least the morula stage were selected and washed as agroup in HEPES–SOF. They were then allocated randomly tothe treatment groups in separate 30-mm dishes, in 50-

µ

L dropsunder mineral oil, with 5 to 7 embryos per drop. Embryos weretransferred in minimal medium using flame pulled pipettes.Treatment groups were (1) SOF conditioned by inflamedendometrium (inflamed), (2) SOF conditioned by non-inflamedendometrium (non-inflamed), and (3) SOF + 10% FCS (SOF +FCS). The SOF + FCS group was included because FCS iscommonly used in culture to improve embryo development.

Embryo evaluation

Embryo morphology was evaluated on Day 7 after fertilisation(144 h in culture) under a stereomicroscope (Leica) and theembryos were grouped according to stage (morula, compactmorula, blastocyst). Poor quality morulas/compact morulas(Grade 4) were classified as degenerate if there was loss of plasmamembrane integrity (lysis) and/or generalised loss of cell forms.

Objective evaluation of embryo development was by comparisonof cell counts for each morphological group. Total cell countswere performed by equilibration of embryos in 0.5 mg/mLHoechst 33342 for 30 min. Compact morulas, morulas anddegenerate embryos were then visualised under UV light using ablue/green filter. Blastocysts were differentially stained to showthe inner cell mass (ICM) and trophectoderm cells to enableevaluation of the respective total numbers, as well as the innercell mass:trophectoderm ratio. Differential staining was accom-plished by disrupting the membrane integrity of the surfacetrophectoderm cells with detergent (Triton X-100) and thenstaining with propidium iodide and Hoechst 33342,

10

whichpermits penetration of propidium iodide into the trophectodermcells, but not into the inner cell mass cells. Because all cells werestained with Hoechst 33342, the inner cell mass stained blue andthe trophectoderm red.

Differential staining was performed in 4-well dishes withapproximately 0.5 mL of HEPES-buffered embryo culturemedium. Prior to differential staining, embryos were equilibratedin 0.5 mg/mL Hoechst 33342 for 30 to 60 min. Embryos werepermeabilised for 4 min in HEPES–SOF containing 0.04%Triton, 1 mg/mL Hoechst 33342 and 1 mg/mL propidium iodide,then washed in HEPES–SOF containing 1 mg/mL Hoechst33342. During Triton X-100 exposure, embryos were gentlyswirled by hand to ensure good mixing. After permeabilisation,embryos were stained in 2 mg/mL propidium iodide in HEPES–SOF for 15 min, then mounted on a glass slide in a small (5

µ

L)drop of medium. A glass coverslip with two beads of vacuumgrease along its long-axis was laid over the drop, and pressureapplied to the coverslip at points along the bead of grease until


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the desired compression of the embryos was achieved. Theembryos were then visualised under ultraviolet light using ablue/green filter.

Statistical analysis

Data was analysed using SigmaStat statistical software (SPSS,Chicago, IL, USA). Development to Day 7 was assessed usingchi-square. Cell counts were compared using one-way ANOVAand the non-parametric Kruskall-Wallis test with Dunn’spairwise multiple comparison. P values < 0.05 were consideredsignificant.

Results

Morphology and development to blastocyst

A total of 1618 oocytes were fertilised over the course of thisexperiment. The mean cleavage rate following fertilisation was60.1%. The experiment was repeated seven times and a total of756 embryos were randomly allocated at Day 5 into the fourgroups. The overall development to blastocyst by Day 7 of all ofthe selected Day 5 embryos averaged 39.9% (302/756).

The culture of selected Day 5 embryos for 2 days in conditionedmedia (exposure to inflamed or non-inflamed endometrium)accelerated development to the blastocyst stage by Day 7 afterfertilisation compared with controls (Figure 1). A greater proportionof embryos cultured in both conditioned media and serum-containing media progressed to blastocyst than did embryoscultured in the control medium (inflamed 42.2%; non-inflamed49.3%, control + serum 50.4%; control 21%; chi square P < 0.001).The inflamed, non-inflamed and control + serum groups werenot significantly different in their proportions of Day 7 blastocysts(P = 0.22). There was no significant difference between anygroups in the proportions of degenerate or morula embryos,although the proportion of control morulas appeared to begreater than in the other groups (P = 0.055). No morphologicaldifference was noted between groups. Hatching rates wereassessed at Day 9 after fertilisation in a separate experiment andwere similar for those embryos that reached the blastocyst stagein each group (SOF 50%; non-Inflamed 75%; inflamed 60%;SOF + FCS 42%).

Objective cell counts

Culture of embryos in media conditioned by inflamedendometrium resulted in a lowered embryo quality comparedwith controls (as determined by a significant reduction in thetotal cell count) at each stage of development (Figure 2). Of mostimportance was the reduction in blastocyst total cell countscompared with the other three groups (Table 1). A reduction incell counts for embryos cultured in media containing serum wasobserved at the morula and compact morula stages whencompared with controls.

When blastocysts were differentially stained to permit countingof inner cell mass and trophectoderm cell numbers, the meantrophectoderm cell number was reduced in the inflamed group(Figure 3; P < 0.001). No difference in mean inner cell mass

Figure 1. Proportion of Day 5 embryos that reached each developmentalstage after culture for 2 days in media conditioned by endometrial lavage(inflamed/non-inflamed) or in control media. A higher proportion ofembryos cultured in either conditioned media or in the control + serummedia developed to blastocyst by Day 7 compared with controls. A greaterproportion of control embryos were at the compact morula stage at Day 7.*Significantly higher proportion (P < 0.001); **significantly lower proportion(P < 0.001).

Figure 2. When the quality of embryos at Day 7 was objectively measuredby total cell counts, embryos cultured in media conditioned by exposure toinflamed endometrium possessed significantly fewer cells than the controlgroup at each stage of morphological development. Embryos wereassessed for morphological stage at Day 7 after fertilisation, then fixed andstained for cell counting. *Significantly lower cell counts compared withcontrols (P < 0.01).


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numbers was found. The inner cell mass:trophectoderm ratiowas significantly elevated in the inflamed group, while the otherthree groups did not differ significantly from one another.

Discussion

This study investigated the effect of bovine endometrialinflammation on the development of in vitro embryos. Theconditioning of embryo culture media by prior infusion into abovine uterus enhanced development of Day 5 embryos to theblastocyst stage, whether culture medium was conditioned by aninflamed or a non-inflamed endometrium. However, embryoscultured in media conditioned by an inflamed endometriumshowed a reduction in blastocyst quality, with lower total cell andtrophectoderm numbers and a resultant increase in the inner cellmass:trophectoderm ratio, which would be expected to result inreduced embryo viability.

Differential staining for the inner cell mass and trophoblasts inblastocysts showed no difference in the inner cell mass counts,but there were fewer trophoblast cells in the inflamed group.Culture conditions that reduced the total number of cells or thenumber of inner cell mass cells in the pre-implantation embryoreduced the viability of embryos after transfer.

11

In the present

study, inner cell mass numbers remained the same between treat-ments, yet trophoblast numbers were reduced in the inflamedgroup, which suggests that the inflammatory products of aninflamed uterus preferentially inhibit the trophectoderm and it isthe trophectoderm that may protect the inner cell mass fromdamage by components such as inflammatory cytokines.

In the inflamed group, the ratio of inner cell mass to trophoblastwas increased. A reduction in trophoblast number would likelyaffect placental size and function. The placenta is the source ofthe bovine maternal recognition signal and a reduction in thequantity produced or an alteration in the time of productionwould likely reduce initial pregnancy rates. Lowered placentalefficiency would inhibit development throughout gestation,including the first trimester when rapid placental growth is requiredto establish the fetal supply of nutrients for later fetal growth.

The infusion of glycogen into the uterus results in an acute, butmild and transient inflammatory response, characterised primarilyby the influx of neutrophils,

8

which our data confirmed. Whenembryos were cultured in the non-cellular components of thisaseptic inflammation the effect was subtle, which may be relatedto only 2 days exposure to the conditioned media. Day 5embryos were used to mimic the time of entry of the bovineembryo into the uterus following fertilisation. No increase in thenumber of degenerate or dead embryos was found for theembryos cultured in inflamed media. With direct damage fromcomplement or inflammatory cells we would expect to see morelysed embryos in the inflamed group, but this did not happen,perhaps because of the filtering of the endometrial lavage toremove cells and also because of the use of a non-septic inflam-mation model. Additionally, the lavage effect may have dilutedthe toxic inflammatory products. The use of endometrial fluidderived from cows with normal or abnormal fertility may be a usefuldiagnostic and research tool in the future. In this study endometritiswas induced aseptically and an active bacterial endometritis islikely to have a much greater affect on embryo quality.

Not all cows with detectable endometrial inflammation areculture-positive for bacteria.

12

Cows that are positive for inflam-mation, but negative for bacteria, have a localised bacterialinfection or the organism is difficult to culture or the animal haseliminated the infection, leaving residual inflammation to persistfor an indeterminate length of time. Endometrial cytology usingsmall-volume lavage or the cytobrush technique may, therefore, be amore sensitive indicator of inflammation than endometrial biopsy.

13

In the present study the improvement in blastocyst rates wassimilar to that achieved using serum-supplemented media.Serum supplementation, together with coculture of embryoswith somatic cells, has been commonly used for development ofbovine embryos past the 8- to 16-cell stage to blastocyst insuboptimal embryo culture media.

14,15

Coculture either suppliesembryotrophic factors or filters out undesirable products in theculture media;

17,18

however, coculture of embryos with somaticcells or serum has been blamed for altering the phenotype of thefetus and offspring. In vitro embryo production in farm animals

Table 1. Cell counts from Day 7 blastocysts cultured in four types of media

Medium Total cell count

Inner cell mass

Trophectoderm Ratio ICM:Troph

Conditioned-inflamed 83.1* 30.1 53.0* 0.38*

Conditioned-non-inflamed 99.8 26.5 73.3 0.28

Culture medium + serum 100.6 27.6 73.0 0.29

Culture medium 110.1 27.2 82.9 0.26

*Significantly different from others within the column at P < 0.001.

Figure 3. Differential staining of the inner cell mass (ICM) and trophoblastcells in a Day 7 control blastocyst in which the ICM appears blue (Hoechst33342 stain) and the trophoblast cells pale red (propidium iodide; Bar = 50 µµµµm).


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has been associated with gestational abnormalities, such as anincrease in abortions and gestation length, altered organ weightsand birth weight, placental anomalies and reduced neonatalsurvival;

19–25

that is, the ‘large offspring syndrome’ (LOS).

22,26,27

The specific cause of LOS has not been elucidated, but the presenceof serum in the embryo culture medium results in a higher pro-portion of abnormal pregnancies and offspring, an effect that hasalso been seen with embryo coculture systems without serum. Ingeneral, it is thought to be a suboptimal culture environmentthat results in LOS,

28

so the early embryonic environment iscrucial for establishing the later fetal phenotype.

In our study the addition of both serum and endometrial fluidenhanced embryonic development. It is likely that endometrialfluid will promote development in a more physiological waythan serum, and further experiments are planned to test this.Uterine lavage fluid was obtained from cows 6 days followingovulation to synchronise the uterine environment with theembryo stage during in vitro culture. This fluid likely providesimportant additives such as growth factors, proteins, amino acidsand antioxidants. We did not study the specific componentsof the inflamed or non-inflamed fluid for concentrations ofglucose, amino acids, triglycerides and free fatty acids, each ofwhich is known to affect embryonic development. However, wehave shown that this system is a valid substitute for coculturewith somatic cells. The routine use of this system would requirecaution because there remains the risk of disease transmission,particularly of viruses. Current methods of culturing embryosuse media with defined components that are specificallydesigned to meet the changing needs of an embryo, so the needfor coculture or serum supplementation has been reduced,

29

buttheir use requires a high level of skill and quality control.

The present study demonstrated that the in vivo conditioning ofembryo culture media through brief exposure to a normal bovineendometrium improved the embryo development rate. Thedegree of improvement was similar to that achieved by addingserum to the culture media. A reduction in embryo quality wasobserved when embryos were cultured in media conditioned byexposure to an inflamed endometrium. In vivo conditioning ofembryo culture media may be useful for improving in vitroembryo production and for investigating the contribution of theuterine environment to subfertility in cows.

Acknowledgments

We thank Marcel Frajblat and Heather Roman for their verysignificant contributions in obtaining samples and conductingthe IVF procedures.

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(Accepted for publication 30 October 2007)


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Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium